Limits...
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.

Show MeSH

Related in: MedlinePlus

Transmembrane Hidden Markov Model posterior probabilities for the sequences of E5 and the mutants M1, M2 and M3. A) Amino acid sequence of the wild-type E5 protein and corresponding mutants. Aminoacids of the AU1-tag are underlined. Arrows show the position of exchanged amino acids. B) Analysis of the wild-type E5 and mutants using the TMHMM 2.0 algorithm (36, 37), showing the three hydrophobic regions predicted to be transmembrane domains, and the corresponding disruptions in the three mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2164959&req=5

Figure 6: Transmembrane Hidden Markov Model posterior probabilities for the sequences of E5 and the mutants M1, M2 and M3. A) Amino acid sequence of the wild-type E5 protein and corresponding mutants. Aminoacids of the AU1-tag are underlined. Arrows show the position of exchanged amino acids. B) Analysis of the wild-type E5 and mutants using the TMHMM 2.0 algorithm (36, 37), showing the three hydrophobic regions predicted to be transmembrane domains, and the corresponding disruptions in the three mutants.

Mentions: To analyze the characteristics of the E5-calnexin binding in more detail, we prepared a series of point mutants -M1, M2 and M3- in which we modified the E5 protein sequence, altering the hydrophobic profile and the local propensity to form helical structures. Leucine and/or isoleucine residues were mutated to proline, aspartate or arginines and then the resulting hydrophobic profile, propensity to helical structure and potential for stably spanning the cellular membrane were analysed and compared with those of the wild-type E5 protein (Fig. 6A, 6B). The point mutations were chosen so that they resulted respetively in the disruption of each of the three putative transmembrane helix within each of the three hydrophobic domains of the E5 protein, without changing the total protein length. All three mutants were based on the codon-optimised version of 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)

Transmembrane Hidden Markov Model posterior probabilities for the sequences of E5 and the mutants M1, M2 and M3. A) Amino acid sequence of the wild-type E5 protein and corresponding mutants. Aminoacids of the AU1-tag are underlined. Arrows show the position of exchanged amino acids. B) Analysis of the wild-type E5 and mutants using the TMHMM 2.0 algorithm (36, 37), showing the three hydrophobic regions predicted to be transmembrane domains, and the corresponding disruptions in the three mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Transmembrane Hidden Markov Model posterior probabilities for the sequences of E5 and the mutants M1, M2 and M3. A) Amino acid sequence of the wild-type E5 protein and corresponding mutants. Aminoacids of the AU1-tag are underlined. Arrows show the position of exchanged amino acids. B) Analysis of the wild-type E5 and mutants using the TMHMM 2.0 algorithm (36, 37), showing the three hydrophobic regions predicted to be transmembrane domains, and the corresponding disruptions in the three mutants.
Mentions: To analyze the characteristics of the E5-calnexin binding in more detail, we prepared a series of point mutants -M1, M2 and M3- in which we modified the E5 protein sequence, altering the hydrophobic profile and the local propensity to form helical structures. Leucine and/or isoleucine residues were mutated to proline, aspartate or arginines and then the resulting hydrophobic profile, propensity to helical structure and potential for stably spanning the cellular membrane were analysed and compared with those of the wild-type E5 protein (Fig. 6A, 6B). The point mutations were chosen so that they resulted respetively in the disruption of each of the three putative transmembrane helix within each of the three hydrophobic domains of the E5 protein, without changing the total protein length. All three mutants were based on the codon-optimised version of 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