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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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Mass spectrometry of non-reduced L2 protein.(A) The full MS spectra of the L2 tryptic peptides T21-K23 and Q24-K35 with C22–C28 disulfide bond intact. The predominant doubly charged and triply charged parent cations are evident. (B) The MS/MS spectra of the doubly charged precursor ion and assignment of internal fragments. Upon collisional activation the disulfide bond was broken, but two major non-reduced species are evident. The non-reduced species were preferentially fragmented at I32 and P34.
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pone-0004463-g004: Mass spectrometry of non-reduced L2 protein.(A) The full MS spectra of the L2 tryptic peptides T21-K23 and Q24-K35 with C22–C28 disulfide bond intact. The predominant doubly charged and triply charged parent cations are evident. (B) The MS/MS spectra of the doubly charged precursor ion and assignment of internal fragments. Upon collisional activation the disulfide bond was broken, but two major non-reduced species are evident. The non-reduced species were preferentially fragmented at I32 and P34.

Mentions: Tandem mass spectrometry (MS/MS) of non-reduced L2 sample was performed to verify the existence of the C22–C28 disulfide. Purified wild type HPV16 virions were reacted with excess N-ethylmaleimide (NEM) to protect any native disulfide bonds from reduction by reactive free thiols. Virion proteins were then separated by denaturing, non-reducing SDS-PAGE and the L2 band was excised for in-gel trypsin digestion and extraction of tryptic peptides. Trypsin cleavage of L2 is predicted to occur following lysine residues K20, K23, and K35, within the immediate vicinity of C22 and C28, in the L2 primary amino acid sequence. K23 is positioned between C22 and C28, so the disulfide will link the adjacent T21-K23 and Q24-K35 tryptic peptides. MS analysis detected masses corresponding to both the doubly and triply charged cations of the disulfide-bonded peptides (Fig. 4A). MS/MS of the doubly charged peptide cation and analysis of the internal fragments further verified its assignment, confirming the existence of the C22–C28 disulfide bond in L2 (Fig. 4B).


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)

Mass spectrometry of non-reduced L2 protein.(A) The full MS spectra of the L2 tryptic peptides T21-K23 and Q24-K35 with C22–C28 disulfide bond intact. The predominant doubly charged and triply charged parent cations are evident. (B) The MS/MS spectra of the doubly charged precursor ion and assignment of internal fragments. Upon collisional activation the disulfide bond was broken, but two major non-reduced species are evident. The non-reduced species were preferentially fragmented at I32 and P34.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636891&req=5

pone-0004463-g004: Mass spectrometry of non-reduced L2 protein.(A) The full MS spectra of the L2 tryptic peptides T21-K23 and Q24-K35 with C22–C28 disulfide bond intact. The predominant doubly charged and triply charged parent cations are evident. (B) The MS/MS spectra of the doubly charged precursor ion and assignment of internal fragments. Upon collisional activation the disulfide bond was broken, but two major non-reduced species are evident. The non-reduced species were preferentially fragmented at I32 and P34.
Mentions: Tandem mass spectrometry (MS/MS) of non-reduced L2 sample was performed to verify the existence of the C22–C28 disulfide. Purified wild type HPV16 virions were reacted with excess N-ethylmaleimide (NEM) to protect any native disulfide bonds from reduction by reactive free thiols. Virion proteins were then separated by denaturing, non-reducing SDS-PAGE and the L2 band was excised for in-gel trypsin digestion and extraction of tryptic peptides. Trypsin cleavage of L2 is predicted to occur following lysine residues K20, K23, and K35, within the immediate vicinity of C22 and C28, in the L2 primary amino acid sequence. K23 is positioned between C22 and C28, so the disulfide will link the adjacent T21-K23 and Q24-K35 tryptic peptides. MS analysis detected masses corresponding to both the doubly and triply charged cations of the disulfide-bonded peptides (Fig. 4A). MS/MS of the doubly charged peptide cation and analysis of the internal fragments further verified its assignment, confirming the existence of the C22–C28 disulfide bond in L2 (Fig. 4B).

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