Negatively charged amino acids at the foot-and-mouth disease virus capsid reduce the virion-destabilizing effect of viral RNA at acidic pH

Figures

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  Figure 1. Growth characteristics and acidic resistance of FMDV mutants with substitutions at residue VP1 17. (A) Plaque size of FMDV mutants. BHK-21 cells were infected in agar semisolid medium, and viral plaques were detected by staining with crystal violet. About 100 viral plaques were analyzed for each virus. Asterisk/s denote statistically significant differences. (B) Single-step growth curve analysis of FMDV mutants. BHK-21 cells were infected (MOI of 1 PFU/cell), and the virus titer in the supernatants was determined by plaque assay at different hours postinfection. Results are expressed as the percentage of the virus titer relative to that of the WT at 24 h postinfection. Data are presented as means ± SD of triplicates. *P < 0.05; **P < 0.005; and ***P < 0.0001. (C) Acid sensitivity profiles of FMDV mutants. Equal PFU amounts of each of the viruses were treated (in triplicate) with different acid buffers (from pH 5.4 to 6.7) or pH 7.2 as a control. Samples were neutralized and plated on BHK-21 cell monolayers and the infectivity was calculated as the percentage of PFU recovered at each pH relative to that obtained at pH 7.2. Data are presented as means ± SD. The data for mutants VP1…

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  Figure 2. Expression and processing of capsid protein and capsid assembly of FMDV empty capsids with substitutions at residue VP1 17. Empty capsids were expressed, radiolabelled, and purified as previously described16. (A) Representative results of Western blot assays showing the expression and processing of capsid polyprotein P1 from WT and the mutant capsids indicated. M: molecular weight markers. The protein bands corresponding to the unprocessed polyprotein P1–2A and the end product VP3 (both determined by reactivity with an anti-VP3 antibody) are indicated. (B) Percentage yields of the mutant mature capsid protein VP3s relative to the WT control. The relative amount of radiolabelled empty capsids (mutants compared to wt) was estimated by densitometry of the corresponding bands. (C) Percentage yields of mutant assembled capsids relative to the WT control. The relative amount of radiolabelled empty capsids (mutants compared to WT) was estimated by determining the radioactivity associated to the 80 S peak observed by centrifugation in sucrose density gradients. For each mutant in (B) and (C), the averaged value obtained from two independent experiments and the corresponding error bar…

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  Figure 3. Analysis of empty capsid integrity after acid treatment. Acid sensitivity profiles of FMDV mutant empty capsids. Equal amounts of radiolabeled empty capsids (A) VP1 N17D (B) VP1 N17D + VP2 H145Y were treated with different acid buffers (from pH 4.5 to 7). Samples were neutralized and the amount of capsids at each pH was estimated by centrifugation in sucrose gradients and determination of the radioactivity present in each fraction (see Materials and Methods). In each experiment, the WT capsid was included as an internal control to compare stability in parallel under exactly the same conditions. The results obtained for each mutant capsid were normalized with respect to those for the WT capsid in the same experiment. The average values and error bars (SD) corresponding to two independent measurements are indicated. (C) Comparison of the acid sensitivity profiles of WT and capsid mutants VP1 N17D, VP1 N17D + VP2 H145Y (filled symbols) and those reported for the RNA-filled WT and mutant VP1 N17D virions22 (empty symbols).