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.

Public Health Relevance

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). 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. Latency is a prerequisite for tumorigenesis and the long-term goal of this work is to understand LANA-dependent virus/host interactions that lead to the establishment of latency. Our model is that LANA recruits cellular enzymes, which usually regulate gene expression by modifying host chromosomes, to organize viral genomes into chromosome-like structures. Understanding the molecular events leading to this could identify new strategies to intervene with or block viral latency. More specifically, we study how LANA supports the replication of the viral genome and how it helps to recruit the above mentioned factors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA088763-13
Application #
8607135
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2000-07-01
Project End
2016-02-29
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
13
Fiscal Year
2014
Total Cost
$207,842
Indirect Cost
$65,970
Name
University of Florida
Department
Genetics
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Hu, Jianhong; Yang, Yajie; Turner, Peter C et al. (2014) LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex. PLoS Pathog 10:e1004240
Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M et al. (2014) A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells. BMC Genomics 15:668
Reese, T A; Wakeman, B S; Choi, H S et al. (2014) Helminth infection reactivates latent ?-herpesvirus via cytokine competition at a viral promoter. Science 345:573-7
Plaisance-Bonstaff, Karlie; Choi, Hong Seok; Beals, Tyler et al. (2014) KSHV miRNAs decrease expression of lytic genes in latently infected PEL and endothelial cells by targeting host transcription factors. Viruses 6:4005-23
Pantry, Shara N; Medveczky, Maria M; Arbuckle, Jesse H et al. (2013) Persistent human herpesvirus-6 infection in patients with an inherited form of the virus. J Med Virol 85:1940-6
Darst, Russell P; Haecker, Irina; Pardo, Carolina E et al. (2013) Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus. Nucleic Acids Res 41:2993-3009
Boss, Isaac W; Renne, Rolf (2011) Viral miRNAs and immune evasion. Biochim Biophys Acta 1809:708-14
Boss, Isaac W; Nadeau, Peter E; Abbott, Jeffrey R et al. (2011) A Kaposi's sarcoma-associated herpesvirus-encoded ortholog of microRNA miR-155 induces human splenic B-cell expansion in NOD/LtSz-scid IL2Rýýnull mice. J Virol 85:9877-86
Han, Soo-Jin; Hu, Jianhong; Pierce, Brian et al. (2010) Mutational analysis of the latency-associated nuclear antigen DNA-binding domain of Kaposi's sarcoma-associated herpesvirus reveals structural conservation among gammaherpesvirus origin-binding proteins. J Gen Virol 91:2203-15
Boss, Isaac W; Renne, Rolf (2010) Viral miRNAs: tools for immune evasion. Curr Opin Microbiol 13:540-5

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