Inarigivir (SB 9200) is a linear dinucleotide antiviral drug designed to act as a RIG-I agonist, activating cellular innate immune responses. Clinical trials in hepatitis C virus (HCV) patients have confirmed the innate immune-activating antiviral activity of Inarigivir. In hepatitis B virus (HBV) patients, clinical trials using Inarigivir have demonstrated significant reductions in HBV DNA and RNA levels, although heterogeneous drug-induced liver injury has been observed at higher doses. To further explore Inarigivir's mechanism of action against HBV, we characterized its antiviral effect in various hepatocyte-derived cell lines, including transiently transfected HepG2 and Huh7 cells, inducible HBV stable cell lines HepAD38 and HepDES19, and HBV-infected human hepatocytes. Our results show that Inarigivir inhibits HBV DNA replication in a dose-dependent manner without affecting HBV RNA levels or pgRNA encapsidation. HBV RNA immunoprecipitation assays revealed that Inarigivir does not affect viral polymerase binding to pgRNA, while the in vitro endogenous polymerase reaction (EPR) assays demonstrated that Inarigivir inhibits HBV DNA synthesis catalyzed by polymerase within the capsid. Additionally, we provided several lines of evidence indicating that Inarigivir's antiviral effect is independent of the RIG-I signalling pathway in hepatocyte-derived cells. Taken together, our study demonstrates a RIG-I-independent antiviral mechanism of Inarigivir by inhibiting HBV DNA replication as a dinucleosidic non-chain-terminating reverse transcriptase inhibitor.
SARS-CoV-2 is a positive-sense single-stranded RNA virus of the Coronaviridae family. Emerging coronaviruses posing pandemic risk and the swift appearance of new antigenic variants of circulating SARS-CoV-2 urge the need for new and diverse effective treatments. The most outstanding targets to inhibit viral replication are its cysteine-proteases, Mpro and PLpro, which are essential for virus life cycle. Here, we investigated the functional consequences of heterologously expressed SARS-CoV-2 proteases in the eukaryotic model Saccharomyces cerevisiae. Both overproduced Mpro and PLpro caused yeast growth inhibition. This phenotype was dependent on their protease activities, since it was abrogated by the expression of catalytically inactive mutants or counteracted by the treatment with known protease inhibitors. Mpro reproduced in yeast the proteolytic cleavage of the pore-forming protein Gasdermin D, previously described in human cell lines. We generated a yeast-based screening platform to detect mutations in Mpro conferring resistance to inhibitors. To this end, we evaluated the behaviour of the purported resistant mutant, MproE166V, and found a resistant pattern to nirmatrelvir. Taking advantage of this model, we performed a random mutagenesis screen to isolate nirmatrelvir-resistant mutations in Mpro. Our screening identified nine clones. Five harbored previously described mutations, either identical (A260V) or involving alternative substitutions (G15N/F159I, G23E/D33N/R60K, C128R/R188K, S139I). Importantly, our study also revealed four novel residues that have not yet been associated with nirmatrelvir resistance (K88R, L220M, Y209F, L262M). In sum, we report a suitable model to screen compounds in search of new Mpro and PLpro inhibitors and to predict mutations associated to drug resistance.
