Abstract
Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.
| Original language | English (US) |
|---|---|
| Article number | 1752 |
| Journal | Nature communications |
| Volume | 13 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2022 |
Funding
We thank members of the Krogan and Marson groups for helpful comments and discussion. This research was supported by a Mathilde Krim amfAR grant using funds raised by generationCURE (109504-61-RKRL, J.F.H.), a Mathilde Krim amfAR grant in biomedical research (1110189-69-RKRL, U.R.), NIH/NIGMS funding for the HIV Accessory & Regulatory Complexes (HARC) Center (P50 GM082250, J.A.D., J.F.H., A.M., and N.J.K.), NIH funding for the study of innate immune responses to intracellular pathogens (R01 AI120694 & P01 AI063302, N.J.K.), NIH funding for the Third Coast Center for AIDS Research (P30 AI117943, J.F.H.), NIH funding for the UCSF-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR, P30 AI027763), NIH funding for the UCSF Medical Scientist Training Program (T32GM007618, J.H.), an NIH/NIDA grant (DP2 DA042423-01, A.M.), several NIH/NIAID grants for HIV research (K22 AI136691, R01 AI165236, and R01 AI150998, J.F.H.), an NIH/NIDA grant (DP2 DA042423-01, A.M.), and funding from Gilead Sciences (A.M.). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is an investigator at the Chan Zuckerberg Biohub, and is a recipient of The Cancer Research Institute (CRI) Lloyd J. Old STAR grant. The Marson lab has received funds from the Innovative Genomics Institute (IGI), the Simons Foundation, and the Parker Institute for Cancer Immunotherapy (PICI). The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HeLa–CD4–LTR–β-gal (Cat #1470) from Dr. Michael Emerman; pBR431eG-nef + (Cat #11349) from Dr. Frank Kirchhoff. Special thanks to A. Roguev for helpful discussion on edge correction and data analysis; M.J. Montano and the Gladstone BSL3 facility; E. Brookes, M. Hall, and O. Cantada at Lonza Bioscience for their support with the electroporation technology; D.H. Chow at Dharmacon for his support with gRNA synthesis; T.W. Brown and the flow-cytometry team at ThermoFisher; C. Jeans and the University of California, Berkeley Macrolab for the production of Cas9 protein; K. Pollard for providing a platform for collaboration; and M. Soucheray, D. Sainz, and G. Ehle for their foresight. We thank members of the Krogan and Marson groups for helpful comments and discussion. This research was supported by a Mathilde Krim amfAR grant using funds raised by generationCURE (109504-61-RKRL, J.F.H.),?a Mathilde Krim amfAR grant in biomedical research (1110189-69-RKRL, U.R.),?NIH/NIGMS funding for the HIV Accessory & Regulatory Complexes (HARC) Center (P50 GM082250, J.A.D., J.F.H., A.M., and N.J.K.), NIH funding for the study of innate immune responses to intracellular pathogens (R01 AI120694 & P01 AI063302, N.J.K.), NIH funding for the Third Coast Center for AIDS Research (P30 AI117943, J.F.H.), NIH funding for the UCSF-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR, P30 AI027763), NIH funding for the UCSF Medical Scientist Training Program (T32GM007618, J.H.), an NIH/NIDA grant (DP2 DA042423-01, A.M.), several NIH/NIAID grants for HIV research (K22 AI136691, R01 AI165236, and R01 AI150998, J.F.H.), an NIH/NIDA grant (DP2 DA042423-01, A.M.), and funding from Gilead Sciences (A.M.). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is an investigator at the Chan Zuckerberg Biohub, and is a recipient of The Cancer Research Institute (CRI) Lloyd J. Old STAR grant. The Marson lab has received funds from the Innovative Genomics Institute (IGI), the Simons Foundation, and the Parker Institute for Cancer Immunotherapy (PICI). The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HeLa?CD4?LTR??-gal (Cat #1470) from Dr. Michael Emerman; pBR431eG-nef?+?(Cat #11349) from Dr. Frank Kirchhoff. Special thanks to A. Roguev for helpful discussion on edge correction and data analysis; M.J. Montano and the Gladstone BSL3 facility; E. Brookes, M. Hall, and O. Cantada at Lonza Bioscience for their support with the electroporation technology; D.H. Chow at Dharmacon for his support with gRNA synthesis; T.W. Brown and the flow-cytometry team at ThermoFisher; C. Jeans and the University of California, Berkeley Macrolab for the production of Cas9 protein; K. Pollard for providing a platform for collaboration; and M. Soucheray, D. Sainz, and G. Ehle for their foresight.
ASJC Scopus subject areas
- General Chemistry
- General Biochemistry, Genetics and Molecular Biology
- General Physics and Astronomy
