Document Type

Dissertation - University Access Only

Award Date

2012

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Ying Fang

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is still causing tremendous economic lose in the swine industry worldwide. Previous study results indicate that immune-suppression might be a major strategy that contributes to the PRRSV pathogenesis and persistent infection. Recent studies from our laboratory and others showed that nsp 1 a/􀂮, nps2, nsp4 and nsp 11 were responsible for inhibition of type I interferon (IFN) expression, however, the exact mechanism of these viral proteins in subverting IFN expression are still unknown. The objective of this dissertation study is to determine the potential mechanism that PRRSV nsp2 involves to inhibit IFN synthesis and signaling. PRRSV nsp2 is a multifunctional protein in the virus life cycle. The N terminal of nsp2 contains an ovarian tumor domain (OTU), which is reported to have trans- and cis cleavage ability to cleave the nsp2/3 site. In this study, the nsp2 N- terminal OTU domain was determined to be responsible for inhibition of IFN expression. The results demonstrated that PRRSV nsp2 OTU domain antagonizes type 1 IFN induction through interfering with NF-kB signaling pathway. Further analysis revealed that the nsp2 OTU domain possess ubiquitin deconjugation ( de-Ub) ability. It inhibits NF-kB activation by interfering with the poly-ubiquitination process of IKBa, which subsequently prevents lKBa degradation. Besides inhibition of IFN synthesis, nsp2 also blocked the downstream IFN stimulated anti-viral signaling protein function. In vitro ISGylation assay results showed that the N-terminal OTU domain of nsp2 had deconjugating (deISGylation) activity towards ISGylated cellular proteins. The nsp2 OTU also suppressed ISG 15 expression at the late stage of infection . Taken together, these data indicate that the nsp2 OTU domain plays pivotal roles in modulating the host antiviral immune response. To determine whether the immune antagonist function mediated by the PRRSV nsp2 OTU domain could be selectively inactivated, a panel of mutants with deletion and/ or mutation was constructed. Most of the mutations and / or deletions were lethal to the virus, however, the three point mutations (D459A, S463A, D465A,) and one deletion (nsp2-d402-420) allowed recovery of viable viruses using a reverse genetic system. This study represents a fundamental step in elucidating the role of nsp2 in PRRSV pathogenesis, and provides an important insight in the future development of modified live attenuated PRRS vaccines.

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome -- Molecular aspects
Viral proteins
Immune response
Swine -- Virus diseases

Publisher

South Dakota State University

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Rights Statement

In Copyright