Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.

Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.     

Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification.

Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.     

VP3 G–H环中氨基酸突变对O型口蹄疫病毒生物学特性的影响

【目的】为了研究O型口蹄疫病毒VP3G–H环中氨基酸突变对其生物学特性的影响。【方法】借助口蹄疫病毒反向遗传操作技术平台拯救出2株定点突变体rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)。进行蚀斑形成试验、一步生长曲线的绘制、TCID_(50)和LD_(50)的测定、间接免疫荧光与激光共聚焦显微镜检测。【结果】结果显示,与骨架病毒rHN相比,虽然rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对BHK-21细胞的感染性及其蚀斑表型和复制动力学无显著性差异;但rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对乳鼠的致病力明显减弱,且均获得了小窝蛋白介导侵染CHO-K1细胞的能力。【结论】VP3上第3174位特征性氨基酸突变影响O型口蹄疫病毒感染宿主细胞的毒力及其内吞作用路径,这有助于我们认知VP3 G–H环在口蹄疫病毒粒子立体空间构象中潜在的作用。     

Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock?

The number of nucleotide (nt) substitutions found in the VP1 gene (encoding viral capsid protein) between any two of 16 closely related isolates of foot-and-mouth disease virus (FMDV) has been quantified as a function of the time interval between isolations [Villaverde et al., J. Mol. Biol. 204 (1988) 771-776]. One of them (isolate C-S12) includes some replacements found in isolates that preceded it and other replacements found in later isolates. The study has revealed alternating periods of rapid evolution and of relative genetic stability of VP1. During a defined period of acute disease, the rate of fixation of replacements at the VP1 coding segment was 6 x 10(-3) substitutions per nt per year. Only small differences in the rate of evolution were observed between subsegments within the VP1 gene. The observation of a relatively constant rate of evolution during a disease episode was unexpected. We propose that such constancy may be a consequence of random sampling of mutants from the FMDV quasispecies, followed by their amplification in susceptible hosts (to generate a new quasispecies). Successive sampling and amplification events may result in a steady accumulation of mutations.     

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.     

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.     

Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering.

Plaque-to-plaque transfers of RNA viruses lead to accumulation of mutations and fitness decrease. To test whether continuing plaque-to-plaque transfers would lead to viral extinction, we have subjected several low fitness foot-and-mouth disease virus (FMDV) clones to up to 130 successive plaque transfers, and have analyzed the evolution of plaque titers and genomic nucleotide sequences. No case of viral extinction could be documented. Some low fitness clones that posses an internal poly(A) tract evaded extinction by modifying the length or base composition of the poly(A) tract. The comparison of entire genomic sequences of FMDV clones at increasing plaque transfer number revealed that mutations accumulated at a uniform rate, and that they were distributed unevenly along the genome. Clusters of mutations were identified at different genomic sites in two plaque transfer lineages. Mutation clustering appears to occur stochastically and could not be related to fixation of compensatory mutations. The results document resistance of viral clones to extinction, and suggest that mutation clustering may be a mechanism of genetic diversification of low fitness virus.     

Rapid selection of genetic and antigenic variants of foot-and-mouth disease virus during persistence in cattle.

Rapid evolution of foot-and-mouth disease virus (FMDV) is documented during persistent infections of cattle. The carrier state was established experimentally with plaque-purified FMDV of serotype C3. Virus was recovered from the esophageal pharyngeal area of the animals up to 539 days postinfection. Analysis of capsid proteins by electrofocusing and by electrophoretic mobility of the genomic poly(C)-rich tract suggested heterogeneity in several isolates and sequential dominance of viral subpopulations. Nucleotide sequences of the VP1-coding region of the parental FMDV C3 clones and of seven isolates from the carrier cattle showed point mutations that represented rates of fixation of mutations of 0.9 X 10(-2) to 7.4 X 10(-2) substitutions per nucleotide per year; 59% of the base changes led to amino acid substitutions, some of which were located within residues 135 to 151, a region involved in neutralization of FMDV. In the esophageal pharyngeal fluid samples, FMDV C3-neutralizing activity was present. Antigenic variation was demonstrated with monoclonal antibodies raised against FMDV C3. Two isolates from carrier cattle differed from the parental virus by 10(2)- or 10(3)-fold decreased reactivity with neutralizing monoclonal antibodies. We suggest that persistent, inapparent infections of ruminants, in addition to being a reservoir of virus, may promote the rapid selection of antigenically variant FMDVs.     

Evidence for positive selection in the capsid protein-coding region of the foot-and-mouth disease virus (FMDV) subjected to experimental passage regimens

We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (d(N)/d(S)) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our analyses also identified a subregion of VP3, part of the fivefold axis of FMDV particles, that also appeared to be subjected to positive selection of amino acid replacements. From these results, we can conclude that under the restrictive conditions imposed either by the presence of the monoclonal antibodies, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in some capsid-coding regions can be positively selected toward an increase of those mutants with a higher capability to infect the cell.     

Sequence of the viral replicase gene from foot-and-mouth disease virus C1-Santa Pau (C-S8)

The nucleotide sequence of the region including the viral replicase gene, the carboxy terminus of protein P18, and the 3'-extracistronic region of foot-and-mouth disease virus (FMDV) type C1-Santa Pau (C-S8) has been determined from previously cloned cDNA fragments [Villanueva et al., Gene 23 (1983) 185-194]. The comparison with the corresponding gene segments of FMDV of serotypes A or O shows base substitutions in 7.2-8.6% of residues in the replicase gene with no insertions or deletions. This is about fourfold lower variation than found for the region encoding capsid protein VP1 of the corresponding viruses. Intermediate variability (substitution at 16.1-23.6% positions) exists in the 3'-extracistronic region, including point mutations, insertions and deletions. The predicted amino acid sequence of the replicase gene indicates that 75.5-82.6% of mutations are silent and that 93.4% of amino acids are conserved in the four FMDV replicases. The frequency of certain types of silent mutations and of rare codon usage is significantly lower for the replicase gene than for the protein VP1 coding region.     

An Increase in Acid Resistance of Foot-and-Mouth Disease Virus Capsid Is Mediated by a Tyrosine Replacement of the VP2 Histidine Previously Associated with VP0 Cleavage

The foot-and-mouth disease virus (FMDV) capsid is highly acid labile, but introduction of amino acid replacements, including an N17D change in VP1, can increase its acid resistance. Using mutant VP1 N17D as a starting point, we isolated a virus with higher acid resistance carrying an additional replacement, VP2 H145Y, in a residue highly conserved among picornaviruses, which has been proposed to be responsible for VP0 cleavage. This mutant provides an example of the multifunctionality of picornavirus capsid residues.     

Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region

We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N(C)) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G+C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an approximately 10-fold range, and virus yields decreased over an approximately 65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced approximately 3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased approximately 18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (approximately 50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.     

Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture.

Virus and cells evolve during serial passage of cloned BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV). These carrier cells, termed C1-BHK-Rc1 (J.C. de la Torre, M. Dávila, F. Sobrino, J. Ortín, and E. Domingo, Virology 145:24-35, 1985), become constitutively resistant to the parental FMDV C-S8c1. Curing of late-passage C1-BHK-Rc1 cells of FMDV by ribavirin treatment (J.C. de la Torre, B. Alarcón, E. Martínez-Salas, L. Carrasco, and E. Domingo, J. Virol. 61:233-235, 1987) did not restore sensitivity to FMDV C-S8c1. The resistance of C1-BHK-Rc1 cells to FMDV C-S8c1 was not due to an impairment of attachment, penetration, or uncoating of the particles but to some intracellular block that resulted in a 100-fold decrease in the amount of FMDV RNA in the infected cells. FMDV R59, the virus isolated from late-passage carrier cells, partly overcame the cellular block and was more cytolytic than FMDV C-S8c1 for BHK-21 cells. Sequencing of the VP1 gene from nine viral clones from C1-BHK-Rc1 cells showed genetic heterogeneity of 5 X 10(-4) substitutions per nucleotide. Mutations were sequentially fixed during persistence. In addition to resistance to FMDV C-S8c1, C1-BHK-Rc1 cells showed a characteristic round cell morphology, and compared with BHK-21 cells, they grew faster in liquid culture, were less subject to contact inhibition of growth, and had an increased ability to form colonies in semisolid agar. Reconstitution of a persistent infection was readily attained with late-passage C1-BHK-Rc1 cells and FMDV C-S8c1 or FMDV R59. The results suggest that coevolution of BHK-21 cells and FMDV contributes to the maintenance of persistence in cell culture.     

Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.     

Resistance to foot-and-mouth disease virus mediated by trans-acting cellular products.

Upon serial passage of BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) C-S8c1, cells with increased resistance to the virus were selected (J. C. de la Torre, E. Martinez-Salas, J. Diez, and E. Domingo, J. Virol. 63:59-63, 1989). Two highly resistant cell clones, 74A11 and 74D12, were transformed to puromycin resistance (Purr) and were fused to BHK-21 cells transformed to neomycin resistance (Neor). The hybrid Neor Purr cells showed the specific resistance to FMDV C-S8c1 characteristic of clones 74A11 and 74D12. The results suggest that resistance to FMDV C-S8c1 is mediated by trans-acting cellular products. The possibility of engineering constitutive resistance to FMDV is discussed.     

Evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo

In this work we analyze the antigenic properties and the stability in cell culture of virus mutants recovered upon challenge of peptide-vaccinated cattle with foot-and-mouth disease virus (FMDV) C3 Arg85. Previously, we showed that a significant proportion of 29 lesions analyzed (41%) contained viruses with single amino acid replacements (R141G, L144P, or L147P) within a major antigenic site located at the G-H loop of VP1, known to participate also in interactions with integrin receptors. Here we document that no replacements at this site were found in viruses from 12 lesions developed in six control animals upon challenge with FMDV C3 Arg85. Sera from unprotected, vaccinated animals exhibited poor neutralization titers against mutants recovered from them. Sequence analyses of the viruses recovered upon 10 serial passages in BHK-21 and FBK-2 cells in the presence of preimmune (nonneutralizing) sera revealed that mutants reverted to the parental sequence, suggesting an effect of the amino acid replacements in the interaction of the viruses with cells. Parallel passages in the presence of subneutralizing concentrations of immune homologous sera resulted in the maintenance of mutations R141G and L147P, while mutation L144P reverted to the C3 Arg85 sequence. Reactivity with a panel of FMDV type C-specific monoclonal antibodies indicated that mutant viruses showed altered antigenicity. These results suggest that the selective pressure exerted by host humoral immune response can play a role in both the selection and stability of antigenic FMDV variants and that such variants can manifest alterations in cell tropism.     

Functional origins of fitness effect-sizes of compensatory mutations in the DNA bacteriophage phiX174

Abstract Epistasis is an important and poorly understood aspect of mutations and strongly influences the evolutionary impact of genetic variation on adaptation and fitness. Although recent studies have begun to characterize the distribution of epistatic effects between mutations affecting fitness, there is currently a lack of empirical information on the underlying biological causes of these epistatic interactions. What are the functional constraints that determine the effectiveness of a compensatory mutation at restoring fitness? We have measured the effect‐sizes of 52 compensatory mutations affecting nine different deleterious mutations in the major capsid and spike proteins of the DNA bacteriophage X174. On average, an experimentally detectable compensatory mutation recovers about two‐thirds of the fitness cost of the preceding deleterious mutation. Variation in fitness effect‐sizes is only weakly associated with measures of the distance separating the deleterious and compensatory mutations in the amino acid sequence or the folded protein structure. However, there is a strong association of fitness effect‐size with the correlation in the effects of the mutations on the biochemical properties of amino acids. A compensatory mutation has the largest effect‐size, on average, when both the compensatory and deleterious mutations have radical effects on the overall biochemical make‐up of the amino acids. By examining the relative contributions of specific biochemical properties to variation in fitness effect‐size, we find that the area and charge of amino acids have a major influence, which suggests that the complexity of the amino acid phenotype is simplified by selection into a reduced number of phenotypic components.     

Systematic Study of the Genetic Response of a Variable Virus to the Introduction of Deleterious Mutations in a Functional Capsid Region

We have targeted the intersubunit interfaces in the capsid of foot-and-mouth disease virus to investigate the genetic response of a variable virus when individual deleterious mutations are systematically introduced along a functionally defined region of its genome. We had previously found that the individual truncation (by mutation to alanine) of 28 of the 42 amino acid side chains per protomer involved in interactions between capsid pentameric subunits severely impaired infectivity. We have now used viral RNAs individually containing each of those 28 deleterious mutations (or a few others) to carry out a total of 96 transfections of susceptible cells, generally followed by passage(s) of the viral progeny in cell culture. The results revealed a very high frequency of fixation in the capsid of second-site, stereochemically diverse substitutions that compensated for the detrimental effect of primary substitutions at many different positions. Most second-site substitutions occurred at or near the capsid interpentamer interfaces and involved residues that are spatially very close to the originally substituted residue. However, others occurred far from the primary substitution, and even from the interpentamer interfaces. Remarkably, most second-site substitutions involved only a few capsid residues, which acted as “second-site hot spots.” Substitutions at these hot spots compensated for the deleterious effects of many different replacements at diverse positions. The remarkable capacity of the virus to respond to the introduction of deleterious mutations in the capsid with the frequent fixation of diverse second-site mutations, and the existence of second-site hot spots, may have important implications for virus evolution.The rapid replication, high mutation rates, and large population sizes of RNA viruses give rise to genetically highly heterogeneous virus populations, termed viral quasispecies (15-18). Viral quasispecies may quickly adapt to different environments through the fixation of some of the multiple mutations present in individual genomes in the population. Accordingly, fixation of second-site mutations able to compensate for the effect of randomly occurring deleterious mutations could be particularly frequent in RNA virus populations. Compensatory mutations have indeed been detected, many times in a fortuitous way, during genetic analyses of different viruses (4, 6, 7, 9, 12, 13, 22, 29, 33, 36-42, 48, 49, 51-54). However, to our knowledge no study has systematically analyzed the frequency, types, and distribution of second-site mutations acquired by a virus in response to many different deleterious mutations introduced along a extended, but functionally defined, region of its genome.The viral capsid provides a particularly interesting target to analyze the fixation of second-site, compensatory mutations in a functional region of a virus. Nonenveloped virus capsids may be subjected to multiple, severe, and even contradictory selective pressures imposed on them to preserve their adaptive physicochemical properties and multiple biological functions (8, 31). Accordingly, accumulating evidence indicates that the capsids of small, nonenveloped viruses have been exquisitely fine-tuned through evolution, probably to a much larger extent than most cellular proteins or protein complexes. Structure-function analyses almost invariably show that the vast majority of amino acid replacements in nonenveloped viral capsids, even in exposed loops with no known function, significantly decreases viral yields, or at least unfavorably affects viral fitness (see, for example, references 28, 31, 32, and 43 and references therein). Thus, despite the dramatic potential for variation in viral quasispecies, strong negative selection could be expected to lead to very high amino acid sequence conservation in the capsid proteins. In fact, such a high sequence conservation is not observed for many nonenveloped RNA virus capsids. This paradox could be resolved by invoking a frequent fixation in the capsid region of compensatory second-site mutations, which could help the virus to evade the immune response and to adapt to new environments and hosts.Foot-and-mouth disease virus (FMDV) is a small, icosahedral, nonenveloped RNA virus whose structure, function, and evolution have been widely studied (27, 46). FMDV populations are quasispecies (19), and the FMDV capsid may provide a good model for the study of compensatory mutations and their role in virus variation and evolution. In addition, this virus is the causative agent of foot-and-mouth disease (FMD), one of the economically most important animal diseases worldwide. Frequent genetic and antigenic variation in the capsid proteins impose severe difficulties for FMD control (27, 31, 46); thus, a profound knowledge of the dynamics of compensatory mutations in the FMDV capsid may also contribute to improve current strategies to fight FMD and other diseases caused by highly variable viruses.The FMDV capsid is formed by 60 copies of each of three proteins (VP1, VP2, and VP3) and a small, internal polypeptide (VP4). During assembly of FMDV and other picornaviruses, one copy of the precursor polyprotein P1 folds and is proteolytically processed to yield a protomeric subunit composed of one copy of each capsid protein. Five protomers oligomerize to yield a pentameric subunit, and twelve pentamers assemble to form an icosahedral capsid (44) (Fig. ​(Fig.1A).1A). The three-dimensional structure of several FMDV isolates (1, 11, 21, 26), including variant C-S8c1, a biological clone from vaccine strain C₁Santa Pau-Spain/70, is known (26). Based on this structural information, we carried out previously a systematic structure-function study of the capsid interpentamer interfaces in FMDV C-S8c1. The analysis revealed that most interfacial side chains were critically required for viral function and infectivity (28). However, partial sequencing fortuitously revealed the occasional fixation in the capsid of a few pseudoreversions and second-site amino acid substitutions that, in some cases, were responsible for restoring the infectivity, thus acting as compensatory mutations (28). These and other observations suggested the possibility that a complex dynamics of compensatory mutations could be operating at the functionally critical intersubunit interfaces during the evolution of FMDV.Open in a separate windowFIG. 1.Primary and second-site substitutions in the FMDV capsid. (A) Schematic quaternary structure of the capsid. Each protein subunit is represented by a trapezoid. The numbers 1, 2, and 3, respectively, denote VP1, VP2, and VP3. The black pentagons, triangles, and ellipses indicate the positions of capsid fivefold, threefold, and twofold symmetry axes, respectively. Three protomeric subunits around a threefold axis, each of them belonging to a different pentameric subunit, are colored deep red, yellow, or blue. Three adjacent pentamers to which those protomers belong are colored in shades of red, yellow, or blue, respectively. (B and C) Primary and second-site substitutions, mapped on a partial model of the FMDV C-S8c1 capsid structure (26). The image represents a wireframe atomic model of the three protomers in the scheme shown in panel A that belong to adjacent pentamers and are deep-colored and labeled, plus one additional VP2 subunit (also labeled in panel A) from each of the three pentamers. A threefold axis is located at the center of the image, and the color code is as in panel A. The interfaces between three pentamers are thus defined by the limits between different colors (compare also panel A). In panel B, the interfacial residues subjected to deleterious substitutions are shown as white space-filling models. In panel C, the capsid residues where second-site substitutions were partially or totally fixed in the viral progeny populations are shown as space-filling models; they are colored either green (residues involved in second-site substitutions that were dominant in the population) or orange (residues involved in nondominant, “third-site” substitutions).In the present study we have systematically analyzed the frequency, types, and patterns of second-site amino acid substitutions fixed in the FMDV capsid in response to the introduction of intrinsically deleterious substitutions of the functionally critical residues at the interpentamer interfaces. The results have revealed a complex dynamics of very frequent and diverse second-site mutations, many of them concentrated in a few capsid hot spots. A working model to integrate the large amount of data obtained on the frequency, type, location and other characteristics of the second-site mutations detected is discussed.     

Big-benefit mutations in a bacteriophage inhibited with heat

High temperature inhibits the growth of the wild-type bacteriophage phiX174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0. 8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.