Integrase (IN) is one of three viral-encoded enzymes that are essential for retroviral replication and, therefore, an important target for the development of strategies for the treatment of AIDS. Although some HIV-1 IN inhibitors with antiviral activity have been identified by us and others, progress has been seriously limited by a lack of critical details concerning the molecular structure of the active IN complex and its interactions with viral and host DNA substrates. The new approaches described in this competing renewal are designed to close this gap, benefiting from experimental systems developed to study ASV IN. The first two Aims address specific features of IN-DNA interactions:
Aim 1 will identify ASV and HIV-1 IN residues that promote or stabilize viral DNA end unpairing, a step required for processing, by using chemical probing, biophysical methods, and mutagenesis.
Aim 2 will map specific contacts between IN and viral and target DNA using new photo crosslinking methods together with molecular models of IN-DNA complexes. An iterative process of modeling and testing will identify all relevant contact sites between IN and substrate DNAs.
In Aim 3 ASV IN proteins and IN-DNA complexes will be analyzed by dynamic light scattering and small angle X-ray scattering methods to determine their composition and conformation in solution. Chemical trapping methods will be used to prepare covalently linked, biologically-relevant IN-DNA complexes for structural analysis. The results of these analyses will confirm the stoichiometry and spatial orientation of IN domains and substrate DNAs in these complexes and support the modeling process of Aim 2. Homogeneous complex preparations will be tested in crystallization trials with the goal of acquiring structural data at atomic resolution. Predictions concerning structure and mechanism derived from these studies will be tested with HIV-1 IN. Experiments in this proposal are designed to provide a detailed understanding of the molecular interactions between HIV-1 IN and its DNA substrates. Such information is crucial for exploiting this viral protein as a target for the design of new therapies against AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI040385-13
Application #
7762728
Study Section
Special Emphasis Panel (ZRG1-AARR-A (03))
Program Officer
Gupta, Kailash C
Project Start
1997-08-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
13
Fiscal Year
2010
Total Cost
$423,683
Indirect Cost
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
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Benleulmi, Mohamed Salah; Matysiak, Julien; Henriquez, Daniel Rodrigo et al. (2015) Intasome architecture and chromatin density modulate retroviral integration into nucleosome. Retrovirology 12:13
Skala, Anna Marie (2014) Retroviral DNA Transposition: Themes and Variations. Microbiol Spectr 2:
Skalka, Anna Marie (2014) Retroviral DNA Transposition: Themes and Variations. Microbiol Spectr 2:MDNA300052014
Bojja, Ravi Shankar; Andrake, Mark D; Merkel, George et al. (2013) Architecture and assembly of HIV integrase multimers in the absence of DNA substrates. J Biol Chem 288:7373-86
Shalginskikh, Natalia; Poleshko, Andrey; Skalka, Anna Marie et al. (2013) Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx. J Virol 87:2137-50
Bojja, Ravi S; Andrake, Mark D; Weigand, Steven et al. (2011) Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking. J Biol Chem 286:17047-59
Katz, Richard A; Merkel, George; Andrake, Mark D et al. (2011) Retroviral integrases promote fraying of viral DNA ends. J Biol Chem 286:25710-8
Peletskaya, Elena; Andrake, Mark; Gustchina, Alla et al. (2011) Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis. PLoS One 6:e27751

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