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 #
2R01AI052845-12
Application #
8467368
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
2013-02-01
Budget End
2014-01-31
Support Year
12
Fiscal Year
2013
Total Cost
$384,974
Indirect Cost
$138,101
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Sherman, Eric; Nobles, Christopher; Berry, Charles C et al. (2017) INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes. Mol Ther Methods Clin Dev 4:39-49
Chehoud, Christel; Stieh, Daniel J; Bailey, Aubrey G et al. (2017) Associations of the vaginal microbiota with HIV infection, bacterial vaginosis, and demographic factors. AIDS 31:895-904
Berry, Charles C; Nobles, Christopher; Six, Emmanuelle et al. (2017) INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions. Mol Ther Methods Clin Dev 4:17-26
Feng, Lei; Dharmarajan, Venkatasubramanian; Serrao, Erik et al. (2016) The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS Chem Biol 11:1313-21
Gupta, Kushol; Turkki, Vesa; Sherrill-Mix, Scott et al. (2016) Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase. PLoS Biol 14:e1002584
Ocwieja, Karen E; Sherrill-Mix, Scott; Liu, Changchun et al. (2015) A reverse transcription loop-mediated isothermal amplification assay optimized to detect multiple HIV subtypes. PLoS One 10:e0117852
Pache, Lars; Dutra, Miriam S; Spivak, Adam M et al. (2015) BIRC2/cIAP1 Is a Negative Regulator of HIV-1 Transcription and Can Be Targeted by Smac Mimetics to Promote Reversal of Viral Latency. Cell Host Microbe 18:345-53
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
Sherrill-Mix, Scott; Ocwieja, Karen E; Bushman, Frederic D (2015) Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats. Retrovirology 12:79
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

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