An essential step during the life cycle of all retroviruses, including human immunodeficiency virus (HIV), is integration of a double-stranded DNA copy of the viral genome into a chromosome of the host cell. Two factors are critical for the integration process: the viral protein, integrase, and sequences at the ends of the linear viral DNA. Integrase removes two nucleotides from the 3' ends of a linear viral DNA molecule, and subsequently mediates a coupled cleavage-ligation reaction during which a staggered cut is made in the target DNA, and the resulting 5' ends of the target DNA are covalently joined to the recessed 3' ends of the viral DNA. The timing of the joining of the viral 5' ends to target DNA is presently not known, and the protein factors involved remain to be characterized. During integration, many sites in the host chromosome can be used as targets, although a wide variation in integration efficiency is observed among the target sites. The mechanism that determines target site specificity is not well understood. Since integration is required for retroviral replication and there is no recognized counterpart to integrase in normal cellular function, integration is an appealing target for developing specific inhibitors against retroviruses. The broad, long-term objective of this proposal is to further the understanding of the mechanism of HIV integration.
The specific aims are (A) to examine target site selection during retroviral DNA integration, and (B) to study the kinetics and mechanisms of joining of the viral 5' end to target DNA, a poorly characterized final step of integration. The experimental design and methods for achieving these goals arc. (1) to construct chimeric proteins between HIV-1 and FIV integrases for determining the protein domain involved in selecting DNA sites for integration, and to use a PCR-based selection and amplification protocol for determining the DNA sequence optimal for integrase recognition, (2) to explore site-directed integration by studying in vitro and in vivo the activities and integration patterns of fusion proteins consisting of integraSe and a sequence-specific DNA binding protein, and (3) to use a novel strategy to study the timing of viral 3'- and 5'-end joining, and to develop an in vitro system for characterizing the 5'-end joining step and the factors required. The study may reveal potential novel targets for inhibitors and provide useful assays for drug screening. Information obtained from studying integration site selection may lead to a new approach for inserting exogenous genes at specific sites.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA068859-08S1
Application #
6559104
Study Section
Special Emphasis Panel (ZRG1 (01))
Program Officer
Rosenfeld, Bobby
Project Start
1995-09-01
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
8
Fiscal Year
2002
Total Cost
$44,162
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Kim, Sanggu; Kim, Namshin; Presson, Angela P et al. (2014) Dynamics of HSPC repopulation in nonhuman primates revealed by a decade-long clonal-tracking study. Cell Stem Cell 14:473-85
Kim, Sanggu; Kim, Yun-Cheol; Qi, Hangfei et al. (2011) Efficient identification of human immunodeficiency virus type 1 mutants resistant to a protease inhibitor by using a random mutant library. Antimicrob Agents Chemother 55:5090-8
Presson, Angela P; Kim, Namshin; Xiaofei, Yan et al. (2011) Methodology and software to detect viral integration site hot-spots. BMC Bioinformatics 12:367
Kim, Sanggu; Rusmevichientong, Alice; Dong, Beihua et al. (2010) Fidelity of target site duplication and sequence preference during integration of xenotropic murine leukemia virus-related virus. PLoS One 5:e10255
Briones, Marisa S; Chow, Samson A (2010) A new functional role of HIV-1 integrase during uncoating of the viral core. Immunol Res 48:14-26
Briones, Marisa S; Dobard, Charles W; Chow, Samson A (2010) Role of human immunodeficiency virus type 1 integrase in uncoating of the viral core. J Virol 84:5181-90
Kim, Sanggu; Kim, Namshin; Presson, Angela P et al. (2010) High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones. J Virol 84:11771-80
Vatakis, Dimitrios N; Kim, Sanggu; Kim, Namshin et al. (2009) Human immunodeficiency virus integration efficiency and site selection in quiescent CD4+ T cells. J Virol 83:6222-33
Woodward, Cora L; Prakobwanakit, Sarin; Mosessian, Sherly et al. (2009) Integrase interacts with nucleoporin NUP153 to mediate the nuclear import of human immunodeficiency virus type 1. J Virol 83:6522-33
Wilkinson, Thomas A; Januszyk, Kurt; Phillips, Martin L et al. (2009) Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase. J Biol Chem 284:7931-9

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