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Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

Panjwani A, Strauss M, Gold S, Wenham H, Jackson T, Chou JJ, Rowlands DJ, Stonehouse NJ, Hogle JM, Tuthill TJ - PLoS Pathog. (2014)

Bottom Line: In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes.Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome.The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom; School of Molecular and Cellular Biology & Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, West Yorkshire, United Kingdom.

ABSTRACT
Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

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Purity and concentration of recombinant VP4 assessed by SDS-PAGE.Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (A) or western blot using antisera to VP4 (B). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (figure S1).
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ppat-1004294-g001: Purity and concentration of recombinant VP4 assessed by SDS-PAGE.Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (A) or western blot using antisera to VP4 (B). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (figure S1).

Mentions: Previous studies have implicated picornavirus VP4 in viral penetration of the cell membrane and virus induced permeability in model membranes [13], [14], [19]. To further investigate the function of VP4 using biophysical approaches it was necessary to work with isolated protein. Although native VP4 can be isolated from purified virus, the amount of the protein derived from laboratory-scale virus preparations was insufficient for biophysical studies. To address this problem we previously produced recombinant HRV16 VP4 as an N-terminally myristoylated VP4GST fusion protein (VP4GST) which was able to induce membrane permeability in model membranes [18]. In the present study, the same bacterial expression system was used to produce myristoylated VP4 with a smaller tag (six-histidines at the C-terminus of VP4; VP4His). VP4His was overexpressed as insoluble inclusion bodies, purified by denaturing nickel affinity chromatography, precipitated and solubilised in DMSO, with a final yield of purified protein in the order of 0.1–0.5 mg per litre of culture. Protein purity was assessed by SDS-PAGE and silver staining and the concentration of recombinant VP4 was confirmed by comparison with native VP4 present in known quantities in purified HRV16 (Fig. 1). The migration of native VP4 and recombinant VP4His was consistent with their predicted molecular weights (approximately 7.4 kDa and 8.2 kDa respectively).


Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

Panjwani A, Strauss M, Gold S, Wenham H, Jackson T, Chou JJ, Rowlands DJ, Stonehouse NJ, Hogle JM, Tuthill TJ - PLoS Pathog. (2014)

Purity and concentration of recombinant VP4 assessed by SDS-PAGE.Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (A) or western blot using antisera to VP4 (B). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (figure S1).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4125281&req=5

ppat-1004294-g001: Purity and concentration of recombinant VP4 assessed by SDS-PAGE.Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (A) or western blot using antisera to VP4 (B). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (figure S1).
Mentions: Previous studies have implicated picornavirus VP4 in viral penetration of the cell membrane and virus induced permeability in model membranes [13], [14], [19]. To further investigate the function of VP4 using biophysical approaches it was necessary to work with isolated protein. Although native VP4 can be isolated from purified virus, the amount of the protein derived from laboratory-scale virus preparations was insufficient for biophysical studies. To address this problem we previously produced recombinant HRV16 VP4 as an N-terminally myristoylated VP4GST fusion protein (VP4GST) which was able to induce membrane permeability in model membranes [18]. In the present study, the same bacterial expression system was used to produce myristoylated VP4 with a smaller tag (six-histidines at the C-terminus of VP4; VP4His). VP4His was overexpressed as insoluble inclusion bodies, purified by denaturing nickel affinity chromatography, precipitated and solubilised in DMSO, with a final yield of purified protein in the order of 0.1–0.5 mg per litre of culture. Protein purity was assessed by SDS-PAGE and silver staining and the concentration of recombinant VP4 was confirmed by comparison with native VP4 present in known quantities in purified HRV16 (Fig. 1). The migration of native VP4 and recombinant VP4His was consistent with their predicted molecular weights (approximately 7.4 kDa and 8.2 kDa respectively).

Bottom Line: In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes.Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome.The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom; School of Molecular and Cellular Biology & Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, West Yorkshire, United Kingdom.

ABSTRACT
Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Show MeSH
Related in: MedlinePlus