This proposal describes genetic and biochemical analyses of the replication of the Moloney murine leukemia virus, the prototype of the simple mammalian retroviruses. We are broadly interested in all aspects of the viral life cycle, but are especially focused on identifying and characterizing the cellular host factors that interact with the virus. In this proposal we will address three host systems that act to limit or restrict virus replication. First, we will characterize the mechanisms by which embryonic stem (ES) cells transcriptionally silence proviral DNAs and maintain the integrity of the germ line. We will study two parallel pathways - a rapid and efficient mechanism targeting a specific DNA element of the Moloney provirus, and a slower, less efficient one acting more broadly on many proviruses -- and determine how these silencing mechanisms are specifically active in ES cells. These experiments will provide important new information about the properties that define """"""""stemness"""""""" - the pluripotent state of ES cells. Second, we will explore the role of SUMOylation, the covalent addition of SUMO (small ubiquitin-related modifier) to particular protein substrates, in virus replication and restriction. We will test for SUMOylation of the viral capsid protein during infection, and explore the potential role of SUMOylation in the capsid-dependent restriction of virus infection by the host genes Fv1 and TRIM5?. Third, we will examine the mechanism of expression of the Gag-Pol precursor protein, which requires the suppression of translational termination at a UAG stop codon at the 3'end of the gag gene. This readthrough event depends on an RNA pseudoknot immediately downstream of the stop codon. We will identify new regulators of readthrough by isolation of RNA-protein complexes from infected cells. Finally, we will explore the mechanisms by which the MuLV mRNA for Gag and Gag-Pol evades the Nonsense-Mediated mRNA Decay (NMD) system. We will test the hypothesis that an interaction between the retroviral reverse transcriptase and the host translational termination factor eRF1 prevents normal recognition of the viral mRNA by NMD and protects them from degradation. All three areas of investigation will advance our understanding of host defense systems and may provide important new targets for antiviral intervention. Most importantly, these experiments will significantly extend our understanding of fundamental aspects of retrovirus replication, and of new cell biological processes that impact on these important viruses.

Public Health Relevance

The goal of this project is to expand our knowledge of the Moloney murine leukemia virus, prototype of the simple mammalian retroviruses, and model for the human retroviruses HTLV-1 and HIV-1. The proposed work is focused on virus-host interactions: the characterization of cellular machinery exploited by the virus, and especially host restriction systems that act to inhibit virus replication. The studies will reveal new aspects of cell biology -- transcription, protein processing, and translation -- and will inform new approaches to treatment of viral leukemias and AIDS.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA030488-34
Application #
8629004
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1981-08-01
Project End
2019-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
34
Fiscal Year
2014
Total Cost
$178,132
Indirect Cost
$64,659
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Wang, Gary Z; Goff, Stephen P (2016) Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells. J Virol :
Tang, Xuhua; Zhu, Yiping; Baker, Stacey L et al. (2016) Structural basis of suppression of host translation termination by Moloney Murine Leukemia Virus. Nat Commun 7:12070
Wang, Gary Z; Wang, Ying; Goff, Stephen P (2016) Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry. Cell Host Microbe 20:798-809
Yang, Bin Xia; El Farran, Chadi A; Guo, Hong Chao et al. (2015) Systematic identification of factors for provirus silencing in embryonic stem cells. Cell 163:230-45
Wang, Gary Z; Goff, Stephen P (2015) Postentry restriction of Mason-Pfizer monkey virus in mouse cells. J Virol 89:2813-9
Wang, Gary Z; Goff, Stephen P (2015) Regulation of Yin Yang 1 by Tyrosine Phosphorylation. J Biol Chem 290:21890-900
Green, Lisa; Goff, Stephen P (2015) Translational readthrough-promoting drugs enhance pseudoknot-mediated suppression of the stop codon at the Moloney murine leukemia virus gag–pol junction. J Gen Virol 96:3411-21
Erazo, Angela; Goff, Stephen P (2015) Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction. Retrovirology 12:57
Schlesinger, Sharon; Goff, Stephen P (2015) Retroviral transcriptional regulation and embryonic stem cells: war and peace. Mol Cell Biol 35:770-7
Schlesinger, Sharon; Meshorer, Eran; Goff, Stephen P (2014) Asynchronous transcriptional silencing of individual retroviral genomes in embryonic cells. Retrovirology 11:31

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