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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 KRcyto poly-ubiquitination. A. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 25 μg of the TDN mutant of Ub his(6)/c-myc-Ub K48/R. Transfected cells were not treated with BFA prior to lysis. Samples were then treated as described in the materials and methods. B. Quantitative analysis of the relative levels of ubiquitinated CD4 conjugates for CD4 wt and CD4 KRcyto in two independent experiments. (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Relative levels of ubiquitinated CD4 conjugates were determined as described in Fig. 2B.
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Figure 4: Effect of Vpu on CD4 KRcyto poly-ubiquitination. A. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 25 μg of the TDN mutant of Ub his(6)/c-myc-Ub K48/R. Transfected cells were not treated with BFA prior to lysis. Samples were then treated as described in the materials and methods. B. Quantitative analysis of the relative levels of ubiquitinated CD4 conjugates for CD4 wt and CD4 KRcyto in two independent experiments. (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Relative levels of ubiquitinated CD4 conjugates were determined as described in Fig. 2B.

Mentions: Given that CD4 KRcyto was still susceptible to Vpu-mediated degradation, we next evaluated whether CD4 KRcyto could undergo ubiquitination in presence of Vpu. To optimize the recovery of CD4-Ub conjugates, Vpu/CD4 or Vpu/CD4 KRcyto HEK 293T transfectants were made to co-express the TDN Ub K48/R mutant. Analysis of CD4-Ub and CD4 KRcyto-Ub conjugates levels in presence or absence of Vpu was performed as described above for Fig. 2B. Fig. 4A reveals that even though CD4 KRcyto is less susceptible to Vpu-mediated degradation as compared to CD4 wt (compare lanes 1 and 3 with lanes 5 and 7, middle panel), it still undergoes enhanced ubiquitination in presence of Vpu (compare lane 6 and lane 8). However, it is important to note that the relative level of recovered CD4 KRcyto-Ub conjugates was decreased as compared to CD4-Ub conjugates. In fact, quantitative analysis of ubiquitinated CD4 conjugate levels reveals that Vpu enhanced ubiquitination of CD4 KR by approximately 3-fold while it increased ubiquitination of wt CD4 by 8-fold. Altogether, these results suggest that lysine residues in the cytosolic domain of CD4 are not absolutely essential for ubiquitination and degradation of the viral receptor in presence of Vpu. Even-though optimal Vpu-mediated CD4 ubiquitination most probably involves cytosolic lysine residues there must be other sites that are also targeted during Vpu-induced ubiquitination.


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 KRcyto poly-ubiquitination. A. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 25 μg of the TDN mutant of Ub his(6)/c-myc-Ub K48/R. Transfected cells were not treated with BFA prior to lysis. Samples were then treated as described in the materials and methods. B. Quantitative analysis of the relative levels of ubiquitinated CD4 conjugates for CD4 wt and CD4 KRcyto in two independent experiments. (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Relative levels of ubiquitinated CD4 conjugates were determined as described in Fig. 2B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2170451&req=5

Figure 4: Effect of Vpu on CD4 KRcyto poly-ubiquitination. A. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto, 10 μg of provirus encoding Vpu- (HxBH10-vpu-) or Vpu+ (HxBH10-vpu+) and 25 μg of the TDN mutant of Ub his(6)/c-myc-Ub K48/R. Transfected cells were not treated with BFA prior to lysis. Samples were then treated as described in the materials and methods. B. Quantitative analysis of the relative levels of ubiquitinated CD4 conjugates for CD4 wt and CD4 KRcyto in two independent experiments. (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Relative levels of ubiquitinated CD4 conjugates were determined as described in Fig. 2B.
Mentions: Given that CD4 KRcyto was still susceptible to Vpu-mediated degradation, we next evaluated whether CD4 KRcyto could undergo ubiquitination in presence of Vpu. To optimize the recovery of CD4-Ub conjugates, Vpu/CD4 or Vpu/CD4 KRcyto HEK 293T transfectants were made to co-express the TDN Ub K48/R mutant. Analysis of CD4-Ub and CD4 KRcyto-Ub conjugates levels in presence or absence of Vpu was performed as described above for Fig. 2B. Fig. 4A reveals that even though CD4 KRcyto is less susceptible to Vpu-mediated degradation as compared to CD4 wt (compare lanes 1 and 3 with lanes 5 and 7, middle panel), it still undergoes enhanced ubiquitination in presence of Vpu (compare lane 6 and lane 8). However, it is important to note that the relative level of recovered CD4 KRcyto-Ub conjugates was decreased as compared to CD4-Ub conjugates. In fact, quantitative analysis of ubiquitinated CD4 conjugate levels reveals that Vpu enhanced ubiquitination of CD4 KR by approximately 3-fold while it increased ubiquitination of wt CD4 by 8-fold. Altogether, these results suggest that lysine residues in the cytosolic domain of CD4 are not absolutely essential for ubiquitination and degradation of the viral receptor in presence of Vpu. Even-though optimal Vpu-mediated CD4 ubiquitination most probably involves cytosolic lysine residues there must be other sites that are also targeted during Vpu-induced ubiquitination.

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