Coronaviruses (CoV) are important emerging human viruses, with multiple cross-species transmission events identified over the past 200 years. Human infections with severe acute respiratory syndrome coronavirus (SARS-CoV), the first newly emerged virus of the 21st century, results in acute and organizing phase diffuse alveolar damage, atypical pneumonia, and acute respiratory distress syndrome (ARDS), leading to mortality rates of 10-50 percent, dependent on age. A major component in the success of the SARS-CoV is its ability to manipulate and subvert the host immune response. Studies by our laboratory and others have revealed numerous genes that antagonize the type I interferon (IFN) response including NSP1, ORF3b, and ORF6. In addition, several groups have generated SARS-CoV deletion mutants and demonstrated various levels of attenuation, possibly due to increased type I IFN sensitivity. A central hypothesis in this application is that highly conserved viral gene functions that target type I IFN responses can serve to provide a universal platform for the rational design of vaccine and drug therapeutics affording rapid responses in outbreak settings. Recently, research has focused on viral components involved in type I RNA capping utilized by SARS, other CoV, and many RNA and DNA viruses. In an ordered process, several CoV proteins contribute to the capping process including NSP13 (RTPase), NSP14 (N7-guanine methylation), and NSP10 (scaffold). However, interest has focused on NSP16, a S-adenosylmethionine (SAM) dependent nucleoside 2'-O-methyltransferase (2'-O-MTase) and its critical role in subverting the type I IFN response. Recent works have implicated 2'O- methylation in distinguishing between self and non-self RNA by MDA-5, a RIG-I like recognition molecule, and the IFIT family of interferon stimulated genes (ISGs). These results suggest that 2'-O-MTases like NSP16 play a critical role in immune antagonism during viral infection. Based on these recent findings, we sought to evaluate the impact of 2'O-methylation on SARS-CoV replication and pathogenesis by generating mutants lacking NSP16 2'O-MTase activity. We hypothesize that deltaNSP16 mutant viruses will be exquisitely sensitive to and attenuated in the presence of type I IFN both in vitro and in vivo. We anticipate the absence of type I IFN signaling or specific ISGs including MDA5 and IFIT family members will restore replication and possibly virulence. Due to broad conservation of this activity across RNA and DNA virus families, targeting the 2'O methylation pathways with either vaccine or drug strategies may provide unique, broadly applicable treatment options that can protect against current and emerging viruses.
To evaluate the impact of 2'O-methylation on SARS-CoV pathogenesis, mutant viruses lacking active NSP16, a 2'O-methyltransferase, will be evaluated in vitro and in vivo. DeltaNSP16 mutants are hypothesized to be sensitive to specific interferon stimulated genes and may have restored virulence in their absence. Targeting the 2'O-methylation pathways may also provide broad-spectrum therapeutics for SARS-CoV and other RNA viruses.
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