KSHV-encoded LANA protects the cellular replication machinery from hypoxia induced degradation

Kaposi's sarcoma associated herpesvirus (KSHV), like all herpesviruses maintains lifelong persistence with its host genome in latently infected cells with only a small fraction of cells showing signatures of productive lytic replication. Modulation of cellular signaling pathways by KSHV-encoded latent antigens, and microRNAs, as well as some level of spontaneous reactivation are important requirements for establishment of viral-associated diseases. Hypoxia, a prominent characteristic of the microenvironment of cancers, can exert specific effects on cell cycle control, and DNA replication through HIF1 alpha-dependent pathways. Furthermore, hypoxia can induce lytic replication of KSHV. The mechanism by which KSHV-encoded RNAs and antigens regulate cellular and viral replication in the hypoxic microenvironment has yet to be fully elucidated. We investigated replication-associated events in the isogenic background of KSHV positive and negative cells grown under normoxic or hypoxic conditions and discovered an indispensable role of KSHV for sustained cellular and viral replication, through protection of critical components of the replication machinery from degradation at different stages of the process. These include proteins involved in origin recognition, pre-initiation, initiation and elongation of replicating genomes. Our results demonstrate that KSHV-encoded LANA inhibits hypoxia-mediated degradation of these proteins to sustain continued replication of both host and KSHV DNA. The present study provides a new dimension to our understanding of the role of KSHV in survival and growth of viral infected cells growing under hypoxic conditions and suggests potential new strategies for targeted treatment of KSHV-associated cancer. Author summary Hypoxia induces cell cycle arrest and DNA replication to minimize energy and macromolecular demands on the ATP stores of cells in this microenvironment. A select set of proteins functions as transcriptional activators in hypoxia. However, transcriptional and translational pathways are negatively regulated in response to hypoxia. This preserves ATP until the cell encounters more favorable conditions. In contrast, the genome of cancer cells replicates spontaneously under hypoxic conditions, and KSHV undergoes enhanced lytic replication. This unique feature by which KSHV genome is reactivated to induce lytic replication is important to elucidate the molecular mechanism by which cells can bypass hypoxia-mediated arrest of DNA replication in cancer cells. Here we provide data which shows that KSHV can manipulate the DNA replication machinery to support replication in hypoxia. We observed that KSHV can stabilize proteins involved in the pre-initiation, initiation and elongation steps of DNA replication. Specifically, KSHV-encoded LANA was responsible for this stabilization, and maintenance of endogenous HIF1 alpha levels was required for stabilization of these proteins in hypoxia. Expression of LANA in KSHV negative cells confers protection of these replication proteins from hypoxia-dependent degradation, and knock-down of LANA or HIF1 alpha showed a dramatic reduction in KSHV-dependent stabilization of replication-associated proteins in hypoxia. These data suggest a role for KSHV-encoded LANA in replication of infected cells, and provides a mechanism for sustained replication of both cellular and viral DNA in hypoxia.