Significant progress towards understanding the mechanisms involved in retrovirus integration is evident, but many facets of retrovirus integration remain unclear. The multimeric state of integrase (IN) subunits, their role in assembly and their molecular interactions with viral DNA in preintegration complexes (PIC) found in virus-infected cells are still undefined. The spatial arrangements of IN subunits near the viral DNA termini necessary to mediate assembly and concerted integration of the viral DNA termini are unknown. The regions of IN responsible for contact between the two viral termini bound by IN mediating concerted integration are unknown. Our initial efforts with non-ionic detergent disrupted HIV-1 virions to characterize concerted integration in vitro; has led to our recent success with newly-folded recombinant HIV-1 IN. Co-expression of HIV-1 IN with a plasmid expressing protein chaperones (GroEL-GroES) in bacteria allows purified recombinant HIV-1 IN to efficiently mediate concerted (full-site) integration without auxiliary viral or cellular proteins. We will use the reconstitution assay to determine what role newly-folded HIV-1 IN plays in specific assembly steps, in IN-LTR DNA interactions at the molecular level, in full-site integration properties of nucleoprotein complexes having characteristics of purified HIV-1 and murine leukemia virus PIC (or intasomes). The interactions of HIV-1 IN with wild type and mutant viral termini for full-site integration will be investigated. We will attempt to establish the multimeric state of HIV-1 IN subunits necessary and sufficient in nucleoprotein complexes mediating full-site integration. We will determine what amino acid residues of HIV-1 IN contribute to the protein-protein and protein-DNA interactions responsible for assembly and full-site integration. We will use the reconstituted HIV-1 IN-LTR donor complexes to investigate the effects of active site inhibitors to HIV-1 IN integration in vivo and in vitro. We will continue to investigate the role HIV-1 IN has in the retrovirus life cycle with emphasis on understanding full-site integration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI031334-14
Application #
6732068
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Program Officer
Gupta, Kailash C
Project Start
1991-09-01
Project End
2006-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
14
Fiscal Year
2004
Total Cost
$257,250
Indirect Cost
Name
Saint Louis University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Bera, Sibes; Pandey, Krishan K; Vora, Ajaykumar C et al. (2009) Molecular Interactions between HIV-1 integrase and the two viral DNA ends within the synaptic complex that mediates concerted integration. J Mol Biol 389:183-98
Grandgenett, Duane P; Bera, Sibes; Pandey, Krishan K et al. (2009) Biochemical and biophysical analyses of concerted (U5/U3) integration. Methods 47:229-36
Pandey, Krishan K; Grandgenett, Duane P (2008) HIV-1 Integrase Strand Transfer Inhibitors: Novel Insights into their Mechanism of Action. Retrovirology 2:11-16
Zahm, Jacob A; Bera, Sibes; Pandey, Krishan K et al. (2008) Mechanisms of human immunodeficiency virus type 1 concerted integration related to strand transfer inhibition and drug resistance. Antimicrob Agents Chemother 52:3358-68
Pandey, Krishan K; Bera, Sibes; Zahm, Jacob et al. (2007) Inhibition of human immunodeficiency virus type 1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate. J Virol 81:12189-99
Pandey, Krishan K; Sinha, Sapna; Grandgenett, Duane P (2007) Transcriptional coactivator LEDGF/p75 modulates human immunodeficiency virus type 1 integrase-mediated concerted integration. J Virol 81:3969-79
Bera, Sibes; Vora, Ajaykumar C; Chiu, Roger et al. (2005) Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study. Biochemistry 44:15106-14
Sinha, Sapna; Grandgenett, Duane P (2005) Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells. J Virol 79:8208-16
Vora, Ajaykumar; Bera, Sibes; Grandgenett, Duane (2004) Structural organization of avian retrovirus integrase in assembled intasomes mediating full-site integration. J Biol Chem 279:18670-8
Sinha, Sapna; Pursley, Michael H; Grandgenett, Duane P (2002) Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors. J Virol 76:3105-13

Showing the most recent 10 out of 22 publications