Characterization of murine gammaherpesvirus 68 v-cyclin interactions with cellular cdks.
All known gamma2-herpesviruses encode a cyclin homolog with significant homology to mammalian D-type cyclins. The murine gammaherpesvirus 68 (gammaHV68) viral cyclin (v-cyclin) has been shown to be oncogenic when expression is targeted to thymocytes in transgenic mice and to be critical for virus reactivation from latency. Here, we investigate the interaction of the gammaHV68 v-cyclin with cellular cyclin-dependent kinases (cdks). We show that, in contrast to the Kaposi's sarcoma-associated herpesvirus (KSHV) v-cyclin, the gammaHV68 v-cyclin preferentially interacts with cdk2 and cdc2 but does not interact with either cdk4 or cdk6. Mutation of conserved residues, predicted to be involved in cdk binding based on the gammaHV68 v-cyclin:cdk2 crystal structure, resulted in the loss of both cdk binding and the ability to mediate phosphorylation of substrates. Like the KSHV v-cyclin, the gammaHV68 v-cyclin appears to confer expanded substrate specificity to the cellular cdk binding partners. As expected, the gammaHV68 v-cyclin:cdk complexes are able to target phosphorylation of histone H1, the retinoblastoma protein (pRb), and p27(Kip1) as assessed using in vitro kinase assays. Notably, hyperphosphorylation of pRb was observed during wt gammaHV68 replication in serum-starved murine fibroblasts, but not in cells that were either mock-infected or infected with a v-cyclin null gammaHV68. In addition, infection of serum-starved murine fibroblasts also results in a v-cyclin-dependent increase in cdk2-associated kinase activity and a concomitant decrease in the levels of p27(Kip1). Taken together, the latter studies served to validate the results of the in vitro kinase assays. Finally, in vitro kinase assays revealed that the gammaHV68 v-cyclin:cdk complexes can also phosphorylate p21(Cip1), Bcl-2, and p53. The latter suggests that, at least in vitro, the gammaHV68 v-cyclin exhibits functional characteristics of both cyclin E and cyclin A.
Upton
Jason W
JW
Center for Emerging Infectious Diseases, Yerkes National Primate Research Center, Emory University School of Medicine, NE Atlanta, GA 30329, USA.
van Dyk
Linda F
LF
Speck
Samuel H
SH
D019943:Amino Acid Substitution; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D005347:Fibroblasts / Q000821:virology; D006657:Histones / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D018144:Rhadinovirus / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D006657:Histones; C411568:M cyclin, gamma-herpesvirus 68; D019253:Proto-Oncogene Proteins c-bcl-2; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010770:Phosphotransferases; D016203:CDC2 Protein Kinase; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; C495912:Cdk6 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases