CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models

Anurag Rathore,Sho Iketani,David D Ho,Vincent Sahi,Pengfei Wang,Manxue Jia

doi:10.1038/s41598-020-62375-3

Anurag Rathore, Sho Iketani + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-62375-3

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Abstract

The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.

Highlights

HIV-1 infection can be durably suppressed by combination antiretroviral therapy, the current standard of treatment[1]
Recent clustered regularly interspersed short palindromic repeats (CRISPR)-based approaches include a report by Li et al that showed enrichment of single guide RNAs in 2D10 cells in a CRISPR interference (CRISPRi) screen that targeted suppressors of HIV-1 transcription (NFKBIA and CYLD), proteasome subunits (PSMD1, PSMD3, PSMD8) and a protein found in a transcriptional corepressor complex with HDAC1 (GON4L)[22]
Further gene enrichment and network analysis highlighted a potential network of HIV-1 latency factors, which were verified in an additional HIV-1 latency cell line, resulting in the identification of the genes IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potentially involved in HIV-1 latency

Summary

Introduction

HIV-1 infection can be durably suppressed by combination antiretroviral therapy (cART), the current standard of treatment[1]. Recent CRISPR-based approaches include a report by Li et al that showed enrichment of single guide RNAs (sgRNAs) in 2D10 cells in a CRISPR interference (CRISPRi) screen that targeted suppressors of HIV-1 transcription (NFKBIA and CYLD), proteasome subunits (PSMD1, PSMD3, PSMD8) and a protein found in a transcriptional corepressor complex with HDAC1 (GON4L)[22] In another example, Huang et al used a CRISPR-Cas[9] knockout screen targeting nuclear proteins in the J-Lat A2 cell line to identify MYC-induced nuclear antigen 53 (MINA53) as a novel latency promoting gene, possibly by altering methylation levels[21]. We have identified several factors which may be involved in HIV-1 latency, and we propose that targeting such factors may serve as novel avenues for HIV-1 latency reversal

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