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Document Type

Thesis - University Access Only

Award Date

2012

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

Xiuqing Wang

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the Arteriviridae family and encompasses a single-stranded positive-sense RNA genome. As an obligate intracellular parasite, PRRSV depends on the host's translation machinery for its protein synthesis. A key regulator of the translation process is the alpha subunit of eukaryotic translation initiation factor 2 (eIF2a). When phosphorylated, eIF2a inhibits the initiation of translation by preventing eIF2 from delivering the initiator Met-tRNA to the 40S ribosomal subunit. During viral infections, double-stranded RNA-dependent protein kinase R (PKR) is known to phosphorylate eIF2a following activation by doublestranded RNA. The status of PKR activation with regards to phosphorylation of eIF2a has yet to be determined in PRRSV-infected cells. Consequently, we sought to reveal the roles of PKR and its substrate, eIF2a, in relation to PRRSV replication. Here, .we show that PRRSV induces the phosphorylation of PKR early in infection; however, the appearance of phosphorylated eIF2a was not correlated with this event. GADD34, a regulatory subunit of the protein phosphatase 1 complex (PP 1 c ), was induced early in infection and it is likely that GADD34 is responsible for the dephosphorylation of eIF2a mediated by activated PKR to facilitate viral protein synthesis during early infection. PRRSV induced the phosphorylation of eIF2a during late infection in a time-dependent manner, which appeared to be independent of PKR activation. Phosphorylation of eIF2a correlated with reduced viral protein synthesis, indicating viral protein synthesis depends on eIF2a. The appearance of cytopathic effects was strongly correlated with eIF2a phosphorylation, suggesting a possible role of eIF2a phosphorylation in PRRSV-induced apoptosIS. Furthermore, inhibiting the phosphorylation of PKR with the PKR-specific inhibitor, 2-aminopurine (2-AP), did not affect the phosphorylation status of eIF2a, indicating that another eIF2a kinase is responsible for the phosphorylation eIF2a in PRRSV-infected MARC-145 cells. Although PRRSV protein synthesis was not affected by 2-AP treatment, PRRSV titers were reduced by approximately 1 log, suggesting that PKR activation may contribute to PRRSV replication in an eIF2a-indepentent manner. Overall, the results obtained in this study reveal that PRRSV activates the phosphorylation of PKR while limiting the phosphorylation of eIF2a during early infection to promote viral protein synthesis. PRRSV induced the phosphorylation of eIF2a during later infection by undefined kinases, which may contribute to the observed cell death and virus release from infected cells. The results will aid in future research regarding control of the host translation machinery with respect to maintaining viral replication.

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome Viral proteins Swine -- Virus diseases

Description

Includes bibliographical references (pages 52-69)

Format

application/pdf

Number of Pages

81

Publisher

South Dakota State University

Rights

In Copyright - Non-Commercial Use Permitted
http://rightsstatements.org/vocab/InC-NC/1.0/

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