| Gammaherpesviruses are large, membrane-enveloped, double-stranded DNA viruses that include Epstein-Barr (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri, and murine gammaherpesvirus 68 (MHV-68, or gammaHV-68). EBV and KSHV are important human pathogens associated with several types of human tumors. Gammaherpesviruses present two distinct phases in their life cycles, i.e. lytic replication and latency, from which viruses can be reactivated into lytic replication under certain stimuli. Both lytic replication and latency play important roles in the pathogenesis of gammaherpesviruses related diseases, including gammaherpesviruses-associated malignancies. How cellular genes regulate the lytic replication and reactivation of gammaherpesviruses is an essential aspect of our knowledge about viral-host interactions and yet is not well defined. In this study, we took the advantage of the robust lytic replication of MHV-68 in cell culture and relative tight maintenance of KSHV latency in cultured primary effusion lymphoma (PEL) cell lines to systematically study the functions of cellular genes during gammaherpesvirus lytic replication and reactivation.First, we screened a library of kinases and identified Tpl2 (tumor progression locus 2, also called Map3k8) as a positive regulator of MHV-68 lytic replication. Through an AP-1 element, Tpl2 transcriptionally upregulates the expression of MHV-68 RTA, the molecular switch that controls the whole cascade of lytic gene expression. Tpl2 also enhances the promoter activities of ORF57 and M3, indicating that Tpl2 can directly enhance the expression of other viral lytic genes independent of the enhancement of RTA expression. In addition, Tpl2 enhances AP-1 activity through upregulating Fos expression, which is required for the enhancement of MHV-68 replication by Tpl2. Moreover, Tpl2 can reactivate KSHV in BC-3 cells via enhancing KSHV RTA expression. Finally, we showed that Myc inhibits the activation of MHV-68 lytic replication by the Tpl2/AP-1 pathway and TGFbeta promotes virus replication through both upregulation of Jun and downregulation of Myc expression. Therefore, our data indicate a network of kinases and transcription factors cooperate to regulate gammaherpesvirus lytic replication.Second, we studied the regulation of KSHV reactivation by cellular genes in PEL cells. KSHV induced primary effusion lymphoma (PEL) is a type of aggressive non-Hodgkin lymphoma that lacks effective therapy. Here we report that inhibition of Myc or Cdk1 induces p53-independent apoptosis and KSHV reactivation in PEL cells. Myc inhibits KSHV reactivation via inhibiting transcriptional activation of KSHV RTA by the endogenous Erk/Ets-1 pathway. Apoptosis is not required for but instead inhibits KSHV reactivation whereas KSHV reactivation inhibits apoptosis upon Cdk1 inhibition, suggesting that early upstream signals lead to two competing downstream processes, i.e. apoptosis and KSHV reactivation that inhibits each other. In addition, Cdk1 inhibition synergizes with p53 activation in inducing PEL cell apoptosis. These findings therefore establish an essential role for Myc and Cdk1 in promoting PEL cell survival and the maintenance of KSHV latency and suggest that targeting such genes might be effective for the treatment of PEL either as a standalone therapy or in the settings of combinatorial therapies.Taken together, through functional genetic screens and detailed biochemical analysis, we not only identified individual cellular genes but also established the functional relationship of multiple cellular genes regulating the lytic replication and reactivation of gammaherpesviruses. In addition, we demonstrated that cellular genes such as Myc and Cdk1 are essential for both the survival of latently infected tumor cells and the maintenance of virus latency and provided basis for exploiting these genes as therapeutic targets for the treatment of gammaherpesvirus-associated malignancies such as primary effusion lymphoma. |
