Directed and global mutagenesis strategies for the identification of gammaHV68 pathogenesis determinants

Murine gammaherpesvirus 68 (γHV68) infection of mice has been used by our lab and others as a small animal model of gammaherpesvirus infection. Unlike other members of the gammaherpesvirus family, γHV68 presents the opportunity to dissect the role of viral gene products in both lytic and latent infection. Using a directed mutagenesis approach, we have determined the role of the viral IL-8 receptor homolog (v-GPCR) and latency-associated nuclear antigen (LANA) in viral infection in vitro and in vivo. Mutant viruses lacking the v-GPCR were found to replicate as well as wild type virus in vitro and in vivo, but reactivated from latency with decreased efficiency from cells of the peritoneum following intraperitoneal infection. Mutant viruses containing disruption of the LANA open reading frame (LANA.STOP) replicated as well as wild type virus in vitro, but replicated less efficiently than wild type virus during lytic infection in both lungs and spleens. LANA.STOP virus was incapable of reactivating from latently infected splenocytes following intranasal infection. The lack of reactivating correlated with a decrease in the number of viral genomes found in the spleen during latency. While the lytic defect of the LANA.STOP virus could be partially overcome by increasing the inoculating dose, the latency defect was absolute, regardless of dose. These results are in agreement with the predicted role of the LANA protein in the viral lifecycle, as closely related proteins in other gammaherpesviruses have been found to be required for episomal maintenance of the viral genome during latent infection.; With the cloning of the γHV68 genome into a bacterial artificial chromosome (γHV68 BAC), global strategies of mutagenesis have also become feasible. We have adapted the signature-tagged mutagenesis (STM) system to generate pools of virus mutants. Since each transposon contains a unique sequence identifier, this allows detection of individual mutant viruses in a large pool of viruses. Using this system we have been able to rapidly determine the role in lytic replication of 31 open reading frames encoded by the virus. Several intergenic regions were also identified and characterized. We are currently applying the same approach to identify viral genes required for reactivation from latency in vivo, and have identified several promising candidate latency-associated genes.