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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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Analysis of infectious Optiprep fractions.(A) Western blot analysis of L1 in highly infectious Optiprep fractions #7 and #8 from Optiprep-fractionated 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. The two rows are replicates with the top being overexposed. Higher and lower molecular weight species of L1 are indicated. (B) Densitometric analysis of the faster migrating L1 species (i.e. 55 kD) in three independent Western blots as shown in A. WT fraction #7 was set to 1.0. (C) Titers per fraction, normalized to the relative amount of L1 protein within fractions. WT fraction #7 was set to 1.0. (D) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. WT is set to 1.0. (E) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. Relative L1 protein content per fraction were used to normalize relative infectivity data within fractions. WT is set to 1.0.The results are means and standard errors for at least three independent experiments. The arrow points to the L1 band with the same motility as seen with L1 from PsV [23]. The asterisk marks the slow mobility L1 band seen only in native HPV16 virion [23].
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pone-0022427-g007: Analysis of infectious Optiprep fractions.(A) Western blot analysis of L1 in highly infectious Optiprep fractions #7 and #8 from Optiprep-fractionated 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. The two rows are replicates with the top being overexposed. Higher and lower molecular weight species of L1 are indicated. (B) Densitometric analysis of the faster migrating L1 species (i.e. 55 kD) in three independent Western blots as shown in A. WT fraction #7 was set to 1.0. (C) Titers per fraction, normalized to the relative amount of L1 protein within fractions. WT fraction #7 was set to 1.0. (D) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. WT is set to 1.0. (E) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. Relative L1 protein content per fraction were used to normalize relative infectivity data within fractions. WT is set to 1.0.The results are means and standard errors for at least three independent experiments. The arrow points to the L1 band with the same motility as seen with L1 from PsV [23]. The asterisk marks the slow mobility L1 band seen only in native HPV16 virion [23].

Mentions: We have previously demonstrated that highly infectious wild-type virus migrates into Optiprep fractions #7 and #8 [22], [23]. Therefore, to further examine the stability of these mutants, 20-day viruses were Optiprep-fractionated and Western blot analyses of L1 were performed on mature virus-containing, fractions #7 and #8 (Fig. 7A–B). It was observed that more L1 protein was found in fractions #7 and #8 of wild-type compared to mutant preparations (Fig. 7A–B). This decrease in L1 protein in mutant fractions occurred while total L1 protein concentration in 20-day viruses appeared equal or higher in mutant viruses compared to wild-type virus (Fig. 4C). We hypothesized that, if mutant capsids were more unstable than wild-type capsids, the majority of mutant L1 protein would migrate to the top fractions of the Optiprep gradient due to the destruction of capsids because of the high forces of ultracentrifugation. Previously published data in our laboratory suggested that such capsid destruction is prevalent in immature, 10-day wild-type HPV16 virions and is mostly absent in the more stable HPV16 20-day wild-type virions [23].


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

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

Analysis of infectious Optiprep fractions.(A) Western blot analysis of L1 in highly infectious Optiprep fractions #7 and #8 from Optiprep-fractionated 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. The two rows are replicates with the top being overexposed. Higher and lower molecular weight species of L1 are indicated. (B) Densitometric analysis of the faster migrating L1 species (i.e. 55 kD) in three independent Western blots as shown in A. WT fraction #7 was set to 1.0. (C) Titers per fraction, normalized to the relative amount of L1 protein within fractions. WT fraction #7 was set to 1.0. (D) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. WT is set to 1.0. (E) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. Relative L1 protein content per fraction were used to normalize relative infectivity data within fractions. WT is set to 1.0.The results are means and standard errors for at least three independent experiments. The arrow points to the L1 band with the same motility as seen with L1 from PsV [23]. The asterisk marks the slow mobility L1 band seen only in native HPV16 virion [23].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022427-g007: Analysis of infectious Optiprep fractions.(A) Western blot analysis of L1 in highly infectious Optiprep fractions #7 and #8 from Optiprep-fractionated 20-day wild-type (WT), C428S, C175S, C185S, and C175,185S viruses. The two rows are replicates with the top being overexposed. Higher and lower molecular weight species of L1 are indicated. (B) Densitometric analysis of the faster migrating L1 species (i.e. 55 kD) in three independent Western blots as shown in A. WT fraction #7 was set to 1.0. (C) Titers per fraction, normalized to the relative amount of L1 protein within fractions. WT fraction #7 was set to 1.0. (D) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. WT is set to 1.0. (E) Infectivity of 20-day wild-type (WT), C428S, C175S, C185S, C175,185S virions within fractions #7 and #8. Relative L1 protein content per fraction were used to normalize relative infectivity data within fractions. WT is set to 1.0.The results are means and standard errors for at least three independent experiments. The arrow points to the L1 band with the same motility as seen with L1 from PsV [23]. The asterisk marks the slow mobility L1 band seen only in native HPV16 virion [23].
Mentions: We have previously demonstrated that highly infectious wild-type virus migrates into Optiprep fractions #7 and #8 [22], [23]. Therefore, to further examine the stability of these mutants, 20-day viruses were Optiprep-fractionated and Western blot analyses of L1 were performed on mature virus-containing, fractions #7 and #8 (Fig. 7A–B). It was observed that more L1 protein was found in fractions #7 and #8 of wild-type compared to mutant preparations (Fig. 7A–B). This decrease in L1 protein in mutant fractions occurred while total L1 protein concentration in 20-day viruses appeared equal or higher in mutant viruses compared to wild-type virus (Fig. 4C). We hypothesized that, if mutant capsids were more unstable than wild-type capsids, the majority of mutant L1 protein would migrate to the top fractions of the Optiprep gradient due to the destruction of capsids because of the high forces of ultracentrifugation. Previously published data in our laboratory suggested that such capsid destruction is prevalent in immature, 10-day wild-type HPV16 virions and is mostly absent in the more stable HPV16 20-day wild-type virions [23].

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