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Among the distinguishing characteristics of members of the gamma-2 herpesvirus family is the expression of a mammalian D-type cyclin homolog, termed v-cyclin. Murine gammaherpesvirus 68 (gammaHV68) is a gamma2-herpesvirus that can infect inbred and outbred strains of mice, providing a genetic system for the study of gammaherpesvirus pathogenesis. Disruption of the v-cyclin gene of gammaHV68 results in a virus that establishes latency in infected mice to wild-type levels, but is severely attenuated for virus reactivation [van Dyk, L.F., Virgin IV, H.W., Speck, S.H., 2000. J. Virol. 74:7451-7461]. Transcriptional regulation of the gammaHV68 v-cyclin has not been defined. We report here the initial characterization of the v-cyclin transcript expressed in permissive murine fibroblasts. Based on 5' mapping of the v-cyclin transcript, we identified a promoter that is involved in driving v-cyclin expression during virus replication. In addition, we determined that the promoter is responsive to the major viral lytic transactivator, Rta, encoded by orf 50. Using reporter plasmids we have analyzed both basal and Rta-induced v-cyclin promoter activity, initially identifying two regions of the v-cyclin promoter important for both basal and Rta-induced activity. Notably, only one of these regions could be shown to confer Rta responsiveness on a reporter construct containing the hsp70 TATA box. The importance of this region in regulating v-cyclin expression during virus replication was confirmed by introducing these mutations into the context of the viral genome and assessing v-cyclin expression following infection of permissive murine fibroblasts in tissue culture. In addition, we show that mutations that severely cripple Rta-induction of v-cyclin expression did not adversely impact virus reactivation from splenocytes recovered from latently infected mice, indicating that alternatively regulated v-cyclin gene expression is required for virus reactivation.