Small and large-scale gene regulation in Kaposi's Sarcoma-associated Herpesvirus

Viruses depend upon host cells for genome replication and propagation. Viral genomes must be labile in order to exist inertly within infectious extracellular virions and also dynamically as self-replicating entities within infected host cells. Herpesviruses are large double-stranded DNA viruses which can establish persistent lifelong infections in vertebrates. Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the most recently discovered human herpesvirus. KSHV infects endothelial and B cells which can lead to three human cancers: Kaposi's Sarcoma, Multicentric Castleman's Disease, and Primary Effusion Lymphoma. KSHV alternates between a latent replication phase, during which viral gene expression is restricted and no progeny virions are produced; and a lytic replication phase, which entails robust viral transcription and genome replication as well as host cell lysis and the release of new infectious daughter virions. Both replication patterns are implicated in KSHV pathology, however nearly all infected cells, including those within tumors, display latent infection.;The balance between lytic and latent KSHV infection is sensitive and complex. Viral latency is characterized by the expression of a multifunctional viral protein called the KSHV Latency-Associated Nuclear Antigen (LANA). LANA maintains the viral genome and modulates gene expression to ensure persistent latent infection. KSHV latency can be reversed upon appropriate stimuli in a process called reactivation. Reactivation of KSHV is coordinated through a virally-encoded transcription factor called RTA. In this dissertation I have characterized a novel small-scale regulatory mechanism by which RTA acts to selectively synthesize either latent (i.e. LANA) or lytic transcripts from within the major KSHV latency locus. This selective bidirectional gene expression results in competitive RTA-mediated transactivation, the potency of which varies in proportion to RTA concentration. The resulting transcriptional circuit may have a role in the early establishment of KSHV latency, in attenuating and fine-tuning viral transcription, or both. In a second study presented here I have investigated how nucleosome depletion, or "open chromatin" is dispersed in latent KSHV genomes. This effort integrates our own work with that of others in the KSHV community, and collectively the data indicate that open chromatin in KSHV may be programmed for latency; with select latent regions accessible and most others insulated by CTCF and Cohesin. CTCF-free and CTCF-enriched subsets of latent open chromatin occur proximal to mapped H3-ac/H3K4-me3 modifications on the KSHV genome. Many of these regions also contain identified RTA Recognition Elements (RRE's) and RNA polymerase II occupancy, including the viral lytic replication origins. This pattern of nucleosome depletion is mostly shared among latent episomes in both B and endothelial cells, implying a common latent open chromatin landscape in the viral genome. These studies of small-scale bidirectional gene induction and of large-scale latent nucleosome depletion in KSHV are connected. This work extends our understanding of KSHV biology and could reveal novel targets of therapeutic intervention.