Overexpression of HSP27 suppresses Vpr-induced cell cycle G2 arrest. (A) Suppression of Vpr-induced G2 arrest by HSP27 in Vpr-transfected HEK293-632 cells. Both the vpr and HSP27 genes were induced by muristerone A as described previously (18) through pZY1-vpr or pZH1-HSP27 plasmids, respectively. The extent of Vpr-induced G2 arrest, measured by flow cytometric analysis, was quantified 72 h after gene induction, by the relative G2 to G1 ratio between gene-repressing and gene-expressing cells. A relative G2/G1 ratio close to one indicates no significant difference between the gene-on and gene-off cultures. A relative ratio > 1 suggests increased proportion of G2 cells in the gene-on culture (18). A representative experiment out of three performed is shown; the following P values have been calculated: (pZY + pZH) vs. (Vpr + pZH), P < 0.05; (Vpr + HSP27) vs. (Vpr + pZH), P < 0.05; (pZY + pZH) vs. (Vpr + HSP27), P = 0.31. (B) Western blot analysis of Vpr and HSP27 on an aliquot of cells collected for flow cytometric analysis in panel A. (C) VSV-G pseudotyped Vpr(+) or Vpr(−) HIV-1_NLHX (22) was used to infect HSP27-expressing cells. Cell cycle distribution was measured by flow cytometry 48 h after viral infection. Note that this experiment was performed together with analysis of Hsp16 activity, which was published previously (13); therefore, the control panels are the same as shown in that paper.

HSP27 inhibits viral replication in CEM-SS and H9 T-lymphocytes in a Vpr-dependent manner. (A) HSP27 expression analysis in stably transfected cells by Western blot. Ctr – mock-transfected cells; lanes 1–3: H9 cells; lanes 4-6: CEM-SS cells; pcDNA3.1 – cells transfected with an empty vector; + HSP27 – cells transfected with HSP27-expressing plasmid. (B) Stable expression of HSP27 reduces viral replication in CD4 + H9 and CEM-SS cells in a Vpr-dependent manner. Viral replication was determined by measuring p24 antigen in culture supernatant at indicated time. *, indicates statistical significance (at the level of P < 0.01) between HIV-1_NLHX-infected cells with or without overexpression of HSP27.

Vpr reduces elevation of HSP27 induced by heat treatment. (A) Western blot analysis of HEK293-262 cells carrying muristerone A-inducible vpr. Cells were treated (vpr-on) or not (vpr-off) with muristerone A, then treated at 43°C for 30 min and analyzed by Western blotting at indicated time intervals after heat shock (HS). (B) Quantification of Western blot analyses in panel A by densitometry; * statistical significance at the level of P < 0.05.

Expression of endogenous HSP27 is responsive to HIV-1 infection in a Vpr-dependent manner. (A) Expression of vpr triggers an increase of HSP27 production in HEK293 cells. The vpr gene expression was induced by muristerone A as described (18). Following indicated times after gene induction (GI), HSP27 protein levels were determined by Western blot analysis. (B) Quantification of results in panel A by densitometry. Results show mean ± SD calculated from three independent experiments. (C) Analysis of HSP27 production in HIV-infected CEM-SS T-lymphocytes. The level of HSP27 expression was examined at indicated time intervals post infection (p.i.) with HIV-1. Top panels show results with Vpr(+) virus; bottom panels – with Vpr(−) HIV-1. (D) Quantification of results in panel C by densitometry. Results show mean ± SD calculated from three independent experi-

Overexpression of HSP27 suppresses Vpr-induced cell death and apoptosis. (A) Suppression of Vpr-induced cell death by HSP27 in vpr-transfected HEK293-632 cells. Vpr and HSP27 expression was induced by muristerone A. Dead cells were detected by trypan blue straining 72 h after gene induction. (B) Anti-apoptotic activity of HSP27 in HEK293-632 cells. Apoptosis was detected by Annexin V assay using a BD ApoAlert Annexin V kit. Annexin V-positive cells that appear in the low right quadrant typically represent early apoptosis and cells in late apoptosis are found in the upper right quadrant (30). (C) HSP27 overexpression reduces cytopathic effect of HIV-1 infection. Dead cells (trypan blue-positive) were counted in HIV-infected H9 T-lymphocytes by trypan blue assay (left panel); cell viability was determined by an MTT assay (right panel). H9 cells were either mock-infected (H9 + mock), infected with HIV-1_LAI without overexpressed HSP27 (H9-vector + HIV-1) or infected with HIV-1_LAI with overexpressed HSP27 (H9-HSP27 + HIV-1). Cell death and cell viability of the infected cultures were examined three, five, seven, and ten days after infection. Percentage of cell death induced by HIV-1 infection was quantified by counting the number of blue cells over total cell population. Cell viability was quantified by measuring optical density at 630 nm using a commercial MTT assay kit. Average and standard errors represent a total of three independent experiments. *, statistical significance at the level of P < 0.001; **, statistical significance at the level of P < 0.0001. (D) Overexpression of HSP27 does not have an effect on cell viability. Cell death and cell viability were examined in un-infected H9 cells as in panel C.

Activation of HSP27 is mediated through HSF1. (A) Analysis of Vpr, HSF1 and HSP27 expression in HEK293 cells carrying muristerone A inducible Vpr by Western blotting at various time points after vpr gene induction (GI). (B) Quantification of Western blot analysis in panel A by densitometry. (C) HSF1 expression was knocked-down using shRNA, and expression of indicated proteins was analyzed after vpr gene induction (GI). Cell extracts without anti-HSF1 shRNA were used as the positive control (Ctr) to show production of HSF1 and HSP27. (D) Analysis of CEM-SS cells infected with Vpr(+) or Vpr(−) HIV-1. Analysis by Western blotting was performed at indicated times after infection. (E) Results in panel D were quantified by densitometry.