Evidence for CDK-Dependent and CDK-Independent Functions of the Murine Gammaherpesvirus 68 v-Cyclin

Abstract

Gamma-2 herpesviruses encode homologues of mammalian D-type cyclins (v-cyclins), which likely function to manipulate the cell cycle, thereby providing a cellular environment conducive to virus replication and/or reactivation from latency. We have previously shown that the v-cyclin of murine gammaherpesvirus 68 is an oncogene that binds and activates cellular cyclin-dependent kinases (CDKs) and is required for efficient reactivation from latency. To determine the contribution of v-cyclin-mediated cell cycle regulation to the viral life cycle, recombinant viruses in which specific point mutations (E133V or K104E) were introduced into the v-cyclin open reading frame were generated, resulting in the disruption of CDK binding and activation. While in vitro growth of these mutant viruses was unaffected, lytic replication in the lungs following low-dose intranasal inoculation was attenuated for both mutants deficient in CDK binding as well as virus in which the entire v-cyclin open reading frame was disrupted by the insertion of a translation termination codon. This replication defect was not apparent in spleens of mice following intraperitoneal inoculation, suggesting a cell type-and/or route-specific dependence on v-cyclin-CDK interactions during the acute phase of virus infection. Notably, although a v-cyclin-null virus was highly attenuated for reactivation from latency, the E133V v-cyclin CDK-binding mutant exhibited only a modest defect in virus reactivation from splenocytes, and neither the E133V nor K104E v-cyclin mutants were compromised in reactivation from peritoneal exudate cells. Taken together, these data suggest that lytic replication and reactivation in vivo are differentially regulated by CDK-dependent and CDK-independent functions of v-cyclin, respectively.The eukaryotic cell cycle comprises a series of coordinated events leading to cellular DNA and the segregation of the replicated DNA to daughter cells. The transitions through this cycle are controlled by the temporal and spatial activation of highly conserved holoenzymes consisting of a short-lived regulatory cyclin subunit and a cyclin-dependent kinase (CDK) (5,54,55,71). Quiescent (G 0 ) cells stimulated by mitogens upregulate the highly redundant D-type cyclins (cyclins D1, D2, and/or D3), which bind and activate CDK4 and/or CDK6 to mediate progression of the cell cycle into and through the G 1 phase. Cyclin D/CDK activity allows for the expression of Etype cyclins, which bind and activate CDK2 to drive progression through the restriction point from the G 1 phase into the S phase. Cyclin A expression allows for the continued activation of CDK2 and subsequently binds and activates CDK1 as S phase progresses and the G 2 /M boundary is reached, at which stage cyclin B levels are maximal for the regulation of mitosis in conjunction with CDK1. The activities, localization, and stability of these holoenzymes are further affected by proteinprotein interactions and posttranslational modifications that act to positively or negatively modulate cyclin-CDK ...

ORF73-Null Murine Gammaherpesvirus 68 Reveals Roles for mLANA and p53 in Virus Replication

“…8). Since v-cyclin is a requirement for MHV68 reactivation following ex vivo plating in some settings (69,72), these data further suggest that cell cycle arrest and/or p53 activation contribute to viral reactivation from latency.…”

“…Cells were harvested by direct lysis for immunoblotting or by freezing at Ϫ80°C for titer determinations. Progeny virions were liberated by freeze-thaw lysis, lysates were serially diluted, and titers were determined by MHV68 plaque assay as described previously (69).…”

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

“…We also assessed induction of the MHV68 cyclin homolog (v-cyclin), which is critical for viral reactivation from latency (61,69). A20-HE cells reactivated in response to PMA treatment and BCR cross-linking (anti-Ig) as evidenced by production of lytic antigens and detection of ORF59 and v-cyclin (Fig.…”

“…1). Also induced by PMA treatment were genes that influence host colonization (M4) (15) or reactivation upon explant of latently infected cells, including M1, v-cyclin (orf72), and v-bcl2 (M11) (13,23,38,61,69). PMA treatment also induced transcription of the multifunctional M2 gene, which influences both latency establishment and reactivation, particularly in B cells, perhaps through modulating host regulatory signaling cascades (24, 26, 34, 35, 5 A20-HE cells per ml were treated as follows: 10 g/ml LPS, 20 ng/ml PMA, 2 mM NaB, 5 g/ml antiimmunoglobulin G (␣-Ig), 2.5 g/ml anti-CD40, or the combinations indicated at the same concentrations.…”

“…For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24, 26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency maintenance (22,42) or orf72 (v-cyclin) in replication and reactivation (61,68,69).…”