HCMV encodes a stable 5 kb RNA (RNA5.0) of unknown function that is conserved across cytomegalovirus species. In vivo studies of the MCMV orthologue, a 7.2 kb RNA (RNA7.2), demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. Current analysis demonstrates that RNA7.2 is expressed with late viral gene kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to

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... roduce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5' end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of RNA7.2 of approximately 28.8 hours. This extraordinarily long half-life is caused by RNA sequence elements that reside primarily within the 3' end of the processed intron. These sequence elements are postulated to prevent debranching of a RNA7.2 intron lariat structure and subsequent exonuclease digestion. Our data indicates that RNA7.2 is protected from debranching and 5' to 3' exonuclease degradation, but iv future studies will need to validate the hypothesized lariat conformation. Stability analysis of the HCMV RNA5.0 also revealed a long half-life of greater than 32 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site. The data presented in this dissertation provides a comprehensive transcriptional analysis of the MCMV RNA7.2 locus. Our studies indicate that both RNA7.2 and RNA5.0 are extremely long-lived RNAs, a feature which is likely to be important in their role promoting viral persistent replication. The form and content of this abstract are approved. I recommend its publication.