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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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Highly conserved HPV16 L2 cysteines C22 and C28 are not reactive with BMCC-biotin in native virions.(A) Primary amino acid sequence alignment of L2 proteins from various members of the Family Papillomaviridae shows conservation of C22 and C28 (HPV16) across divergent genera. (B) Amine and thiol biotinylation assays of HPV16 virions. Purified virions were either mock treated (native) or DTT treated (reduced) prior to incubation with amine reactive NHS-biotin or thiol reactive BMCC-biotin. Biotinylated proteins were separated by SDS-PAGE, transferred to membranes, and detected with streptavidin-HRP. Note that L2 cysteines are only reactive when the virions are reduced prior to labeling.
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pone-0004463-g001: Highly conserved HPV16 L2 cysteines C22 and C28 are not reactive with BMCC-biotin in native virions.(A) Primary amino acid sequence alignment of L2 proteins from various members of the Family Papillomaviridae shows conservation of C22 and C28 (HPV16) across divergent genera. (B) Amine and thiol biotinylation assays of HPV16 virions. Purified virions were either mock treated (native) or DTT treated (reduced) prior to incubation with amine reactive NHS-biotin or thiol reactive BMCC-biotin. Biotinylated proteins were separated by SDS-PAGE, transferred to membranes, and detected with streptavidin-HRP. Note that L2 cysteines are only reactive when the virions are reduced prior to labeling.

Mentions: Sequence alignment of diverse papillomavirus types shows that C22 and C28 of HPV16 L2 are absolutely conserved throughout the Family Papillomaviridae (Fig. 1A), prompting us to further examine the role of these two cysteines in HPV infection. To examine the native redox state of the two highly conserved L2 cysteines within the structural context of virions, we performed a thiol-specific biotinylation assay. Purified HPV16 virions were kept in their native state or reduced by DTT prior to chemical biotinylation with either amine-specific NHS-biotin or thiol-specific BMCC-biotin reagents. Biotinylated proteins were then detected by western blot and streptavidin-HRP staining. NHS-biotin labeling resulted in robust biotinylation of the amine groups of both L1 and L2 capsid proteins, irrespective of the redox status of the virions (Fig. 1B, lanes 1–2). In contrast, L2 was only labeled detectably with BMCC-biotin if the virions were first reduced by DTT (Fig. 1B, lane 4). This suggests that within the context of intact particles, C22 and C28 of HPV16 L2 are either buried and inaccessible to BMCC-biotin or they are bound up as disulfides, S-nitrosothiols (S-NO), or some other S-alkyl modification. These possibilities are not mutually exclusive, as the cysteines could be both modified and buried.


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)

Highly conserved HPV16 L2 cysteines C22 and C28 are not reactive with BMCC-biotin in native virions.(A) Primary amino acid sequence alignment of L2 proteins from various members of the Family Papillomaviridae shows conservation of C22 and C28 (HPV16) across divergent genera. (B) Amine and thiol biotinylation assays of HPV16 virions. Purified virions were either mock treated (native) or DTT treated (reduced) prior to incubation with amine reactive NHS-biotin or thiol reactive BMCC-biotin. Biotinylated proteins were separated by SDS-PAGE, transferred to membranes, and detected with streptavidin-HRP. Note that L2 cysteines are only reactive when the virions are reduced prior to labeling.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636891&req=5

pone-0004463-g001: Highly conserved HPV16 L2 cysteines C22 and C28 are not reactive with BMCC-biotin in native virions.(A) Primary amino acid sequence alignment of L2 proteins from various members of the Family Papillomaviridae shows conservation of C22 and C28 (HPV16) across divergent genera. (B) Amine and thiol biotinylation assays of HPV16 virions. Purified virions were either mock treated (native) or DTT treated (reduced) prior to incubation with amine reactive NHS-biotin or thiol reactive BMCC-biotin. Biotinylated proteins were separated by SDS-PAGE, transferred to membranes, and detected with streptavidin-HRP. Note that L2 cysteines are only reactive when the virions are reduced prior to labeling.
Mentions: Sequence alignment of diverse papillomavirus types shows that C22 and C28 of HPV16 L2 are absolutely conserved throughout the Family Papillomaviridae (Fig. 1A), prompting us to further examine the role of these two cysteines in HPV infection. To examine the native redox state of the two highly conserved L2 cysteines within the structural context of virions, we performed a thiol-specific biotinylation assay. Purified HPV16 virions were kept in their native state or reduced by DTT prior to chemical biotinylation with either amine-specific NHS-biotin or thiol-specific BMCC-biotin reagents. Biotinylated proteins were then detected by western blot and streptavidin-HRP staining. NHS-biotin labeling resulted in robust biotinylation of the amine groups of both L1 and L2 capsid proteins, irrespective of the redox status of the virions (Fig. 1B, lanes 1–2). In contrast, L2 was only labeled detectably with BMCC-biotin if the virions were first reduced by DTT (Fig. 1B, lane 4). This suggests that within the context of intact particles, C22 and C28 of HPV16 L2 are either buried and inaccessible to BMCC-biotin or they are bound up as disulfides, S-nitrosothiols (S-NO), or some other S-alkyl modification. These possibilities are not mutually exclusive, as the cysteines could be both modified and buried.

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