Favored sites for HIV cDNA integration in the human genome. Abstract Replication of HIV requires integration of a DNA copy of the viral RNA genome into a host cell chromosome. The basic chemical steps mediating action of the viral integrase (IN) protein are now well understood, so interest turns to understanding the roles of cellular factors in the integration process in vivo, and use of such information to develop inhibitors. Raltegravir, the first FDA-approved integrase (IN) inhibitor, binds the enzyme active site and blocks DNA strand transfer. Evolution of HIV in the presence of raltegravir elicits resistance mutants, however, motivating studies of potential new drug targets in the preintegration complex. IN binds the host cell protein PSIP1/LEDGF/p75 (henceforth LEDGF), which is important for efficient integration, and previous work under this award showed that LEDGF targets HIV integration to active transcription units via a tethering mechanism. We also reported that the LEDGF binding site on IN can be bound by small molecule inhibitors, and now highly active inhibitors targeting this site are available. This application thus focuses on identifying interactions between HIV IN and host factors important in integration (Aim 1), and studies of integrase inhibitors (Aim 2) with the dual goals of optimizing therapy and using inhibitors to understand mechanism. Research in both aims will be driven in part by our infrastructure for studying retroviral integration targeting, which consists of well established bioinformatic pipelines and an archive of over one billion integration site sequence reads. Progress from the previous funding cycle allows us to propose several exciting new hypotheses to investigate in the renewal. 1) We hypothesize that HIV PICs engage in an orderly series of interactions with cellular proteins, beginning in the cytoplasm, that ultimately delivers PICs to favored integration sites in the nucleus. 2) We hypothesize that an inhibitor that binds to the LEDGF interacting site on integrase can have wider effects on the HIV replication cycle than previously appreciated, sending us in new directions in the study of LEDGF. 3) We hypothesize that raltegravir alters integration targeting by increasing the proportion of events mediated by the cellular NHEJ machinery. Thus our Specific Aims are:
Aim 1. Mechanistic studies of interactions between PICs and cellular factors to identify potential inhibitor targets.
Aim 2. Probing the mechanism of integrase inhibitors, both those targeting the LEDGF binding site and the active site. !

Public Health Relevance

Studies of HIV integration have led to the discovery of novel inhibitors that have now achieved FDA approval. This grant application centers on studies of integration mechanisms, with particular focus on potential targets for new classes of inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052845-13
Application #
8595275
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
2002-06-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
13
Fiscal Year
2014
Total Cost
$354,231
Indirect Cost
$117,863
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Berry, Charles C; Ocwieja, Karen E; Malani, Nirav et al. (2014) Comparing DNA integration site clusters with scan statistics. Bioinformatics 30:1493-500
Manganaro, Lara; Pache, Lars; Herrmann, Tobias et al. (2014) Tumor suppressor cylindromatosis (CYLD) controls HIV transcription in an NF-?B-dependent manner. J Virol 88:7528-40
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
Gupta, Kushol; Brady, Troy; Dyer, Benjamin M et al. (2014) Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J Biol Chem 289:20477-88
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
Petersen, Josiah; Drake, Mary Jane; Bruce, Emily A et al. (2014) The major cellular sterol regulatory pathway is required for Andes virus infection. PLoS Pathog 10:e1003911
Sherrill-Mix, Scott; Lewinski, Mary K; Famiglietti, Marylinda et al. (2013) HIV latency and integration site placement in five cell-based models. Retrovirology 10:90
Li, Xianghong; Ewis, Hosam; Hice, Robert H et al. (2013) A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture. Proc Natl Acad Sci U S A 110:E478-87
Bushman, Frederic D; Barton, Spencer; Bailey, Aubrey et al. (2013) Bringing it all together: big data and HIV research. AIDS 27:835-8
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

Showing the most recent 10 out of 51 publications