Characterization of Murine Cytomegalovirus m142 and m143 Essential Gene Products

CHARACTERIZATION OF MURINE CYTOMEGALOVIRUS m l42 AND ml43 ESSENTIAL GENE PRODUCTS Bridget Dalton Eastern Virginia Medical School and Old Dominion University, 2001 Director: Dr. Ann Campbell 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 o f 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, ml42 and ml43, are members of the US22 gene family of HCMV, some of which are transcriptional transactivators. The m l42 and ml43 genes are also essential for viral replication in fibroblasts. The purpose of this dissertation was to characterize the m l42 and ml43 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 m l42 and ml43 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, ml42 and ml43, encode a 1.8 and 3.8 kb transcript, respectively. The m l42 and ml43 proteins (designated pm l42 and pml43, respectively) encoded by these transcripts were present by 3 hours post infection and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. remained abundant at 12 and 24 hours post infection. Interestingly, pm 142 and pm 143 could not be designated as immediate early proteins, because they were not expressed in the presence o f drugs that block viral growth at this specific stage within the viral replication cycle. Therefore, the m l42 and ml43 immediate early genes do not express detectable levels of protein until early times post infection. The pm 142 protein is 43 kD protein, and the pml43 protein is 53 kD. The pm l42 and pm 143 proteins localized to both the nucleus and the cytoplasm at 3, 4 and 24 hours post infection. In transactivation studies, we tested the ability o f pm 142 and pm 143 to activate the MCMV major immediate early promoter (M122-123) and the early el promoter (M l 12-113). In combination, MCMV pml42 and pml43 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 pm l42 or pml43 failed to transactivate the MIEPE. Together, pml42 and pml43 also exhibited noncooperative effects with IE1 and IE3 in the activation o f the MCMV MIEPE. In combination, pm l42 and pml43 failed to activate the early e l promoter, but pm l42 and pml43 cooperated with IE 1 and IE3 to activate this promoter. Activation o f the e 1 promoter by IE1 and IE3 increased from 30 fold above basal levels in the absence of pm l42 and pm l43, to as high as 100 fold above basal levels in the presence o f pm l42 and pml43. This was the first time it has been shown that other MCMV viral proteins assist in IE1/IE3 mediated transactivation o f the e l promoter or any other promoter, and these findings have important implications in MCMV replication. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.