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1.
Virol Sin ; 2022 Jun 30.
Article in English | MEDLINE | ID: covidwho-1915075

ABSTRACT

Several variants of concern (VOCs) have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated in January 2020. Due to mutations in the spike (S) protein, these VOCs have evolved to enhance viral infectivity and immune evasion. However, whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure. The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle. Although several SARS-CoV-2 replicons have been developed, none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA (sgRNA) transcription. In this study, SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs (the Beta and Delta variants) were prepared by removing the S gene from their genomes, while other structural genes remained untouched. These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles, as manifested by electron microscopic assays. Thus, the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs, including inhibitors of virion assembly and release. Through the quantification of replicon RNA released into the supernatant, we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.

2.
Front Microbiol ; 13: 907422, 2022.
Article in English | MEDLINE | ID: covidwho-1903085

ABSTRACT

Understanding the process of replication and transcription of SARS-CoV-2 is essential for antiviral strategy development. The replicase polyprotein is indispensable for viral replication. However, whether all nsps derived from the replicase polyprotein of SARS-CoV-2 are indispensable is not fully understood. In this study, we utilized the SARS-CoV-2 replicon as the system to investigate the role of each nsp in viral replication. We found that except for nsp16, all the nsp deletions drastically impair the replication of the replicon, and nsp14 could recover the replication deficiency caused by its deletion in the viral replicon. Due to the unsuccessful expressions of nsp1, nsp3, and nsp16, we could not draw a conclusion about their in trans-rescue functions. Our study provided a new angle to understand the role of each nsp in viral replication and transcription, helping the evaluation of nsps as the target for antiviral drug development.

3.
Topics in Antiviral Medicine ; 30(1 SUPPL):180, 2022.
Article in English | EMBASE | ID: covidwho-1880232

ABSTRACT

Background: Molnupiravir (MOV), the orally administered prodrug of the antiviral ribonucleoside analogue, N-hydroxycytidine (NHC) has received emergency use authorization for treatment of COVID-19. NHC inhibits viral replication by introduction of random transition errors across the viral genome, resulting in non-infectious virus. In the Phase II/III (MOVe-OUT) study, non-hospitalized participants received MOV or placebo (PBO) for 5 days and followed to Day 29. Viral RNA was sequenced to determine the rate, distribution and type of viral errors observed. Methods: SARS-CoV-2 RNA isolated from nasopharyngeal swabs was quantified by RT-PCR followed by complete genome NGS using the Ion AmpliSeq SARS-CoV-2 Research panel and Ion Torrent sequencing. To distinguish between nucleotide errors resulting from the mechanism of action of MOV and those potentially associated with reduced susceptibility to NHC, two different analyses were used. To measure impact of MOV on accumulation of low-frequency errors in the viral quasispecies, nucleotide variants were identified using VarScan 2.4 mutation caller with 0.4% minimum variant allele frequency cut-off. Resistance-associated changes were identified as amino acid substitutions occurring in D3 or D5 samples from ≥2 participants with a frequency of ≥5% of NGS reads. Phenotypic analysis of selected amino acid substitutions was performed using a replicon model. Results: NGS results showed a relationship between the number of random errors across the viral genome with increasing MOV dose. By Day 5 the mean number of viral genome errors were 21, 83, 129 and 223 in the PBO, 200, 400 and 800 mg groups, respectively. Among the sequence changes observed, the majority were transitions errors, consistent with MOV's mechanism of action. After MOV treatment, few treatment-emergent amino acid substitutions were identified in the viral replicase genes. These included nsp12 (T731I) and nsp14 (A220S/T/V, V466I, S503L/P);none associated with loss of susceptibility to MOV. Changes in spike protein in both PBO and MOV groups were at sites previously described in circulating variants. Conclusion: Consistent with the mechanism of action, MOV treatment resulted in a dose-dependent increase in transition errors across the SARS-CoV-2 genome. No resistance-associated mutations were identified in the viral replicase and no evidence that MOV treatment selected for unique mutations in spike protein not previously observed in circulating variants.

4.
Viruses ; 14(6)2022 05 27.
Article in English | MEDLINE | ID: covidwho-1869816

ABSTRACT

Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.


Subject(s)
COVID-19 , Flavivirus , RNA Viruses , Viruses , Antiviral Agents/pharmacology , Humans , SARS-CoV-2
5.
Front Immunol ; 13: 884862, 2022.
Article in English | MEDLINE | ID: covidwho-1855363

ABSTRACT

The mRNA vaccines from Pfizer/BioNTech and Moderna were granted emergency approval in record time in the history of vaccinology and played an instrumental role in limiting the pandemic caused by SARS-CoV-2. The success of these vaccines resulted from over 3 decades of research from many scientists. However, the development of orally administrable mRNA vaccine development is surprisingly underexplored. Our group specializing in Salmonella-based vaccines explored the possibility of oral mRNA vaccine development. Oral delivery was made possible by the exploitation of the Semliki Forest viral replicon and Salmonella vehicle for transgene amplification and gene delivery, respectively. Herein we highlight the prospect of developing oral replicon-based mRNA vaccines against infectious diseases based on our recent primary studies on SARS-CoV-2. Further, we discuss the potential advantages and limitations of bacterial gene delivery.


Subject(s)
COVID-19 , Communicable Diseases , Bacteria , COVID-19/prevention & control , COVID-19 Vaccines , Humans , RNA, Messenger/genetics , SARS-CoV-2/genetics , Vaccines, Synthetic , mRNA Vaccines
6.
Antiviral Res ; 199: 105268, 2022 03.
Article in English | MEDLINE | ID: covidwho-1850634

ABSTRACT

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-ß but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-ß. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , High-Throughput Screening Assays , Humans , Replicon , SARS-CoV-2/genetics , Virus Replication
7.
Viruses ; 14(5)2022 05 05.
Article in English | MEDLINE | ID: covidwho-1820426

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.


Subject(s)
Replicon , SARS-CoV-2 , Antiviral Agents/pharmacology , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Promoter Regions, Genetic , RNA, Guide , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Replication/drug effects
8.
Elife ; 112022 Feb 22.
Article in English | MEDLINE | ID: covidwho-1776585

ABSTRACT

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.


Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , COVID-19/prevention & control , COVID-19 Vaccines , Cricetinae , Humans , Mice , RNA, Viral , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Synthetic , mRNA Vaccines
9.
Journal of Virology ; 96(3):10, 2022.
Article in English | Web of Science | ID: covidwho-1755893

ABSTRACT

Research activities with infectious severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (Delta S-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [Delta S-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from Delta S-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with Delta S-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of Delta S-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose-and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and anti-viral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.

10.
Journal of Virology ; 96(3):1-10, 2022.
Article in English | Academic Search Complete | ID: covidwho-1678986

ABSTRACT

Research activities with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARSCoV-2 mutant (DS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [DSVRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from DS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with DS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of DS-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose- and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. [ FROM AUTHOR] Copyright of Journal of Virology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

11.
Mol Ther ; 30(5): 1926-1940, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1665550

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolution has resulted in many variants, contributing to the striking drop in vaccine efficacy and necessitating the development of next-generation vaccines to tackle antigenic diversity. Herein we developed a multivalent Semliki Forest virus replicon-based mRNA vaccine targeting the receptor binding domain (RBD), heptad repeat domain (HR), membrane protein (M), and epitopes of non-structural protein 13 (nsp13) of SARS-CoV-2. The bacteria-mediated gene delivery offers the rapid production of large quantities of vaccine at a highly economical scale and notably allows needle-free mass vaccination. Favorable T-helper (Th) 1-dominated potent antibody and cellular immune responses were detected in the immunized mice. Further, immunization induced strong cross-protective neutralizing antibodies (NAbs) against the B.1.617.2 delta variant (clade G). We recorded a difference in induction of immunoglobulin (Ig) A response by the immunization route, with the oral route eliciting a strong mucosal secretory IgA (sIgA) response, which possibly has contributed to the enhanced protection conferred by oral immunization. Hamsters immunized orally were completely protected against viral replication in the lungs and the nasal cavity. Importantly, the vaccine protected the hamsters against SARS-CoV-2-induced pneumonia. The study provides proof-of-principle findings for the development of a feasible and efficacious oral mRNA vaccine against SARS-CoV-2 and its variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , Antibodies, Viral , Bacteria , COVID-19/prevention & control , COVID-19 Vaccines/genetics , Cricetinae , Humans , Mice , Replicon , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Synthetic , mRNA Vaccines
12.
Viruses ; 14(2)2022 01 18.
Article in English | MEDLINE | ID: covidwho-1625168

ABSTRACT

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.


Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Reverse Genetics/methods , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Virus Replication/genetics , Amino Acid Substitution , Animals , Chlorocebus aethiops , Chromosomes, Artificial, Bacterial/genetics , Humans , Polymorphism, Genetic , Replicon/drug effects , Replicon/genetics , Vero Cells
13.
J Virol ; 96(3): e0183721, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1546443

ABSTRACT

Research activities with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [ΔS-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose- and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.


Subject(s)
Genetic Engineering , Recombination, Genetic , Replicon , SARS-CoV-2/genetics , COVID-19/virology , Cell Culture Techniques , Cell Line , Containment of Biohazards/standards , Genes, Reporter , Humans , Laboratories/standards , Viral Proteins/genetics , Virus Replication
14.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1518611

ABSTRACT

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Interferon-alpha/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Exoribonucleases/genetics , Genetic Vectors , HeLa Cells , Humans , Interferon-alpha/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Naphthyridines/administration & dosage , Naphthyridines/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , RNA, Viral/drug effects , Replicon
15.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1481965

ABSTRACT

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.


Subject(s)
Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , RNA, Viral/administration & dosage , Replicon , Viral Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Defective Viruses/genetics , Defective Viruses/immunology , Female , Gene Deletion , Genes, env , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral/genetics , RNA, Viral/immunology , Vaccines, DNA , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics , Virulence/immunology
16.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1488612

ABSTRACT

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Interferon-alpha/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Exoribonucleases/genetics , Genetic Vectors , HeLa Cells , Humans , Interferon-alpha/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Naphthyridines/administration & dosage , Naphthyridines/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , RNA, Viral/drug effects , Replicon
17.
Viruses ; 12(6)2020 05 30.
Article in English | MEDLINE | ID: covidwho-1389514

ABSTRACT

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Subject(s)
Antiviral Agents/pharmacology , Drug Discovery/methods , Genes, Reporter/physiology , RNA Viruses/drug effects , Replicon/physiology , Animals , Antiviral Agents/chemistry , Cell Line , Chlorocebus aethiops , Cricetinae , Humans , RNA Viruses/genetics , Transfection , Vero Cells
18.
J Virol ; 95(18): e0068721, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1373942

ABSTRACT

The emerging coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, resulting in global public health emergencies and economic crises. In the present study, a noninfectious and biosafety level 2 (BSL2)-compatible SARS-CoV-2 replicon expressing a nano luciferase (nLuc) reporter was constructed in a bacterial artificial chromosomal (BAC) vector by reverse genetics. The nLuc reporter is highly sensitive, easily quantifiable, and high throughput adaptable. Upon transfecting the SARS-CoV-2 replicon BAC plasmid DNA into Vero E6 cells, we could detect high levels of nLuc reporter activity and viral RNA transcript, suggesting the replication of the replicon. The replicon replication was further demonstrated by the findings that deleting nonstructural protein 15 or mutating its catalytic sites significantly reduced replicon replication, whereas providing the nucleocapsid protein in trans enhanced replicon replication in a dose-dependent manner. Finally, we showed that remdesivir, a U.S. Food and Drug Administration-approved antiviral drug, significantly inhibited the replication of the replicon, providing proof of principle for the application of our replicon as a useful tool for developing antivirals. Taken together, this study established a sensitive and BSL2-compatible reporter system in a single BAC plasmid for investigating the functions of SARS-CoV-2 proteins in viral replication and evaluating antiviral compounds. This should contribute to the global effort to combat this deadly viral pathogen. IMPORTANCE The COVID-19 pandemic caused by SARS-CoV-2 is having a catastrophic impact on human lives. Combatting the pandemic requires effective vaccines and antiviral drugs. In the present study, we developed a SARS-CoV-2 replicon system with a sensitive and easily quantifiable reporter. Unlike studies involving infectious SARS-CoV-2 virus that must be performed in a biosafety level 3 (BSL3) facility, the replicon is noninfectious and thus can be safely used in BSL2 laboratories. The replicon will provide a valuable tool for testing antiviral drugs and studying SARS-CoV-2 biology.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Evaluation, Preclinical , Green Fluorescent Proteins/metabolism , Replicon , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , COVID-19/virology , Chlorocebus aethiops , Green Fluorescent Proteins/genetics , HEK293 Cells , High-Throughput Screening Assays , Humans
19.
J Control Release ; 338: 201-210, 2021 10 10.
Article in English | MEDLINE | ID: covidwho-1364213

ABSTRACT

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.


Subject(s)
COVID-19 , Influenza Vaccines , Nanoparticles , Humans , Lipids , Polymers , RNA , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
20.
Virol Sin ; 36(5): 913-923, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1230296

ABSTRACT

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.


Subject(s)
Antiviral Agents , COVID-19 , Antiviral Agents/pharmacology , Containment of Biohazards , Humans , Replicon , SARS-CoV-2 , Virus Replication
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