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Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes.

Campos SK, Ozbun MA - PLoS ONE (2009)

Bottom Line: Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope.Denaturing/non-reducing gel analysis and thiol labeling experiments of wild type and cysteine mutant HPV16 virion particles strongly support the existence of a buried intramolecular C22-C28 disulfide bond.L2 residues C22 and C28 are bound as an intramolecular disulfide bond in HPV16 virions and are necessary for infectivity.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.

ABSTRACT

Background: Minor capsid protein L2 performs an indispensable but uncharacterized role in human papillomavirus infections. A neutralizing B cell epitope has recently been mapped to the N-terminus of HPV16 L2, residues 17-36, and exposure of this region of L2 has been implicated in translocation of incoming virions from the endo/lysosomal compartment to the cellular cytoplasm. Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope. We also investigate the infectivity of virions containing L2 single and double cysteine point mutants.

Methodology and principal findings: Denaturing/non-reducing gel analysis and thiol labeling experiments of wild type and cysteine mutant HPV16 virion particles strongly support the existence of a buried intramolecular C22-C28 disulfide bond. The disulfide was confirmed by tandem mass spectrometry of L2 protein from non-reduced virions. Single C22S and C28S and the double C22/28S mutants were non-infectious but had no apparent defects in cell binding, endocytosis, or trafficking to lysosomes by 8 h post infection. During infection with L2 mutant particles, there was a marked decrease in L2 levels compared to wild type L2-containing virions, suggesting a failure of mutant L2/genome complexes to exit the endo/lysosomal compartment.

Conclusions and significance: L2 residues C22 and C28 are bound as an intramolecular disulfide bond in HPV16 virions and are necessary for infectivity. Previous work has suggested that the furin-dependent exposure of the 17-36 epitope and subsequent interaction of this region with an unknown receptor is necessary for egress from the endo/lysosomal compartment and infection. Identification of the C22-C28 disulfide suggests that reduction of this disufide bond may be necessary for exposure of 17-36 and HPV16 infection.

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Thiol biotinylation assay of HPV16 L2 cysteine mutants.(A) Lack of L2 biotinylation by BMCC-biotin of normal and mutant virions in their native state suggest that L2 cysteines are either buried and inaccessible to BMCC-biotin or bound up and not present as free thiols. (B) L2 cysteines become accessible and are biotinylated in DTT-reduced virions. (C) Denaturation of virions by SDS results in L2 biotinylation of C22S and C28S single mutants only (lanes 2,3), indicative of free but buried cysteines in these mutants. Lack of L2 biotinylation in denatured wild type virions implies that although the cysteines are exposed, they are bound as an intramolecular disulfide, becoming available for modification in the single mutants only.
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pone-0004463-g003: Thiol biotinylation assay of HPV16 L2 cysteine mutants.(A) Lack of L2 biotinylation by BMCC-biotin of normal and mutant virions in their native state suggest that L2 cysteines are either buried and inaccessible to BMCC-biotin or bound up and not present as free thiols. (B) L2 cysteines become accessible and are biotinylated in DTT-reduced virions. (C) Denaturation of virions by SDS results in L2 biotinylation of C22S and C28S single mutants only (lanes 2,3), indicative of free but buried cysteines in these mutants. Lack of L2 biotinylation in denatured wild type virions implies that although the cysteines are exposed, they are bound as an intramolecular disulfide, becoming available for modification in the single mutants only.

Mentions: Thiol-specific biotinylation experiments were performed to further elucidate the nature of HPV16 L2 C22 and C28 residues. Virions were kept in their native state, reduced by DTT, or denatured with SDS prior to BMCC-biotin labeling. As seen previously, L2 molecules from virions in their native state were not labeled by BMCC-biotin (Fig. 3A) and reduction of virions was required for L2 biotinylation (Fig. 3B). For the reduced samples, L2 biotinylation was less pronounced for the C22S and C28S mutants than for virions containing wild type HPV16 L2 proteins (Fig. 3B, lanes 2–3), presumably because single mutants have only half the number of cysteines available for BMCC-biotin modification. As expected, no labeling was observed for the double C22/28S mutant, which lacks cysteine residues (Fig. 3B, lane 4). If C22 and C28 were normally bound together as an intramolecular disulfide one might predict that the lone cysteines of the single C22S and C28S mutants in their native virion states would be free and available to react with BMCC-biotin. Yet, biotinylation was only observed upon virion reduction with DTT (Fig. 3A & B, lanes 2–3). Inaccessibility of the L2 cysteines in native virion paticles could explain this lack of BMCC-biotin labeling. To determine if C22 and C28 are simply buried in the context of native virions, virions were denatured with SDS treatment prior to BMCC-biotin labeling. Only the C22S and C28S single mutants exhibited robust L2 biotinylation upon denaturation with SDS, indicating that a majority of the lone cysteines of the C22S and C28S single mutants exist as free thiols (Fig. 3C, lanes 2–3). L2 proteins of wild type HPV16 virions were only minimally labeled after denaturation, suggesting that although C22 and C28 are accessible, they are disulfide bonded (Fig. 3C, lane 1). Again no labeling was seen for the C22/28S double mutant because no cysteines are present (Fig. 3C, lane 4).


Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes.

Campos SK, Ozbun MA - PLoS ONE (2009)

Thiol biotinylation assay of HPV16 L2 cysteine mutants.(A) Lack of L2 biotinylation by BMCC-biotin of normal and mutant virions in their native state suggest that L2 cysteines are either buried and inaccessible to BMCC-biotin or bound up and not present as free thiols. (B) L2 cysteines become accessible and are biotinylated in DTT-reduced virions. (C) Denaturation of virions by SDS results in L2 biotinylation of C22S and C28S single mutants only (lanes 2,3), indicative of free but buried cysteines in these mutants. Lack of L2 biotinylation in denatured wild type virions implies that although the cysteines are exposed, they are bound as an intramolecular disulfide, becoming available for modification in the single mutants only.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636891&req=5

pone-0004463-g003: Thiol biotinylation assay of HPV16 L2 cysteine mutants.(A) Lack of L2 biotinylation by BMCC-biotin of normal and mutant virions in their native state suggest that L2 cysteines are either buried and inaccessible to BMCC-biotin or bound up and not present as free thiols. (B) L2 cysteines become accessible and are biotinylated in DTT-reduced virions. (C) Denaturation of virions by SDS results in L2 biotinylation of C22S and C28S single mutants only (lanes 2,3), indicative of free but buried cysteines in these mutants. Lack of L2 biotinylation in denatured wild type virions implies that although the cysteines are exposed, they are bound as an intramolecular disulfide, becoming available for modification in the single mutants only.
Mentions: Thiol-specific biotinylation experiments were performed to further elucidate the nature of HPV16 L2 C22 and C28 residues. Virions were kept in their native state, reduced by DTT, or denatured with SDS prior to BMCC-biotin labeling. As seen previously, L2 molecules from virions in their native state were not labeled by BMCC-biotin (Fig. 3A) and reduction of virions was required for L2 biotinylation (Fig. 3B). For the reduced samples, L2 biotinylation was less pronounced for the C22S and C28S mutants than for virions containing wild type HPV16 L2 proteins (Fig. 3B, lanes 2–3), presumably because single mutants have only half the number of cysteines available for BMCC-biotin modification. As expected, no labeling was observed for the double C22/28S mutant, which lacks cysteine residues (Fig. 3B, lane 4). If C22 and C28 were normally bound together as an intramolecular disulfide one might predict that the lone cysteines of the single C22S and C28S mutants in their native virion states would be free and available to react with BMCC-biotin. Yet, biotinylation was only observed upon virion reduction with DTT (Fig. 3A & B, lanes 2–3). Inaccessibility of the L2 cysteines in native virion paticles could explain this lack of BMCC-biotin labeling. To determine if C22 and C28 are simply buried in the context of native virions, virions were denatured with SDS treatment prior to BMCC-biotin labeling. Only the C22S and C28S single mutants exhibited robust L2 biotinylation upon denaturation with SDS, indicating that a majority of the lone cysteines of the C22S and C28S single mutants exist as free thiols (Fig. 3C, lanes 2–3). L2 proteins of wild type HPV16 virions were only minimally labeled after denaturation, suggesting that although C22 and C28 are accessible, they are disulfide bonded (Fig. 3C, lane 1). Again no labeling was seen for the C22/28S double mutant because no cysteines are present (Fig. 3C, lane 4).

Bottom Line: Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope.Denaturing/non-reducing gel analysis and thiol labeling experiments of wild type and cysteine mutant HPV16 virion particles strongly support the existence of a buried intramolecular C22-C28 disulfide bond.L2 residues C22 and C28 are bound as an intramolecular disulfide bond in HPV16 virions and are necessary for infectivity.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.

ABSTRACT

Background: Minor capsid protein L2 performs an indispensable but uncharacterized role in human papillomavirus infections. A neutralizing B cell epitope has recently been mapped to the N-terminus of HPV16 L2, residues 17-36, and exposure of this region of L2 has been implicated in translocation of incoming virions from the endo/lysosomal compartment to the cellular cytoplasm. Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope. We also investigate the infectivity of virions containing L2 single and double cysteine point mutants.

Methodology and principal findings: Denaturing/non-reducing gel analysis and thiol labeling experiments of wild type and cysteine mutant HPV16 virion particles strongly support the existence of a buried intramolecular C22-C28 disulfide bond. The disulfide was confirmed by tandem mass spectrometry of L2 protein from non-reduced virions. Single C22S and C28S and the double C22/28S mutants were non-infectious but had no apparent defects in cell binding, endocytosis, or trafficking to lysosomes by 8 h post infection. During infection with L2 mutant particles, there was a marked decrease in L2 levels compared to wild type L2-containing virions, suggesting a failure of mutant L2/genome complexes to exit the endo/lysosomal compartment.

Conclusions and significance: L2 residues C22 and C28 are bound as an intramolecular disulfide bond in HPV16 virions and are necessary for infectivity. Previous work has suggested that the furin-dependent exposure of the 17-36 epitope and subsequent interaction of this region with an unknown receptor is necessary for egress from the endo/lysosomal compartment and infection. Identification of the C22-C28 disulfide suggests that reduction of this disufide bond may be necessary for exposure of 17-36 and HPV16 infection.

Show MeSH
Related in: MedlinePlus