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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 ubiquitination. A. Vpu-mediated ubiquitination of CD4 wt when CD4 is retained in the ER through treatment with BFA. HEK 293T cells were mock-transfected or co-transfected with 1 μg of SVCMV CD4 wt, 8 μg of SVCMV Vpu+ or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N and 8 μg of the TDN mutant his(6)/c-myc-Ub K48/R. Samples were then treated as described in the materials and methods section. CD4 molecules were immunoprecipitated with anti-CD4 polyclonal antibodies prior to western-blot analysis with anti-myc monoclonal antibodies. (triangle) indicates the position of the heavy chains of anti-CD4 antibodies. B. Quantitative analysis of ubiquitinated CD4 conjugates. (asterisk) represents the area of the autoradiogram that was used for quantitation of CD4-Ub conjugates. The histogram shows the relative levels of ubiquitinated CD4 conjugates in presence or absence of a functional Vpu. Relative CD4-Ub conjugate levels were evaluated by quantitation of the signal detected in the area delineated on the autoradiogram relative to total CD4 as determined by quantitation of the band detected with the anti-CD4 antibodies on whole cell lysate. The relative level of ubiquitinated CD4 detected in absence of Vpu was arbitrarily set at 1. The data represent results from seven experiments. C. Vpu-mediated ubiquitination of CD4 wt in condition where CD4 is retained in the ER through binding with HIV-1 Env. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 20 μg of his(6)/c-myc-Ub K48/R. Samples were then treated as in A but in absence of BFA. D. Quantitative analysis showing the relative levels of ubiquitinated CD4 detected in two independent experiments. Relative levels of ubiquitinated CD4 conjugates were determined as described in B.
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Figure 2: Effect of Vpu on CD4 ubiquitination. A. Vpu-mediated ubiquitination of CD4 wt when CD4 is retained in the ER through treatment with BFA. HEK 293T cells were mock-transfected or co-transfected with 1 μg of SVCMV CD4 wt, 8 μg of SVCMV Vpu+ or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N and 8 μg of the TDN mutant his(6)/c-myc-Ub K48/R. Samples were then treated as described in the materials and methods section. CD4 molecules were immunoprecipitated with anti-CD4 polyclonal antibodies prior to western-blot analysis with anti-myc monoclonal antibodies. (triangle) indicates the position of the heavy chains of anti-CD4 antibodies. B. Quantitative analysis of ubiquitinated CD4 conjugates. (asterisk) represents the area of the autoradiogram that was used for quantitation of CD4-Ub conjugates. The histogram shows the relative levels of ubiquitinated CD4 conjugates in presence or absence of a functional Vpu. Relative CD4-Ub conjugate levels were evaluated by quantitation of the signal detected in the area delineated on the autoradiogram relative to total CD4 as determined by quantitation of the band detected with the anti-CD4 antibodies on whole cell lysate. The relative level of ubiquitinated CD4 detected in absence of Vpu was arbitrarily set at 1. The data represent results from seven experiments. C. Vpu-mediated ubiquitination of CD4 wt in condition where CD4 is retained in the ER through binding with HIV-1 Env. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 20 μg of his(6)/c-myc-Ub K48/R. Samples were then treated as in A but in absence of BFA. D. Quantitative analysis showing the relative levels of ubiquitinated CD4 detected in two independent experiments. Relative levels of ubiquitinated CD4 conjugates were determined as described in B.

Mentions: Having established that over-expression of Ub K48/R inhibited Vpu-mediated CD4 degradation in HEK 293T cells, we investigated whether we could isolate and directly detect ubiquitinated forms of CD4 that are expected to accumulate under these conditions. Towards this goal, we first analyzed CD4 expression at steady state in Vpu/CD4 HEK 293T transfectants in presence or absence of tagged-Ub K48/R (Fig. 2A). In these experiments, transfected cells were treated with BFA during 2 h prior to lysis to retain newly synthesized CD4 in the ER. Fig. 2A reveals that CD4 levels at steady-state were significantly reduced in presence of Vpu (compare lanes 2 and 4). As expected, expression of tagged-Ub K48/R suppressed the effect of Vpu on CD4 and re-established the amounts of CD4 to levels comparable to those detected in absence of Vpu (compare lanes 5 and 2). To detect CD4-Ub conjugates, cell lysates were first immunoprecipitated with anti-CD4 polyclonal antibodies and the resulting CD4-containing immunocomplexes were subsequently analyzed for the presence of CD4-Ub conjugates by western-blot using anti-myc antibodies. Ubiquitinated forms of CD4 were detected as a typical smear in presence of Vpu (lane 5). Although background high molecular weight ubiquitinated forms of CD4 could still be detected in absence of Vpu (lane 3) or in presence of the non-functional Vpu S52,56/N mutant (lane 7), their levels were not as elevated as in presence of wt Vpu (lane 5). Indeed, quantitative analysis revealed that levels of CD4-Ub conjugates were approximately 6-fold higher in presence than in absence of a functional Vpu (Fig. 2B). The detection of a smear of high molecular weight proteins in presence of Vpu is suggestive of poly-ubiquitination of CD4. Poly-ubiquitination is still possible even if Ub K48/R is over-expressed because cells are expressing endogenous wt Ub that can initiate poly-Ub chains before a molecule of Ub K48/R can prematurely terminate the chain.


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 ubiquitination. A. Vpu-mediated ubiquitination of CD4 wt when CD4 is retained in the ER through treatment with BFA. HEK 293T cells were mock-transfected or co-transfected with 1 μg of SVCMV CD4 wt, 8 μg of SVCMV Vpu+ or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N and 8 μg of the TDN mutant his(6)/c-myc-Ub K48/R. Samples were then treated as described in the materials and methods section. CD4 molecules were immunoprecipitated with anti-CD4 polyclonal antibodies prior to western-blot analysis with anti-myc monoclonal antibodies. (triangle) indicates the position of the heavy chains of anti-CD4 antibodies. B. Quantitative analysis of ubiquitinated CD4 conjugates. (asterisk) represents the area of the autoradiogram that was used for quantitation of CD4-Ub conjugates. The histogram shows the relative levels of ubiquitinated CD4 conjugates in presence or absence of a functional Vpu. Relative CD4-Ub conjugate levels were evaluated by quantitation of the signal detected in the area delineated on the autoradiogram relative to total CD4 as determined by quantitation of the band detected with the anti-CD4 antibodies on whole cell lysate. The relative level of ubiquitinated CD4 detected in absence of Vpu was arbitrarily set at 1. The data represent results from seven experiments. C. Vpu-mediated ubiquitination of CD4 wt in condition where CD4 is retained in the ER through binding with HIV-1 Env. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 20 μg of his(6)/c-myc-Ub K48/R. Samples were then treated as in A but in absence of BFA. D. Quantitative analysis showing the relative levels of ubiquitinated CD4 detected in two independent experiments. Relative levels of ubiquitinated CD4 conjugates were determined as described in B.
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Figure 2: Effect of Vpu on CD4 ubiquitination. A. Vpu-mediated ubiquitination of CD4 wt when CD4 is retained in the ER through treatment with BFA. HEK 293T cells were mock-transfected or co-transfected with 1 μg of SVCMV CD4 wt, 8 μg of SVCMV Vpu+ or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N and 8 μg of the TDN mutant his(6)/c-myc-Ub K48/R. Samples were then treated as described in the materials and methods section. CD4 molecules were immunoprecipitated with anti-CD4 polyclonal antibodies prior to western-blot analysis with anti-myc monoclonal antibodies. (triangle) indicates the position of the heavy chains of anti-CD4 antibodies. B. Quantitative analysis of ubiquitinated CD4 conjugates. (asterisk) represents the area of the autoradiogram that was used for quantitation of CD4-Ub conjugates. The histogram shows the relative levels of ubiquitinated CD4 conjugates in presence or absence of a functional Vpu. Relative CD4-Ub conjugate levels were evaluated by quantitation of the signal detected in the area delineated on the autoradiogram relative to total CD4 as determined by quantitation of the band detected with the anti-CD4 antibodies on whole cell lysate. The relative level of ubiquitinated CD4 detected in absence of Vpu was arbitrarily set at 1. The data represent results from seven experiments. C. Vpu-mediated ubiquitination of CD4 wt in condition where CD4 is retained in the ER through binding with HIV-1 Env. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 20 μg of his(6)/c-myc-Ub K48/R. Samples were then treated as in A but in absence of BFA. D. Quantitative analysis showing the relative levels of ubiquitinated CD4 detected in two independent experiments. Relative levels of ubiquitinated CD4 conjugates were determined as described in B.
Mentions: Having established that over-expression of Ub K48/R inhibited Vpu-mediated CD4 degradation in HEK 293T cells, we investigated whether we could isolate and directly detect ubiquitinated forms of CD4 that are expected to accumulate under these conditions. Towards this goal, we first analyzed CD4 expression at steady state in Vpu/CD4 HEK 293T transfectants in presence or absence of tagged-Ub K48/R (Fig. 2A). In these experiments, transfected cells were treated with BFA during 2 h prior to lysis to retain newly synthesized CD4 in the ER. Fig. 2A reveals that CD4 levels at steady-state were significantly reduced in presence of Vpu (compare lanes 2 and 4). As expected, expression of tagged-Ub K48/R suppressed the effect of Vpu on CD4 and re-established the amounts of CD4 to levels comparable to those detected in absence of Vpu (compare lanes 5 and 2). To detect CD4-Ub conjugates, cell lysates were first immunoprecipitated with anti-CD4 polyclonal antibodies and the resulting CD4-containing immunocomplexes were subsequently analyzed for the presence of CD4-Ub conjugates by western-blot using anti-myc antibodies. Ubiquitinated forms of CD4 were detected as a typical smear in presence of Vpu (lane 5). Although background high molecular weight ubiquitinated forms of CD4 could still be detected in absence of Vpu (lane 3) or in presence of the non-functional Vpu S52,56/N mutant (lane 7), their levels were not as elevated as in presence of wt Vpu (lane 5). Indeed, quantitative analysis revealed that levels of CD4-Ub conjugates were approximately 6-fold higher in presence than in absence of a functional Vpu (Fig. 2B). The detection of a smear of high molecular weight proteins in presence of Vpu is suggestive of poly-ubiquitination of CD4. Poly-ubiquitination is still possible even if Ub K48/R is over-expressed because cells are expressing endogenous wt Ub that can initiate poly-Ub chains before a molecule of Ub K48/R can prematurely terminate the chain.

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