Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated virus (KSHV) are human oncogenic herpesviruses that cause a wide variety of lymphomas and malignancies of epithelial and endothelial origin. Lytic reactivation from latent infection and expression of lytic cycle genes ar important in pathogenesis of both viruses. While viral factors important for replication have been extensively studied, major gaps exist in our knowledge of host factors that control KSHV and EBV reactivation. We have shown that cohesin and CTCF, two cellular proteins that bind to chromatin and modulate transcription, potently repress KSHV lytic replication and virus production. Cohesin is removed from the KSHV genome when it begins to replicate and depletion of either CTCF or cohesin leads to widespread de-repression of KSHV gene transcription. CTCF and cohesin are chromosome remodelers that mediate DNA looping and three-dimensional changes in conformation. Based on these known functions of CTCF and cohesin, we hypothesize that they impose topological constraints on KSHV and EBV circular latent genomes that prevent efficient transcription and DNA replication until they are removed. In contrast, a select subset of KSHV genes is poorly expressed when cohesin or CTCF is depleted. Many of these genes have promoters with paused RNA pol II, a characteristic of promoters that are positively regulated by cohesin. This cluster of KSHV genes also encodes proteins that have unique immunoevasive and growth promoting functions. Several of them are immunomodulatory and act as viral cytokines that blunt the host antiviral response. Others downregulate surface molecules on KSHV-infected cells rendering them less prone to cell-mediated immune recognition. These proteins are rapidly expressed early during primary infection. We hypothesize that KSHV utilizes cohesin and CTCF to efficiently express these particular genes early in infection and reactivation. We first propose to expand our studies to EBV, thus establishing these cohesin/CTCF regulatory mechanisms as a general paradigm for host control of gammaherpes virus reactivation. These mechanisms will be confirmed and characterized in unique EBV-infected cell lines from patients with mutations in the cohesin pathway. We will determine the molecular mechanisms by which cohesin and CTCF inhibit KSHV transcription and the extent to which they directly inhibit the physical process of viral DNA replication. We will investigate the molecular pathways by which cohesin is removed from latent viral genomes to permit lytic replication - systematically studying the role of proteins that modif cohesin and load and release it from human chromosomes. Compounds that inhibit removal or enhance loading of cohesin will be employed to validate these pathways as therapeutic targets. Finally, we will define the contribution of cohesin and CTCF to KSHV's ability to express immune evasion genes. We will determine the extent to which depletion of CTCF and cohesin impairs KSHV immune modulator expression and function. We will confirm these findings by constructing and characterizing KSHV mutant viruses which cannot bind cohesin and CTCF at the immunonomodulatory gene locus and the origin of replication. This multi-faceted approach to investigating host control of KSHV and EBV should yield many novel insights into the host-pathogen balance in the broad areas of viral reactivation and immune evasion.
Epstein-Barr virus (EBV), and a related virus, Kaposi's sarcoma-associated herpesvirus (KSHV), common viruses that infect humans, contribute to the development of lymphoma and other cancers of blood cells. Both viruses establish lifelong infections in humans and reactivate intermittently. Understanding how the host cell controls these viruses is critical for devising new treatments. Certain lymphomas are increasing in the elderly, and as the population of veterans ages, this becomes an increasingly important medical problem. Prognosis is also poorer among the elderly. In addition, patients who receive organ transplants are particularly susceptible to cancers associated with EBV and KSHV. Transplant recipients are another unique component of the veteran patient population for whom innovative treatments are needed. This proposal builds on new findings in the area of virus replication to increase our understanding of how virus reactivation might be controlled.
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| Baglio, S Rubina; van Eijndhoven, Monique A J; Koppers-Lalic, Danijela et al. (2016) Sensing of latent EBV infection through exosomal transfer of 5'pppRNA. Proc Natl Acad Sci U S A 113:E587-96 |
| Li, Da-Jiang; Verma, Dinesh; Mosbruger, Tim et al. (2014) CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription. PLoS Pathog 10:e1003880 |
