Tpl2/AP-1 Enhances Murine Gammaherpesvirus 68 Lytic Replication

How cellular factors regulate gammaherpesvirus lytic replication is not well understood. Here, through functional screening of a cellular kinase expression library, we identified mitogen-activated protein kinase kinase kinase 8 (MAP3K8/Tpl2) as a positive regulator of murine gammaherpesvirus 68 (MHV-68 or γHV-68) lytic gene expression and replication. Tpl2 enhances MHV-68 lytic replication by upregulating lytic gene expression and promoter activities of viral lytic genes, including RTA and open reading frame 57 (ORF57). By screening a cellular transcription factor library, we identified the Fos AP-1 transcription factor as a downstream factor that is both necessary and sufficient for mediating the enhancement of MHV-68 lytic replication by Tpl2. In addition, Tpl2 stimulates the promoter activities of key viral lytic genes, including RTA and ORF57, in an AP-1-dependent manner. We identified an AP-1-responsive element on the MHV-68 RTA promoter as the cis element mediating the upregulation of RTA promoter activity by Tpl2. MHV-68 lytic infection upregulates Fos expression, AP-1 activity, and RTA promoter activity in a Tpl2-dependent manner. We constructed a mutant MHV-68 virus that abolished this AP-1-responsive element. This mutant virus exhibited attenuated lytic replication kinetics, indicative of a critical role of this AP-1-responsive element during lytic replication. Moreover, Tpl2 knockdown inhibited the lytic replication of wild-type MHV-68 (MHV-68-WT) but not that of the MHV-68 mutant virus, indicating that endogenous Tpl2 promotes efficient virus lytic replication through AP-1-dependent upregulation of RTA expression. In summary, through tandem functional screens, we identified the Tpl2/AP-1 signaling transduction pathway as a positive regulator of MHV-68 lytic replication.Gammaherpesviruses are a family of large, membrane-enveloped, double-stranded DNA viruses, including Epstein-Barr virus (EBV), Kaposi''s sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri, and murine gammaherpesvirus 68 (MHV-68 or γHV-68). Human gammaherpesviruses EBV and KSHV are associated with a number of malignancies. EBV is associated with Burkitt''s lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin''s disease (14). KSHV is the etiological agent of three types of human tumors: Kaposi''s sarcoma (KS), primary effusion lymphoma (PEL), and a plasmablastic variant of multicentric Castleman disease (MCD) (7, 8, 17).Gammaherpesviruses, like other herpesviruses, have two phases in their life cycles, i.e., latency and lytic replication. Although latent infection is essential for gammaherpesvirus-associated tumorigenesis, lytic reactivation and lytic replication are also believed to play important roles in the persistent infection by gammaherpesviruses and their associated pathogeneses (15). Upon de novo lytic infection or reactivation from latency, a cascade of viral lytic genes is expressed. Herpesvirus lytic genes are classified as immediate early (IE), early (E), and late (L) (28). Viral IE transcription factors, including ZTA and RTA in EBV (13, 19, 38) and RTA in KSHV (38, 52) and MHV-68 (62), control the transcription of other viral lytic genes and are therefore important for initiating the whole lytic cascade. Early genes encode proteins important for viral genomic DNA replication, which is required for the expression of late genes, many of which encode structural proteins (27, 40). Virus assembly and egress complete the virus lytic replication cycle.Many questions still remain unanswered regarding the regulation of gammaherpesvirus lytic replication, one critical aspect of which is the roles that cellular genes play. As with all other viruses, gammaherpesviruses rely on cellular machineries for replication and propagation. For example, several cellular genes have been shown to mediate KSHV entry in different types of cells (1, 31, 46). Other cellular genes, such as those for topoisomerase I, topoisomerase II, and poly(ADP-ribose) polymerase 1 (PARP-1), were shown to function during KSHV lytic DNA replication (58).Gammaherpesviruses have a complex life cycle and therefore critically depend on their ability to sense specific cellular contexts to undergo different phases of their life cycle accordingly. Thus, cellular factors may play an even bigger role in influencing the fate of gammaherpesviruses than they do for other viruses that have simpler life cycles. Because of the critical role that RTA and/or ZTA plays in initiating the whole lytic replication cascade, a number of studies have focused on cellular factors that regulate RTA and ZTA. For example, several cellular factors, such as NF-κB, PARP-1, and KSHV-RTA-binding protein (K-RBP), were shown to inhibit gammaherpesvirus lytic replication through inhibiting RTA expression or activity (4, 24, 64), whereas RBP-Jκ (CSL or CBF1), CREB-binding protein (CBP), SWI/SNF, and CCAAT/enhancer-binding protein-α (C/EBPα) have been found to upregulate RTA''s transcriptional activity and lytic replication (22, 23, 35, 36, 57).Despite the progress made, there is little doubt that the majority of the cellular genes that regulate gammaherpesvirus lytic replication have yet to be discovered. The identification and study of such cellular factors are hampered by the lack of a cell culture system that can support robust lytic replication of EBV or KSHV. MHV-68, which shares sequence homology with EBV and KSHV, is able to undergo efficient lytic replication in a number of common cell lines, including those of human origin, and therefore provides a system to effectively study gammaherpesvirus lytic replication in vitro and in vivo (44, 49, 51, 55).Kinases and transcription factors are critical cellular proteins that regulate many aspects of cell homeostasis, including cell survival, proliferation, differentiation, and metabolism. Therefore, gammaherpesviruses are likely to be regulated by kinases and transcription factors. Here, we utilized tandem functional genetic screens to identify cellular kinases and transcription factors regulating MHV-68 lytic replication, establishing the role of the Tpl2/AP-1 pathway in regulating MHV-68 lytic gene expression and lytic replication.