Degree Type

Thesis

Date of Award

2013

Degree Name

Master of Science

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Cathy L. Miller

Abstract

A clinically benign member of the dsRNA virus family Reoviridae, Mammalian Orthoreovirus (MRV) serves as a model system to determine the nuances of replication within the family. MRV is composed of ten dsRNA gene segments that are contained in a double capsid structure and encode 12-13 viral proteins. It has previously been shown that at early times in MRV infection, granules form throughout the cytoplasm of the host cells that have been termed stress granules (SGs). MRV induces the formation of these SGs by activation of the kinase Protein Kinase R (PKR) which subsequently phosphorylates the initiation factor eIF2α to trigger the accumulation of the various factors found within SGs. SGs consist of cellular proteins, ribosomal subunits, silenced mRNAs, and initiation factors and their presence effectively results in the shutoff of translation of both the host and viral proteins. It has been previously shown that at early times in infection, viral cores associate with SGs and that translation is shutoff. However, at late times in infection SGs are no longer present in the cell and translation of viral, but not host, protein occurs. In order to determine how MRV is able to disrupt SGs, we mapped the association of a viral protein, µNS, to SGs using a series of deletion and substitution mutants. Using these mutants, we mapped the necessary amino acids in µNS to amino acids 78 and 79. We then used an amino acid 78-79 substitution mutant to determine whether these amino acids were important for interaction with other MRV proteins and if a virus containing these mutations was replication competent. To determine how MRV proteins are able to be preferentially translated at late times in infection, we created plasmids that contained different combinations of viral or non-viral sequence, UTR lengths, and mRNA modifications (cap, poly A tail). Using these plasmids and a non-radioactive protein labeling system, we viewed the ability of samples transfected with our plasmids to escape translational shutoff in the presence of either the oxidative stressor sodium arsenite or viral infection.

Copyright Owner

Kate Carroll

Language

en

File Format

application/pdf

File Size

87 pages

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