Limits...
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.

Show MeSH

Related in: MedlinePlus

VP4-induced membrane permeability is enhanced by low pH and VP4 myristoylation.Carboxyfluorescein (CF)-containing liposomes at pH 7.0, pH 6.5, and pH 5.5, were mixed with the following peptides or proteins and membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. A) The pH-independent pore-forming peptide melittin at 10 µM. AU, arbitrary units. CF fluorescence is known to be less efficient at low pH and the apparent reduction in melittin-induced signal at low pH values (pH 6.5 and pH 5.5) could therefore be restored to the same level as the pH 7 sample by adjusting released CF in supernatants to neutral pH (data not shown), thus confirming that melittin-induced permeability was unaffected by pH and demonstrating a requirement to compensate for the reduced efficiency of CF fluorescence at low pH. Therefore, data in the remaining panels (B–F) was normalized to the maximum signal induced by melittin at each pH value. B) the acid-dependent pore-forming peptide GALA at 1 µM. C) VP4His at 5 µM. D) ΔVP4His (unmyristoylated) at 5 µM. E) End point and F) initial rates are shown to summarise the data in panels A–D. Data shown is representative of multiple experiments (n>3). Error bars represent standard error of the mean of values from 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125281&req=5

ppat-1004294-g003: VP4-induced membrane permeability is enhanced by low pH and VP4 myristoylation.Carboxyfluorescein (CF)-containing liposomes at pH 7.0, pH 6.5, and pH 5.5, were mixed with the following peptides or proteins and membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. A) The pH-independent pore-forming peptide melittin at 10 µM. AU, arbitrary units. CF fluorescence is known to be less efficient at low pH and the apparent reduction in melittin-induced signal at low pH values (pH 6.5 and pH 5.5) could therefore be restored to the same level as the pH 7 sample by adjusting released CF in supernatants to neutral pH (data not shown), thus confirming that melittin-induced permeability was unaffected by pH and demonstrating a requirement to compensate for the reduced efficiency of CF fluorescence at low pH. Therefore, data in the remaining panels (B–F) was normalized to the maximum signal induced by melittin at each pH value. B) the acid-dependent pore-forming peptide GALA at 1 µM. C) VP4His at 5 µM. D) ΔVP4His (unmyristoylated) at 5 µM. E) End point and F) initial rates are shown to summarise the data in panels A–D. Data shown is representative of multiple experiments (n>3). Error bars represent standard error of the mean of values from 3 experiments.

Mentions: We therefore used the well-characterised pH independent pore-forming peptide melittin [23], [24] as a control for the liposome dye-release assay. The permeability induced by melittin appeared reduced at low pH due to the quenching of CF fluorescence at low pH (Fig. 3A). However, when samples were separated from liposomes and adjusted to neutral pH as described above, the signal in all samples became equivalent to the pH 7.0 sample. This confirmed that melittin induced permeability was indeed pH independent and that dye-release data generated at a given pH could be expressed as a proportion of the maximum release by a melittin control at that same pH. This provided a method for normalisation of data which corrected for the low-pH quenching of CF and allowed real-time data generated at different pH values to be directly compared.


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-induced membrane permeability is enhanced by low pH and VP4 myristoylation.Carboxyfluorescein (CF)-containing liposomes at pH 7.0, pH 6.5, and pH 5.5, were mixed with the following peptides or proteins and membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. A) The pH-independent pore-forming peptide melittin at 10 µM. AU, arbitrary units. CF fluorescence is known to be less efficient at low pH and the apparent reduction in melittin-induced signal at low pH values (pH 6.5 and pH 5.5) could therefore be restored to the same level as the pH 7 sample by adjusting released CF in supernatants to neutral pH (data not shown), thus confirming that melittin-induced permeability was unaffected by pH and demonstrating a requirement to compensate for the reduced efficiency of CF fluorescence at low pH. Therefore, data in the remaining panels (B–F) was normalized to the maximum signal induced by melittin at each pH value. B) the acid-dependent pore-forming peptide GALA at 1 µM. C) VP4His at 5 µM. D) ΔVP4His (unmyristoylated) at 5 µM. E) End point and F) initial rates are shown to summarise the data in panels A–D. Data shown is representative of multiple experiments (n>3). Error bars represent standard error of the mean of values from 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g003: VP4-induced membrane permeability is enhanced by low pH and VP4 myristoylation.Carboxyfluorescein (CF)-containing liposomes at pH 7.0, pH 6.5, and pH 5.5, were mixed with the following peptides or proteins and membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. A) The pH-independent pore-forming peptide melittin at 10 µM. AU, arbitrary units. CF fluorescence is known to be less efficient at low pH and the apparent reduction in melittin-induced signal at low pH values (pH 6.5 and pH 5.5) could therefore be restored to the same level as the pH 7 sample by adjusting released CF in supernatants to neutral pH (data not shown), thus confirming that melittin-induced permeability was unaffected by pH and demonstrating a requirement to compensate for the reduced efficiency of CF fluorescence at low pH. Therefore, data in the remaining panels (B–F) was normalized to the maximum signal induced by melittin at each pH value. B) the acid-dependent pore-forming peptide GALA at 1 µM. C) VP4His at 5 µM. D) ΔVP4His (unmyristoylated) at 5 µM. E) End point and F) initial rates are shown to summarise the data in panels A–D. Data shown is representative of multiple experiments (n>3). Error bars represent standard error of the mean of values from 3 experiments.
Mentions: We therefore used the well-characterised pH independent pore-forming peptide melittin [23], [24] as a control for the liposome dye-release assay. The permeability induced by melittin appeared reduced at low pH due to the quenching of CF fluorescence at low pH (Fig. 3A). However, when samples were separated from liposomes and adjusted to neutral pH as described above, the signal in all samples became equivalent to the pH 7.0 sample. This confirmed that melittin induced permeability was indeed pH independent and that dye-release data generated at a given pH could be expressed as a proportion of the maximum release by a melittin control at that same pH. This provided a method for normalisation of data which corrected for the low-pH quenching of CF and allowed real-time data generated at different pH values to be directly compared.

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