CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells

Ujjwal Rathore; Paige Haas; Vigneshwari Easwar Kumar; Joseph Hiatt; Kelsey M. Haas; Mehdi Bouhaddou; Danielle L. Swaney; Erica Stevenson; Lorena Zuliani-Alvarez; Michael J. McGregor; Autumn Turner-Groth; Charles Ochieng Olwal; Yaw Bediako; Hannes Braberg; Margaret Soucheray; Melanie Ott; Manon Eckhardt; Judd F. Hultquist; Alexander Marson; Robyn M. Kaake; Nevan J. Krogan

doi:10.1128/mbio.02222-23

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells

Ujjwal Rathore, Paige Haas, Vigneshwari Easwar Kumar, Joseph Hiatt, Kelsey M. Haas, Mehdi Bouhaddou, Danielle L. Swaney, Erica Stevenson, Lorena Zuliani-Alvarez, Michael J. McGregor, Autumn Turner-Groth, Charles Ochieng Olwal, Yaw Bediako, Hannes Braberg, Margaret Soucheray, Melanie Ott, Manon Eckhardt, Judd F. Hultquist, Alexander Marson, Robyn M. Kaake^*Nevan J. Krogan^*

^*Corresponding author for this work

Medicine, Infectious Diseases Division

Research output: Contribution to journal › Article › peer-review

Abstract

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.

Original language	English (US)
Journal	mBio
Volume	15
Issue number	4
DOIs	https://doi.org/10.1128/mbio.02222-23
State	Published - Apr 2024

Funding

This research was supported by a Mathilde Krim amfAR fellowship in biomedical research grant (1110189-69-RKRL, U.R.), NIH/NIAID funding for the HIV Accessory & Regulatory Complexes (HARC) Center (P50 AI150476, 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 UCSF-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR, P30 AI027763), NIH/NIDA grant (DP2 DA042423-01, A.M.), California HIV/AIDS Research Program (CHRP) Basic Biomedical Sciences Discovery research grant (H22BD4486, U.R.), funding from Gilead Sciences (A.M.), and funding from James B. Pendleton Charitable Trust to the Gladstone Institutes Flow Cytometry Core Facility. A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, was 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).

Keywords

CRISPR screen
HIV latency
human immunodeficiency virus
resting primary T cells
ubiquitin E3 ligases

ASJC Scopus subject areas

Microbiology
Virology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1128/mbio.02222-23

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Rathore, U., Haas, P., Kumar, V. E., Hiatt, J., Haas, K. M., Bouhaddou, M., Swaney, D. L., Stevenson, E., Zuliani-Alvarez, L., McGregor, M. J., Turner-Groth, A., Olwal, C. O., Bediako, Y., Braberg, H., Soucheray, M., Ott, M., Eckhardt, M., Hultquist, J. F., Marson, A., ... Krogan, N. J. (2024). CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells. mBio, 15(4). https://doi.org/10.1128/mbio.02222-23

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title = "CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells",

abstract = "During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.",

keywords = "CRISPR screen, HIV latency, human immunodeficiency virus, resting primary T cells, ubiquitin E3 ligases",

author = "Ujjwal Rathore and Paige Haas and Kumar, {Vigneshwari Easwar} and Joseph Hiatt and Haas, {Kelsey M.} and Mehdi Bouhaddou and Swaney, {Danielle L.} and Erica Stevenson and Lorena Zuliani-Alvarez and McGregor, {Michael J.} and Autumn Turner-Groth and Olwal, {Charles Ochieng} and Yaw Bediako and Hannes Braberg and Margaret Soucheray and Melanie Ott and Manon Eckhardt and Hultquist, {Judd F.} and Alexander Marson and Kaake, {Robyn M.} and Krogan, {Nevan J.}",

note = "Publisher Copyright: {\textcopyright} 2024 Rathore et al.",

year = "2024",

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doi = "10.1128/mbio.02222-23",

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Rathore, U, Haas, P, Kumar, VE, Hiatt, J, Haas, KM, Bouhaddou, M, Swaney, DL, Stevenson, E, Zuliani-Alvarez, L, McGregor, MJ, Turner-Groth, A, Olwal, CO, Bediako, Y, Braberg, H, Soucheray, M, Ott, M, Eckhardt, M, Hultquist, JF, Marson, A, Kaake, RM & Krogan, NJ 2024, 'CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells', mBio, vol. 15, no. 4. https://doi.org/10.1128/mbio.02222-23

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T1 - CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells

AU - Rathore, Ujjwal

AU - Haas, Paige

AU - Kumar, Vigneshwari Easwar

AU - Hiatt, Joseph

AU - Haas, Kelsey M.

AU - Bouhaddou, Mehdi

AU - Swaney, Danielle L.

AU - Stevenson, Erica

AU - Zuliani-Alvarez, Lorena

AU - McGregor, Michael J.

AU - Turner-Groth, Autumn

AU - Olwal, Charles Ochieng

AU - Bediako, Yaw

AU - Braberg, Hannes

AU - Soucheray, Margaret

AU - Ott, Melanie

AU - Eckhardt, Manon

AU - Hultquist, Judd F.

AU - Marson, Alexander

AU - Kaake, Robyn M.

AU - Krogan, Nevan J.

PY - 2024/4

Y1 - 2024/4

N2 - During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.

AB - During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.

KW - CRISPR screen

KW - HIV latency

KW - human immunodeficiency virus

KW - resting primary T cells

KW - ubiquitin E3 ligases

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ER -

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells

Abstract

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UN SDGs

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