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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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(A) Expression of  Vpu by recombinant vaccinia viruses. HeLa cells were infected  with rVVs expressing either  wild-type Vpu (VV-Vpu) or the  mutant VpuDEL1 (VV-UDEL1). 3 h  after infection, HeLa cells were  pulse labeled with [35S]methionine for 25 min and cells expressing wild-type Vpu were chased  for up to 3 h. Cell lysates were  immunoprecipitated with antiVpu sera (sheep and rabbit), separated in a 12.5% acryl aide gel,  and analyzed by fluorography.  Positions of 14C-labeled molecular weight marker proteins are  indicated on the left (M). (B)  Mouse L929 cells were co- infected with rVVs expressing either human CD4 (vCB-3) or wild-type Vpu (VV-Vpu). 3 h after infection, cells were pulse labeled with [35S]methionine for 7 min and chased for up to  4 h. CD4 molecules were recovered with anti-CD4 serum, separated in a 10% acryl aide gel, and analyzed by fluorography. Only a part of the fluorogram demonstrating CD4-specific bands in the range of 55 kD is shown. Stability of CD4 present at different times during the chase period were calculated relative to the levels of CD4 present at the end of the pulse labeling (0), which was empirically defined as 100%.
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Figure 3: (A) Expression of Vpu by recombinant vaccinia viruses. HeLa cells were infected with rVVs expressing either wild-type Vpu (VV-Vpu) or the mutant VpuDEL1 (VV-UDEL1). 3 h after infection, HeLa cells were pulse labeled with [35S]methionine for 25 min and cells expressing wild-type Vpu were chased for up to 3 h. Cell lysates were immunoprecipitated with antiVpu sera (sheep and rabbit), separated in a 12.5% acryl aide gel, and analyzed by fluorography. Positions of 14C-labeled molecular weight marker proteins are indicated on the left (M). (B) Mouse L929 cells were co- infected with rVVs expressing either human CD4 (vCB-3) or wild-type Vpu (VV-Vpu). 3 h after infection, cells were pulse labeled with [35S]methionine for 7 min and chased for up to 4 h. CD4 molecules were recovered with anti-CD4 serum, separated in a 10% acryl aide gel, and analyzed by fluorography. Only a part of the fluorogram demonstrating CD4-specific bands in the range of 55 kD is shown. Stability of CD4 present at different times during the chase period were calculated relative to the levels of CD4 present at the end of the pulse labeling (0), which was empirically defined as 100%.

Mentions: Using a Vpu-specific antiserum, a protein with the predicted mobility of Vpu in SDS-PAGE was recovered from HeLa cells infected with VV-Vpu (Fig. 3 A), but not with VV-UDEL1 or from the uninfected culture. The stability of Vpu expressed by VV-Vpu was comparable to the half life of Vpu previously reported for HIV-1–infected or –transfected human cell lines (25, 44, 46). To characterize the biological activity of Vpu in VV-infected cells, we examined its effects on CD4 biogenesis. Mouse L929 cells were coinfected with VV-Vpu and vCB-3 (expressing wild-type human CD4; reference 51), radiolabeled for 7 min with [35S]methionine, and then chased at 37°C for up to 240 min (Fig. 3 B). Detergent extracts were immunoprecipitated with an anti-CD4 antiserum, and analyzed by SDSPAGE (Fig. 3 B, inset). The kinetics of CD4 decay were determined by calculating the levels of CD4 present at different times relative to the levels of CD4 present at the end of the pulse (0 min), which was defined as 100% (Fig. 3 B). In the presence of rVV expressed Vpu, the half-life of CD4 was ∼22 min. Similar Vpu activity was observed in CD4+ T cell lines and HeLa cells co-infected with VV-Vpu and vCB-3 (not shown). In the absence of Vpu, CD4 exhibited a t1/2 ∼4 h which is consistent with previously reported half-lives of CD4 in human cell lines (39, 46). Therefore, rVV-expressed Vpu has biological activity comparable to Vpu expressed in human cell lines transfected with vpu+ HIV-1 subgenomic expression vectors (38, 44, 46).


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)

(A) Expression of  Vpu by recombinant vaccinia viruses. HeLa cells were infected  with rVVs expressing either  wild-type Vpu (VV-Vpu) or the  mutant VpuDEL1 (VV-UDEL1). 3 h  after infection, HeLa cells were  pulse labeled with [35S]methionine for 25 min and cells expressing wild-type Vpu were chased  for up to 3 h. Cell lysates were  immunoprecipitated with antiVpu sera (sheep and rabbit), separated in a 12.5% acryl aide gel,  and analyzed by fluorography.  Positions of 14C-labeled molecular weight marker proteins are  indicated on the left (M). (B)  Mouse L929 cells were co- infected with rVVs expressing either human CD4 (vCB-3) or wild-type Vpu (VV-Vpu). 3 h after infection, cells were pulse labeled with [35S]methionine for 7 min and chased for up to  4 h. CD4 molecules were recovered with anti-CD4 serum, separated in a 10% acryl aide gel, and analyzed by fluorography. Only a part of the fluorogram demonstrating CD4-specific bands in the range of 55 kD is shown. Stability of CD4 present at different times during the chase period were calculated relative to the levels of CD4 present at the end of the pulse labeling (0), which was empirically defined as 100%.
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Related In: Results  -  Collection

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Figure 3: (A) Expression of Vpu by recombinant vaccinia viruses. HeLa cells were infected with rVVs expressing either wild-type Vpu (VV-Vpu) or the mutant VpuDEL1 (VV-UDEL1). 3 h after infection, HeLa cells were pulse labeled with [35S]methionine for 25 min and cells expressing wild-type Vpu were chased for up to 3 h. Cell lysates were immunoprecipitated with antiVpu sera (sheep and rabbit), separated in a 12.5% acryl aide gel, and analyzed by fluorography. Positions of 14C-labeled molecular weight marker proteins are indicated on the left (M). (B) Mouse L929 cells were co- infected with rVVs expressing either human CD4 (vCB-3) or wild-type Vpu (VV-Vpu). 3 h after infection, cells were pulse labeled with [35S]methionine for 7 min and chased for up to 4 h. CD4 molecules were recovered with anti-CD4 serum, separated in a 10% acryl aide gel, and analyzed by fluorography. Only a part of the fluorogram demonstrating CD4-specific bands in the range of 55 kD is shown. Stability of CD4 present at different times during the chase period were calculated relative to the levels of CD4 present at the end of the pulse labeling (0), which was empirically defined as 100%.
Mentions: Using a Vpu-specific antiserum, a protein with the predicted mobility of Vpu in SDS-PAGE was recovered from HeLa cells infected with VV-Vpu (Fig. 3 A), but not with VV-UDEL1 or from the uninfected culture. The stability of Vpu expressed by VV-Vpu was comparable to the half life of Vpu previously reported for HIV-1–infected or –transfected human cell lines (25, 44, 46). To characterize the biological activity of Vpu in VV-infected cells, we examined its effects on CD4 biogenesis. Mouse L929 cells were coinfected with VV-Vpu and vCB-3 (expressing wild-type human CD4; reference 51), radiolabeled for 7 min with [35S]methionine, and then chased at 37°C for up to 240 min (Fig. 3 B). Detergent extracts were immunoprecipitated with an anti-CD4 antiserum, and analyzed by SDSPAGE (Fig. 3 B, inset). The kinetics of CD4 decay were determined by calculating the levels of CD4 present at different times relative to the levels of CD4 present at the end of the pulse (0 min), which was defined as 100% (Fig. 3 B). In the presence of rVV expressed Vpu, the half-life of CD4 was ∼22 min. Similar Vpu activity was observed in CD4+ T cell lines and HeLa cells co-infected with VV-Vpu and vCB-3 (not shown). In the absence of Vpu, CD4 exhibited a t1/2 ∼4 h which is consistent with previously reported half-lives of CD4 in human cell lines (39, 46). Therefore, rVV-expressed Vpu has biological activity comparable to Vpu expressed in human cell lines transfected with vpu+ HIV-1 subgenomic expression vectors (38, 44, 46).

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