PEBP 1 suppresses HIV transcription and induces latency by inactivating MAPK / NF ‐κB signaling
Authors: Yinzhong Shen
Zhengtao Jiang
Huanzhang Zhu
Hao Wu
Lin Zhao
Guochun Jiang
Jianqing Xu
Yuqi Zhu
Xinyi Yang
Zhiming Liang
Xiaoting Shen
Panpan Lu
Daru Lu
Shibo Jiang
He Yang
Jing Wang
Yangcheng Zhong
Yanan Wang
Xiaying Zhao
Hanyu Pan
Hongzhou Lu
Journal: EMBO reports (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: CRISPR-Cas9; HIV latency; NF-κB; PEBP1; genome-wide screening.
Affiliations: State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology Ministry of Education Institute of Genetics School of Life Sciences Fudan University Shanghai China, Department of Infectious Disease Key Laboratory of Medical Molecular Virology of Ministry of Education/Health School of Basic Medical Sciences and Shanghai Public Health Clinical Center Fudan University Shanghai China, Center for Infectious Diseases Beijing You'an Hospital Capital Medical University Beijing China, UNC HIV Cure Center Institute of Global Health and Infectious Diseases &amp; Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill Chapel Hill NC USA

Abstract:  The latent HIV-1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV-1 establishes latency is incomplete. Here, by performing a genome-wide CRISPR-Cas9 knockout library screen, we identify phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV-1 in multiple models of latency. Mechanistically, PEBP1 de-phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF-κB in the cytoplasm, which transcriptionally inactivates HIV-1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin-3-gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF-κB, thereby suppressing HIV-1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF-κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future. 

Retracted: No

PubPeer comments:
 1.tIn Figure 6F, the authors claimed that they knocked out PEBP1 in the primary CD4+ T-cell model of latency by lentiviral-vectored CRISPR Cas9. However, this experiment could not have been possible as the lentiviral transduction rates and knockout efficiency is very low in primary CD4+ T cells via LV-CRISPR Cas9[1-4]. In addition, since primary cells do not differentiation or grow, it will be extremely difficult to screen out mutant clones. 2.tMoreover, the nonactivated human T cells used in Figure 6F are very difficult to infect with lentiviruses and let alone to be efficiently edited by lentiviral-vectored CRISPR Cas9. ![file] Reference: [1] Liu Z , Chen S , Jin X , et al. Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4 + T cells from HIV-1 infection[J]. Cell and Bioence, 2017, 7(1):47. [2] Yu S , Yao Y , Xiao H , et al. Simultaneous knockout of CXCR4 and CCR5 genes in CD4+ T cells via CRISPR Cas9 confers resistance to both X4- and R5-tropic HIV-1 infection[J]. Human Gene Therapy, 2017:hum.2017.032. [3] Schumann K , Lin S , Boyer E , et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins[J]. Proceedings of the National Academy of ences, 2015, 112(33):10437-10442. [4] Seki A , Rutz S . Optimized RNP transfection for highly efficient CRISPR Cas9-mediated gene knockout in primary T cells[J]. Journal of Experimental Medicine, 2018:jem.20171626. , 
 #1 Thank you very much for your attention. when we designed this experiment, we referenced Dr. Kamel Khaliliu2019s research[1]. In this paper, Dr. Kamel Khaliliu2019s research group used lentivirus-based CRISPR to infect primary CD4+ T lymphocytes, targeted to knock out HIV-1 in the cells, and showed good excision efficiency (Figure 5). The experimental procedure we designed is almost identical to the experimental procedure of this article (except for the targeted cleavage site and the time point of virus infection). During we carried this experiment, we did find that the efficiency of gene editing after the Cas9-expressing lentivirus infects primary CD4+ T lymphocytes is low. Therefore, we have made improvements in many ways, including high MOI, using the latest reagents to promote infection instead of plybrene, improving the primary cell culture system so that the cells are in a better state, etc., At the same time, we still use nanoluciferase with higher detection sensitivity as the detection index. In addition, after sorting the primary CD4+ T lymphocytes, we also used CD3 CD28 magnetic beads for cell activation. The related content is also mentioned in the material method. [1]Kaminski R , Chen Y , Fischer T , et al. Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR Cas9 Gene Editing[J]. entific Reports, 2016, 6:22555. , 
 #1 Thank you very much for your attention. when we designed this experiment, we referenced Dr. Kamel Khaliliu2019s research[1]. In this paper, Dr. Kamel Khaliliu2019s research group used lentivirus-based CRISPR to infect primary CD4+ T lymphocytes, targeted to knock out HIV-1 in the cells, and showed good excision efficiency (Figure 5). The experimental procedure we designed is almost identical to the experimental procedure of this article (except for the targeted cleavage site and the time point of virus infection). During we carried this experiment, we did find that the efficiency of gene editing after the Cas9-expressing lentivirus infects primary CD4+ T lymphocytes is low. Therefore, we have made improvements in many ways, including high MOI, using the latest reagents to promote infection instead of plybrene, improving the primary cell culture system so that the cells are in a better state, etc., At the same time, we still use nanoluciferase with higher detection sensitivity as the detection index. In addition, after sorting the primary CD4+ T lymphocytes, we also used CD3 CD28 magnetic beads for cell activation. The related content is also mentioned in the material method. ,