A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid lability and confer resistance to acid-dependent uncoating inhibition

Abstract

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH(4)Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH(4)Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH(4)Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.

FIG. 1.

Effects of NH₄Cl on FMDV infection. (A) NH₄Cl significantly inhibits FMDV C-S8c1 infection when added preinfection but has no significant effects when added p.i. Monolayers of infected (MOI of 0.5) BHK-21 cells were treated with increasing concentrations of NH₄Cl added 1 h preinfection or 3 h p.i. The total virus yield was determined 8 h p.i. by plaque assay. (B) NH₄Cl does not inhibit FMDV cell binding. Control and NH₄Cl-treated cells were incubated for 25 min with FMDV (MOI of 100), fixed, and stained for immunofluorescence using MAb 5C4 to stain FMDV capsid proteins and a secondary antibody coupled to AF488. Bar, 20 μm; bar for inset, 5 μm. (C) NH₄Cl inhibits expression of FMDV nonstructural proteins. Confocal images of cells infected (4 h p.i.) with FMDV (MOI of 100) in the presence or in the absence of NH₄Cl and immunostained with rabbit serum to 3A protein in combination with MAb 5C4. AF488 (green) anti-rabbit and AF555 (red) anti-mouse IgGs were used as secondary antibodies. Colocalization is shown in yellow in the merged image. Bar, 20 μm. (D) FMDV particles are retained in endosomal compartments in NH₄Cl-treated cells. Confocal images of cells treated with NH₄Cl and infected as described for panel C, immunostained with rabbit anti-EEA1 antibody and MAb 5C4. AF488 (green) anti-rabbit and AF555 (red) anti-mouse IgGs were used as secondary antibodies. Arrowheads denote spots positive for EEA1 and 5C4 markers. Bar, 20 μm.

FIG. 2.

NH₄Cl-resistant FMDVs use integrins as cellular receptors. (A) RGD-containing peptides block attachment to the cell by NH₄Cl-resistant FMDVs. Cells incubated with an RGD-containing peptide or a control non-RGD-containing peptide were infected with the different NH₄Cl-resistant viruses (c1, c2, and c3), parental FMDV (C-S8c1), and MARLS FMDV. After 25 min, cells were fixed and immunostained with MAb 5C4 to detect FMDV capsid proteins and a secondary, AF555-labeled antibody. Phalloidin staining was included as an indicator of cell shape. Bar, 20 μm. (B) NH₄Cl-resistant FMDVs do not grow in CHO cells. Cells were infected (MOI of 0.5) with the different FMDV variants, and supernatant virus titers were measured at different times p.i.

FIG. 3.

Characterization of resistance to NH₄Cl and acid sensitivity of FMDV mutants. (A) Differential NH₄Cl sensitivity of FMDV mutants c1, c2, and c3. Monolayers of BHK-21 cells treated or not treated with NH₄Cl were infected with the mutants and the parental virus C-S8c1 (MOI of 0.5). The total virus yield was determined 8 h p.i. (B) Acid sensitivity of NH₄Cl-resistant FMDVs. Equal amounts of the different viruses were treated with acid buffers of different pHs for 30 min, neutralized, and plated. Infectivity was calculated as the percentage of PFU recovered relative to that obtained at pH 7.5. BEV, an acid-resistant virus, was included as a control.

FIG. 4.

Mutant c2 is resistant to NH₄Cl and to ConA. (A) NH₄Cl does not prevent the expression of the 3A FMDV nonstructural protein in cells infected with mutant c2. BHK-21 cells treated or not treated with NH₄Cl were infected (MOI of 10) and fixed 4 h p.i. The percentage of 3A-positive cells was determined by immunostaining using anti-3A rabbit serum and secondary antibodies labeled with AF555. (B) NH₄Cl does not reduce VP1 production in cells infected with c2. Western blot analysis of VP1 production in BHK-21 cells treated or not treated with NH₄Cl and infected (MOI of 0.5). Cells were lysed at 8 h p.i., and the amount of FMDV VP1 protein was determined by Western blotting using MAb SD6. Membranes were reprobed with a MAb against β-actin as a control for protein loading. (C) NH₄Cl does not inhibit infection of mutant c2 in IBRS-2 cells. Monolayers were processed as described for panel B, and the total virus yield was determined 8 h p.i. (D) ConA does not inhibit infection of mutant c2 in IBRS-2 cells. Monolayers were processed and analyzed as described for panel B.

FIG. 5.

Locations on the structure of the C-S8c1 capsid (36) of amino acid residues found to be substituted in NH₄Cl-resistant FMDV variants. (A) Outside and inside schematic views of a pentameric subunit in the capsid. VP1 is colored green, VP2 magenta, VP3 cyan, and VP4 yellow. H140 and H143 are also indicated. (B) Close-up of a section of the interface among three neighboring pentamers in the capsid. Dashed lines represent interpentameric interfaces. VP2 and VP3, VP2′ and VP3′, and VP2′′ and VP3′′ correspond to protein subunits belonging to the three different pentameric subunits. Residues A118 and A123 found mutated in viruses c1 and c2 are, respectively, indicated in red and blue. H140 and H143 are indicated in black and gray, respectively.

FIG. 6.

The single amino acid substitution A118V in VP3 is responsible for the NH₄Cl resistance and increased acid sensitivity of mutant c2. (A) Differential sensitivity to NH₄Cl of viruses derived from infectious clones carrying the mutations found in the capsid from virus c2. BHK-21 cells treated or not treated with NH₄Cl were infected (MOI of 0.5), and the total virus yield was determined 8 h p.i. (B) Acid sensitivity of viruses carrying the mutations found in the c2 virus. Equal amounts of the viruses were treated with acid buffers of different pHs for 30 min, neutralized, and plated. Infectivity was calculated as the percentage of PFU recovered in BHK-21 cells relative to that obtained at pH 7.5. (C) Immunofluorescence analysis of BHK-21 cells treated or not treated with NH₄Cl and infected (MOI of 10). Cells were fixed 6 h p.i., and 3A protein was detected using anti-3A rabbit serum and secondary antibodies labeled with AF594. (D) Western blot analysis of VP1 production of BHK-21 cells treated and infected as described for panel A. Cells were lysed at 8 h p.i., and the amount of FMDV VP1 protein was determined by Western blotting using MAb SD6. Membranes were reprobed with a MAb against β-actin as a control for protein loading. (E) Infection by viruses carrying the mutation VP3 A118V is not inhibited by NH₄Cl in IBRS-2 cells. Cells were processed as described for panel A. (F) Infection by viruses carrying mutation VP3 A118V is not inhibited by ConA in IBRS-2 cells. Cells treated or not treated with ConA were infected, and the total virus yield was determined as described for panel A.

FIG. 7.

Sedimentation profiles of radiolabeled C-S8c1 and VP3 A118V/VP1 N47D and VP3 A118V mutant virions. (A) Sedimentation profile obtained during the last purification step of FMDV virions by sedimentation in 7.5% to 30% sucrose gradients. (B) Analytical sedimentation profile for purified, dialyzed virions in 10% to 45% sucrose density gradients. Circles, C-S8c1 virus (nonmutated); diamonds, VP3 A118V/VP1 N47D mutant; triangles, VP3 A118V mutant. The peak on the right side of each profile corresponds to intact (nondissociated) virions (sedimentation coefficient, 140S), and the peak on the left corresponds to dissociated pentameric subunits (sedimentation coefficient, 12S).