Deterministic, compensatory mutational events in the capsid of foot-and-mouth disease virus in response to the introduction of mutations found in viruses from persistent infections

Abstract

The evolution of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) in persistently infected cells led to the emergence of a variant (R100) that displayed increased virulence, reduced stability, and other modified phenotypic traits. Some mutations fixed in the R100 genome involved a cluster of highly conserved residues around the capsid pores that participate in interactions with each other and/or between capsid protomers. We have investigated phenotypic and genotypic changes that occurred when these replacements were introduced into the C-S8c1 capsid. The C3007V and M3014L mutations exerted no effect on plaque size or viral yield during lytic infections, or on virion stability, but led to a reduction in biological fitness; the D3009A mutation caused drastic reductions in plaque size and viability. Remarkably, competition of the C3007V mutant with the nonmutated virus invariably resulted in the fixation of the D3009A mutation in the C3007V capsid. In turn, the presence of the D3009A mutation invariably led to the fixation of the M3014L mutation. In both cases, two individually disadvantageous mutations led, together, to an increase in fitness, as the double mutants outcompeted the nonmutated genotype. The higher fitness of C3007V/D3009A was related to a faster multiplication rate. These observations provide evidence for a chain of linked, compensatory mutational events in a defined region of the FMDV capsid. Furthermore, they indicate that the clustering of unique amino acid replacements in viruses from persistent infections may also occur in cytolytic infections in response to changes caused by previous mutations without an involvement of the new mutations in the adaptation to a different environment.

FIG. 1.

Location in the virus capsid of a cluster of residues found to be mutated in FMDV R100. (A) Cα tracing model of a pentameric subunit in the FMDV C-S8c1 capsid viewed from the exterior of the virion. Five copies of each of the capsid proteins VP1, VP2, VP3, and VP4 are depicted in different colors. The residues around the fivefold axis pore (center) that were found to be mutated in FMDV R100 (16) are represented as space-filling models and are color coded (green, Cys3007; red, Asp3009; yellow, Asn3013; magenta, Met3014). (B) Close-up view of the N termini of five symmetry-related copies of VP3 around the fivefold axis pore in a pentamer shown in the same orientation depicted in A. The tracing of the main-chain backbone is indicated by ribbons. The residues that were found to be mutated in FMDV R100 are represented as in A. The five cysteine residues (green) constrict the pore (center). (C). Side view of the fivefold axis region in a pentamer. The outer surface is up. The β-annulus formed by β-strands belonging to the five VP3 subunits (colored arrows) is shown below the residues found mutated in FMDV R100 (shown as colored space-filling models as in A and B).

FIG. 2.

Thermal inactivation kinetics of the parental and mutant FMDVs. (A) Inactivation of undiluted parental virus (circles) and mutant C3007V (squares) (using the first type of inactivation assay described in Materials and Methods) at 42°C. Linear fitting of the logarithmic values is indicated. (B) Inactivation of the diluted parental virus (circles), C3007V (squares), M3014L (triangles), and D3009A/M3014L (inverted triangles) (using the second type of inactivation assay described in Materials and Methods) at 42°C. Fitting of the data to a single exponential is indicated.

FIG. 3.

Approximate percentage of FMDV genomes carrying a specific residue at a defined position during serial passages in competition experiments. The global sequence of the viral populations obtained after each passage was determined by automated sequencing, and the relative abundance of each residue type at the positions of interest was estimated from the ratio between the integrated areas of the corresponding bands in the chromatograms, as described in Materials and Methods. (A) Competition between the M3014L mutant and the parental virus. Circles, Met; squares, Leu at position 3014. (B) Competition between the D3009A/M3014L mutant and the parental virus. (B.1) Circles, Ala; squares, Asp at position 3009; black inverted triangles, Leu; black triangles, Met at position 3014. (B.2) Squares, Thr; circles, Pro at position 1104. (C) competition between the C3007V mutant and the parental virus. (C.1) Squares, Val; circles, Cys at position 3007. (C.2) Squares, Ala; circles, Asp at position 3009. All three competition assays were carried out in duplicate or triplicate with very similar results.

FIG. 4.

Kinetics of extracellular virus production of nonmutated and mutant FMDVs. (A and B) Absolute virus titers at different times after infection. (A) Circles, nonmutated virus; squares, C3007V mutant. (B) Circles, nonmutated virus; squares, C3007V/D3009A mutant. (C and D) Relative infectivity of mutant viruses with respect to the infectivity of the nonmutated virus at different times after infection. The infectivity of the nonmutated virus at each time after infection has been given the reference value of 100. (C) White bars, nonmutated virus; black bars, C3007V mutant. (D) White bars, nonmutated virus; black bars, C3007V/D3009A mutant. Standard deviations are indicated in each case.