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The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells.

Gruener M, Bravo IG, Momburg F, Alonso A, Tomakidi P - Virol. J. (2007)

Bottom Line: The molecular mechanisms underlying this effect are so far unknown.In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein.On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Differentiation, German Cancer Research Center, Heidelberg, Germany. m.gruener@dkfz.de

ABSTRACT
The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

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HPV16 E5 expression down-regulates HLA-I surface molecules. HEK-293T cells were transfected either with (A) pEGFP-HPV16-E5 or empty pEGFP vector, (B) pCI-HPV16-E5-HA or empty pCI vector. HLA-I molecules were then detected by immunostaining and flow cytometry using mouse monoclonal anti-HLA-A, B, C (mAb B9.12). Differences between the HLA-I surface expression levels were assessed by Kolmogorov-Smirnov test. This statistic defines the maximum vertical deviation between the two curves (pEGFP-E5 and GFP, pCI-E5-HA and pCI) as the statistic D. The p value of each single experiment was in all cases ≤ 0.001.
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Figure 1: HPV16 E5 expression down-regulates HLA-I surface molecules. HEK-293T cells were transfected either with (A) pEGFP-HPV16-E5 or empty pEGFP vector, (B) pCI-HPV16-E5-HA or empty pCI vector. HLA-I molecules were then detected by immunostaining and flow cytometry using mouse monoclonal anti-HLA-A, B, C (mAb B9.12). Differences between the HLA-I surface expression levels were assessed by Kolmogorov-Smirnov test. This statistic defines the maximum vertical deviation between the two curves (pEGFP-E5 and GFP, pCI-E5-HA and pCI) as the statistic D. The p value of each single experiment was in all cases ≤ 0.001.

Mentions: Experimental results have shown that BPV E5 as well as HPV16 E5 and HPV2 E5 proteins down-regulate surface expression of HLA-I molecules [22,24,41,42]. To evaluate this effect under our experimental conditions, we transfected pEGFP-HPV16-E5 or pCI-HPV16-E5-HA into HEK-293T cells and analysed cell surface expression of HLA-I by flow cytometry. Both constructs lead to a significant down-regulation of HLA-I surface expression (p ≤ 0.001, Kolmogorov-Smirnov test, Fig. 1). For the pEGFP-HPV16-E5 and pEGFP constructs, the intracellular GFP-dependent fluorescence allowed us to gate GFP-expressing transfected cells making it possible to compare GFP-E5 with GFP positive populations in respect to their HLA-I signals (Fig. 1A). Further, in our hands the anti-HA antibody did not render sharp results differentiating transfected from untransfected cells. For this reason, the effects for the pCI-HPV16-E5-HA and pCI constructs were assessed by comparing total living cell populations (Fig. 1B). Since transfection efficiency never reached 100 %, reduction in relative values of the HLA-I surface expression tended to be more discrete in HPV16E5-HA than in pEGFP-HPV16-E5 transfected cells, leading to clearly significant though smaller values in the statistical analyses (Fig. 1A and 1B). These results therefore demonstrate that HPV16 E5 can down-regulate cell surface expression under our experimental conditions. Further, they also show that neither the small HA (10 amino acids) nor the large EGFP (239 amino acids) used for tagging the viral protein impairs the ability of HPV16 E5 to down-regulate HLA-I cell surface expression.


The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells.

Gruener M, Bravo IG, Momburg F, Alonso A, Tomakidi P - Virol. J. (2007)

HPV16 E5 expression down-regulates HLA-I surface molecules. HEK-293T cells were transfected either with (A) pEGFP-HPV16-E5 or empty pEGFP vector, (B) pCI-HPV16-E5-HA or empty pCI vector. HLA-I molecules were then detected by immunostaining and flow cytometry using mouse monoclonal anti-HLA-A, B, C (mAb B9.12). Differences between the HLA-I surface expression levels were assessed by Kolmogorov-Smirnov test. This statistic defines the maximum vertical deviation between the two curves (pEGFP-E5 and GFP, pCI-E5-HA and pCI) as the statistic D. The p value of each single experiment was in all cases ≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164959&req=5

Figure 1: HPV16 E5 expression down-regulates HLA-I surface molecules. HEK-293T cells were transfected either with (A) pEGFP-HPV16-E5 or empty pEGFP vector, (B) pCI-HPV16-E5-HA or empty pCI vector. HLA-I molecules were then detected by immunostaining and flow cytometry using mouse monoclonal anti-HLA-A, B, C (mAb B9.12). Differences between the HLA-I surface expression levels were assessed by Kolmogorov-Smirnov test. This statistic defines the maximum vertical deviation between the two curves (pEGFP-E5 and GFP, pCI-E5-HA and pCI) as the statistic D. The p value of each single experiment was in all cases ≤ 0.001.
Mentions: Experimental results have shown that BPV E5 as well as HPV16 E5 and HPV2 E5 proteins down-regulate surface expression of HLA-I molecules [22,24,41,42]. To evaluate this effect under our experimental conditions, we transfected pEGFP-HPV16-E5 or pCI-HPV16-E5-HA into HEK-293T cells and analysed cell surface expression of HLA-I by flow cytometry. Both constructs lead to a significant down-regulation of HLA-I surface expression (p ≤ 0.001, Kolmogorov-Smirnov test, Fig. 1). For the pEGFP-HPV16-E5 and pEGFP constructs, the intracellular GFP-dependent fluorescence allowed us to gate GFP-expressing transfected cells making it possible to compare GFP-E5 with GFP positive populations in respect to their HLA-I signals (Fig. 1A). Further, in our hands the anti-HA antibody did not render sharp results differentiating transfected from untransfected cells. For this reason, the effects for the pCI-HPV16-E5-HA and pCI constructs were assessed by comparing total living cell populations (Fig. 1B). Since transfection efficiency never reached 100 %, reduction in relative values of the HLA-I surface expression tended to be more discrete in HPV16E5-HA than in pEGFP-HPV16-E5 transfected cells, leading to clearly significant though smaller values in the statistical analyses (Fig. 1A and 1B). These results therefore demonstrate that HPV16 E5 can down-regulate cell surface expression under our experimental conditions. Further, they also show that neither the small HA (10 amino acids) nor the large EGFP (239 amino acids) used for tagging the viral protein impairs the ability of HPV16 E5 to down-regulate HLA-I cell surface expression.

Bottom Line: The molecular mechanisms underlying this effect are so far unknown.In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein.On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Differentiation, German Cancer Research Center, Heidelberg, Germany. m.gruener@dkfz.de

ABSTRACT
The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

Show MeSH
Related in: MedlinePlus