Kaposi's sarcoma Associated Herpesvirus (KSHV) or Human Herpesvirus 8 (HHV-8) is an oncogenic gammaherpesvirus known to be the causative agent of Kaposi's sarcoma (KS), and contributes to body cavity based lymphomas (BCBLs) or pleural effusion lymphomas (PELs) in AIDS patients. It is also associated with Multicentric Castleman's Disease (MCD). KSHV infects endothelial and human B cells with expression of a limited repertoire of genes that are linked to latent infection including the major latency associated nuclear antigen (LANA). KSHV undergoes two major replication modes; a lytic mode and a latent replication mode and in some instances there is an underlying low level of lytic replication that is seen during latency. This may be critical for the pathogenesis associated with the virus. KSHV latent replication is dependent on expression of LANA and initiates at the terminal repeats (TRs). LANA binding sites have been mapped to the TR elements and these sites recruit replication proteins ORCs and MCMs. We have shown that additional sites on the KSHV genome can initiate replication at other regions shown to also recruit ORC and MCMs. A unique technology referred to as single molecule analysis of replicated DNA (SMARD) was used to identify other regions capable of incorporating fluorescent nucleoside analogs during cell proliferation. We have also shown that the replication initiation zone is independent of the presence of LANA demonstrating that the KSHV genome is capable of initiating replication during latency at multiple sites along the genome. In this proposed application we will focus our efforts on understanding genome replication of the KSHV virus after de novo infection by focusing on the major regions of the genome that are activated for replication on infection of primary cells. We will determine the epigenetic programming of the genome, and higher order conformations which dictates genome sites containing firing capabilities for successful replication of the genome. Infected cells will be harvested at different time points of infection and the replication zones monitored by SMARD. We will compare these zones after infection of primary B- and endothelial cells. We will also quantitate the semi-conservative replication using a Meselson Stahl modified approach with real-time PCR. ChIP/ChIP-Seq and ChIA-PET-sequencing will be used to identify the genome regions associated with replication proteins ORCs, MCMs, chromatin modifying factors, and viral antigens. The analysis will determine the time points after the viral genome enters the nucleus to obtain a temporal picture of the transitional epigenetic marks that are determinants for replication. Furthermore, we will monitor the long range interactions, and conformation changes that occur on the viral genome during de novo infection to understand the contribution of epigenetics, higher order interactions and the viral and cellular antigens required for replication of the KSHV genome after de novo infection and establishment of latency. This will identify potential targets and development of intervention strategies for treatment of KSHV associated diseases.

Public Health Relevance

KSHV is a major human pathogen associated with a number of human malignancies including AIDS associated pleural effusion lymphomas and Kaposi's sarcoma. This study will focus on the specific replication events that happen during KSHV infection. This is unique because for the first time we explore the epigenetic requirements for KSHV replication during primary cell infection. Results obtained will provide new possibilities for strategies which can thwart infection in humans.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
HIV Coinfections and HIV Associated Cancers Study Section (HCAC)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pennsylvania
Schools of Medicine
United States
Zip Code