Date of Award

Spring 2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Ann E. Campbell

Committee Member

Julie A. Kerry

Committee Member

Kenneth Somers

Committee Member

Timothy J. Bos

Abstract

Cytomegalovirus (CMV) is a species-specific virus belonging to the Herpesviridae family. This DNA virus causes severe disease or even death in newborns and immunosuppressed patients. Murine CMV (MCMV) provides an opportunity to study the role of various viral products in replication and pathology in the natural host. Successful replication of CMV in the host depends upon the expression of a cascade of viral genes: immediate early (IE), early (E), and late (L). To date only three MCMV IE proteins have been characterized (IE1, IE2 and IE3). Our laboratory recently identified a novel IE gene region within the Hind III I region of MCMV. These genes, m142 and m143, are members of the US22 gene family of HCMV, some of which are transcriptional transactivators. The m142 and m143 genes are also essential for viral replication in fibroblasts.

The purpose of this dissertation was to characterize the m142 and m143 transcripts and proteins, as well as determine if the gene products function as transcriptional transactivators like some of the other US22 gene family members. Although the m142 and m143 transcripts were present during IE times, levels of these two transcripts increased during early times of viral infection and remained abundant during late times. The genes, m142 and m143, encode a 1.8 and 3.8 kb transcript, respectively. The m142 and m143 proteins (designated pm142 and pm143, respectively) encoded by these transcripts were present by 3 hours post infection and remained abundant at 12 and 24 hours post infection. Interestingly, pm142 and pm143 could not be designated as immediate early proteins, because they were not expressed in the presence of drugs that block viral growth at this specific stage within the viral replication cycle. Therefore, the m142 and m143 immediate early genes do not express detectable levels of protein until early times post infection. The pm142 protein is 43 kD protein, and the pm143 protein is 53 kD. The pm142 and pm143 proteins localized to both the nucleus and the cytoplasm at 3, 4 and 24 hours post infection.

In transactivation studies, we tested the ability of pm142 and pm143 to activate the MCMV major immediate early promoter (M122–123) and the early e1 promoter (M112–113). In combination, MCMV pm142 and pm143 transactivated the MCMV major immediate early promoter and enhancer (MIEPE) in a dose dependent manner to at least 3 fold above basal levels. However, individually pm142 or pm143 failed to transactivate the MIEPE. Together, pm142 and pm143 also exhibited noncooperative effects with IE1 and 1E3 in the activation of the MCMV MIEPE. In combination, pm142 and pm143 failed to activate the early e1 promoter, but pm142 and pm143 cooperated with IE1 and 1E3 to activate this promoter. Activation of the e1 promoter by IE1 and IE3 increased from 30 fold above basal levels in the absence of pm142 and pm143, to as high as 100 fold above basal levels in the presence of pm142 and pm143. This was the first time it has been shown that other MCMV viral proteins assist in IE1/IE3 mediated transactivation of the e1 promoter or any other promoter, and these findings have important implications in MCMV replication.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/5fzs-xa03

ISBN

9780493177809

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