Fig 1.
JIB-04 inhibits HIV-1 Tat expression in activated 2D10 T cells.
(A) Immunoblot analysis of HIV-1 Tat expression from the integrated HIV-1 provirus in 2D10 T cells induced with TNFα for 0–24 h, as indicated above each lane. Cells were treated with DMSO (lanes 1–6) or 3 μM JIB-04 (lanes 7–12) overnight. Cyclin T1 served as loading control. (B) As in part A, except that 2D10 cells were activated with PHA (10 μg/mL) and PMA (50 ng/mL) for the times indicated above each lane. (C) Immunoblot analysis of HIV-1 Tat protein levels in 2D10 cells treated with different concentrations of JIB-04 (0.1 μM-50 μM) for 24 h, as indicated. Cyclin T1 and TAF4 (TFIID subunit) served as loading controls. (D) As in part C, except that cells were exposed to the histone demethylase inhibitor GSKJ1, at the concentrations listed above each lane. Cyclin T1 and p65 (NF-κB subunit) served as loading controls. (E) Quantification of Tat and eGFP protein levels in TNFα-stimulated 2D10 cells treated with the JIB-04 at the concentrations indicated in the X-axis. The signals were calculated by Image J and averaged from three independent experiments. (F) qRT-PCR analysis d2EGFP (HIV-1 reporter gene), HIV-1 Env, or host cell CXCL10 and CDK9 mRNAs extracted from 2D10 cells pre-treated with either DMSO or 3 μM JIB-04 for 16 h and stimulated by TNFα (10 ng/ml), for the times indicated below each graph. Values on the Y-axis for d2EGFP, Env, CXCL10 and CDK9 mRNAs prior to TNFα stimulation were normalized to 1. Differences between DMSO and 3 μM-JIB-04 treated samples were calculated by Student’s T-test at each time point (*p<0.05, **p<0.005, ***p<0.0005). Shown is a representative result from three independent experiments.
Fig 2.
ChIP analysis of the effect of JIB-04 on the binding of transcription factors to the HIV-1 genome.
ChIP analysis comparison of transcription factor binding to the single integrated HIV-1 genome in 2D10 T cells treated either with DMSO (A,C) or with 3 μM JIB-04 (B,D). Cells were treated with TNFα (10 ng/ml) for 0 h (blue line), 0.5 h (pink line), 2 h (yellow line), or 6 h (light blue line). The ChIP values in the Y-axis are expressed as percentage input. The ChIP primers (A-G) that were used are indicated on the X-axis, and their relative location on the HIV-1 genome are shown in the schematic at the bottom left. Antisera used for ChIP antibodies are NF-κB (p65 subunit), Cyclin T1, CDK9, NELF-A, RNAPII CTD, and phosphorylated RNAPII CTD-Ser2 (Ser2P), CTD-Ser5 (Ser5P) and CTD-Ser7 (Ser7P), as indicated above each panel.
Fig 3.
JIB-04 has a limited effect on host cell transcription.
(A) Shown is a graph of normalized RNA-seq reads mapped to HIV-1 and eGFP in 2D10 cells. Cells were pre-treated with DMSO or 3 μM JIB-04 for 16 h, and stimulated by TNFα (10 ng/ml) for 0h or 6h, as indicated. (B) RNA-seq heatmap of 100 genes that showed the largest differences in expression in DMSO versus JIB-04-treated 2D10 cells. The effect of JIB-04 on host cell gene expression was monitored both in the absence (0h) or presence (6h) of TNFα. Data were derived from minimum and the maximum rlog values of duplicate RNA-seq experiments in 2D10 cells. Genes are grouped as JIB-04-repressed, JIB-04-activated and TNFα-inducible groups, as indicated on the right. (C) Analysis of mRNA by qRT-PCR for HIV-Env, eGFP and selected TNFα-inducible (top) and non-inducible (bottom) genes in 2D10 cells in DMSO or JIB-04-treated cells. Significant differences between samples treated by DMSO or 3 μM JIB-04 at 6 h TNFα stimulation were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005; ns = non-significant) for each gene.
Fig 4.
JIB-04 inhibits HIV replication in HeLa P4.R5 MAGI. indicator cells.
(A) Images of two representative fields from single-cycle infectivity imaging assays for HeLa P4.R5 MAGI cells subjected to HIV-1 infection and treated with either DMSO or JIB-04. The numbers above each panel refer to the percentage of HIV-infected blue cells exposed to DMSO, 1 μM JIB-04, or 5 μM JIB-04. Shown are two representative photos taken from each of three replicate plates, with a 10X10 amplification. (B) Graph plotting the average numbers of total (pink bar), blue (blue bar) or white (white bar) cells from at least three representative photos in the presence of DMSO or different concentrations of JIB-04 (1–5 μM). The percentage HIV-infected (blue) cells is indicated above the blue bars. Significant differences between cell numbers treated by DMSO or different concentrations of JIB-04 for total, blue and white cells were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005; ns = non-significant), respectively.
Fig 5.
DiffPOP analysis identifies the SHMT2 serine hydroxymethyltransferase as a target of JIB-04.
(A) Schematic diagram of the DiffPOP protocol. Soluble proteins in 2D10 whole cell extracts were precipitated in fractions containing increasing concentrations of methanol, in the presence and absence of the JIB-04 compound. The protein composition of the different precipitates was then assessed using mass-spectrometry proteomics analysis. (B) Pearson Correlation coefficients were calculated to identify factors that show altered protein solubility in methanol when exposed to DMSO or 200 μM JIB-04. (C) The top five candidate host cell proteins targeted by JIB-04, defined as those with the smallest Pearson’s R value. The complete protein list is shown in S2 File. (D) Manual verification of DiffPOP results for SHMT2. Extracts precipitated at different methanol concentrations in the presence or absence of JIB-04 were analyzed by immunoblot for endogenous SHMT2. The results also show that the methanol solubility of HIV-1 Tat, Csn3, and Cyclin T1 proteins did not change in response to JIB-04. The peak of SHMT2 shifted from fractions 4–6 in the DMSO-treated fractions to fractions 2–5 in the extracts exposed to JIB-04.
Fig 6.
JIB-04 increases Tat K63Ub and proteolytic destruction.
(A) Left, immunoblot analysis of HIV-1 Tat expression in 2D10 cells exposed to DMSO, JIB-04, MG132 or JIB-04+MG132. Cells were pre-treated with or without TNFα (10 ng/ml) for 16 h. The COP9 signalosome complex subunit 3 (Csn3) served as loading control. Right, qRT-PCR analysis of Tat, GFP and Env mRNA levels in these cells. Values shown in the Y-axis were normalized to mRNAs from 2D10 cells without TNFα-stimulation. (B) Dual-Luc (HIV-LTR-Luc/SV40-Renilla-Luc) reporter gene analysis in FLAG-Tat101 transfected HeLa P4 cells. Left, immunoblot analysis of FLAG-Tat101 protein levels in cells treated with DMSO or 2 μM JIB-04. Right panels show dual-luc reporter gene activity in these cells. Significant differences between HIV-Luc activity treated by DMSO or 2 μM JIB-04 were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005). (C) Dual-Luc reporter gene analysis, as in part B, in Tet-on-Tat-off HeLa cells. Left, immunoblot analysis of HA-Tat86 protein levels in 2D10 cells treated by DMSO or 2.5 μM JIB-04. Right, dual-Luc reporter gene activity in these cells. Significant differences between HIV-Luc activity treated by DMSO or 2.5 μM JIB-04 were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005). (D) Analysis of the effect of JIB-04 on endogenous Tat K63Ub levels in HeLa cells. HIV-1 Tat was immunoprecipitated from lysates of HeLa cells exposed to DMSO or JIB-04 (1 μM and 3 μM), and endogenous ubiquitylation was monitored using the antisera indicated to the left of each panel (HC = antibody heavy chain). (E) Immunoprecipitation of FLAG-Tat-101 from lysates of HeLa cells treated with DMSO or JIB-04 (1μM and 3μM). Ubiquitination of FLAG-Tat-101 in the presence of ectopically expressed HA-ubiquitin-WT, HA-ubiquitin-K63-only or HA-ubiquitin-K48-only was assessed using anti-HA antisera.
Fig 7.
JIB-04 promotes destruction of HIV-1 Tat by selective autophagy.
(A) MudPIT proteomics analysis of HA-Tat immunoaffinity purified complexes in HeLa cells. The table lists factors present in HA-Tat complexes, ranked by sequence coverage. The list includes several autophagy regulators, as well as well-known Tat-associated proteins (Cyclin T1, SSU72). (B) Immunoblot analysis to confirm the association of SQSMT1/p62 with HIV-1 Tat. (C) Immunoblot of Tat protein levels in HeLa cells depleted of the autophagy regulator SQSTM1/p62. Cyclin T1 served as loading control. (D) Immunoblot analysis of Tat protein levels in TNFα-stimulated 2D10 cells depleted of SQSTM1/p62. Cyclin T1 served as loading control. (E) Immunoblot analysis of HIV-1 Tat protein levels in 2D10 cells exposed to an inhibitor of lysosomal proteases, hydroxychloroquine (HCQ). Cyclin T1 served as loading control. (F) Immunoblot analysis of the effect of JIB-04 on Tat protein levels in 2D10 cells treated with the autophagy inhibitor HCQ. Cyclin T1 served as loading control.
Fig 8.
SHMT2 and the BRCC36/BRISC deubiquitinase controls Tat K63Ub and destruction by autophagy.
(A) Immunoblot analysis of Tat and GFP protein levels in 2D10 cells depleted of SHMT2 or Cyclin T1, as indicated. TAF4 served as loading control. (B) Immunoblot analysis of Tat protein expression in 2D10 cells depleted of BRCC36 or Cyclin T1. NF-κB2 protein levels were monitored to assess any change in T cell signaling. TAF4 served as loading control. (C) Immunoblot analysis of Tat expression in Tet-on-Tat-off HeLa cells depleted of SHMT1, SHMT2 or BRCC36, as indicated above each lane. TAF4 served as loading control. (D) Top, immunoblot analysis of the effects of overexpression of Flag-SHMT1 and FH-BRCC36 proteins on Tat protein levels. TAF4 served as loading control. Bottom, Dual:Luc reporter activity of the HIV-Luc reporter. Plasmids expressing the Flag-vector, Flag-SHMT1 and FH-BRCC36 proteins were tested at 0 ng (blue bar), 20 ng (purple bar), 100 ng (yellow bar) and 500 ng (light blue bar), as indicated. (E) Analysis of FLAG-Tat-101 immunoprecipitates from lysates of BRCC36- or SHMT1- knockdown cells. FLAG-Tat-101 proteins were labelled with HA-tagged ubiquitin, either wild-type Ub (WT), or ubiquitin mutants that selectively support only K63 or K48 ubiquitylation, and Tat proteins were monitored using anti-HA antisera. (F) Immunoblot analysis of the effect of JIB-04 on Tat protein levels in 2D10 cells depleted of SHMT2 or BRCC36. (G) The effect of JIB-04 on wildtype or Tat ΔK (+K41) STREP-tagged Tat proteins was shown by immunoblot. TAF4 served as loading control. (H) Model of the role of SHMT2 and BRCC36 in the release of Tat-K63Ub from destruction through chaperone-mediated autophagy or SQSMT1/p62-dependent macroautophagy.