KSHV is the causative agent of Kaposi's sarcoma (KS) that occurs most frequently and severely in HIV-AIDS. KSHV has also been implicated in several lymphoid disorders, including pleural effusion lymphoma (PEL) and Castleman's disease. Long-term latent infection in B-lymphocytes and persistent infection in endothelial cells is thought to be a major driving force for KSHV-associated pathogenesis. Latent infection is characterized by the transcription of several viral genes associated with viral genome stability and host-cell survival. The major latency transcript is a multicistronic message that consists of the LANA, vCyclin, and vFLIP genes, which can be alternatively spliced to generate the viral microRNAs (vmiRNAs) and Kaposin genes. The latency transcriptional control is also responsible for the regulation of the vGPCR gene, which has been implicated in KS pathogenesis. Proper regulation of these viral transcripts is critical for viral genome persistence during latency and for pathogenesis in KSHV infected lesions. Our preliminary data indicates that this region of the viral chromosome is protected from epigenetic silencing by the chromatin insulator protein CTCF and colocalization with cohesin subunits (e.g. SMC1, SMC3, Rad21). In addition, changes in CTCF-cohesin interactions contribute to the cell cycle activation of vGPCR in latently infected PEL cells. We have also found that these CTCF sites in the latency control region are important for the programming of RNA polymerase II elongation and mRNA processing.
In Aim 1, we will determine how the CTCF-cohesin complex contributes to the regulation of latency transcription, and protects the major latency control region from epigenetic silencing. We have also found that viral encoded miRNAs contribute to the epigenetic programming of the viral chromosome during latency. We propose in Aim 2 of this proposal to determine whether CTCF-cohesin complex regulates vmiRNA expression, and additionally, whether vmiRNAs coregulate the function of CTCF- cohesins in regulating the epigenetic state of the latent viral chromosome. Finally, we propose to investigate the mechanism of a small molecule inhibitor that selectively deregulates KSHV latency transcription. Glychyrrizic acid (GA) is a bioactive natural product derived from licorice that selectively inhibits KSHV positive PEL cell proliferation. We have found that GA deregulates RNA polymerase II interactions with the CTCF- cohesin complex at the LANA 5'UTR. We have used chemical affinity to identify candidate target molecules of GA.
In Aim 3, we propose to further characterize the mechanism of GA deregulation of LANA transcription and its potential targeting of the CTCF-cohesin complex. Together, these aims focus on a novel mechanism of gene regulation that maintains latency gene expression during latency, restricts the pathogenic expression of vGPCR, and may serve as a potential target for small molecule intervention in KSHV-associated disease.

Public Health Relevance

Human herpesvirus 8 (HHV8), more commonly referred to as Kaposi's Sarcoma (KS)-Associated Herpesvirus (KSHV) is the etiological agent of HIV-associated and endemic forms of KS. KSHV has also been linked to two other lymphoproliferative disorders, primary effusion lymphoma (PELs) and multicentric Castleman's disease. Additional diseases may also be associated with KSHV, including autoimmune disorders like multiple sclerosis and system lupus erythematosis. In all cases of KSHV, the infection is a complex mixture of latent episomes and lytic productive infection. In this application, we investigate the mechanisms regulating gene expression from KSHV latent genomes, and how small molecule inhibitors may be developed to disrupt this process. Further elucidation of the molecular mechanisms regulating latency transcription will provide useful information for understanding and treating viral pathogenesis and disease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA117830-08
Application #
8403631
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2005-07-01
Project End
2015-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
8
Fiscal Year
2013
Total Cost
$278,661
Indirect Cost
$116,649
Name
Wistar Institute
Department
Type
DUNS #
075524595
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Martínez, Francisco Puerta; Cruz, Ruth; Lu, Fang et al. (2014) CTCF binding to the first intron of the major immediate early (MIE) gene of human cytomegalovirus (HCMV) negatively regulates MIE gene expression and HCMV replication. J Virol 88:7389-401
Domsic, John F; Chen, Horng-Shen; Lu, Fang et al. (2013) Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA. PLoS Pathog 9:e1003672
Chen, Horng-Shen; Lu, Fang; Lieberman, Paul M (2013) Epigenetic regulation of EBV and KSHV latency. Curr Opin Virol 3:251-9
Knipe, David M; Lieberman, Paul M; Jung, Jae U et al. (2013) Snapshots: chromatin control of viral infection. Virology 435:141-56
Kang, Hyojeung; Cho, Hyosun; Sung, Gi-Ho et al. (2013) CTCF regulates Kaposi's sarcoma-associated herpesvirus latency transcription by nucleosome displacement and RNA polymerase programming. J Virol 87:1789-99
Dheekollu, Jayaraju; Chen, Horng-Shen; Kaye, Kenneth M et al. (2013) Timeless-dependent DNA replication-coupled recombination promotes Kaposi's Sarcoma-associated herpesvirus episome maintenance and terminal repeat stability. J Virol 87:3699-709
Lu, Fang; Tsai, Kevin; Chen, Horng-Shen et al. (2012) Identification of host-chromosome binding sites and candidate gene targets for Kaposi's sarcoma-associated herpesvirus LANA. J Virol 86:5752-62
Kang, Hyojeung; Lieberman, Paul M (2011) Mechanism of glycyrrhizic acid inhibition of Kaposi's sarcoma-associated herpesvirus: disruption of CTCF-cohesin-mediated RNA polymerase II pausing and sister chromatid cohesion. J Virol 85:11159-69
Kang, Hyojeung; Wiedmer, Andreas; Yuan, Yan et al. (2011) Coordination of KSHV latent and lytic gene control by CTCF-cohesin mediated chromosome conformation. PLoS Pathog 7:e1002140
Lu, Fang; Stedman, William; Yousef, Malik et al. (2010) Epigenetic regulation of Kaposi's sarcoma-associated herpesvirus latency by virus-encoded microRNAs that target Rta and the cellular Rbl2-DNMT pathway. J Virol 84:2697-706

Showing the most recent 10 out of 16 publications