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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.

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Infectivity of the 16PVs that have bound to BPEOIA, NEM, DTNB, MBTA, or MTSET. The 16PVs were incubated with the thiol-reactive reagent indicated at 37°C for 2 h. The samples were diluted by 1000-fold and added to HeLa cells. The cells were incubated for 2 days and harvested. The cells expressing EGFP were counted by a FACS. BPEOIA: biotin polyethyleneoxide iodoacetamid, NEM: N-ethylmaleimide, DTNB: 5,5'-dithiobis(2-nitrobenzoic acid), MBTA: 4-(N-maleimido)benzyl-trimethylammonium iodide, MTSET: [2-(trimethylammonium)ethyl] methanethiosulfonate bromide.
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Figure 2: Infectivity of the 16PVs that have bound to BPEOIA, NEM, DTNB, MBTA, or MTSET. The 16PVs were incubated with the thiol-reactive reagent indicated at 37°C for 2 h. The samples were diluted by 1000-fold and added to HeLa cells. The cells were incubated for 2 days and harvested. The cells expressing EGFP were counted by a FACS. BPEOIA: biotin polyethyleneoxide iodoacetamid, NEM: N-ethylmaleimide, DTNB: 5,5'-dithiobis(2-nitrobenzoic acid), MBTA: 4-(N-maleimido)benzyl-trimethylammonium iodide, MTSET: [2-(trimethylammonium)ethyl] methanethiosulfonate bromide.

Mentions: The 16PVs was found to lose their infectivity for HeLa cells after binding to 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). 16PVs were incubated with BPEOIA (1 mM), DTNB (2 mM), NEM (2 mM), MBTA (2 mM), or MTSET (2 mM) for 2 h at 37°C. After dilution at 1 to 1,000 the 16PVs were inoculated to the cells. The number of the infected cells, which expressed EGFP, was counted 2 days later. The HeLa cells inoculated with the 16PVs incubated with these thiol-reactive reagents did not express EGFP (Fig. 2). Like HeLa cells, SiHa and 293TT cells inoculated with the 16PVs that had incubated with DTNB did not express EGFP (data not presented). The data indicate that these thiol-reactive reagents inhibited infectivity of the 16PVs.


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)

Infectivity of the 16PVs that have bound to BPEOIA, NEM, DTNB, MBTA, or MTSET. The 16PVs were incubated with the thiol-reactive reagent indicated at 37°C for 2 h. The samples were diluted by 1000-fold and added to HeLa cells. The cells were incubated for 2 days and harvested. The cells expressing EGFP were counted by a FACS. BPEOIA: biotin polyethyleneoxide iodoacetamid, NEM: N-ethylmaleimide, DTNB: 5,5'-dithiobis(2-nitrobenzoic acid), MBTA: 4-(N-maleimido)benzyl-trimethylammonium iodide, MTSET: [2-(trimethylammonium)ethyl] methanethiosulfonate bromide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Infectivity of the 16PVs that have bound to BPEOIA, NEM, DTNB, MBTA, or MTSET. The 16PVs were incubated with the thiol-reactive reagent indicated at 37°C for 2 h. The samples were diluted by 1000-fold and added to HeLa cells. The cells were incubated for 2 days and harvested. The cells expressing EGFP were counted by a FACS. BPEOIA: biotin polyethyleneoxide iodoacetamid, NEM: N-ethylmaleimide, DTNB: 5,5'-dithiobis(2-nitrobenzoic acid), MBTA: 4-(N-maleimido)benzyl-trimethylammonium iodide, MTSET: [2-(trimethylammonium)ethyl] methanethiosulfonate bromide.
Mentions: The 16PVs was found to lose their infectivity for HeLa cells after binding to 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). 16PVs were incubated with BPEOIA (1 mM), DTNB (2 mM), NEM (2 mM), MBTA (2 mM), or MTSET (2 mM) for 2 h at 37°C. After dilution at 1 to 1,000 the 16PVs were inoculated to the cells. The number of the infected cells, which expressed EGFP, was counted 2 days later. The HeLa cells inoculated with the 16PVs incubated with these thiol-reactive reagents did not express EGFP (Fig. 2). Like HeLa cells, SiHa and 293TT cells inoculated with the 16PVs that had incubated with DTNB did not express EGFP (data not presented). The data indicate that these thiol-reactive reagents inhibited infectivity of the 16PVs.

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