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Nikolaitchik OA, Chameettachal A, Islam S, Cheng Z, Delviks-Frankenberry K, Keele BF, Pathak VK, Hu WS. How to recover from a bad start: adaptation of HIV-1 transcription start site mutants during serial passaging in culture. J Virol 2025; 99:e0015925. [PMID: 40331822 DOI: 10.1128/jvi.00159-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2025] [Accepted: 03/14/2025] [Indexed: 05/08/2025] Open
Abstract
HIV-1 uses neighboring sequences as transcription start sites and generates multiple unspliced transcripts, including two major transcripts with three guanosines (3G) or one guanosine (1G) at the 5' end. Although only differing by 2-nt, 3G RNA and 1G RNA are functionally distinct. We have previously generated two mutants: the TTG virus mainly expresses 1G RNA, whereas the plusAC virus predominantly expresses 3G RNA. Both mutant viruses are replication-competent but exhibit fitness defects. Here, we passaged the plusAC virus in T cells and characterized the changes near the transcription start sites. We observed the rapid loss of the plusAC virus genotype and the emergence of multiple revertants. All major revertants that dominated the cultures had a 1- to 3-nt deletion that compensated for the dinucleotide insertion in the plusAC virus. These major revertants express more than one major transcript, preferentially package 1G RNA, and have improved replication kinetics compared with the plusAC virus. Most major revertants likely arose through errors during reverse transcription, including misalignments during minus-strand DNA transfer, nucleotide deletion in a homopolymer run, or deletion of a short direct repeat. Additionally, we have determined that a T-to-G substitution near transcription start sites occurs at ~5% per replication cycle by copying the guanosine cap. Converting a base to guanosine through cap copying has been observed in multiple positions, but always directly upstream of a major transcription start site. Taken together, our findings demonstrate the selection pressure for expressing functionally distinct unspliced RNA species to optimize replication fitness.IMPORTANCEHIV-1 unspliced RNA serves as the mRNA to translate Gag/Gag-Pol polyproteins and as the virion genome. HIV-1 produces two major RNA species: 1G RNA is preferentially packaged and 3G RNA is favorably translated, although each transcript can perform both functions. We have previously generated a replication-competent mutant virus that mainly expresses 3G RNA and observed that this mutant has replication fitness defects. We found that the mutant virus improved its replication kinetics after passaging, indicating adaptation. Our analyses showed that, through mutations occurring during DNA synthesis, multiple revertants arose rapidly to replace the input mutant virus. The major revertants regained the ability to generate more than one major transcript and preferentially package 1G RNA. These results highlight the importance of expressing HIV-1 RNA species that serve distinct functions and the ability of HIV-1 to adapt through mutations in the genome.
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Affiliation(s)
| | | | - Saiful Islam
- Viral Recombination Section, Frederick, Maryland, USA
| | - Zetao Cheng
- Viral Recombination Section, Frederick, Maryland, USA
| | - Krista Delviks-Frankenberry
- Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute at Frederick, Frederick, Maryland, USA
| | - Brandon F Keele
- AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA
| | - Vinay K Pathak
- Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute at Frederick, Frederick, Maryland, USA
| | - Wei-Shau Hu
- Viral Recombination Section, Frederick, Maryland, USA
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2
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Greber UF. Clicking viruses-with chemistry toward mechanisms in infection. J Virol 2025; 99:e0047125. [PMID: 40366176 DOI: 10.1128/jvi.00471-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/15/2025] Open
Abstract
Viruses subvert cells and evade host defense. They emerge unpredictably and threaten humans and livestock through their genetic and phenotypic diversity. Despite more than 100 years since the discovery of viruses, the molecular underpinnings of virus infections are incompletely understood. The introduction of new methodologies into the field, such as that of click chemistry some 10 years ago, keeps uncovering new facets of viruses. Click chemistry uses bio-orthogonal reactions on chemical probes and couples nucleic acids, proteins, and lipids with tractable labels, such as fluorophores for single-cell and single-molecule imaging, or biotin for biochemical profiling of infections. Its applications in single cells often achieve single-molecule resolution and provide important insights into the widely known phenomenon of cell-to-cell infection variability. This review describes click chemistry advances to unravel infection mechanisms of a select set of enveloped and nonenveloped DNA and RNA viruses, including adenovirus, herpesvirus, and human immunodeficiency virus. It highlights recent click chemistry breakthroughs with viral DNA, viral RNA, protein, as well as host-derived lipid functions in both live and chemically fixed cells. It discusses new insights on specific processes including virus entry, uncoating, transcription, replication, packaging, and assembly and provides a perspective for click chemistry to explore viral cell biology, infection variability, and genome organization in the particle.
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Affiliation(s)
- Urs F Greber
- Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
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3
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Chaudhuri E, Jang S, Chakraborty R, Radhakrishnan R, Arnarson B, Prakash P, Cornish D, Rohlfes N, Singh PK, Shi J, Aiken C, Campbell E, Hultquist J, Balsubramaniam M, Engelman AN, Dash C. CPSF6 promotes HIV-1 preintegration complex function. J Virol 2025; 99:e0049025. [PMID: 40202316 PMCID: PMC12090733 DOI: 10.1128/jvi.00490-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2025] [Accepted: 03/25/2025] [Indexed: 04/10/2025] Open
Abstract
Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as an HIV-1 capsid (CA) binding host factor to promote viral DNA integration targeting into gene-dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC)-the sub-viral machinery that carries out viral DNA integration-are unknown. To study CPSF6's role in HIV-1 PIC function, we extracted PICs from cells that are either depleted of CPSF6 or express a mutant form that cannot bind to CA. These PICs exhibited significantly lower viral DNA integration activity when compared to the control PICs. The addition of purified recombinant CPSF6 restored the integration activity of PICs extracted from the CPSF6-mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6's role in PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in integration in these cells was detected, and this reduction was not a consequence of lower reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6-mutant cells. Finally, sequencing analysis revealed that HIV-1 integration into CPSF6-mutant cell genomes was significantly redirected away from gene-dense regions of chromatin compared to the control cells. Collectively, these results suggest that the CPSF6-CA interaction promotes PIC function both in vitro and in infected cells.IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with cellular host factors. However, the molecular details of HIV-host factor interactions are not fully understood. For instance, the HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 to restrict HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes HIV-1 integration targeting into gene-dense regions of the host genome. Here, we provide evidence that CPSF6-CA interaction stimulates the activity of HIV-1 preintegration complexes (PICs). We also describe that disruption of CPSF6-CA binding in target cells significantly reduces viral DNA integration and redirects integration targeting away from gene-dense regions into regions of low transcriptional activity. These findings identify a critical role for the CPSF6-CA interaction in PIC function and integration targeting.
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Affiliation(s)
- Evan Chaudhuri
- Center for AIDS Health Disparities Research, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Pharmacology, and Neuroscience, Meharry Medical College, Nashville, Tennessee, USA
- School of Graduate Studies, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Sooin Jang
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA
| | - Rajasree Chakraborty
- Center for AIDS Health Disparities Research, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Rajalingam Radhakrishnan
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
| | - Bjarki Arnarson
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
| | - Prem Prakash
- Center for AIDS Health Disparities Research, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Daphne Cornish
- Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Nicholas Rohlfes
- Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA
| | - Parmit K. Singh
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA
| | - Jiong Shi
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Christopher Aiken
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Edward Campbell
- Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA
| | - Judd Hultquist
- Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Muthukumar Balsubramaniam
- Center for AIDS Health Disparities Research, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Alan N. Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA
| | - Chandravanu Dash
- Center for AIDS Health Disparities Research, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
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Pereira-Montecinos C, Pittet-Díaz I, Morales-Vejar I, Millan-Hidalgo C, Rojas-Celis V, Vallejos-Vidal E, Reyes-López FE, Fuenzalida LF, Reyes-Cerpa S, Toro-Ascuy D. Involvement of lncRNAs NEAT1 and ZBTB11-AS1 in Active and Persistent HIV-1 Infection in C20 Human Microglial Cell Line. Int J Mol Sci 2025; 26:4745. [PMID: 40429887 PMCID: PMC12112671 DOI: 10.3390/ijms26104745] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2025] [Revised: 05/09/2025] [Accepted: 05/11/2025] [Indexed: 05/29/2025] Open
Abstract
HIV-1 infection in microglia induces HIV-associated neurocognitive disorder (HAND). Recent evidence suggests that microglia can be infected with HIV-1 in the active, persistent, or latent replication stages. The molecular mechanisms governing these stages of infection are still the subject of continuous study. In this study, we analyzed the relationship between HIV-1 infection and two lncRNAs, NEAT1 and ZBTB11-AS1, on different days post-infection. We found that on days 1 and 4 post-infection, HIV-1 was actively replicating; meanwhile, by day 21, HIV-1 had entered a persistent stage. We also noted that the expression levels of NEAT1 and ZBTB11-AS1 varied during these different stages of HIV-1 infection in microglia, as did their subcellular localization. We performed an interaction network analysis and identified DDX3X and ZC3HAV1 as hypothetically related to NEAT1 and ZBTB11-AS1 in the C20 human microglial cell line. Additionally, we determined that IL-6, a cytokine regulated by DDX3X and ZC3HAV1, exhibits changes in protein expression levels during both active and persistent HIV-1 infection.
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Affiliation(s)
- Camila Pereira-Montecinos
- Centro de Genómica y Bioinformática, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago 8580745, Chile;
- Escuela de Biotecnología, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago 8580745, Chile;
| | - Isidora Pittet-Díaz
- Virology Laboratory, Department of Biology, Faculty of Sciences, Universidad of Chile, Santiago 7800003, Chile; (I.P.-D.); (I.M.-V.); (V.R.-C.)
| | - Isidora Morales-Vejar
- Virology Laboratory, Department of Biology, Faculty of Sciences, Universidad of Chile, Santiago 7800003, Chile; (I.P.-D.); (I.M.-V.); (V.R.-C.)
| | - Catalina Millan-Hidalgo
- Escuela de Biotecnología, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago 8580745, Chile;
- Virology Laboratory, Department of Biology, Faculty of Sciences, Universidad of Chile, Santiago 7800003, Chile; (I.P.-D.); (I.M.-V.); (V.R.-C.)
| | - Victoria Rojas-Celis
- Virology Laboratory, Department of Biology, Faculty of Sciences, Universidad of Chile, Santiago 7800003, Chile; (I.P.-D.); (I.M.-V.); (V.R.-C.)
| | - Eva Vallejos-Vidal
- Núcleo de Investigación en Producción y Salud de Especies Acuáticas (NIP-SEA), Facultad de Medicina Veterinaria y Agronomía, Universidad De Las Américas, La Florida, Santiago 8250122, Chile;
- Centro de Nanociencia y Nanotecnología (CEDENNA), Universidad de Santiago de Chile, Santiago 9170002, Chile
- Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 9170002, Chile;
| | - Felipe E. Reyes-López
- Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 9170002, Chile;
| | - Loreto F. Fuenzalida
- Facultad de Ciencias de la Salud, Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Santiago 8910060, Chile;
| | - Sebastián Reyes-Cerpa
- Centro de Genómica y Bioinformática, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago 8580745, Chile;
- Escuela de Biotecnología, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago 8580745, Chile;
| | - Daniela Toro-Ascuy
- Virology Laboratory, Department of Biology, Faculty of Sciences, Universidad of Chile, Santiago 7800003, Chile; (I.P.-D.); (I.M.-V.); (V.R.-C.)
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5
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Rangaraj S, Agarwal A, Banerjee S. Bird's Eye View on Mycobacterium tuberculosis-HIV Coinfection: Understanding the Molecular Synergism, Challenges, and New Approaches to Therapeutics. ACS Infect Dis 2025; 11:1042-1063. [PMID: 40229972 DOI: 10.1021/acsinfecdis.4c00870] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/16/2025]
Abstract
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is the most common secondary infection in the Human Immunodeficiency Virus (HIV) infected population, accounting for more than one-fourth of deaths in people living with HIV (PLWH). Reciprocally, HIV infection increases the susceptibility to primary TB or reactivation of latent TB by several folds. The synergistic interactions between M.tb and HIV not only potentiate their deleterious impact but also complicate the clinical management of both the diseases. M.tb-HIV coinfected patients have a high risk of failure of accurate diagnosis, treatment inefficiency for both TB and HIV, concurrent nontuberculous mycobacterial infections, other comorbidities such as diabetes mellitus, severe cytotoxicity due to drug overburden, and immune reconstitution inflammatory syndrome (IRIS). The need of the hour is to understand M.tb-HIV coinfection biology and their collective impact on the host immunocompetence and to think of out-of-the-box treatment perspectives, including host-directed therapy under the rising view of homeostatic medicines. This review aims to highlight the molecular players, both from the pathogens and host, that facilitate the synergistic interactions and host-associated proteins/enzymes regulating immunometabolism, underlining potential targets for designing and screening chemical inhibitors to reduce the burden of both pathogens concomitantly during M.tb-HIV coinfection. To appreciate the necessity of revisiting therapeutic approaches and research priorities, we provide a glimpse of anti-TB and antiretroviral drug-drug interactions, project the gaps in our understanding of coinfection biology, and also enlist some key research initiatives that will help us deal with the synergistic epidemic of M.tb-HIV coinfection.
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Affiliation(s)
- Siranjeevi Rangaraj
- Laboratory of Molecular Pathogenesis, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana 500046, India
| | - Anushka Agarwal
- Laboratory of Molecular Pathogenesis, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana 500046, India
| | - Sharmistha Banerjee
- Laboratory of Molecular Pathogenesis, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana 500046, India
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Panchapakesan A, Ranga U. A Model of Non-Homologous Recombination Mediated by HIV-1 Reverse Transcriptase Explaining Sequence Motif Duplications That Confer a Replication Fitness Advantage. Viruses 2025; 17:680. [PMID: 40431692 PMCID: PMC12115494 DOI: 10.3390/v17050680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2025] [Revised: 04/29/2025] [Accepted: 04/30/2025] [Indexed: 05/29/2025] Open
Abstract
The Reverse Transcriptase of the Human Immunodeficiency Virus (HIV) is distinguished by its high rate of homologous recombination. A less-studied consequence of this phenomenon is the increased occurrence of non-homologous recombination, which results in length polymorphism. While most of these genome-wide variations are sporadic, some provide a replication advantage to variant strains, such as those in the Long Terminal Repeat (LTR) and p6-Gag regions. By analyzing sequences from these two regions in the HIV-1 databases, we categorize all types of non-homologous recombination into four groups based on the presence or absence of two molecular features. Additionally, drawing on established models of homologous recombination, we propose a model that describes the process of sequence duplication. This model can also be applied to explain non-homologous recombination in different types of HIV and other viruses.
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Affiliation(s)
- Arun Panchapakesan
- HIV-AIDS Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India;
- Molecular Biology Laboratory, Y R Gaitonde Centre for AIDS Research and Education (YRG CARE), Chennai 600031, India
| | - Udaykumar Ranga
- HIV-AIDS Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India;
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Zhang S, Chen J, Wang Y, Sun W, Lv C. Poly-guanine RNAs form ultra-stable intermolecular G-quadruplexes to suppress RNA function at low environmental temperature. Int J Biol Macromol 2025; 305:141110. [PMID: 39956242 DOI: 10.1016/j.ijbiomac.2025.141110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2024] [Revised: 02/06/2025] [Accepted: 02/13/2025] [Indexed: 02/18/2025]
Abstract
Intramolecular G-quadruplexes (G4) are four-stranded nucleic acid structures formed by guanine-rich sequences that play crucial roles in various biological processes, such as gene regulation and genome stability. However, the formation and function of intermolecular G4, especially in RNA, remain less understood. In this study, it was demonstrated that poly-guanine (poly-G) RNA sequences can form ultra-stable intermolecular G4 structures, particularly when the guanine content exceeds five. The stability and formation of RNA intermolecular G4 were further investigated under various physicochemical conditions, with temperature identified as a critical influencing factor. Furthermore, stable intermolecular G4 was shown to significantly inhibit RNA reverse transcription, and poly-G miRNAs were found to form stable G4 with poly-G mRNAs. Further analysis revealed a significant correlation between guanine content and environmental temperature, based on nucleic acid sequence analysis across organisms with different environmental temperatures and evolutionary simulations involving bacterial resistance genes. We proposed that the formation of intermolecular G4 to inhibit RNA function is a potential molecular mechanism by which genetic material responds to changes in the organism's environmental temperature. This study enhances our understanding of RNA G4 biology and suggests that poly-G sequences may play a critical role in the molecular response of organisms to environmental temperature fluctuations, offering new insights into the adaptation mechanisms of genetic material to climate and environmental changes.
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Affiliation(s)
- Shun Zhang
- Department of Emergency Medicine Center, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610000, PR China
| | - Jiao Chen
- Department of Emergency Medicine Center, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610000, PR China
| | - Yang Wang
- Department of Emergency Medicine Center, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610000, PR China.
| | - Wen Sun
- Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610000, PR China.
| | - Chuanzhu Lv
- Department of Emergency Medicine Center, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610000, PR China.
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8
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Gurgo C, Fenizia C, McKinnon K, Hsia RC, Franchini G. Expression of HIV from a 1-LTR circular DNA in the absence of integration. Retrovirology 2025; 22:2. [PMID: 40098202 PMCID: PMC11912779 DOI: 10.1186/s12977-025-00658-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2024] [Accepted: 02/05/2025] [Indexed: 03/19/2025] Open
Abstract
BACKGROUND Like all retroviruses, two kinds of viral DNA are present in the nucleus of HIV-infected cells: integrated DNA and a pool of unintegrated DNA containing linear and circular forms. For the most part, it has been difficult to examine the role of the unintegrated DNA forms in the viral life cycle in the presence of the integrated form, or to distinguish the respective contributions of the two circular DNA forms in the context of the unintegrated DNA. RESULTS In our approach, we constructed a 1-LTR circular form of HIV in order to study its expression in isolation from the other forms; we derived a linear genomic HIV DNA lacking the 5'-LTR (1-LTRHIV) from a molecular clone of HIV. This linear form is transcriptionally incompetent, but via circularization becomes a transcriptionally competent 1-LTR circle. When transfected into cells lacking CD4 where neither the spread of virus nor reinfection can occur, the linear or in vitro circularized form produces a fully infectious HIV. Virus expression is stable throughout cell division as measured on a per cell basis by flow cytometry. A progressive accumulation of copies of the circular form is observed in the presence of the cell growth inhibitor aphidicolin, suggestive of episomal amplification, for which we propose a model. CONCLUSION We demonstrate in this study that production of infectious virus is initiated and completed by the 1-LTR episomal form of HIV DNA in the absence of reinfection and integration. In addition, we show that the 1-LTR episomal form replicates in the absence of an origin of replication, and we propose a model for its amplification. In line with the work of others but following a different approach, we provide support for a potential role of episomal forms in HIV persistence. Our data highlight the biological complexity of HIV replication and the potential of the episomal form to contribute to the persistence of HIV.
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Affiliation(s)
- Corrado Gurgo
- Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
| | - Claudio Fenizia
- Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
- Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy
| | - Katherine McKinnon
- Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Ru-Ching Hsia
- Electron Microscopy Laboratory, Frederick National Laboratory for Cancer Research, NCI, NIH, Bethesda, MD, USA
| | - Genoveffa Franchini
- Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
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9
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Huang Y, Abdelgawad A, Gololobova O, Liao Z, Cong X, Batish M, Zheng L, Witwer KW. Enhanced packaging of U6 small nuclear RNA and splicing-related proteins into extracellular vesicles during HIV infection. SCIENCE ADVANCES 2025; 11:eadq6557. [PMID: 40073117 PMCID: PMC11900857 DOI: 10.1126/sciadv.adq6557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/24/2024] [Accepted: 02/06/2025] [Indexed: 03/14/2025]
Abstract
U6 small nuclear RNA (U6 snRNA), a critical spliceosome component primarily found in the nucleus, plays a vital role in RNA splicing. Our previous study, using the simian immunodeficiency virus (SIV) macaque model, revealed an increase of U6 snRNA in plasma extracellular vesicles (EVs) in acute retroviral infection. Given the limited understanding of U6 snRNA dynamics across cells and EVs, particularly in SIV infection, this research explores U6 snRNA trafficking and its association with splicing proteins in the nucleus, cytoplasm, and EVs. We observed a redistribution of U6 snRNA from the nucleus to EVs post-infection, accompanied by distinct protein profile changes and alterations in nucleic acid metabolism and spliceosome pathways. In addition, U6 machinery proteins changed in cells and EVs in a contrasting manner. The redistribution of U6 and related proteins we observed could be part of a viral strategy to redirect host splicing machinery, suggesting that U6 may have regulatory roles and be part of retroviral infection signature.
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Affiliation(s)
- Yiyao Huang
- Department of Laboratory Medicine, Guangdong Provincial Key Laboratory of Precision Medical Diagnostics, Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Guangdong Provincial Key Laboratory of Single-cell and Extracellular Vesicles, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Ahmed Abdelgawad
- Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, USA
| | - Olesia Gololobova
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Zhaohao Liao
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Xinyu Cong
- Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, University of Cincinnati, Cincinnati, OH, USA
| | - Mona Batish
- Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, USA
| | - Lei Zheng
- Department of Laboratory Medicine, Guangdong Provincial Key Laboratory of Precision Medical Diagnostics, Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Guangdong Provincial Key Laboratory of Single-cell and Extracellular Vesicles, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Kenneth W. Witwer
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Richman Family Precision Medicine Center of Excellence in Alzheimer’s Disease, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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10
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Oberdoerffer S, Gilbert WV. All the sites we cannot see: Sources and mitigation of false negatives in RNA modification studies. Nat Rev Mol Cell Biol 2025; 26:237-248. [PMID: 39433914 DOI: 10.1038/s41580-024-00784-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/12/2024] [Indexed: 10/23/2024]
Abstract
RNA modifications are essential for human health - too much or too little of them leads to serious illnesses ranging from neurodevelopmental disorders to cancer. Technical advances in RNA modification sequencing are beginning to uncover the RNA targets of diverse RNA-modifying enzymes that are dysregulated in disease. However, the emerging transcriptome-wide maps of modified nucleosides installed by these enzymes should be considered as first drafts. In particular, a range of technical artefacts lead to false negatives - modified sites that are overlooked owing to technique-dependent, and often sequence-context-specific, 'blind spots'. In this Review, we discuss potential sources of false negatives in sequencing-based RNA modification maps, propose mitigation strategies and suggest guidelines for transparent reporting of sensitivity to detect modified sites in profiling studies. Important considerations for recognition and avoidance of false negatives include assessment and reporting of position-specific sequencing depth, identification of protocol-dependent RNA capture biases and applying controls for false negatives as well as for false positives. Despite their limitations, emerging maps of RNA modifications reveal exciting and largely uncharted potential for post-transcriptional control of all aspects of RNA function.
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Affiliation(s)
- Shalini Oberdoerffer
- Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA.
| | - Wendy V Gilbert
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
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11
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Shannon A, Canard B. Nucleotide analogues and mpox: Repurposing the repurposable. Antiviral Res 2025; 234:106057. [PMID: 39694420 DOI: 10.1016/j.antiviral.2024.106057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2024] [Revised: 12/05/2024] [Accepted: 12/09/2024] [Indexed: 12/20/2024]
Abstract
While the COVID-19 crisis is still ongoing, a new public health threat has emerged with recent outbreaks of monkeypox (mpox) infections in Africa. Mass vaccination is not currently recommended by the World Health Organization (WHO), and antiviral treatments are yet to be specifically approved for mpox, although existing FDA-approved drugs (Tecovirimat, Brincidofovir, and Cidofovir) may be used in severe cases or for immunocompromised patients. A first-line of defense is thus drug repurposing, which was heavily attempted against SARS-CoV-2 - albeit with limited success. This review focuses on nucleoside analogues as promising antiviral candidates for targeting of the viral DNA-dependent DNA polymerase. In contrast to broad-spectrum screening approaches employed for SARS-CoV-2, we emphasize the importance of understanding the structural specificity of viral polymerases for rational selection of potential candidates. By comparing DNA-dependent DNA polymerases with other viral polymerases, we highlight the unique features that influence the efficacy and selectivity of nucleoside analogues. These structural insights provide a framework for the preselection, repurposing, optimization, and design of nucleoside analogues, aiming to accelerate the development of targeted antiviral therapies for mpox and other viral infections.
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Affiliation(s)
- Ashleigh Shannon
- Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, UMR7257, Marseille, France
| | - Bruno Canard
- Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, UMR7257, Marseille, France.
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12
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Nuwagaba J, Li JA, Ngo B, Sutton RE. 30 years of HIV therapy: Current and future antiviral drug targets. Virology 2025; 603:110362. [PMID: 39705895 PMCID: PMC11788039 DOI: 10.1016/j.virol.2024.110362] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Revised: 12/09/2024] [Accepted: 12/16/2024] [Indexed: 12/23/2024]
Abstract
Significant advances in treatment have turned HIV-1 into a manageable chronic condition. This has been achieved due to highly active antiretroviral therapy (HAART), involving a combination regimen of medications, including drugs that target Reverse Transcriptase, Protease, Integrase, and viral entry, explored in this review. This paper also highlights novel therapies, such as Lenacapavir, and avenues toward functional cure targeting the CCR5 co-receptor, including the Δ32 mutation. Challenges of HAART include lifelong adherence, toxicity, drug interactions, and drug resistance. Future therapeutic strategies may focus on underexplored antiviral targets. HIV-1 Tat and Rev proteins have essential HIV-1 regulatory functions of transcriptional elongation of the viral long terminal repeat and nuclear export of intron-containing HIV-1 RNA, respectively. These non-enzymatic proteins should thus be investigated to identify small molecules that inhibit HIV-1 replication, without causing undue toxicity. Continued innovation is essential to address therapeutic gaps and bring us closer to a potential HIV-1 cure.
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Affiliation(s)
- Julius Nuwagaba
- Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT, 06510, USA
| | - Jessica A Li
- Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT, 06510, USA
| | - Brandon Ngo
- Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT, 06510, USA
| | - Richard E Sutton
- Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT, 06510, USA.
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13
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González SA, Affranchino JL. The life cycle of feline immunodeficiency virus. Virology 2025; 601:110304. [PMID: 39561619 DOI: 10.1016/j.virol.2024.110304] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2024] [Revised: 11/10/2024] [Accepted: 11/13/2024] [Indexed: 11/21/2024]
Abstract
Feline immunodeficiency virus (FIV) is a retrovirus of worldwide distribution that can cause an acquired immunodeficiency disease in domestic cats. FIV and the primate lentiviruses, human and simian immunodeficiency viruses (HIV and SIV, respectively) share structural and biological features but also exhibit important differences, which reflect both their evolutionary relationship and divergence. Given that FIV is not only an important cat pathogen but also a useful model for certain aspects of HIV-1 infections in humans, the study of FIV biology is highly relevant. In this review we provide an updated description of the molecular mechanisms involved in each stage of the FIV life cycle.
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Affiliation(s)
- Silvia A González
- Laboratorio de Virología, Facultad de Ciencias Exactas y Naturales, Universidad de Belgrano (UB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
| | - José L Affranchino
- Centro de Virología Humana y Animal (CEVHAN), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Abierta Interamericana (UAI), Buenos Aires, Argentina
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14
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Ding H, Nguyen HT, Li W, Deshpande A, Zhang S, Jiang F, Zhang Z, Anang S, Mothes W, Sodroski J, Kappes JC. Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation. J Virol 2024; 98:e0172024. [PMID: 39508605 PMCID: PMC11650979 DOI: 10.1128/jvi.01720-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Accepted: 10/16/2024] [Indexed: 11/15/2024] Open
Abstract
During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.
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Affiliation(s)
- Haitao Ding
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Hanh T. Nguyen
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Wenwei Li
- Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, USA
| | - Ashlesha Deshpande
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Shijian Zhang
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Fan Jiang
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Zhiqing Zhang
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Saumya Anang
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Walther Mothes
- Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, USA
| | - Joseph Sodroski
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - John C. Kappes
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
- Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, Alabama, USA
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15
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Azzouzi M, Ouchaoui AA, Azougagh O, El Hadad SE, Abou-Salama M, Oussaid A, Pannecouque C, Rohand T. Synthesis, crystal structure, and antiviral evaluation of new imidazopyridine-schiff base derivatives: in vitro and in silico anti-HIV studies. RSC Adv 2024; 14:36902-36918. [PMID: 39569129 PMCID: PMC11574953 DOI: 10.1039/d4ra07561g] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2024] [Accepted: 11/13/2024] [Indexed: 11/22/2024] Open
Abstract
A series of Imidazo[1,2-a]pyridine-Schiff base derivatives were synthesized and characterized using 1H NMR, 13C NMR, Mass Spectrometry and FTIR techniques, and the structure of 4a was further confirmed through single-crystal X-ray diffraction analysis. Density Functional Theory (DFT) has been used to investigate the structural and electronic properties. The synthesized compounds were evaluated in vitro for their antiviral activity against human immunodeficiency virus type-1 (HIV-1) and human immunodeficiency virus type-2 (HIV-2) in MT-4 cells. Compound 4a displayed EC50 values of 82,02 and 47,72 μg ml-1 against HIV-1 and HIV-2, respectively. Molecular docking studies were conducted to gain insights into the interaction mechanism of the synthesized compounds with HIV-1 reverse transcriptase. ADME analysis suggested acceptable pharmacokinetic profiles, though solubility remains a limitation for these compounds, highlighting the need for further structural modifications to enhance bioavailability and therapeutic potential.
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Affiliation(s)
- Mohamed Azzouzi
- Laboratory of Molecular Chemistry, Materials and Environment (LCM2E), Department of Chemistry, Multidisciplinary Faculty of Nador, University Mohamed I Nador 60700 Morocco
| | - Abderrahim Ait Ouchaoui
- Mohammed VI University of Sciences and Health (UM6SS) Casablanca Morocco
- Mohammed VI Center for Research and Innovation (CM6) Rabat 10000 Morocco
| | - Omar Azougagh
- Laboratory of Molecular Chemistry, Materials and Environment (LCM2E), Department of Chemistry, Multidisciplinary Faculty of Nador, University Mohamed I Nador 60700 Morocco
| | - Salah Eddine El Hadad
- Chemical and Biochemical Sciences-Green Process Engineering, University Mohammed VI Polytechnic Ben Guerir Morocco
| | - Mohamed Abou-Salama
- Laboratory of Molecular Chemistry, Materials and Environment (LCM2E), Department of Chemistry, Multidisciplinary Faculty of Nador, University Mohamed I Nador 60700 Morocco
| | - Adyl Oussaid
- Laboratory of Molecular Chemistry, Materials and Environment (LCM2E), Department of Chemistry, Multidisciplinary Faculty of Nador, University Mohamed I Nador 60700 Morocco
| | - Christophe Pannecouque
- Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U. Leuven Leuven B-3000 Belgium
| | - Taoufik Rohand
- Laboratory of Molecular Chemistry, Materials and Environment (LCM2E), Department of Chemistry, Multidisciplinary Faculty of Nador, University Mohamed I Nador 60700 Morocco
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16
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Padron A, Dwivedi R, Chakraborty R, Prakash P, Kim K, Shi J, Ahn J, Pandhare J, Luban J, Aiken C, Balasubramaniam M, Dash C. Cyclophilin A facilitates HIV-1 integration. J Virol 2024; 98:e0094724. [PMID: 39480090 PMCID: PMC11575316 DOI: 10.1128/jvi.00947-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Accepted: 09/25/2024] [Indexed: 11/02/2024] Open
Abstract
Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5α. Interestingly, recent studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior to integration. We have previously reported that the capsid protein regulates HIV-1 integration. Therefore, we probed whether CypA-capsid interaction also regulates this post-nuclear entry step. First, we challenged CypA-expressing (CypA+/+) and CypA-depleted (CypA-/-) cells with HIV-1 and quantified the levels of provirus. CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral integration in CypA+/+ cells but not in CypA-/- cells. HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at the integration step in CypA+/+ cells but not in CypA-/- cells. Then, to understand the mechanism, we assessed the integration activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected cells. PICs from CypA-/- cells retained lower integration activity in vitro compared to those from CypA+/+ cells. PICs from cells depleted of both CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC was independent of TRIM5α. Finally, CypA protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA. Collectively, these results provide strong evidence that CypA directly promotes HIV-1 integration, a previously unknown role of this host factor in the nucleus of an infected cell. IMPORTANCE Interaction between the HIV-1 capsid and host cellular factors is essential for infection. However, the molecular details and functional consequences of viral-host factor interactions during HIV-1 infection are not fully understood. Over 30 years ago, Cyclophilin A (CypA) was identified as the first host protein to bind to the HIV-1 capsid. Now it is established that CypA-capsid interaction promotes reverse transcription and nuclear entry of HIV-1. In addition, CypA blocks TRIM5α-mediated restriction of HIV-1. In this report, we show that CypA promotes the post-nuclear entry step of HIV-1 integration by binding to the viral capsid. Notably, we show that CypA stimulates the viral DNA integration activity of the HIV-1 preintegration complex. Collectively, our studies identify a novel role of CypA during the early steps of HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.
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Affiliation(s)
- Adrian Padron
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- School of Graduate Studies, Meharry Medical College, Nashville, Tennessee, USA
| | - Richa Dwivedi
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Rajasree Chakraborty
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Prem Prakash
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Kyusik Kim
- Program in Molecular Medicine University of Massachusetts Medical School, Worcester, Massachusetts, USA
| | - Jiong Shi
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Jinwoo Ahn
- Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Jui Pandhare
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
| | - Jeremy Luban
- Program in Molecular Medicine University of Massachusetts Medical School, Worcester, Massachusetts, USA
| | - Christopher Aiken
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Muthukumar Balasubramaniam
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Pharmacology, and Neuroscience, Meharry Medical College, Nashville, Tennessee, USA
| | - Chandravanu Dash
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
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17
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Martínez Del Río J, Frutos-Beltrán E, Sebastián-Martín A, Lasala F, Yasukawa K, Delgado R, Menéndez-Arias L. HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells. J Mol Biol 2024; 436:168815. [PMID: 39384034 DOI: 10.1016/j.jmb.2024.168815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Revised: 10/02/2024] [Accepted: 10/02/2024] [Indexed: 10/11/2024]
Abstract
Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg2+) using an in vitro-transcribed RNA were around 7.3 × 10-5. In agreement with previous reports, an 8-fold increase in RT's accuracy was observed after reducing Mg2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10-5). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg2+ (7.4 × 10-5) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8 × 10-5 in the presence of 0.5 mM Mg2+. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.
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Affiliation(s)
- Javier Martínez Del Río
- Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid 28049, Spain
| | - Estrella Frutos-Beltrán
- Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid 28049, Spain
| | - Alba Sebastián-Martín
- Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid 28049, Spain
| | - Fátima Lasala
- Laboratory of Molecular Microbiology, Instituto de Investigación Hospital 12 de Octubre (lmas12), Madrid 28041, Spain
| | - Kiyoshi Yasukawa
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
| | - Rafael Delgado
- Laboratory of Molecular Microbiology, Instituto de Investigación Hospital 12 de Octubre (lmas12), Madrid 28041, Spain; CIBERINFEC, Instituto de Salud Carlos III, Madrid, Spain; School of Medicine, Universidad Complutense, Madrid 28040, Spain
| | - Luis Menéndez-Arias
- Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid 28049, Spain.
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18
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Cheng Z, Islam S, Kanlong JG, Sheppard M, Seo H, Nikolaitchik OA, Kearse MG, Pathak VK, Musier-Forsyth K, Hu WS. Translation of HIV-1 unspliced RNA is regulated by 5' untranslated region structure. J Virol 2024; 98:e0116024. [PMID: 39315813 PMCID: PMC11494990 DOI: 10.1128/jvi.01160-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2024] [Accepted: 09/03/2024] [Indexed: 09/25/2024] Open
Abstract
HIV-1 must generate infectious virions to spread to new hosts and HIV-1 unspliced RNA (HIV-1 RNA) plays two central roles in this process. HIV-1 RNA serves as an mRNA that is translated to generate proteins essential for particle production and replication, and it is packaged into particles as the viral genome. HIV-1 uses several transcription start sites to generate multiple RNAs that differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. The virus relies on host machinery to translate its RNAs in a cap-dependent manner. Here, we demonstrate that the 5' context of HIV-1 RNA affects the efficiency of translation both in vitro and in cells. Although both RNAs are competent for translation, 3G RNA is translated more efficiently than 1G RNA. The 5' untranslated region (UTR) of 1G and 3G RNAs has previously been shown to fold into distinct structural ensembles. We show that HIV-1 mutants in which the 5' UTR of 1G and 3G RNAs fold into similar structures were translated at similar efficiencies. Thus, the host machinery translates two 99.9% identical HIV-1 RNAs with different efficiencies, and the translation efficiency is regulated by the 5' UTR structure.IMPORTANCEHIV-1 unspliced RNA contains all the viral genetic information and encodes virion structural proteins and enzymes. Thus, the unspliced RNA serves distinct roles as viral genome and translation template, both critical for viral replication. HIV-1 generates two major unspliced RNAs with a 2-nt difference at the 5' end (3G RNA and 1G RNA). The 1G transcript is known to be preferentially packaged over the 3G transcript. Here, we showed that 3G RNA is favorably translated over 1G RNA based on its 5' untranslated region (UTR) RNA structure. In HIV-1 mutants in which the two major transcripts have similar 5' UTR structures, 1G and 3G RNAs are translated similarly. Therefore, HIV-1 generates two 9-kb RNAs with a 2-nt difference, each serving a distinct role dictated by differential 5' UTR structures.
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Affiliation(s)
- Zetao Cheng
- Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA
| | - Saiful Islam
- Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA
| | - Joseph G. Kanlong
- Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, Ohio State University, Columbus, Ohio, USA
| | - Madeline Sheppard
- Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, Ohio State University, Columbus, Ohio, USA
| | - Heewon Seo
- Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, Ohio State University, Columbus, Ohio, USA
| | - Olga A. Nikolaitchik
- Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA
| | - Michael G. Kearse
- Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State University, Columbus, Ohio, USA
| | - Vinay K. Pathak
- Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA
| | - Karin Musier-Forsyth
- Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, Ohio State University, Columbus, Ohio, USA
| | - Wei-Shau Hu
- Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA
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19
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Yang Z, Shan Y, Liu X, Chen G, Pan Y, Gou Q, Zou J, Chang Z, Zeng Q, Yang C, Kong J, Sun Y, Li S, Zhang X, Wu WC, Li C, Peng H, Holmes EC, Guo D, Shi M. VirID: Beyond Virus Discovery-An Integrated Platform for Comprehensive RNA Virus Characterization. Mol Biol Evol 2024; 41:msae202. [PMID: 39331699 PMCID: PMC11523140 DOI: 10.1093/molbev/msae202] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2024] [Revised: 09/10/2024] [Accepted: 09/24/2024] [Indexed: 09/29/2024] Open
Abstract
RNA viruses exhibit vast phylogenetic diversity and can significantly impact public health and agriculture. However, current bioinformatics tools for viral discovery from metagenomic data frequently generate false positive virus results, overestimate viral diversity, and misclassify virus sequences. Additionally, current tools often fail to determine virus-host associations, which hampers investigation of the potential threat posed by a newly detected virus. To address these issues we developed VirID, a software tool specifically designed for the discovery and characterization of RNA viruses from metagenomic data. The basis of VirID is a comprehensive RNA-dependent RNA polymerase database to enhance a workflow that includes RNA virus discovery, phylogenetic analysis, and phylogeny-based virus characterization. Benchmark tests on a simulated data set demonstrated that VirID had high accuracy in profiling viruses and estimating viral richness. In evaluations with real-world samples, VirID was able to identify RNA viruses of all types, but also provided accurate estimations of viral genetic diversity and virus classification, as well as comprehensive insights into virus associations with humans, animals, and plants. VirID therefore offers a robust tool for virus discovery and serves as a valuable resource in basic virological studies, pathogen surveillance, and early warning systems for infectious disease outbreaks.
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Affiliation(s)
- Ziyue Yang
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Yongtao Shan
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Xue Liu
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Guowei Chen
- Department of Electrical Engineering, City University of Hong Kong, Kowloon, Hong Kong (SAR), China
| | - Yuanfei Pan
- Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Qinyu Gou
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Jie Zou
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Zilong Chang
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Qiang Zeng
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Chunhui Yang
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Jianbin Kong
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Yanni Sun
- Department of Electrical Engineering, City University of Hong Kong, Kowloon, Hong Kong (SAR), China
| | - Shaochuan Li
- Goodwill Institute of Life Sciences, Guangzhou, China
| | - Xu Zhang
- Goodwill Institute of Life Sciences, Guangzhou, China
| | - Wei-chen Wu
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Chunmei Li
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Hong Peng
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
| | - Edward C Holmes
- School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia
- Laboratory of Data Discovery for Health Limited, Hong Kong (SAR), China
| | - Deyin Guo
- Guangzhou National Laboratory, Guangzhou International Bio-Island, Guangzhou, China
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
| | - Mang Shi
- State Key Laboratory for Biocontrol, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China
- Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China
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20
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Hossain MS, Alom MS, Kader MS, Hossain MA, Halim MA. Structure-Guided Antiviral Peptides Identification Targeting the HIV-1 Integrase. ACS PHYSICAL CHEMISTRY AU 2024; 4:464-475. [PMID: 39346608 PMCID: PMC11428276 DOI: 10.1021/acsphyschemau.4c00006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/21/2024] [Revised: 06/25/2024] [Accepted: 06/25/2024] [Indexed: 10/01/2024]
Abstract
HIV-1 integrase (IN), a major protein in the HIV life cycle responsible for integrating viral cDNA into the host DNA, represents a promising drug target. Small peptides have emerged as antiviral therapeutics for HIV because of their facile synthesis, highly selective nature, and fewer side effects. However, selecting the best candidates from a vast pool of peptides is a daunting task. In this study, multistep virtual screening was employed to identify potential peptides from a list of 280 HIV inhibitory peptides. Initially, 80 peptides were selected based on their minimum inhibitory concentrations (MIC). Then, molecular docking was performed to evaluate their binding scores compared to HIP000 and HIP00N which are experimentally validated HIV-1 integrase binding peptides that were used as a positive and negative control, respectively. The top-scoring docked complexes, namely, IN-HIP1113, IN-HIP1140, IN-HIP1142, IN-HIP678, IN-HIP776, and IN-HIP777, were subjected to initial 500 ns molecular dynamics (MD) simulations. Subsequently, HIP776, HIP777, and HIP1142 were selected for an in-depth mechanistic study of peptide interactions, with multiple simulations conducted for each complex spanning one microsecond. Independent simulations of the peptides, along with comparisons to the bound state, were performed to elucidate the conformational dynamics of the peptides. These peptides exhibit strong interactions with specific residues, as revealed by snapshot interaction analysis. Notably, LYS159, LYS156, VAL150, and GLU69 residues are prominently involved in these interactions. Additionally, residue-based binding free energy (BFE) calculations highlight the significance of HIS67, GLN148, GLN146, and SER147 residues within the binding pocket. Furthermore, the structure-activity relationship (SAR) analysis demonstrated that aromatic amino acids and the overall volume of peptides are the two major contributors to the docking scores. The best peptides will be validated experimentally by incorporating SAR properties, aiming to develop them as therapeutic agents and structural models for future peptide-based HIV-1 drug design, addressing the urgent need for effective HIV treatments.
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Affiliation(s)
- Md Shahadat Hossain
- Division of Infectious Diseases and Division of Computer Aided Drug Design, The Red-Green Research Centre, BICCB, 16 Tejkunipara, Tejgaon, Dhaka 1215, Bangladesh
- Department of Pharmacy, Faculty of Life Science, Mawlana Bhashani Science & Technology University, Tangail 1902, Bangladesh
| | - Md Siddik Alom
- Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States
- Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, United States
| | | | | | - Mohammad A Halim
- Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia 30144, United States
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21
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Liu Y, Luo Z, Chen X, Yang X, Qi Q, Alifu M, Tao C, Cui W, Liu M, Wang W. Determinants of the interaction between the 5'-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process. Biochem Biophys Res Commun 2024; 725:150252. [PMID: 38878758 DOI: 10.1016/j.bbrc.2024.150252] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2024] [Revised: 06/04/2024] [Accepted: 06/10/2024] [Indexed: 07/06/2024]
Abstract
Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5' leader (5'L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5'L responsible for LysRS binding, how the conformations and various hairpin structures of 5'L regulate 5'L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5'L significantly influences its binding affinity with LysRS. The 5'L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5'L conformation that is not selected for packaging. Additionally, dimerization of 5'L impairs LysRS-5'L interaction. Furthermore, among various regions of 5'L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5'L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.
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Affiliation(s)
- Yong Liu
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Zhi Luo
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Xiang Chen
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Xin Yang
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Qi Qi
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Mailikezhati Alifu
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Chengcheng Tao
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Wen Cui
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Mengmeng Liu
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China.
| | - Wei Wang
- Institute of Life Sciences, Chongqing Medical University, Chongqing, China.
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22
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Bakhanashvili M. The Role of Tumor Suppressor p53 Protein in HIV-Host Cell Interactions. Cells 2024; 13:1512. [PMID: 39329696 PMCID: PMC11429533 DOI: 10.3390/cells13181512] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Revised: 09/02/2024] [Accepted: 09/06/2024] [Indexed: 09/28/2024] Open
Abstract
The virus-host relationship is indispensable for executing successful viral infection. The pathogenesis of HIV is determined by an intricate interaction between the host and the virus for the regulation of HIV infection, thereby influencing various aspects, including the regulation of signaling pathways. High mutation rates and population heterogeneity characterize HIV with consequences for viral pathogenesis and the potential to escape the immune system and anti-viral inhibitors used in therapy. The origin of the high mutation rates exhibited by HIV may be attributed to a limited template-copied fidelity that likely operates in the cytoplasm. HIV-1 infection induces upregulation and activation of tumor suppressor p53 protein in the early stages of HIV-1 infection. p53 plays a multifaceted role in the context of HIV infection, thereby affecting viral replication. p53 is involved in maintaining genetic integrity, actively participating in various DNA repair processes through its various biochemical activities and via its ability to interact with components of the repair machinery. This report focuses on the impact of the p53 protein on the HIV-1 reverse transcription process while incorporating various incorrect and non-canonical nucleotides. The presence of functional host-coded p53 protein with proofreading-repair activities in the cytoplasm may lead to various biological outcomes.
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Affiliation(s)
- Mary Bakhanashvili
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
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23
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Nugraha Y, Laksmi FA, Nuryana I, Helbert, Khasna FN. Production of reverse transcriptase from Moloney murine Leukemia virus in Escherichia coli expression system. Prep Biochem Biotechnol 2024; 54:1079-1087. [PMID: 38411149 DOI: 10.1080/10826068.2024.2317311] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/28/2024]
Abstract
Reverse transcriptase (RT) is one of the most important enzymes used in molecular biology applications, enabling the conversion of RNA into complementary DNA (cDNA) that is used in reverse transcription-polymerase chain reaction (RT-PCR). The high demand of RT enzymes in biotechnological applications making the production optimization of RT is crucial for meeting the growing demand in industrial settings. Conventionally, the expression of recombinant RT is T7-induced promoter using IPTG in Escherichia coli expression systems, which is not cost-efficient. Here, we successfully made an alternative procedure for RT expression from Moloney murine leukemia virus (M-MLV) using autoinduction method in chemically defined medium. The optimization of carbon source composition (glucose, lactose, and glycerol) was analyzed using Response Surface Methodology (RSM). M-MLV RT was purified for further investigation on its activity. A total of 32.8 mg/L purified M-MLV RT was successfully obtained when glucose, glycerol, and lactose were present at concentration of 0.06%, 0.9%, and 0.5% respectively, making a 3.9-fold improvement in protein yield. In addition, the protein was produced in its active form by displaying 7462.50 U/mg of specific activity. This study provides the first step of small-scale procedures of M-MLV RT production that make it a cost-effective and industrially applicable strategy.
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Affiliation(s)
- Yudhi Nugraha
- Research Center for Molecular Biology Eijkman, National Research and Innovation Agency, Cibinong, Bogor, West Java, Indonesia
| | - Fina Amreta Laksmi
- Research Center for Applied Microbiology, National Research and Innovation Agency, Cibinong, Bogor, West Java, Indonesia
| | - Isa Nuryana
- Research Center for Applied Microbiology, National Research and Innovation Agency, Cibinong, Bogor, West Java, Indonesia
| | - Helbert
- Research Center for Ecology and Ethnobiology, National Research and Innovation Agency, Cibinong, Bogor, West Java, Indonesia
| | - Firyal Nida Khasna
- Research Center for Applied Microbiology, National Research and Innovation Agency, Cibinong, Bogor, West Java, Indonesia
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Prakash P, Khodke P, Balasubramaniam M, Davids BO, Hollis T, Davis J, Kumbhar B, Dash C. Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties. J Biol Chem 2024; 300:107438. [PMID: 38838778 PMCID: PMC11259700 DOI: 10.1016/j.jbc.2024.107438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Revised: 05/17/2024] [Accepted: 05/24/2024] [Indexed: 06/07/2024] Open
Abstract
HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.
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Affiliation(s)
- Prem Prakash
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
| | - Purva Khodke
- Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be-) University, Mumbai, Maharashtra, India
| | - Muthukumar Balasubramaniam
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
| | - Benem-Orom Davids
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York City, New York, USA
| | - Thomas Hollis
- Department of Biochemistry and Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA
| | - Jamaine Davis
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
| | - Bajarang Kumbhar
- Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be-) University, Mumbai, Maharashtra, India
| | - Chandravanu Dash
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA; Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA; Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA.
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25
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Arribas L, Menéndez-Arias L, Betancor G. May I Help You with Your Coat? HIV-1 Capsid Uncoating and Reverse Transcription. Int J Mol Sci 2024; 25:7167. [PMID: 39000271 PMCID: PMC11241228 DOI: 10.3390/ijms25137167] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2024] [Revised: 06/26/2024] [Accepted: 06/27/2024] [Indexed: 07/16/2024] Open
Abstract
The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes.
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Affiliation(s)
- Laura Arribas
- Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad de Las Palmas de Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain;
| | - Luis Menéndez-Arias
- Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), 28049 Madrid, Spain;
| | - Gilberto Betancor
- Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad de Las Palmas de Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain;
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26
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Padron A, Dwivedi R, Chakraborty R, Prakash P, Kim K, Shi J, Ahn J, Pandhare J, Luban J, Aiken C, Balasubramaniam M, Dash C. Cyclophilin A Facilitates HIV-1 DNA Integration. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.15.599180. [PMID: 38948800 PMCID: PMC11212919 DOI: 10.1101/2024.06.15.599180] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/02/2024]
Abstract
Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA +/+ ) and CypA-depleted (CypA -/- ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA +/+ cells but not in CypA -/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA +/+ cells but not in CypA -/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA -/- cells had lower activity in vitro compared to those from CypA +/+ cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA +/+ and CypA -/- cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. Importance HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.
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27
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Yoon J, Lee J, Kim J, Lee SM, Kim S, Park HG. A novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension reaction. Biosens Bioelectron 2024; 253:116174. [PMID: 38432074 DOI: 10.1016/j.bios.2024.116174] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Revised: 02/16/2024] [Accepted: 02/27/2024] [Indexed: 03/05/2024]
Abstract
We herein present a novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction. The detection probe employed as a key component in this technique serves as a substrate for RNase H and triggers the PS-THSP reaction upon the RNase H-mediated degradation of the probe. As a consequence, a large number of long concatemeric amplification products could be produced and used to identify the RNase H activity through the fluorescence signals produced by the nucleic acid-specific fluorescent dye, SYTO 9. Importantly, the use of the gp32 protein allowed the PS-THSP reaction to be performed at 37 °C, ultimately enabling an isothermal one-step RNase H assay. Based on this sophisticated design principle, the RNase H activity was very sensitively detected, down to 0.000237 U mL-1 with high specificity. We further verified its practical applicability through its successful application to the screening of RNase H inhibitors. With its operational convenience and excellent analytical performance, this technique could serve as a new platform for RNase H assay in a wide range of biological applications.
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Affiliation(s)
- Junhyeok Yoon
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jinhwan Lee
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jaemin Kim
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Sang Mo Lee
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Soohyun Kim
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Hyun Gyu Park
- Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
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28
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Garcia AK, Almodovar S. The Intersection of HIV and Pulmonary Vascular Health: From HIV Evolution to Vascular Cell Types to Disease Mechanisms. JOURNAL OF VASCULAR DISEASES 2024; 3:174-200. [PMID: 39464800 PMCID: PMC11507615 DOI: 10.3390/jvd3020015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/29/2024]
Abstract
People living with HIV (PLWH) face a growing burden of chronic diseases, owing to the combinations of aging, environmental triggers, lifestyle choices, and virus-induced chronic inflammation. The rising incidence of pulmonary vascular diseases represents a major concern for PLWH. The study of HIV-associated pulmonary vascular complications ideally requires a strong understanding of pulmonary vascular cell biology and HIV pathogenesis at the molecular level for effective applications in infectious diseases and vascular medicine. Active HIV infection and/or HIV proteins disturb the delicate balance between vascular tone and constriction, which is pivotal for maintaining pulmonary vascular health. One of the defining features of HIV is its high genetic diversity owing to several factors including its high mutation rate, recombination between viral strains, immune selective pressures, or even geographical factors. The intrinsic HIV genetic diversity has several important implications for pathogenic outcomes of infection and the overall battle to combat HIV. Challenges in the field present themselves from two sides of the same coin: those imposed by the virus itself and those stemming from the host. The field may be advanced by further developing in vivo and in vitro models that are well described for both pulmonary vascular diseases and HIV for mechanistic studies. In essence, the study of HIV-associated pulmonary vascular complications requires a multidisciplinary approach, drawing upon insights from both infectious diseases and vascular medicine. In this review article, we discuss the fundamentals of HIV virology and their impact on pulmonary disease, aiming to enhance the understanding of either area or both simultaneously. Bridging the gap between preclinical research findings and clinical practice is essential for improving patient care. Addressing these knowledge gaps requires interdisciplinary collaborations, innovative research approaches, and dedicated efforts to prioritize HIV-related pulmonary complications on the global research agenda.
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Affiliation(s)
- Amanda K. Garcia
- Department of Immunology & Molecular Microbiology, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX 79430, USA
| | - Sharilyn Almodovar
- Department of Immunology & Molecular Microbiology, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX 79430, USA
- Center for Tropical Medicine & Infectious Diseases, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX 79430, USA
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Kilroy JM, Leal AA, Henderson AJ. Chronic HIV Transcription, Translation, and Persistent Inflammation. Viruses 2024; 16:751. [PMID: 38793632 PMCID: PMC11125830 DOI: 10.3390/v16050751] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2024] [Revised: 05/02/2024] [Accepted: 05/07/2024] [Indexed: 05/26/2024] Open
Abstract
People with HIV exhibit persistent inflammation that correlates with HIV-associated comorbidities including accelerated aging, increased risk of cardiovascular disease, and neuroinflammation. Mechanisms that perpetuate chronic inflammation in people with HIV undergoing antiretroviral treatments are poorly understood. One hypothesis is that the persistent low-level expression of HIV proviruses, including RNAs generated from defective proviral genomes, drives the immune dysfunction that is responsible for chronic HIV pathogenesis. We explore factors during HIV infection that contribute to the generation of a pool of defective proviruses as well as how HIV-1 mRNA and proteins alter immune function in people living with HIV.
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Affiliation(s)
- Jonathan M. Kilroy
- Department of Virology, Immunology, Microbiology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA 02118, USA; (J.M.K.); (A.A.L.)
| | - Andrew A. Leal
- Department of Virology, Immunology, Microbiology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA 02118, USA; (J.M.K.); (A.A.L.)
| | - Andrew J. Henderson
- Department of Virology, Immunology, Microbiology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA 02118, USA; (J.M.K.); (A.A.L.)
- Department of Medicine and Virology, Immunology, Microbiology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA 02118, USA
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Mondal A, Sarkar A, Das D, Sengupta A, Kabiraj A, Mondal P, Nag R, Mukherjee S, Das C. Epigenetic orchestration of the DNA damage response: Insights into the regulatory mechanisms. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2024; 387:99-141. [PMID: 39179350 DOI: 10.1016/bs.ircmb.2024.03.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/26/2024]
Abstract
The DNA damage response (DDR) is a critical cellular mechanism that safeguards genome integrity and prevents the accumulation of harmful DNA lesions. Increasing evidence highlights the intersection between DDR signaling and epigenetic regulation, offering profound insights into various aspects of cellular function including oncogenesis. This comprehensive review explores the intricate relationship between the epigenetic modifications and DDR activation, with a specific focus on the impact of viral infections. Oncogenic viruses, such as human papillomavirus, hepatitis virus (HBV or HCV), and Epstein-Barr virus have been shown to activate the DDR. Consequently, these DNA damage events trigger a cascade of epigenetic alterations, including changes in DNA methylation patterns, histone modifications and the expression of noncoding RNAs. These epigenetic changes exert profound effects on chromatin structure, gene expression, and maintenance of genome stability. Importantly, elucidation of the viral-induced epigenetic alterations in the context of DDR holds significant implications for comprehending the complexity of cancer and provides potential targets for therapeutic interventions.
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Affiliation(s)
- Atanu Mondal
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | | | - Dipanwita Das
- Virus Unit [NICED-ICMR], ID and BG Hospital, Kolkata, India
| | - Amrita Sengupta
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
| | - Aindrila Kabiraj
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Payel Mondal
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Rachayita Nag
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Shravanti Mukherjee
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
| | - Chandrima Das
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India.
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Hardy J, Demecheleer E, Schauvliege M, Staelens D, Mortier V, Verhofstede C. Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming. Microbiol Spectr 2024; 12:e0387223. [PMID: 38442427 PMCID: PMC10986323 DOI: 10.1128/spectrum.03872-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2023] [Accepted: 02/05/2024] [Indexed: 03/07/2024] Open
Abstract
In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5' end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5' end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.
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Affiliation(s)
- Jarryt Hardy
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
| | - Els Demecheleer
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
| | - Marlies Schauvliege
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
| | - Delfien Staelens
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
| | - Virginie Mortier
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
| | - Chris Verhofstede
- Aids Reference Laboratory, Department of Diagnostic Sciences, Ghent University, Ghent, Belgium
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Wang Q, Kline EC, Gilligan-Steinberg SD, Lai JJ, Hull IT, Olanrewaju AO, Panpradist N, Lutz BR. Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2024:2024.03.29.24305085. [PMID: 38633802 PMCID: PMC11023665 DOI: 10.1101/2024.03.29.24305085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/19/2024]
Abstract
Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25μL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.
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Affiliation(s)
- Qin Wang
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Enos C. Kline
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | | | - James J. Lai
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
| | - Ian T. Hull
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Ayokunle O. Olanrewaju
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - Nuttada Panpradist
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Barry R. Lutz
- Department of Bioengineering, University of Washington, Seattle, WA, USA
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Nurjannah, Jayanti S, Tanoerahardjo FS, Al Musyahadah US, Sukowati CHC, Massi MN. Major Drug Resistance Mutations on Reverse Transcriptase Gene in Human Immunodeficiency Virus Type-1 in Indonesia: A Systematic Review. Curr HIV/AIDS Rep 2024; 21:31-39. [PMID: 38244171 DOI: 10.1007/s11904-023-00687-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/29/2023] [Indexed: 01/22/2024]
Abstract
PURPOSE OF REVIEW The prevalence of HIV-1 in Indonesia is on a concerning upward trajectory, with a concurrent rise in the development of drug-resistant strains, challenging the efficacy of antiretroviral therapy (ART). Many mutations have been found in the pol gene that makes HIV resistant to ART. We aim to review the major drug resistance mutations (DRMs) of reverse transcriptase (RT) of pol gene in HIV-1 cases in Indonesia. RECENT FINDINGS A total of eleven articles reporting DRMs in HIV-1 subjects from various regions between 2015-2020 in Indonesia are included. The prevalence of major DRMs on the RT gene in studies included varies from 3.4% to 34%. The CRF01_AE subtype stands out as the predominant variant. Notably, the prevalence of major DRMs in ART-experienced individuals is 22.1%, while ART-naïve individuals show a lower rate of 4.4%. Among the RT gene mutations, M184I/V emerges as the most prevalent (10.5%) within the nucleos(t)ide reverse transcriptase inhibitors (NRTI) group, while K103N leads among the non-NRTI (NNRTI) group, with a frequency of 6.4%. Regionally, North Sulawesi records the highest prevalence of major DRMs in the RT gene at 21.1%, whereas Riau and Central Papua exhibit the lowest rates at 3.4%. Significant variations in drug resistance mutations within the RT gene across Indonesian regions highlight the importance of closely monitoring and evaluating the effectiveness of current antiretroviral therapy (ART) regimens. Considerably, more studies are needed to understand better and overcome the emergence of DRMs on HIV-1 patients in Indonesia.
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Affiliation(s)
- Nurjannah
- Postgraduate School, Hasanuddin University, Makassar, 90245, Indonesia
- Ministry of Health of Republic Indonesia, Jakarta Selatan, 12950, Indonesia
| | - Sri Jayanti
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, 16911, Indonesia
| | | | - Ummu Syauqah Al Musyahadah
- Department of Bioinformatics, Faculty of Health Technology, University of Megarezky, Makassar, 90234, Indonesia
| | - Caecilia Hapsari Ceriapuri Sukowati
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, 16911, Indonesia
- Liver Cancer Unit, Fondazione Italiana Fegato ONLUS, AREA Science Park, 34149, Trieste, Italy
| | - Muhammad Nasrum Massi
- Department of Clinical Microbiology, Hasanuddin University, Makassar, 90245, Indonesia.
- Microbiology Laboratory, Hasanuddin University Hospital, Makassar, 90245, Indonesia.
- Hasanuddin University Medical Research Center Laboratory, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia.
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Prakash P, Khodke P, Balasubramaniam M, Davids BO, Hollis T, Davis J, Pandhare J, Kumbhar B, Dash C. Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.19.585766. [PMID: 38562877 PMCID: PMC10983988 DOI: 10.1101/2024.03.19.585766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/04/2024]
Abstract
HIV-1 integration into the human genome is dependent on 3'-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3'-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3'-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.
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Affiliation(s)
- Prem Prakash
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, 37208, USA
| | - Purva Khodke
- Sunandan Divatia School of Science, NMIMS University, Mumbai, 400056, India
| | - Muthukumar Balasubramaniam
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, 37208, USA
| | - Benem-Orom Davids
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York City, New York, 10032, USA
| | - Thomas Hollis
- Department of Biochemistry and Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA
| | - Jamaine Davis
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, 37208, USA
| | - Jui Pandhare
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, 37208, USA
| | - Bajarang Kumbhar
- Sunandan Divatia School of Science, NMIMS University, Mumbai, 400056, India
| | - Chandravanu Dash
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, 37208, USA
- Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, 37208, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, 37208, USA
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35
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Azzman N, Gill MSA, Hassan SS, Christ F, Debyser Z, Mohamed WAS, Ahemad N. Pharmacological advances in anti-retroviral therapy for human immunodeficiency virus-1 infection: A comprehensive review. Rev Med Virol 2024; 34:e2529. [PMID: 38520650 DOI: 10.1002/rmv.2529] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 01/23/2024] [Accepted: 03/09/2024] [Indexed: 03/25/2024]
Abstract
The discovery of anti-retroviral (ARV) drugs over the past 36 years has introduced various classes, including nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitor, fusion, and integrase strand transfer inhibitors inhibitors. The introduction of combined highly active anti-retroviral therapies in 1996 was later proven to combat further ARV drug resistance along with enhancing human immunodeficiency virus (HIV) suppression. As though the development of ARV therapies was continuously expanding, the variation of action caused by ARV drugs, along with its current updates, was not comprehensively discussed, particularly for HIV-1 infection. Thus, a range of HIV-1 ARV medications is covered in this review, including new developments in ARV therapy based on the drug's mechanism of action, the challenges related to HIV-1, and the need for combination therapy. Optimistically, this article will consolidate the overall updates of HIV-1 ARV treatments and conclude the significance of HIV-1-related pharmacotherapy research to combat the global threat of HIV infection.
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Affiliation(s)
- Nursyuhada Azzman
- School of Pharmacy, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia
- Faculty of Pharmacy, Universiti Teknologi MARA, Cawangan Pulau Pinang Kampus Bertam, Permatang Pauh, Pulau Pinang, Malaysia
| | - Muhammad Shoaib Ali Gill
- Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
| | - Sharifah Syed Hassan
- Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia
| | - Frauke Christ
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Zeger Debyser
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Wan Ahmad Syazani Mohamed
- Nutrition Unit, Nutrition, Metabolism and Cardiovascular Research Centre (NMCRC), Level 3, Block C, Institute for Medical Research (IMR), National Institutes of Health (NIH) Complex, Ministry of Health Malaysia (MOH), Shah Alam, Selangor, Malaysia
| | - Nafees Ahemad
- School of Pharmacy, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia
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Siew ZY, Asudas E, Khoo CT, Cho GH, Voon K, Fang CM. Fighting nature with nature: antiviral compounds that target retroviruses. Arch Microbiol 2024; 206:130. [PMID: 38416180 DOI: 10.1007/s00203-024-03846-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Revised: 01/10/2024] [Accepted: 01/11/2024] [Indexed: 02/29/2024]
Abstract
The human immunodeficiency virus (HIV) is a type of lentivirus that targets the human immune system and leads to acquired immunodeficiency syndrome (AIDS) at a later stage. Up to 2021, there are millions still living with HIV and many have lost their lives. To date, many anti-HIV compounds have been discovered in living organisms, especially plants and marine sponges. However, no treatment can offer a complete cure, but only suppressing it with a life-long medication, known as combined antiretroviral therapy (cART) or highly active antiretroviral therapy (HAART) which are often associated with various adverse effects. Also, it takes many years for a discovered compound to be approved for clinical use. Thus, by employing advanced technologies such as automation, conducting systematic screening and testing protocols may boost the discovery and development of potent and curative therapeutics for HIV infection/AIDS. In this review, we aim to summarize the antiretroviral therapies/compounds and their associated drawbacks since the discovery of azidothymidine. Additionally, we aim to provide an updated analysis of the most recent discoveries of promising antiretroviral candidates, along with an exploration of the current limitations within antiretroviral research. Finally, we intend to glean insightful perspectives and propose future research directions in this crucial area of study.
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Affiliation(s)
- Zhen Yun Siew
- Division of Biomedical Sciences, School of Pharmacy, University of Nottingham Malaysia, Jalan Broga, 43500, Semenyih, Selangor, Malaysia.
| | - Elishea Asudas
- Division of Biomedical Sciences, School of Pharmacy, University of Nottingham Malaysia, Jalan Broga, 43500, Semenyih, Selangor, Malaysia
| | - Chia Ting Khoo
- School of Biosciences, University of Nottingham Malaysia, 43500, Semenyih, Selangor, Malaysia
| | - Gang Hyeon Cho
- School of Pharmacy, University of Nottingham Malaysia, 43500, Semenyih, Selangor, Malaysia
| | - Kenny Voon
- Division of Biomedical Sciences, School of Pharmacy, University of Nottingham Malaysia, Jalan Broga, 43500, Semenyih, Selangor, Malaysia
| | - Chee-Mun Fang
- Division of Biomedical Sciences, School of Pharmacy, University of Nottingham Malaysia, Jalan Broga, 43500, Semenyih, Selangor, Malaysia.
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37
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Navasardyan I, Miwalian R, Petrosyan A, Yeganyan S, Venketaraman V. HIV-TB Coinfection: Current Therapeutic Approaches and Drug Interactions. Viruses 2024; 16:321. [PMID: 38543687 PMCID: PMC10974211 DOI: 10.3390/v16030321] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2023] [Revised: 02/16/2024] [Accepted: 02/17/2024] [Indexed: 05/23/2024] Open
Abstract
The co-occurrence of human immunodeficiency virus (HIV) and tuberculosis (TB) infection poses a significant global health challenge. Treatment of HIV and TB co-infection often necessitates combination therapy involving antiretroviral therapy (ART) for HIV and anti-TB medications, which introduces the potential for drug-drug interactions (DDIs). These interactions can significantly impact treatment outcomes, the efficacy of treatment, safety, and overall patient well-being. This review aims to provide a comprehensive analysis of the DDIs between anti-HIV and anti-TB drugs as well as potential adverse effects resulting from the concomitant use of these medications. Furthermore, such findings may be used to develop personalized therapeutic strategies, dose adjustments, or alternative drug choices to minimize the risk of adverse outcomes and ensure the effective management of HIV and TB co-infection.
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Affiliation(s)
| | | | | | | | - Vishwanath Venketaraman
- College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766, USA; (I.N.); (R.M.); (A.P.); (S.Y.)
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Zhitkevich A, Bayurova E, Avdoshina D, Zakirova N, Frolova G, Chowdhury S, Ivanov A, Gordeychuk I, Palefsky JM, Isaguliants M. HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype. Viruses 2024; 16:193. [PMID: 38399969 PMCID: PMC10892743 DOI: 10.3390/v16020193] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 01/22/2024] [Accepted: 01/24/2024] [Indexed: 02/25/2024] Open
Abstract
The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.
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Affiliation(s)
- Alla Zhitkevich
- Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, 119991 Moscow, Russia; (E.B.); (D.A.); (G.F.); (I.G.)
| | - Ekaterina Bayurova
- Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, 119991 Moscow, Russia; (E.B.); (D.A.); (G.F.); (I.G.)
- Gamaleya National Research Center for Epidemiology and Microbiology, 123098 Moscow, Russia;
| | - Darya Avdoshina
- Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, 119991 Moscow, Russia; (E.B.); (D.A.); (G.F.); (I.G.)
| | - Natalia Zakirova
- Centre for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia;
| | - Galina Frolova
- Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, 119991 Moscow, Russia; (E.B.); (D.A.); (G.F.); (I.G.)
| | - Sona Chowdhury
- Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143, USA; (S.C.); (J.M.P.)
| | - Alexander Ivanov
- Gamaleya National Research Center for Epidemiology and Microbiology, 123098 Moscow, Russia;
- Centre for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia;
| | - Ilya Gordeychuk
- Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, 119991 Moscow, Russia; (E.B.); (D.A.); (G.F.); (I.G.)
- Gamaleya National Research Center for Epidemiology and Microbiology, 123098 Moscow, Russia;
| | - Joel M. Palefsky
- Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143, USA; (S.C.); (J.M.P.)
| | - Maria Isaguliants
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden
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Abstract
IMPORTANCE HIV-1 capsid protein (CA)-independently or by recruiting host factors-mediates several key steps of virus replication in the cytoplasm and nucleus of the target cell. Research in the recent years have established that CA is multifunctional and genetically fragile of all the HIV-1 proteins. Accordingly, CA has emerged as a validated and high priority therapeutic target, and the first CA-targeting antiviral drug was recently approved for treating multi-drug resistant HIV-1 infection. However, development of next generation CA inhibitors depends on a better understanding of CA's known roles, as well as probing of CA's novel roles, in HIV-1 replication. In this timely review, we present an updated overview of the current state of our understanding of CA's multifunctional role in HIV-1 replication-with a special emphasis on CA's newfound post-nuclear roles, highlight the pressing knowledge gaps, and discuss directions for future research.
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Affiliation(s)
- Richa Dwivedi
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
| | - Prem Prakash
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
| | - Bajarang Vasant Kumbhar
- Department of Biological Sciences, Sunandan Divatia School of Science, NMIMS (Deemed to be) University, Mumbai, Maharashtra, India
| | - Muthukumar Balasubramaniam
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
| | - Chandravanu Dash
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, Tennessee, USA
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Yue Z, Zhang X, Gu Y, Liu Y, Lan LM, Liu Y, Li Y, Yang G, Wan P, Chen X. Regulation and functions of the NLRP3 inflammasome in RNA virus infection. Front Cell Infect Microbiol 2024; 13:1309128. [PMID: 38249297 PMCID: PMC10796458 DOI: 10.3389/fcimb.2023.1309128] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2023] [Accepted: 11/30/2023] [Indexed: 01/23/2024] Open
Abstract
Virus infection is one of the greatest threats to human life and health. In response to viral infection, the host's innate immune system triggers an antiviral immune response mostly mediated by inflammatory processes. Among the many pathways involved, the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome has received wide attention in the context of viral infection. The NLRP3 inflammasome is an intracellular sensor composed of three components, including the innate immune receptor NLRP3, adaptor apoptosis-associated speck-like protein containing CARD (ASC), and the cysteine protease caspase-1. After being assembled, the NLRP3 inflammasome can trigger caspase-1 to induce gasdermin D (GSDMD)-dependent pyroptosis, promoting the maturation and secretion of proinflammatory cytokines such as interleukin-1 (IL-1β) and interleukin-18 (IL-18). Recent studies have revealed that a variety of viruses activate or inhibit the NLRP3 inflammasome via viral particles, proteins, and nucleic acids. In this review, we present a variety of regulatory mechanisms and functions of the NLRP3 inflammasome upon RNA viral infection and demonstrate multiple therapeutic strategies that target the NLRP3 inflammasome for anti-inflammatory effects in viral infection.
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Affiliation(s)
- Zhaoyang Yue
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Xuelong Zhang
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Yu Gu
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Ying Liu
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Lin-Miaoshen Lan
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Yilin Liu
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Yongkui Li
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
| | - Ge Yang
- Foshan Institute of Medical Microbiology, Foshan, China
| | - Pin Wan
- Foshan Institute of Medical Microbiology, Foshan, China
- Wuhan Institute of Biomedical Sciences, School of Medicine, Jianghan University, Wuhan, China
| | - Xin Chen
- Institute of Medical Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China
- Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, China
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41
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Romero EV, Feder AF. Elevated HIV Viral Load is Associated with Higher Recombination Rate In Vivo. Mol Biol Evol 2024; 41:msad260. [PMID: 38197289 PMCID: PMC10777272 DOI: 10.1093/molbev/msad260] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 11/21/2023] [Accepted: 11/27/2023] [Indexed: 01/11/2024] Open
Abstract
HIV's exceptionally high recombination rate drives its intrahost diversification, enabling immune escape and multidrug resistance within people living with HIV. While we know that HIV's recombination rate varies by genomic position, we have little understanding of how recombination varies throughout infection or between individuals as a function of the rate of cellular coinfection. We hypothesize that denser intrahost populations may have higher rates of coinfection and therefore recombination. To test this hypothesis, we develop a new approach (recombination analysis via time series linkage decay or RATS-LD) to quantify recombination using autocorrelation of linkage between mutations across time points. We validate RATS-LD on simulated data under short read sequencing conditions and then apply it to longitudinal, high-throughput intrahost viral sequencing data, stratifying populations by viral load (a proxy for density). Among sampled viral populations with the lowest viral loads (<26,800 copies/mL), we estimate a recombination rate of 1.5×10-5 events/bp/generation (95% CI: 7×10-6 to 2.9×10-5), similar to existing estimates. However, among samples with the highest viral loads (>82,000 copies/mL), our median estimate is approximately 6 times higher. In addition to co-varying across individuals, we also find that recombination rate and viral load are associated within single individuals across different time points. Our findings suggest that rather than acting as a constant, uniform force, recombination can vary dynamically and drastically across intrahost viral populations and within them over time. More broadly, we hypothesize that this phenomenon may affect other facultatively asexual populations where spatial co-localization varies.
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Affiliation(s)
- Elena V Romero
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Alison F Feder
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
- Herbold Computational Biology Program, Fred Hutchinson Cancer Center, Seattle, WA, USA
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42
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Balaji S, Chakraborty R, Aggarwal S. Neurological Complications Caused by Human Immunodeficiency Virus (HIV) and Associated Opportunistic Co-infections: A Review on their Diagnosis and Therapeutic Insights. CNS & NEUROLOGICAL DISORDERS DRUG TARGETS 2024; 23:284-305. [PMID: 37005520 DOI: 10.2174/1871527322666230330083708] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Revised: 12/28/2022] [Accepted: 01/25/2023] [Indexed: 04/04/2023]
Abstract
Neurocognitive disorders associated with human immunodeficiency virus (HIV) infected individuals increase the risk of mortality and morbidity that remain a prevalent clinical complication even in the antiretroviral therapy era. It is estimated that a considerable number of people in the HIV community are developing neurological complications at their early stages of infection. The daily lives of people with chronic HIV infections are greatly affected by cognitive declines such as loss of attention, learning, and executive functions, and other adverse conditions like neuronal injury and dementia. It has been found that the entry of HIV into the brain and subsequently crossing the blood-brain barrier (BBB) causes brain cell damage, which is the prerequisite for the development of neurocognitive disorders. Besides the HIV replication in the central nervous system and the adverse effects of antiretroviral therapy on the BBB, a range of opportunistic infections, including viral, bacterial, and parasitic agents, augment the neurological complications in people living with HIV (PLHIV). Given the immuno-compromised state of PLHIV, these co-infections can present a wide range of clinical syndromes with atypical manifestations that pose challenges in diagnosis and clinical management, representing a substantial burden for the public health system. Therefore, the present review narrates the neurological complications triggered by HIV and their diagnosis and treatment options. Moreover, coinfections that are known to cause neurological disorders in HIV infected individuals are highlighted.
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Affiliation(s)
- Sivaraman Balaji
- Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research-Headquarters, Ansari Nagar, New Delhi, 110029, India
| | - Rohan Chakraborty
- Department of Medical Elementology and Toxicology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, India
| | - Sumit Aggarwal
- Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research-Headquarters, Ansari Nagar, New Delhi, 110029, India
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43
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Levintov L, Vashisth H. Structural and computational studies of HIV-1 RNA. RNA Biol 2024; 21:1-32. [PMID: 38100535 PMCID: PMC10730233 DOI: 10.1080/15476286.2023.2289709] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 11/15/2023] [Accepted: 11/21/2023] [Indexed: 12/17/2023] Open
Abstract
Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA genome, which is reverse transcribed into viral DNA and further integrated into the host-cell DNA for viral replication and proliferation. The RNA structures from the HIV-1 genome provide valuable insights into the mechanisms underlying the viral replication cycle. Moreover, these structures serve as models for designing novel therapeutic approaches. Here, we review structural data on RNA from the HIV-1 genome as well as computational studies based on these structural data. The review is organized according to the type of structured RNA element which contributes to different steps in the viral replication cycle. This is followed by an overview of the HIV-1 transactivation response element (TAR) RNA as a model system for understanding dynamics and interactions in the viral RNA systems. The review concludes with a description of computational studies, highlighting the impact of biomolecular simulations in elucidating the mechanistic details of various steps in the HIV-1's replication cycle.
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Affiliation(s)
- Lev Levintov
- Department of Chemical Engineering & Bioengineering, University of New Hampshire, Durham, USA
| | - Harish Vashisth
- Department of Chemical Engineering & Bioengineering, University of New Hampshire, Durham, USA
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44
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Sun L, Nie P, Luan L, Herdewijn P, Wang YT. Synthetic approaches and application of clinically approved small-molecule Anti-HIV drugs: An update. Eur J Med Chem 2023; 261:115847. [PMID: 37801826 DOI: 10.1016/j.ejmech.2023.115847] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2023] [Revised: 09/18/2023] [Accepted: 09/29/2023] [Indexed: 10/08/2023]
Abstract
Application of chemotherapeutic agents to inhibit the HIV replication process has brought about a significant metamorphosis in the landscape of AIDS. Substantial declines in morbidity and mortality rates have been attained, accompanied by notable decreases in healthcare resource utilization. However, treatment modalities do not uniformly inhibit HIV replication in every patient, while the emergence of drug-resistant viral strains poses a substantial obstacle to subsequent therapeutic interventions. Furthermore, chronic administration of therapy may lead to the manifestation of toxicities. These challenges necessitate the exploration of novel pharmacological agents and innovative therapeutic approaches aimed at effectively managing the persistent viral replication characteristic of chronic infection. This review examines the role of clinically approved small-molecule drugs in the treatment of HIV/AIDS, which provides an in-depth analysis of the major classes of small-molecule drugs, including nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors, entry inhibitors, and pharmacokinetic enhancers. The review mainly discusses the application, synthetic routes, and mechanisms of action of small-molecule drugs employed in the treatment of HIV, as well as their use in combination with antiretroviral therapy, presenting viewpoints on forthcoming avenues in the development of novel anti-HIV drugs.
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Affiliation(s)
- Lu Sun
- Zhongshan Hospital Affiliated to Dalian University, Dalian, 116001, China
| | - Peng Nie
- Medicinal Chemistry, Rega Institute of Medical Research, KU Leuven, Herestraat 49, 3000, Leuven, Belgium
| | - Li Luan
- Zhongshan Hospital Affiliated to Dalian University, Dalian, 116001, China.
| | - Piet Herdewijn
- Medicinal Chemistry, Rega Institute of Medical Research, KU Leuven, Herestraat 49, 3000, Leuven, Belgium.
| | - Ya-Tao Wang
- First People's Hospital of Shangqiu, Henan Province, Shangqiu, 476100, China; Medicinal Chemistry, Rega Institute of Medical Research, KU Leuven, Herestraat 49, 3000, Leuven, Belgium.
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45
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Batran RZ, Sabt A, Khedr MA, Allayeh AK, Pannecouque C, Kassem AF. 4-Phenylcoumarin derivatives as new HIV-1 NNRTIs: Design, synthesis, biological activities, and computational studies. Bioorg Chem 2023; 141:106918. [PMID: 37866206 DOI: 10.1016/j.bioorg.2023.106918] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 10/05/2023] [Accepted: 10/13/2023] [Indexed: 10/24/2023]
Abstract
A series of 4-phenylcoumarin derivatives was synthesized and evaluated for their cellular anti-HIV-1 and HIV-2 activities as well as their inhibitory effects against HIV-1 reverse transcriptase (RT). The hydrazone compound 8b and the ethylthiosemicarbazide derivative 4c showed the best inhibition activity against wild-type (WT) HIV-1. The promising compounds were further evaluated against HIV-1 RT and exhibited significant inhibitory activity with compound 8b showing comparable effect to the reference NNRTI Efavirenz (IC50 = 9.01 nM). Structure activity relationship study revealed the importance of 6-chloro and 4-phenyl substituents for optimum activity, as well as the 5-atoms linker (=N-NH-CO-CH2-O-) at position 7 of coumarin scaffold that can support the rotation and flexibility of compound 8b to fit well in the binding pocket. The molecular docking of compound 8b demonstrated a typical seahorse binding mode with better binding interactions that covered more residues when compared to Efavirenz.
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Affiliation(s)
- Rasha Z Batran
- Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, Dokki, Cairo 12622, Egypt.
| | - Ahmed Sabt
- Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, Dokki, Cairo 12622, Egypt
| | - Mohammed A Khedr
- Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Kuwait
| | - Abdou K Allayeh
- Water Pollution Research Department, Environment and Climate Change Institute, National Research Centre, Dokki, Cairo 12622, Egypt
| | | | - Asmaa F Kassem
- Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, Dokki, Cairo 12622, Egypt
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Abstract
PURPOSE OF REVIEW This review aims to elucidate the multifaceted role of the tumor suppressor protein p53 in the context of HIV infection. We explore how p53, a pivotal regulator of cellular processes, interacts with various facets of the HIV life cycle. Understanding these interactions could provide valuable insights into potential therapeutic interventions and the broader implications of p53 in viral infections. RECENT FINDINGS Recent research has unveiled a complex interplay between p53 and HIV. Several reports have highlighted the involvement of p53 in restricting the replication of HIV within both immune and nonimmune cells. Various mechanisms have been suggested to unveil how p53 enforces this restriction on HIV replication. However, HIV has developed strategies to manipulate p53, benefiting its replication and evading host defenses. In summary, p53 plays a multifaceted role in HIV infection, impacting viral replication and disease progression. Recent findings underscore the importance of understanding the intricate interactions between p53 and HIV for the development of innovative therapeutic approaches. Manipulating p53 pathways may offer potential avenues to suppress viral replication and ameliorate immune dysfunction, ultimately contributing to the management of HIV/AIDS. Further research is warranted to fully exploit the therapeutic potential of p53 in the context of HIV infection.
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Affiliation(s)
- Mahmoud Mohammad Yaseen
- Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts, Jordan University of Science and Technology, P.O. Box 3030, Irbid, 22110, Jordan.
| | - Nizar Mohammad Abuharfeil
- Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts, Jordan University of Science and Technology, P.O. Box 3030, Irbid, 22110, Jordan
| | - Homa Darmani
- Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts, Jordan University of Science and Technology, P.O. Box 3030, Irbid, 22110, Jordan
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47
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Padron A, Prakash P, Pandhare J, Luban J, Aiken C, Balasubramaniam M, Dash C. Emerging role of cyclophilin A in HIV-1 infection: from producer cell to the target cell nucleus. J Virol 2023; 97:e0073223. [PMID: 37843371 PMCID: PMC10688351 DOI: 10.1128/jvi.00732-23] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2023] Open
Abstract
The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.
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Affiliation(s)
- Adrian Padron
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- School of Graduate Studies, Meharry Medical College, Nashville, Tennessee, USA
| | - Prem Prakash
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Pharmacology and Neuroscience, Meharry Medical College, Nashville, Tennessee, USA
| | - Jui Pandhare
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- School of Graduate Studies, Meharry Medical College, Nashville, Tennessee, USA
| | - Jeremy Luban
- Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
| | - Chris Aiken
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Muthukumar Balasubramaniam
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Pharmacology and Neuroscience, Meharry Medical College, Nashville, Tennessee, USA
| | - Chandravanu Dash
- The Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, Tennessee, USA
- Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, Tennessee, USA
- Department of Biochemistry, Cancer Biology, Pharmacology and Neuroscience, Meharry Medical College, Nashville, Tennessee, USA
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48
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Periferakis A, Periferakis AT, Troumpata L, Periferakis K, Scheau AE, Savulescu-Fiedler I, Caruntu A, Badarau IA, Caruntu C, Scheau C. Kaempferol: A Review of Current Evidence of Its Antiviral Potential. Int J Mol Sci 2023; 24:16299. [PMID: 38003488 PMCID: PMC10671393 DOI: 10.3390/ijms242216299] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Revised: 11/07/2023] [Accepted: 11/12/2023] [Indexed: 11/26/2023] Open
Abstract
Kaempferol and its derivatives are flavonoids found in various plants, and a considerable number of these have been used in various medical applications worldwide. Kaempferol and its compounds have well-known antioxidant, anti-inflammatory and antimicrobial properties among other health benefits. However, the antiviral properties of kaempferol are notable, and there is a significant number of experimental studies on this topic. Kaempferol compounds were effective against DNA viruses such as hepatitis B virus, viruses of the alphaherpesvirinae family, African swine fever virus, and pseudorabies virus; they were also effective against RNA viruses, namely feline SARS coronavirus, dengue fever virus, Japanese encephalitis virus, influenza virus, enterovirus 71, poliovirus, respiratory syncytial virus, human immunodeficiency virus, calicivirus, and chikungunya virus. On the other hand, no effectiveness against murine norovirus and hepatitis A virus could be determined. The antiviral action mechanisms of kaempferol compounds are various, such as the inhibition of viral polymerases and of viral attachment and entry into host cells. Future research should be focused on further elucidating the antiviral properties of kaempferol compounds from different plants and assessing their potential use to complement the action of antiviral drugs.
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Affiliation(s)
- Argyrios Periferakis
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
- Akadimia of Ancient Greek and Traditional Chinese Medicine, 16675 Athens, Greece
- Elkyda, Research & Education Centre of Charismatheia, 17675 Athens, Greece
| | - Aristodemos-Theodoros Periferakis
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
- Elkyda, Research & Education Centre of Charismatheia, 17675 Athens, Greece
| | - Lamprini Troumpata
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Konstantinos Periferakis
- Akadimia of Ancient Greek and Traditional Chinese Medicine, 16675 Athens, Greece
- Pan-Hellenic Organization of Educational Programs (P.O.E.P), 17236 Athens, Greece
| | - Andreea-Elena Scheau
- Department of Radiology and Medical Imaging, Fundeni Clinical Institute, 022328 Bucharest, Romania
| | - Ilinca Savulescu-Fiedler
- Department of Internal Medicine, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
- Department of Internal Medicine and Cardiology, Coltea Clinical Hospital, 030167 Bucharest, Romania
| | - Ana Caruntu
- Department of Oral and Maxillofacial Surgery, “Carol Davila” Central Military Emergency Hospital, 010825 Bucharest, Romania
- Department of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, “Titu Maiorescu” University, 031593 Bucharest, Romania
| | - Ioana Anca Badarau
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Constantin Caruntu
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
- Department of Dermatology, “Prof. N.C. Paulescu” National Institute of Diabetes, Nutrition and Metabolic Diseases, 011233 Bucharest, Romania
| | - Cristian Scheau
- Department of Physiology, The “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
- Department of Radiology and Medical Imaging, “Foisor” Clinical Hospital of Orthopaedics, Traumatology and Osteoarticular TB, 021382 Bucharest, Romania
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49
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Jennings J, Bracey H, Nguyen DT, Dasgupta R, Rivera AV, Sluis-Cremer N, Shi J, Aiken C. The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.08.566350. [PMID: 37986899 PMCID: PMC10659366 DOI: 10.1101/2023.11.08.566350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2023]
Abstract
The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 particles can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, indicating that gp41 permits the entry of nucleotides into virions. Protease treatment of virions markedly reduced NERT suggesting the presence of a proteinaceous membrane channel. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.
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Affiliation(s)
- Jordan Jennings
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Harrison Bracey
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Danny T. Nguyen
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Rishav Dasgupta
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Alondra Vázquez Rivera
- Division of Infectious Disease, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America
| | - Nicolas Sluis-Cremer
- Division of Infectious Disease, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America
| | - Jiong Shi
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Christopher Aiken
- Department of Pathology, Microbiology, and Immunology and Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
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Romero EV, Feder AF. Elevated HIV viral load is associated with higher recombination rate in vivo. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.05.539643. [PMID: 37873119 PMCID: PMC10592651 DOI: 10.1101/2023.05.05.539643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
HIV's exceptionally high recombination rate drives its intra-host diversification, enabling immune escape and multi-drug resistance within people living with HIV. While we know that HIV's recombination rate varies by genomic position, we have little understanding of how recombination varies throughout infection or between individuals as a function of the rate of cellular coinfection. We hypothesize that denser intra-host populations may have higher rates of coinfection and therefore recombination. To test this hypothesis, we develop a new approach (Recombination Analysis via Time Series Linkage Decay, or RATS-LD) to quantify recombination using autocorrelation of linkage between mutations across time points. We validate RATS-LD on simulated data under short read sequencing conditions and then apply it to longitudinal, high-throughput intra-host viral sequencing data, stratifying populations by viral load (a proxy for density). Among sampled viral populations with the lowest viral loads (< 26,800 copies/mL), we estimate a recombination rate of 1.5 × 10-5 events/bp/generation (95% CI: 7 × 10-6 - 2.9 × 10-5), similar to existing estimates. However, among samples with the highest viral loads (> 82,000 copies/mL), our median estimate is approximately 6 times higher. In addition to co-varying across individuals, we also find that recombination rate and viral load are associated within single individuals across different time points. Our findings suggest that rather than acting as a constant, uniform force, recombination can vary dynamically and drastically across intra-host viral populations and within them over time. More broadly, we hypothesize that this phenomenon may affect other facultatively asexual populations where spatial co-localization varies.
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Affiliation(s)
- Elena V. Romero
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Alison F. Feder
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
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