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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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Related in: MedlinePlus

VP4 forms a multimeric complex.A. VP4His was incubated in the presence of the membrane-mimetic detergent DHPC or liposomes, or mock-treated (−), resolved by native PAGE and visualised by silver staining. The position of bands shown by arrows indicates potential migration of VP4His into the gel. B. VP4 was incubated with (+) or without (−) liposomes and crosslinked by the addition of DSP at 0.5 mM (+) or 1 mM (++). Samples were resolved by non-reducing SDS-PAGE and detected by western blot using antisera to VP4. DTT was used in some samples to reverse the crosslinking prior to SDS-PAGE. Arrows indicate VP4 multimers. Molecular mass markers (in kilodaltons) are indicated on the left.
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ppat-1004294-g005: VP4 forms a multimeric complex.A. VP4His was incubated in the presence of the membrane-mimetic detergent DHPC or liposomes, or mock-treated (−), resolved by native PAGE and visualised by silver staining. The position of bands shown by arrows indicates potential migration of VP4His into the gel. B. VP4 was incubated with (+) or without (−) liposomes and crosslinked by the addition of DSP at 0.5 mM (+) or 1 mM (++). Samples were resolved by non-reducing SDS-PAGE and detected by western blot using antisera to VP4. DTT was used in some samples to reverse the crosslinking prior to SDS-PAGE. Arrows indicate VP4 multimers. Molecular mass markers (in kilodaltons) are indicated on the left.

Mentions: Membrane pores formed by small pore-forming proteins or peptides often have a homo-oligomeric structure. Multimerization seemed the likely mechanism for VP4 pore formation. We therefore investigated the ability of VP4 to form a homomultimeric complex by analysing samples by PAGE. Purified VP4His was difficult to detect when analysed directly by native PAGE (non-denaturing conditions) and silver staining (Fig. 5A) perhaps due to solubility or charge which did not permit efficient electrophoresis. However, after mixing with liposome membranes or the membrane mimetic detergent DHPC, VP4 appeared to be readily detected (Fig. 5A), suggesting that interaction with micelles or multimerization was able to facilitate migration into the gel.


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)

VP4 forms a multimeric complex.A. VP4His was incubated in the presence of the membrane-mimetic detergent DHPC or liposomes, or mock-treated (−), resolved by native PAGE and visualised by silver staining. The position of bands shown by arrows indicates potential migration of VP4His into the gel. B. VP4 was incubated with (+) or without (−) liposomes and crosslinked by the addition of DSP at 0.5 mM (+) or 1 mM (++). Samples were resolved by non-reducing SDS-PAGE and detected by western blot using antisera to VP4. DTT was used in some samples to reverse the crosslinking prior to SDS-PAGE. Arrows indicate VP4 multimers. Molecular mass markers (in kilodaltons) are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g005: VP4 forms a multimeric complex.A. VP4His was incubated in the presence of the membrane-mimetic detergent DHPC or liposomes, or mock-treated (−), resolved by native PAGE and visualised by silver staining. The position of bands shown by arrows indicates potential migration of VP4His into the gel. B. VP4 was incubated with (+) or without (−) liposomes and crosslinked by the addition of DSP at 0.5 mM (+) or 1 mM (++). Samples were resolved by non-reducing SDS-PAGE and detected by western blot using antisera to VP4. DTT was used in some samples to reverse the crosslinking prior to SDS-PAGE. Arrows indicate VP4 multimers. Molecular mass markers (in kilodaltons) are indicated on the left.
Mentions: Membrane pores formed by small pore-forming proteins or peptides often have a homo-oligomeric structure. Multimerization seemed the likely mechanism for VP4 pore formation. We therefore investigated the ability of VP4 to form a homomultimeric complex by analysing samples by PAGE. Purified VP4His was difficult to detect when analysed directly by native PAGE (non-denaturing conditions) and silver staining (Fig. 5A) perhaps due to solubility or charge which did not permit efficient electrophoresis. However, after mixing with liposome membranes or the membrane mimetic detergent DHPC, VP4 appeared to be readily detected (Fig. 5A), suggesting that interaction with micelles or multimerization was able to facilitate migration into the gel.

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