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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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Recombinant VP4 induces dose-dependent membrane permeability.Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.
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ppat-1004294-g002: Recombinant VP4 induces dose-dependent membrane permeability.Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.

Mentions: The ability of VP4His to induce permeability in model membranes was investigated by mixing purified recombinant protein with carboxyfluorescein (CF)-containing liposomes at neutral pH and room temperature, and detecting the VP4-induced release of encapsulated dye. VP4His induced a robust signal for membrane permeability and both rate and extent of dye-release were dose-dependent (Fig. 2).


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)

Recombinant VP4 induces dose-dependent membrane permeability.Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g002: Recombinant VP4 induces dose-dependent membrane permeability.Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.
Mentions: The ability of VP4His to induce permeability in model membranes was investigated by mixing purified recombinant protein with carboxyfluorescein (CF)-containing liposomes at neutral pH and room temperature, and detecting the VP4-induced release of encapsulated dye. VP4His induced a robust signal for membrane permeability and both rate and extent of dye-release were dose-dependent (Fig. 2).

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