PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling

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.

Keywords: CRISPR-Cas9; HIV latency; NF-κB; PEBP1; genome-wide screening.

Figure 1. A pooled, genome‐wide CRISPR screen for candidate genes involved in HIV‐1 latency

1. 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.

2. 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.

3. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection.

4. Before next generation sequencing, the GFP expression in two‐round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm.

5. Fold change (Log₂) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted.

Figure 2. Validation of the candidate genes screened from the lentiCRISPR v2.0 library in HIV‐1 latently infected cell lines

1. 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.

2. 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.

3. 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.

4. 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.

Figure EV1. Monoclonal analysis of PEBP1‐KO‐C11 cell line

1. 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.

2. The expression of PEBP1 was detected by Western blot in individual monoclonal cell lines.

3. The proportion of GFP‐positive cells was detected by flow cytometry after PEBP1 knockout.

4. 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.

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

1. Cell proliferation of PEBP1‐KO-C11 cells and mock C11 cells was analyzed by CCK‐8 levels.

2. Apoptosis of PEBP1‐KO-C11 cells and mock C11 cells was measured by TUNEL staining followed by flow cytometry.

3. 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.

4. 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).

Figure 3. PEBP1 inhibits HIV‐1 reactivation by inactivating Raf1/IKK/NF‐κB signaling pathways

1. A

   PEBP1 expression was measured by qPCR (left panel) and Western blot (right panel) in the latently infected C11 cells and the parental Jurkat cells.

2. B

   The immunoprecipitation assay was performed in C11 cell lysates with anti‐PEBP1 antibody followed by Western blot with anti‐Raf1 or anti‐IKK antibodies.

3. C, D

   Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) or the levels of NF‐κB/p65 protein in nucleus (D) were analyzed by Western blot in C11‐PEBP1‐KO cells and mock C11 cells.

4. E

   NF‐κB/p65 protein recruitment into HIV LTR was analyzed by ChIP‐qPCR with specific primers targeting the HIV LTR after normalization to the input.

5. F

   The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells. 293T cells were co‐transfected with PEBP1‐sg1 alone or PEBP1‐sg1 with HIV‐1 LTR‐empty plasmids, HIV‐1 wild‐type LTR‐luciferase plasmids, HIV‐1 LTR‐Δsp1‐luciferase, HIV‐1 LTR‐ΔNF‐κB‐luciferase, HIV‐1 LTR‐ΔAp1‐luciferase, or HIV‐1 LTR‐ΔYY1‐luciferase. Transcription of HIV‐1 was determined by luciferase reporter assay.

Data information: Data are normalized to the HIV LTR‐empty plasmid transfection group. Data represent the mean ± SD of three independent experiments (n = 3) and were analyzed with t‐test. *P < 0.05, ***P < 0.001.Source data are available online for this figure.

Figure 4. PEBP1 restricts HIV‐1 transcription to induce HIV latency

1. A

   The expression of PEBP1 was analyzed by Western blot in TZM‐bl cells infected with mock, Lv‐PCDH-empty, or Lv‐PCDH-PEBP1 plasmids.

2. B, C

   Overexpression of PEBP1 suppressed HIV‐1 replication. TZM‐bl cells were transfected with Lv-PCDH‐empty or Lv‐PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV‐1 was evaluated 72 h post‐infection by luciferase activity (B) and levels of p24 in the supernatants (C).

3. D

   The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.

4. E

   Induction of PEBP1 by EGCG or DHA in HIV‐1 infected primary CD4⁺ T cells. Primary CD4⁺ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV‐1 infection. Similar to Panel B, HIV‐1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy‐two hours post‐treatment with 10 μM EGCG or DHA, the expression of PEBP1 was detected by qPCR.

5. F

   The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4⁺ T cells. The primary CD4⁺ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV‐1 infection. Similar to Panel B and E, HIV‐1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV‐1-infected CD4⁺ T cell were collected 1, 2, 3 or 4 days post‐infection. Replication of HIV‐1 was analyzed by p24 ELISA.

Data information: Data represent the mean ± SD of three independent experiments (n = 3) and were analyzed with t‐test and compared with TZM‐bl cells infected with Lv‐PCDH‐empty vector or mock. **P < 0.01; ***P < 0.001.Source data are available online for this figure.

Figure 5. PEBP1 induction by EGCG inhibits the reactivation of latent HIV in primary CD4⁺ T cells isolated from patients receiving ART

1. EGCG inhibited α‐CD3/CD28 reactivation of latent HIV‐1. Primary CD4⁺ T cells were isolated from HIV‐1-positive patients on antiretroviral therapy (n = 5, P1–P5). Primary CD4⁺ T cells were treated with α‐CD3/CD28 alone or α‐CD3/CD28 and 10 μM EGCG. Cell‐associated RNA was extracted 7 days post‐treatment. The transcription of HIV‐1 was determined by real‐time qPCR.

2. PEBP1 mRNA was induced by EGCG in patient primary CD4⁺ T cells. Primary CD4⁺ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.

3. PEBP1 protein was induced by EGCG in patient primary CD4⁺ T cells, which was determined by Western blot of whole cell lysate from primary CD4⁺ T cells.

4. EGCG‐induced PEBP1 suppressed nuclear entry of NF‐κB/p65. This was measured by Western blot of nuclear protein of primary CD4⁺ T cells treated with α‐CD3/CD28 or α‐CD3/CD28 plus 10 μM EGCG.

Data information: Data represent the mean ± SD of three independent experiments (n = 3) and were analyzed with t‐test. *P < 0.05; **P < 0.01, compared with mock treatment.Source data are available online for this figure.

Figure 6. PEBP1 reactivates latent HIV‐1 in the primary CD4⁺ T model of latency

1. A

   Outline of latency establishment in the primary CD4⁺ T cells. Human primary CD4⁺ T cells were activated and expanded with α‐CD3/CD28 beads at day 1. The α‐CD3/CD28 beads were removed at day 3. Cells were then infected with HIV‐1 NL4.3‐nanoluc at 3^rd day after expansion and maintained over 7 days with a decreasing concentration of IL‐2 to establish latency until day 12. At day 12, cells were infected with CRISPR/Cas9 and PEBP1‐sgRNA lentivirus.

2. B

   Transcription of HIV‐1 in the primary CD4⁺ T cells was determined by nanoluc‐luciferase assays during HIV‐1 infection.

3. C

   After infection with VSVG pseudotyped HIV‐1 NL4.3‐nanoluc, the mRNA expression of PEBP1 was measured by qPCR.

4. D, E

   Jurkat CD4⁺ T cells were treated with IFN‐α, IFN‐β, and IFN‐γ for 24 h. The mRNA (A) or protein (B) expression levels of PEBP1 were measured by qPCR or Western blot.

5. F

   PEBP1 knockout enhanced HIV‐1 transcription in the primary CD4⁺ T‐cell model of latency. The expression of HIV‐1 was measured by nanoluc after gene knockout where α‐CD3/CD28 stimulation served as a positive control.

6. G

   PEBP1 gene deletion after Lv‐PEBP1‐sg1 knockout in the primary CD4⁺ T cells. The PCR products of PEBP1 were cloned and then sequenced. PEBP1‐sg1 target gene sequences are shown in red letters. Dashes indicate deleted bases relative to the wild‐type sequence.

Data information: Data represent the mean ± SD of three independent experiments (n = 3) and were analyzed with t‐test. *P < 0.05; ***P < 0.001.Source data are available online for this figure.

Figure EV3. Differential activation of MAPK, IKK, and NF‐κB signaling pathways in uninfected Jurkat CD4⁺ T cells, HIV‐1 latently infected C11 CD4⁺ T cells, and HIV‐1 actively infected YA CD4⁺ T cells

1. A, B

   Activation of Raf1/ERK/IκB (A) or IKK/IκB (B) signaling pathway was analyzed in total protein lysates by Western blot in Jurkat, C11, and YA cells.

2. C

   Nuclear levels of NF‐κB/p65 protein were analyzed by Western blot in Jurkat, C11, and YA cells.

Source data are available online for this figure.

Figure 7. A working model of the role of PEBP1 in the establishment of HIV latency

PEBP1 interacts and inhibits the activity of Raf1 and IKK kinases. This blocks the phosphorylation of downstream proteins ERK1, IKK, and IκBα, resulting in the sequestration of p65/p50 heterodimer in the cytoplasm, thereby silencing HIV‐1 transcription. When PEBP1 is knocked out, Raf1 and IKK signaling pathways are activated to phosphorylate IκBα, thereby releasing p65/p50 heterodimer to translocate into the nucleus to drive HIV‐1 transcription.