Transcriptional and functional characterisation of the murine cytomegalovirus m15 locus

Cytomegalovirus (CMV) is a large DNA virus of the Herpesviridae family. This species-specific virus is a ubiquitous opportunistic pathogen that generally causes asymptomatic infection in healthy individuals, but can cause severe disease in newborns and immunocompromised patients. Murine CMV (MCMV) is widely used as a model of HCMV, allowing the study of pathogenesis in a natural host. Our laboratory has recently identified the novel m15 open reading frame (ORF) as one of the most variable in the MCMV genome. m15 is a member of the unique m02 gene family, where known and putative immune evasion genes are located. The purpose of this thesis was to characterise the transcription of m15 and to define the role m15 plays in MCMV pathogenesis. A recombinant virus, K181-Δm15, with a targeted disruption of the m15 ORF was constructed and used for pathogenesis studies in inbred mice. K181-Δm15 replicated to slightly higher titres in the spleens of acutely infected SJL, BALB/c and CBA mice. Somewhat paradoxically, K181-Δm15 was significantly attenuated during persistent infection of the salivary glands in these mice. This attenuation was apparent during viremia and from first seeding of virus into the salivary glands. Reduced viral titres in the salivary glands was accompanied by reduced shedding into the saliva. These data demonstrated that the m15 ORF is responsible for normal dissemination to, and replication of, MCMV in the salivary glands. To identify the 5’- and 3’-ends of the m15 transcript, RACE analysis was employed, with the studies extended to include adjacent ORFs, m14 and m16. Five separate 5’- transcripts were detected over the m14-m16 region, all of which were 3’-co-terminal, with a consensus polyadenylation site downstream of the m16 stop codon.