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 multimeric structure visualized by electron microscopy.A. VP4GST was reconstituted in the membrane mimetic detergent DPC, applied to carbon coated grids and stained with uranyl formate. Scale bar = 20 nm. B. Individual ring-like structures were manually selected and cropped from unprocessed digital images. Scale bar = 10 nm. C. Class average images of particles automatically picked from a large image data set and classified into 7 classes. Scale bar = 5 nm. D. Class averages 3 and 7 (top-down views) analyzed by rotational harmonic analysis.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g006: VP4 multimeric structure visualized by electron microscopy.A. VP4GST was reconstituted in the membrane mimetic detergent DPC, applied to carbon coated grids and stained with uranyl formate. Scale bar = 20 nm. B. Individual ring-like structures were manually selected and cropped from unprocessed digital images. Scale bar = 10 nm. C. Class average images of particles automatically picked from a large image data set and classified into 7 classes. Scale bar = 5 nm. D. Class averages 3 and 7 (top-down views) analyzed by rotational harmonic analysis.

Mentions: To facilitate detection of multimeric VP4 complexes by electron microscopy, we used recombinant VP4 fused to a larger tag, the 26 kDa glutathione S-transferase (VP4GST). This protein was previously shown to be functional for interaction with and induction of permeability in model membranes [18]. In the study reported here, myristoylated VP4GST was mixed with the membrane mimetic zwitterionic detergent dodecylphosphocholine (DPC), protein-micelle complexes were purified by size exclusion chromatography and examined by negative stain transmission electron microscopy. Ring-like structures were readily observed (Fig. 6a and 6b) consistent with ‘top-down’ views of multimeric pores. We also initially tried to image VP4 in liposomes but in our hands the negative stain images were of poor quality while reconstitution of VP4 in detergent gave far superior results. We therefore took forward the latter strategy to generate the data shown in the manuscript. An image data set representing all detected structures (n = 5929) was separated into seven class averages (Fig. 6c). Several different classification schemes were tested with varying numbers of classes, and a K-means classification with seven classes gave the most consistent and interpretable classes. We believe the overall micelle-pore complexes (VP4GST+micelle) would in fact be wider than they are high, therefore forming an overall disc shape which would preferentially lie flat on the grid perhaps favouring orientation of the pore facing upwards and reducing the number of alternate views. Of the seven classes, two were selected (class 3 and 7 in Fig. 6c) as clear top-down views (the remainder appeared to represent mis-alignments or side views of the complex) and analysed by rotational harmonic analysis which indicated 5- or 6-fold symmetry (Fig. 6d), consistent with a pentameric or hexameric complex of VP4GST molecules. The initial appearance of the class 3 average as a complex with 3-fold symmetry may be due to the propensity of the GST fusion partner to form dimers, thus initially presenting six GST moieties as three dimers. However, the appearance of the class 7 average as a pentameric structure suggested that GST dimerization was not controlling the stoichiometry of the complex. It is conceivable that recombinant VP4 is able to form pore structures with different numbers of multimers; perhaps a single preferred structure for the native virus-derived pore may be influenced by additional factors.


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 multimeric structure visualized by electron microscopy.A. VP4GST was reconstituted in the membrane mimetic detergent DPC, applied to carbon coated grids and stained with uranyl formate. Scale bar = 20 nm. B. Individual ring-like structures were manually selected and cropped from unprocessed digital images. Scale bar = 10 nm. C. Class average images of particles automatically picked from a large image data set and classified into 7 classes. Scale bar = 5 nm. D. Class averages 3 and 7 (top-down views) analyzed by rotational harmonic analysis.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g006: VP4 multimeric structure visualized by electron microscopy.A. VP4GST was reconstituted in the membrane mimetic detergent DPC, applied to carbon coated grids and stained with uranyl formate. Scale bar = 20 nm. B. Individual ring-like structures were manually selected and cropped from unprocessed digital images. Scale bar = 10 nm. C. Class average images of particles automatically picked from a large image data set and classified into 7 classes. Scale bar = 5 nm. D. Class averages 3 and 7 (top-down views) analyzed by rotational harmonic analysis.
Mentions: To facilitate detection of multimeric VP4 complexes by electron microscopy, we used recombinant VP4 fused to a larger tag, the 26 kDa glutathione S-transferase (VP4GST). This protein was previously shown to be functional for interaction with and induction of permeability in model membranes [18]. In the study reported here, myristoylated VP4GST was mixed with the membrane mimetic zwitterionic detergent dodecylphosphocholine (DPC), protein-micelle complexes were purified by size exclusion chromatography and examined by negative stain transmission electron microscopy. Ring-like structures were readily observed (Fig. 6a and 6b) consistent with ‘top-down’ views of multimeric pores. We also initially tried to image VP4 in liposomes but in our hands the negative stain images were of poor quality while reconstitution of VP4 in detergent gave far superior results. We therefore took forward the latter strategy to generate the data shown in the manuscript. An image data set representing all detected structures (n = 5929) was separated into seven class averages (Fig. 6c). Several different classification schemes were tested with varying numbers of classes, and a K-means classification with seven classes gave the most consistent and interpretable classes. We believe the overall micelle-pore complexes (VP4GST+micelle) would in fact be wider than they are high, therefore forming an overall disc shape which would preferentially lie flat on the grid perhaps favouring orientation of the pore facing upwards and reducing the number of alternate views. Of the seven classes, two were selected (class 3 and 7 in Fig. 6c) as clear top-down views (the remainder appeared to represent mis-alignments or side views of the complex) and analysed by rotational harmonic analysis which indicated 5- or 6-fold symmetry (Fig. 6d), consistent with a pentameric or hexameric complex of VP4GST molecules. The initial appearance of the class 3 average as a complex with 3-fold symmetry may be due to the propensity of the GST fusion partner to form dimers, thus initially presenting six GST moieties as three dimers. However, the appearance of the class 7 average as a pentameric structure suggested that GST dimerization was not controlling the stoichiometry of the complex. It is conceivable that recombinant VP4 is able to form pore structures with different numbers of multimers; perhaps a single preferred structure for the native virus-derived pore may be influenced by additional factors.

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