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. 2004 Sep;78(18):9697-704.
doi: 10.1128/JVI.78.18.9697-9704.2004.

Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R

Sergey Iordanskiy  1 , Yuqi Zhao, Larisa Dubrovsky, Tatiana Iordanskaya, Mongzhong Chen, Dong Liang, Michael Bukrinsky
Affiliations

Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R

Sergey Iordanskiy et al. J Virol. 2004 Sep.

Abstract

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis. This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2 arrest and apoptosis of target T cells. A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest. Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress. In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells. Overexpression of Hsp70 reduced the Vpr-dependent G2 arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1. Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1. Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished. Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells. Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr.

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Figures

FIG. 1.
FIG. 1.
Hsp70 suppresses Vpr-induced cell cycle G2 arrest. (A) Suppression of Vpr-induced G2 arrest in fission yeast. Vpr-induced cell elongation was used here as an indication of cell cycle G2 arrest (11). RE076 is a fission yeast strain that carries a single integrated copy of F34Ivpr (11, 56). RE076+Vector denotes the RE076 strain transformed with the pYZ1N vector control. RE076+spHsp70 and RE076+mHsp70 are RE076 strains transformed with fission yeast or mammalian Hsp70, respectively. vpr-Off, vpr gene expression suppressed; vpr-On, vpr gene expression induced. (B) Hsp70-mediated suppression of Vpr-induced G2 arrest in 293T-632 cells. Vpr-induced G2 arrest was measured by flow cytometric analysis after staining DNA with propidium iodide. The extent of G2 arrest was evaluated by the relative G2/G1 ratio (shown in the right-hand panels) between cells cultured in the presence and absence of the inducer (muristerone A): R = [G2/G1 (+ inducer)]/[G2/G1 (− inducer)] (57). A Western blot (bottom) shows coexpression of HIV-1 vpr and mammalian Hsp70 genes in muristerone A-induced 293T-632 cells. pZY-1 is a vector used for vpr expression, and pZH-1 was used for mHsp70 expression. Both vectors are induced by addition of 1 μM muristerone A (57). Results are shown for one representative experiment out of three performed. (C) Hsp70 suppresses Vpr-induced G2 arrest in HIV-1-infected cells. MAGI cells were transfected with an Hsp70-expressing vector and 24 h after transfection were infected with a Vpr-positive or Vpr-negative MLV-pseudotyped HIV-1. G2 arrest was analyzed 48 h after infection by flow cytometry as in panel B. The effect of Hsp70 was evaluated by relative G2/G1 ratio (shown in left panels) in cells transfected with Hsp70-expressing vector and control cells: R = [G2/G1 (Hsp70−)]/[G2/G1 (Hsp70+)]. Results are shown for one representative experiment out of two performed.
FIG. 2.
FIG. 2.
RNAi-mediated suppression of Hsp70 expression enhances Vpr-specific apoptosis of HIV-1-infected cells. MAGI cells were transfected with Hsp70-specific or control siDNA/RNA duplexes and then infected with Vpr-negative (Vpr−) or Vpr-positive (Vpr+) HIV-1 or mock infected. (A) Hsp70 and Hsp27 were quantified in cell lysates by ELISA 24 h after infection. Results are the mean ± standard error of triplicate wells. (B) Cell cycle distribution was analyzed by FACS 48 h after infection as in Fig. 1B. The effect of siDNA/RNA was evaluated by the ratio of G2/G1 in cultures transfected with control siDNA/RNA to G2/G1 in cultures transfected with Hsp70-specific siDNA/RNA (shown in the left column panels). Results are representative of two independent experiments. (C) Apoptotic cells were stained with Annexin V-FITC and analyzed by flow cytometry 24 h after infection. The effect of siDNA/RNA was evaluated by the ratio of apoptotic cells in cultures transfected with control and Hsp70-specific siDNA/RNA (shown in left column panels). Results are representative of two independent experiments.
FIG. 3.
FIG. 3.
Analysis of Hsp70 effects on HIV-1 replication. (A) MAGI cells were transfected with control or Hsp70 siDNA/RNA. Twenty-four hours after transfection, cells were infected with HIV-1 or mock infected. HIV-1 replication was analyzed 48 h after infection by staining intracellular p24 essentially as described previously (32). The percentage of p24-positive cells is shown. For mock-infected cells, only results with Hsp70 siDNA/RNA-transfected cells are presented. (B) The experiment was performed as in panel A, except that cells were infected with Vpr-positive (Vpr+) or Vpr-negative (Vpr−) HIV-1 and extracellular p24 was measured by ELISA. Results show mean ± standard deviation of three independent wells. (C) Triplicate cultures of MAGI cells, transfected with either an Hsp70-expressing (vector+Hsp70) or an empty vector, were infected with Vpr-positive or Vpr-negative HIV-1. Virus replication was measured at indicated times after infection by reverse transcriptase activity in culture supernatants. Results are presented as means ± standard deviation. (D) Triplicate cultures of H9 cells stably transfected with Hsp70-expressing (vector+Hsp70) or an empty vector were infected with Vpr-positive or Vpr-negative HIV-1. p24 in culture supernatants was measured at indicated time points after infection by ELISA. Results are presented as the mean ± standard deviation. The inset in the left panel shows Western blot analysis of Hsp70 and β-actin proteins in transfected H9 cultures.
FIG. 4.
FIG. 4.
Hsp70 coimmunoprecipitates with Vpr from HIV-1-infected cells. (A) MAGI cells were infected with Vpr-positive (Vpr+) or Vpr-negative (Vpr−) HIV-1. Forty-eight hours after infection, cells were lysed and immunoprecipitated with a polyclonal anti-Hsp70 antibody. The immunoprecipitates were analyzed for the presence of Hsp70 (using anti-Hsp70 MAb) and Vpr (using polyclonal anti-Vpr antibody) by Western blotting (WB). (B). MAGI cells were transfected with Myc-tagged Hsp70 and then infected with Vpr-positive or Vpr-negative HIV-1. Immunoprecipitation was performed with anti-Vpr polyclonal antibody and revealed by anti-Myc MAb.
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