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
Schlesinger, Sharon; Meshorer, Eran; Goff, Stephen P (2014) Asynchronous transcriptional silencing of individual retroviral genomes in embryonic cells. Retrovirology 11:31
Wang, Gary Z; Wolf, Daniel; Goff, Stephen P (2014) EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney murine leukemia virus in embryonic cells. J Virol 88:1825-9
Beyer, Andrea R; Bann, Darrin V; Rice, Breanna et al. (2013) Nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein L9. J Virol 87:1069-82
Schlesinger, Sharon; Lee, Andreia H; Wang, Gary Z et al. (2013) Proviral silencing in embryonic cells is regulated by Yin Yang 1. Cell Rep 4:50-8
Studamire, Barbara; Goff, Stephen P (2008) Host proteins interacting with the Moloney murine leukemia virus integrase: multiple transcriptional regulators and chromatin binding factors. Retrovirology 5:48
Evans, Matthew J; Bacharach, Eran; Goff, Stephen P (2004) RNA sequences in the Moloney murine leukemia virus genome bound by the Gag precursor protein in the yeast three-hybrid system. J Virol 78:7677-84
Gao, Guangxia; Goff, Stephen P (2004) Isolation of suppressor genes that restore retrovirus susceptibility to a virus-resistant cell line. Retrovirology 1:30
Goff, Stephen P (2004) Retrovirus restriction factors. Mol Cell 16:849-59
Towers, Greg J; Goff, Stephen P (2003) Post-entry restriction of retroviral infections. AIDS Rev 5:156-64
Wang, Margaret Q; Goff, Stephen P (2003) Defects in virion production caused by mutations affecting the C-terminal portion of the Moloney murine leukemia virus capsid protein. J Virol 77:3339-44

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