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Kuo YS, Chiang PC, Kuo CY, Huang CG, Kuo ML, Chiu YF. Inhibition of influenza A virus proliferation in mice via universal RNA interference. Antiviral Res 2025; 238:106149. [PMID: 40147537 DOI: 10.1016/j.antiviral.2025.106149] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2024] [Revised: 03/01/2025] [Accepted: 03/24/2025] [Indexed: 03/29/2025]
Abstract
Influenza A virus (IAV) is a respiratory pathogen that causes seasonal outbreaks and periodic pandemics. As frequent mutations in the IAV viral genome often render vaccines ineffective or inefficient in preventing the latest outbreak, there is a need to explore other preventive strategies to control the disease. This study sought to investigate the use of antiviral short hairpin RNA (shRNA), delivered by a recombinant adeno-associated virus (AAV), for the prevention of IAV infections. Conserved regions with less than 10 % of variation were identified from IAV genome sequences deposited in the National Center for Biotechnology Information (NCBI) database between 2000 and 2023. The shRNA targeting these conserved sequences was transcribed from the human RNA polymerase III U6 promoter in an AAV system. This study demonstrates that AAV delivery of shRNA against IAV genes encoding two of the viral RNA-dependent RNA polymerase subunits, PB1 and PB2, inhibits the replication of IAV H1N1 and H3N2 viruses in Madin-Darby canine kidney (MDCK) cells. Delivery of shPB1 to lung tissue in mice through AAV also provided effective protection against IAV infection. These results offer support for a shRNA-based strategy of influenza prevention.
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Affiliation(s)
- Yu-Shen Kuo
- Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan
| | - Pei-Chuan Chiang
- Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan
| | - Chieh-Ying Kuo
- Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan
| | - Chung-Guei Huang
- Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Ming-Ling Kuo
- Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan
| | - Ya-Fang Chiu
- Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan; Division of Infectious Diseases, Department of Medicine, New Taipei Municipal Tucheng Hospital (Built and Operated By Chang Gung Medical Foundation), New Taipei City, Taiwan; Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan.
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Taibe NS, Mahmoud SH, Kord MA, Badawy MA, Shehata M, Elhefnawi M. In Silico and In Vitro development of novel small interfering RNAs (siRNAs) to inhibit SARS-CoV-2. Comput Struct Biotechnol J 2025; 27:1460-1471. [PMID: 40256168 PMCID: PMC12008453 DOI: 10.1016/j.csbj.2025.03.034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 03/14/2025] [Accepted: 03/20/2025] [Indexed: 04/22/2025] Open
Abstract
SARS-CoV-2 is causing severe to moderate respiratory tract infections, posing global health, social life, and economic threats. Our design strategy for siRNAs differs from existing studies through a step-by-step filtration process utilizing integrative bioinformatics protocols and web tools. Stage one: Multiple Sequence Alignment was employed to identify the most conserved areas. Stage two involves using various online tools, among the most reputable tools for building siRNA. The first filtration step of siRNA uses the Huesken dataset, estimating a 90 % experimental inhibition. The second filtration stage involves choosing the most suitable and targeted siRNA by utilizing thermodynamics and Target Accessibility of siRNAs. The final filtration step is off-target filtration using BLAST with specific parameters. Four of the 258 siRNAs were chosen for their potency and specificity, targeting conserved regions (NSP8, NSP12, and NSP14) with minimal human transcripts off-targets. We conducted in-vitro experiments, including cytotoxicity, TCID50, and RT-PCR assays. When tested on the SARS-CoV-2 strain hCoV-19/Egypt/NRC-03/2020 at 100 nM, none showed cellular toxicity. The TCID50 assay confirmed viral replication reduction at 12 h.p.i; the efficacy of the four siRNAs and their P value were highly significant. siRNA2 maintaining efficacy at 24, 36, and 48 h.p.i, while siRNA4 had a significant P value (≤0.0001) at 48 h.p.i. At 24 h.p.i, siRNA2 and siRNA4 showed statistical significance in viral knockdown of the virus's S gene and ORF1b gene by 95 %, 89 %, and 96 %, 97 %, respectively. Our computational method and experimental assessment of specific siRNAs have led us to conclude that siRNA2 and siRNA4 could be promising new therapies for SARS-CoV-2 that need further development.
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Affiliation(s)
- Noha Samir Taibe
- Biotechnology Department, Faculty of Science, Cairo University, Giza, Egypt
| | - Sara H. Mahmoud
- Center of Scientific Excellence for Influenza Viruses (CSEIV), National Research Centre, Cairo 12622, Egypt
| | - Maimona A. Kord
- Botany Department, Faculty of Science, Cairo University, Giza, Egypt
| | | | - Mahmoud Shehata
- Center of Scientific Excellence for Influenza Viruses (CSEIV), National Research Centre, Cairo 12622, Egypt
| | - Mahmoud Elhefnawi
- Biomedical Informatics and Cheminformatics Group, Informatics and Systems Department, National Research Centre, Cairo, Egypt
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Luong QXT, Hoang PT, Ho PT, Ayun RQ, Lee TK, Lee S. Potential Broad-Spectrum Antiviral Agents: A Key Arsenal Against Newly Emerging and Reemerging Respiratory RNA Viruses. Int J Mol Sci 2025; 26:1481. [PMID: 40003946 PMCID: PMC11855616 DOI: 10.3390/ijms26041481] [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: 11/10/2024] [Revised: 12/05/2024] [Accepted: 12/16/2024] [Indexed: 02/27/2025] Open
Abstract
Respiratory viral infections present significant global health challenges, causing substantial morbidity and mortality, particularly among highly susceptible components of the population. The emergence of pandemics and epidemics, such as those caused by influenza viruses and coronaviruses, emphasizes the urgent need for effective antiviral therapeutics. In this review, we explore the potential of broad-spectrum antiviral agents targeting respiratory RNA viruses, including influenza viruses, coronaviruses, respiratory syncytial virus, human metapneumovirus, human parainfluenza viruses, and rhinoviruses. Various broad-spectrum direct-acting and host-targeting antivirals are discussed, including monoclonal antibodies targeting conserved regions of viral surface proteins, molecules interfering with host cell receptors or viral replication machinery, viral protease inhibitors, siRNA therapies, ribonuclease, and 3D8 scFv. Advancements in host-targeting approaches to reduce resistance and RNA-based therapeutics offer significant potential for combating respiratory viral threats. Despite challenges, broad-spectrum antiviral agents represent a crucial strategy, particularly when specific viral pathogens are unidentified or rapid intervention is essential, such as during pandemics or outbreaks.
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Affiliation(s)
- Quynh Xuan Thi Luong
- Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea; (Q.X.T.L.); (P.T.H.); (P.T.H.); (R.Q.A.)
| | - Phuong Thi Hoang
- Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea; (Q.X.T.L.); (P.T.H.); (P.T.H.); (R.Q.A.)
| | - Phuong Thi Ho
- Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea; (Q.X.T.L.); (P.T.H.); (P.T.H.); (R.Q.A.)
| | - Ramadhani Qurrota Ayun
- Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea; (Q.X.T.L.); (P.T.H.); (P.T.H.); (R.Q.A.)
| | - Taek Kyun Lee
- Risk Assessment Research Center, Korea Institute of Ocean Science & Technology, Geoje 53201, Republic of Korea
| | - Sukchan Lee
- Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea; (Q.X.T.L.); (P.T.H.); (P.T.H.); (R.Q.A.)
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Rasizadeh R, Shiri Aghbash P, Mokhtarzadeh A, Poortahmasebi V, Ahangar Oskouee M, Sadri Nahand J, Amini M, Zahra Bahojb Mahdavi S, Hossein Yari A, Bannazadeh Baghi H. Novel strategies in HPV‑16‑related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy. Gene 2025; 932:148904. [PMID: 39218415 DOI: 10.1016/j.gene.2024.148904] [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: 04/09/2024] [Revised: 07/31/2024] [Accepted: 08/28/2024] [Indexed: 09/04/2024]
Abstract
BACKGROUND Cervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. METHODS The CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. RESULTS E5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10-8 M (45.36 μg/ml) to 6.71 × 10-8 M (26.66 μg/ml) and Ifosfamide from 12.52 × 10-5 M (32.7 μg/ml) to 8.206 × 10-5 M (21.43 μg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. CONCLUSION The study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.
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Affiliation(s)
- Reyhaneh Rasizadeh
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Parisa Shiri Aghbash
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ahad Mokhtarzadeh
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Genetic, Higher Education Institute of Rab-Rashid, Tabriz, Iran
| | - Vahdat Poortahmasebi
- Department of Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mahin Ahangar Oskouee
- Department of Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Javid Sadri Nahand
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Amini
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Amir Hossein Yari
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hossein Bannazadeh Baghi
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
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Repkova M, Mazurkov O, Filippova E, Protsenko M, Mazurkova N, Meschaninova M, Levina A, Zarytova V. Effect of modification of siRNA molecules delivered with aminopropylsilanol nanoparticles on suppression of A/H5N1 virus in cell culture. Biochim Biophys Acta Gen Subj 2024; 1868:130727. [PMID: 39437973 DOI: 10.1016/j.bbagen.2024.130727] [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: 07/02/2024] [Revised: 10/16/2024] [Accepted: 10/18/2024] [Indexed: 10/25/2024]
Abstract
The application of siRNAs as antiviral agents is limited by several obstacles including their poor penetration into cells and instability in biological media. To overcome these problems, we used non-agglomerated aminopropylsilanol nanoparticles (NP) to deliver siRNA into cells. All studied siRNAs had identical nucleoside sequences comprising phosphodiester or phosphorothioate (PS) internucleotide groups and the 2'-OMe and/or 2'-F groups in nucleoside units at different positions of RNA. The siRNA molecules were attached to NP, thus forming the NP-siRNA nanocomplexes. We studied the effect of siRNA modification in the nanocomplexes on suppressing the highly pathogenic influenza A/H5N1 virus replication. The results demonstrated that all siRNA-containing nanocomplexes inhibited the replication of the A/H5N1 virus by 1-3 orders of magnitude. The nanocomplexes containing partially modified siRNAs exhibited the most pronounced inhibition with an efficacy of 900-fold. This result was achieved by using siRNA consisting of the canonical 19-bp RNA duplex with the 3'-dTdT dangling ends, with the antisense strand in this duplex being protected from endonucleases (one UMeA site within the strand). The additional modifications of siRNA reduce their antiviral activity. Promising sense strands for loading into the RISC complex are likely to be phosphodiester sequences that contain dTdT at the 3' end (such as S4) to be protected against exonucleases. The sense strands of this type can probably be the most suitable for designing siRNAs as therapeutic agents. The proposed NP-siRNA nanocomplexes that consisted of low toxic and non-agglomerated aminopropylsilanol nanoparticles and siRNA molecules could be hopeful agents for gene silencing.
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Affiliation(s)
- Marina Repkova
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, pr. Lavrent'eva 8, Novosibirsk 630090, Russia
| | - Oleg Mazurkov
- FBRI State Research Center of Virology and Biotechnology "Vector" Rospotrebnadzor, Koltsovo, Novosibirsk 630559, Russia
| | - Ekaterina Filippova
- FBRI State Research Center of Virology and Biotechnology "Vector" Rospotrebnadzor, Koltsovo, Novosibirsk 630559, Russia
| | - Maria Protsenko
- FBRI State Research Center of Virology and Biotechnology "Vector" Rospotrebnadzor, Koltsovo, Novosibirsk 630559, Russia
| | - Natalia Mazurkova
- FBRI State Research Center of Virology and Biotechnology "Vector" Rospotrebnadzor, Koltsovo, Novosibirsk 630559, Russia
| | - Maria Meschaninova
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, pr. Lavrent'eva 8, Novosibirsk 630090, Russia
| | - Asya Levina
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, pr. Lavrent'eva 8, Novosibirsk 630090, Russia
| | - Valentina Zarytova
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, pr. Lavrent'eva 8, Novosibirsk 630090, Russia.
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Jiang M, Laine L, Kolehmainen P, Kakkola L, Avelin V, Väisänen E, Poranen MM, Österlund P, Julkunen I. Virus-specific Dicer-substrate siRNA swarms inhibit SARS-CoV-2 infection in TMPRSS2-expressing Vero E6 cells. Front Microbiol 2024; 15:1432349. [PMID: 39611095 PMCID: PMC11602746 DOI: 10.3389/fmicb.2024.1432349] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Accepted: 10/21/2024] [Indexed: 11/30/2024] Open
Abstract
After 4 years of the COVID-19 pandemic, SARS-CoV-2 continues to circulate with epidemic waves caused by evolving new variants. Although the rapid development of vaccines and approved antiviral drugs has reduced virus transmission and mitigated the symptoms of infection, the continuous emergence of new variants and the lack of simple-use (non-hospitalized, easy timing, local delivery, direct acting, and host-targeting) treatment modalities have limited the effectiveness of COVID-19 vaccines and drugs. Therefore, novel therapeutic approaches against SARS-CoV-2 infection are still urgently needed. As a positive-sense single-stranded RNA virus, SARS-CoV-2 is highly susceptible to RNA interference (RNAi). Accordingly, small interfering (si)RNAs targeting different regions of SARS-CoV-2 genome can effectively block the expression and replication of the virus. However, the rapid emergence of new SARS-CoV-2 variants with different genomic mutations has led to the problem of viral escape from the targets of RNAi strategy, which has increased the potential of off-target effects by siRNA and decreased the efficacy of long-term use of siRNA treatment. In our study, we enzymatically generated a set of Dicer-substrate (D)siRNA swarms containing DsiRNAs targeting single or multiple conserved sequences of SARS-CoV-2 genome by using in vitro transcription, replication and Dicer digestion system. Pre-transfection of these DsiRNA swarms into Vero E6-TMPRSS2 cells inhibited the replication of several SARS-CoV-2 variants, including the recent Omicron subvariants BQ.1.1 and XBB.1.5. This in vitro investigation of novel DsiRNA swarms provides solid evidence for the feasibility of this new RNAi strategy in the prevention and treatment of SARS-CoV-2 infection.
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Affiliation(s)
- Miao Jiang
- Microbiology Unit, Finnish Institute for Health and Welfare, Helsinki, Finland
- Infection and Immunity, Institute of Biomedicine, University of Turku, Turku, Finland
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Larissa Laine
- Microbiology Unit, Finnish Institute for Health and Welfare, Helsinki, Finland
| | - Pekka Kolehmainen
- Infection and Immunity, Institute of Biomedicine, University of Turku, Turku, Finland
| | - Laura Kakkola
- Infection and Immunity, Institute of Biomedicine, University of Turku, Turku, Finland
- Clinical Microbiology Unit, Turku University Central Hospital, Turku, Finland
| | - Veera Avelin
- Microbiology Unit, Finnish Institute for Health and Welfare, Helsinki, Finland
| | - Elina Väisänen
- Microbiology Unit, Finnish Institute for Health and Welfare, Helsinki, Finland
- Infection and Immunity, Institute of Biomedicine, University of Turku, Turku, Finland
| | - Minna M. Poranen
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Pamela Österlund
- Microbiology Unit, Finnish Institute for Health and Welfare, Helsinki, Finland
| | - Ilkka Julkunen
- Infection and Immunity, Institute of Biomedicine, University of Turku, Turku, Finland
- Clinical Microbiology Unit, Turku University Central Hospital, Turku, Finland
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Sharma SP, Chawla-Sarkar M, Sandhir R, Dutta D. Decoding the role of RNA sequences and their interactions in influenza A virus infection and adaptation. WILEY INTERDISCIPLINARY REVIEWS. RNA 2024; 15:e1871. [PMID: 39501458 DOI: 10.1002/wrna.1871] [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: 11/12/2023] [Revised: 08/19/2024] [Accepted: 08/27/2024] [Indexed: 04/10/2025]
Abstract
Influenza viruses (types A, B, C, and D) belong to the family orthomyxoviridae. Out of all the influenza types, influenza A virus (IAV) causes human pandemic outbreaks. Its pandemic potential is predominantly attributed to the genetic reassortment favored by a broad spectrum of host species that could lead to an antigenic shift along with a high rate of mutations in its genome, presenting a possibility of subtypes with heightened pathogenesis and virulence in humans (antigenic drift). In addition to antigenic shift and drift, there are several other inherent properties of its viral RNA species (vRNA, vmRNA, and cRNA) that significantly contribute to the success of specific stages of viral infection. In this review, we compile the key features of IAV RNA, such as sequence motifs and secondary structures, their functional significance in the infection cycle, and their overall impact on the virus's adaptive and evolutionary fitness. Because many of these motifs and folds are conserved, we also assess the existing antiviral approaches focused on targeting IAV RNA. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.
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Affiliation(s)
- Satya P Sharma
- Department of Biochemistry, Panjab University, Chandigarh, India
| | - Mamta Chawla-Sarkar
- ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India
| | - Rajat Sandhir
- Department of Biochemistry, Panjab University, Chandigarh, India
| | - Dipanjan Dutta
- School of Biological Sciences, Amity University, Punjab, India
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Miao Z, Li J, Wang Y, Shi M, Gu X, Zhang X, Wei F, Tang X, Zheng L, Xing Y. Hsa_circ_0136666 stimulates gastric cancer progression and tumor immune escape by regulating the miR-375/PRKDC Axis and PD-L1 phosphorylation. Mol Cancer 2023; 22:205. [PMID: 38093288 PMCID: PMC10718020 DOI: 10.1186/s12943-023-01883-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2023] [Accepted: 10/17/2023] [Indexed: 12/17/2023] Open
Abstract
BACKGROUND Targeted drugs are not quite effective for prolonging the survival of patients with gastric cancer due to off-target effects as well as tumor immune escape mechanisms. Circular RNAs widely exist in tumor regions as biomarkers and can be developed as effective drug targets. METHODS Western blot, QRT-PCR, fluorescence in situ hybridization, and flow cytometry were used to investigate the function of hsa_circ_0136666 in promoting the proliferation of gastric cancer cells. Tissue immunofluorescence, enzyme-linked immunosorbent assay (ELISA), as well as flow cytometric analysis, was conducted to explore the process of tumor immune evasion in tumor-bearing mice. The differences of circRNA expression in clinical samples were analyzed through tissue microarray FISH. The effect of siRNA on improving the efficacy of anti-PDL1 drugs and suppressing the immune microenvironment was evaluated by the coadministration model. RESULTS We demonstrated that hsa_circ_0136666 was widely and highly expressed in gastric cancer tissues and cells. Functionally, hsa_circ_0136666 promoted gastric cancer tumor proliferation and tumor microenvironment formation, leading to tumorigenesis immune escape, and this effect was dependent on CD8 + T cells. Mechanistically, we confirmed that hsa_circ_0136666 competitively upregulated PRKDC expression by sponging miR-375-3p, regulating immune checkpoint proteins, prompting phosphorylation of PD-L1 to preventing its degradation, driving PD-L1 aggregation and suppressing immune function, thereby impairing cancer immune responses. In terms of application, we found that LNP-siRNA effectively improved anti-PDL1 drug efficacy and inhibited immune escape. CONCLUSION Our results reveal an oncogenic role played by hsa_circ_0136666 in gastric cancer, driving PD-L1 phosphorylation via the miR-375/PRKDC signaling axis, prompting immune escape. This work proposes a completely new pathogenic mechanism of gastric cancer, uncovers a novel role for hsa_circ_0136666 as an immune target, and provides a rationale for enhancing the efficacy of anti-PD-L1 therapy for gastric cancer.
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Affiliation(s)
- Zhenyan Miao
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Jifei Li
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Yu Wang
- State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Mingqin Shi
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Xiao Gu
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Xuanqi Zhang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Fang Wei
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China
| | - Xinying Tang
- State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
- Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
| | - Lufeng Zheng
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
| | - Yingying Xing
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
- Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
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Kang H, Ga YJ, Kim SH, Cho YH, Kim JW, Kim C, Yeh JY. Small interfering RNA (siRNA)-based therapeutic applications against viruses: principles, potential, and challenges. J Biomed Sci 2023; 30:88. [PMID: 37845731 PMCID: PMC10577957 DOI: 10.1186/s12929-023-00981-9] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 10/04/2023] [Indexed: 10/18/2023] Open
Abstract
RNA has emerged as a revolutionary and important tool in the battle against emerging infectious diseases, with roles extending beyond its applications in vaccines, in which it is used in the response to the COVID-19 pandemic. Since their development in the 1990s, RNA interference (RNAi) therapeutics have demonstrated potential in reducing the expression of disease-associated genes. Nucleic acid-based therapeutics, including RNAi therapies, that degrade viral genomes and rapidly adapt to viral mutations, have emerged as alternative treatments. RNAi is a robust technique frequently employed to selectively suppress gene expression in a sequence-specific manner. The swift adaptability of nucleic acid-based therapeutics such as RNAi therapies endows them with a significant advantage over other antiviral medications. For example, small interfering RNAs (siRNAs) are produced on the basis of sequence complementarity to target and degrade viral RNA, a novel approach to combat viral infections. The precision of siRNAs in targeting and degrading viral RNA has led to the development of siRNA-based treatments for diverse diseases. However, despite the promising therapeutic benefits of siRNAs, several problems, including impaired long-term protein expression, siRNA instability, off-target effects, immunological responses, and drug resistance, have been considerable obstacles to the use of siRNA-based antiviral therapies. This review provides an encompassing summary of the siRNA-based therapeutic approaches against viruses while also addressing the obstacles that need to be overcome for their effective application. Furthermore, we present potential solutions to mitigate major challenges.
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Affiliation(s)
- Hara Kang
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Yun Ji Ga
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Soo Hyun Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Young Hoon Cho
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Jung Won Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
- Convergence Research Center for Insect Vectors, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Chaeyeon Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Jung-Yong Yeh
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- Research Institute for New Drug Development, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- Convergence Research Center for Insect Vectors, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- KU Center for Animal Blood Medical Science, College of Veterinary Medicine, Konkuk University, 120 Neungdong-Ro, Gwangjin-Gu, Seoul, 05029, South Korea.
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10
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Low ZY, Wong KH, Wen Yip AJ, Choo WS. The convergent evolution of influenza A virus: Implications, therapeutic strategies and what we need to know. CURRENT RESEARCH IN MICROBIAL SCIENCES 2023; 5:100202. [PMID: 37700857 PMCID: PMC10493511 DOI: 10.1016/j.crmicr.2023.100202] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/14/2023] Open
Abstract
Influenza virus infection, more commonly known as the 'cold flu', is an etiological agent that gives rise to recurrent annual flu and many pandemics. Dated back to the 1918- Spanish Flu, the influenza infection has caused the loss of many human lives and significantly impacted the economy and daily lives. Influenza virus can be classified into four different genera: influenza A-D, with the former two, influenza A and B, relevant to humans. The capacity of antigenic drift and shift in Influenza A has given rise to many novel variants, rendering vaccines and antiviral therapies useless. In light of the emergence of a novel betacoronavirus, the SARS-CoV-2, unravelling the underpinning mechanisms that support the recurrent influenza epidemics and pandemics is essential. Given the symptom similarities between influenza and covid infection, it is crucial to reiterate what we know about the influenza infection. This review aims to describe the origin and evolution of influenza infection. Apart from that, the risk factors entail the implication of co-infections, especially regarding the COVID-19 pandemic is further discussed. In addition, antiviral strategies, including the potential of drug repositioning, are discussed in this context. The diagnostic approach is also critically discussed in an effort to understand better and prepare for upcoming variants and potential influenza pandemics in the future. Lastly, this review encapsulates the challenges in curbing the influenza spread and provides insights for future directions in influenza management.
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Affiliation(s)
- Zheng Yao Low
- School of Science, Monash University Malaysia, 47500 Subang Jaya, Selangor, Malaysia
| | - Ka Heng Wong
- School of Science, Monash University Malaysia, 47500 Subang Jaya, Selangor, Malaysia
| | - Ashley Jia Wen Yip
- School of Science, Monash University Malaysia, 47500 Subang Jaya, Selangor, Malaysia
| | - Wee Sim Choo
- School of Science, Monash University Malaysia, 47500 Subang Jaya, Selangor, Malaysia
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11
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Ahmed EM, Boseila AA, Hanora AS, Solyman SM. Antiviral and protective effect of small interfering RNAs against rift valley fever virus in vitro. Mol Biol Rep 2023:10.1007/s11033-023-08455-9. [PMID: 37231214 DOI: 10.1007/s11033-023-08455-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Accepted: 04/12/2023] [Indexed: 05/27/2023]
Abstract
BACKGROUND Rift Valley Fever Virus (RVFV) is an arbovirus, a zoonotic disease that resurfaces as a potential hazard beyond geographic boundaries. Fever that can proceed to encephalitis, retinitis, hemorrhagic fever, and death is the main manifestation observed in human infections. RVFV has no authorized medication. The RNA interference (RNAi) gene silencing pathway is extremely well conserved. By targeting specific genes, small interfering RNA (siRNA) can be used to suppress viral replication. The aim of this study was to design specific siRNAs against RVFV and evaluate their prophylactic and antiviral effects on the Vero cells. METHODS AND RESULTS Various siRNAs were designed using different bioinformatics tools. Three unique candidates were tested against an Egyptian sheep cell culture-adapted strain BSL-2 that suppressed RVFV N mRNA expression. SiRNAs were transfected a day before RVFV infection (pre-transfection), and 1 h after the viral infection (post-transfection), and were evaluated to detect the silencing activity and gene expression decrease using real-time PCR and a TCID50 endpoint test. The degree of N protein expression was determined by western blot 48 h after viral infection. D2 which targets the (488-506 nucleotides), the middle region of RVFV N mRNA was the most effective siRNA at 30 nM concentration, it almost eliminates N mRNA expression when utilized as antiviral or preventive therapy. siRNAs had a stronger antiviral silencing impact when they were post-transfected into Vero cells. CONCLUSION Pre and post-transfection of siRNAs significantly reduced RVFV titer in cell lines, offering novel and potentially effective anti-RVFV epidemics and epizootics therapy.
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Affiliation(s)
- Engy M Ahmed
- Egyptian Drug Authority (EDA), Giza, Egypt
- Microbiology & Immunology Department, College of Pharmacy, Suez Canal University, Ismailia, Egypt
| | | | - Amro S Hanora
- Microbiology & Immunology Department, College of Pharmacy, Suez Canal University, Ismailia, Egypt.
| | - Samar M Solyman
- Microbiology & Immunology Department, College of Pharmacy, Suez Canal University, Ismailia, Egypt
- Microbiology & Immunology Department, Faculty of Pharmacy, Sinai University Kantara branch, Ismailia, Egypt
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12
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Villa R, Renzi S, Dotti S, Lucchini F. siRNAs pools generated in Escherichia coli exhibit strong RNA-interference activity against influenza virus genomic sequences. Virology 2023; 579:38-45. [PMID: 36599198 DOI: 10.1016/j.virol.2022.12.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 12/27/2022] [Accepted: 12/27/2022] [Indexed: 01/01/2023]
Abstract
Due to the recurrent pandemic outbreaks that occurred during the last century, Influenza A viruses are considered a serious potential danger to human health. Among the innate immune pathways in eukaryotes, RNA interference plays a significant role in the interaction between viruses and host cells. RNA interference is addressed by small dsRNA molecules produced by the host itself (miRNAs, i.e. "micro-RNAs") but can be triggered also by the administration of exogenous short RNAs (siRNAs, "short interfering RNAs"). In this work, artificial siRNA pools targeting NP and PB genomic regions of the Influenza virus were produced in engineered Escherichia coli, adapting a published protocol. In a MDCK cell in vitro model, these preparations were challenged against reporter vectors bearing viral genomic sequences. A strong and specific RNA interference activity was observed, and the details of this action were indagated.
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Affiliation(s)
- Riccardo Villa
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Laboratorio di Controllo di Prodotti Biologici, Farmaceutici e Convalida di Processi Produttivi, via A. Bianchi 9, Brescia, Italy
| | - Sabrina Renzi
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Laboratorio di Controllo di Prodotti Biologici, Farmaceutici e Convalida di Processi Produttivi, via A. Bianchi 9, Brescia, Italy
| | - Silvia Dotti
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Laboratorio di Controllo di Prodotti Biologici, Farmaceutici e Convalida di Processi Produttivi, via A. Bianchi 9, Brescia, Italy
| | - Franco Lucchini
- Università Cattolica del Sacro Cuore, Facoltà di Scienze Agrarie, Alimentari ed Ambientali, DiSTAS - Dipartimento di Scienze e Tecnologie Alimentari per una filiera agro-alimentare sostenibile, via L. Bissolati 74, Cremona, Italy.
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13
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Wong KH, Lal SK. Alternative antiviral approaches to combat influenza A virus. Virus Genes 2023; 59:25-35. [PMID: 36260242 PMCID: PMC9832087 DOI: 10.1007/s11262-022-01935-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Accepted: 09/06/2022] [Indexed: 01/14/2023]
Abstract
Influenza A (IAV) is a major human respiratory pathogen that contributes to a significant threat to health security, worldwide. Despite vaccinations and previous immunisations through infections, humans can still be infected with influenza several times throughout their lives. This phenomenon is attributed to the antigenic changes of hemagglutinin (HA) and neuraminidase (NA) proteins in IAV via genetic mutation and reassortment, conferring antigenic drift and antigenic shift, respectively. Numerous findings indicate that slow antigenic drift and reassortment-derived antigenic shift exhibited by IAV are key processes that allow IAVs to overcome the previously acquired host immunity, which eventually leads to the annual re-emergence of seasonal influenza and even pandemic influenza, in rare occasions. As a result, current therapeutic options hit a brick wall quickly. As IAV remains a constant threat for new outbreaks worldwide, the underlying processes of genetic changes and alternative antiviral approaches for IAV should be further explored to improve disease management. In the light of the above, this review discusses the characteristics and mechanisms of mutations and reassortments that contribute to IAV's evolution. We also discuss several alternative RNA-targeting antiviral approaches, namely the CRISPR/Cas13 systems, RNA interference (RNAi), and antisense oligonucleotides (ASO) as potential antiviral approaches against IAV.
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Affiliation(s)
- Ka Heng Wong
- School of Science, Monash University Malaysia, 47500, Bandar Sunway, Selangor DE, Malaysia
| | - Sunil K Lal
- School of Science, Monash University Malaysia, 47500, Bandar Sunway, Selangor DE, Malaysia.
- Tropical Medicine & Biology Multidisciplinary Platform, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
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14
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Tagore R, Alagarasu K, Patil P, Pyreddy S, Polash SA, Kakade M, Shukla R, Parashar D. Targeted in vitro gene silencing of E2 and nsP1 genes of chikungunya virus by biocompatible zeolitic imidazolate framework. Front Bioeng Biotechnol 2022; 10:1003448. [PMID: 36601387 PMCID: PMC9806579 DOI: 10.3389/fbioe.2022.1003448] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Accepted: 12/02/2022] [Indexed: 12/15/2022] Open
Abstract
Chikungunya fever caused by the mosquito-transmitted chikungunya virus (CHIKV) is a major public health concern in tropical, sub-tropical and temperate climatic regions. The lack of any licensed vaccine or antiviral agents against CHIKV warrants the development of effective antiviral therapies. Small interfering RNA (siRNA) mediated gene silencing of CHIKV structural and non-structural genes serves as a potential antiviral strategy. The therapeutic efficiency of siRNA can be improved by using an efficient delivery system. Metal-organic framework biocomposits have demonstrated an exceptional capability in protecting and efficiently delivering nucleic acids into cells. In the present study, carbonated ZIF called ZIF-C has been utilized to deliver siRNAs targeted against E2 and nsP1 genes of CHIKV to achieve a reduction in viral replication and infectivity. Cellular transfection studies of E2 and nsP1 genes targeting free siRNAs and ZIF-C encapsulated siRNAs in CHIKV infected Vero CCL-81 cells were performed. Our results reveal a significant reduction of infectious virus titre, viral RNA levels and percent of infected cells in cultures transfected with ZIF-C encapsulated siRNA compared to cells transfected with free siRNA. The results suggest that delivery of siRNA through ZIF-C enhances the antiviral activity of CHIKV E2 and nsP1 genes directed siRNAs.
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Affiliation(s)
- Rajarshee Tagore
- Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune, India
| | - Kalichamy Alagarasu
- Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune, India
| | - Poonam Patil
- Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune, India
| | - Suneela Pyreddy
- Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, RMIT University, Melbourne, VIC, Australia,Centre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne, VIC, Australia
| | - Shakil Ahmed Polash
- Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, RMIT University, Melbourne, VIC, Australia,Centre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne, VIC, Australia
| | - Mahadeo Kakade
- Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune, India
| | - Ravi Shukla
- Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, RMIT University, Melbourne, VIC, Australia,Centre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne, VIC, Australia,*Correspondence: Ravi Shukla, ; Deepti Parashar,
| | - Deepti Parashar
- Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune, India,*Correspondence: Ravi Shukla, ; Deepti Parashar,
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15
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Hussain M, Ahmed F, Henzeler B, Husain M. Anti-microbial host factor HDAC6 is antagonised by the influenza A virus through host caspases and viral PA. FEBS J 2022; 290:2744-2759. [PMID: 36516338 DOI: 10.1111/febs.16703] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Revised: 11/13/2022] [Accepted: 12/13/2022] [Indexed: 12/15/2022]
Abstract
Histone deacetylase 6 (HDAC6), through the repertoire of its substrate proteins, plays a critical role in human physiology, and an aberrant function of HDAC6 contributes to various pathophysiological conditions. HDAC6 is also known to be an anti-microbial host factor and has been implicated in restricting or clearing the infection of various human viral and bacterial pathogens. However, the state and the mechanisms of its antagonism in infected cells are not understood. Here, we demonstrate that influenza A virus (IAV) antagonises HDAC6 by recruiting both viral and host components. We found that HDAC6 mRNA expression, and consequently, the HDAC6 polypeptide expression is downregulated in human lung epithelial cells during early stage of IAV infection but can be rescued by depleting the expression of viral polymerase acidic (PA) protein, a subunit of IAV RNA polymerase. In addition, during later stage of the infection, the HDAC6 polypeptide undergoes caspase-mediated cleavage at two sites, generating two cleaved fragments. Both these fragments disappeared when the expression of caspase 3 was depleted in infected cells, whereas only second fragment disappeared when the expression of caspase 6 was depleted. But both fragments disappeared and the level of full-length HDAC6 polypeptide was rescued when the expression of PA was depleted in infected cells. Collectively, these data indicated that IAV antagonises the HDAC6 by decreasing its expression level in infected cells, both at mRNA and polypeptide level via PA gene, which has been implicated in auxiliary functions like degradation of host mRNA and induction of apoptosis.
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Affiliation(s)
- Mazhar Hussain
- Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
| | - Farjana Ahmed
- Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
| | - Bennett Henzeler
- Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
| | - Matloob Husain
- Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
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16
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A Novel Approach of Antiviral Drugs Targeting Viral Genomes. Microorganisms 2022; 10:microorganisms10081552. [PMID: 36013970 PMCID: PMC9414836 DOI: 10.3390/microorganisms10081552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 07/25/2022] [Accepted: 07/29/2022] [Indexed: 11/16/2022] Open
Abstract
Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.
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17
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Gao J, Xia Z, Vohidova D, Joseph J, Luo JN, Joshi N. Progress in non-viral localized delivery of siRNA therapeutics for pulmonary diseases. Acta Pharm Sin B 2022; 13:1400-1428. [PMID: 37139423 PMCID: PMC10150162 DOI: 10.1016/j.apsb.2022.07.010] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Revised: 05/10/2022] [Accepted: 06/13/2022] [Indexed: 11/01/2022] Open
Abstract
Emerging therapies based on localized delivery of siRNA to lungs have opened up exciting possibilities for treatment of different lung diseases. Localized delivery of siRNA to lungs has shown to result in severalfold higher lung accumulation than systemic route, while minimizing non-specific distribution in other organs. However, to date, only 2 clinical trials have explored localized delivery of siRNA for pulmonary diseases. Here we systematically reviewed recent advances in the field of pulmonary delivery of siRNA using non-viral approaches. We firstly introduce the routes of local administration and analyze the anatomical and physiological barriers towards effective local delivery of siRNA in lungs. We then discuss current progress in pulmonary delivery of siRNA for respiratory tract infections, chronic obstructive pulmonary diseases, acute lung injury, and lung cancer, list outstanding questions, and highlight directions for future research. We expect this review to provide a comprehensive understanding of current advances in pulmonary delivery of siRNA.
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18
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Semple SL, Au SKW, Jacob RA, Mossman KL, DeWitte-Orr SJ. Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells. Front Immunol 2022; 13:859749. [PMID: 35603190 PMCID: PMC9120774 DOI: 10.3389/fimmu.2022.859749] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2022] [Accepted: 04/04/2022] [Indexed: 12/20/2022] Open
Abstract
In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.
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Affiliation(s)
- Shawna L. Semple
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, ON, Canada
| | - Sarah K. W. Au
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, ON, Canada
| | - Rajesh A. Jacob
- Department of Medicine, McMaster University, Hamilton, ON, Canada
- Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada
- McMaster Immunology Research Centre, McMaster University, Hamilton, ON, Canada
| | - Karen L. Mossman
- Department of Medicine, McMaster University, Hamilton, ON, Canada
- Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada
- McMaster Immunology Research Centre, McMaster University, Hamilton, ON, Canada
| | - Stephanie J. DeWitte-Orr
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, ON, Canada
- *Correspondence: Stephanie J. DeWitte-Orr,
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19
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Influenza A Virus Production in Mouse Lung Is Inhibited by Inhalation of Aerosol Polyethylenimine/Short Hairpin RNA Plasmid Complexes. DISEASE MARKERS 2022; 2022:7404813. [PMID: 35493304 PMCID: PMC9054431 DOI: 10.1155/2022/7404813] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Accepted: 03/24/2022] [Indexed: 11/17/2022]
Abstract
Influenza pandemics are a global threat to human health, with existing vaccines and antiviral drugs providing limited protection. There is an urgent need for new prophylactic and treatment strategies. In this study, 12 short hairpin (sh)RNAs targeting conserved regions of influenza A virus (IAV) matrix protein (M)2, nucleocapsid protein (NP), nonstructural protein (NS), and polymerase acidic (PA) were synthesized, and their effects on IAV replication in cells were investigated using Madin-Darby canine kidney (MDCK) cells transfected with the shRNA plasmids. Additionally, mice were administered a polyethyleneimine PEI/pLKD-NP-391 complex in aerosol form and then infected with AIV, and viral particles in the mouse lung were detected. IAV production was markedly lower in MDCK cells transfected with pLKD-M-121, pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD-PA-1645 plasmids than in control cells (p < 0.01). The viral load in MDCK cells was decreased by transfection of plasmids pLKD-M-121 (p < 0.05) and pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD PA-1645 (p < 0.01) compared to an empty plasmid. The viral load was significantly lower in the lungs of mice transfected with pLKD-NP-391 than in the control plasmid and mock transfection groups (p < 0.01 and p < 0.05, respectively). Thus, IAV production was inhibited by shRNAs targeting matrix IAV components; moreover, inhalation of a PEI/pLKD-NP-391 complex in aerosol form suppressed IAV production in infected mice. Thus, these shRNAs can be effective for the prevention and treatment of influenza virus infection.
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Aripova T, Muratkhodjaev J. A novel concept of human antiviral protection: It's all about RNA (Review). Biomed Rep 2022; 16:29. [PMID: 35251616 PMCID: PMC8889527 DOI: 10.3892/br.2022.1512] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Accepted: 01/10/2022] [Indexed: 11/06/2022] Open
Abstract
The comparative analysis of the antiviral protective mechanisms, including protozoa and RNA interference in multicellular organisms, has revealed their similarity and provided a basic understanding of adaptive immunity. The present article summarizes the latest studies on RNA-guided gene regulation in human antiviral protection, and its importance. Additionally, the role of both neutralizing antibodies and the interferon system in viral invasion is considered. The interferon system is an additional mechanism for suppressing viral infections in humans, which shifts cells into an 'alarm' mode to attempt to prevent further contagion. The primary task of the human central immune system is to maintain integrity and to protect against foreign organisms. In this review, a novel concept is proposed: Antiviral protection in all organisms can be achieved through an intracellular RNA-guided mechanism. A simple and effective defence against viruses is incorporation of a part of a virus's DNA (spacer) into the hosts chromosomes. Following reinfection, RNA transcripts of this spacer are created to direct nuclease enzymes to destroy the viral genome. This is an example of real-time adaptive immunity potentially possessed by every cell with a full complement of chromosomes, and an indicator that antiviral immunity is not only mediated by the presence of neutralizing antibodies and memory B- and T-cells, but also by the presence of specific spacers in the DNA of individuals who have recovered from a viral infection.
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Affiliation(s)
- Tamara Aripova
- Institute of Immunology and Human Genomics, Academy of Sciences of Uzbekistan, Tashkent 100060, Uzbekistan
| | - Javdat Muratkhodjaev
- Institute of Immunology and Human Genomics, Academy of Sciences of Uzbekistan, Tashkent 100060, Uzbekistan
- GENEX LLC Pharmaceutical Company, Tashkent 100052, Uzbekistan
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21
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Friedrich M, Pfeifer G, Binder S, Aigner A, Vollmer Barbosa P, Makert GR, Fertey J, Ulbert S, Bodem J, König EM, Geiger N, Schambach A, Schilling E, Buschmann T, Hauschildt S, Koehl U, Sewald K. Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2. Front Bioeng Biotechnol 2022; 10:801870. [PMID: 35309990 PMCID: PMC8925020 DOI: 10.3389/fbioe.2022.801870] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Accepted: 02/07/2022] [Indexed: 12/16/2022] Open
Abstract
In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.
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Affiliation(s)
- Maik Friedrich
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
- Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
- *Correspondence: Maik Friedrich,
| | - Gabriele Pfeifer
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
| | - Stefanie Binder
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
| | - Achim Aigner
- Rudolf Boehm Institute for Pharmacology and Toxicology, Clinical Pharmacology, Leipzig University, Faculty of Medicine, Leipzig, Germany
| | | | - Gustavo R. Makert
- Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
| | - Jasmin Fertey
- Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
| | - Sebastian Ulbert
- Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
| | - Jochen Bodem
- Institute of Virology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
| | - Eva-Maria König
- Institute of Virology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
| | - Nina Geiger
- Institute of Virology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
- Boston Children’s Hospital, Harvard Medical School, Boston, MA, United States
| | - Erik Schilling
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
| | - Tilo Buschmann
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
| | | | - Ulrike Koehl
- Institute of Clinical Immunology, Faculty of Leipzig University of Leipzig, Max-Bürger-Forschungszentrum (MBFZ), Leipzig, Germany
- Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
- REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
- Institute for Cellular Therapeutics, Hannover Medical School, Hannover, Germany
| | - Katherina Sewald
- Fraunhofer Institute of Toxicology and Experimental Medicine, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH) of the German Center for Lung Research (DZL), Hannover, Germany
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22
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Saadat KASM. RNAi-mediated siRNA sequences to combat the COVID-19 pandemic with the inhibition of SARS-CoV2. GENE REPORTS 2022; 26:101512. [PMID: 35071824 PMCID: PMC8760737 DOI: 10.1016/j.genrep.2022.101512] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2021] [Accepted: 01/11/2022] [Indexed: 11/29/2022]
Affiliation(s)
- Khandakar A S M Saadat
- Department of Medical Biology, Faculty of Medicine, Gaziantep University, Gaziantep 27310, Turkey
- Department of Medical Biology and Genetics, Institute of Health Sciences, Gaziantep University, Gaziantep 27310, Turkey
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23
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Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells. Methods Protoc 2022; 5:mps5010018. [PMID: 35200534 PMCID: PMC8879667 DOI: 10.3390/mps5010018] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 02/03/2022] [Accepted: 02/07/2022] [Indexed: 12/01/2022] Open
Abstract
Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14–15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.
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24
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Hartawan R, Pujianto DA, Dharmayanti NLPI, Soebandrio A. Improving siRNA design targeting nucleoprotein gene as antiviral against the Indonesian H5N1 virus. J Vet Sci 2022; 23:e24. [PMID: 35187881 PMCID: PMC8977538 DOI: 10.4142/jvs.21174] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 11/08/2021] [Accepted: 12/07/2021] [Indexed: 12/05/2022] Open
Abstract
Background Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.
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Affiliation(s)
- Risza Hartawan
- Doctoral Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
- Indonesian Research Center for Veterinary Science, Ministry of Agriculture, Bogor 16114, Indonesia
| | - Dwi Ari Pujianto
- Department of Medical Biology Pre Clinic, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
| | | | - Amin Soebandrio
- Eijkman Institute, Ministry of Research, Technology and Higher Education, Jakarta 10430, Indonesia
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25
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Endogenous Feline Leukemia Virus (FeLV) siRNA Transcription May Interfere with Exogenous FeLV Infection. J Virol 2021; 95:e0007021. [PMID: 34495702 DOI: 10.1128/jvi.00070-21] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Endogenous retroviruses (ERVs) are increasingly recognized for biological impacts on host cell function and susceptibility to infectious agents, particularly in relation to interactions with exogenous retroviral progenitors (XRVs). ERVs can simultaneously promote and restrict XRV infections using mechanisms that are virus and host specific. The majority of endogenous-exogenous retroviral interactions have been evaluated in experimental mouse or chicken systems, which are limited in their ability to extend findings to naturally infected outbred animals. Feline leukemia virus (FeLV) has a relatively well-characterized endogenous retrovirus with a coexisting virulent exogenous counterpart and is endemic worldwide in domestic cats. We have previously documented an association between endogenous FeLV (enFeLV) long terminal repeat (LTR) copy number and abrogated exogenous FeLV in naturally infected cats and experimental infections in tissue culture. Analyses described here examine limited FeLV replication in experimentally infected peripheral blood mononuclear cells, which correlates with higher enFeLV transcripts in these cells compared to fibroblasts. We further examine NCBI Sequence Read Archive RNA transcripts to evaluate enFeLV transcripts and RNA interference (RNAi) precursors. We find that lymphoid-derived tissues, which are experimentally less permissive to exogenous FeLV infection, transcribe higher levels of enFeLV under basal conditions. Transcription of enFeLV-LTR segments is significantly greater than that of other enFeLV genes. We documented transcription of a 21-nucleotide (nt) microRNA (miRNA) just 3' to the enFeLV 5'-LTR in the feline miRNAome of all data sets evaluated (n = 27). Our findings point to important biological functions of enFeLV transcription linked to solo LTRs distributed within the domestic cat genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. IMPORTANCE Endogenous retroviruses (ERVs) are increasingly implicated in host cellular processes and susceptibility to infectious agents, specifically regarding interactions with exogenous retroviral progenitors (XRVs). Exogenous feline leukemia virus (FeLV) and its endogenous counterpart (enFeLV) represent a well-characterized, naturally occurring XRV-ERV dyad. We have previously documented an abrogated FeLV infection in both naturally infected cats and experimental fibroblast infections that harbor higher enFeLV proviral loads. Using an in silico approach, we provide evidence of miRNA transcription that is produced in tissues that are most important for FeLV infection, replication, and transmission. Our findings point to important biological functions of enFeLV transcription linked to solo-LTRs distributed within the feline genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. This body of work provides additional evidence of RNA interference (RNAi) as a mechanism of viral interference and is a demonstration of ERV exaptation by the host to defend against related XRVs.
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26
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Mousavi SR, Sajjadi MS, Khosravian F, Feizbakhshan S, Salmanizadeh S, Esfahani ZT, Beni FA, Arab A, Kazemi M, Shahzamani K, Sami R, Hosseinzadeh M, Salehi M, Lotfi H. Dysregulation of RNA interference components in COVID-19 patients. BMC Res Notes 2021; 14:401. [PMID: 34715923 PMCID: PMC8554738 DOI: 10.1186/s13104-021-05816-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Accepted: 10/19/2021] [Indexed: 12/21/2022] Open
Abstract
OBJECTIVE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. RESULTS Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.
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Affiliation(s)
- Seyyed Reza Mousavi
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Maryam Sadat Sajjadi
- Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Farinaz Khosravian
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Sara Feizbakhshan
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Sharareh Salmanizadeh
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Zahra Taherian Esfahani
- Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Faeze Ahmadi Beni
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Ameneh Arab
- Noor Educational and Medical Center،Isfahan University of Medical Sciences, Isfahan, Iran
| | - Mohammad Kazemi
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Kiana Shahzamani
- Isfahan Gastroenterology and Hepatology Research Center (lGHRC), Isfahan University of Medical Sciences, Isfahan, Iran
| | - Ramin Sami
- Department of Pulmonology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Majid Hosseinzadeh
- Craniofacial and Cleft Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Mansoor Salehi
- Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran.
- Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran.
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
| | - Hajie Lotfi
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
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27
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Challagulla A, Schat KA, Doran TJ. In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells. Methods Protoc 2021; 4:40. [PMID: 34201194 PMCID: PMC8293360 DOI: 10.3390/mps4020040] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 06/04/2021] [Accepted: 06/05/2021] [Indexed: 12/12/2022] Open
Abstract
Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.
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Affiliation(s)
- Arjun Challagulla
- CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong 3220, Australia;
| | - Karel A. Schat
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA;
| | - Timothy J. Doran
- CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong 3220, Australia;
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28
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Bappy SS, Shibly AZ, Sultana S, Mohiuddin AKM, Kabir Y. Designing potential siRNA molecule for the nucleocapsid(N) gene silencing of different SARS-CoV-2 strains of Bangladesh: Computational approach. Comput Biol Chem 2021; 92:107486. [PMID: 33984653 PMCID: PMC8099544 DOI: 10.1016/j.compbiolchem.2021.107486] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Revised: 03/18/2021] [Accepted: 04/08/2021] [Indexed: 02/08/2023]
Abstract
SARS-CoV-2 is a single-stranded RNA (+) virus first identified in China and then became an ongoing global outbreak. In most cases, it is fatal in humans due to respiratory malfunction. Extensive researches are going to find an effective therapeutic technique for the treatment of SARS-CoV-2 infected individuals. In this study, we attempted to design a siRNA molecule to silence the most suitable nucleocapsid(N) gene of SARS-CoV-2, which play a major role during viral pathogenesis, replication, encapsidation and RNA packaging. At first, 270 complete N gene sequences of different strains in Bangladesh of these viruses were retrieved from the NCBI database. Different computational methods were used to design siRNA molecules. A siRNA molecule was built against these strains using the SiDirect 2.0 server. Using Mfold and the OligoCalc server, the siRNA molecule was tested for its secondary structure and GC material. The Clustal Omega tool was employed to evaluate any off-target harmony of the planned siRNA molecule. Herein, we proposed a duplex siRNA molecule that does not fit any off-target sequences for the gene silencing of SARS-CoV-2. To treat SARS-CoV-2 infections, currently, any effective therapy is not available. Our engineered siRNA molecule could give an alternative therapeutic approach against various sequenced SARS-CoV-2 strains in Bangladesh.
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Affiliation(s)
- Syed Shahariar Bappy
- Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail, 1902, Bangladesh; Research and Development, Incepta Vaccine Ltd, Zirabo, Savar, Dhaka, 1341, Bangladesh
| | - Abu Zaffar Shibly
- Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail, 1902, Bangladesh
| | - Sorna Sultana
- Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail, 1902, Bangladesh
| | - A K M Mohiuddin
- Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail, 1902, Bangladesh
| | - Yearul Kabir
- Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka, 1000, Bangladesh.
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29
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Phan T, Fay EJ, Lee Z, Aron S, Hu WS, Langlois RA. Segment-specific kinetics of mRNA, cRNA and vRNA accumulation during influenza infection. J Virol 2021; 95:JVI.02102-20. [PMID: 33658346 PMCID: PMC8139675 DOI: 10.1128/jvi.02102-20] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Accepted: 02/18/2021] [Indexed: 12/11/2022] Open
Abstract
Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication.ImportanceInfluenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.
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Affiliation(s)
- Thu Phan
- Department of Chemical Engineering and Materials Sciences, University of Minnesota, Minneapolis, MN, United States of America
| | - Elizabeth J Fay
- Biochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis MN, United States of America
| | - Zion Lee
- Department of Chemical Engineering and Materials Sciences, University of Minnesota, Minneapolis, MN, United States of America
| | - Stephanie Aron
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis MN, United States of America
| | - Wei-Shou Hu
- Department of Chemical Engineering and Materials Sciences, University of Minnesota, Minneapolis, MN, United States of America
| | - Ryan A Langlois
- Biochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis MN, United States of America
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis MN, United States of America
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30
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Soobramoney C, Parboosing R. siRNAs and viruses: The good, the bad and the way forward. Curr Mol Pharmacol 2021; 15:143-158. [PMID: 33881977 DOI: 10.2174/1874467214666210420113427] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 01/08/2021] [Accepted: 02/08/2021] [Indexed: 11/22/2022]
Abstract
There are no available antivirals for many viruses or strains, while current antivirals are limited by toxicity and drug resistance. Therefore, alternative strategies, such as RNA interference (RNAi) are required. RNAi suppresses gene expression of any mRNA, making it an attractive candidate for antiviral therapeutics. Studies have evaluated siRNAs in a range of viruses, with some showing promising results. However, issues with stability and delivery of siRNAs remain. These may be minimized by modifying the siRNA structure, using an efficient delivery vector and targeting multiple regions of a virus's genome in a single dose. Finding these solutions could accelerate the progress of RNAi-based antivirals. This review highlights selected examples of antiviral siRNAs, limitations of RNAi and strategies to overcome these limitations.
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Affiliation(s)
| | - Raveen Parboosing
- Department of Virology, University of KwaZulu Natal/ National Health Laboratory Services, Durban, South Africa
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31
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Hasan M, Ashik AI, Chowdhury MB, Tasnim AT, Nishat ZS, Hossain T, Ahmed S. Computational prediction of potential siRNA and human miRNA sequences to silence orf1ab associated genes for future therapeutics against SARS-CoV-2. INFORMATICS IN MEDICINE UNLOCKED 2021; 24:100569. [PMID: 33846694 PMCID: PMC8028608 DOI: 10.1016/j.imu.2021.100569] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Revised: 03/26/2021] [Accepted: 03/31/2021] [Indexed: 12/12/2022] Open
Abstract
The coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by an RNA virus termed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 possesses an almost 30kbp long genome. The genome contains open-reading frame 1ab (ORF1ab) gene, the largest one of SARS-CoV-2, encoding polyprotein PP1ab and PP1a responsible for viral transcription and replication. Several vaccines have already been approved by the respective authorities over the world to develop herd immunity among the population. In consonance with this effort, RNA interference (RNAi) technology holds the possibility to strengthen the fight against this virus. Here, we have implemented a computational approach to predict potential short interfering RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs), which are presumed to be intrinsically active against SARS-CoV-2. In doing so, we have screened miRNA library and siRNA library targeting the ORF1ab gene. We predicted the potential miRNA and siRNA candidate molecules utilizing an array of bioinformatic tools. By extending the analysis, out of 24 potential pre-miRNA hairpins and 131 siRNAs, 12 human miRNA and 10 siRNA molecules were sorted as potential therapeutic agents against SARS-CoV-2 based on their GC content, melting temperature (Tm), heat capacity (Cp), hybridization and minimal free energy (MFE) of hybridization. This computational study is focused on lessening the extensive time and labor needed in conventional trial and error based wet lab methods and it has the potential to act as a decent base for future researchers to develop a successful RNAi therapeutic.
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Key Words
- ACE-2, Angiotensin-converting enzyme 2
- COVID-19
- COVID-19, coronavirus disease 2019
- Cp, heat capacity
- Gene silencing
- ORF, open reading frame
- Posttranscriptional regulation
- RNAi Therapeutics
- RNAi, RNA interference
- SARS-CoV-2
- SARS-CoV-2, severe acute respiratory syndrome coronavirus-2
- TMPRSS2, transmembrane protease serine 2
- Tm, melting temperature
- UTR, untranslated region
- hsa-miR, human microRNA
- miRNA
- miRNA, microRNA
- sgRNA, sub-genomic RNA
- siRNA
- siRNA, small interfering RNA
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Affiliation(s)
- Mahedi Hasan
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Arafat Islam Ashik
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Md Belal Chowdhury
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Atiya Tahira Tasnim
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Zakia Sultana Nishat
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Tanvir Hossain
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
| | - Shamim Ahmed
- Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
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Mehta A, Michler T, Merkel OM. siRNA Therapeutics against Respiratory Viral Infections-What Have We Learned for Potential COVID-19 Therapies? Adv Healthc Mater 2021; 10:e2001650. [PMID: 33506607 PMCID: PMC7995229 DOI: 10.1002/adhm.202001650] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 01/06/2021] [Indexed: 12/30/2022]
Abstract
Acute viral respiratory tract infections (AVRIs) are a major burden on human health and global economy and amongst the top five causes of death worldwide resulting in an estimated 3.9 million lives lost every year. In addition, new emerging respiratory viruses regularly cause outbreaks such as SARS-CoV-1 in 2003, the "Swine flu" in 2009, or most importantly the ongoing SARS-CoV-2 pandemic, which intensely impact global health, social life, and economy. Despite the prevalence of AVRIs and an urgent need, no vaccines-except for influenza-or effective treatments were available at the beginning of the COVID-19 pandemic. However, the innate RNAi pathway offers the ability to develop nucleic acid-based antiviral drugs. siRNA sequences against conserved, essential regions of the viral genome can prevent viral replication. In addition, viral infection can be averted prophylactically by silencing host genes essential for host-viral interactions. Unfortunately, delivering siRNAs to their target cells and intracellular site of action remains the principle hurdle toward their therapeutic use. Currently, siRNA formulations and chemical modifications are evaluated for their delivery. This progress report discusses the selection of antiviral siRNA sequences, delivery techniques to the infection sites, and provides an overview of antiviral siRNAs against respiratory viruses.
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Affiliation(s)
- Aditi Mehta
- Department of PharmacyPharmaceutical Technology and BiopharmaceuticsLudwig‐Maximilians‐Universität MünchenButenandtstraße 5Munich81377Germany
| | - Thomas Michler
- Institute of VirologyTechnische Universität MünchenTrogerstr. 30Munich81675Germany
| | - Olivia M. Merkel
- Department of PharmacyPharmaceutical Technology and BiopharmaceuticsLudwig‐Maximilians‐Universität MünchenButenandtstraße 5Munich81377Germany
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Blanchard EL, Vanover D, Bawage SS, Tiwari PM, Rotolo L, Beyersdorf J, Peck HE, Bruno NC, Hincapie R, Michel F, Murray J, Sadhwani H, Vanderheyden B, Finn MG, Brinton MA, Lafontaine ER, Hogan RJ, Zurla C, Santangelo PJ. Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents. Nat Biotechnol 2021; 39:717-726. [PMID: 33536629 DOI: 10.1038/s41587-021-00822-w] [Citation(s) in RCA: 123] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 12/11/2022]
Abstract
Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.
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Affiliation(s)
- Emmeline L Blanchard
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Daryll Vanover
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Swapnil Subhash Bawage
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Pooja Munnilal Tiwari
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Laura Rotolo
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Jared Beyersdorf
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Hannah E Peck
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
| | - Nicholas C Bruno
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
| | - Robert Hincapie
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
| | - Frank Michel
- Department of Veterinary Biosciences and Diagnostic Imaging, College of Veterinary Medicine University of Georgia, Athens, GA, USA
| | - Jackelyn Murray
- Department of Infectious Diseases, College of Veterinary Medicine University of Georgia, Athens, GA, USA
| | - Heena Sadhwani
- Department of Biology, Georgia State University, Atlanta, GA, USA
| | - Bob Vanderheyden
- Analytics and Data Science Institute, Kennesaw State University, Kennesaw, GA, USA
| | - M G Finn
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
| | - Margo A Brinton
- Department of Biology, Georgia State University, Atlanta, GA, USA
| | - Eric R Lafontaine
- Department of Infectious Diseases, College of Veterinary Medicine University of Georgia, Athens, GA, USA
| | - Robert J Hogan
- Department of Veterinary Biosciences and Diagnostic Imaging, College of Veterinary Medicine University of Georgia, Athens, GA, USA.,Department of Infectious Diseases, College of Veterinary Medicine University of Georgia, Athens, GA, USA
| | - Chiara Zurla
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
| | - Philip J Santangelo
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
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Zhou J, Krishnan N, Jiang Y, Fang RH, Zhang L. Nanotechnology for virus treatment. NANO TODAY 2021; 36:101031. [PMID: 33519948 PMCID: PMC7836394 DOI: 10.1016/j.nantod.2020.101031] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Revised: 11/09/2020] [Accepted: 11/11/2020] [Indexed: 04/14/2023]
Abstract
The continued emergence of novel viruses poses a significant threat to global health. Uncontrolled outbreaks can result in pandemics that have the potential to overburden our healthcare and economic systems. While vaccination is a conventional modality that can be employed to promote herd immunity, antiviral vaccines can only be applied prophylactically and do little to help patients who have already contracted viral infections. During the early stages of a disease outbreak when vaccines are unavailable, therapeutic antiviral drugs can be used as a stopgap solution. However, these treatments do not always work against emerging viral strains and can be accompanied by adverse effects that sometimes outweigh the benefits. Nanotechnology has the potential to overcome many of the challenges facing current antiviral therapies. For example, nanodelivery vehicles can be employed to drastically improve the pharmacokinetic profile of antiviral drugs while reducing their systemic toxicity. Other unique nanomaterials can be leveraged for their virucidal or virus-neutralizing properties. In this review, we discuss recent developments in antiviral nanotherapeutics and provide a perspective on the application of nanotechnology to the SARS-CoV-2 outbreak and future virus pandemics.
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Affiliation(s)
- Jiarong Zhou
- Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093, USA
| | - Nishta Krishnan
- Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093, USA
| | - Yao Jiang
- Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093, USA
| | - Ronnie H Fang
- Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093, USA
| | - Liangfang Zhang
- Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093, USA
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Aghamollaei H, Sarvestani R, Bakherad H, Zare H, Guest PC, Ranjbar R, Sahebkar A. Emerging Technologies for the Treatment of COVID-19. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1321:81-96. [PMID: 33656715 DOI: 10.1007/978-3-030-59261-5_7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), turned into a pandemic affecting more than 200 countries. Due to the high rate of transmission and mortality, finding specific and effective treatment options for this infection is currently of urgent importance. Emerging technologies have created a promising platform for developing novel treatment options for various viral diseases such as the SARS-CoV-2 virus. Here, we have described potential novel therapeutic options based on the structure and pathophysiological mechanism of the SARS-CoV-2 virus, as well as the results of previous studies on similar viruses such as SARS and MERS. Many of these approaches can be used for controlling viral infection by reducing the viral damage or by increasing the potency of the host response. Owing to their high sensitivity, specificity, and reproducibility, siRNAs, aptamers, nanobodies, neutralizing antibodies, and different types of peptides can be used for interference with viral replication or for blocking internalization. Receptor agonists and interferon-inducing agents are also potential options to balance and enhance the innate immune response against SARS-CoV-2. Solid evidence on the efficacy and safety of such novel technologies is yet to be established although many well-designed clinical trials are underway to address these issues.
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Affiliation(s)
- Hossein Aghamollaei
- Chemical Injuries Research Center, Systems biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
| | - Rahim Sarvestani
- Research and Development Department, PersisGen Par Biopharma Accelerator, Tehran, Iran
| | - Hamid Bakherad
- Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Hamed Zare
- Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
| | - Paul C Guest
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil
| | - Reza Ranjbar
- Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
| | - Amirhossein Sahebkar
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
- Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Halal Research Center of IRI, FDA, Tehran, Iran.
- Polish Mother's Memorial Hospital Research Institute (PMMHRI), Lodz, Poland.
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Földes F, Madai M, Papp H, Kemenesi G, Zana B, Geiger L, Gombos K, Somogyi B, Bock-Marquette I, Jakab F. Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro. Molecules 2020; 25:molecules25235771. [PMID: 33297527 PMCID: PMC7731286 DOI: 10.3390/molecules25235771] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 11/26/2020] [Accepted: 12/04/2020] [Indexed: 11/24/2022] Open
Abstract
Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.
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Affiliation(s)
- Fanni Földes
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Mónika Madai
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Henrietta Papp
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Gábor Kemenesi
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Brigitta Zana
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Lili Geiger
- Department of Laboratory Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary; (L.G.); (K.G.)
| | - Katalin Gombos
- Department of Laboratory Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary; (L.G.); (K.G.)
| | - Balázs Somogyi
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
| | - Ildikó Bock-Marquette
- Regenerative Science, Sport and Medicina Research Group, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary;
| | - Ferenc Jakab
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
- Correspondence: ; Tel.: +36-72-501-668 (ext. 29044)
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Ullah A, Qazi J, Rahman L, Kanaras AG, Khan WS, Hussain I, Rehman A. Nanoparticles-assisted delivery of antiviral-siRNA as inhalable treatment for human respiratory viruses: A candidate approach against SARS-COV-2. NANO SELECT 2020; 1:612-621. [PMID: 34485978 PMCID: PMC7675679 DOI: 10.1002/nano.202000125] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 09/25/2020] [Indexed: 12/13/2022] Open
Abstract
The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has challenged healthcare structures across the globe. Although a few therapies are approved by FDA, the search for better treatment options is continuously on rise. Clinical management includes infection prevention and supportive care such as supplemental oxygen and mechanical ventilatory support. Given the urgent nature of the pandemic and the number of companies and researchers developing COVID-19 related therapies, FDA has created an emergency program to move potential treatments with already approved drugs to patients as quickly as possible in parallel to the development of new drugs that must first pass the clinical trials. In this manuscript, we have reviewed the available literature on the use of sequence-specific degradation of viral genome using short-interfering RNA (siRNA) suggesting it as a possible treatment against SARS-CoV-2. Delivery of siRNA can be promoted by the use of FDA approved lipids, polymers or lipid-polymer hybrids. These nanoparticulate systems can be engineered to exhibit increased targetability and formulated as inhalable aerosols.
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Affiliation(s)
- Ata Ullah
- National Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
| | - Javaria Qazi
- Department of BiotechnologyQuaid‐i‐Azam UniversityIslamabadPakistan
| | - Lutfur Rahman
- National Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
| | - Antonios G. Kanaras
- Physics and AstronomyInstitute for Life SciencesUniversity of SouthamptonSouthamptonSO171BJUK
| | - Waheed S. Khan
- National Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
| | - Irshad Hussain
- Department of Chemistry and Chemical EngineeringSBA School of Science & Engineering (SBASSE)Lahore University of Management Sciences (LUMS)LahorePakistan
| | - Asma Rehman
- National Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
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de Carvalho OV, Rebouças Santos M, Lopes Rangel Fietto J, Pires Moraes M, de Almeida MR, Costa Bressan G, José Pena L, Silva-Júnior A. Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus. BMC Vet Res 2020; 16:448. [PMID: 33213424 PMCID: PMC7676405 DOI: 10.1186/s12917-020-02671-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Accepted: 11/06/2020] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV. RESULTS In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication. CONCLUSIONS We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.
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Affiliation(s)
- Otávio Valério de Carvalho
- Laboratory of Immunobiological and Animal Virology, Department of Veterinary Medicine, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
- Department of Virology and Experimental Therapy, Oswaldo Cruz Foundation (FIOCRUZ), Aggeu Magalhães Research Center, Av. Moraes Rego, s/n, Campus UFPE, Cidade Universitária, Recife, PE, 50670-420, Brazil
| | - Marcus Rebouças Santos
- Laboratory of Immunobiological and Animal Virology, Department of Veterinary Medicine, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
| | - Juliana Lopes Rangel Fietto
- Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
| | - Mauro Pires Moraes
- Laboratory of Immunobiological and Animal Virology, Department of Veterinary Medicine, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
| | - Márcia Rogéria de Almeida
- Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
| | - Gustavo Costa Bressan
- Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG, 36570-000, Brazil
| | - Lindomar José Pena
- Department of Virology and Experimental Therapy, Oswaldo Cruz Foundation (FIOCRUZ), Aggeu Magalhães Research Center, Av. Moraes Rego, s/n, Campus UFPE, Cidade Universitária, Recife, PE, 50670-420, Brazil.
| | - Abelardo Silva-Júnior
- Department of Virology and Experimental Therapy, Oswaldo Cruz Foundation (FIOCRUZ), Aggeu Magalhães Research Center, Av. Moraes Rego, s/n, Campus UFPE, Cidade Universitária, Recife, PE, 50670-420, Brazil.
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Szabat M, Lorent D, Czapik T, Tomaszewska M, Kierzek E, Kierzek R. RNA Secondary Structure as a First Step for Rational Design of the Oligonucleotides towards Inhibition of Influenza A Virus Replication. Pathogens 2020; 9:pathogens9110925. [PMID: 33171815 PMCID: PMC7694947 DOI: 10.3390/pathogens9110925] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 11/05/2020] [Accepted: 11/05/2020] [Indexed: 02/06/2023] Open
Abstract
Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome (viral RNA—vRNA) consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.
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40
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Panda S, Banik U, Adhikary AK. Bioinformatics analysis reveals four major hexon variants of human adenovirus type-3 (HAdV-3) as the potential strains for development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2020; 85:104439. [PMID: 32585339 PMCID: PMC7308778 DOI: 10.1016/j.meegid.2020.104439] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 04/24/2020] [Accepted: 06/20/2020] [Indexed: 11/25/2022]
Abstract
Human adenovirus type 3 (HAdV-3) encompasses 15-87% of all adenoviral respiratory infections. The significant morbidity and mortality, especially among the neonates and immunosuppressed patients, demand the need for a vaccine or a targeted antiviral against this type. However, due to the existence of multiple hexon variants (3Hv-1 to 3Hv-25), the selection of vaccine strains of HAdV-3 is challenging. This study was designed to evaluate HAdV-3 hexon variants for the selection of potential vaccine candidates and the use of hexon gene as a target for designing siRNA that can be used as a therapy. Based on the data of worldwide distribution, duration of circulation, co-circulation and their percentage among all the variants, 3Hv-1 to 3Hv-4 were categorized as the major hexon variants. Phylogenetic analysis and the percentage of homology in the hypervariable regions followed by multi-sequence alignment, zPicture analysis and restriction enzyme analysis were carried out. In the phylogram, the variants were arranged in different clusters. The HVR encoding regions of hexon of 3Hv-1 to 3Hv-4 showed 16 point mutations resulting in 12 amino acids substitutions. The homology in HVRs was 81.81-100%. Therefore, the major hexon variants are substantially different from each other which justifies their inclusion as the potential vaccine candidates. Interestingly, despite the significant differences in the DNA sequence, there were many conserved areas in the HVRs, and we have designed functional siRNAs form those locations. We have also designed immunogenic vaccine peptide epitopes from the hexon protein using bioinformatics prediction tool. We hope that our developed siRNAs and immunogenic vaccine peptide epitopes could be used in the future development of siRNA-based therapy and designing a vaccine against HAdV-3.
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Affiliation(s)
- Somnath Panda
- Unit of Microbiology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia.
| | - Urmila Banik
- Unit of Pathology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia
| | - Arun K. Adhikary
- Unit of Microbiology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia
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Pashkov EA, Faizuloev EB, Svitich OA, Sergeev OV, Zverev VV. [The potential of synthetic small interfering RNA-based antiviral drugs for influenza treatment]. Vopr Virusol 2020; 65:182-190. [PMID: 33533221 DOI: 10.36233/0507-4088-2020-65-4-182-190] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Accepted: 09/16/2020] [Indexed: 06/12/2023]
Abstract
Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.
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Affiliation(s)
- E A Pashkov
- I.M. Sechenov First Moscow State Medical University (Sechenov University); I.I. Mechnikov Research Institute for Vaccines and Sera
| | - E B Faizuloev
- I.I. Mechnikov Research Institute for Vaccines and Sera
| | - O A Svitich
- I.M. Sechenov First Moscow State Medical University (Sechenov University); I.I. Mechnikov Research Institute for Vaccines and Sera
| | - O V Sergeev
- I.M. Sechenov First Moscow State Medical University (Sechenov University); National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - V V Zverev
- I.M. Sechenov First Moscow State Medical University (Sechenov University); I.I. Mechnikov Research Institute for Vaccines and Sera
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Piyush R, Rajarshi K, Chatterjee A, Khan R, Ray S. Nucleic acid-based therapy for coronavirus disease 2019. Heliyon 2020; 6:e05007. [PMID: 32984620 PMCID: PMC7501848 DOI: 10.1016/j.heliyon.2020.e05007] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Revised: 08/02/2020] [Accepted: 09/17/2020] [Indexed: 12/14/2022] Open
Abstract
The coronavirus disease 2019 (COVID-19), the pandemic that originated in China has already spread into more than 190 countries, resulting in huge loss of human life and many more are at the stake of losing it; if not intervened with the best therapeutics to contain the disease. For that aspect, various scientific groups are continuously involved in the development of an effective line of treatment to control the novel coronavirus from spreading rapidly. Worldwide scientists are evaluating various biomolecules and synthetic inhibitors against COVID-19; where the nucleic acid-based molecules may be considered as potential drug candidates. These molecules have been proved potentially effective against SARS-CoV, which shares high sequence similarity with SARS-CoV-2. Recent advancements in nucleic acid-based therapeutics are helpful in targeted drug delivery, safely and effectively. The use of nucleic acid-based molecules also known to regulate the level of gene expression inside the target cells. This review mainly focuses on various nucleic acid-based biologically active molecules and their therapeutic potentials in developing vaccines for SARS-CoV-2.
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Affiliation(s)
- Ravikant Piyush
- School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu 625021, India
| | - Keshav Rajarshi
- School of Community Science and Technology (SOCSAT) Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Howrah, West Bengal 711103, India
| | - Aroni Chatterjee
- Indian Council of Medical Research (ICMR)-Virus Research Laboratory, NICED, Kolkata, India
| | - Rajni Khan
- Motihari College of Engineering, Bariyarpur, Motihari, NH 28A, Furshatpur, Motihari, Bihar 845401, India
| | - Shashikant Ray
- Department of Biotechnology, Mahatma Gandhi Central University Motihari, 845401, India
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Computational Identification of Small Interfering RNA Targets in SARS-CoV-2. Virol Sin 2020; 35:359-361. [PMID: 32297156 PMCID: PMC7157830 DOI: 10.1007/s12250-020-00221-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Accepted: 04/03/2020] [Indexed: 01/08/2023] Open
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Kumar S, Yeo D, Harur Muralidharan N, Lai SK, Tong C, Tan BH, Sugrue RJ. Impaired Nuclear Export of the Ribonucleoprotein Complex and Virus-Induced Cytotoxicity Combine to Restrict Propagation of the A/Duck/Malaysia/02/2001 (H9N2) Virus in Human Airway Cells. Cells 2020; 9:cells9020355. [PMID: 32028682 PMCID: PMC7072679 DOI: 10.3390/cells9020355] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Revised: 01/20/2020] [Accepted: 01/29/2020] [Indexed: 01/02/2023] Open
Abstract
In humans, (A549) cells impaired H9N2 virus nuclear export of the ribonucleoprotein (RNP) complex contrasted with the early and efficient nuclear export of the H1N1/WSN and pH1N1 virus RNP complexes. Although nuclear export of the RNP complex occurred via the nuclear pore complex, H9N2 virus infection also induced modifications in the nuclear envelope and induced cell cytotoxicity. Reduced PA protein levels in H9N2 virus-infected A549 cells occurred, and this phenomenon was independent of virus infection. Silencing the H1N1/WSN PA protein expression leads to impaired nuclear export of RNP complexes, suggesting that the impaired nuclear export of the H9N2 virus RNP complex may be one of the consequences of reduced PA protein levels. Early and efficient export of the RNP complex occurred in H9N2 virus-infected avian (CEF) cells, although structural changes in the nuclear envelope also occurred. Collectively our data suggest that a combination of delayed nuclear export and virus-induced cell cytotoxicity restricts H9N2 virus transmission in A549 cells. However, the early and efficient export of the RNP complex mitigated the effects of virus-induced cytotoxicity on H9N2 virus transmission in CEF cells. Our findings highlight the multi-factorial nature of host-adaptation of the polymerase proteins of avian influenza viruses in non-avian cell environments.
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Affiliation(s)
- Sriram Kumar
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
| | - Dawn Yeo
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
- Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510, Singapore;
| | - Nisha Harur Muralidharan
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
| | - Soak Kuan Lai
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
| | - Cathlyn Tong
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
| | - Boon Huan Tan
- Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510, Singapore;
| | - Richard J. Sugrue
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; (S.K.); (D.Y.); (N.H.M.); (S.K.L.); (C.T.)
- Correspondence:
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RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference. MOLECULAR THERAPY. NUCLEIC ACIDS 2019; 19:627-642. [PMID: 31945726 PMCID: PMC6965531 DOI: 10.1016/j.omtn.2019.12.018] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Revised: 12/16/2019] [Accepted: 12/16/2019] [Indexed: 12/31/2022]
Abstract
The influenza A virus is a human pathogen that poses a serious public health threat due to rapid antigen changes and emergence of new, highly pathogenic strains with the potential to become easily transmitted in the human population. The viral genome is encoded by eight RNA segments, and all stages of the replication cycle are dependent on RNA. In this study, we designed small interfering RNA (siRNA) targeting influenza segment 5 nucleoprotein (NP) mRNA structural motifs that encode important functions. The new criterion for choosing the siRNA target was the prediction of accessible regions based on the secondary structure of segment 5 (+)RNA. This design led to siRNAs that significantly inhibit influenza virus type A replication in Madin-Darby canine kidney (MDCK) cells. Additionally, chemical modifications with the potential to improve siRNA properties were introduced and systematically validated in MDCK cells against the virus. A substantial and maximum inhibitory effect was achieved at concentrations as low as 8 nM. The inhibition of viral replication reached approximately 90% for the best siRNA variants. Additionally, selected siRNAs were compared with antisense oligonucleotides targeting the same regions; this revealed that effectiveness depends on both the target accessibility and oligonucleotide antiviral strategy. Our new approach of target-site preselection based on segment 5 (+)RNA secondary structure led to effective viral inhibition and a better understanding of the impact of RNA structural motifs on the influenza replication cycle.
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Galvin HD, Husain M. Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4. J Biol Chem 2019; 294:20207-20221. [PMID: 31757810 DOI: 10.1074/jbc.ra119.010650] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2019] [Revised: 11/05/2019] [Indexed: 12/22/2022] Open
Abstract
Influenza A virus (IAV) effectively manipulates host machinery to replicate. There is a growing evidence that an optimal acetylation environment in the host cell is favorable to IAV proliferation and vice versa. The histone deacetylases (HDACs), a family of 18 host enzymes classified into four classes, are central to negatively regulating the acetylation level, hence the HDACs would not be favorable to IAV. Indeed, by using the RNAi and overexpression strategies, we found that human HDAC4, a class II member, possesses anti-IAV properties and is a component of host innate antiviral response. We discovered that IAV multiplication was augmented in HDAC4-depleted cells and abated in HDAC4-supplemented cells. Likewise, the expression of IFITM3, ISG15, and viperin, some of the critical markers of host anti-IAV response was abated in HDAC4-depleted cells and augmented in HDAC4-supplemented cells. In turn, IAV strongly antagonizes the HDAC4, by down-regulating its expression both at the mRNA level via viral RNA endonuclease PA-X and at the polypeptide level by inducing its cleavage via host caspase 3 in infected cells. Such HDAC4 polypeptide cleavage resulted in a ∼30 kDa fragment that is also observed in some heterologous systems and may have a significant role in IAV replication.
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Affiliation(s)
- Henry D Galvin
- Department of Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand
| | - Matloob Husain
- Department of Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand
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47
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Valdés JJ, Miller AD. New opportunities for designing effective small interfering RNAs. Sci Rep 2019; 9:16146. [PMID: 31695077 PMCID: PMC6834666 DOI: 10.1038/s41598-019-52303-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2019] [Accepted: 10/10/2019] [Indexed: 12/13/2022] Open
Abstract
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.
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MESH Headings
- Argonaute Proteins/chemistry
- Argonaute Proteins/metabolism
- Base Pairing
- Crystallography, X-Ray
- Drug Design
- Humans
- Hydrolysis
- Magnesium
- Molecular Docking Simulation
- Monte Carlo Method
- Nucleic Acid Conformation
- Nucleic Acid Hybridization
- RNA Interference
- RNA, Messenger/chemistry
- RNA, Messenger/metabolism
- RNA, Small Interfering/chemistry
- RNA, Small Interfering/genetics
- RNA, Small Interfering/metabolism
- RNA, Viral/antagonists & inhibitors
- RNA, Viral/chemistry
- RNA-Induced Silencing Complex
- Structure-Activity Relationship
- Thermodynamics
- RNA, Guide, CRISPR-Cas Systems
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Affiliation(s)
- James J Valdés
- Veterinary Research Institute, Hudcova 70, CZ-62100, Brno, Czech Republic.
- Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Branišovska 1160/31, CZ-37005, České Budějovice, Czech Republic.
| | - Andrew D Miller
- Veterinary Research Institute, Hudcova 70, CZ-62100, Brno, Czech Republic.
- KP Therapeutics Ltd, 86 Deansgate, Manchester, M3 2ER, UK.
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Fan J, Yuan L, Liu Q, Tong C, Wang W, Xiao F, Liu B, Liu X. An ultrasensitive and simple assay for the Hepatitis C virus using a reduced graphene oxide-assisted hybridization chain reaction. Analyst 2019; 144:3972-3979. [PMID: 31140473 DOI: 10.1039/c9an00179d] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
Hepatitis C virus (HCV) is a major cause of chronic liver disease, which affects 2-3% of the world population. Until now, the early detection of HCV has been a great challenge, especially for those who live in developing countries. In this study, we developed a novel and ultrasensitive assay for the detection of HCV RNA based on the reduced graphene oxide nanosheets (rGONS) and hybridization chain reaction (HCR) amplification technique. This detection system contains a pair of single fluorophore-labeled hairpin probes that can freely exist in the solution in the absence of target RNA. The introduction of target RNA can robustly trigger a HCR with the two probes and produce long nanowires containing a double-stranded structure. The weak adsorption to rGONS makes the long nanowires emit a strong fluorescence. Using this enzyme-free amplification strategy, we developed a new method for the HCV RNA assay with a detection limit of 10 fM, which is far more sensitive than the common GO-based fluorescence method. Furthermore, the new method exhibits high selectivity for the discrimination of perfectly complementary and mismatched sequences. Finally, the new method was successfully used as a HCV RNA assay in biological samples with a strong anti-interference capability in complicated environments. In summary, these remarkable characteristics of the new method highlight its potential use in a clinical sample primary screening.
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Affiliation(s)
- Jialong Fan
- College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha, 410082, China.
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Liu M, Wang B, Wang F, Yang Z, Gao D, Zhang C, Ma L, Yu X. Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner. Appl Microbiol Biotechnol 2019; 103:6071-6079. [PMID: 31175428 DOI: 10.1007/s00253-019-09925-6] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Revised: 05/09/2019] [Accepted: 05/09/2019] [Indexed: 12/12/2022]
Abstract
Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.
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Affiliation(s)
- Min Liu
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Bin Wang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Fei Wang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Zhi Yang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Dan Gao
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Chenyao Zhang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Lixin Ma
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China.
| | - Xiaolan Yu
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China.
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50
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Maillard PV, van der Veen AG, Poirier EZ, Reis e Sousa C. Slicing and dicing viruses: antiviral RNA interference in mammals. EMBO J 2019; 38:e100941. [PMID: 30872283 PMCID: PMC6463209 DOI: 10.15252/embj.2018100941] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Revised: 01/17/2019] [Accepted: 01/25/2019] [Indexed: 12/15/2022] Open
Abstract
To protect against the harmful consequences of viral infections, organisms are equipped with sophisticated antiviral mechanisms, including cell-intrinsic means to restrict viral replication and propagation. Plant and invertebrate cells utilise mostly RNA interference (RNAi), an RNA-based mechanism, for cell-intrinsic immunity to viruses while vertebrates rely on the protein-based interferon (IFN)-driven innate immune system for the same purpose. The RNAi machinery is conserved in vertebrate cells, yet whether antiviral RNAi is still active in mammals and functionally relevant to mammalian antiviral defence is intensely debated. Here, we discuss cellular and viral factors that impact on antiviral RNAi and the contexts in which this system might be at play in mammalian resistance to viral infection.
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Affiliation(s)
- Pierre V Maillard
- Division of Infection and Immunity, University College London, London, UK
| | | | - Enzo Z Poirier
- Immunobiology Laboratory, The Francis Crick Institute, London, UK
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