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Hyde VR, Zhou C, Fernandez JR, Chatterjee K, Ramakrishna P, Lin A, Fisher GW, Çeliker OT, Caldwell J, Bender O, Sauer PJ, Lugo-Martinez J, Bar DZ, D'Aiuto L, Shemesh OA. Anti-herpetic tau preserves neurons via the cGAS-STING-TBK1 pathway in Alzheimer's disease. Cell Rep 2025; 44:115109. [PMID: 39753133 DOI: 10.1016/j.celrep.2024.115109] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Revised: 08/06/2024] [Accepted: 12/03/2024] [Indexed: 02/01/2025] Open
Abstract
Alzheimer's disease (AD) diagnosis relies on the presence of extracellular β-amyloid (Aβ) and intracellular hyperphosphorylated tau (p-tau). Emerging evidence suggests a potential link between AD pathologies and infectious agents, with herpes simplex virus 1 (HSV-1) being a leading candidate. Our investigation, using metagenomics, mass spectrometry, western blotting, and decrowding expansion pathology, detects HSV-1-associated proteins in human brain samples. Expression of the herpesvirus protein ICP27 increases with AD severity and strongly colocalizes with p-tau but not with Aβ. Modeling in human brain organoids shows that HSV-1 infection elevates tau phosphorylation. Notably, p-tau reduces ICP27 expression and markedly decreases post-infection neuronal death from 64% to 7%. This modeling prompts investigation into the cGAS-STING-TBK1 pathway products, nuclear factor (NF)-κB and IRF-3, which colocalizes with ICP27 and p-tau in AD. Furthermore, experimental activation of STING enhances tau phosphorylation, while TBK1 inhibition prevents it. Together, these findings suggest that tau phosphorylation acts as an innate immune response in AD, driven by cGAS-STING.
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Affiliation(s)
- Vanesa R Hyde
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Chaoming Zhou
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Juan R Fernandez
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Krishnashis Chatterjee
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Pururav Ramakrishna
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Amanda Lin
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Gregory W Fisher
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Orhan Tunç Çeliker
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Jill Caldwell
- Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Omer Bender
- Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Peter Joseph Sauer
- Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Jose Lugo-Martinez
- Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Daniel Z Bar
- Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Leonardo D'Aiuto
- Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Or A Shemesh
- School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 9112102, Israel; Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
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Xiang J, Fan C, Dong H, Ma Y, Xu P. A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1. eLife 2023; 12:e85412. [PMID: 37702383 PMCID: PMC10522339 DOI: 10.7554/elife.85412] [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: 12/06/2022] [Accepted: 09/12/2023] [Indexed: 09/14/2023] Open
Abstract
The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an 'Escaping' effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for 'Escaping'. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.
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Affiliation(s)
- Juan Xiang
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Chaoyang Fan
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Hongchang Dong
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Yilei Ma
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Pei Xu
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
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Harrell TL, Davido DJ, Bertke AS. Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 0 (ICP0) Targets of Ubiquitination during Productive Infection of Primary Adult Sensory Neurons. Int J Mol Sci 2023; 24:2931. [PMID: 36769256 PMCID: PMC9917815 DOI: 10.3390/ijms24032931] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Revised: 01/18/2023] [Accepted: 01/26/2023] [Indexed: 02/05/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons.
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Affiliation(s)
- Telvin L. Harrell
- Biomedical and Veterinary Science, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
| | - David J. Davido
- Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
| | - Andrea S. Bertke
- Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
- Center for Emerging Zoonotic and Arthropod-Borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
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Agelidis A, Turturice BA, Suryawanshi RK, Yadavalli T, Jaishankar D, Ames J, Hopkins J, Koujah L, Patil CD, Hadigal SR, Kyzar EJ, Campeau A, Wozniak JM, Gonzalez DJ, Vlodavsky I, Li JP, Perkins DL, Finn PW, Shukla D. Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival. JCI Insight 2021; 6:144255. [PMID: 33621216 PMCID: PMC8119219 DOI: 10.1172/jci.insight.144255] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 02/18/2021] [Indexed: 01/03/2023] Open
Abstract
The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.
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Affiliation(s)
- Alex Agelidis
- Department of Microbiology and Immunology
- Department of Ophthalmology and Visual Sciences, and
| | - Benjamin A. Turturice
- Department of Microbiology and Immunology
- Division of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA
| | | | | | - Dinesh Jaishankar
- Department of Ophthalmology and Visual Sciences, and
- Department of Dermatology, Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA
| | - Joshua Ames
- Department of Microbiology and Immunology
- Department of Ophthalmology and Visual Sciences, and
| | - James Hopkins
- Department of Microbiology and Immunology
- Department of Ophthalmology and Visual Sciences, and
| | - Lulia Koujah
- Department of Microbiology and Immunology
- Department of Ophthalmology and Visual Sciences, and
| | | | | | - Evan J. Kyzar
- Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois, USA
| | - Anaamika Campeau
- Department of Pharmacology and
- Skaggs School of Pharmacy, UCSD, San Diego, La Jolla, California, USA
| | - Jacob M. Wozniak
- Department of Pharmacology and
- Skaggs School of Pharmacy, UCSD, San Diego, La Jolla, California, USA
| | - David J. Gonzalez
- Department of Pharmacology and
- Skaggs School of Pharmacy, UCSD, San Diego, La Jolla, California, USA
| | - Israel Vlodavsky
- Technion Integrated Cancer Center (TICC), Rappaport Faculty of Medicine, Technion, Haifa, Israel
| | - Jin-ping Li
- Department of Medical Biochemistry and Microbiology, University of Uppsala, Uppsala, Sweden
| | - David L. Perkins
- Division of Nephrology, Department of Medicine, and
- Department of Surgery, University of Illinois at Chicago, Chicago, Illinois, USA
| | - Patricia W. Finn
- Department of Microbiology and Immunology
- Division of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA
| | - Deepak Shukla
- Department of Microbiology and Immunology
- Department of Ophthalmology and Visual Sciences, and
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5
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Dogrammatzis C, Waisner H, Kalamvoki M. "Non-Essential" Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review. Viruses 2020; 13:E17. [PMID: 33374862 PMCID: PMC7824580 DOI: 10.3390/v13010017] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 12/19/2022] Open
Abstract
Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.
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Affiliation(s)
| | | | - Maria Kalamvoki
- Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA; (C.D.); (H.W.)
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Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay. J Virol 2019; 93:JVI.00619-19. [PMID: 30996104 DOI: 10.1128/jvi.00619-19] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 04/12/2019] [Indexed: 01/23/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.
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7
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Khalique H, López Marco J, Lim F. A haploid HSV-1 genome platform for vector development: testing of the tetracycline-responsive switch shows interference by infected cell protein 0. J Gene Med 2018; 18:302-311. [PMID: 27672733 DOI: 10.1002/jgm.2929] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 09/23/2016] [Accepted: 09/23/2016] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Although herpes simplex virus type 1 (HSV-1) has outstanding properties for gene delivery vectors and its genome is available in bacterial artificial chromosomes (BACs) for mutagenesis studies, one impediment is the presence of approximately 15.4 kb of DNA sequences that are duplicated in the HSV-1 genome, complicating vector construction and stability. METHODS As a useful platform for building HSV-1 vectors, we have constructed a fully haploid HSV-1 genome BAC by deletion of one of these repeats, confirming that viral propagation in culture is not impaired. We used this ΔIR mutant to subsequently investigate whether the insertion of tetracycline-responsive tetO elements into the ICP34.5-ICP0 gene region can be used to control HSV-1 lytic replication. RESULTS The results of the present study show that ΔIR mutants deleted for ICP34.5 are viable for replication but not when the ICP0 promoter is also disrupted, thus indicating that regulation of infected cell protein 0 (ICP0) levels in the absence of ICP34.5 could be a viable means for controlling growth of HSV-1 vectors. Surprisingly, however, the tetO elements inserted into the ICP0 promoter did not confer ligand responsiveness to growth or ICP0 expression. Further analysis by transfection experiments revealed that ICP0 itself interferes with the tetracycline switch and reduces the the inducibility of this system. CONCLUSIONS Our new haploid HSV-1 BAC is a useful platform for building multiply deleted HSV-1 vectors. Deletion of the gene for ICP34.5 in this backbone renders viral growth dependent on ICP0, although ICP0 expression could not be regulated by tet-responsive transcriptional regulators.
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Affiliation(s)
- Hena Khalique
- Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
| | - Jorge López Marco
- Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
| | - Filip Lim
- Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
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Cbl E3 Ligase Mediates the Removal of Nectin-1 from the Surface of Herpes Simplex Virus 1-Infected Cells. J Virol 2017; 91:JVI.00393-17. [PMID: 28381567 DOI: 10.1128/jvi.00393-17] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2017] [Accepted: 03/28/2017] [Indexed: 01/24/2023] Open
Abstract
The Cbl E3 ligase has been linked to the down-modulation of surface signaling responses by inducing internalization of surface receptors. The adaptor protein CIN85 is a partner of Cbl that augments many of these interactions. Previously, an interaction was demonstrated between ICP0 and CIN85, which results in the removal of epidermal growth factor receptor (EGFR) from the surface of the infected cells with a concomitant attenuation of EGFR signaling. Here, we examined whether Cbl mediates the removal of the herpes simplex virus 1 (HSV-1) entry receptor Nectin-1 from the surface of infected cells. We found the following: (i) that Cbl, Nectin-1, and the viral glycoprotein D (gD) form a complex in infected cells; (ii) that during infection Nectin-1 is removed from the surface of the infected cells but is retained on the surface of cells that have been depleted of Cbl; and (iii) that in cells infected with a ΔICP0 mutant virus, Nectin-1 remained on the cell surface. Thus, Cbl is necessary but not sufficient for the removal of Nectin-1 from the cell surface. In addition, we observed that in Cbl-depleted cells there was enhanced entry after infection. These cells were susceptible to secondary infections by HSV-1. Viral entry in CIN85-depleted cells was only moderately enhanced compared to that in the Cbl-depleted cells, suggesting that the Cbl-Nectin-1 interaction is likely the key to the downregulation of surface Nectin-1. The removal of the HSV-1 entry receptor Nectin-1 from the surface of the infected cells may be part of the strategy of the virus to efficiently spread to uninfected cells.IMPORTANCE The Cbl E3 ligase suppresses surface signaling responses by inducing internalization of surface components. The targets of Cbl include such components as immune system receptors, growth factor receptors, adhesion, and cell-to-cell contact molecules. The immediate early protein ICP0 of herpes simplex virus 1 (HSV-1) interacts with CIN85, an adaptor protein that augments Cbl functions. The consequence of this interaction is the removal of the epidermal growth factor receptor (EGFR) from the surface of the infected cells with concomitant suppression of the EGF ligand signaling. The viral entry receptor Nectin-1 is also internalized during HSV-1 infection in a Cbl-dependent mechanism, and that increases the opportunity of the virus to spread to uninfected cells. The diversion of the Cbl/CIN85 endocytic machinery may be a strategy utilized by the virus to alter the cell surface pattern to prevent detrimental host responses.
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9
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Gu H. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication. World J Virol 2016; 5:1-13. [PMID: 26870669 PMCID: PMC4735549 DOI: 10.5501/wjv.v5.i1.1] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2015] [Revised: 10/28/2015] [Accepted: 12/08/2015] [Indexed: 02/05/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host.
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10
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Hogue IB, Bosse JB, Engel EA, Scherer J, Hu JR, Del Rio T, Enquist LW. Fluorescent Protein Approaches in Alpha Herpesvirus Research. Viruses 2015; 7:5933-61. [PMID: 26610544 PMCID: PMC4664988 DOI: 10.3390/v7112915] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 10/12/2015] [Accepted: 10/14/2015] [Indexed: 12/28/2022] Open
Abstract
In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.
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Affiliation(s)
- Ian B Hogue
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Jens B Bosse
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Esteban A Engel
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Julian Scherer
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Jiun-Ruey Hu
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Tony Del Rio
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
| | - Lynn W Enquist
- Department of Molecular Biology & Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA.
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11
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The stability of herpes simplex virus 1 ICP0 early after infection is defined by the RING finger and the UL13 protein kinase. J Virol 2014; 88:5437-43. [PMID: 24574411 DOI: 10.1128/jvi.00542-14] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
UNLABELLED Herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a multifunctional protein that plays a key role in overcoming numerous facets of host innate immunity. A key function of ICP0 that requires an intact RING finger domain is that of an ubiquitin E3 ligase: ICP0 interacts with at least three ubiquitin-conjugating enzymes of which one, UbcH5a, is required for degradation of PML and SP100. A preceding report showed that ICP0 is highly unstable at very early times after infection but becomes stable at later times. We report here that (i) the degradation of ICP0 is not infected cell specific, (ii) the degradation does not require the interaction of ICP0 with either UbcH5a, UbcH6, or UbcH9, (iii) ICP0 is degraded both early and late in cells infected with a mutant lacking the UL13 protein kinase, (iv) ICP0 encoded by wild-type virus or the ΔUL13 mutant is stable in cells transfected with a plasmid encoding UL13 before infection, (v) ICP0 carrying mutations in the RING finger domain is stable both early and late in infection, and, finally, (vi) in cells infected with both wild type and RING finger mutant only the wild-type ICP0 is rapidly degraded at early times. The results suggest that the stability of ICP0 is mediated by the UL13 protein kinase and that the target of proteolysis is a site at or near the RING domain of ICP0. IMPORTANCE ICP0, a major regulatory protein of HSV-1, turns over rapidly early in infection but becomes stable at late times. We report that stabilization requires the presence of UL13 protein kinase and that an ICP0 with mutations in RING finger is stable. In mixed infections mutant ICP0 is stable, whereas the wild-type ICP0 is degraded. Our findings suggest that the lifestyle of HSV-1 requires an ICP0 that turns over rapidly if late proteins are absent.
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12
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HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation. Proc Natl Acad Sci U S A 2014; 111:E611-7. [PMID: 24449861 DOI: 10.1073/pnas.1323414111] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
STING (stimulator of IFN genes) activates the IFN pathway in response to cytosolic DNA. Knockout of STING in mice was reported to exacerbate the pathogenicity of herpes simplex virus 1 (HSV-1). Here we report the following: (i) STING is stable in cancer-derived HEp-2 or HeLa cells infected with wild-type HSV-1 but is degraded in cells infected with mutants lacking the genes encoding functional infected cell protein 0 (ICP0), ICP4, or the US3 protein kinase (US3-PK). In HEp-2 cells, depletion of STING by shRNA results in a decrease in the yields of wild-type or ΔICP0 viruses. (ii) STING is stable throughout infection with either wild-type or ICP0 mutant viruses in human embryonic lung cells (HEL) or HEK293T cells derived from normal tissues. In these cells, depletion of STING results in higher yields of both wild-type and ΔICP0 viruses. (iii) The US3-PK is also required for stabilization of IFI16, a nuclear DNA sensor. However, the stability of IFI16 does not correlate positively or negatively with that of STING. IFI16 is stable in STING-depleted HEL cells infected with wild-type virus. In contrast to HEL cells, IFI16 was undetectable in STING-depleted HEp-2 cells, and hence the role of HSV-1 in maintaining IFI16 could not be ascertained. The results indicate that in HSV-1-infected cells the stability of IFI16 and the function and stability of STING are dependent on cell derivation, the functional integrity of ICP0, and US3-PK, an indication that in wild-type virus-infected cells both proteins are actively stabilized. In HEp-2 cells, the stability of IFI16 requires STING.
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Interaction of herpes simplex virus ICP0 with ND10 bodies: a sequential process of adhesion, fusion, and retention. J Virol 2013; 87:10244-54. [PMID: 23864622 DOI: 10.1128/jvi.01487-13] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
On entry into the nucleus, herpes simplex virus 1 (HSV-1) DNA localizes to nuclear bodies known as ND10. Gene repression imposed by ND10 is released by a viral protein, ICP0, via degradation of the ND10 constituents promyelocytic leukemia protein (PML) and Sp100 and the subsequent dispersal of ND10 bodies. In order to understand the dynamic interaction between ICP0 and ND10, we carried out deletion mapping to identify the domains of ICP0 responsible for its association with ND10. Here, we report the following. (i) An ND10 entry signal (ND10-ES), located between residues 245 and 474, is required for ICP0 to penetrate and fuse with ND10. ICP0 lacking ND10-ES adheres to the surface of ND10 but fails to enter. (ii) In the absence of ND10-ES, the E3 ubiquitin ligase of ICP0 facilitates the transient adhesion of the truncated ICP0 to the ND10 surface, whereas the presence of ND10-ES in ICP0 renders ND10 fusion regardless of the E3 ligase activity. (iii) The C terminus of ICP0 is required for retention of ICP0 in ND10 but plays no role in the recruitment process. (iv) The adverse effects of an inactive RING domain on viral replication are partially reversed by deleting either ND10-ES or the C-terminal retention domain, suggesting that additional ICP0 functions require the release of ICP0 from ND10. Based on these results, we conclude that association of ICP0 and ND10 is a dynamic process, in which three sequential steps--adhesion, fusion, and retention--are adopted to stabilize the interaction. A faithful execution of these steps defines the ultimate productivity of the virus.
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Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the UL47 tegument protein. Proc Natl Acad Sci U S A 2013; 110:E1669-75. [PMID: 23589852 DOI: 10.1073/pnas.1305475110] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHS-RNase degrades stable cellular mRNAs and α (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral β or γ mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, UL47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral α mRNAs and effectively blocks the degradation of β and γ mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of UL47 suggest that it, along with the α protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and UL47 overexpress α genes and delay the expression of β and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.
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Overexpression of the ubiquitin-specific protease 7 resulting from transfection or mutations in the ICP0 binding site accelerates rather than depresses herpes simplex virus 1 gene expression. J Virol 2012; 86:12871-8. [PMID: 22993145 DOI: 10.1128/jvi.01981-12] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.
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HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons. PLoS Pathog 2012; 8:e1002852. [PMID: 22912575 PMCID: PMC3415458 DOI: 10.1371/journal.ppat.1002852] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2012] [Accepted: 06/26/2012] [Indexed: 02/04/2023] Open
Abstract
Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called “Single” and “Multiple”, which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. “Single” and “Multiple” patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the “Multiple” pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction. After an initial lytic infection, many viruses establish a lifelong latent infection that hides them from the host immune system activity until reactivation. To understand the resurgence of the associated diseases, it is indispensable to acquire a better knowledge of the different mechanisms involved in the antiviral defense. During latency, viral genomes of nuclear-replicative viruses, such as herpes simplex virus type 1 (HSV-1), are stored in the nucleus of host cells in a non-integrated form. Latency establishment is associated with a drastic change in HSV-1 gene expression program that is maintained until reactivation occurs. The last two decades of research has revealed that the functional organization of the cell nucleus, so-called nuclear architecture, is a major factor of regulation of cellular genes expression. Nonetheless, the role of nuclear architecture on HSV-1 gene expression has been widely overlooked. Here we describe that the genome of HSV-1 selectively interacts with two major nuclear structures, the promyelocytic nuclear bodies (PMLNBs or ND10) and the centromeres. We provide evidence supporting that these nuclear domains directly influence the behavior of latent viral genomes and their transcriptional activity. Overall, this study demonstrates that nuclear architecture is a major parameter driving the highly complex HSV-1 latency process.
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The checkpoints of viral gene expression in productive and latent infection: the role of the HDAC/CoREST/LSD1/REST repressor complex. J Virol 2011; 85:7474-82. [PMID: 21450817 DOI: 10.1128/jvi.00180-11] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
At the portal of entry into the body, herpes simplex viruses (HSV) vigorously multiply and spread until curtailed by the adaptive immune response. At the same time, HSV invades nerve ending-abutting infected cells and is transported in a retrograde manner to the neuronal nucleus, where it establishes a latent (silent) infection. At intervals, as a consequence of physical or metabolic stress, the virus is activated and transported in an anterograde manner to the body surface. The progression of infection is regulated at four checkpoints. In cell culture or at the portal of entry into the body, HSV uses components of the HDAC1- or HDAC2/CoREST/LSD1/REST repressor complex to activate α genes (checkpoint 1) and then uses an α protein, ICP0, to suppress the same repressor complex from silencing post-α gene expression (checkpoint 2). In neurons destined to harbor latent virus (checkpoint 3), HSV hijacks the same repressor complex to silence itself as a first step in the establishment of the latent state. Suppression of histone deacetylases (HDACs) plays a key role in the reactivation from latency (checkpoint 4). HSV has evolved a strategy of using the same host repressor complex to meet its diverse lifestyle needs.
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Interwoven roles of cyclin D3 and cdk4 recruited by ICP0 and ICP4 in the expression of herpes simplex virus genes. J Virol 2010; 84:9709-17. [PMID: 20660182 DOI: 10.1128/jvi.01050-10] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Elsewhere this laboratory reported that (i) ICP0 interacts with cyclin D3 but not D1 or D2. The 3 cyclins independently partially rescue DeltaICP0 mutants. (ii) Interaction with cyclin D3 is required for the switch from nuclear to cytoplasmic accumulation of ICP0. (iii) In infected cells cdk4 is activated whereas cdk2 is not. Inhibition of cdk4 results in nuclear retention of ICP0. Overexpression of cyclin D3 reverses the effect of the inhibitor. Here we report the following. (i) cdk4 interacts with ICP0, ICP4, and possibly with ICP8. This interaction is required to recruit cdk4 initially to ND10 and later to the viral replication compartments. (ii) cdk4 inhibitor I reduced or delayed the transcription and ultimately translation of mRNAs of ICP4, ICP27, or ICP8 and to a lesser extent that of the ICP0 gene in wild-type virus-infected cells. (iii) Overexpression of cyclin D3 resulted in a more rapid transcription of these genes. In the presence of inhibitor, the rates of accumulation of the products of these genes resemble those of wild-type virus in the absence of inhibitor. (iv) Overexpression of cyclin D3 also results in mobilization of cdk6 in nuclei of infected cells. We conclude that ICP0 encodes a function that enhances the recruitment of cyclin D3 to ND10 structures to activate cdk4 and that ICP0 along with other viral proteins recruits cdk4 to ND10 structures and ultimately to replication compartments for enhanced expression of viral genes and viral DNA synthesis.
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