Rolf Renne - US grants

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) also called Human herpes virus type 8 (HHV-8) is associated with Kaposis's sarcoma (KS), and two lymphoproliferative diseases: Primary effusion lymphomas (PEL) and a subset of Multicentric Castlemen's disease (MCD). Common to these malignancies is that the majority of tumor cells are latently infected and express only a small number of viral genes. One of these genes, ORF73, encodes the latency-associated nuclear antigen (LANA). LANA is highly expressed in all KS tumor cells and plays an important role in the biology of KSHV. LANA is the only viral protein required for latent DNA replication and genome maintenance during latency. In addition, LANA interacts with a variety of cellular proteins including the tumor suppressors p53 and Rb thereby modulating gene expression in latently infected cells. To analyze LANA's ability to modulate cellular gene expression, we performed gene expression profiling and found that LANA regulates a number of genes in the Rb/E2F pathway which, as a consequence can protect lymphoid cells from p161NK4a-induced cell cycle arrest. To study LANA's role in DNA replication we performed a detailed biochemical analysis of its DNA binding specificity and activity. We identified two binding sites within the terminal repeat of the KSHV genome and demonstrated that both sites contribute to the ability of LANA to support replication of TR-containing plasmids. We hypothesize that LANA and/or cis-regulatory sequences within TR interact with cellular proteins that facilitate DNA replication. To address this hypothesis we propose the following three aims that are focused on deciphering mechanisms by which LANA supports DNA replication: 1) Mapping cis-regulatory sequences of the putative origin of replication within TR 2) Mapping trans-requirements for DNA binding and LANA-dependent DNA replication 3) Identification of cellular proteins that interact with LANA-C and/or a minimal origin of replication.

The goal of the Molecular Virology and Gene Expression (MVGE) core is to provide technical support, training, and instrumentation for techniques that can be utilized by a broad group of CFAR investigators whose research focus is on molecular virology. The establishment of this new core is a direct response of our CFAR leadership to a CFAR-wide survey that demonstrated the need for support of investigators performing AIDS-related basic science and translational research projects. Based on the survey results, the MVGE Core will provide technical support and training for: 1. Recombinant protein expression and purification in prokaryotic and eukaryotic systems 2. Retrovirus and Adenovirus vector-based gene deliver techniques 3. Cost-effective access to and training in CFAR sponsored technologies such as real-time PCR During the core implementation phase we will first focus on support services for protein expression and purification utilizing prokaryotic and eukaryotic systems. These services will be offered at different levels ranging from training of CFAR lab members to providing full service. After successfully establishing these protein expression services, the next goal is to provide support for gene delivery systems such as recombinant Retrovirus and Adenovirus vectors that are commonly used in applications where transfection is not an option. These techniques will be supported by providing individualized and or small group training and access to key reagents with the goal to significantly lower time and effort for laboratories to establish these techniques in their research programs. Additionally, the MVGE core will provide training on CFAR equipment. We will ensure easy access and cost-efficient usage of CFAR-sponsored equipment to all CFAR members and, based on capacity, to other CWRU/UH research groups by (a) organizing a discounted reagent program and (b) by an outreach program designed to maximize core utilization.

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a plasmablastic variant of multicentric Castleman's disease (MCD). Common to these malignancies is that a majority of cells in these lesions are latently infected and express only a small subset of viral genes. One of these genes, the latency-associated nuclear antigen (LANA) has consistently shown to be expressed in all KSHV -associated malignancies. LANA is a multifunctional nuclear protein that interacts with a variety of host cellular proteins including- transcriptional modulators such as mSin3 and the tumor suppressors p53 and RB, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA genome during latency in-dividing cells. Ballestas, et.al., demonstrated that LANA expressing cells efficiently maintain plasmids containing terminal repeat (TR) over many cell divisions. This suggested that the latent origin of replication is located within TR and that interaction of LANA and TR sequences are crucial for DNA replication. By performing detailed mapping and footprinting studies, we have identified two LANA-binding sites within TR that are bound by LANA in a cooperative fashion. Furthermore, in congruence with data from other laboratories, we have shown that LANA is required for supporting DNA replication of TR-containing plasmids. Based on the fact that LANA is required not only for long-term maintenance but also for DNA replication, we hypothesize that LANA is an attractive target for the development of antiviral compounds. Such potential drugs would specifically act on latently infected cells and conceivably, could also prevent the establishment of latency in de novo infected cells. To directly test our hypothesis, we propose to develop a fluorescence anisotropy-based high-throughput screening assay (HTS) to identify small molecule inhibitors of LANA function. We further propose to develop cell-based assays in order to investigate the ability of candidate compounds to interfere with DNA replication and long-term maintenance.

DESCRIPTION (provided by applicant): MicroRNAs are small non-coding regulatory RNA molecules that bind to 3'UTRs of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by DNA viruses. The human ?-herpesvirus Epstein-Barr Virus has been shown to encode miRNAs which potentially regulate both viral and cellular genes. To determine whether Kaposi's Sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV positive primary effusion lymphoma derived cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align to KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-associated region of KSHV (Samols et al., Journal of Virology 2005). While our work was in review, two other laboratories reported the identification of KSHV-encoded miRNAs. Hence, these data suggest that virally-encoded miRNAs represent a novel mechanism by which KSHV may regulate viral and/or cellular gene expression during both latent and lytic KSHV replication. In human cells, over 450 miRNAs have been identified. Targets and functions of only a few miRNAs have been experimentally determined thus far, yet some miRNAs such as human hsa-miR-14 and hsa-miR-181 regulate fundamental biological processes like apoptosis, cell proliferation, and hematopoiesis and very recently have been implicated in tumorigenesis. Based on our preliminary results we hypothesize that miRNAs play an important role in the KSHV life cycle and may contribute to KSHV pathogenesis. To directly address this hypothesis we propose the following specific aims: SA1: Analysis of KSHV miRNA expression during latent and lytic replication in cultured cells and tissues samples of different origins. SA2: Identification of cellular and viral targets regulated by KSHV-encoded miRNAs. SA3: Evaluate the role of miRNA expression in the context of the viral genome. In support of our hypothesis we provide new preliminary data on identifying cellular miRNA targets in stably miRNA-expressing 293 cells (Samols et al., PLoS Pathogens, 2007, May 11). In summary, the identification of virally encoded miRNAs within the latency-associated region of KSHV is novel and very exciting as it suggests an entirely new level of regulating gene expression by which KSHV can potentially reprogram the host cell environment.

DESCRIPTION (provided by applicant): This meeting, which has been held annually since 1998, is the premier forum for the presentation and exchange of advances in KSHV and related agents. Special emphasis has always been on the presentation and discussion of unpublished new data with the goal to facilitate the dissemination and exchange of new research findings, ideas, and developments among an international group of scientists representing basic, translational, and clinical research who cover a broad spectrum of disciplines and focus on KSHV and related agents. The long-term goal of these studies is to enhance our understanding on how KSHV and related agents contribute to pathogenesis and to integrate this new knowledge from different research fields into the identification and development of new treatment, prevention, and vaccination strategies to alleviate KSHV- associated diseases. Public Health Relevance: This project provides partial funding for a meeting, which has been held annually since 1998, that is the premier forum for the presentation and exchange of advances in KSHV and related agents.

DESCRIPTION (provided by applicant): The aim of the proposed Biodefense and Emerging Infectious Diseases (BEID) training program is to produce independent investigators capable of sustaining productive research programs working with class A - C agents or agents of emerging diseases. This begins at the predoctoral level with broad, rigorous research training in the biology of infectious agents and the mechanisms by which these agents cause disease in animal models. Key strengths of the proposed training program include: 1) strong institutional intellectual and fiscal support for biodefense training from the central administration of the University of Florida (UF) plus additional, independent support from the three component colleges (Medicine, Dentistry and Veterinary Medicine);2) established and recognized faculty with productive research programs in fundamental areas of microbial pathogenesis and animal models of human disease;and 3) a track record of training students who are currently conducting research in the areas of biodefense and emerging pathogens. The program comprises 11 faculty preceptors (bacteriologists, virologists, and parasitologists) from 5 departments within 3 colleges. The program faculty includes clinician scientists, providing integration of animal models as applied to human infectious disease. Predoctoral trainees will be recruited and initially trained in collaboration with the UF Interdisciplinary Program (IDP) in Biomedical Sciences. For BEID trainees, specialized training will be tailored to the scientific, legal, and regulatory aspects of working with Class A, B, and C agents and emerging disease pathogens. Predoctoral training encompasses approximately 5 years, including a first year of support from UF, an average of 2 years of support from the training grant and the remainder of support from the mentor's research grant or other external sources. The focus of the program will be to augment existing strengths at UF and provide trainees with additional background and skill sets necessary to conduct research directed towards biodefense and emerging diseases.

DESCRIPTION (provided by applicant): Consistent with the ability of herpesviruses to establish persistent and latent infections, about 25% of all herpesvirus genes modulate and/or abrogate host cellular responses to viral infection. Recently, 140 microRNAs have been identified in a-, b-, and g-herpesvirus genomes which for CMV and KSHV also target host immune functions. MicroRNAs are short 22 + 3 nt RNAs that post-transcriptionally regulate gene expression by binding to 3'UTRs of mRNAs and inducing translational silencing or RNA degradation. To date, only a few genes have been experimentally proven to be miRNA targets. From these limited studies it is clear that viral miRNAs regulate fundamental cellular processes including innate and adaptive immunity, angiogenesis, and apoptosis, and key steps in the herpesvirus life cycle, latency and the switch from latent to lytic replication. Modulation of these processes by microRNAs is likely to affect host/virus interactions and thereby directly contribute to viral pathogenesis. In addition, understanding how miRNAs target innate and adaptive immune function will be critically important for herpesvirus vaccine development. To study the role of human herpesvirus-encoded miRNAs, we propose to establish a core laboratory with the mission to systematically generate a library of herpesvirus genomes that carry single and multiple miRNA mutations. To eliminate the possibility of secondary mutations, recombinant genomes will be re-sequenced using our well established massively parallel sequencing facility. This concerted effort will create highly valuable reagents that will enable studies on how miRNAs function in the context of viral infection, and where appropriate systems exist, in the context of animal models. Importantly, after quality control, each mutant virus will be freely shared with the research community. Creating a core laboratory for this specialized task will have high impact and significantly increase research output in this novel and highly significant field of investigation. Furthermore, in the future, such a core laboratory can be expanded towards targeted mutagenesis of additional genes that can be requested on a for fee basis by investigators at any US research site. PUBLIC HEALTH RELEVANCE: Recently, a novel class of non-coding RNAs (microRNAs) has been identified in human viruses associated with cancer. First insights into their function suggest important roles in immune evasion and pathogenesis like their human counterparts which are involved in diseases including many cancers. This proposal aims to establish a service laboratory that would create viruses with specific mutations in these microRNA genes, a task which is laborious and technically challenging. The newly created laboratory, which will employ five scientists, will then provide these valuable reagents to all investigators in the field. As a result, this project would dramatically accelerate progress towards closing this knowledge gap, which is highly significant for human disease.

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV), also called Human herpesvirus type 8, is associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and a subset of Multicentric Castleman's disease (MCD) (1-3). LANA is a multifunctional protein, which regulates host and viral gene expression and is the only viral protein required for latent DNA replication and episome segregation during latency. Many mechanistic details about LANA's role, latent DNA replication, episome tethering and transcription have been revealed. However, the complex molecular events leading to the establishment of latency, which allow viral episomes to be stably maintained in an epigenetic configuration permissive to reactivation, are still poorly understood. We hypothesize that early events after infection are crucial to establish latency during which viral episomes are replicated and segregated in a LANA-dependent fashion. We found that LANA specifically interacts with SSRP1 a chromatin remodeling factor that is important for latent DNA replication. LANA interaction with SSRP and other known epigenetic modifiers suggest that LANA functions in shaping both the viral and host cellular epigenomes. In aim 1, we propose to further study SSRP1/LANA interaction and its possible role in chromatin remodeling. In aim 2, we propose to first generate a detailed genome-wide map of the KSHV epigenome in latently infected cells and then to follow the occurrence of these epigenetic modifications after de novo infection of endothelial cells. SA1: Determine the molecular mechanisms by which LANA supports latent DNA replication through interactions with chromatin remodeling factors. Based on our recent finding that LANA specifically interacts with SSRP1, a subunit of FACT, we will map LANA and SSRP1 interaction domains and determine whether this complex also contributes to LANA-dependent transcriptional regulation. In addition, we will utilize reconstituted histone chromatin preparations to determine whether LANA/SSRP1 complexes have nucleosome disassembly activity. We also identified several additional LANA/origin interacting cellular proteins and will determine whether they play a role in LANA-dependent DNA replication and latency. SA2: Study LANA's contribution to the epigenetic state of the viral genome and transcriptional regulation of both host and viral genes during the establishment of latency. Using chromatin immunoprecipitations (ChIPs) in combination with High Throughput Sequencing (HTS), we propose to study histone modifications, pol II status, and LANA occupancy in PEL cells and long-term infected SLK and TIVE cells with high resolution on a genome-wide scale. We also propose to perform a genome-wide Methyltransferase Accessibility Protocol for Individual Templates (MAP-IT) analysis, a novel technique which probes methylation status, chromatin accessibility, and nucleosome positioning on a single molecule basis when combined with HTS sequencing. After first characterizing the KSHV epigenome during latency in cells of lymphoid and endothelial origin, we propose to study how the latent KSHV epigenome is established after de novo infection of endothelial TIVE and SLK cells. In summary, this highly innovative approach is aimed at increasing our understanding of what constitutes a latent genome and how LANA contributes to the complex events leading to the establishment of latency, a central question to both the biology and pathogenesis of KSHV.

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs that are highly expressed in KS lesions and KSHV- associated lymphoproliferative malignancies. Ectopic expression and miRNA inhibition approaches, combined with gene expression profiling, have identified target mRNAs that are involved in the regulation of critical biological processes such as angiogenesis, apoptosis, endothelial cell differentiation, and immune surveillance. In addition, several miRNAs may regulate latency by targeting viral mRNAs. Preliminary analyses of the mRNAs targeted by KSHV miRNAs in PEL cells using HITS-CLIP technology to enrich RISC-associated RNAs have identified hundreds of new mRNA targets and confirmed previously identified targets. Moreover, our analysis showed that a high percentage of RISC complexes within PEL cells contain KSHV- encoded miRNAs. Based on these data, we hypothesize that the KSHV miRNAs cause significant and tissue-specific changes in gene expression that contribute to pathogenesis and tumorigenesis. To directly address this hypothesis we propose to 1) Comprehensively identify KSHV miRNA targets by HITS-CLIP in KSHV-infected PEL and endothelial cells; 2) Characterize in vitro phenotypes and their associated regulatory pathways with respect to viral replication, the establishment and maintenance of latency, cell cycle control and resistance to apoptosis by using KSHV mutants lacking single or multiple miRNAs. In summary, combining ribonomics approaches for miRNA target identification with viral genetics, and the ability to determine viral phenotypes in cultured cells, will provide a detailed analysis of KSHV-encoded miRNA functions and may lead to the development of antiviral/antitumor strategies.

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS) is the most common oral cancer in human immunodeficiency virus (HIV)-infected patients. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS, and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). AIDS-associated KS is highly frequent in the oral cavity which is also the major site for KSHV shedding via saliva. KSHV infected tumor cells are predominately latently infected. The viral encoded latency-associated nuclear antigen (LANA) is a multifunctional protein required for latency. Our preliminary data demonstrate that histone variant H3.3, which is frequently mutated in human malignancies and plays important roles in transcriptional regulation, occupies viral episomes at specific regions that correlate with LANA binding. We also demonstrate that LANA associates with Daxx and SSRP1, chaperone proteins responsible for the deposition of H3.3. We hypothesize that H3.3 deposition onto viral episomes is crucial for viral gene expression during latency, and furthermore that this process is mediated by LANA's interactions with H3.3 chaperones. Locus specific H3.3 incorporation may account for the tightly controlled gene expression pattern during latency and hence also affect the balance between latent and lytic replication, which may be crucial with respect to tumorigenesis and shedding in the oral cavity. Importantly, this study will not only utilize in vitro tissue cultre systems but for the first time propose a highly innovative approach to analyze complex chromatin architecture analysis in vivo using primary oral KS biopsies. To this end we provide preliminary data on our ability to detect endogenous histone H3.3 by both ChIP and IFA. The long-term goal of this interdisciplinary pilot project is to create proof of concept data that modulating histone variant deposition can be harnessed as a novel KSHV-specific therapeutic strategy.

Summary The Administrative Core supports productive interactions between the different projects and cores, and facilitates communication and data exchange between all associated personnel and the external advisory board (EAB). The Administrative Core will monitor the progress of each project, and the effective utilization of service core resources. Importantly, the core will provide fiscal management ensuring that all funds are utilized efficiently to support the Program Project's mission. A key core activity is to organize an annual review by the EAB, which will be done in the context of a Program Project retreat, where project leaders and trainees will report on scientific progress. This event will complement the bi-monthly project leader and IEC member meetings, and will foster integrated scientific interactions between scientists working in the project leaders' laboratories and core facilities. In addition, the core will facilitate training sessions enabling trainees at Tulane to receive hands-on training at UF in Recombinant Viral Genetics (Core C), and to provide hands-on bioinformatics training at Tulane (Core B) for UF trainees. We note that travel costs for these training-specific activities will be provided as institutional support by the University of Florida. Core A will support the timely publication of findings from the P01 components. Provision of Biostatistics services will be coordinated by Core A. Finally, the core will securely archive research data, share data between groups, and disseminate results with the scientific community after publication. To facilitate these goals, the core will create a Program Project-specific website in close coordination with Core B and Core C.

Summary The purpose of the Recombinant Virus Core C is to generate quality controlled recombinant herpesviruses for KSHV, EBV, and MHV68 (Projects 1, 2 and 3). The services of Core C will aid the overall goal of the program project to comprehensively analyze the role of herpesviral non-coding RNAs as well as studies on host cellular lncRNAs that are perturbed in response to viral infection. In addition to constructing recombinant viruses, Core C will perform genome-wide sequencing of viral mutants. Building upon years of experience in making a complete set of KSHV miRNA deletion mutants, our group has developed and implemented techniques for making bacmid-derived mutants in both KSHV and MHV68, and will readily extend this to EBV.

PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is the causative agent of AIDS malignancies like KS and primary effusion lymphomas (PEL). In recent years it became clear that pathogenic herpesviruses including EBV, KSHV, and MHV68 express numerous long non-coding RNAs (lncRNAs) many of which are in antisense orientation to protein coding transcripts. The function and structure of these RNAs is largely unknown. In addition, these viruses express microRNAs (miRNAs). While characterizing the KSHV miRNA targetome, we identified several hundred host cellular lncRNAs as putative miRNA targets. Furthermore, infection of endothelial cells with wtKSHV induced dramatic dys-regulation of host lncRNAs including the down-regulation of 533 lncRNAs. Of these 126 were rescued when cells were infected with a KSHV recombinant that lacked 10 of 12 KSHV miRNAs. Together these data strongly suggest that both KSHV encoded proteins and miRNAs contribute to dysregulation of host lncRNAs. Importantly, ten lncRNAs that are perturbed following KSHV infection, including HOTTIP, ANRIL, Meg3, and UCA1 are reported to be associated with human cancers. We demonstrate that up-regulation of UCA1 affects proliferation and migration of KSHV infected endothelial cells. Additionally, we provide experimental evidence that the anti-sense LANA transcript is bound by EZH2/PRC2 complexes and may contribute to the regulation of viral latency. These data suggest that viral lncRNAs are important regulators of viral gene expression and hence may be important for viral pathogenesis and/or tumorigenesis. To understand the role of lncRNAs in viral biology we propose to functionally study viral lncRNAs and to determine underlying mechanisms and phenotypical consequences of host lncRNA dysregulation in KS pathogenesis. Importantly, experimental findings observed in appropriate tissue culture models of lymphoid and endothelial origin, will be validated using clinical specimens from AIDS malignancies in collaboration with Dr. Chris Parsons. Moreover, this project will be performed in close collaboration with Projects 2 (EBV) and 3 (MHV68), with the goal of identifying pathways that are commonly regulated by cancer-associated gamma-herpesvirus lncRNAs. Identifying such common regulatory pathways may point to novel virus-specific therapeutic strategies. We note that to date several miRNA and lncRNA based therapeutics are in different stages of clinical development.

SUMMARY The unifying postulate of this P01 proposal is that comparative studies of KSHV, EBV, and MHV68 will accelerate the discovery of pathways regulated by long non-coding RNAs (lncRNAs) that contribute to gammaherpesvirus latency and tumorigenesis. Specifically, we hypothesize A) that herpesviruses utilize lncRNAs to regulate both virus and host gene expression, and B) that viral lncRNAs and/or virus-perturbed host lncRNAs directly contribute to the genesis of virus-associated AIDS malignancies. To address these hypotheses we propose three highly integrated projects with the goal of functionally analyzing lncRNAs and their mechanisms of action. Project 1, led by Dr. Renne (University of Florida, UF), will investigate KSHV- encoded lncRNAs and alteration of host lncRNA expression in the context of HIV-associated KSHV malignancies. Project 2, led by Dr. Flemington (Tulane University) will interrogate Epstein-Barr virus lncRNAs and alteration of host lncRNA expression in the context of HIV-associated EBV malignancies. Project 3 led by Dr. Tibbetts (UF) will investigate function of MHV68 lncRNAs and alteration of host lncRNAs in the context of latency and lymphomagenesis in a facile murine model. Importantly, all projects are supported by strong novel preliminary data, including the discovery of new gammaherpesvirus lncRNAs using a novel multi-platform genomics approach and implicating some of these lncRNAs in viral biology and pathogenesis. The well- organized Administrative core (Core A, Core Leader: Rolf Renne) will maintain oversight and organization of the program, including biostatisical consultation and adherence to reproducibility and transparency standards. Two additional service cores, which are already established and very productive, will support sequencing, bioinformatics and recombinant virus generation needs across the three projects: The Virus RNA-seq and Bioinformatics Core will be maintained at Tulane University (Core B, Core Leader: Erik Flemington). The Recombinant Virus Core, which was established with an NCI-funded RC2 award in 2009, will be maintained at UF (Core C, Core Leader: Rolf Renne). In addition, Dr. Parsons (LSUHSC, New Orleans), who leads the NIH- supported LSUHSC/LCRC HIV Clinical and Biospecimen Repository, will facilitate the acquisition of valuable Kaposi's sarcoma (KS) and lymphoma tumor specimens from HIV-infected patients. Our proposal is significant and highly innovative in terms of the field of study ? ?understanding virus and host lncRNA function in gammaherpesvirus tumorigenesis? ? and in applying numerous state-of-the-art techniques across all three projects. Finally, studying pathogenesis-relevant tissue culture and animal models, complemented by the analysis of human tumor tissues from EBV- and KSHV-associated malignancies, will greatly increase our understanding of both viral and host lncRNAs in the context of AIDS malignancies.

Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KS is the most common oral cancer in human immunodeficiency virus (HIV)-infected patients. The oral cavity is the major site for virus transmission via saliva. Although the incidence of AIDS-KS cases has been reduced in the US due to combination antiretroviral therapy (cART), recent studies suggest recurrence of oral KS in long-term treated AIDS patients. While KS tumor cells are mostly latently infected, and express a limited number of viral genes, gingival epithelial cells in the oral cavity actively replicate KSHV which is important for a) seeding the latency reservoir in B cells after primary infection and b) shedding in the oral cavity and transmission via saliva. Epigenetics and chromatin structure play a central role in the regulation of both transcription and replication. Deregulation, aberrant deposition, and mutations of histone variants such as H3.3, CENP-A, and H2A.Z and their associated chaperones have been implicated in multiple human cancers including head and neck. After de novo infection, KSHV genomes rapidly associate with nucleosomes which acquire specific epigenetic modifications that partition the viral episome into transcriptionally active and silenced domains. While multiple KSHV epigenetic marks have been mapped in lymphoid, epithelial and endothelial cells, the processes and potential host- viral interactions leading to the formation of latency permissive episomes are still poorly understood. Given that the KSHV latency-associated nuclear antigen (LANA) interacts with both chromatin remodelers and H3.3 histone chaperones Daxx, HIRA, and DEK, we hypothesize that H3.3 deposition plays an important role in the establishment and maintenance of KSHV latency. Our preliminary data demonstrate that H3.3 deposition on KSHV episomes can be detected early after de novo infection on episomes of long-term infected cells. Moreover, we demonstrated that genetically disrupting the H3.3 chaperone pathways HIRA and Daxx by CRISPR/Cas9 leads to marked changes in KSHV latency control, associated with alterations in the epigenetic status. We further hypothesize that latency-associated gene products including LANA modulate and or de-regulate histone chaperone pathways during de novo infection and latency. To directly address this hypothesis we propose to mechanistically study histone H3.3 deposition and the formation of histone post translational modifications (PTMs), with the goal of understanding their relationship with respect to viral gene expression and latent/lytic replication. A main aspect of these studies is to investigate how viral gene products interact with and modulate histone variant chaperone pathways. In addition, we use human oral primary epithelial cells which are more lytic than transformed epithelial cell lines to characterize epigenetic changes responsible for their lytic phenotype. Importantly, through a collaboration with Dr. Donald Cohen, College of Dentistry, we propose to establish in vivo epigenomes of oral KS in both formalin-fixed and snap frozen tumor samples. The overall goal is to investigate how histone variant deposition determines latent and lytic infection in a cell type-specific manner. The results may point to KSHV-specific novel therapeutic targets for oral KS.