In vitro studies have established that the latency-associated nuclear antigen encoded by human Kaposi's sarcoma-associated herpesvirus and the related ORF73 gene product of herpesvirus saimiri interact with virus origins of replication to facilitate maintenance of episomal DNA. Such a function implies a critical role for ORF73 in the establishment and maintenance of latency in vivo. To determine the role of ORF73 in virus pathogenesis, the ORF73 gene product encoded by murine herpesvirus-68 (MHV-68) was disrupted by making an ORF73 deletion mutant, D73, and an independent ORF73 frameshift mutant, FS73. The effect of the mutations introduced in ORF73 on MHV-68 pathogenesis was analysed in vivo using a well-characterized murine model system. These studies have revealed that ORF73 is not required for efficient lytic replication either in vitro or in vivo. In contrast, a severe latency deficit is observed in splenocytes of animals infected with an ORF73 mutant, as assessed by infectious centre reactivation assay or by in situ hybridization detection of latent virus. Assessment of viral genome-positive cells in sorted splenocyte populations confirmed the absence of ORF73 mutant virus from splenic latency reservoirs, including germinal centre B cells. These data indicate a crucial role for ORF73 in the establishment of latency and for virus persistence in the host. INTRODUCTIONGammaherpesviruses characteristically establish lifelong latency in lymphoid cell populations and are commonly associated with malignant lymphoproliferative disorders. Critical for the maintenance of latent gammaherpesviral genomes in dividing cells are episome maintenance functions, which are required for the replication of viral genomes during mitosis and their accurate segregation into daughter cells . In the case of Epstein-Barr virus (EBV), the virus-encoded EBNA-1 protein facilitates episome maintenance via interaction with the origin of plasmid replication (Yates et al., 1984(Yates et al., , 2000Lupton & Levine, 1985), and in vitro studies have demonstrated that EBNA-1 is essential for the immortalization of B cells and the establishment of latency (Lee et al., 1999). Similarly, Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a multifunctional latencyassociated nuclear antigen (LANA-1) encoded by ORF73, which binds directly to the terminal repeats of the viral genome to mediate stable episome replication and segregation (Ballestas et al., 1999;Cotter & Robertson, 1999;Ballestas & Kaye, 2001;Grundhoff & Ganem, 2003). A similar function has also been demonstrated for the ORF73 gene product encoded by the related gamma-2 herpesvirus herpesvirus saimiri (HVS) (Smith et al., 2001;.From in vitro studies, it is clear that the trans-acting plasmid maintenance proteins encoded by gammaherpesviruses are essential for the stable maintenance of viral genomes during latency. However, the contribution of virus-encoded maintenance functions to virus persistence in vivo is not known. To directly address this question, we have focussed our studie...

A murine gammaherpesvirus (MHV-68) containing a deletion of the putative plasmid maintenance protein ORF73 exhibits a severe latency deficit. In this study the ability of an ORF73 deletion mutant (D73) to confer in vivo protection against subsequent challenge with wild-type virus has been examined. Vaccination studies have shown that D73 vaccination reduced latent infection of wild-type challenge virus to a level below the limit of detection. These results indicate that a live-attenuated gammaherpesvirus that cannot persist is an effective vaccine.