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Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

Binette J, Dubé M, Mercier J, Halawani D, Latterich M, Cohen EA - Retrovirology (2007)

Bottom Line: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD).Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment.Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montreal, Quebec H2W 1R7, Canada. julie.binette@ircm.qc.ca

ABSTRACT

Background: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex.

Results: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation.

Conclusion: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

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Effect of Vpu on CD4 molecules lacking lysine residues in the cytoplasmic tail. A. Analysis of CD4 wt and CD4 KRcyto turnover in presence or absence of functional Vpu by pulse-chase labeling and immunoprecipitation. HEK 293T cells were mock-transfected or co-transfected with 2 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 20 μg of provirus encoding Vpu+ (HxBH10-vpu+) or phosphorylation-defective Vpu mutant (HxBH10-vpu S52,56/D). Cells were pulse-labeled with [35S]methionine and [35S]cysteine and chased in complete medium for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 antibodies first (polyclonal and monoclonal) and then with anti-Vpu antibodies. B. Using quantitative scanning of CD4 bands from two independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. Effect of Vpu on steady-state CD4 wt and CD4 KRcyto levels. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 10 μg of proviruses encoding Vpu- or Vpu+ in addition to 25 μg of the his(6)/c-myc-Ub K48/R expressor. In the left panel (Env-), a similar experiment was performed except that HEK 293T cells were co-transfected with 10 μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and treated with BFA for 2 h prior to lysis. Cell lysates were then treated as described in the materials and methods section. D. Quantitative analysis of steady-state CD4 levels. CD4 levels in presence of absence of his(6)/c-myc-Ub K48/R were arbitrarily set at 100%. The levels of CD4 in presence of Vpu are shown relative to the corresponding controls. These results are representative of the data obtained in three independent experiments for Env- and five independent experiments for Env+.
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Figure 3: Effect of Vpu on CD4 molecules lacking lysine residues in the cytoplasmic tail. A. Analysis of CD4 wt and CD4 KRcyto turnover in presence or absence of functional Vpu by pulse-chase labeling and immunoprecipitation. HEK 293T cells were mock-transfected or co-transfected with 2 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 20 μg of provirus encoding Vpu+ (HxBH10-vpu+) or phosphorylation-defective Vpu mutant (HxBH10-vpu S52,56/D). Cells were pulse-labeled with [35S]methionine and [35S]cysteine and chased in complete medium for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 antibodies first (polyclonal and monoclonal) and then with anti-Vpu antibodies. B. Using quantitative scanning of CD4 bands from two independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. Effect of Vpu on steady-state CD4 wt and CD4 KRcyto levels. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 10 μg of proviruses encoding Vpu- or Vpu+ in addition to 25 μg of the his(6)/c-myc-Ub K48/R expressor. In the left panel (Env-), a similar experiment was performed except that HEK 293T cells were co-transfected with 10 μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and treated with BFA for 2 h prior to lysis. Cell lysates were then treated as described in the materials and methods section. D. Quantitative analysis of steady-state CD4 levels. CD4 levels in presence of absence of his(6)/c-myc-Ub K48/R were arbitrarily set at 100%. The levels of CD4 in presence of Vpu are shown relative to the corresponding controls. These results are representative of the data obtained in three independent experiments for Env- and five independent experiments for Env+.

Mentions: CD4 contains four potential Ub acceptor lysine residues in its cytoplasmic domain. To determine whether ubiquitination of the cytosolic tail was required for Vpu-mediated CD4 degradation, we analyzed a CD4 mutant, CD4 KRcyto, in which all four cytoplasmic lysine residues were replaced by arginines. The stability of CD4 wt and CD4 KRcyto was first assessed in cells expressing a provirus encoding either wt Vpu (HxBH10-vpu+) or Vpu S52,56/D (HxBH10-vpu S52,56/D) as described above in Fig. 2C. Results of Fig. 3A clearly show that both CD4 wt and CD4 KRcyto were unstable in Vpu expressing cells as observed by the decreased recovery of CD4 molecules over the chase period (lanes 9–12 and lanes 13–16). Quantification of CD4 turnover over several experiments indicated an attenuation of the degradation kinetic of CD4 KRcyto as compared to CD4 wt but the protein was clearly susceptible to Vpu-induced degradation (Fig. 3B). In contrast, both CD4 wt and CD4 KRcyto remained stable over the entire 7 h chase period in cells expressing the phosphorylation mutant Vpu S52,56/D (Fig. 3A, lanes 1–4 and lanes 5–8 and Fig. 3B).


Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

Binette J, Dubé M, Mercier J, Halawani D, Latterich M, Cohen EA - Retrovirology (2007)

Effect of Vpu on CD4 molecules lacking lysine residues in the cytoplasmic tail. A. Analysis of CD4 wt and CD4 KRcyto turnover in presence or absence of functional Vpu by pulse-chase labeling and immunoprecipitation. HEK 293T cells were mock-transfected or co-transfected with 2 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 20 μg of provirus encoding Vpu+ (HxBH10-vpu+) or phosphorylation-defective Vpu mutant (HxBH10-vpu S52,56/D). Cells were pulse-labeled with [35S]methionine and [35S]cysteine and chased in complete medium for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 antibodies first (polyclonal and monoclonal) and then with anti-Vpu antibodies. B. Using quantitative scanning of CD4 bands from two independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. Effect of Vpu on steady-state CD4 wt and CD4 KRcyto levels. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 10 μg of proviruses encoding Vpu- or Vpu+ in addition to 25 μg of the his(6)/c-myc-Ub K48/R expressor. In the left panel (Env-), a similar experiment was performed except that HEK 293T cells were co-transfected with 10 μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and treated with BFA for 2 h prior to lysis. Cell lysates were then treated as described in the materials and methods section. D. Quantitative analysis of steady-state CD4 levels. CD4 levels in presence of absence of his(6)/c-myc-Ub K48/R were arbitrarily set at 100%. The levels of CD4 in presence of Vpu are shown relative to the corresponding controls. These results are representative of the data obtained in three independent experiments for Env- and five independent experiments for Env+.
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Related In: Results  -  Collection

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Figure 3: Effect of Vpu on CD4 molecules lacking lysine residues in the cytoplasmic tail. A. Analysis of CD4 wt and CD4 KRcyto turnover in presence or absence of functional Vpu by pulse-chase labeling and immunoprecipitation. HEK 293T cells were mock-transfected or co-transfected with 2 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 20 μg of provirus encoding Vpu+ (HxBH10-vpu+) or phosphorylation-defective Vpu mutant (HxBH10-vpu S52,56/D). Cells were pulse-labeled with [35S]methionine and [35S]cysteine and chased in complete medium for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 antibodies first (polyclonal and monoclonal) and then with anti-Vpu antibodies. B. Using quantitative scanning of CD4 bands from two independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. Effect of Vpu on steady-state CD4 wt and CD4 KRcyto levels. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 10 μg of proviruses encoding Vpu- or Vpu+ in addition to 25 μg of the his(6)/c-myc-Ub K48/R expressor. In the left panel (Env-), a similar experiment was performed except that HEK 293T cells were co-transfected with 10 μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and treated with BFA for 2 h prior to lysis. Cell lysates were then treated as described in the materials and methods section. D. Quantitative analysis of steady-state CD4 levels. CD4 levels in presence of absence of his(6)/c-myc-Ub K48/R were arbitrarily set at 100%. The levels of CD4 in presence of Vpu are shown relative to the corresponding controls. These results are representative of the data obtained in three independent experiments for Env- and five independent experiments for Env+.
Mentions: CD4 contains four potential Ub acceptor lysine residues in its cytoplasmic domain. To determine whether ubiquitination of the cytosolic tail was required for Vpu-mediated CD4 degradation, we analyzed a CD4 mutant, CD4 KRcyto, in which all four cytoplasmic lysine residues were replaced by arginines. The stability of CD4 wt and CD4 KRcyto was first assessed in cells expressing a provirus encoding either wt Vpu (HxBH10-vpu+) or Vpu S52,56/D (HxBH10-vpu S52,56/D) as described above in Fig. 2C. Results of Fig. 3A clearly show that both CD4 wt and CD4 KRcyto were unstable in Vpu expressing cells as observed by the decreased recovery of CD4 molecules over the chase period (lanes 9–12 and lanes 13–16). Quantification of CD4 turnover over several experiments indicated an attenuation of the degradation kinetic of CD4 KRcyto as compared to CD4 wt but the protein was clearly susceptible to Vpu-induced degradation (Fig. 3B). In contrast, both CD4 wt and CD4 KRcyto remained stable over the entire 7 h chase period in cells expressing the phosphorylation mutant Vpu S52,56/D (Fig. 3A, lanes 1–4 and lanes 5–8 and Fig. 3B).

Bottom Line: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD).Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment.Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montreal, Quebec H2W 1R7, Canada. julie.binette@ircm.qc.ca

ABSTRACT

Background: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex.

Results: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation.

Conclusion: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Show MeSH
Related in: MedlinePlus