Date of Award

Summer 8-14-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Nora M. Chapman, Ph.D.

Abstract

Following natural or experimental infection, and in cell culture, coxsackie B virus (CVB) RNA can persist for weeks in the absence of CPE yet with detectable viral RNA. Earlier studies in our laboratory demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the 5’ genomic terminus, partially degrading the cloverleaf (or domain I), an RNA structure that is required for efficient viral replication. A cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] templates the addition of two uridine residues to the virus-encoded RNA replication primer, VPg, prior to genomic replication. Because our previous work also demonstrated that 5’ terminally deleted CVB (CVB-TD) genomes have VPg covalently linked despite rarely terminating in the canonical UU donated by CRE(2C) mediated uridylylation of VPg, it was hypothesized that a functional (uridylylating) CRE(2C) would be unnecessary for CVB-TD replication.

Using the same 16 mutations in the CVB3 CRE(2C) structure that were considered lethal for this virus by others, this work demonstrates that the wild type (wt) and the CVB3-TD virus carrying these mutations are viable with a non-uridylylating CRE(2C) in infected cell cultures and in mice. While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to that observed in a CVB3-TD strain, mutation of CRE(2C) function in a CVB3-TD strain does not further decrease replication. Further, this work shows that replication of the parental CVB3 strain containing the mutated CRE(2C) drives de novo generation of 5' terminal genomic deletions, and that the 16 mutations introduced into the CRE(2C) region can revert completely to wt sequence after only eight days of replication in cell culture. Cumulatively, these findings demonstrate that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in loss of 5' terminal sequence information. Additionally, these findings demonstrate that high selective pressure exists during replication which drives the complete reversion of the CRE(2C) to wt sequence, from which we propose that it has functions other than the experimentally defined VPg uridylylation template.

A final, unrelated finding of this dissertation shows that creatinine-hydrochloride effectively suppresses bacterial growth while allowing outgrowth of slower growing fungi.

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