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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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Characterization of L2 cysteine mutant virions.(A) Morphology of the L2 wild type and cysteine mutant virions. CsCl-purified virion preparations were negatively stained and visualized by transmission electron microscopy. Scale bar represents 100 nm. (B) Reducing and denaturing SDS-PAGE of wild type and mutant L2-containing virions, immunoblotted for L1 (left panel) and L2 (right panel) capsid proteins. Note the absence of L2 size shift for any of the cysteine mutants, suggesting no large cysteine modifications are present. (C) Analysis of HPV16 L2 wild type and mutant virions by denaturing SDS-PAGE under non-reducing conditions. L1 immunoblot (left panel) shows disulfide-linked multimers of L1 for wild type and mutant virions. L2 immunoblot (right panel) shows no detectable L2-L2 dimers for wild type or C22/28S virions (lanes 1,4), but a fraction of L2 in the single cysteine mutants exist as disulfide-linked L2-L2 dimers (lanes 2,3).
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pone-0004463-g002: Characterization of L2 cysteine mutant virions.(A) Morphology of the L2 wild type and cysteine mutant virions. CsCl-purified virion preparations were negatively stained and visualized by transmission electron microscopy. Scale bar represents 100 nm. (B) Reducing and denaturing SDS-PAGE of wild type and mutant L2-containing virions, immunoblotted for L1 (left panel) and L2 (right panel) capsid proteins. Note the absence of L2 size shift for any of the cysteine mutants, suggesting no large cysteine modifications are present. (C) Analysis of HPV16 L2 wild type and mutant virions by denaturing SDS-PAGE under non-reducing conditions. L1 immunoblot (left panel) shows disulfide-linked multimers of L1 for wild type and mutant virions. L2 immunoblot (right panel) shows no detectable L2-L2 dimers for wild type or C22/28S virions (lanes 1,4), but a fraction of L2 in the single cysteine mutants exist as disulfide-linked L2-L2 dimers (lanes 2,3).

Mentions: Single C22S and C28S and the double C22/28S cysteine-to-serine point mutations were generated in the L2 gene of the HPV16 capsid expression plasmid. Mutant virions were produced by transfection of 293TT cells [40], [41] and purified by CsCl density gradient centrifugation as previously described [11]. Virions containing mutant L2 proteins were obtained in yields comparable to normal HPV16 virions containing wild type L2. SDS-PAGE and Coomassie staining-based densitometry indicated that each of the virus preparations contained an average of ∼22 molecules L2 per virion (data not shown). Visualization by transmission electron microscopy revealed normal capsid morphology among all L2 mutant virions (Fig. 2A) and dot blot analysis showed no decrease in genome encapsidation for any of the mutants (data not shown). SDS-PAGE and immunoblotting of virion preparations for L1 and L2 revealed no defects in L2 encapsidation (Fig. 2B, lanes 5–8). The absence of any size shift in the L2 mutants suggested that no large modifications (palmitoylation, prenylation, etc.) were present on the wild type L2 cysteines.


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)

Characterization of L2 cysteine mutant virions.(A) Morphology of the L2 wild type and cysteine mutant virions. CsCl-purified virion preparations were negatively stained and visualized by transmission electron microscopy. Scale bar represents 100 nm. (B) Reducing and denaturing SDS-PAGE of wild type and mutant L2-containing virions, immunoblotted for L1 (left panel) and L2 (right panel) capsid proteins. Note the absence of L2 size shift for any of the cysteine mutants, suggesting no large cysteine modifications are present. (C) Analysis of HPV16 L2 wild type and mutant virions by denaturing SDS-PAGE under non-reducing conditions. L1 immunoblot (left panel) shows disulfide-linked multimers of L1 for wild type and mutant virions. L2 immunoblot (right panel) shows no detectable L2-L2 dimers for wild type or C22/28S virions (lanes 1,4), but a fraction of L2 in the single cysteine mutants exist as disulfide-linked L2-L2 dimers (lanes 2,3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636891&req=5

pone-0004463-g002: Characterization of L2 cysteine mutant virions.(A) Morphology of the L2 wild type and cysteine mutant virions. CsCl-purified virion preparations were negatively stained and visualized by transmission electron microscopy. Scale bar represents 100 nm. (B) Reducing and denaturing SDS-PAGE of wild type and mutant L2-containing virions, immunoblotted for L1 (left panel) and L2 (right panel) capsid proteins. Note the absence of L2 size shift for any of the cysteine mutants, suggesting no large cysteine modifications are present. (C) Analysis of HPV16 L2 wild type and mutant virions by denaturing SDS-PAGE under non-reducing conditions. L1 immunoblot (left panel) shows disulfide-linked multimers of L1 for wild type and mutant virions. L2 immunoblot (right panel) shows no detectable L2-L2 dimers for wild type or C22/28S virions (lanes 1,4), but a fraction of L2 in the single cysteine mutants exist as disulfide-linked L2-L2 dimers (lanes 2,3).
Mentions: Single C22S and C28S and the double C22/28S cysteine-to-serine point mutations were generated in the L2 gene of the HPV16 capsid expression plasmid. Mutant virions were produced by transfection of 293TT cells [40], [41] and purified by CsCl density gradient centrifugation as previously described [11]. Virions containing mutant L2 proteins were obtained in yields comparable to normal HPV16 virions containing wild type L2. SDS-PAGE and Coomassie staining-based densitometry indicated that each of the virus preparations contained an average of ∼22 molecules L2 per virion (data not shown). Visualization by transmission electron microscopy revealed normal capsid morphology among all L2 mutant virions (Fig. 2A) and dot blot analysis showed no decrease in genome encapsidation for any of the mutants (data not shown). SDS-PAGE and immunoblotting of virion preparations for L1 and L2 revealed no defects in L2 encapsidation (Fig. 2B, lanes 5–8). The absence of any size shift in the L2 mutants suggested that no large modifications (palmitoylation, prenylation, etc.) were present on the wild type L2 cysteines.

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