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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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Mutant M1 binds less calnexin than wild-type E5 protein. HEK-293T cells were transfected with either (A-C) AU1-tagged codon-optimised HPV16 E5, the mutants M1, M2 and M3 or pcDNA 3.1 empty vector as control, (D and E) pEGFP-tagged HPV16 E5, mutant pEGFPM1, mock-control or pEGFP empty vector and lysed at 24 h posttransfection with CHAPS lysis buffer. A) Similar expression levels of all HPV16 E5 and the mutants M1, M2 and M3. B) Immunoprecipitations were performed using monoclonal anti-AU1, and proteins in the immune complex were detected using anti-AU1 and anti-calnexin. C) Quantification of co-precipitated calnexin for wild-type HPV16E5 protein, the mutants M1, M2, M3 and the vector control. The wild-type expression level was set to 100%. Data shown represent six independent experiments 2 plus standard errors of the mean. P values were calculated with paired two-tailed Student's t-test. D) Similar expression levels of pEGFP-HPV16-E5 and the mutant pEGFP-M1. E) Immunoprecipitations were performed using monoclonal anti-GFP, and proteins in the immune complex were detected using anti-GFP and anti-calnexin. Molecular-mass markers in kDa are indicated at the left of the blots.
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Figure 7: Mutant M1 binds less calnexin than wild-type E5 protein. HEK-293T cells were transfected with either (A-C) AU1-tagged codon-optimised HPV16 E5, the mutants M1, M2 and M3 or pcDNA 3.1 empty vector as control, (D and E) pEGFP-tagged HPV16 E5, mutant pEGFPM1, mock-control or pEGFP empty vector and lysed at 24 h posttransfection with CHAPS lysis buffer. A) Similar expression levels of all HPV16 E5 and the mutants M1, M2 and M3. B) Immunoprecipitations were performed using monoclonal anti-AU1, and proteins in the immune complex were detected using anti-AU1 and anti-calnexin. C) Quantification of co-precipitated calnexin for wild-type HPV16E5 protein, the mutants M1, M2, M3 and the vector control. The wild-type expression level was set to 100%. Data shown represent six independent experiments 2 plus standard errors of the mean. P values were calculated with paired two-tailed Student's t-test. D) Similar expression levels of pEGFP-HPV16-E5 and the mutant pEGFP-M1. E) Immunoprecipitations were performed using monoclonal anti-GFP, and proteins in the immune complex were detected using anti-GFP and anti-calnexin. Molecular-mass markers in kDa are indicated at the left of the blots.

Mentions: To test whether the mutants M1, M2 and M3 were expressed at similar levels, HEK-293T cells were transfected with the original codon-optimised E5 sequences or with each of the mutants, and the protein content was analysed by immunoblotting. As shown in Fig. 7A, all recombinants showed similar levels of expression, being differences in SDS-PAGE migration attributable to the different hydrophobicity of the proteins.


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)

Mutant M1 binds less calnexin than wild-type E5 protein. HEK-293T cells were transfected with either (A-C) AU1-tagged codon-optimised HPV16 E5, the mutants M1, M2 and M3 or pcDNA 3.1 empty vector as control, (D and E) pEGFP-tagged HPV16 E5, mutant pEGFPM1, mock-control or pEGFP empty vector and lysed at 24 h posttransfection with CHAPS lysis buffer. A) Similar expression levels of all HPV16 E5 and the mutants M1, M2 and M3. B) Immunoprecipitations were performed using monoclonal anti-AU1, and proteins in the immune complex were detected using anti-AU1 and anti-calnexin. C) Quantification of co-precipitated calnexin for wild-type HPV16E5 protein, the mutants M1, M2, M3 and the vector control. The wild-type expression level was set to 100%. Data shown represent six independent experiments 2 plus standard errors of the mean. P values were calculated with paired two-tailed Student's t-test. D) Similar expression levels of pEGFP-HPV16-E5 and the mutant pEGFP-M1. E) Immunoprecipitations were performed using monoclonal anti-GFP, and proteins in the immune complex were detected using anti-GFP and anti-calnexin. 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 7: Mutant M1 binds less calnexin than wild-type E5 protein. HEK-293T cells were transfected with either (A-C) AU1-tagged codon-optimised HPV16 E5, the mutants M1, M2 and M3 or pcDNA 3.1 empty vector as control, (D and E) pEGFP-tagged HPV16 E5, mutant pEGFPM1, mock-control or pEGFP empty vector and lysed at 24 h posttransfection with CHAPS lysis buffer. A) Similar expression levels of all HPV16 E5 and the mutants M1, M2 and M3. B) Immunoprecipitations were performed using monoclonal anti-AU1, and proteins in the immune complex were detected using anti-AU1 and anti-calnexin. C) Quantification of co-precipitated calnexin for wild-type HPV16E5 protein, the mutants M1, M2, M3 and the vector control. The wild-type expression level was set to 100%. Data shown represent six independent experiments 2 plus standard errors of the mean. P values were calculated with paired two-tailed Student's t-test. D) Similar expression levels of pEGFP-HPV16-E5 and the mutant pEGFP-M1. E) Immunoprecipitations were performed using monoclonal anti-GFP, and proteins in the immune complex were detected using anti-GFP and anti-calnexin. Molecular-mass markers in kDa are indicated at the left of the blots.
Mentions: To test whether the mutants M1, M2 and M3 were expressed at similar levels, HEK-293T cells were transfected with the original codon-optimised E5 sequences or with each of the mutants, and the protein content was analysed by immunoblotting. As shown in Fig. 7A, all recombinants showed similar levels of expression, being differences in SDS-PAGE migration attributable to the different hydrophobicity of the proteins.

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