Behera, P. | bioRxiv Science

bioRxiv2024

TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7-/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

microbiology↗

bioRxiv2023

Single Chain Fragment Variable (scFv) corresponding to a novel epitope within HCV envelope protein restricts virus entry into hepatocytes

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis. The use of neutralizing antibodies could be a more effective therapeutic option. Previously we reported the discovery of a novel epitope at the C terminus of HCV-E2 protein, that induced potent neutralizing antibodies in the infected patients. Furthermore, monoclonal antibodies generated against this epitope could also significantly reduce virus replication in a cell culture system. In this study, we have focused on the generation of single chain variable fragments of this unique neutralizing monoclonal antibody A8A11 raised against the conserved epitope. The nucleotide sequence of the neutralizing monoclonal antibody A8A11 was determined and the scFv gene was constructed followed by cloning into the expression plasmid for recombinant protein expression. The scFv mimicked the antibody in binding to the hepatitis C virus like particles (HCV-LP). As expected, the scFv inhibited HCV-LP binding to hepatocytes and could effectively reduce viral replication in the cell culture system. More importantly, scFv A8A11 could restrict serum HCV RNA levels in HCV-infected chimeric mice harboring human hepatocytes. Results provide a basis for developing a promising scFv-based entry inhibitor, which could be more effective against viruses refractory to drugs targeting viral enzymes.

microbiology↗