Limits...
Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.

Ishii Y, Kondo K, Matsumoto T, Tanaka K, Shinkai-Ouchi F, Hagiwara K, Kanda T - Virol. J. (2007)

Bottom Line: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)].HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET.Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Pathogen Genomics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. yishii@nih.go.jp

ABSTRACT

Background: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity.

Results: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region.

Conclusion: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

Show MeSH

Related in: MedlinePlus

Sedimentation and morphology of the 16PVs that have bound to DTNB or NEM. (A) The 16PVs were incubated with DTNB (2 mM) or NEM (2 mM) at 37°C for 2 h. The sample was loaded on the top of a linear sucrose-density gradient (5 to 40%) and centrifuged. L1 in the fractions obtained by a bottom puncture was detected by immunoblotting with mouse anti-HPV16L1 antibody. (B) The 16PVs were incubated with NEM (2 mM) at 37°C for 2 h and observed under a transmission electron microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2147014&req=5

Figure 5: Sedimentation and morphology of the 16PVs that have bound to DTNB or NEM. (A) The 16PVs were incubated with DTNB (2 mM) or NEM (2 mM) at 37°C for 2 h. The sample was loaded on the top of a linear sucrose-density gradient (5 to 40%) and centrifuged. L1 in the fractions obtained by a bottom puncture was detected by immunoblotting with mouse anti-HPV16L1 antibody. (B) The 16PVs were incubated with NEM (2 mM) at 37°C for 2 h and observed under a transmission electron microscope.

Mentions: The 16PVs that had been incubated with DTNB (2 mM) or NEM (2 mM) sedimented through the sucrose gradient (5–40%) as the normal 16PVs did (Fig. 5A). The 16PVs pre-incubated with MBTA or MTSET sedimented similarly (data not presented). These results strongly suggest that the 16PVs that had bound to these reagents were morphologically similar to the normal 16PVs and did not make aggregates.


Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.

Ishii Y, Kondo K, Matsumoto T, Tanaka K, Shinkai-Ouchi F, Hagiwara K, Kanda T - Virol. J. (2007)

Sedimentation and morphology of the 16PVs that have bound to DTNB or NEM. (A) The 16PVs were incubated with DTNB (2 mM) or NEM (2 mM) at 37°C for 2 h. The sample was loaded on the top of a linear sucrose-density gradient (5 to 40%) and centrifuged. L1 in the fractions obtained by a bottom puncture was detected by immunoblotting with mouse anti-HPV16L1 antibody. (B) The 16PVs were incubated with NEM (2 mM) at 37°C for 2 h and observed under a transmission electron microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2147014&req=5

Figure 5: Sedimentation and morphology of the 16PVs that have bound to DTNB or NEM. (A) The 16PVs were incubated with DTNB (2 mM) or NEM (2 mM) at 37°C for 2 h. The sample was loaded on the top of a linear sucrose-density gradient (5 to 40%) and centrifuged. L1 in the fractions obtained by a bottom puncture was detected by immunoblotting with mouse anti-HPV16L1 antibody. (B) The 16PVs were incubated with NEM (2 mM) at 37°C for 2 h and observed under a transmission electron microscope.
Mentions: The 16PVs that had been incubated with DTNB (2 mM) or NEM (2 mM) sedimented through the sucrose gradient (5–40%) as the normal 16PVs did (Fig. 5A). The 16PVs pre-incubated with MBTA or MTSET sedimented similarly (data not presented). These results strongly suggest that the 16PVs that had bound to these reagents were morphologically similar to the normal 16PVs and did not make aggregates.

Bottom Line: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)].HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET.Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Pathogen Genomics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. yishii@nih.go.jp

ABSTRACT

Background: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity.

Results: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region.

Conclusion: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

Show MeSH
Related in: MedlinePlus