Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
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
2.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Article in English | MEDLINE | ID: covidwho-1467657

ABSTRACT

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Subject(s)
RNA, Viral/genetics , Replicon/physiology , SARS-CoV-2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Humans , Interferons/pharmacology , Microbial Sensitivity Tests , Mutation , Plasmids , RNA, Viral/metabolism , Replicon/genetics , Reverse Genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/genetics , Virion/physiology , Virus Replication
3.
J Gen Virol ; 102(5)2021 05.
Article in English | MEDLINE | ID: covidwho-1218063

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Replicon/drug effects , SARS-CoV-2/drug effects , Animals , COVID-19/drug therapy , Chlorocebus aethiops , Drug Discovery , High-Throughput Screening Assays/methods , Humans , Replicon/genetics , SARS-CoV-2/genetics , Vero Cells , Virus Replication/drug effects
4.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Article in English | MEDLINE | ID: covidwho-1152940

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , High-Throughput Screening Assays/methods , Replicon/drug effects , SARS-CoV-2/drug effects , A549 Cells , Animals , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/genetics , HEK293 Cells , Humans , Replicon/genetics , SARS-CoV-2/genetics , Vero Cells , Virus Replication/drug effects
5.
mBio ; 12(1)2021 01 19.
Article in English | MEDLINE | ID: covidwho-1066818

ABSTRACT

The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , RNA, Viral/biosynthesis , Replicon/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Drug Evaluation, Preclinical/methods , Female , Genetic Engineering , HEK293 Cells , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Mutation , Pandemics , RNA, Viral/genetics , Replicon/genetics , SARS-CoV-2/metabolism , Virus Replication/drug effects
6.
Sci Transl Med ; 12(555)2020 08 05.
Article in English | MEDLINE | ID: covidwho-655207

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.


Subject(s)
Alphavirus/genetics , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , RNA, Viral/genetics , Replicon/genetics , T-Lymphocytes/immunology , Viral Vaccines/immunology , Animals , Antibody Formation/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/prevention & control , Inorganic Chemicals/chemistry , Lipids/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nanoparticles/chemistry , Pandemics , Primates , SARS-CoV-2
SELECTION OF CITATIONS
SEARCH DETAIL