HRV-induced membrane permeability is enhanced by low pH.

<p><b>A</b>) Carboxyfluorescein (CF)-containing liposomes at pH 6.5 and pH 5.5 were mixed with HRV (0.5 µg) 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 (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004294#ppat-1004294-g003" target="_blank">figure 3</a>). <b>B</b>) End points of HRV induced membrane permeability. Data in panels A and B was normalized to the maximum signal induced by melittin at each pH value. Data shown is representative of multiple independent experiments (n>3). Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05).</p

VP4 multimeric structure visualized by electron microscopy.

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

Purity and concentration of recombinant VP4 assessed by SDS-PAGE.

<p>Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (<b>A</b>) or western blot using antisera to VP4 (<b>B</b>). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004294#ppat.1004294.s001" target="_blank">figure S1</a>).</p

Recombinant VP4 induces dose-dependent membrane permeability.

<p>Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.</p

VP4-induced permeability is comparable to that of virus.

<p>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 (<b>A</b>) or 37°C (<b>B</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).</p

VP4-induced membrane permeability is enhanced by low pH and VP4 myristoylation.

<p>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. <b>A</b>) 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>B</b>) the acid-dependent pore-forming peptide GALA at 1 µM. <b>C</b>) VP4His at 5 µM. <b>D</b>) ΔVP4His (unmyristoylated) at 5 µM. <b>E</b>) End point and <b>F</b>) 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.</p

HRV-induced permeability is size-selective.

<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004294#ppat-1004294-g004" target="_blank">figure 4</a>). 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.</p

VP4-induced permeability is size-selective.

<p>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 (<b>A</b>) or 10 µM melittin (<b>B</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.</p

VP4 forms a multimeric complex.

<p><b>A.</b> VP4His was incubated in the presence of the membrane-mimetic detergent DHPC or liposomes, or mock-treated (−), resolved by native PAGE and visualised by silver staining. The position of bands shown by arrows indicates potential migration of VP4His into the gel. <b>B</b>. VP4 was incubated with (+) or without (−) liposomes and crosslinked by the addition of DSP at 0.5 mM (+) or 1 mM (++). Samples were resolved by non-reducing SDS-PAGE and detected by western blot using antisera to VP4. DTT was used in some samples to reverse the crosslinking prior to SDS-PAGE. Arrows indicate VP4 multimers. Molecular mass markers (in kilodaltons) are indicated on the left.</p

PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process.

<p><b>A)</b> Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm. <b>B)</b> Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control. <b>C)</b> Scintillation counting of internalized vs unattached ³H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. <b>D)</b> Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of ³H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. <b>E)</b> Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of ³H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.</p