HIV-1 Vpu induces the ubiquitination of proteins in BST-2 immunoprecipitates. (A) 293T cells were transfected to express transiently BST-2, ubiquitin (Ub)-HA, and either empty plasmid or a codon-optimized version of HIV-1_NL4-3 Vpu. Where indicated, the mutant version of Vpu, Vpu-S52/56N (2/6) was used. At 24 h posttransfection, cells were lysed under nondenaturing conditions, and BST-2 was immunoprecipitated using a monoclonal antibody to the ectodomain of BST-2. The IPs were eluted in Laemmli buffer without β-mercaptoethanol ([β-ME] upper panel), which was added later to half of the eluates to produce reduced samples (lower panel). The IPs were analyzed by Western blotting (WB) using anti-BST-2 antibody for an internal loading control, anti-Vpu antibody to assess the interaction with Vpu, and anti-HA antibody for ubiquitin-HA. Cells not transfected with BST-2 were used as a negative control. One percent of the input was loaded in the first lane from the left. The results shown are representative of five independent experiments. (B) 293T cells were transfected to express transiently BST-2, β-TrCP-HA, and one of the following: empty plasmid, plasmid encoding Vpu, or plasmid encoding Vpu2/6. The IP was performed as described for panel A. Monoclonal anti-HA antibody was used to detect β-TrCP-HA, whereas monoclonal antibody to ubiquitin was used to detect ubiquitin. The results shown are representative of five independent experiments.

Mutated residues in the cytoplasmic domain of BST-2 and the expression of potential ubiquitination site mutants. (A) Sequence alignment of the CDs of BST-2 proteins from humans, African green monkeys, pigtail macaques, and rhesus macaques. The conserved residues are indicated with dashes. The responsiveness of BST-2 proteins to HIV-1 Vpu as reported previously is indicated to the right (23, 39, 40). S, sensitive; R, resistant. The mutated residues are highlighted. (B) 293T cells were transfected in a six-well format with 3 μg of each BST-2- and Vpu-expressing plasmid. At 24 h posttransfection, the cells were lysed and cleared as described in Materials and Methods. The lysates were boiled in Laemmli buffer and analyzed by Western blotting for Vpu, actin, and BST-2.

Colocalization of the BST-2 mutants with CD63 and Vpu. HT-1080 cells were transfected with a plasmid expressing either wild-type (WT) BST-2 or the BST-2 mutants, together with a plasmid expressing either wild-type Vpu or Vpu2/6. BST-2 mutant proteins were coexpressed only with wild-type Vpu. BST-2, Vpu, and endogenous CD63 were visualized as described in Materials and Methods, using monoclonal mouse anti-BST-2, rabbit antiserum to HIV-1 Vpu, and FITC-conjugated mouse monoclonal anti-CD63 antibodies. The distribution of the proteins in the same field is shown separately in black and white, as well as together as merged images in which BST-2 is red, CD63 is green, and Vpu is blue. Scale bar, 10 μm. The results shown are representative of two independent experiments.

The STS sequence is required for optimal counteraction of restricted virion release by Vpu. 293T cells were transfected in duplicate to express transiently wild-type (WT), vpu-negative (Δ), or Vpu2/6-containing HIV-1 proviral DNA along with either an empty plasmid (not shown) or a plasmid expressing BST-2, as well as a plasmid expressing GFP as a transfection marker, as described in Materials and Methods. The supernatants of the cultures were collected 24 h posttransfection and analyzed by ELISA for p24 capsid antigen after centrifugation through a 20% sucrose cushion and by an infectious-center assay using HeLa cells that express CD4 as targets (infectivity). The values graphed are the percentages of the values obtained from the cells expressing HIV-1/ΔVpu and not expressing BST-2. The error bars are the average deviation obtained from two independent measurements of the duplicate transfections. The cell pellets were lysed in Laemmli buffer and subjected to Western blotting using anti-p24/55, anti-Vpu, anti-actin, and rabbit polyclonal anti-BST-2 antibodies. The results shown are representative of two independent experiments.

Models for the ubiquitin-mediated downregulation of BST-2 by Vpu and the role of the STS sequence. In model I, Vpu recruits the β-TrCP-containing E3 ubiquitin ligase complex to directly ubiquitinate the BST-2 CD at S and T residues. This triggers the removal of BST-2 from the cell surface and the consequent relief of the restricted virion release. In model II, Vpu recruits the β-TrCP-containing E3 ubiquitin ligase complex to ubiquitinate factor X, which interacts with the STS sequence in the CD of BST-2. The ubiquitination of factor X triggers the downregulation of BST-2 from the cell surface and the consequent relief of the restricted virion release.

The ubiquitinated species in BST-2 immunoprecipitates are likely BST-2 itself. (A) The transfection of 293T cells and IP was performed as described in the legend of Fig. 1A. Sodium phosphate (to a concentration of 50 mM) and NP-40 (to a concentration of 1%) were added directly to the IPs in Laemmli buffer before treatment with PNGase F. For each 10-cm² dish, 2.5 units of PNGase F (Sigma) was added, followed by incubation at 37°C for 6 h. (B) The transfection of 293T cells and IP were performed as described in the legend of Fig. 1A, except that EGFP-BST-2 was used. Cells transfected with empty EGFP plasmid were used as a no-BST-2 negative control. (C) The transfection of 293T cells and IP were performed as described in the legend of Fig. 1A. IPs were eluted in Laemmli buffer, with boiling. Half of the sample was diluted 35 times with lysis buffer and reimmunoprecipitated using monoclonal anti-BST-2 antibody prebound to anti-mouse IgG magnetic beads. (D) The transfection of 293T cells was performed as described in the legend of Fig. 1B, and IP was performed as described in the legend of Fig. 2C, except that two-thirds of the sample was used for reimmunoprecipitation.

Subcellular distribution and colocalization of the BST-2 mutants with transferrin receptor (TfR). HT-1080 cells were transfected with a plasmid expressing either wild-type BST-2 (WT) or the BST-2 mutants. BST-2 and endogenous TfR were visualized as described in Materials and Methods, using polyclonal rabbit anti-BST-2 and monoclonal mouse anti-TfR antibodies. The distribution of the proteins in the same field is shown separately in black and white, as well as together as merged images in which BST-2 is red and TfR is green. Scale bar, 10 μm.

The STS sequence in the CD of human BST-2 is required for optimal downregulation from the cell surface by Vpu. 293T cells were transfected with 60 ng of BST-2 and 500 ng of either empty plasmid or plasmid expressing Vpu, as well as 250 ng of plasmid expressing GFP as a transfection marker, in a 12-well format. At 24 h posttransfection, cells were stained for surface BST-2 using mouse monoclonal anti-BST-2 antibody and analyzed by two-color flow cytometry. Dot plots represent the amount of BST-2 on the cell surface versus the amount of GFP signal for the individual cells. Contour plots represent the distribution of cells expressing Vpu2/6. Histograms represent the relative cell number versus BST-2 fluorescence intensity for the population of cells shown in the dot plots. In the histograms, the far left peaks represent the IgG2a antibody isotype negative control, thick lines represent cells not expressing Vpu, and gray-shaded peaks represent cells expressing Vpu. Thin lines represent cells expressing Vpu2/6. The MFI values for each distribution are to the right of each histogram. The results shown are representative of three independent experiments. Max, maximum.

All potential acceptor sites in the CD of BST-2 are ubiquitinated by Vpu. (A) 293T cells were transfected, and BST-2 was immunoprecipitated as described in the legend of Fig. 1A (coexpression of ubiquitin-HA), except that the BST-2 mutants KK-CC, STS, and KCST-less were tested in addition to wild-type BST-2. The input samples shown in the right panel are the cell lysates before IP. The amount of actin in the input samples was assessed as a loading control. The immunoprecipitated proteins were probed for BST-2, ubiquitin-HA, and Vpu. The results shown are representative of two independent experiments. (B) 293T cells were transfected, and BST-2 was immunoprecipitated as described in the legend of Fig. 1B (coexpression of β-TrCP-HA) except that the BST-2 mutants KK-CC, STS, and KCST-less were tested in addition to wild-type BST-2. The immunoprecipitated proteins were probed for BST-2, endogenous ubiquitin, β-TrCP-HA, and Vpu. A band that is induced by Vpu but disappears in the case of KCST-less BST-2 is indicated by an open arrow; a band that is increased by Vpu but remains detectable in the case of KCST-less BST-2 is indicated by a filled arrow. The results shown are representative of two independent experiments. (C) 293T cells stably expressing BST-2 WT or the BST-2 mutants KK-CC, STS, and KCST-less were transfected with empty plasmid or Vpu- or Vpu2/6-expressing plasmid, along with a plasmid expressing ubiquitin-HA, and BST-2 was immunoprecipitated as described in Materials and Methods. The input samples shown in the right panel are the cell lysates before IP. The amount of actin in the input samples was assessed as a loading control. The results shown are representative of two independent experiments.