Roles of the Coronavirus 3'-to-5' Exoribonuclease and N7-Methyltransferase in Counteracting Innate Immunity

Abstract

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode many innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV non-structural protein 14 (nsp14) is a multifunctional protein that encodes 3’-to-5’ exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. CoV ExoN activity performs a proofreading function and is required for high-fidelity replication of the uniquely large CoV RNA genome. Furthermore, eukaryotic mRNAs possess a methylated 5’-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. CoV nsp14 N7-MTase activity is implicated in viral RNA 5’ capping resulting in structures that mimic cellular 5’ caps. In this dissertation research, I tested the hypothesis that nsp14 ExoN and N7-MTase activities function to antagonize the innate immune response. Using site directed mutagenesis to ablate CoV nsp14 ExoN activity, I demonstrate that ExoN activity is required for resistance to the innate immune response and that viruses lacking ExoN activity that were generated during an antiviral state are less viable to establish a subsequent infection. In addition, I demonstrate that CoV nsp14 N7-MTase activity prevents detection of viral RNAs by innate sensors, confers resistance to the IFN-β-mediated innate immune response, and directs efficient translation of viral proteins. Altogether, this dissertation research demonstrates that CoV nsp14 ExoN and N7-MTase activities are required for viral resistance to the effects of the IFN-β-mediated innate immune response and represent highly conserved targets for the attenuation of CoVs.