Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Coronaviruses are positive-sense, single-stranded RNA viruses. The majority of the RNA encodes non-structural proteins (nsps) that are translated as a large polyprotein, which is cleaved by the papain-like (PLP) and picornavirus 3C-like (3CLpro) proteases. The nsps modify host membranes to produce double membrane vesicles (DMVs) upon which the replicase-transcriptase assembles and synthesizes viral RNA. nsp3, nsp4, and nsp6 are integral membrane proteins believed to be involved in DMV formation. Work presented here demonstrates that nsp4 is subjected to N-linked glycosylation and mutation of N258 to threonine in nsp4 confers a temperature sensitive phenotype to MHV-A59 infectious clone virus. This virus (Alb ts6 icv) was significantly inhibited when incubated at the non-permissive temperature of 39.5°C. Processing of the p150 intermediate (nsp4-11) was unaffected; however, DMV formation was impaired. Interestingly, at 39.5°C, mitochondrial morphology was altered in Alb ts6 icv infected cells, and nsp3 and nsp4 had increased localization with the mitochondria. Complementation studies suggested that the ts lesion may affect the p150 precursor as well as nsp4. These data demonstrate a critical role for p150 and/or nsp4 in the assembly of DMVs and the viral replication complex. In addition, nsp3 from MHV, HCoV-NL63, and SARS-CoV has been shown to possess deubiquitinating activity, which negatively influences the induction of type I IFNs. Consistently, the PLP domains were shown to inhibit the induction of IFN-β-, IRF-3- and NF-κB-luciferase reporters. In contrast to MHV, the PLPs from HCoV-NL63 (PLP2) and SARS-CoV (PLpro) were able to inhibit type I IFN induction independently of catalytic activity albeit to a lesser degree than wildtype. Further investigation revealed that the PLPs associated with and inhibited the dimerization of a key protein involved in IRF-3 activation, stimulator of IFN genes (STING). The PLPs inhibited STING-mediated activation and nuclear translocation of IRF-3. These data suggest that the PLPs antagonize type I IFN induction by deubiquitinating key proteins in the IFN induction cascade as well as associating with and inhibiting the dimerization of STING thereby negatively affecting the activity of IRF-3.

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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