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Gammaherpesvirus pathogenesis is dependent on the ability of these viruses to establish a lifelong latent infection and the ability to reactivate from latency. Immediate-early genes of theses viruses are thought to be critical regulators of lytic replication and reactivation from latency. The gene 50-encoded Rta is the only immediate-early gene product that appears to be conserved among all characterized gammaherpesviruses. Previous studies have demonstrated that, in Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus, and gammaherpesvirus 68 (␥HV68, also referred to as murine gammaherpesvirus 68), ectopic expression of Rta in latently infected cell lines can lead to induction of the viral cycle. Recently, studies employing null mutants of EBV have provided a formal demonstration that both Rta and the BZLF1 gene product, Zta, the two EBV immediate-early gene products, are essential for EBV replication. Here we generate and characterize a gene 50-null mutant ␥HV68 and demonstrate that the gene 50 product Rta is essential for virus replication. Providing ␥HV68 Rta in trans was sufficient to restore replication of the gene 50-null virus. Notably, Rta expressed from the spliced form of the gene 50 transcript was sufficient to complement growth of the gene 50-null virus. In addition, we provide evidence that loss of Rta expression leads to a complete defect in viral DNA replication and a significant defect in late antigen expression. This work lays the foundation for characterizing the role of Rta in ␥HV68 chronic infection of mice.

The Rta homolog encoded by murine gammaherpesvirus 68 (gammaHV68) gene 50 is essential for virus replication and is capable of driving virus reactivation from the S11 latently infected B lymphoma cell line. Here we characterize Rta activation of gammaHV68 gene 57, which is abundantly transcribed during the early phase of virus replication. Infection of murine fibroblasts with an Rta null virus demonstrated that transcription of gene 57 is dependent on Rta expression. Analysis of the gene 57 promoter identified 2 distinct regions that are Rta responsive, either in the context of the gene 57 promoter or when cloned upstream of a heterologous promoter. Sequence analysis of these regions revealed homology to known Rta-responsive cis-elements in the closely related Kaposi's sarcoma-associated viral (KSHV) genome. In addition, two candidate binding sites for the cellular transcription factor RBP-Jkappa/CBF1 were also identified in one of the Rta-responsive regions, which may play a role in mediating Rta transactivation similar to that observed in some KSHV Rta-responsive genes. Overall, analysis of the gammaHV68 gene 57 promoter suggests that mechanisms of Rta activation are conserved among gamma2-herpesviruses.