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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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Co-localization of HPV16 E5 with calnexin. HaCaT cells were transfected with AU1- tagged codon-optimised E5 or pEGFP-E5 and analysed after 24 h by confocal laser scanning microscopy using a monoclonal anti-AU1 and/or polyclonal anti-calnexin Abs.
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Figure 5: Co-localization of HPV16 E5 with calnexin. HaCaT cells were transfected with AU1- tagged codon-optimised E5 or pEGFP-E5 and analysed after 24 h by confocal laser scanning microscopy using a monoclonal anti-AU1 and/or polyclonal anti-calnexin Abs.

Mentions: To further corroborate this finding at the intracellular level we next sought to demonstrate co-localization of both proteins in human keratinocytes expressing the E5 protein. HaCaT cells were transiently transfected with AU1-tagged codon-adapted E5 and co-localization with calnexin was analysed by laser confocal double immunofluorescence microscopy. As shown in Fig. 5A we observed a sharp colocalization of both proteins, confirming already published results for retroviral transduced keratinocytes [48]. Similar results were obtained when the GFP fusion protein was expressed instead of the AU1-tagged codon-optimised E5 protein (Fig. 5B), indicating that the subcellular localization of the E5 protein does not depend on the nature of the tag used to label E5.


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)

Co-localization of HPV16 E5 with calnexin. HaCaT cells were transfected with AU1- tagged codon-optimised E5 or pEGFP-E5 and analysed after 24 h by confocal laser scanning microscopy using a monoclonal anti-AU1 and/or polyclonal anti-calnexin Abs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Co-localization of HPV16 E5 with calnexin. HaCaT cells were transfected with AU1- tagged codon-optimised E5 or pEGFP-E5 and analysed after 24 h by confocal laser scanning microscopy using a monoclonal anti-AU1 and/or polyclonal anti-calnexin Abs.
Mentions: To further corroborate this finding at the intracellular level we next sought to demonstrate co-localization of both proteins in human keratinocytes expressing the E5 protein. HaCaT cells were transiently transfected with AU1-tagged codon-adapted E5 and co-localization with calnexin was analysed by laser confocal double immunofluorescence microscopy. As shown in Fig. 5A we observed a sharp colocalization of both proteins, confirming already published results for retroviral transduced keratinocytes [48]. Similar results were obtained when the GFP fusion protein was expressed instead of the AU1-tagged codon-optimised E5 protein (Fig. 5B), indicating that the subcellular localization of the E5 protein does not depend on the nature of the tag used to label E5.

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