Kaposi's sarcoma-associated herpesvirus (KSHV) infection manifests in AIDS patients and leads to several lymphoproliferative diseases as well as Kaposi's sarcoma. KSHV can establish a persistent infection, and high-titer virus can be produced in the oral cavity and transmitted via saliva. In the past decade, studies of KSHV replication have been primarily conducted in endothelial cells, B-cells, and kidney epithelial cells, through which a general framework of KSHV replication has been established. Through recent efforts, we now have oral epithelial cells, in which KSHV replicates and produces high titers of viral particles without exogenous stimulation. This tissue culture system will allow us t establish knowledge about the relationship between the oral biology and replication of KSHV. With this application, we would like to focus on an important question; why does KSHV replication occur in a tissue specific manner? As thoughtfully presented by recent reviews, tissue-specific cellular gene expression is regulated by the formation of active chromatin hubs (ACHs) at enhancer regions of the genome, where many tissue specific-gene promoters are brought into proximity. Protein concentration of many nuclear factors is lower than the dissociation constant of protein-protein or protein-DNA interactions. Chromatin-associated proteins find their binding sites by diffusion and three-dimensional scanning of nuclear space, while the formation of productive transcription complexes on DNA is expected to take place via a stop and go mechanism. Transcriptional factors bind to their binding sites and will drop off if another factor does not bind within certain occupancy cycling time. This makes protein concentration a very sensitive limiting factor in the efficiency of transcription. Accordingly, spaial clustering of their cognate binding sites by assembling at ACH is necessary to effectively increase the concentration of transcriptional factors essential for the high transcriptional rate o the genes. A high concentration of factors ensures efficient binding to the DNA templates and dissociating factors could rapidly re-associate to the same or neighboring sequences, which significantly facilitates RNA polymerase II recycling and high-frequency reutilization of stable pre-initiation complexes; this mechanism enables effective transcription. Tissue-specific replication of KSHV, as well as robust and synchronous gene expression, led us to hypothesize that the KSHV genome encodes ACHs that control viral promoter activation. The viral ACH may be highly activated only in specific cells such as oral epithelial cells; this mechanism allows KSHV to replicate in the oral cavity. Importantly, our studies indicated that KSHV indeed forms ACH for effective viral gene expression. In this proposal, we will study viral gene expression by focusing on KSHV genomic enhancer regions. We will examine how a KSHV enhancer is differentially regulated between oral epithelial cells and cells that are non-permissive to lytic replication. We think that regulation of KSHV ACHs associates with tissue-specific viral replication. Interactions between a viral genomic enhancer and cellular promoters will also be examined. This study should provide fundamental knowledge regarding KSHV replication in the oral cavity.

Public Health Relevance

High titers of Kaposi's sarcoma-associated herpesvirus (KSHV) are shed into saliva. Accordingly, knowledge about KSHV replication in oral epithelial cells holds the key to our understanding of KSHV transmission. However, due to the lack of tissue culture models, this remains as a critically important, but under-explored area. The present proposal is specifically designed to reveal molecular mechanisms of KSHV replication in oral epithelial cells with newly adopted tissue culture methods. We will focus on regulation and contents of an active chromatin hub at a KSHV genomic enhancer region.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Special Emphasis Panel (ZDE1-CF (10))
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Lunsford, Dwayne
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University of California Davis
Schools of Medicine
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Campbell, Mel; Watanabe, Tadashi; Nakano, Kazushi et al. (2018) KSHV episomes reveal dynamic chromatin loop formation with domain-specific gene regulation. Nat Commun 9:49
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