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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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Titers in 10 and 20-day wild-type and mutant viruses.Titers were measured using a SYBR-green based genome encapsidation assay. (A) SYBR green-based DNA encapsidation assay relative quantification of 10-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (B) SYBR green-based DNA encapsidation assay relative quantification of 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (C) Side-by-side comparison of 10 (black bars) and 20-day (white bars) raw data from A and B. Relative y-axis values represent copy number controls used in the standard curve. The results are means and standard errors for at least three independent experiments.
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pone-0022427-g005: Titers in 10 and 20-day wild-type and mutant viruses.Titers were measured using a SYBR-green based genome encapsidation assay. (A) SYBR green-based DNA encapsidation assay relative quantification of 10-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (B) SYBR green-based DNA encapsidation assay relative quantification of 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (C) Side-by-side comparison of 10 (black bars) and 20-day (white bars) raw data from A and B. Relative y-axis values represent copy number controls used in the standard curve. The results are means and standard errors for at least three independent experiments.

Mentions: HPV titers are measured similar to other viruses such as the Kaposi's sarcoma virus (KSHV), quantifying viral genome equivalents [28], [29], [30], [31], [32]. In Fig. 5A, the relative amounts of HPV titers in 10-day viral preparations are shown. Ten-day C428S, C175S, C185S, and C175,185S mutants had titers that were 54%±14, 20%±15, 110%±30, and 145%±51, respectively, of wild-type titers (Table 1). This suggests that C428 was mildly and C175 was largely important for early viral genome encapsidation. The observation that 10-day C428S viruses had titers that were 54% of 10-day wild-type virus was surprising since the C428S mutation previously led to complete capsid disassembly when integrated into HPV16 and HPV33 VLPs, and HPV16 PsV [6], [16], [18]. We theorize that proper covalent bonding prevents exposure of viral genomes to benzonase.


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

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

Titers in 10 and 20-day wild-type and mutant viruses.Titers were measured using a SYBR-green based genome encapsidation assay. (A) SYBR green-based DNA encapsidation assay relative quantification of 10-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (B) SYBR green-based DNA encapsidation assay relative quantification of 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (C) Side-by-side comparison of 10 (black bars) and 20-day (white bars) raw data from A and B. Relative y-axis values represent copy number controls used in the standard curve. The results are means and standard errors for at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022427-g005: Titers in 10 and 20-day wild-type and mutant viruses.Titers were measured using a SYBR-green based genome encapsidation assay. (A) SYBR green-based DNA encapsidation assay relative quantification of 10-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (B) SYBR green-based DNA encapsidation assay relative quantification of 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. WT is set to 1.0. (C) Side-by-side comparison of 10 (black bars) and 20-day (white bars) raw data from A and B. Relative y-axis values represent copy number controls used in the standard curve. The results are means and standard errors for at least three independent experiments.
Mentions: HPV titers are measured similar to other viruses such as the Kaposi's sarcoma virus (KSHV), quantifying viral genome equivalents [28], [29], [30], [31], [32]. In Fig. 5A, the relative amounts of HPV titers in 10-day viral preparations are shown. Ten-day C428S, C175S, C185S, and C175,185S mutants had titers that were 54%±14, 20%±15, 110%±30, and 145%±51, respectively, of wild-type titers (Table 1). This suggests that C428 was mildly and C175 was largely important for early viral genome encapsidation. The observation that 10-day C428S viruses had titers that were 54% of 10-day wild-type virus was surprising since the C428S mutation previously led to complete capsid disassembly when integrated into HPV16 and HPV33 VLPs, and HPV16 PsV [6], [16], [18]. We theorize that proper covalent bonding prevents exposure of viral genomes to benzonase.

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