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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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Vpu-mediated CD4 degradation involves dislocation of ubiquitinated CD4 conjugates from the ER membrane to the cytosol. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and 15 μg of his(6)/c-myc-Ub K48/R expression plasmid where indicated. Cells were treated with BFA for 2 h before mechanical lysis. CD4-Ub conjugates were immunoprecipitated with anti-myc monoclonal antibodies prior to western-blot analysis with anti-CD4 polyclonal antibodies while control proteins in each fraction were revealed by western-blot. Actin and calnexin were used as cytosolic and membrane controls, respectively. A. Membrane (M) and cytosolic (C) fractions were separated and treated as described in the materials and methods section. B. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Relative levels of ubiquitinated CD4 conjugates were determined as described in the legend of Fig. 2B. Error bars reflect standard deviations from duplicate independent experiments. C. Membrane (M) fractions were treated with Na2CO3 (pH 11) as described in materials and methods. Treated membrane and supernatant (S) were subsequently recovered by centrifugation. Fractions were analyzed as described above in A. D. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules (as described in the legend of Fig. 2B) present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Error bars reflect standard deviations from duplicate independent experiments.
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Figure 5: Vpu-mediated CD4 degradation involves dislocation of ubiquitinated CD4 conjugates from the ER membrane to the cytosol. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and 15 μg of his(6)/c-myc-Ub K48/R expression plasmid where indicated. Cells were treated with BFA for 2 h before mechanical lysis. CD4-Ub conjugates were immunoprecipitated with anti-myc monoclonal antibodies prior to western-blot analysis with anti-CD4 polyclonal antibodies while control proteins in each fraction were revealed by western-blot. Actin and calnexin were used as cytosolic and membrane controls, respectively. A. Membrane (M) and cytosolic (C) fractions were separated and treated as described in the materials and methods section. B. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Relative levels of ubiquitinated CD4 conjugates were determined as described in the legend of Fig. 2B. Error bars reflect standard deviations from duplicate independent experiments. C. Membrane (M) fractions were treated with Na2CO3 (pH 11) as described in materials and methods. Treated membrane and supernatant (S) were subsequently recovered by centrifugation. Fractions were analyzed as described above in A. D. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules (as described in the legend of Fig. 2B) present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Error bars reflect standard deviations from duplicate independent experiments.

Mentions: To examine whether CD4 undergoes a process of dislocation across the ER membrane during Vpu-mediated degradation, we conducted subcellular fractionation studies. To optimize recovery and detection of dislocated forms of CD4 targeted for degradation by the cytosolic proteasome, we performed these cell fractionation experiments in conditions where CD4 degradation was inhibited by over-expression of the TDN Ub K48/R mutant. BFA-treated HEK 293T cells expressing CD4/Ub K48/R and Vpu or CD4/Ub K48/R alone were fractionated by mechanical lysis into membrane and cytosolic fractions and each resulting fraction was directly analyzed for the presence of CD4, Vpu and membrane or cytosolic markers, such as calnexin and actin respectively, by western-blot as described in materials and methods. Furthermore, the presence of poly-ubiquitinated forms of CD4 in membrane or cytosolic fractions was determined by immunoprecipitation/western-blot analysis. In contrast to Fig. 2 and 4 and because of technical reasons, ubiquitinated CD4 molecules were detected in these experiments by performing immunoprecipitation using anti-Myc antibodies followed by western-blot using anti-CD4 antibodies. As expected, Vpu and calnexin were detected exclusively in association with membrane fractions (Fig. 5A, lane 5 for Vpu and lanes 1, 3, 5 and 7 for calnexin) whereas actin (lanes 2, 4, 6, 8) or Ub (lanes 4, 6, 8) were recovered in a very large proportion in the cytosolic fractions, thus demonstrating that the fractionation procedure was almost free of membrane or cytosolic contaminations. CD4 molecules were found in the membrane fraction in presence or absence of Vpu (lanes 3 and 5). We could repeatedly recover and detect CD4-Ub conjugates, represented as a smear signal, predominantly in the membrane fraction but also in the cytosolic fraction in absence and in presence of Vpu (Fig. 5A); in some instances, depending on the experiments, we also detected discrete high molecular bands in addition to the smear signal [lane 5 of Additional file 2A and lane 6 of Additional file 2B]. Interestingly, the absolute signal associated with membrane and cytosolic fractions was always more intense in presence than in absence of Vpu (Fig. 5A, compare lanes 3, 5 and 7 as well as lanes 4, 6 and 8, upper panel). The specific levels of CD4-Ub conjugates associated with membrane and cytosolic fractions in absence and in presence of Vpu were calculated relative to the amount of CD4 detected directly by western-blot. As shown in Fig. 5B, quantitative analysis revealed that in presence of Vpu there was approximately a six-fold increase in membrane-associated CD4-Ub conjugate levels relative to the negative control without Vpu (Vpu-); in the cytosolic fractions, the levels of CD4-Ub conjugates detected in presence of Vpu were approximately two-fold higher relative to the Vpu- control (Fig. 5B).


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)

Vpu-mediated CD4 degradation involves dislocation of ubiquitinated CD4 conjugates from the ER membrane to the cytosol. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and 15 μg of his(6)/c-myc-Ub K48/R expression plasmid where indicated. Cells were treated with BFA for 2 h before mechanical lysis. CD4-Ub conjugates were immunoprecipitated with anti-myc monoclonal antibodies prior to western-blot analysis with anti-CD4 polyclonal antibodies while control proteins in each fraction were revealed by western-blot. Actin and calnexin were used as cytosolic and membrane controls, respectively. A. Membrane (M) and cytosolic (C) fractions were separated and treated as described in the materials and methods section. B. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Relative levels of ubiquitinated CD4 conjugates were determined as described in the legend of Fig. 2B. Error bars reflect standard deviations from duplicate independent experiments. C. Membrane (M) fractions were treated with Na2CO3 (pH 11) as described in materials and methods. Treated membrane and supernatant (S) were subsequently recovered by centrifugation. Fractions were analyzed as described above in A. D. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules (as described in the legend of Fig. 2B) present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Error bars reflect standard deviations from duplicate independent experiments.
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Figure 5: Vpu-mediated CD4 degradation involves dislocation of ubiquitinated CD4 conjugates from the ER membrane to the cytosol. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10μg of envelope-defective provirus (HxBc2-pr-, vpu-, env- or HxBH10-pr-, vpu+, env-) and 15 μg of his(6)/c-myc-Ub K48/R expression plasmid where indicated. Cells were treated with BFA for 2 h before mechanical lysis. CD4-Ub conjugates were immunoprecipitated with anti-myc monoclonal antibodies prior to western-blot analysis with anti-CD4 polyclonal antibodies while control proteins in each fraction were revealed by western-blot. Actin and calnexin were used as cytosolic and membrane controls, respectively. A. Membrane (M) and cytosolic (C) fractions were separated and treated as described in the materials and methods section. B. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Relative levels of ubiquitinated CD4 conjugates were determined as described in the legend of Fig. 2B. Error bars reflect standard deviations from duplicate independent experiments. C. Membrane (M) fractions were treated with Na2CO3 (pH 11) as described in materials and methods. Treated membrane and supernatant (S) were subsequently recovered by centrifugation. Fractions were analyzed as described above in A. D. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules (as described in the legend of Fig. 2B) present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Error bars reflect standard deviations from duplicate independent experiments.
Mentions: To examine whether CD4 undergoes a process of dislocation across the ER membrane during Vpu-mediated degradation, we conducted subcellular fractionation studies. To optimize recovery and detection of dislocated forms of CD4 targeted for degradation by the cytosolic proteasome, we performed these cell fractionation experiments in conditions where CD4 degradation was inhibited by over-expression of the TDN Ub K48/R mutant. BFA-treated HEK 293T cells expressing CD4/Ub K48/R and Vpu or CD4/Ub K48/R alone were fractionated by mechanical lysis into membrane and cytosolic fractions and each resulting fraction was directly analyzed for the presence of CD4, Vpu and membrane or cytosolic markers, such as calnexin and actin respectively, by western-blot as described in materials and methods. Furthermore, the presence of poly-ubiquitinated forms of CD4 in membrane or cytosolic fractions was determined by immunoprecipitation/western-blot analysis. In contrast to Fig. 2 and 4 and because of technical reasons, ubiquitinated CD4 molecules were detected in these experiments by performing immunoprecipitation using anti-Myc antibodies followed by western-blot using anti-CD4 antibodies. As expected, Vpu and calnexin were detected exclusively in association with membrane fractions (Fig. 5A, lane 5 for Vpu and lanes 1, 3, 5 and 7 for calnexin) whereas actin (lanes 2, 4, 6, 8) or Ub (lanes 4, 6, 8) were recovered in a very large proportion in the cytosolic fractions, thus demonstrating that the fractionation procedure was almost free of membrane or cytosolic contaminations. CD4 molecules were found in the membrane fraction in presence or absence of Vpu (lanes 3 and 5). We could repeatedly recover and detect CD4-Ub conjugates, represented as a smear signal, predominantly in the membrane fraction but also in the cytosolic fraction in absence and in presence of Vpu (Fig. 5A); in some instances, depending on the experiments, we also detected discrete high molecular bands in addition to the smear signal [lane 5 of Additional file 2A and lane 6 of Additional file 2B]. Interestingly, the absolute signal associated with membrane and cytosolic fractions was always more intense in presence than in absence of Vpu (Fig. 5A, compare lanes 3, 5 and 7 as well as lanes 4, 6 and 8, upper panel). The specific levels of CD4-Ub conjugates associated with membrane and cytosolic fractions in absence and in presence of Vpu were calculated relative to the amount of CD4 detected directly by western-blot. As shown in Fig. 5B, quantitative analysis revealed that in presence of Vpu there was approximately a six-fold increase in membrane-associated CD4-Ub conjugate levels relative to the negative control without Vpu (Vpu-); in the cytosolic fractions, the levels of CD4-Ub conjugates detected in presence of Vpu were approximately two-fold higher relative to the Vpu- control (Fig. 5B).

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