Role of porcine reproductive and respiratory syndrome virus non-structural protein 1 in viral replication and pathogenesis.

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious agent of significant concern to the global swine industry. PRRSV infection of pigs is initiated by a long viremia after which the virus enters in an extended persistent phase of 3–4 months that ultimately is resolved by the immune system. The delayed and weak host adaptive response is responsible for the protracted convalescence period. An initial sub-optimal innate immune response is postulated to be the reason behind such meager adaptive immune response. A major focus of the studies undertaken in this dissertation was to identify the viral non-structural proteins (nsps) involved in antagonizing cellular interferon (IFN) production, a principal component of host innate defense system. Among the four different nsps identified from initial cursory screening, nsp1 exhibited the strongest suppression of IFN induction. Nsp1α and nsp1β, the proteolytic products of nsp1 were both found to down-regulate dsRNA-induced activation of interferon regulatory factor 3 (IRF3). Furthermore, Nsp1β specifically antagonized IRF3 phosphorylation and nuclear translocation. In order to determine the amino acid residues responsible for the anti-IFN property of nsp1α and nsp1β, we prepared alanine-scanning mutants of both proteins. These mutants were probed for their ability to alleviate the IFN-suppression in reporter assays. Several candidates with variable degrees of relief were identified. A recombinant PRRSV with the respective mutations incorporated in nsp1β induced higher level of type I IFN and was attenuated in vitro. However, in infected swine the mutant virus quickly reverted to acquire the wild type IFN-suppression phenotype. To further understand the role of nsp1β in PRRSV life cycle, we employed co-immunoprecipitation coupled with mass spectrometry analysis to identify host cellular factors that interact with nsp1β. Here, we characterized nsp1β's interaction with cellular poly(C) binding protein (PCBP)-1 and 2. Both PCBP1 and PCBP2 associated with several components of viral replication and transcription apparatus. Using various biochemical assays, I demonstrated that both PCBP1 and PCBP2 are important for transcription and/or replication of the viral genome.