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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 forms a ternary complex with calnexin and the HLA-I heavy chain. HeLa cells were transiently transfected with AU1-tagged codon-optimised HPV16 E5, M1, or empty vector. 24 h later CHAPS lysates were immunoprecipitated with antibodies against the E5-tag (anti-AU1). Precipitated immune complexes were separated by SDS-PAGE and Western blotted using anti-calnexin and anti-HLA-B, -C mAb (HC10), respectively (band marked with *). Molecular-mass markers in kDa are indicated at the left of the blots.
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Figure 9: HPV16 E5 forms a ternary complex with calnexin and the HLA-I heavy chain. HeLa cells were transiently transfected with AU1-tagged codon-optimised HPV16 E5, M1, or empty vector. 24 h later CHAPS lysates were immunoprecipitated with antibodies against the E5-tag (anti-AU1). Precipitated immune complexes were separated by SDS-PAGE and Western blotted using anti-calnexin and anti-HLA-B, -C mAb (HC10), respectively (band marked with *). Molecular-mass markers in kDa are indicated at the left of the blots.

Mentions: Recent results have shown that HPV16 E5 may co-precipitate with the heavy chain of HLA-I [21]. In the light of our results presented above, and together with the fact that HLA-I and calnexin associate during HLA maturation, we hypothesized that the formation of a trimeric complex between HLA-I heavy chain, calnexin and E5 might be involved in the retention of HLA-I in the ER/Golgi apparatus of the cells expressing E5. To address this question, HeLa cells were transfected with AU1-tagged codon-optimised E5 or with mutant M1, and protein extracts were immunoprecipitated with anti-AU1. Immunoprecipitates separated in SDS-PAGE, were blotted onto PVDF membrane and probed either with anti-HC10, recognizing HLA-B, C heavy chains [49], or with anti-calnexin antibodies. As shown in Fig. 9, both HLA-I heavy chain and calnexin could be co-immunoprecipitated with anti-AU1 antibodies, which target E5. More important, the E5 mutant M1 previously shown to be deficient in immunoprecipitation of calnexin, also failed to co-precipitate the HLA-I heavy chain. These results demonstrate that HPV16 E5 forms a complex with calnexin and HLA-I heavy chain and that this complex depends on the interaction of the first hydrophobic region of E5 with calnexin.


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 forms a ternary complex with calnexin and the HLA-I heavy chain. HeLa cells were transiently transfected with AU1-tagged codon-optimised HPV16 E5, M1, or empty vector. 24 h later CHAPS lysates were immunoprecipitated with antibodies against the E5-tag (anti-AU1). Precipitated immune complexes were separated by SDS-PAGE and Western blotted using anti-calnexin and anti-HLA-B, -C mAb (HC10), respectively (band marked with *). Molecular-mass markers in kDa are indicated at the left of the blots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: HPV16 E5 forms a ternary complex with calnexin and the HLA-I heavy chain. HeLa cells were transiently transfected with AU1-tagged codon-optimised HPV16 E5, M1, or empty vector. 24 h later CHAPS lysates were immunoprecipitated with antibodies against the E5-tag (anti-AU1). Precipitated immune complexes were separated by SDS-PAGE and Western blotted using anti-calnexin and anti-HLA-B, -C mAb (HC10), respectively (band marked with *). Molecular-mass markers in kDa are indicated at the left of the blots.
Mentions: Recent results have shown that HPV16 E5 may co-precipitate with the heavy chain of HLA-I [21]. In the light of our results presented above, and together with the fact that HLA-I and calnexin associate during HLA maturation, we hypothesized that the formation of a trimeric complex between HLA-I heavy chain, calnexin and E5 might be involved in the retention of HLA-I in the ER/Golgi apparatus of the cells expressing E5. To address this question, HeLa cells were transfected with AU1-tagged codon-optimised E5 or with mutant M1, and protein extracts were immunoprecipitated with anti-AU1. Immunoprecipitates separated in SDS-PAGE, were blotted onto PVDF membrane and probed either with anti-HC10, recognizing HLA-B, C heavy chains [49], or with anti-calnexin antibodies. As shown in Fig. 9, both HLA-I heavy chain and calnexin could be co-immunoprecipitated with anti-AU1 antibodies, which target E5. More important, the E5 mutant M1 previously shown to be deficient in immunoprecipitation of calnexin, also failed to co-precipitate the HLA-I heavy chain. These results demonstrate that HPV16 E5 forms a complex with calnexin and HLA-I heavy chain and that this complex depends on the interaction of the first hydrophobic region of E5 with calnexin.

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