Epstein-Barr virus (EBV) is a human oncogenic virus associated with carcinomas of global importance, such as gastric and nasopharyngeal cancer, several lymphomas, including Burkitt, Hodgkin and diffuse large cell lymphoma that occur in immunocompetent individuals and polyclonal lymphomas in immunodeficient hosts. Our laboratory focuses on the EBV lytic cycle that is essential for transmission of virus among cells and between individuals and directly contributes to viral oncogenicity. The EBV lytic cycle is initiated by expression of two viral proteins, ZEBRA and Rta, transcription factors that also play distinct direct roles in lytic viral DNA replication. Our proposed work is based on newly recognized functions of ZEBRA, the major lytic cycle activator protein originally discovered in our laboratory.
In AIM1, in collaboration with the Steitz lab, we will explore mechanisms underlying the novel role of ZEBRA in mediating viral host shut off of cellular gene expression. We will investigate how ZEBRA controls the translocation of cytoplasmic poly A binding protein (PABPC) to the nucleus, how ZEBRA selectively distributes PABPC within the nucleus, how ZEBRA retains poly-adenylated mRNA in the nucleus and how ZEBRA selectively inhibits synthesis of cellular but not viral proteins.
In AIM2, together with the lab of Yong Xiong, we will study the structural basis of preferential recognition of methylated DNA by ZEBRA. We will investigate the methylation haplotype of promoters of viral and cellular genes targeted by ZEBRA. We will uncover features of the methylated ZEBRA response elements in viral and cellular DNA and properties of the ZEBRA protein itself that mediate preferential recognition of methylated DNA. These studies will eventually yield a crystal structure of ZEBRA bound to methylated DNA. Our proposed experiments will elucidate molecular mechansims that govern two previously unknown functions of the major EBV lytic cycle activator protein, namely viral host shutoff and transcriptional activation via recognition of methylated DNA. These proposed studies have particular relevance for EBV-positive gastric cancer where the presence of the EBV genome induces a state of cellular gene hypermethylation.

Public Health Relevance

Project 1, Narrative There is still no preventive or curative therapy for Epstein-Barr virus, EBV, a human cancer virus discovered more than 50 years ago. Our laboratory has pioneered molecular analysis of viral proteins that control lytic replication of EBV. In this proposal we address two related questions about how the EBV replication activator protein ZEBRA controls gene expression: How does ZEBRA promote viral gene expression while generally inhibiting cellular gene expression? How does ZEBRA activate gene expression by binding to methylated DNA? Our studies have special relevance for gastric cancer, numerically the most prevalent cancer associated with EBV.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA016038-44
Application #
9512683
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
44
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Yale University
Department
Type
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
Zhang, Pengwei; Monteiro da Silva, Gabriel; Deatherage, Catherine et al. (2018) Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking. Cell 174:1465-1476.e13
Inoue, Takamasa; Zhang, Pengwei; Zhang, Wei et al. (2018) ?-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection. J Cell Biol 217:3545-3559
Vallery, Tenaya K; Withers, Johanna B; Andoh, Joana A et al. (2018) Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA. J Virol 92:
Hayes, Karen E; Barr, Jamie A; Xie, Mingyi et al. (2018) Immunoprecipitation of Tri-methylated Capped RNA. Bio Protoc 8:
Park, Richard; Miller, George (2018) Epstein-Barr Virus-Induced Nodules on Viral Replication Compartments Contain RNA Processing Proteins and a Viral Long Noncoding RNA. J Virol 92:
Lipovsky, Alex; Erden, Asu; Kanaya, Eriko et al. (2017) The cellular endosomal protein stannin inhibits intracellular trafficking of human papillomavirus during virus entry. J Gen Virol 98:2821-2836
Singh, Gatikrushna; Fritz, Sarah M; Ranji, Arnaz et al. (2017) Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution. J Vis Exp :
Martinez, Ivan; Hayes, Karen E; Barr, Jamie A et al. (2017) An Exportin-1-dependent microRNA biogenesis pathway during human cell quiescence. Proc Natl Acad Sci U S A 114:E4961-E4970
Brown, Jessica A; Kinzig, Charles G; DeGregorio, Suzanne J et al. (2016) Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA. Proc Natl Acad Sci U S A 113:14013-14018
Zhang, Wei; Xie, Mingyi; Shu, Mei-Di et al. (2016) A proximity-dependent assay for specific RNA-protein interactions in intact cells. RNA 22:1785-1792

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