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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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HRV-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 0.5 µg HRV or preheated HRV. Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant (as in figure 4). 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 three independent experiments.
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ppat-1004294-g009: HRV-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 0.5 µg HRV or preheated HRV. Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant (as in figure 4). 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 three independent experiments.

Mentions: The membrane permeability induced by recombinant VP4 was enhanced at low pH (Fig. 3). We therefore investigated the effect of pH on virus-induced permeability by comparing the permeability induced by virus at pH 6.5 (representing early endosomes) and pH 5.5 (representing late endosomes) (Fig. 8). This demonstrated that virus-induced permeability was significantly enhanced at pH 5.5, consistent with this lower pH contributing to the triggering of particle alterations involved in membrane interactions and uncoating. In addition, we carried out dextran release studies to investigate if virus-induced permeability was size-selective, as shown earlier for recombinant VP4 (Fig. 4). Virus-induced permeability was indeed also size-selective, with the smaller dextrans being released to a significantly greater extent than the larger dextran (Fig. 9). This confirmed that virus-induced permeability was not via the large scale disruption of the membrane but was also likely to involve a defined pore. However, while the results for the VP4 pore suggested limiting the transfer of molecules with Stokes' radius approaching ∼60 Å, the virus-induced permeability permitted partial release of these molecules, appearing to have a larger size cut-off, perhaps consistent with the involvement of additional capsid components in the virus-induced pore such as the N-terminus of VP1.


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)

HRV-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 0.5 µg HRV or preheated HRV. Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant (as in figure 4). 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 three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004294-g009: HRV-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 0.5 µg HRV or preheated HRV. Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant (as in figure 4). 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 three independent experiments.
Mentions: The membrane permeability induced by recombinant VP4 was enhanced at low pH (Fig. 3). We therefore investigated the effect of pH on virus-induced permeability by comparing the permeability induced by virus at pH 6.5 (representing early endosomes) and pH 5.5 (representing late endosomes) (Fig. 8). This demonstrated that virus-induced permeability was significantly enhanced at pH 5.5, consistent with this lower pH contributing to the triggering of particle alterations involved in membrane interactions and uncoating. In addition, we carried out dextran release studies to investigate if virus-induced permeability was size-selective, as shown earlier for recombinant VP4 (Fig. 4). Virus-induced permeability was indeed also size-selective, with the smaller dextrans being released to a significantly greater extent than the larger dextran (Fig. 9). This confirmed that virus-induced permeability was not via the large scale disruption of the membrane but was also likely to involve a defined pore. However, while the results for the VP4 pore suggested limiting the transfer of molecules with Stokes' radius approaching ∼60 Å, the virus-induced permeability permitted partial release of these molecules, appearing to have a larger size cut-off, perhaps consistent with the involvement of additional capsid components in the virus-induced pore such as the N-terminus of VP1.

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