Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4

FIG 1. Inhibition of endosomal acidification interferes with an early step in the AiV life cycle. (A) Growth of AiV is prevented by treatment of cells with NH4Cl. Titer of virus 24 h after infection of cells treated without, or with, 2 mM, 10 mM, 20 mM, or 40 mM NH4Cl for 2 h before infection with AiV at an MOI of 1. (B) Time of addition of NH4Cl shows effect early in infection. Titer of virus 24 h after infection of cells treated with 40 mM NH4Cl 1 h before infection (−1) or 0, 1, 2, 3, or 4 h postinfection. Experiments were performed in triplicate.

FIG 2. Low pH enhances AiV capsid alterations. AiV analyzed by PaSTRy over a range of pH values (pH 7.0, 6.2, 5.9, 5.6, 5.0, and 4.9) with incremental increases of temperature of 1°C every 30 s. Experiments were either put in at room temperature (A and D) or heated at either 56°C (B and E) or 59°C (C and F) for 10 min and then chilled on ice for 2 min prior to being put on the Stratagene MX3005p quantitative PCR (qPCR) system. (A to C) Relative fluorescence of SYTO9 nucleic acid-binding dye, where exposure is inferred from increasing signal of viral RNA. (D to F) Relative fluorescence of SYPRO orange hydrophobic protein residue-binding dye, where increasing signal infers exposure of hydrophobic capsid components. All results are normalized to maximum signal for each experiment, representing 100% signal. All experiments were performed in triplicate; this is a representative trace.

FIG 3. AiV-induced membrane permeability is enhanced by low pH. (A) Permeability assay showing CF released from liposomes after mixing with AiV at pH 7.0, 6.2, 5.9, 5.6, 5.0, and 4.9. CF was detected by fluorescence measurements (excitation, 492 nm/emission, 512 nm) recorded every 30 s for 1 h. (B) Endpoints of AiV-induced membrane permeability. Data in panel A were normalized to the maximum signal induced by 0.1% NP-40 at each pH value. (C and D) FD released after addition of 1 μg of purified AiV capsid liposomes containing FD of 4 kDa (FD4), 10 kDa (FD10), 70 kDa (FD70), or 250 kDa (FD250) at pH 7 (C) and pH 5 (D). Experiments were performed in triplicate, and error is measured by standard error of the mean (SEM). *, P ≥ 0.01. Low pH is known to quench CF dye, so all results are normalized to 100% release as determined by incubation with NP-40 and 0% release as determined by liposome incubated alone. As low pH quenches CF/FITC fluorescence, the values were calculated as a percentage of full release by normalizing to untreated liposomes (0% release) and 0.1% NP-40 or Triton (100% release).

FIG 4. Alignments of VP4- and VP0-encoding picornavirus sequences. Alignments of picornavirus VP4 sequences (A) and VP0 sequences (B) were carried out using the MUSCLE sequence alignment tool (55). The amino acids are colored using the Zappo color scheme as follows: aliphatic/hydrophobic (pale pink), aromatic (orange), positively charged (purple), negatively charged (red), hydrophilic (green), conformationally special (magenta), and cysteine residues (yellow). Peptide sequence regions for AiV, LV, and RV are highlighted with a black box.

FIG 5. AiV VP0 N-terminal peptide is able to permeabilize membranes and form a size-selective pore. Permeability assays showing CF or fluorescent dextrans (FD) released from liposomes after mixing with peptides. CF or FD was detected by fluorescence measurements (excitation, 492 nm/emission, 512 nm) and displayed as percentage of total release by detergent-induced lysis. Assays were carried out at pH 7.0 unless otherwise stated. (A) CF release over time after addition of peptide AiV-VP0-N50 at concentrations of 0.5 μM, 5 μM, and 14 μM. (B) CF release over time after addition of peptide Ljungan virus (LV) LV-VP0-N50 at concentrations of 0.5 μM, 5 μM, 14 μM, and 50 μM. (C) CF release over time after addition of peptide AiV-VP0-N50 or RV16-VP4-N45 at concentrations of 5 μM. (D) FD released after addition of 5 μM peptide AiV-VP0-N50 to liposomes containing FD of 4 kDa (FD4), 10 kDa (FD10), 70 kDa (FD70), or 250 kDa (FD250). (E) FD released after addition of 5 μM peptide RV16-VP4-N45 to liposomes containing FD of 4 kDa (FD4), 10 kDa (FD10), 70 kDa (FD70), or 250 kDa (FD250). (F) CF release over time after addition of 0.5 μM peptide AiV-VP0-N50 at pH 5 or pH 7. All experiments were performed…