Project 1: KSHV reprograms replication and metabolic activities in hypoxia

Project #1 Abstract KSHV-associated cancers occur in hypoxic microenvironments. The effects of hypoxia on viral and host DNA replication in the context of KSHV infection are poorly understood. We have previously investigated the replication of KSHV in normoxia cell culture systems. We now propose an in-depth analysis of KSHV and infected host cell replication in the hypoxic environment. KSHV, like many members of the herpesvirus family undergoes both a latent and a lytic replication cycle. In cell culture systems, latency is maintained through synchronization of the viral genome replication with that of the host cell replication, which is cell cycle dependent. Therefore, most of the latent replication activities utilize the host machinery once per cell cycle. These latently infected cells are typically induced to lytic replication through treatment with chemical inducers and requires the expression of a number of viral encoded genes. However, little is known regarding replication of the virus genome in hypoxia, and the viral antigens needed to regulate the ongoing metabolic changes required for persistence of the viral genome through replication in infected cells that allow for propagation into new daughter cells. We will approach these questions through a number of aims geared towards understanding the changes in the cellular and viral replication proteins that occur in hypoxia compared to that seen in normoxia. We will examine the changes in cellular replication proteins at different phases of the cell cycle, and their post-translational modification. Their levels in hypoxia will be analyzed to determine the changes in the essential components of the replication machinery. In addition, we will look at their transcription to determine if these changes are due to changes in the overall transcription of the genes or if the changes are due to transcription modulation by HIF1α in hypoxia. The changes in metabolic enzymes that are induced in KSHV infected cells compared to KSHV negative cells in hypoxia will also be analyzed to determine if the changes are transcriptionally reprogrammed. We will determine the metabolic genes that are dysregulated on KSHV infection in hypoxia and if the replication genes are regulated by HIF1α, the master transcription regulator in hypoxia. We will determine the epigenetic changes that occur during hypoxia that will determine the expression of the replication genes. We will also perform these assays in 3D culture systems to mimic the associated pathologies. We will also generate shRNA knockdown or CRISPR knockout of selected replication genes, and Tet-regulated genes to determine the contribution of these proteins in KSHV replication in the hypoxic microenvironment. 14