PEBP 1 suppresses HIV transcription and induces latency by inactivating MAPK / NF ‐κB signaling
2020; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.15252/embr.201949305
ISSN1469-3178
AutoresXinyi Yang, Yanan Wang, Panpan Lu, Yinzhong Shen, Xiaying Zhao, Yuqi Zhu, Zhengtao Jiang, He Yang, Hanyu Pan, Lin Zhao, Yangcheng Zhong, Jing Wang, Zhiming Liang, Xiaoting Shen, Daru Lu, Shibo Jiang, Jianqing Xu, Hao Wu, Hongzhou Lu, Guochun Jiang, Huanzhang Zhu,
Tópico(s)Immune Cell Function and Interaction
ResumoArticle14 September 2020Open Access Source DataTransparent process PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling Xinyi Yang Xinyi Yang 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 Search for more papers by this author Yanan Wang Yanan Wang 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 Search for more papers by this author Panpan Lu Panpan Lu 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 Search for more papers by this author Yinzhong Shen Yinzhong Shen 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 Search for more papers by this author Xiaying Zhao Xiaying Zhao 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 Search for more papers by this author Yuqi Zhu Yuqi Zhu 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 Search for more papers by this author Zhengtao Jiang Zhengtao Jiang 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 Search for more papers by this author He Yang He Yang 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 Search for more papers by this author Hanyu Pan Hanyu Pan 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 Search for more papers by this author Lin Zhao Lin Zhao 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 Search for more papers by this author Yangcheng Zhong Yangcheng Zhong 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 Search for more papers by this author Jing Wang Jing Wang 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 Search for more papers by this author Zhiming Liang Zhiming Liang 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 Search for more papers by this author Xiaoting Shen Xiaoting Shen 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 Search for more papers by this author Daru Lu Daru Lu 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 Search for more papers by this author Shibo Jiang Shibo Jiang 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 Search for more papers by this author Jianqing Xu Jianqing Xu 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 Search for more papers by this author Hao Wu Hao Wu Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, China Search for more papers by this author Hongzhou Lu Hongzhou Lu 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 Search for more papers by this author Guochun Jiang Guochun Jiang UNC HIV Cure Center, Institute of Global Health and Infectious Diseases & Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Huanzhang Zhu Corresponding Author Huanzhang Zhu [email protected] orcid.org/0000-0001-5797-0292 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 Search for more papers by this author Xinyi Yang Xinyi Yang 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 Search for more papers by this author Yanan Wang Yanan Wang 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 Search for more papers by this author Panpan Lu Panpan Lu 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 Search for more papers by this author Yinzhong Shen Yinzhong Shen 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 Search for more papers by this author Xiaying Zhao Xiaying Zhao 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 Search for more papers by this author Yuqi Zhu Yuqi Zhu 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 Search for more papers by this author Zhengtao Jiang Zhengtao Jiang 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 Search for more papers by this author He Yang He Yang 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 Search for more papers by this author Hanyu Pan Hanyu Pan 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 Search for more papers by this author Lin Zhao Lin Zhao 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 Search for more papers by this author Yangcheng Zhong Yangcheng Zhong 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 Search for more papers by this author Jing Wang Jing Wang 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 Search for more papers by this author Zhiming Liang Zhiming Liang 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 Search for more papers by this author Xiaoting Shen Xiaoting Shen 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 Search for more papers by this author Daru Lu Daru Lu 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 Search for more papers by this author Shibo Jiang Shibo Jiang 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 Search for more papers by this author Jianqing Xu Jianqing Xu 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 Search for more papers by this author Hao Wu Hao Wu Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, China Search for more papers by this author Hongzhou Lu Hongzhou Lu 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 Search for more papers by this author Guochun Jiang Guochun Jiang UNC HIV Cure Center, Institute of Global Health and Infectious Diseases & Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Huanzhang Zhu Corresponding Author Huanzhang Zhu [email protected] orcid.org/0000-0001-5797-0292 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 Search for more papers by this author Author Information Xinyi Yang1, Yanan Wang1, Panpan Lu1, Yinzhong Shen2, Xiaying Zhao1, Yuqi Zhu1, Zhengtao Jiang1, He Yang1, Hanyu Pan1, Lin Zhao1, Yangcheng Zhong1, Jing Wang1, Zhiming Liang1, Xiaoting Shen1, Daru Lu1, Shibo Jiang2, Jianqing Xu2, Hao Wu3, Hongzhou Lu2, Guochun Jiang4 and Huanzhang Zhu *,1 1State 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 2Department 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 3Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, China 4UNC HIV Cure Center, Institute of Global Health and Infectious Diseases & Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA *Corresponding author. Tel: +86 021 31246728; E-mail: [email protected] EMBO Reports (2020)21:e49305https://doi.org/10.15252/embr.201949305 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis The latent HIV-1 reservoir prevents efficient viral eradication. The phosphatase PEBP1 plays a critical role in the regulation of NF-κB-dependent pathways governing HIV-1 transcription, and its targeting could control HIV reservoirs in patients. Depletion of PEBP1 leads to the reactivation of HIV-1 in multiple HIV latency models. EGCG induces PEBP1 protein expression and prevents latency reversal in patients receiving antiretroviral therapy. PEBP1 is induced by IFN signaling during HIV-1 infection and promotes HIV latency in primary CD4+ T cells. Introduction Current antiretroviral therapy (ART) has succeeded in reducing human immunodeficiency virus type 1 (HIV-1) to undetectable levels in HIV-1-infected patients (Ruelas & Greene, 2013; Margolis et al, 2016). However, ART alone cannot cure AIDS. The viral reservoirs comprised of latently infected and long-lived resting CD4+ T cells can survive for decades, thereby preventing our current efforts to cure HIV (Finzi et al, 1997; ;Chun et al, 1998; Richman et al, 2009; ;Barouch & Deeks, 2014). These latent HIV reservoirs are considered as the main barrier for viral eradication (Ho et al, 2013; Kim et al, 2018; Pitman et al, 2018; Rojas et al, 2019). In the last few decades, progress has been made to elucidate the molecular mechanisms underlying the establishment of HIV-1 latency, mostly acting at the level of transcriptional suppression of the viral promoter-long terminal repeats (LTR) (Verdin et al, 1993; Bieniasz et al, 1999; Blazkova et al, 2009; Archin et al, 2014; Kumar et al, 2015; Elsheikh et al, 2019). It has been shown that transcriptional blocks to productive HIV-1 replication are associated with multiple layers of regulation, including epigenetic modifications at the HIV-1 LTR, inadequate availability of transcription factors at the HIV LTR, such as NF-κB, positive transcription elongation factor b (P-TEFb or CDK9/CycinT1), HIV-1 Tat, and among others (Mancebo et al, 1997; Bieniasz et al, 1999; Fiume et al, 2012). Many small molecule compounds to target these signaling pathways have been tested to directly reactivate latent HIV-1 (Alexaki et al, 2007; Imai et al, 2010; Beans et al, 2013; Li et al, 2013; Spivak et al, 2014; Søgaard et al, 2015; Boehm et al, 2017; Wang et al, 2017). Unfortunately, none of these latency reversal agents (LRAs) can effectively reduce the reservoir size in patients although latent HIV-1 can be disrupted in vivo (Archin et al, 2012; Søgaard et al, 2015). While an opposing intervention strategy has been proposed to deeply silence the HIV reservoirs (Mousseau et al, 2015; Elsheikh et al, 2019), an effective prevention of viral rebound has not yet been achieved in a clinical or pre-clinical setting (Kessing et al, 2017), indicating that our current understanding of how HIV-1 establishes and maintains its latency remains limited. In recent years, the methodology behind genome editing has greatly expanded with the emergence of the CRISPR/Cas9 system. In 2014, Dr. Zhang's laboratory constructed a lentivirus library to target human genome using array libraries (Shalem et al, 2014). By using the CRISPR/Cas9 library, researchers successfully identified the host factors necessary for viral infection, such as flavivirus, Zika virus, and HIV-1, to invade host cells for their own replication (Ma et al, 2015; Marceau et al, 2016; Savidis et al, 2016; Park et al, 2017; Jin et al, 2018; Huang et al, 2019; Li et al, 2019), which greatly helped us understand the host–pathogen interaction during viral infection. Here, we carried out a CRISPR-based genetic screen in a latently HIV-infected CD4+ T-cell model of latency using a high complexity and whole genome-wide sgRNA library. Among the enriched genes, we identified that PEBP1 or RKIP is associated with the suppression of HIV replication and promotes the establishment of HIV latency. The knockout of PEBP1 gene reactivated latent HIV-1 by inducing Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways in several HIV latency models, including a primary CD4+ T-cell model of HIV latency. Importantly, PEBP1 can directly inhibit HIV-1 infection and induce HIV latency in primary CD4+ T cells. When PEBP1 was induced by a small molecule extracted from Chinese green tea, epigallocatechin-3-gallate (EGCG), the reactivation of HIV latency was effectively blocked in the primary CD4+ T cells isolated from HIV-positive individuals receiving suppressive ART. To our knowledge, this is the first report to elucidate how upstream signaling of the NF-κB pathway is controlled by PEBP1 or RKIP during the establishment of HIV latency. Our study has discovered mechanistically novel insights of HIV latency. The EGCG compound identified in this study could be further investigated as a new tool for therapeutic intervention of HIV latency in the future. Results Genome-wide CRISPR/Cas9 library screening enriches host factors associated with the establishment of HIV latency To identify host factors associated with HIV-1 latency, we conducted a GECKO library screen (Shalem et al, 2014) in the CD4+ T model of HIV latency, C11, which was previously established in our laboratory (Qu et al, 2013; Wang et al, 2017). This GECKO library contains over 120,000 gRNAs targeting 19,050 human genes. The C11 cell line of HIV latency model is derived from CD4+ T cells (Jurkat), which harbors an HIV-1 proviral DNA with a reporter gene encoding green fluorescent protein (GFP). In the latent state, HIV-1 expression in C11 cells is silenced with an expression level of GFP below 2% (Qu et al, 2013; Wang et al, 2017). We prepared the GECKO lentivirus library to infect the C11 HIV latency model with a multiplicity of infection (MOI) of 0.2. The cell line was screened with puromycin (2 μg/ml) selection for 14 days (Fig 1A). Then, roughly 10% of GFP-positive C11 cells were enriched after two rounds of cell sorting (Fig 1A and B). For the positive knockout cells, the targeted sgRNA sequence was confirmed by PCR. Our data showed that the integrated sgRNA was found in both the unselected group and the positive cells sorted in the first or second round of screening (Fig 1C). Under fluorescence microscopy, we confirmed that the sorted C11 cells were GFP positive after gene knockout (Fig 1D). Following genomic DNA (gDNA) extraction from both sorted and unsorted control cells and PCR amplification of each sgRNA sequence, we performed Illumina sequencing to generate read counts for each gene-targeting GECKO construct. The gene of interest was compared with the distribution of the log2 enrichment values of the negative control sgRNAs and the initial control sgRNAs. We found that several genes such as UBB, SERBP1, ZDHHC1,CNTNAP1, and PEBP1 (Beshir et al, 2010; Lin et al, 2010; Oh et al, 2013; Laquerriere et al, 2014; Bolger, 2017) are among the hotspot candidate genes with high abundance of sgRNAs, along with BRD2 and BRD4 genes which are known as HIV latency-associated genes (Boehm et al, 2013) (Fig 1E and Dataset EV1). Figure 1. A pooled, genome-wide CRISPR screen for candidate genes involved in HIV-1 latency The outline of the genome-wide CRISPR screen strategy. C11 cells were infected with a lentiviral library containing Cas9 proteins and sgRNAs that target 19,050 human genes. After fourteen days of puromycin selection, genomic DNA was extracted from GFP-positive cells after two rounds of sorting. The candidate genes were then identified by next generation sequencing. Flow cytometry of cells infected with the lentiCRISPR v2.0 library where the expression of GFP indicates latency reactivation. Target genes were enriched through two-round sorting. Continuously cultured C11 latent cells infected with the lentiCRISPRv2.0 served as control. Blue dots and the encircled area represent the GFP-positive cells for flow cytometry analysis. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection. Before next generation sequencing, the GFP expression in two-round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm. Fold change (Log2) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted. Download figure Download PowerPoint Validation of candidate HIV latency inducing genes To validate whether these candidate genes are related to HIV-1 transcription, we directly infected HIV latency model of C11 cells with CRISPR/Cas9 lentiviruses containing sgRNAs which target these candidate genes, followed by clone selection with puromycin (2 μg/ml) for 14 days. Among these top-hit genes, we found that the knockout of PEBP1 gene significantly induced the reactivation of latent HIV-1 (roughly 20%), which was higher than the cells with the known HIV latency-related gene BRD2 or BRD4 knockout (Fig 2A), supporting our idea that PEBP1 gene is associated with HIV latency. A similar effect was observed after PEBP1 was knocked out in two other HIV latency models, J-Lat 10.6, and ACH2 cells (Fig 2B and C). To prove that PEBP1 gene was indeed knocked out in these latency disrupted C11 cells, we sequenced the genomic targeting sites in these PEBP1 knockout cell clones by genomic DNA sequencing. We found that PEBP1 gene was deleted in the target sites of PEBP1 sgRNA1 with different forms of indels (Fig 2D). The PEBP1 protein was nearly undetectable after the knockout of PEBP1 gene targeted with PEBP1-specific guide RNAs (Fig 2E). In order to further determine the effect of PEBP1 on HIV-1 latency, a random PEBP1 monoclonal cell line was obtained by flow sorting (Streaming data is not shown). We sequenced the genomic target sites of five different monoclonal cell lines by genomic DNA sequencing and found that PEBP1 gene was successfully knocked out in all five monoclonal cell lines (Fig EV1A). With Western blot, we confirmed that PEBP1 protein expression significantly decreased (Fig EV1B). The levels of HIV-1 transcription were similar among these monoclonal cell lines and cells without clone screening after gene knockout (Figs 2A and EV1C). Cell proliferation and apoptosis were also evaluated in PEBP1-KO-C11 cells and mock C11 cells by CCK8 assay and TUNEL staining. We found the proliferation rate was slightly higher in PEBP1-KO-C11 cells than that of mock knockout C11; however, PEBP1 knockout did not affect apoptosis (Fig EV2A and B). Taken together, our data suggest that PEBP1 is a new HIV latency-associated gene. Figure 2. Validation of the candidate genes screened from the lentiCRISPR v2.0 library in HIV-1 latently infected cell lines A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 μg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation. B, C. The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) and ACH2 (C) models of HIV latency. GFP expression in J-Lat 10.6 cells and p24 in ACH2 cells was analyzed by flow cytometry and ELISA, respectively. D. PEBP1 was deleted after CRISPR/Cas9 knockout. PCR products related to PEBP1 from control or PEBP1 knockout cells were cloned and then sequenced. PEBP1-sg1 target sites are shown in red letters. Dashes indicate the deleted bases relative to the wild-type PEBP1 gene sequence. E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control. Data information: Data represent the mean ± SD of three independent experiments (n = 3) and were analyzed with t-test. ***P < 0.001. Source data are available online for this figure. Source Data for Figure 2 [embr201949305-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Monoclonal analysis of PEBP1-KO-C11 cell line Sequencing of PEBP1 PCR products after monoclonal sorting. The PCR products of PEBP1 gene were cloned then sequenced. PEBP1-sg1 target sites were shown in red letters. Dashes indicate the deleted bases relative to the wild-type sequence. 1-1, 1-2, 1-3, 1-4, and 1-5 represent different monoclonal cell lines, respectively. The expression of PEBP1 was detected by Western blot in individual monoclonal cell lines. The proportion of GFP-positive cells was detected by flow cytometry after PEBP1 knockout. The levels of nuclear NF-κB/p65 protein were analyzed by Western blot in different monoclonal cell lines and the mock C11 cells. Data information: Data represent the mean ± SD of three independent experiments (n = 3). Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Effect of PEBP1 knockout on the proliferation and apoptosis of latently infected C11 cells. The NF-κB nuclear entry inhibitor SC75741 prevents the reactivation of HIV-1 after PEBP1 knockout in C11 cell model of latency Cell proliferation of PEBP1-KO-C11 cells and mock C11 cells was analyzed by CCK-8 levels. Apoptosis of PEBP1-KO-C11 cells and mock C11 cells was measured by TUNEL staining followed by flow cytometry. The expression of GFP in the cells was measured by flow cytometry in C11-PEBP1-KO and mock C11 cells after treatment with NF-κB inhibitor SC75741 for 48 h. The expression of GFP in the cells was detected by flow cytometry in C11-PEBP1-KO and mock C11 cells after treatment with 1 μM of NF-κB inhibitor SC75741 at different time points. Data information: Data represent the mean ± SD of three independent experiments and were analyzed with t-test (n = 3). Download figure Download PowerPoint The PEBP1/Raf1 protein complex promotes HIV-1 latency through inactivation of MAPK and NF-κB signaling pathways in CD4+ T cells Previous studies indicated that PEBP1, also known as Raf1 kinase inhibitor protein RKIP, is involved in MAPK and NF-κB signaling pathways via interaction with Raf1 and IKK in cancer cells (Yeung et al, 2001; Lee et al, 2006; Tavel et al, 2012; Wei et al, 2015). When PEBP1 is defective, the MAPK and NF-κB signaling pathways are activated, leading to the translocation of NF-κB from the cytoplasm to the nucleus (Lin et al, 2010). It has been demonstrated that inactivation of NF-κB signaling is essential for the establishment of HIV latency while activation of NF-κB signaling pathway disrupts latent HIV-1 (Fiume et al, 2012). However, it is not clear how cytoplasmic sequestration of NF-κB is regulated during the establishment of HIV latency in CD4+ T cells. We hypothesized that PEBP1 gene is essential for the upstream signaling of NF-κB by preventing its translocation into the nucleus, therefore turning off the transcription of HIV for transcriptional silence. We found that both the expression of PEBP1 mRNA and protein in the C11 HIV latent cell line were significantly higher than its parental Jurkat cell line (Fig 3A). To see whether the expression of PEBP1 causes silencing of HIV by inactivating NF-κB signaling pathway and whether this is through its interacting with Raf1 and IKK, we performed a co-immunoprecipitation (Co-IP) assay and revealed that PEBP1 not only interacted with Raf1 but also with IKK in C11 HIV latency cell model (Fig 3B). After PEBP1 was knocked out, the expression levels of MEK1/2, RSK, ERK1/2, IKKβ, and IKBα was marginally changed; however, their phosphorylation levels increased significantly, indicating that the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways were indeed activated when PEBP1 gene was knocked out (Fig 3C). The level of NF-κB/p65 significantly increased in the nucleus when PEBP1 gene was knocked out (Fig 3D), leading to a significant binding of NF-κB/p65 to HIV-1 LTR in C11-PEBP1-KO cells but not in the control C11 cells (Fig 3E). In addition
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