CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells
ABSTRACTDuring 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 activate...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2024-04-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mbio.02222-23 |
| _version_ | 1797215306432643072 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Ujjwal Rathore |
| collection | DOAJ |
| description | ABSTRACTDuring 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.IMPORTANCEHIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell’s ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or “latent” cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells. |
| first_indexed | 2024-04-24T11:27:58Z |
| format | Article |
| id | doaj.art-a68bf9f80cd74600b676bdd895184026 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-24T11:27:58Z |
| publishDate | 2024-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-a68bf9f80cd74600b676bdd8951840262024-04-10T13:01:15ZengAmerican Society for MicrobiologymBio2150-75112024-04-0115410.1128/mbio.02222-23CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cellsUjjwal Rathore0Paige Haas1Vigneshwari Easwar Kumar2Joseph Hiatt3Kelsey M. Haas4Mehdi Bouhaddou5Danielle L. Swaney6Erica Stevenson7Lorena Zuliani-Alvarez8Michael J. McGregor9Autumn Turner-Groth10Charles Ochieng' Olwal11Yaw Bediako12Hannes Braberg13Margaret Soucheray14Melanie Ott15Manon Eckhardt16Judd F. Hultquist17Alexander Marson18Robyn M. Kaake19Nevan J. Krogan20Gladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAWest African Centre for Cell Biology of Infectious Pathogens (WACCBIP), College of Basic and Applied Sciences, University of Ghana, Accra, GhanaWest African Centre for Cell Biology of Infectious Pathogens (WACCBIP), College of Basic and Applied Sciences, University of Ghana, Accra, GhanaGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USADivision of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAGladstone Institutes, San Francisco, California, USAABSTRACTDuring 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.IMPORTANCEHIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell’s ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or “latent” cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.https://journals.asm.org/doi/10.1128/mbio.02222-23human immunodeficiency virusubiquitin E3 ligasesHIV latencyCRISPR screenresting primary T cells |
| spellingShingle | 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 CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells mBio human immunodeficiency virus ubiquitin E3 ligases HIV latency CRISPR screen resting primary T cells |
| title | CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells |
| title_full | CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells |
| title_fullStr | CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells |
| title_full_unstemmed | CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells |
| title_short | CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells |
| title_sort | crispr cas9 screen of e3 ubiquitin ligases identifies traf2 and uhrf1 as regulators of hiv latency in primary human t cells |
| topic | human immunodeficiency virus ubiquitin E3 ligases HIV latency CRISPR screen resting primary T cells |
| url | https://journals.asm.org/doi/10.1128/mbio.02222-23 |
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