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The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

Kerkau T, Bacik I, Bennink JR, Yewdell JW, Húnig T, Schimpl A, Schubert U - J. Exp. Med. (1997)

Bottom Line: This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4.Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains.VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunobiology, University of Würzburg, Germany.

ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.

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Vpu disturbs an early process in MHC-I biogenesis. Parallel  cultures of HeLa cells were co-infected with VV-Kd expressing mouse H  chain Kd together with VV-Vpu (+Vpu), VV-UDEL1 (−Vpu), or VVICP47 (+ICP47). 2.5 h after infection cells were pulse labeled with  [35S]methionine for 4 min, aliquoted in ice-cold medium, and chased at  37°C for up to 2 h. Half of the cell lysates were immunoprecipitated with  mAb 215 (A) or with anti-Kd serum pAb-ex8 (B). Two rounds of immunocollection were conducted and H chain molecules collected were analyzed by SDS-PAGE followed by fluorography. Only bands corresponding to H chains are demonstrated in the upper part; the quantitation of H  chains detected after sequential collection (1st and 2nd) by means of a  PhosphorImager is demonstrated in the lower part, left histograms. Stability of H chains recovered is demonstrated in the right histograms. Arrows  indicate mature glycosylated H chains detected after 2 h of chase period.
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Figure 5: Vpu disturbs an early process in MHC-I biogenesis. Parallel cultures of HeLa cells were co-infected with VV-Kd expressing mouse H chain Kd together with VV-Vpu (+Vpu), VV-UDEL1 (−Vpu), or VVICP47 (+ICP47). 2.5 h after infection cells were pulse labeled with [35S]methionine for 4 min, aliquoted in ice-cold medium, and chased at 37°C for up to 2 h. Half of the cell lysates were immunoprecipitated with mAb 215 (A) or with anti-Kd serum pAb-ex8 (B). Two rounds of immunocollection were conducted and H chain molecules collected were analyzed by SDS-PAGE followed by fluorography. Only bands corresponding to H chains are demonstrated in the upper part; the quantitation of H chains detected after sequential collection (1st and 2nd) by means of a PhosphorImager is demonstrated in the lower part, left histograms. Stability of H chains recovered is demonstrated in the right histograms. Arrows indicate mature glycosylated H chains detected after 2 h of chase period.

Mentions: Notably, the effects of Vpu in class I biogenesis were evident immediately upon pulse labeling, indicating that Vpu affects early events in the assembly of class I molecules. To facilitate biochemical evaluation of Vpu effects on H chain synthesis, we examined the biosynthesis of mouse Kd class I H chain expressed in HeLa cells co-infected with VV-Kd, VV-UDEL1, or VV-Vpu (Fig. 5). The relatively high levels of Kd expression allowed for shorter labeling times with [35S]methionine. Cells were radiolabeled for 4 min, and immediately placed on ice to stop cellular metabolism. Cells were then either maintained on ice or incubated for up to 2 h at 37°C. Cell lysates were equalized for [35S]methionine incorporation, and class I molecules were collected with either mAb 215 specific for conformed Kd (Fig. 5 A, mAB 215; reference 54) or with rabbit serum specific for exon 8 (Fig. 5 B, pAb-ex8) that reacts with both folded and unfolded Kd molecules (54). To ensure complete recovery of H chains, supernatants from the first round of immunocollection were subjected to a second round of precipitation with the same Ab (Fig. 5, A and B, 1st and 2nd). The relative amounts of H chains recovered after sequential collection were determined by PhosphorImager analysis (Fig. 5, A and B, left histograms). In the absence of Vpu, the amount of Kd recovered by mAb 215 doubled over the first 20 min of chase, and then decayed slowly. This ∼10 min half-life of Kd folding is consistent with previous findings regarding class I folding (3). We also detected a similar, but less marked, increase in Kd class I molecules collected by pAb-ex8. The increase in this case may be due to initial masking of exon 8 by interaction with molecular chaperones (60). We observed a number of remarkable effects of Vpu on Kd biogenesis. First, approximately two- to threefold less Kd reactive with either mAb 215 or pAb-ex8 was recovered from the unchased samples. Second, instead of an initial increase, the amount of Kd recovered precipitously declined over the first 20 min of chase period. Vpu had no significant effect on the stability of Kd that survived the initial effect, which are transported through the Golgi complex with kinetics similar to Kd synthesized in the control infected cells, as indicated by the recovery of a Kd form with lower mobility on SDS-PAGE (indicated by arrows in Fig. 5, A and B) that probably represents Kd with terminally sialyated N-linked oligosaccharides.


The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

Kerkau T, Bacik I, Bennink JR, Yewdell JW, Húnig T, Schimpl A, Schubert U - J. Exp. Med. (1997)

Vpu disturbs an early process in MHC-I biogenesis. Parallel  cultures of HeLa cells were co-infected with VV-Kd expressing mouse H  chain Kd together with VV-Vpu (+Vpu), VV-UDEL1 (−Vpu), or VVICP47 (+ICP47). 2.5 h after infection cells were pulse labeled with  [35S]methionine for 4 min, aliquoted in ice-cold medium, and chased at  37°C for up to 2 h. Half of the cell lysates were immunoprecipitated with  mAb 215 (A) or with anti-Kd serum pAb-ex8 (B). Two rounds of immunocollection were conducted and H chain molecules collected were analyzed by SDS-PAGE followed by fluorography. Only bands corresponding to H chains are demonstrated in the upper part; the quantitation of H  chains detected after sequential collection (1st and 2nd) by means of a  PhosphorImager is demonstrated in the lower part, left histograms. Stability of H chains recovered is demonstrated in the right histograms. Arrows  indicate mature glycosylated H chains detected after 2 h of chase period.
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Figure 5: Vpu disturbs an early process in MHC-I biogenesis. Parallel cultures of HeLa cells were co-infected with VV-Kd expressing mouse H chain Kd together with VV-Vpu (+Vpu), VV-UDEL1 (−Vpu), or VVICP47 (+ICP47). 2.5 h after infection cells were pulse labeled with [35S]methionine for 4 min, aliquoted in ice-cold medium, and chased at 37°C for up to 2 h. Half of the cell lysates were immunoprecipitated with mAb 215 (A) or with anti-Kd serum pAb-ex8 (B). Two rounds of immunocollection were conducted and H chain molecules collected were analyzed by SDS-PAGE followed by fluorography. Only bands corresponding to H chains are demonstrated in the upper part; the quantitation of H chains detected after sequential collection (1st and 2nd) by means of a PhosphorImager is demonstrated in the lower part, left histograms. Stability of H chains recovered is demonstrated in the right histograms. Arrows indicate mature glycosylated H chains detected after 2 h of chase period.
Mentions: Notably, the effects of Vpu in class I biogenesis were evident immediately upon pulse labeling, indicating that Vpu affects early events in the assembly of class I molecules. To facilitate biochemical evaluation of Vpu effects on H chain synthesis, we examined the biosynthesis of mouse Kd class I H chain expressed in HeLa cells co-infected with VV-Kd, VV-UDEL1, or VV-Vpu (Fig. 5). The relatively high levels of Kd expression allowed for shorter labeling times with [35S]methionine. Cells were radiolabeled for 4 min, and immediately placed on ice to stop cellular metabolism. Cells were then either maintained on ice or incubated for up to 2 h at 37°C. Cell lysates were equalized for [35S]methionine incorporation, and class I molecules were collected with either mAb 215 specific for conformed Kd (Fig. 5 A, mAB 215; reference 54) or with rabbit serum specific for exon 8 (Fig. 5 B, pAb-ex8) that reacts with both folded and unfolded Kd molecules (54). To ensure complete recovery of H chains, supernatants from the first round of immunocollection were subjected to a second round of precipitation with the same Ab (Fig. 5, A and B, 1st and 2nd). The relative amounts of H chains recovered after sequential collection were determined by PhosphorImager analysis (Fig. 5, A and B, left histograms). In the absence of Vpu, the amount of Kd recovered by mAb 215 doubled over the first 20 min of chase, and then decayed slowly. This ∼10 min half-life of Kd folding is consistent with previous findings regarding class I folding (3). We also detected a similar, but less marked, increase in Kd class I molecules collected by pAb-ex8. The increase in this case may be due to initial masking of exon 8 by interaction with molecular chaperones (60). We observed a number of remarkable effects of Vpu on Kd biogenesis. First, approximately two- to threefold less Kd reactive with either mAb 215 or pAb-ex8 was recovered from the unchased samples. Second, instead of an initial increase, the amount of Kd recovered precipitously declined over the first 20 min of chase period. Vpu had no significant effect on the stability of Kd that survived the initial effect, which are transported through the Golgi complex with kinetics similar to Kd synthesized in the control infected cells, as indicated by the recovery of a Kd form with lower mobility on SDS-PAGE (indicated by arrows in Fig. 5, A and B) that probably represents Kd with terminally sialyated N-linked oligosaccharides.

Bottom Line: This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4.Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains.VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunobiology, University of Würzburg, Germany.

ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.

Show MeSH
Related in: MedlinePlus