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Emerg Microbes Infect ; : 1-28, 2022 Jan 16.
Article in English | MEDLINE | ID: covidwho-1625432


AbstractThe extremely high transmission rate of SARS-CoV-2 and severe cases of COVID-19 pose the two critical challenges in the battle against COVID-19. Increasing evidence has shown that the viral spike (S) protein-driven syncytia may be responsible for these two events. Intensive attention has thus been devoted to seeking S-guided syncytium inhibitors. However, the current screening campaigns mainly rely on either live virus-based or plasmid-based method, which are always greatly limited by the shortage of high-level biosafety BSL-3 facilities or too much labor-intensive work. Here, we constructed a new hybrid VEEV-SARS-CoV-2-S-eGFP reporter vector through replacement of the structural genes of Venezuelan equine encephalitis virus (VEEV) with the S protein of SARS-CoV-2 as the single structural protein. VEEV-SARS-CoV-2-S-eGFP can propagate steadily through cell-to-cell transmission pathway in S- and ACE2-dependent manner, forming GFP positive syncytia. In addition, a significant dose-dependent decay in GFP signals was observed in VEEV-SARS-CoV-2-S-eGFP replicating cells upon treatment with SARS-CoV-2 antiserum or entry inhibitors, providing further evidence that VEEV-SARS-CoV-2-S-eGFP system is highly sensitive to characterize the anti-syncytium-formation activity of antiviral agents. More importantly, the assay is able to be performed in a BSL-2 laboratory without manipulation of live SARS-CoV-2. Taken together, our work establishes a more convenient and efficient VEEV-SARS-CoV-2-S-eGFP replicating cells-based method for rapid screening of inhibitors blocking syncytium formation.

Signal Transduct Target Ther ; 6(1): 369, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1483125


The lung is the prophylaxis target against SARS-CoV-2 infection, and neutralizing antibodies are a leading class of biological products against various infectious viral pathogen. In this study, we develop a safe and cost-effective platform to express neutralizing antibody in the lung with replicating mRNA basing on alphavirus replicon particle (VRP) delivery system, to prevent SARS-CoV-2 infections. First, a modified VEEV replicon with two subgenomic (sg) promoters was engineered to translate the light and heavy chains of antibody simultaneously, for expression and assembly of neutralizing anti-SARS-CoV-2 antibody CB6. Second, the feasibility and protective efficacy of replicating mRNA against SARS-CoV-2 infection were demonstrated through both in vitro and in vivo assays. The lung target delivery with the help of VRP system resulted in efficiently block SARS-CoV-2 infection with reducing viral titer and less tissue damage in the lung of mice. Overall, our data suggests that expressing neutralizing antibodies in the lungs with the help of self-replicating mRNA could potentially be a promising prophylaxis approach against SARS-CoV-2 infection.

Alphavirus , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , Replicon , SARS-CoV-2/metabolism , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/genetics , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , COVID-19/genetics , COVID-19/metabolism , Chlorocebus aethiops , Cricetinae , Female , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Vero Cells
Signal Transduct Target Ther ; 5(1): 218, 2020 10 03.
Article in English | MEDLINE | ID: covidwho-1387198

Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Cardiac Glycosides/pharmacology , Gene Expression Regulation/drug effects , Host-Pathogen Interactions/drug effects , Animals , Antiviral Agents/chemistry , Betacoronavirus/pathogenicity , Biological Products/chemistry , Biological Products/pharmacology , Bufanolides/chemistry , Bufanolides/pharmacology , COVID-19 , Cardiac Glycosides/chemistry , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Digoxin/chemistry , Digoxin/pharmacology , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Humans , Janus Kinases/antagonists & inhibitors , Janus Kinases/genetics , Janus Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Pandemics , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Signal Transduction , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Vero Cells , Virus Replication/drug effects
Nucleic Acids Res ; 48(3): 1392-1405, 2020 02 20.
Article in English | MEDLINE | ID: covidwho-1332861


The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5'-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.

Enterovirus A, Human/ultrastructure , Multiprotein Complexes/ultrastructure , Protein Conformation , RNA-Dependent RNA Polymerase/ultrastructure , Antiviral Agents/chemistry , Binding Sites , Crystallography, X-Ray , Enterovirus A, Human/chemistry , Enterovirus A, Human/genetics , Genome, Viral , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Nucleotides/chemistry , RNA, Viral/chemistry , RNA, Viral/ultrastructure , RNA-Dependent RNA Polymerase/chemistry , Virus Replication/genetics
J Gen Virol ; 102(5)2021 05.
Article in English | MEDLINE | ID: covidwho-1218063


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.

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
Emerg Microbes Infect ; 9(1): 1170-1173, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-324574


The emerging SARS-CoV-2 infection associated with the outbreak of viral pneumonia in China is ongoing worldwide. There are no approved antiviral therapies to treat this viral disease. Here we examined the antiviral abilities of three broad-spectrum antiviral compounds gemcitabine, lycorine and oxysophoridine against SARS-CoV-2 in cell culture. We found that all three tested compounds inhibited viral replication in Vero-E6 cells at noncytotoxic concentrations. The antiviral effect of gemcitabine was suppressed efficiently by the cytidine nucleosides. Additionally, combination of gemcitabine with oxysophoridine had an additive antiviral effect against SARS-CoV-2. Our results demonstrate that broad-spectrum antiviral compounds may have a priority for the screening of antiviral compounds against newly emerging viruses to control viral infection.

Alkaloids/pharmacology , Amaryllidaceae Alkaloids/pharmacology , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Deoxycytidine/analogs & derivatives , Phenanthridines/pharmacology , Virus Replication/drug effects , Animals , Betacoronavirus/growth & development , Betacoronavirus/metabolism , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/pharmacology , Deoxycytidine/pharmacology , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , SARS-CoV-2 , Vero Cells