Modulation of cytomegalovirus replication by the viral temperance factor RL13 - Project Summary Human cytomegalovirus (CMV) is a β-herpesvirus exquisitely adapted to horizontally transmit in the face of robust preexisting immunity, replicate while causing minimal damage, maintain life-long latency, and reactivate sub-clinically to reach new hosts. Central to this strategy of coexistence with the host are virally-encoded “temperance factors” that actively restrict CMV replication and spread. The RL13 temperance factor, a virion- associated envelope glycoprotein, is clearly important in vivo as an intact RL13 gene is present in all CMV genomes sequenced from clinical materials. In cell culture RL13WT CMV isolates initially form small foci that grow slowly with little or no release of infectious virus into the culture medium. However, within a few cell culture passages mutations that disrupt RL13 invariably emerge and quickly outgrow the RL13WT parental virus. Consequently, the vast majority of CMV strains used for research are RL13null mutants. How and why RL13 represses CMV replication remain unknown. Our overarching hypothesis is that in vivo RL13 is turned on or off to modulate CMV replication in different settings. Thus, RL13 may be turned off at sites of entry to enhance spread and promote dissemination, then turned on during the convalescent phase to limit the production of cell-free virus that would be rapidly inactivated by neutralizing antibodies, then again turned off at sites of virus shedding (kidney and salivary glands) in order to drive high level release of cell-free virus into urine or saliva for transmission to new hosts. Recent studies have identified amino acid substitutions in the CMV glycoprotein M (gM) and Immediate Early 2 (IE2) proteins that independently counteract the temperance effects of RL13. We hypothesize (i) that RL13 repression is mediated through interactions with gM that alter virion morphogenesis, egress, or infectivity; and (ii) that under different circumstances or cell types in vivo, RL13 transcription is activated or repressed by cell type-specific expression of certain IE2 isoforms. These hypotheses will be explored through the following Aims: AIM 1. Determine the mechanism by which RL13 restricts CMV replication. Genetic and molecular techniques will be used to explore potential interactions between RL13 and gM to elucidate the molecular mechanisms whereby RL13 impairs assembly and/or release of infectious CMV virions. AIM 2. Determine how IE2 isoforms modulate RL13 temperance activities. Genetic and molecular approaches will determine how amino acid substitutions in IE2 or altered expression of IE2 isoforms impact RL13 expression or repression. RL13 has a seemingly paradoxical but clearly important role in the natural history of CMV infections. The proposed studies may reveal that RL13 is a “master regulator” that can be activated or repressed to control virus production as circumstances in vivo require. The results will be among the first to elucidate the functional basis of viral proteins that restrict, rather than enhance, viral replication and spread.
