Kaposi's sarcoma associated herpes virus (KSHV/HHV-8) is the second identified gammaherpesvirus associated with at least 3 major proliferative diseases. These include Kaposi's sarcoma and body cavity based lymphomas (BCBLs) or Pleural Effusion Lymphomas (PELs). One of the major latent antigens, latency- associated nuclear antigen (LANA) is constitutively expressed in all latently infected KSHV cells and is involved in the long erm maintenance of the dsDNA KSHV genome as well as transcriptional regulation. Studies have also shown an important role in regulation of viral latency in association with the viral immediate early transactivator Rta. LANA binds to cis-acting elements within the KSHV terminal repeats (TR) and is associated with a number of chromosomal proteins which are important for tethering the viral proteins and segregation of the viral genome to new daughter cells. Furthermore, LANA can induce chromosomal abberations when expressed in human cells in vitro suggesting a role for LANA in induction of oncogenesis in KSHV infected cells.
The specific aims of this new proposal is to focus our studies investigating KSHV latent infection, through persistence and mechanism of segregation of the viral genome which is passed from parental cell to progeny cells which are latently infected. There has been no direct studies which comprehensively describes a specific mechanism by which this occurs. We have shown associations of LANA with cellular proteins that are necessary for tethering and segregation of the viral genome in the infected cells. We have also shown an association with the nuclear mitosis apparatus protein NuMA, as well as Bub1 and a number of CenP proteins. Here we will explore the direct connections with LANA and cellular proteins associated with the centromere and kinetochore using state of the art in vivo visualizatin strategies combined with targeted shRNA knockdown of specific cellular targets, to understand the function of these proteins in mediating or controlling the transitional states of the KSHV genomic DNA as it passes from one daughter cell to the next without loss of the genome. We will use direct biochemical, genetics and in vivo FRET visualization analyses to focus on the interaction of LANA with the kinetochore protein Bub1 and the APC proteins known to regulate the stability of this protein complex, and to investigate the mechanism of stability of this protein complex whereby LANA and the KSHV genome can progress through the stages of mitosis without loss of the genome thus allowing segregation of the viral genomes to the new daughter cells.

Public Health Relevance

Human herpes virus 8 (HHV-8), also referred to as Kaposi's sarcoma associated herpesvirus (KSHV), is an important etiological agent involved in initiation and development of a number of human cancers. In this proposed study we will examine the mechanism by which the viral genome is maintained and segregated from one daughter cell to the next with the hope that understanding this process will have implications for targeted disruption of this process and development of therapeutic approaches which will result in loss of KSHV and elimination of the associated cancers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA171979-01A1
Application #
8467401
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2013-01-01
Project End
2017-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
1
Fiscal Year
2013
Total Cost
$298,800
Indirect Cost
$112,050
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Zhang, Liming; Zhu, Caixia; Guo, Yi et al. (2014) Inhibition of KAP1 enhances hypoxia-induced Kaposi's sarcoma-associated herpesvirus reactivation through RBP-J?. J Virol 88:6873-84
Lu, Jie; Jha, Hem C; Verma, Subhash C et al. (2014) Kaposi's sarcoma-associated herpesvirus-encoded LANA contributes to viral latent replication by activating phosphorylation of survivin. J Virol 88:4204-17
Sun, Zhiguo; Xiao, Bingyi; Jha, Hem Chandra et al. (2014) Kaposi's sarcoma-associated herpesvirus-encoded LANA can induce chromosomal instability through targeted degradation of the mitotic checkpoint kinase Bub1. J Virol 88:7367-78
Cai, Qiliang; Banerjee, Shuvomoy; Cervini, Amanda et al. (2013) IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation. PLoS Pathog 9:e1003751
Verma, Subhash C; Cai, Qiliang; Kreider, Edward et al. (2013) Comprehensive analysis of LANA interacting proteins essential for viral genome tethering and persistence. PLoS One 8:e74662