Analysis of the role of the gammaherpesvirus 68 immediate early gene 50 for the lytic cycle

Gammaherpesviruses are lymphotropic viruses that establish a lifelong infection of the host and are associated with cellular transformation and tumor formation in immunosuppressed hosts. Studies of the important human pathogens, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), have been hampered by their strict species specificity. Murine gammaherpesvirus 68 (gammaHV68, MHV68) is a natural pathogen of the bank vole and field mouse. The ability of gammaHV68 to infect laboratory strains of mice and to generate viral recombinants, has allowed the integration of host and viral genetics studies to study gammaherpesvirus pathogenesis. Gene 50 is an immediate early gene, which is thought to be a critical regulator of the viral lytic cascade. To define the genetic requirement for gene 50 for lytic replication, we generated a null gene 50 recombinant gammaHV68. Analysis of the role of gene 50 during lytic replication in vitro revealed the following: (i) the gene 50 null mutant is incapable of virion production in fibroblast cells, (ii) providing gene 50 in trans is sufficient to restore normal levels of virus production of the gene 50 null virus, (iii) the spliced gene 50 form is sufficient to restore virus production of the gene 50 null virus, (iv) the gene 50 null virus has a complete defect in DNA replication, (v) the gene 50 null virus has a severe defect in viral gene expression, as measured by viral transcript and protein levels. Thus, we conclude that gene 50 is essential for lytic virus replication in fibroblast cells. Additionally, we have analyzed the ability of gene 50 to act as a transcriptional activator of the viral gene 57 promoter. These studies have revealed that gene 50 can transactivate the gene 57 promoter to very high levels in the absence of other viral factors. Furthermore, we have defined cis-elements within the gene 57 promoter that are necessary and sufficient for gene 50 transactivation. In summary, we have generated the first gammaHV68 recombinant in an essential gene, and have provided genetic evidence for the requirement for gene 50 in vitro. Further studies using infection of laboratory mice with gene 50 null virus will provide valuable information on the role of the immediate early gene 50 for in vivo pathogenesis and the requirement of lytic replication and reactivation from latency for the establishment and maintenance of latency. Additionally, the gene 50 null virus might aid in the development of a gammaHV68 tumorigenesis model in mice.