Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May;83(9):4574-90.
doi: 10.1128/JVI.01800-08. Epub 2009 Feb 25.

Suppression of Tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of Vpu in the trans-Golgi network

Mathieu Dubé  1 , Bibhuti Bhusan Roy, Pierre Guiot-Guillain, Johanne Mercier, Julie Binette, Grace Leung, Eric A Cohen
Affiliations

Suppression of Tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of Vpu in the trans-Golgi network

Mathieu Dubé et al. J Virol. 2009 May.

Abstract

Vpu promotes the efficient release of human immunodeficiency virus type 1 (HIV-1) by overcoming the activity of tetherin, a host cell restriction factor that retains assembled virions at the cell surface. In this study, we analyzed the intracellular localization and trafficking of subtype B Vpu in HIV-1-producing human cells. We found that mutations of conserved positively charged residues (R30 and K31) within the putative overlapping tyrosine- and dileucine-based sorting motifs of the Vpu hinge region affected both the accumulation of the protein in the trans-Golgi network (TGN) and its efficient delivery to late endosomal degradative compartments. A functional characterization of this mutant revealed that the mislocalization of Vpu from the TGN correlated with an attenuation of HIV-1 release. Interestingly, clathrin light chain small interfering RNA-directed disruption of Vpu trafficking from the TGN to the endosomal system slightly stimulated Vpu-mediated HIV-1 release and completely restored the activity of the Vpu R30A,K31A mutant. An analysis of the C-terminal deletion mutants of Vpu identified an additional determinant in the second helical structure of the protein, which regulated TGN retention/localization, and further revealed the functional importance of Vpu localization in the TGN. Finally, we show that a large fraction of Vpu colocalizes with tetherin in the TGN and provide evidence that the degree of Vpu colocalization with tetherin in the TGN is important for efficient HIV-1 release. Taken together, our results reveal that Vpu traffics between the TGN and the endosomal system and suggest that the proper distribution of Vpu in the TGN is critical to overcome the restricting activity of tetherin on HIV-1 release.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Analysis of Vpu subcellular localization. HeLa cells expressing HxBH10-vpu wt were costained with the anti-Vpu serum (red) and anti-TGN46, anti-CD63, anti-LAMP1 or anti-calreticulin antibodies (green). Nuclei were counterstained with DAPI (blue). Cells were observed by deconvolution fluorescence microscopy. Pictures show representative examples of Vpu association with TGN46 (A and B) and with CD63 (B). Enlarged pictures are shown beside panel B. White arrows indicate noticeable examples of punctate colocalization. White bars represent a distance of 10 μm.
FIG. 2.
FIG. 2.
Mutations in the hinge region between the TM and cytoplasmic domains of Vpu affect its subcellular localization. (A) Schematic representation of the Vpu structural domains with the amino acid sequence of the hinge region derived from several strains of HIV-1 group M. Representative consensus amino acid sequences of the Vpu hinge region for each HIV-1 subtype are indicated. The red rectangle highlights the overlapping tyrosine-based (YXXΦ) and dileucine-based ([D/E]XXXL[L/I]) sorting motifs. Among sequences of all HIV-1 strains from the HIV databases of the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH; www.hiv.lanl.gov/content/index), 65% of the sequences show RK residues at positions 30 and 31 (red letters), while 96% of the sequences show a conservation of positively charged residues (RR, KR, or KK) at those positions within the hinge region. Yellow circled amino acids indicate phosphoacceptor sites at serines 52 and 56 (bold). −, amino acid identical to Vpu from HxBH10; αH, α-helix. (B) HeLa cells expressing HxBH10-vpu R30A,K31A were costained with the anti-Vpu serum (red) and anti-TGN46, anti-CD63, anti-LAMP1, or anti-calreticulin antibodies (green). Nuclei were counterstained with DAPI (blue). Pictures show representative examples of Vpu R30A,K31A-expressing cells. Enlarged pictures are shown beside the panels. White arrows indicate noticeable examples of punctate colocalization. White bars represent a distance of 10 μm. (C) Quantitation of Vpu wt and Vpu R30A,K31A accumulation in the TGN as determined by the Vpu signal measured in the TGN (region 1) relative to the total Vpu signal (region 2) in the cell. Error bars indicate the standard deviations of the means from the quantitative analysis of at least 25 distinct Vpu-expressing cells.
FIG. 3.
FIG. 3.
Abrogation of lysosomal function or targeting increases Vpu stability. (A) Vpu wt-expressing HEK 293T (top panels; SVCMV-vpu+) or HeLa cells (bottom panels; HxBH10-vpu wt) were treated for 20 h with the indicated increasing concentrations of CQ prior to lysis. Vpu was subsequently resolved on SDS-PAGE and detected by Western blotting using anti-Vpu antibodies. Actin served as a loading control. (B) HEK 293T cells were cotransfected with SVCMV-vpu wt and the pEGFP-Rab7 plasmids as indicated. Proteins were resolved on SDS-PAGE and detected by Western blotting using anti-Vpu, anti-actin, and anti-GFP antibodies. Note that the blots in panels A and B are underexposed to clearly show Vpu stabilization upon CQ treatment or pEGFP-Rab7 N125I coexpression. (C) HeLa cells were cotransfected with the HxBH10-vpu wt proviral construct and either the pEGFP-Rab7 wt or pEGFP-Rab7 N125I plasmids. Transfected cells were stained with anti-Vpu rabbit serum (red). Nuclei were counterstained with DAPI (blue). White arrows indicate noticeable colocalization of Vpu with EGFP-Rab7 N125I in vesicular structures. The white bar represents a distance of 20 μm. (D) Quantitation of Vpu accumulation in the TGN was performed as described in the legend to Fig. 2.
FIG. 4.
FIG. 4.
Mutations in the hinge region of Vpu interfere with the Vpu-mediated enhancement of HIV-1 particle release. (A) HeLa cells were mock transfected (lane 1) or transfected with either the HxBH10-vpu- (lane 2), HxBH10-vpu wt (lane 3), or HxBH10-vpu R30A,K31A (lane 4) proviral plasmids as indicated. Radiolabeled cells and supernatant-containing viral particles were harvested 48 h posttransfection and analyzed for the presence of Vpu and Gag proteins by immunoprecipitation as described in Materials and Methods. (B) Densitometric quantitation of viral release efficiency. Bands corresponding to Gag products in cells and viral particles were scanned by laser densitometry. The release efficiency of HxBH10-vpu wt was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of the results from four separate experiments. (C) HeLa cells were transfected with HxBH10-vpu- alone (lane 1) or with increasing quantities of HxBH10-vpu wt (lanes 2 to 4) or HxBH10-vpu R30A,K31A (lanes 5 to 7). The total amount of proviral DNA transfected in each condition was identical. Cells and supernatant-containing viral particles were harvested 48 h posttransfection, and Gag proteins in cell and virus lysates were analyzed by Western blotting using anti-p24 antibodies. Vpu was detected by Western blotting using anti-Vpu antibodies. The asterisk indicates a nonspecific band. (D) Same as described for panel C except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells and a chemiluminescence assay in relative light units (RLU), as described in Materials and Methods. The maximal RLU value obtained with HxBH10-vpu wt-expressing cells (lane 4) was arbitrarily set at 100%. Note that the data shown in lanes 4 and 5 correspond to the activities obtained with comparable levels of Vpu wt and Vpu R30A,K31A (panel C). (E) HeLa cells were cotransfected with the HxBH10-vpu- proviral construct, the indicated pEGFP-Rab7 plasmids, and the SVCMV-vpu wt or control constructs. Cells and virus particles were processed and analyzed as described for panel C. (F) Densitometric quantitation of the results shown in panel E. Viral particle release efficiency obtained in cells coexpressing HxBH10-vpu- and SVCMV-vpu wt was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of the results from three separate experiments.
FIG. 5.
FIG. 5.
Effect of the depletion of CLCs on the subcellular localization of Vpu and its viral particle release enhancing function. (A) HeLa cells were transfected either with control siRNA (Scrambled) (lanes 1 to 3) or specific siRNA against CLCa and CLCb (CLCs) (lanes 4 to 6). Seventy-two hours post-siRNA transfection, cells were transfected with similar amounts of the HxBH10-vpu- (lanes 1 and 4), HxBH10-vpu wt (lanes 2 and 5), or HxBH10-vpu R30A,K31A (lanes 3 and 6) proviral constructs as indicated. Cells and supernatant-containing viral particles were harvested 96 h post-siRNA transfection. Gag proteins in cell and virus lysates were analyzed by Western blotting using anti-p24 antibodies. Vpu and CLC levels were determined using specific antibodies. Actin served as a loading control. (B) Same as described for panel A except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells and a chemiluminescence assay in relative light units (RLU) as described in Materials and Methods. The RLU value obtained in HxBH10-vpu wt-expressing cells transfected with the nontargeting control siRNA was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of the results from four independent experiments. (C) HeLa cells were transfected with either the scrambled siRNA or with CLC siRNA. Cells were transfected with the HxBH10-vpu wt or HxBH10-vpu R30A,K31A proviral plasmid 72 h later. Twenty-four hours later, cells were costained for Vpu (red) and TGN46 (green). Nuclei were counterstained with DAPI (blue). The white bar represents a distance of 10 μm. (D) Quantitation of Vpu accumulation in the TGN was performed as described in the legend to Fig. 2.
FIG. 6.
FIG. 6.
Mutations in the putative overlapping tyrosine- and dileucine-based trafficking signals do not interfere with Vpu subcellular localization or Vpu-mediated enhancement of HIV-1 particle release. (A) HeLa cells transfected with the SVCMV-vpu wt, SVCMV-vpu Y29A or SVCMV-vpu I32A,L33A plasmid were costained with the anti-Vpu serum (red) and anti-TGN46 antibodies (green) 48 h posttransfection and observed by confocal microscopy. Pictures show representative examples of Vpu localization patterns. The white bar represents a distance of 10 μm. (B) Quantitation of Vpu accumulation in the TGN as determined by the Vpu signal measured in the TGN relative to the total Vpu signal in the cell. Error bars indicate the standard deviations of the means from the quantitative analysis of at least 25 distinct Vpu-expressing cells. (C) HeLa cells were cotransfected with the specified HxBH10 proviral constructs. Cells and supernatant-containing viral particles were analyzed as described in the legend to Fig. 4C. (D) Same as described for panel C except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells and a chemiluminescence assay. The RLU value obtained in Vpu wt-expressing cells (lane 2) was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of the results from four independent experiments.
FIG. 7.
FIG. 7.
Effect of deletions of the Vpu cytosolic tail on Vpu subcellular localization. (A) Schematic representation of the Vpu-EYFP deletion constructs. Yellow circled amino acids indicate phosphoacceptor sites at serines 52 and 56 (bold). αH, α-helix. (B) HeLa cells transfected with plasmids encoding the indicated Vpu-EYFP deletion mutant were costained with the anti-GFP (green) and anti-TGN46 (red) antibodies. Cells were observed by confocal microscopy. Pictures show representative examples of the localization pattern observed for each deletion mutant. (C) Quantitation of Vpu accumulation in the TGN was performed as described in the legend to Fig. 2. (D) HeLa cells expressing Vpu-EYFP or VpuΔ23-EYFP were costained with anti-GFP (green) and anti-CD63 or anti-Rab5 (red) antibodies. Cells were observed by confocal microscopy. Enlarged pictures are shown beside the panels. White arrows indicate noticeable examples of punctate colocalization. All the white bars represent a distance of 10 μm.
FIG. 8.
FIG. 8.
Effect of deletions of the Vpu cytosolic tail on Vpu-mediated enhancement of viral HIV-1 release. (A) HeLa cells were mock transfected (lane 1) or transfected with HxBH10-vpu- and plasmids encoding EYFP (lane 2) or Vpu-EYFP (lane 8) or the specified Vpu-EYFP deletion mutants (lanes 3 to 7). Cells and supernatants were analyzed as described in the legend to Fig. 4C. EYFP or Vpu-EYFP derivatives were detected using anti-GFP antibodies. (B) Same as described for panel A except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells and a chemiluminescence assay as described in Materials and Methods. The RLU measured with the Vpu-EYFP wt was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of the results from four independent experiments.
FIG. 9.
FIG. 9.
Analysis of the subcellular localization of native Vpu and tetherin. HeLa cells expressing HxBH10-vpu-, HxBH10-vpu wt, HxBH10-vpu R30A,K31A (A) or Vpu-EYFP or VpuΔ23-EYFP (B) were costained for Vpu (A) or GFP (B) (green) as well as for tetherin (blue) and TGN46 (red). Cells were observed by confocal microscopy. Enlarged pictures are shown beside panels A and B. White arrows indicate noticeable examples of punctate Vpu localization. White bars represent a distance of 10 μm. (C) Quantitation of the extent of Vpu colocalization with tetherin using the Zeiss LSM510 software. The values (%) represent the fraction of Vpu (green pixels) that overlapped with tetherin (blue pixels) relative to the total Vpu in the cell. Error bars indicate the standard deviations of the means from the quantitative analysis of at least 25 distinct Vpu-expressing cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Binette, J., and E. A. Cohen. 2004. Recent advances in the understanding of HIV-1 Vpu accessory protein functions. Curr. Drug Targets Immune Endocr. Metabol. Disord. 4297-307. - PubMed
    1. Binette, J., M. Dube, J. Mercier, and E. A. Cohen. 2007. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum. Retrovirology 475. - PMC - PubMed
    1. Bonifacino, J. S., and L. M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72395-447. - PubMed
    1. Bos, K., C. Wraight, and K. K. Stanley. 1993. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 122219-2228. - PMC - PubMed
    1. Bucci, C., P. Thomsen, P. Nicoziani, J. McCarthy, and B. van Deurs. 2000. Rab7: a key to lysosome biogenesis. Mol. Biol. Cell 11467-480. - PMC - PubMed

Publication types

MeSH terms

Substances

-