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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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GSSG treatment alters viral titers and infectivity of tissues.(A) Relative RT-qPCR infectivity assay of wild-type (WT), C428S, C175S, C185S, and C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. (B) SYBR green-based DNA encapsidation assay of wild-type (WT), C428S, C175S, C185S, C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. Results are means and standard errors for three independent experiments.
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pone-0022427-g009: GSSG treatment alters viral titers and infectivity of tissues.(A) Relative RT-qPCR infectivity assay of wild-type (WT), C428S, C175S, C185S, and C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. (B) SYBR green-based DNA encapsidation assay of wild-type (WT), C428S, C175S, C185S, C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. Results are means and standard errors for three independent experiments.

Mentions: Previous studies have shown that treatment of HPV PsV or HPV-infected organotypic raft cultures with 5 mM GSSG enhances capsid maturation. However, work with PsV suggests that GSSG treatment also results in an increase in misfolded capsids since they are more susceptible to partial tryptic proteolysis than untreated capsids [6], [23]. Our previous research has shown that treatment of HPV16-infected organotypic raft cultures with 5 mM GSSG starting on day 8 and ending on day 10, followed by harvesting of the tissue, leads to enhanced infectivity [23]. Similar results have also been observed through the addition of low concentrations of the mildly oxidizing agent DMSO (data not shown). Since previous research utilizing HPV PsV showed a role for C428S and C175S in capsid maturation, we tested whether or not C428S, C175S, C185S, or C175,185S mutant viruses would respond to GSSG-treatment as compared to wild-type virus (Fig. 9A–B) [6]. Early treatment of wild-type HPV16-infected organotypic tissues resulted in an approximately 3.5-fold increase in infectivity and a 2-fold increase in total endonuclease-resistant genomes (Fig. 9A–B). As previously reported, this phenomenon only occurred when treated with GSSG starting on day 8 and ending on day 10 (Fig. 9A–B) [23]. It did not occur when treated with GSSG starting on day 15 and ending on day 20 (Fig. 9A–B). None of the mutant viruses C428S, C175S, C185S, or C175,185S responded to GSSG-treatment when starting on day 8 and ending on day 10 (Fig. 9A–B). In fact, similar to data in Fig. 5C which showed a decrease in titer of 20-day mutant viruses compared to 10-day mutant viruses, titers and infectivity appeared to decrease in most instances upon treatment with 5 mM GSSG (Fig. 9A–B). These results suggest that under the influence of the natural tissue-spanning redox gradient between days 10 and 20, or via early treatment of virions with 5 mM GSSG, mutant virions appear to be destabilized either by a lack of stabilizing disulfide bonds or through incorrectly formed intra- and/or interpentameric disulfide bonds between the mutant L1 proteins.


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

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

GSSG treatment alters viral titers and infectivity of tissues.(A) Relative RT-qPCR infectivity assay of wild-type (WT), C428S, C175S, C185S, and C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. (B) SYBR green-based DNA encapsidation assay of wild-type (WT), C428S, C175S, C185S, C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. Results are means and standard errors for three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139651&req=5

pone-0022427-g009: GSSG treatment alters viral titers and infectivity of tissues.(A) Relative RT-qPCR infectivity assay of wild-type (WT), C428S, C175S, C185S, and C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. (B) SYBR green-based DNA encapsidation assay of wild-type (WT), C428S, C175S, C185S, C175,185S 10 and 20-day viruses made from untreated (white) versus 5 mM GSSG-treated (black) organotypic cultures treated during days 8 through 10 or during days 15 through 20. Untreated 10-day WT was set to 1.0. Results are means and standard errors for three independent experiments.
Mentions: Previous studies have shown that treatment of HPV PsV or HPV-infected organotypic raft cultures with 5 mM GSSG enhances capsid maturation. However, work with PsV suggests that GSSG treatment also results in an increase in misfolded capsids since they are more susceptible to partial tryptic proteolysis than untreated capsids [6], [23]. Our previous research has shown that treatment of HPV16-infected organotypic raft cultures with 5 mM GSSG starting on day 8 and ending on day 10, followed by harvesting of the tissue, leads to enhanced infectivity [23]. Similar results have also been observed through the addition of low concentrations of the mildly oxidizing agent DMSO (data not shown). Since previous research utilizing HPV PsV showed a role for C428S and C175S in capsid maturation, we tested whether or not C428S, C175S, C185S, or C175,185S mutant viruses would respond to GSSG-treatment as compared to wild-type virus (Fig. 9A–B) [6]. Early treatment of wild-type HPV16-infected organotypic tissues resulted in an approximately 3.5-fold increase in infectivity and a 2-fold increase in total endonuclease-resistant genomes (Fig. 9A–B). As previously reported, this phenomenon only occurred when treated with GSSG starting on day 8 and ending on day 10 (Fig. 9A–B) [23]. It did not occur when treated with GSSG starting on day 15 and ending on day 20 (Fig. 9A–B). None of the mutant viruses C428S, C175S, C185S, or C175,185S responded to GSSG-treatment when starting on day 8 and ending on day 10 (Fig. 9A–B). In fact, similar to data in Fig. 5C which showed a decrease in titer of 20-day mutant viruses compared to 10-day mutant viruses, titers and infectivity appeared to decrease in most instances upon treatment with 5 mM GSSG (Fig. 9A–B). These results suggest that under the influence of the natural tissue-spanning redox gradient between days 10 and 20, or via early treatment of virions with 5 mM GSSG, mutant virions appear to be destabilized either by a lack of stabilizing disulfide bonds or through incorrectly formed intra- and/or interpentameric disulfide bonds between the mutant L1 proteins.

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