(A) HeLa cells were treated without (mock) or with siRNAs to GAPDH (negative control) or β-TrCP1/2 (positive control). Cells were then transfected with plasmids encoding human CD4 and no Vpu (empty vector), wild-type Vpu or Vpu-S52,56N. At 12 h after transfection, cell lysates were analyzed by SDS-PAGE and immunoblotting with antibodies to the CD4 ectodomain and actin (used as a loading control). (B) HeLa cells treated with different siRNAs were processed as in A. CD4 levels in the presence of Vpu were quantified by densitometry and expressed as percentage of the total amount of CD4 in the absence of Vpu (100% control). Values represent the mean ± SEM from three independent screens. (C, E) HeLa cells were treated as in A with siRNAs to VCP, UFD1L or NPL4, and then transfected with plasmids encoding human CD4, plus or minus Vpu. At 12 h after transfection, cells were labeled with [³⁵S]methionine-cysteine for 2 min and chased for the indicated times at 37°C. Cell extracts were subjected to immunoprecipitation of CD4 using a conformation-independent (C) or conformation-dependent (E) antibody. Immunoprecipitated proteins were resolved by SDS-PAGE and fluorography. (D, F) Percentage of CD4 at each chase time relative to CD4 at time 0 (100% control). (G) A postnuclear supernatant of HeLa cells treated as in C and E was separated into cytosolic and membrane fractions. The membranes were washed with 0.2 M Na₂CO₃ pH 11.3, and pellet (P) and supernatant (S) fractions were collected. Fractions were analyzed by SDS-PAGE and immunoblotting with antibodies to the CD4 ectodomain and actin (used as a cytosolic marker).

(A–L) HeLa cells were treated without (mock) (A–H) or with siRNAs to NPL4 (I–L). Cells were then transfected with plasmids encoding human CD4 without (A–D) or with Vpu (E–L). At 12 h after transfection, cells were fixed, permeabilized and labeled with a rabbit polyclonal antibody to Vpu (green channel; A, E, I), IgG_2a mouse monoclonal antibody to CD4 (red channel; B, F, J) and IgG₁ mouse monoclonal antibody to calnexin (blue channel; C, G, K). Stained cells were examined by confocal fluorescence microscopy. Bars: 10 µm. (M–O) HeLa cells expressing Vpu were fixed and processed for immunoelectron microscopy as previously described [14]. Notice that enhanced nanogold particles indicative of Vpu are mainly associated with the ER and nuclear envelope (NE). In contrast, untransfected cells show no enhanced nanogold, confirming the specificity of the staining (Fig. S3). N: nucleus; PM: plasma membrane. Bars: 1 µm. (P) Total lysates from HeLa cells treated as in A–L were digested with Endo H, PNGase F or left untreated before immunoblotting with an antibody to the CD4 ectodomain. CHO: N-linked carbohydrate chain. (Q) Data are represented as mean ± SEM from three independent experiments like that in P. (R) HeLa cells treated as in A–L were analyzed for cell surface CD4 by FACS. (S) Bar graphs represent percentage of surface CD4 levels in cells expressing Vpu relative to CD4-surface levels in the absence of Vpu (100%). Values are expressed as mean ± SEM from three independent experiments.

(A) Sequence of the CD4 TMD and cytosolic tail with residues targeted for mutagenesis underlined. (B) HeLa cells were transfected with plasmids encoding Vpu-S52,56N and no CD4 (empty vector), human wild-type CD4, CD4 with all four cytosolic lysine residues mutated to arginine (CD4-K-less), or CD4 with all cytosolic lysine, serine and threonine residues mutated to arginine, alanine and isoleucine, respectively (CD4-KST-less). At 12 h after transfection, cells were lysed and subjected to immunoprecipitation with an antibody to the CD4 ectodomain and immunoblotting with an antibody to Vpu. (C) HeLa cells were transfected with plasmids encoding no CD4 (empty vector), human wild-type CD4, CD4-K-less or CD4-KST-less and FLAG-tagged Ub, plus or minus Vpu (1∶0.5∶1 ratio of CD4, Ub and Vpu, respectively). At 12 h after transfection, equivalent amounts of cell lysates made under denaturing conditions were subjected to immunoprecipitation with a conformation-independent antibody to the CD4 ectodomain. Ubiquitination of CD4 was detected by immunoblotting with a polyclonal antibody to the FLAG epitope. (D) CD4 levels in the presence of Vpu from C were quantified by densitometry and expressed as percentage of the total amount of CD4 in the absence of Vpu (100% control). Data are represented as the mean ± SEM from three independent experiments. (E) Ub_n-CD4 levels in the presence of Vpu from C were quantified by densitometry and expressed as percentage of the total amount of Ub_n-CD4 in the absence of Vpu (100% control). These values were normalized for the remaining CD4 in C (i.e., 1 for Ub_n-CD4 in the absence of Vpu). Data are the mean ± SEM from three independent experiments. (F) Cell lysates from HeLa cells expressing human CD4, CD4-K-less or CD4-KST-less, plus or minus Vpu, were digested with Endo H, PNGase F or left untreated before immunoblotting with an antibody to the CD4 ectodomain. The arrowhead indicates Endo H-sensitive species corresponding to the CD4-K-less mutant in the presence of Vpu. (G) Data from three independent experiments such as that in F are represented as the mean ± SEM.

(A) Amino acid sequence of the TMD and cytosolic tail of wild-type Vpu and Vpu-VSV-G-TMD. (B) HeLa cells were transfected with plasmids encoding FLAG-tagged human β-TrCP1, plus no Vpu (empty vector), wild-type Vpu, Vpu-S52,56N or Vpu-VSV-G-TMD. At 12 h after transfection, cells were lysed and subjected to immunoprecipitation using an antibody to the FLAG epitope. Co-precipitation of the different Vpu variants with β-TrCP1 was detected by immunoblotting with an antibody to Vpu. Notice that only phosphorylatable wild-type Vpu and Vpu-VSV-G-TMD co-precipitate with β-TrCP1. (C) Detergent lysates from HeLa cells expressing human CD4 in the absence or presence of wild-type Vpu, Vpu-S52,56N or Vpu-VSV-G-TMD were immunoprecipitated with an antibody to the CD4 ectodomain. Vpu co-precipitation with CD4 was detected as described in B. (D) CD4 levels in the presence of wild-type Vpu or Vpu-VSV-G-TMD were quantified by densitometry and expressed as percentage of the total amount of CD4 in the absence of Vpu (100% control). Values are the mean ± SEM from three independent experiments. (E–P) HeLa cells were transfected with plasmids encoding human CD4 and no Vpu (empty vector; E–H), wild-type Vpu (I–L) or Vpu-VSV-G-TMD (M–P). At 12 h after transfection, cells were fixed, permeabilized and labeled with a rabbit polyclonal antibody to Vpu (green channel; E, I, M), IgG_2a mouse monoclonal antibody to CD4 (red channel; F, J, N) and IgG₁ mouse monoclonal antibody to calnexin (blue channel; G, K, O). Stained cells were examined by confocal fluorescence microscopy. Bars: 10 µm. (Q) Total lysates from HeLa cells treated as in E–P were digested with Endo H, PNGase F or left untreated before immunoblotting with an antibody to the CD4 ectodomain. (R) Data are represented as mean ± SEM from three independent experiments like that in Q.

(A) Domain structure of mouse VCP, mouse UFD1L and rat NPL4 used in this study. (B, G, I) HeLa cells were transfected with plasmids encoding human CD4, Vpu and wild-type VCP or mutant VCP constructs (VCP-ΔN, VCP-AA, VCP-QQ) (B), wild-type UFD1L or mutant UFD1L constructs (UFD1L-ΔUT3, UFD1L-ΔUT6) (G), or wild-type NPL4 or mutant NPL4 constructs (NPL4-ΔUBD, NPL4-ΔZFD) (I). At 12 h after transfection, cells were labeled with [³⁵S]methionine-cysteine for 2 min and chased for the indicated times at 37°C. (D) HeLa cells expressing human CD4 and Vpu were pulse-labeled as described above and chased in normal (+ATP) or ATP-depleted (-ATP) medium. (B, D, G, I) Cell extracts were subjected to immunoprecipitation using an antibody to the CD4 cytosolic tail. Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. (C, E, H, J) Percentage of CD4 at each chase time relative to CD4 at time 0 (100% control). (F) HeLa cells were transfected with plasmids encoding human CD4 and RGS-His-tagged VCP-QQ, plus no Vpu (empty vector), wild-type Vpu or Vpu-S52,56N. At 12 h after transfection, cells were lysed and subjected to immunoprecipitation with an antibody to the CD4 ectodomain. Co-precipitation of RGS-His-tagged VCP-QQ with CD4 in the presence of wild-type Vpu was detected by immunoblotting with an antibody to the RGS-His epitope. (K) HeLa cells were treated without (mock) or with siRNAs to VCP, UFD1L or NPL4. Cell extracts were prepared and subjected to immunoblotting with antibodies to VCP, UFD1L or NPL4.

(A) HeLa cells were treated without (mock) or with siRNAs to β-TrCP1/2. Cells were then transfected with plasmids encoding human CD4 and no Vpu (empty vector), wild-type Vpu or Vpu-S52,56N. The glycosylation state of CD4 was analyzed as described in the legend to Fig. 3P. (B) Data are represented as mean ± SEM from three independent experiments like that in A. (C) HeLa cells expressing human CD4 in the absence or presence of wild-type Vpu or Vpu-S52,56N were labeled with [³⁵S]methionine-cysteine for 2 min and chased for the indicated times at 37°C. Cell extracts were subjected to immunoprecipitation with an antibody to the CD4 cytosolic tail. Immunoprecipitated proteins were digested with Endo H or left untreated before analysis by SDS-PAGE and fluorography. (D) Data from three independent experiments like that in C are represented as mean ± SEM. (E) HeLa cells treated as in A were analyzed for cell surface CD4 by FACS. (F) Bar graphs represent percentage of surface CD4 levels in cells expressing wild-type Vpu or Vpu-S52,56N relative to CD4-surface levels in the absence of Vpu (100%). Values are expressed as mean ± SEM from three independent experiments.

(A) HeLa cells were transfected with plasmids encoding human CD4 or CD4-Δcyto together with no Vpu (empty vector), wild-type Vpu or Vpu-S52,56N. At 12 h after transfection, cell extracts were prepared and subjected to immunoblotting with an antibody to the CD4 ectodomain. (B) CD4 levels in the presence of wild-type Vpu were quantified by densitometry and expressed as percentage of the total amount of CD4 in the absence of Vpu (100% control). Data are represented as the mean ± SEM from three independent experiments. (C) HeLa cells expressing human CD4 or CD4-Δcyto in the absence of Vpu were labeled with [³⁵S]methionine-cysteine for 2 min and chased for the indicated times at 37°C. Cell extracts were subjected to immunoprecipitation with an antibody to the CD4 ectodomain. Immunoprecipitated proteins were digested with Endo H or left untreated before analysis by SDS-PAGE and fluorography. The increased detection of both CD4 constructs from 0 to 30 min of chase is due to the development of a conformational epitope recognized by this particular antibody. (D) Data from C are plotted as percentage of Endo H-resistant (Endo H^R) CD4 as a function of the chase time. (E) Total cell lysates from HeLa cells treated as in A were digested with Endo H, PNGase F or left untreated before immunoblotting with an antibody to the CD4 ectodomain. (F) Data from three independent experiments like that in E are represented as mean ± SEM. (G) HeLa cells treated as in A were analyzed for cell surface CD4 by FACS. (H) Bar graphs represent percentage of surface CD4 levels in cells expressing wild-type Vpu or Vpu-S52,56N relative to CD4-surface levels in the absence of Vpu (100%). Values are expressed as mean ± SEM from three independent experiments.

(A) Amino acid sequence of the TMD and cytosolic tail of wild-type CD4 and CD4-VSV-G-cyto. (B, C) The effect of Vpu on human CD4, CD4-Δcyto or CD4-VSV-G-cyto was analyzed as described for Fig. 6, A and B. (D, E) The carbohydrate processing of CD4 and CD4-VSV-G-cyto in the presence or absence of Vpu was analyzed as described for Fig. 6, E and F. (F, G) HeLa cells expressing human CD4 or CD4-VSV-G-cyto, plus or minus Vpu, were analyzed for cell surface CD4 by FACS as described for Fig. 6, G and H. (H) Model for the dual role of Vpu in ER retention and ERAD targeting of CD4. See Discussion for details.