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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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VP4-induced permeability is comparable to that of virus.Carboxyfluorescein-containing liposomes were mixed with VP4His at 5 µM (equivalent to approximately 5 µg/assay) or 1 µg HRV16 (equivalent to 50 ng VP4/assay) and membrane permeability detected by fluorescence measurements recorded every 30 seconds. Assays were conducted at 25°C (A) or 37°C (B). Only a minority proportion of recombinant protein is thought to take part in the reaction. Data is presented as % of total end-point release observed by lysis of liposomes by addition of detergent. Data shown is representative of multiple experiments (n>3).
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ppat-1004294-g007: VP4-induced permeability is comparable to that of virus.Carboxyfluorescein-containing liposomes were mixed with VP4His at 5 µM (equivalent to approximately 5 µg/assay) or 1 µg HRV16 (equivalent to 50 ng VP4/assay) and membrane permeability detected by fluorescence measurements recorded every 30 seconds. Assays were conducted at 25°C (A) or 37°C (B). Only a minority proportion of recombinant protein is thought to take part in the reaction. Data is presented as % of total end-point release observed by lysis of liposomes by addition of detergent. Data shown is representative of multiple experiments (n>3).

Mentions: We wished to confirm that the function of recombinant VP4 was relevant to the membrane interactions of virus particles. Previous studies demonstrated that PV was able to induce electrical conductance channels in planar model membranes [19], [28]. We therefore compared the membrane permeability induced by VP4His and purified HRV16, by measuring CF leakage from liposomes in the dye-release assay. Recombinant VP4 and purified virus induced membrane permeability with broadly similar characteristics (Fig. 7). However, virus-induced permeability was more efficient at 37°C than at 25°C, while the recombinant VP4-induced permeability was relatively unaffected by the change in temperature. These observations demonstrate a temperature dependence for HRV-membrane interactions, consistent with previous studies with PV [19] and with a model of temperature dependent particle breathing which controls the exposure and membrane interactions of internal capsid components such as VP4 [29], [30]. Although studies with PV demonstrated breathing only at physiological temperatures approaching 37°C [29], studies with HRV have shown particle breathing at lower temperatures such as 25°C [30]. Importantly, virus samples pre-treated at 60°C to convert virions into empty particles induced only low levels of permeability, most likely because empty particles would no longer contain VP4.


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 permeability is comparable to that of virus.Carboxyfluorescein-containing liposomes were mixed with VP4His at 5 µM (equivalent to approximately 5 µg/assay) or 1 µg HRV16 (equivalent to 50 ng VP4/assay) and membrane permeability detected by fluorescence measurements recorded every 30 seconds. Assays were conducted at 25°C (A) or 37°C (B). Only a minority proportion of recombinant protein is thought to take part in the reaction. Data is presented as % of total end-point release observed by lysis of liposomes by addition of detergent. Data shown is representative of multiple experiments (n>3).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g007: VP4-induced permeability is comparable to that of virus.Carboxyfluorescein-containing liposomes were mixed with VP4His at 5 µM (equivalent to approximately 5 µg/assay) or 1 µg HRV16 (equivalent to 50 ng VP4/assay) and membrane permeability detected by fluorescence measurements recorded every 30 seconds. Assays were conducted at 25°C (A) or 37°C (B). Only a minority proportion of recombinant protein is thought to take part in the reaction. Data is presented as % of total end-point release observed by lysis of liposomes by addition of detergent. Data shown is representative of multiple experiments (n>3).
Mentions: We wished to confirm that the function of recombinant VP4 was relevant to the membrane interactions of virus particles. Previous studies demonstrated that PV was able to induce electrical conductance channels in planar model membranes [19], [28]. We therefore compared the membrane permeability induced by VP4His and purified HRV16, by measuring CF leakage from liposomes in the dye-release assay. Recombinant VP4 and purified virus induced membrane permeability with broadly similar characteristics (Fig. 7). However, virus-induced permeability was more efficient at 37°C than at 25°C, while the recombinant VP4-induced permeability was relatively unaffected by the change in temperature. These observations demonstrate a temperature dependence for HRV-membrane interactions, consistent with previous studies with PV [19] and with a model of temperature dependent particle breathing which controls the exposure and membrane interactions of internal capsid components such as VP4 [29], [30]. Although studies with PV demonstrated breathing only at physiological temperatures approaching 37°C [29], studies with HRV have shown particle breathing at lower temperatures such as 25°C [30]. Importantly, virus samples pre-treated at 60°C to convert virions into empty particles induced only low levels of permeability, most likely because empty particles would no longer contain VP4.

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