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 (γHV68) 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 γHV68 v-cyclin mutant (v-cyclin.LacZ) that is unable to express a functional v-cyclin protein. Notably, although the γHV68 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 γHV68-induced chronic disease in mice lacking the gamma interferon receptor (IFNγR(-/-)), v-cyclin.LacZ virus was similar to wt γHV68 in terms of the incidence of mortality and vasculitis. Further analysis revealed that the frequencies of splenocytes and peritoneal cells harboring the latent γHV68 genome in normal and B-cell-deficient mice infected with wt γHV68 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 γHV68; 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 lyric cycle gene, the pro-proliferative γHV68 v-cyclin is not required for γHV68 replication either in vitro or during acute infection in vivo but rather is a critical determinant of reactivation from latency.