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. 2004 Oct;78(20):11016-29.
doi: 10.1128/JVI.78.20.11016-11029.2004.

Anti-Vpr activity of a yeast chaperone protein

Zsigmond Benko  1 , Dong Liang, Emmanuel Agbottah, Jason Hou, Karen Chiu, Min Yu, Scott Innis, Patrick Reed, William Kabat, Robert T Elder, Paola Di Marzio, Lorena Taricani, Lee Ratner, Paul G Young, Michael Bukrinsky, Richard Yuqi Zhao
Affiliations

Anti-Vpr activity of a yeast chaperone protein

Zsigmond Benko et al. J Virol. 2004 Oct.

Abstract

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G(2) arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

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Figures

FIG. 1.
FIG. 1.
Overexpression of hsp16 suppresses Vpr-induced cell cycle G2 arrest in fission yeast and human noninfected and HIV-infected cells. (A) Suppression of Vpr-induced G2 arrest by Hsp16 as measured in fission yeast by Vpr-induced cell elongation (a) and growth delay (b) in RE076 cells. Cell length was measured after 21 h of gene induction at 30°C. Growth kinetics was measured at 25°C. (B) Suppression of Vpr-induced G2 arrest by Hsp16 in human 293T cells. (a) The extent of Vpr-induced G2 arrest, measured by flow cytometric analysis, was quantified by the relative G2 to G1 ratio between gene-repressing and gene-expressing cells 72 h after gene induction. A relative G2/G1 ratio close to 1 indicates no significant difference between the gene-on and gene-off cultures. A relative ratio of >1 suggests increased G2 cells in the gene-on culture (66). (b) Western blotting shows specific expression of HIV-1 vpr and the fission yeast hsp16 gene in 293T-632 cells. GI, gene induction. (C) Suppression of Vpr-induced G2 arrest by Hsp16 in 293T cells infected with VSV-G pseudotyped HIV-1NL4-3. Vpr-positive or Vpr-negative HIV-1NL4-3 (57) was used to infect hsp16-expressing cells. Cell cycle profiles were measured by flow cytometric analysis 48 h after viral infection. Cells were infected with viruses adjusted to 3 × 106 cpm of RT activity per 106 cells.
FIG. 2.
FIG. 2.
Suppression of Vpr-induced cell cycle G2 arrest by Hsp16 mimics cellular heat shock responses. (A) Induction of cellular heat shock responses suppresses Vpr-induced G2 arrest as indicated by prevention of the Vpr-induced cell elongation (a) and growth delay (b). Cellular heat shock responses were triggered either by an acute heat treatment (45°C for 15 min) or constant high temperature at 36°C. (B) Induction of cellular heat shock responses does not affect the protein levels of Vpr as indicated by Western blot analysis. GI, gene induction.
FIG. 3.
FIG. 3.
Overexpression of hsp16 suppresses Vpr-induced cell death in fission yeast and human noninfected and HIV-infected cells. (A) Suppression of Vpr-induced cell death in RE007 cells as shown by colony forming ability on EMM selective agar under the vpr-inducing (vpr-on) and vpr-repressing (vpr-off) conditions (a). Plates are shown after 3 to 4 days of incubation at 30°C. Overproduction of Hsp16 in RE007 does not affect the protein levels of Vpr as detected by anti-Vpr serum in a Western blot analysis (b). RE007 cells expressing hsp16 were collected 24 h after gene induction at 30°C. GI, gene induction; LC, protein loading control. (B) Suppression of Vpr-induced cell death by Hsp16 in human 293T cells. Deadcells were detected 72 h after gene induction by trypan blue straining. Gene-off, no muristerone A was added; Gene-on, 1.0 μM muristerone A was added to induce gene expression. (C) Suppressive effects of Hsp16 on cytopathicity induced by HIV-1 infection in H9 cells. Top panel shows cell death induced by HIV-1 infection. Bottom panel measures cell viability during the course of the experiments. H9 cells were either mock infected (left columns) or HIV-1LAI-infected cells carrying empty vector (middle columns) or a vector expressing hsp16 (right columns). Cell death and viability of the infected cells were examined 3, 5, 7, and 10 days after infection. The percentage of cell death induced by HIV-1 infection was quantified by trypan blue straining. Cell viability was determined by measuring the optical density at 630 nm (OD630) by using a commercial MTT assay (Boehringer Mannheim).
FIG.4.
FIG.4.
Overexpression of hsp16 interferes with nuclear membrane localization and nuclear import of Vpr through direct protein-protein interaction. (A) Hsp16 diminishes nuclear membrane localization of Vpr in HeLa cells (a) and fission yeast (b). Note that Vpr localization on the nuclear membrane in HeLa cells can be distinguished as a clear ring surrounding the nucleus in the GFP-Vpr panel. However, this nuclear ring formed by GFP-Vpr was not clearly visible in GFP alone or GFP-Vpr+Hsp16 panels. Single-cell inserts in each panel show a magnified view of the presence or absence of nuclear membrane localization of Vpr. Nuclear membrane localization of Vpr was visualized by the expression of the GFP-Vpr fusion protein 24 h after transfection in HeLa cells. Cells containing the pYZ1N vector show Vpr localization on the nuclear membrane (panel b, top); cells containing pYZ1N-hsp16 show enhanced distribution of Vpr in the nucleus and cytoplasm (panel b, bottom). Nuclear membrane localization of Vpr was visualized 24 h after gene induction in fission yeast. DAPI staining was used to show nuclei. (B) Hsp16 blocks nuclear entry of Vpr in 293T cells. Nuclear import of Vpr was examined 24 h after transfection and 1 h after the addition of 400 nM LMB. (C) Potential interaction of Vpr with Hsp16. Panel a shows the effect of Vpr on subcellular localization of GFP-Hsp16 in strain Q1649. Note that expression of HIV-1 vpr changes the subcellular distribution of GFP-Hsp16 from evenly distributed (left) to a pattern that is similar to Vpr distribution, as indicated by arrows (right). Panel b shows the coimmunoprecipitation of Hsp16 with HA-tagged Vpr. Both anti-HA and anti-Hsp16 antibodies were used in Western blot analysis. LC, protein loading control.
FIG.5.
FIG.5.
Stable expression of Hsp16 in H9 and CEM-SS cells prior to HIV infection reduces viral replication and HIV-induced cytopathicity in a Vpr-dependent manner. (A) Western blots show high levels of Hsp16 in CD4+ H9 and CEM-SS cells. Lane 1, mock-infected cells; lane 2, HIV-infected cells carrying plasmid pcDNA3.1; lane 3, HIV-infected cells expressing hsp16. Blots show 16-kDa bands reacted to anti-Hsp16 antibody. LC, protein loading control. (B) Stable expression of Hsp16 in H9 and CEM-SS cells blocks the gross enlargement of cells typically induced by Vpr-positive HIV infection (29). Images were taken with equal amount of cells. Gross enlarged cells are of typical effect caused by Vpr (29). (C) Stable expression of Hsp16 in CD4+ H9 and CEM-SS cells inhibits viral replication in Vpr-positive HIV infection; Hsp16 has no effect on viral replication in Vpr-negative HIV infection.
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