Abstract

Gene therapy protocols risk the potential of gene activation due to retroviral insertional activation. The ability to retarget or limit the sites of retroviral integration though cannot be successfully achieved in the absence of the basic understanding of the mechanism of retroviral integration. This application studies the basic mechanism of integration of murine leukemia virus (MuLV), the most common retroviral vector used in gene therapy protocols. Integration is a critical step in the viral life cycle which results in the stable insertion of the viral genome throughout the host DNA. There is no mechanism of excision of an integrated provirus and integration can lead to the disruption and/or activation of cellular genes. The application utilizes biochemical and genetic approaches to define the interactions which orchestrate the assembly of protein-DNA integrative complex.
Three specific aims are proposed which examine individual components of the integration complex.
The first aim focuses on the viral Integrase (IN) protein, which catalyzes the transesterification process. These studies aim at defining the domains of the protein required to assemble a synaptic complex with the viral and target DNAs. The second specific aim defines the structural and/or sequence requirements of the viral substrate, the inverted terminal repeats at the ends of the LTRs. Novel polyamides which sequence specifically bind within the minor groove the MuLV LTR termini have been developed. The effect of these compounds on the structure of the MuLV LTR will be examined. The third specific aim modifies the MuLV IN protein to include sequences found to direct integration to heterochromatin, considered to be transcriptionally inactive. This approach will adapt the mechanism identified for the yeast Ty5 system to the retroviral particles. The potential of the Ty5 tag to target retroviral integration will be examined. These studies examine critical aspects of retroviral integration needed to understand, manipulate, and inhibit this requisite step in the viral life cycle. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM070837-01A1S1
Application #
7114750
Study Section
Virology Study Section (VR)
Program Officer
Anderson, Richard A
Project Start
2005-02-01
Project End
2009-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
1
Fiscal Year
2005
Total Cost
$19,500
Indirect Cost

  Institution

Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854

  Publications

Guan, Rongjin; Aiyer, Sriram; Cote, Marie L et al. (2017) X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition. Proteins 85:647-656
Aiyer, Sriram; Rossi, Paolo; Malani, Nirav et al. (2015) Structural and sequencing analysis of local target DNA recognition by MLV integrase. Nucleic Acids Res 43:5647-63
Larue, Ross C; Plumb, Matthew R; Crowe, Brandon L et al. (2014) Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes. Nucleic Acids Res 42:4868-81
Aiyer, Sriram; Swapna, G V T; Malani, Nirav et al. (2014) Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction. Nucleic Acids Res 42:5917-28
Sharma, Amit; Larue, Ross C; Plumb, Matthew R et al. (2013) BET proteins promote efficient murine leukemia virus integration at transcription start sites. Proc Natl Acad Sci U S A 110:12036-41
Schneider, William M; Brzezinski, Jonathon D; Aiyer, Sriram et al. (2013) Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag. Proc Natl Acad Sci U S A 110:9487-92
Schneider, William M; Wu, Dai-tze; Amin, Vaibhav et al. (2012) MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA. Virology 426:188-96
Farias, Richard V; Vargas, Deborah A; Castillo, Andres E et al. (2011) Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening. Antimicrob Agents Chemother 55:4735-41
Schneider, William M; Tang, Yuefeng; Vaiphei, S Thangminlal et al. (2010) Efficient condensed-phase production of perdeuterated soluble and membrane proteins. J Struct Funct Genomics 11:143-154
Tang, Yuefeng; Schneider, William M; Shen, Yang et al. (2010) Fully automated high-quality NMR structure determination of small (2)H-enriched proteins. J Struct Funct Genomics 11:223-32

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