Epigenetic Reprogramming of Kaposi’s Sarcoma-Associated Herpesvirus during Hypoxic Reactivation

SIMPLE SUMMARY: Epigenetic reprogramming of the KSHV genome plays a central role in lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV). Hypoxia, a common phenotype of cancer cells, exerts a negative effect on DNA replication. Interestingly, KSHV is known to bypass this repression and undergo lytic replication. Therefore, we investigated epigenetic reprogramming of the KSHV genome under hypoxic conditions. The KSHV genome was enriched with both transcriptional activator and repressor modifications of histones due to the upregulated expression of the modifying enzymes in KSHV-positive cells grown in hypoxia. KSHV-encoded antigens were responsible for this increase in modified histone proteins. The differential enrichment of KSHV-encoded LANA and RTA in hypoxia were suggestive of their role in transcriptional regulation. Furthermore, analysis investigating enrichment of DNA polymerase 1α on the KSHV genome in conjunction with single molecule analysis of replicated DNA identified specific regions of the KSHV genome that may be critical for DNA replication in hypoxia. ABSTRACT: The biphasic life cycle (latent and lytic) of Kaposi’s sarcoma-associated Herpesvirus (KSHV) is regulated by epigenetic modification of its genome and its associated histone proteins. The temporal events driving epigenetic reprogramming of the KSHV genome on initial infection to establish latency has been well studied, but the reversal of these epigenetic changes during lytic replication, especially under physiological conditions such as hypoxia, has not been explored. In this study, we investigated epigenetic reprogramming of the KSHV genome during hypoxic reactivation. Hypoxia induced extensive enrichment of both transcriptional activators and repressors on the KSHV genome through H3K4Me3, H3K9Me3, and H3K27Me3, as well as histone acetylation (H3Ac) modifications. In contrast to uniform quantitative enrichment with modified histones, a distinct pattern of RTA and LANA enrichment was observed on the KSHV genome. The enrichment of modified histone proteins was due to their overall higher expression levels, which was exclusively seen in KSHV-positive cells. Multiple KSHV-encoded factors such as LANA, RTA, and vGPCR are involved in the upregulation of these modified histones. Analysis of ChIP-sequencing for the initiator DNA polymerase (DNAPol1α) combined with single molecule analysis of replicated DNA (SMARD) demonstrated the involvement of specific KSHV genomic regions that initiate replication in hypoxia.