The murine gammaherpesvirus 68 v-cyclin is a critical regulator of reactivation from latency

Gamma-2 herpesviruses encode a homolog of mammalian D-type cyclins. The v-cyclin encoded by murine gammaherpesvirus 68 (gamma HV68) induces cell cycle progression and is an oncogene (L. F. van Dyk, J. L. Hess, J. D. Katz, M. Jacoby, S. H. Speck, and H. W. Virgin IV. J. Virol. 73:5110-5122, 1999). However, the role of the pro-proliferative v-cyclins in gamma-2 herpesvirus pathogenesis is not known. Here we report the generation and characterization of a gamma HV68 v-cyclin mutant (v-cyclin.LacZ) that is unable to express a functional v-cyclin protein. Notably, although the gamma HV68 v-cyclin is, expressed from an early-late lytic transcript, v-cyclin.lacZ replicated normally in fibroblasts in vitro and during acute infection in the spleen, liver, and lungs in vivo. Moreover, v-cyclin.lacZ exhibited wild-type (wt) virulence in mice with severe combined immunodeficiency. In addition, in a model of gamma HV68-induced chronic disease in mice lacking the gamma interferon receptor (IFN gamma R-/-), v-cydin.LacZ virus was similar to wt gamma HV68 in terms of the incidence of mortality and vasculitis. Further analysis revealed that the frequencies of splenocytes and peritoneal cells harboring the latent gamma HV68 genome in normal and B-cell-deficient mice infected with wt gamma HV68 or v-cyclin.lacZ were very similar. However, v-cyclin.lacZ was significantly compromised in its capacity to reactivate from latency. This phenotype was conclusively mapped to the v-cyclin gene by (i) generating a marker rescue virus (v-cyclin.MR) from the v-cyclin.lacZ mutant, which restored the frequency of cells in which virus reactivated from latency to the levels observed with wt gamma HV68; and (ii) generating a second v-cyclin mutant virus containing a translation stop codon within the v-cyclin gene (v-cyclin.stop), which was compromised in reactivation from latency. These studies demonstrate that despite expression as a lytic cycle gene, the pro-proliferative gamma HV68 v-cyclin is not required for gamma HV68 replication either in vitro or during acute infection in vivo but rather is a critical determinant of reactivation from latency.