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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 size-selective.Liposomes containing FITC-labelled dextrans of 4 kD (FD4), 10 kD (FD10), 70 kD (FD70) or 250 kD (FD250) were mixed with 5 µM VP4His (A) or 10 µM melittin (B). Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant. Data is presented as percentage of total release observed by lysis of liposomes by addition of detergent. Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05). Data is representative of multiple independent experiments.
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ppat-1004294-g004: VP4-induced permeability is size-selective.Liposomes containing FITC-labelled dextrans of 4 kD (FD4), 10 kD (FD10), 70 kD (FD70) or 250 kD (FD250) were mixed with 5 µM VP4His (A) or 10 µM melittin (B). Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant. Data is presented as percentage of total release observed by lysis of liposomes by addition of detergent. Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05). Data is representative of multiple independent experiments.

Mentions: We hypothesised that VP4-induced membrane permeability was the result of formation of discrete pores with dimensions sufficient for transfer of macro-molecules such as RNA across the membrane. This was investigated by measuring the VP4-induced release from liposomes of FITC-labelled dextrans of different sizes. Smaller dextrans of 4 kDa (Stokes' radius of 14 Å) and 10 kDa (Stokes' radius of 23 Å) were indeed released from liposomes with higher efficiency than 70 kDa (Stokes' radius of 60 Å) and 250 kDa (Fig. 4A). The pore forming toxin melittin was included as a control for these studies and released only the smallest dextran tested (Fig. 4B), in agreement with previous reports [24], [27]. These experiments indicate that VP4 permeability is via formation of size-selective membrane pores which limit the transfer of molecules with Stokes' radius approaching ∼60 Å. Such a pore, with a lumen diameter ≤12 nm would be consistent with the size required for passage of single-stranded nucleic acid.


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 size-selective.Liposomes containing FITC-labelled dextrans of 4 kD (FD4), 10 kD (FD10), 70 kD (FD70) or 250 kD (FD250) were mixed with 5 µM VP4His (A) or 10 µM melittin (B). Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant. Data is presented as percentage of total release observed by lysis of liposomes by addition of detergent. Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05). Data is representative of multiple independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g004: VP4-induced permeability is size-selective.Liposomes containing FITC-labelled dextrans of 4 kD (FD4), 10 kD (FD10), 70 kD (FD70) or 250 kD (FD250) were mixed with 5 µM VP4His (A) or 10 µM melittin (B). Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant. Data is presented as percentage of total release observed by lysis of liposomes by addition of detergent. Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05). Data is representative of multiple independent experiments.
Mentions: We hypothesised that VP4-induced membrane permeability was the result of formation of discrete pores with dimensions sufficient for transfer of macro-molecules such as RNA across the membrane. This was investigated by measuring the VP4-induced release from liposomes of FITC-labelled dextrans of different sizes. Smaller dextrans of 4 kDa (Stokes' radius of 14 Å) and 10 kDa (Stokes' radius of 23 Å) were indeed released from liposomes with higher efficiency than 70 kDa (Stokes' radius of 60 Å) and 250 kDa (Fig. 4A). The pore forming toxin melittin was included as a control for these studies and released only the smallest dextran tested (Fig. 4B), in agreement with previous reports [24], [27]. These experiments indicate that VP4 permeability is via formation of size-selective membrane pores which limit the transfer of molecules with Stokes' radius approaching ∼60 Å. Such a pore, with a lumen diameter ≤12 nm would be consistent with the size required for passage of single-stranded nucleic acid.

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