Emerging human and zoonotic RNA viruses including SARS-CoV cause significant global morbidity, mortality, and social disruption. The current model for RNA virus host-species movement, adaptation, evolution, disease, and resistance to vaccines and antivirals is that they generate vast diversity of related mutants around the central consensus genome, also referred to as quasispecies diversity or "mutant swarms". This allows for rapid selection of adaptive change, and until recently was thought to result solely from the action of RNA- dependent RNA polymerases with low fidelity (high basal mutation rate) and no error repair - proofreading - during RNA synthesis. Coronaviruses (CoVs) are broadly represented in humans, bats, and other mammals and avian species, and are adept at host-species movement and adaptation, as demonstrated by SARS-CoV and the novel HCoV-EMC/2012. CoVs contain the largest positive-strand RNA genomes, up to 32 kb, posing unique challenges to models of intrinsic low-fidelity replication. CoVs encode a DEDDh family 3'-to-5'exoribonuclease in nonstructural protein 14 (nsp14-ExoN), and inactivating mutations in ExoN (ExoN-) of murine hepatitis virus (MHV) and SARS-CoV result in viable mutator phenotypes (20-fold decreased fidelity) in vitro and in vivo. CoV ExoN- mutator viruses manifest decreased fitness compared to ExoN+ (WT) viruses, are sensitive to RNA mutagens, and are stably attenuated in mouse models of lethal SARS-CoV infection. Thus, nsp14-ExoN is likely the first known RNA virus proofreading exonuclease, and is critically involved in virus replication, fitness, and pathogenesis. However, how it regulates fidelity is unknown. Nsp14-ExoN is a distinct protein from the RNA dependent RNA polymerase (nsp12) and also has been shown to have enhanced exoribonuclease activity in vitro by interactions with nsp10. The contributions of these and other CoV proteins to fidelity regulation also is undetermined. Experiments in this proposal will test the hypothesis that CoVs encode multiple proteins, including nsp14-ExoN, nsp12, and nsp10, that together regulate replication fidelity.
The specific aims of the proposal are: 1) To define fidelity determinants in nsp14 and the impact on viral fitness and RNA synthesis;2) To identify proteins and determinants within the CoV fidelity complex;and 3) To determine the effect of altered fidelity on in vivo replication and pathogenesis. The proposed aims build on the complementary strengths and collaborations of the Denison and Baric labs in CoV (MHV and SARS-CoV) reverse genetics, replicase protein mutagenesis and functions, pathogenesis, immune response, and synthetic genomics. The results of the proposed experiments will identify fidelity determinants critical for replication, pathogenesis and virulence, define the range of tolerated fidelity, and identify novel approaches for CoV attenuation and inhibition.
Coronaviruses (CoVs) including SARS-CoV encode a novel exoribonuclease (ExoN) activity that is required for replication fidelity, a process unprecedented in RNA virus biology. We will identify key determinants in ExoN and other replicase proteins that regulate CoV replication fidelity, and will determine the role of fidelity in CoV replication and pathogenesis. Results from these experiments will increase our understanding of how CoVs adapt to new hosts, will establish fidelity-based strategies for CoV attenuation, and will identify novel targets for inhibition of replication fidelity.
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