Favored Sites for HIV cDNA Integration in the Human Genome Abstract In order to replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. The question of how retroviruses select genomic sites for DNA integration has long interested retrovirologists, because studies in this area provide a unique window on replication mechanism and inform design of potential antiviral therapy. We have investigated integration targeting in detail, taking advantage of new high-throughput sequencing and bioinformatic methods. Studies of integration targeting have emphasized the importance of a new cellular factor LEDGF/p75, which has in turn suggested new routes to designing inhibitors. Highlights of published data from the previous funding period include the findings that 1) HIV favors integration in active transcription units, 2) other retroviruses, in contrast, show different favored genomic integration sites, 3) the cellular protein PSIP1/LEDGF/p75, which binds tightly to HIV integrase (IN), helps guide HIV integration into transcription units, 4) swapping the MLV IN coding region into HIV causes the HIVmIN chimera to integrate with the specificity of MLV, 5) applying the ultra-high throughput pyrosequencing method has allowed 40,761 unique HIV integration sites to be determined, revealing that integration takes place on histones in chromosomal DNA and that histone post-translational modifications are important determinants of HIV integration site selection in vivo.
Our Specific Aims for the renewal are as follows.
In Aim 1, we will study integration site selection by tethering of integration complexes to cellular factors. We will extend our studies of the HIV IN binding protein PSIP1/LEDGF/p75 to investigate the mechanisms by which this factor promotes integration efficiency and guides integration targeting. We will also use genomic methods to identify additional candidate factors important in HIV integration, and biochemical methods to study their mechanisms.
In Aim 2, we will investigate the influence of chromatin structure on integration, with a focus on extending the observations from pyrosequencing data mentioned above.
In Aim 3, we will investigate the possible role of cell cycle progression in integration targeting. In the previous funding cycle, our data sets and methods were used extensively by other laboratories--in the renewal, we aim to contribute an even richer set of resources to the field.

Public Health Relevance

The attached grant application is designed to improve our understanding of HIV DNA integration. The integration system is a target for antiviral agents, and the proposed experiments should provide information directly useful for designing inhibitors.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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AIDS Molecular and Cellular Biology Study Section (AMCB)
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Sharma, Opendra K
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University of Pennsylvania
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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
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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
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
Manganaro, Lara; Pache, Lars; Herrmann, Tobias et al. (2014) Tumor suppressor cylindromatosis (CYLD) controls HIV transcription in an NF-κB-dependent manner. J Virol 88:7528-40
Craigie, Robert; Bushman, Frederic D (2014) Host Factors in Retroviral Integration and the Selection of Integration Target Sites. Microbiol Spectr 2:

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