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Differentiation-dependent interpentameric disulfide bond stabilizes native human papillomavirus type 16.

Conway MJ, Cruz L, Alam S, Christensen ND, Meyers C - PLoS ONE (2011)

Bottom Line: All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE.Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids.Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America.

ABSTRACT
Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.

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Expression of L1 in wild-type and mutant 10 day tissues.Western blot analyses of equal volumes from WT, C428, C175, C185S, and C175,185S 10-day virus preparations. A representative Western blot is shown below while a graph depicting the relative L1 protein expression per viral preparation via densitometry is shown above. The results are means and standard deviations from three independent experiments.
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pone-0022427-g003: Expression of L1 in wild-type and mutant 10 day tissues.Western blot analyses of equal volumes from WT, C428, C175, C185S, and C175,185S 10-day virus preparations. A representative Western blot is shown below while a graph depicting the relative L1 protein expression per viral preparation via densitometry is shown above. The results are means and standard deviations from three independent experiments.

Mentions: To develop producer cell lines which can synthesize organotypic culture-derived native virions from differentiating epithelia, primary human foreskin keratinocytes (HFKs) were electroporated with linearized wild-type and site-directed mutagenized HPV16(114/B) DNA [9]. The recircularization and maintenance of episomal HPV16 viral genomes for representative HPV16 L1 C428S, C175S, C185S, and C175,185S cell lines can be seen in Figure 1. Data for wild-type and C428S HPV16-containing cell lines have been published previously [22], [25]. The total number of episomal copies per cell was less in mutant cell lines (∼50–200 copies/cell) than the wild-type cell line utilized (>1,000 copies/cell). Importantly, the lower copy numbers observed in monolayer culture in the mutant cell lines are not significant regarding productivity of tissues since previous reports have suggested that copy number does not directly correlate with the titer of HPV-infected organotypic tissues [25], [26], [27]. Quantifying genome equivalents and the detection of the major capsid protein via Western blot allowed for the analysis of the relative infectivity of viral stocks produced by HPV-infected organotypic tissues. For each mutant genome, multiple episomal DNA-containing cell lines were produced and utilized in experiments. In addition, L1 ORFs were sequenced to verify the existence of the intended mutation, and the absence of erroneous mutations. Only then were stable cell lines allowed to grow as stratified and differentiated epithelial tissues in organotypic culture (Fig. 2). Importantly, mutant-infected tissues did not show a significantly different phenotype than wild-type-infected tissues (Fig. 2) and total L1 protein concentration was similar in wild-type compared to mutant in 10-day tissues (Fig. 3A and 4C).


Differentiation-dependent interpentameric disulfide bond stabilizes native human papillomavirus type 16.

Conway MJ, Cruz L, Alam S, Christensen ND, Meyers C - PLoS ONE (2011)

Expression of L1 in wild-type and mutant 10 day tissues.Western blot analyses of equal volumes from WT, C428, C175, C185S, and C175,185S 10-day virus preparations. A representative Western blot is shown below while a graph depicting the relative L1 protein expression per viral preparation via densitometry is shown above. The results are means and standard deviations from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139651&req=5

pone-0022427-g003: Expression of L1 in wild-type and mutant 10 day tissues.Western blot analyses of equal volumes from WT, C428, C175, C185S, and C175,185S 10-day virus preparations. A representative Western blot is shown below while a graph depicting the relative L1 protein expression per viral preparation via densitometry is shown above. The results are means and standard deviations from three independent experiments.
Mentions: To develop producer cell lines which can synthesize organotypic culture-derived native virions from differentiating epithelia, primary human foreskin keratinocytes (HFKs) were electroporated with linearized wild-type and site-directed mutagenized HPV16(114/B) DNA [9]. The recircularization and maintenance of episomal HPV16 viral genomes for representative HPV16 L1 C428S, C175S, C185S, and C175,185S cell lines can be seen in Figure 1. Data for wild-type and C428S HPV16-containing cell lines have been published previously [22], [25]. The total number of episomal copies per cell was less in mutant cell lines (∼50–200 copies/cell) than the wild-type cell line utilized (>1,000 copies/cell). Importantly, the lower copy numbers observed in monolayer culture in the mutant cell lines are not significant regarding productivity of tissues since previous reports have suggested that copy number does not directly correlate with the titer of HPV-infected organotypic tissues [25], [26], [27]. Quantifying genome equivalents and the detection of the major capsid protein via Western blot allowed for the analysis of the relative infectivity of viral stocks produced by HPV-infected organotypic tissues. For each mutant genome, multiple episomal DNA-containing cell lines were produced and utilized in experiments. In addition, L1 ORFs were sequenced to verify the existence of the intended mutation, and the absence of erroneous mutations. Only then were stable cell lines allowed to grow as stratified and differentiated epithelial tissues in organotypic culture (Fig. 2). Importantly, mutant-infected tissues did not show a significantly different phenotype than wild-type-infected tissues (Fig. 2) and total L1 protein concentration was similar in wild-type compared to mutant in 10-day tissues (Fig. 3A and 4C).

Bottom Line: All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE.Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids.Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America.

ABSTRACT
Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.

Show MeSH
Related in: MedlinePlus