Despite 30 years of AIDS research, there is still no robust, reverse-genetic system for studying the in vivo function of human genes that regulate HIV-1 replication, pathogenesis, and immunity. As the number of human genes suspected of influencing HIV-1 skyrockets, the need for such technology has never been greater. This proposal will address technical hurdles that must be overcome before such an experimental system can be realized. RNAi revolutionized how molecular biologists study human gene function but lack of reproducibility and the inability to assess allelic variants are some of the limitations with this methodology. Mouse gene knockout technology is superb at unambiguously assigning function to particular mammalian genes. Unfortunately, many human genes are not shared with the mouse, including APOBEC3G and TRIM5alpha, two genes that potently restrict HIV-1 replication in tissue culture. Additionally, HIV-1 does not replicate in mouse cells. The project proposed here will develop tools for targeted gene replacement by homologous recombination in cells of the human immune system and for the functional assessment of these modified cells within the context of an in vivo model for HIV-1 transmission, replication, immunity, and AIDS pathogenesis. Towards this end, we will exploit technical developments from the past ten years, including derivation of human embryonic stem cells, reprogramming of human somatic cells into induced pluripotent stem cells, improved reconstitution of immunodeficient mice with human hematopoietic stem cells, development of lentiviral vectors that permit efficient delivery of DNA to transfection-resistant cells, and designer nucleases to stimulate homologous recombination. Development of a perpetual source of isogenic hematopoietic stem cells that can be genetically modified, along with improvements in humanized mouse models, will permit us to draw firm conclusions concerning the function of particular human genes - or of particular alleles - in hematopoietic development, immune function, and in HIV-1 replication and pathogenesis.

Public Health Relevance

Our proposal addresses the critical need for tools that allow targeted disruption of select human genes, such that the importance of these genes can be tested in experimental models of HIV-1 infection and HIV-1-associated pathology. The proposed experiments are expected to yield critical information concerning how HIV-1 interacts with the innate immune system, avoids elimination by the acquired immune system, and causes AIDS. Such information is critical for preventing disease progression in HIV-1-infected drug abusers and for developing vaccination strategies that protect them from HIV-1 acquisition.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
Application #
Study Section
Special Emphasis Panel (ZDA1)
Program Officer
Satterlee, John S
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
Zip Code
Derr, Alan; Yang, Chaoxing; Zilionis, Rapolas et al. (2016) End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data. Genome Res 26:1397-1410
Chen, Nan-Yu; Zhou, Lihong; Gane, Paul J et al. (2016) HIV-1 capsid is involved in post-nuclear entry steps. Retrovirology 13:28
Diehl, William E; Lin, Aaron E; Grubaugh, Nathan D et al. (2016) Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic. Cell 167:1088-1098.e6
Bilbao, Aivett; Zhang, Ying; Varesio, Emmanuel et al. (2015) Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS. J Proteome Res 14:4581-93
Pizzato, Massimo; McCauley, Sean Matthew; Neagu, Martha R et al. (2015) Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration. PLoS Pathog 11:e1005050
Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique et al. (2015) Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies. Anal Biochem 484:40-50
Lascano, Josefina; Uchil, Pradeep D; Mothes, Walther et al. (2015) TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling. J Virol 90:308-16
Rosa, Annachiara; Chande, Ajit; Ziglio, Serena et al. (2015) HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 526:212-7
De Iaco, Alberto; Luban, Jeremy (2014) Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA. Retrovirology 11:11
Singh, Ravesh; Patel, Vinod; Mureithi, Marianne W et al. (2014) TRIM5α and TRIM22 are differentially regulated according to HIV-1 infection phase and compartment. J Virol 88:4291-303

Showing the most recent 10 out of 20 publications