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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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Relative stabilities of 20-day wild-type versus mutant virions.20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses were Optiprep-fractionated. Fractions were assayed by a SYBR green-based DNA encapsidation assay to detect total endonuclease-resistant genomes per fraction. As a measure of capsid stability, the sum of genomes in the uppermost gradient fractions (fractions #1 through #4) was described as “unstable” (US), which were genomes either not associated or disassociated from an intact capsid during ultracentrifugation. The sum of genomes flanking highly infectious fractions (fractions #6 through #9) was described as “stable” (S) since they were associated with intact capsids. Relative capsid stability is described by the ratio US/S, whereby higher values indicate more unstable virions [23].
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pone-0022427-g008: Relative stabilities of 20-day wild-type versus mutant virions.20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses were Optiprep-fractionated. Fractions were assayed by a SYBR green-based DNA encapsidation assay to detect total endonuclease-resistant genomes per fraction. As a measure of capsid stability, the sum of genomes in the uppermost gradient fractions (fractions #1 through #4) was described as “unstable” (US), which were genomes either not associated or disassociated from an intact capsid during ultracentrifugation. The sum of genomes flanking highly infectious fractions (fractions #6 through #9) was described as “stable” (S) since they were associated with intact capsids. Relative capsid stability is described by the ratio US/S, whereby higher values indicate more unstable virions [23].

Mentions: The instability of mutant capsids was compared to wild-type capsids by assessing the relative stability of virus within the Optiprep gradient (Fig. 8). The relative stability of virus was measured by the ratio of unstably encapsidated viral genomes (sum of fractions #1 through #4) over stably encapsidated viral genomes (sum of fractions #6 through #9) [23]. We have shown previously that 10-day wild-type HPV16 virions have a stability ratio of 26.0, while their 20-day counterparts have a stability ratio of 1.0, suggesting that HPV16 virions are stabilized over time [23]. While 20-day wild-type virions had a relative stability of 1.0, 20-day C428S, C175, C185S, and C175,185S virions had relative stabilities of 7.3, 13.8, 16.1, and 11.4, respectively, suggesting that these mutations rendered capsids less stable than 20-day wild-type virions (Fig. 8).


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

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

Relative stabilities of 20-day wild-type versus mutant virions.20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses were Optiprep-fractionated. Fractions were assayed by a SYBR green-based DNA encapsidation assay to detect total endonuclease-resistant genomes per fraction. As a measure of capsid stability, the sum of genomes in the uppermost gradient fractions (fractions #1 through #4) was described as “unstable” (US), which were genomes either not associated or disassociated from an intact capsid during ultracentrifugation. The sum of genomes flanking highly infectious fractions (fractions #6 through #9) was described as “stable” (S) since they were associated with intact capsids. Relative capsid stability is described by the ratio US/S, whereby higher values indicate more unstable virions [23].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022427-g008: Relative stabilities of 20-day wild-type versus mutant virions.20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses were Optiprep-fractionated. Fractions were assayed by a SYBR green-based DNA encapsidation assay to detect total endonuclease-resistant genomes per fraction. As a measure of capsid stability, the sum of genomes in the uppermost gradient fractions (fractions #1 through #4) was described as “unstable” (US), which were genomes either not associated or disassociated from an intact capsid during ultracentrifugation. The sum of genomes flanking highly infectious fractions (fractions #6 through #9) was described as “stable” (S) since they were associated with intact capsids. Relative capsid stability is described by the ratio US/S, whereby higher values indicate more unstable virions [23].
Mentions: The instability of mutant capsids was compared to wild-type capsids by assessing the relative stability of virus within the Optiprep gradient (Fig. 8). The relative stability of virus was measured by the ratio of unstably encapsidated viral genomes (sum of fractions #1 through #4) over stably encapsidated viral genomes (sum of fractions #6 through #9) [23]. We have shown previously that 10-day wild-type HPV16 virions have a stability ratio of 26.0, while their 20-day counterparts have a stability ratio of 1.0, suggesting that HPV16 virions are stabilized over time [23]. While 20-day wild-type virions had a relative stability of 1.0, 20-day C428S, C175, C185S, and C175,185S virions had relative stabilities of 7.3, 13.8, 16.1, and 11.4, respectively, suggesting that these mutations rendered capsids less stable than 20-day wild-type virions (Fig. 8).

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