Determinants of Coronavirus Fidelity in Replication and Pathogenesis

PROJECT SUMMARYViruses in the Coronaviridae family (CoVs) have emerged as zoonoses with pandemic potential twice in the21st century, causing severe human disease. Middle East respiratory syndrome (MERS)-CoV continues tocause new cases of lethal respiratory infections with 35% mortality. Further, severe acute respiratory syndrome(SARS)-like bat CoVs currently circulating are capable of infecting human cells, establishing the risk for futureemergence of zoonotic CoVs. There are no approved vaccines or antivirals for any human or zoonotic CoV,emphasizing the importance of identifying vulnerable and broadly conserved CoV targets for therapeuticintervention and vaccine development. Most RNA viruses generate genetic diversity required for interspeciesmovement and adaptation via error-prone RNA-dependent RNA polymerases (RdRps) that lack proofreading.In contrast, all CoVs encode a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that isa key driver of CoV evolution and adaptation via RNA-dependent RNA proofreading. During the four years offunding for this program, we have shown that CoV nsp14-ExoN mediates high-fidelity replication and that CoVslacking ExoN activity (ExoN(-)) are less fit during infection in cell culture, more sensitive to RNA mutagens, andattenuated in a murine model of SARS-CoV infection. Our findings suggest that divergent β-CoVs - MERS-CoV,SARS-CoV, and murine hepatitis virus (MHV) - have differential requirements for ExoN to sustain viability andoverall fitness. Finally, ExoN may play important and previously unpredicted functions in CoV resistance tohost innate immune surveillance. Thus, our published and preliminary studies support the scientific premisethat nsp14-ExoN is a master regulator of CoV fitness, evolution, and pathogenesis via functions in viralreplication, fidelity, and evasion of host innate immune responses. Specific aims of this proposal will define: 1)Sequence and structural determinants of nsp14-ExoN-mediated functions in CoV replication, fidelity, andinterferon sensitivity; 2) Adaptations in nsp14, nsp12-RdRp, and elsewhere in the CoV replicase thatcompensate for loss of ExoN-mediated fidelity; and 3) Mechanisms of ExoN regulation of the innate antiviralimmune response in vitro and in vivo. The availability of a high-resolution structure of nsp14; facile reversegenetics systems for MHV, SARS-CoV, and MERS-CoV; and robust, relevant animal models for SARS-CoVand MERS-CoV will allow us to address these questions, resulting in a comprehensive understanding of ExoNroles and mechanisms in CoV replication, adaptation, and pathogenesis. These studies will catalyzeapproaches targeting ExoN as a basis for stably attenuated CoV vaccines and novel antiviral drugs.