Host Determinants of Viral Entry and Replication

Abstract

As obligate intracellular parasites, viruses rely on the biological machinery of their hosts to replicate. Each step of the viral life cycle, from entry, to protein translation and genome replication, to egress, involves host factors and their associated mechanisms to function. The presence of these factors, and the virus’s ability to exploit them by mimicking the host, can determine the success of viral infection. Cells in turn have evolved mechanisms to defeat viral subversion, including self/non-self recognition by the innate immune system, and cell-intrinsic responses induced by interferons. Using vesicular stomatitis virus (VSV), we found that the host N6-adenosine messenger RNA (mRNA) cap methyltransferase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response to interferon-beta (IFN-β). PCIF1 N6-methylates the cap-proximal nucleotide of VSV mRNA to m7Gpppm6Am, and we define substrate requirements for this modification. PCIF1-dependent N6-methylation had no impact on mRNA stability, translation, or viral infectivity. However, cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following IFN-β pretreatment. We further demonstrate that mRNA cap structures of rabies and measles viruses are also modified by PCIF1. This identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to viral infection. Host factors, specifically host proteases, are required to trigger membrane fusion and entry of coronaviruses into cells, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The serine protease TMPRSS2 fulfills this function in lung cells, and is an attractive therapeutic target for antiviral drugs against COVID-19. Using rational structure-based drug design, substrate specificity screening of TMPRSS2, and entry inhibition screening using infectious chimeric VSV-SARS-CoV-2, we discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have improved activity over existing inhibitors Camostat and Nafamostat. Lead compound MM3122 has a half-maximal inhibitory concentration (IC50) of 430 pM in blocking entry of VSV-SARS-CoV-2 into Calu-3 human lung epithelial cells, and a similar IC50 of 870 pM against a chimeric VSV Middle East respiratory syndrome coronavirus (VSV-MERS-CoV). Excellent metabolic stability, safety, and pharmacokinetics in mice make it a promising drug candidate for COVID-19 treatment.