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Short Communication |
Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Correspondence
Mauricio G. Mateu
mgarcia{at}cbm.uam.es
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ABSTRACT |
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MAIN TEXT |
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; Domingo et al., 1999, 2001, 2004; Domingo, 2006). However, severe limitations to fully realize such a dramatic potential for adaptation probably occur, because mutations in vast regions of the sequence space of RNA virus genomes may lead to significant decreases in biological fitness, and can be difficult to exploit by these viruses. Among the substantial body of evidence that supports this view, a number of extensive structure–function studies indicate that very few single mutations are accepted in viral capsids, especially those of small, non-enveloped viruses, without a negative effect on one or several functions needed for infectivity (e.g. Mateu, 1995 and references therein; Verdaguer et al., 1995; Smith et al., 1996; Chow et al., 1997; Wu et al., 2000; Limn et al., 2000; Mateo et al., 2003; Reguera et al., 2004, 2005; Carreira & Mateu, 2006; unpublished results). A compromise between the many complex, sometimes contradictory, functions that a small virus particle must perform and its minimalist architecture appears to have been achieved by tuning the virion structure to extraordinary extents (Mateu, 1995; Mateo et al., 2003; Carrasco et al., 2006). The identification and understanding of specific structural and functional restrictions to variation may provide powerful weapons against many viral diseases.
Physical stability is one important adaptive property of a virus particle. A compromise between extracellular virion stability and intracellular lability to allow the release of the genome has been repeatedly invoked; however, not enough experimental data are available to decide whether a virus may generally accept a substantial increase in thermal stability without compromising its infectivity. A clear understanding of the relationship between thermostability and biological fitness is also relevant for the engineering of viral particles of increased thermostability, as a basis for more stable vaccines against important human or animal viral diseases, such as poliomyelitis or foot-and-mouth disease (FMD) (Terry et al., 1983; Doel, 1985; Milstien et al., 1997; Shiomi et al., 2004).
Early pioneering studies (Bachrach et al., 1960; Pringle, 1964) described the selection of thermostable variants from some populations of an RNA virus, Foot-and-mouth disease virus (FMDV), but the genotypic changes involved were not determined, and these variants were genetically unstable and/or showed a reduced fitness. Recently, thermostable poliovirus variants that carry a single mutation in their capsid and appear to be genetically stable have been selected (Shiomi et al., 2004). We have started to explore the possibility of obtaining FMDV of increased stability without compromising viral function. In the present study the detection and selection of thermostable variants from widely different FMDV populations was attempted. We expected that identification of specific mutations responsible for an increase in thermostability could provide new insights into the molecular basis of capsid stability and its relationship with fitness; it could also help our attempts for the rational engineering of a thermostable and genetically stable FMDV virion through the introduction of appropriate, additional interactions between the capsid subunits (Mateo et al., 2003; unpublished results).
For the selection from FMDV quasispecies of virus variants of increased resistance against heat inactivation, viral populations derived from a serotype C field variant (C-S8; Sobrino et al., 1983) were analysed first. Two uncloned populations of isolate C-S8, originally obtained from infected swine and subjected to a limited number of passages in cell culture, were used. In addition, two clonal populations of FMDV whose capsid corresponded to that of a biological clone of C-S8 (C-S8c1; Sobrino et al., 1983) were obtained by electroporation of cells with an infectious molecular clone (Zibert et al., 1990; Baranowski et al., 1998; Mateo et al., 2003). The four populations were heated for 1 h at different temperatures. Titration in standard plaque assays showed that heating at 60–70 °C generally left no detectable infectious virions (less than 50 p.f.u. ml–1), while heating between 50 and 59 °C led to high reductions in titre (about 2–5 orders of magnitude), without inactivating all of the virions. We reasoned that these latter conditions would lead to an important enrichment in those virus variants with a substantially higher thermal resistance, if present in the original populations. To evaluate this possibility, thermal inactivation kinetic assays of the unheated and heated populations were carried out at 42 °C; the inactivation rate constants were calculated by fitting the data to a single exponential decay as previously described (Mateo et al., 2003) (Fig. 1a; Table 1). No significant decrease in the thermal inactivation rate constant was found for any population, even in those instances in which only about 0.001 % of the virions remained infectious after heating. We considered the possibility that, in the heated population, thermostable variants could be present in a sizeable proportion, but that they were only slightly more stable than average, leading to no substantial increase in the rate constant of the population. To attempt the isolation of some of those variants, if they were present at all, 23 biological clones from one of the previously heated C-S8 populations were obtained by plaque purification. Their thermal resistance was first qualitatively assessed by incubating them for 90 min at 42 °C. The titres of the individual clones were reduced to 3–19 % of the original titre, compared to 12 % for the uncloned population (data not shown). The eight clones with the lowest reductions in infectivity were then tested in quantitative thermal inactivation assays (Table 2). No significant difference in the inactivation rate constant, relative to the unheated population, was obtained for any of them. These results suggest that, in the FMDV C-S8 populations analysed, the proportion of variants with increased thermostability was nil or very low.
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; Table 2); the differences detected may be statistically not significant. We then considered the possibility that heat-resistant variants were present, but that they had a reduced fitness and were outcompeted by less stable virions in the population when the selective pressure by heat was removed during amplification at 37 °C. Thus, we compared the inactivation rate of heated FMDV populations before and after amplification. The rate constants obtained were indistinguishable (0.009±0.001 vs 0.011±0.001 for passage 1, or 0.014±0.001 vs 0.012±0.002 for passage 3 of series A in Table 2).
A somewhat different procedure for selection and analysis (Bachrach et al., 1960) was also used on uncloned and cloned C-S8 populations. The original virus preparations (about 108 p.f.u. ml–1) were incubated at 59 °C and the remaining titres at different times were obtained, which yielded the corresponding inactivation kinetics. The survivors present after 1 h were amplified, and the progeny population was subjected to the same heat treatment. This cycle of inactivation and amplification was repeated up to four times. This procedure led to results equivalent to those obtained using the procedure first described, i.e. no significant increase in the thermostability of the population could be detected, even after four rounds of selection by heat (Fig. 1c).
The results described above indicate that no variants with substantially increased stability are found, even in very minor proportions, in the C-S8 populations analysed. To ascertain whether the same could apply to populations of other FMDV strains and serotypes, uncloned populations derived from field isolates A5Westerwald (serotype A, A5Ww) and O1Kaufbeuren (serotype O, O1K) were subjected to the same heat treatment (59 °C for 1 h) and analysed in thermal-inactivation assays. Again, no significant difference in the inactivation rate constant was found between the unheated and heated populations (Table 1).
In contrast to the results above, obtained with field FMDV populations that had been subjected to just a few passages in cell culture, early studies had shown that the heat-resistance of a laboratory population obtained from FMDV strain 119 (serotype A) after extensive passaging (76 times) in cell culture could be readily and dramatically increased (Bachrach et al., 1960). Thus, we considered the possibility that adaptation of FMDV to cell culture could alter the quasispecies equilibrium, leading to the presence of highly thermostable variants. We tested three laboratory populations that, like the virus population used in the early study by Bachrach et al. (1960), had been previously subjected to extensive replication in cultured cells. FMDV R100 was recovered from BHK-21 cells that had been persistently infected with C-S8 and passaged 100 times in culture (Díez et al., 1990). FMDV C-S8c1 p51 and C-S8c1 p114 are C-S8 populations that were obtained after 51 or 114 passages, respectively, of C-S8c1 in cytolytic infections of cultured cells (García-Arriaza et al., 2004). We expected that these two latter populations would have adapted to cell culture in a way similar to the specific population analysed by Bachrach and colleagues several decades ago. Interestingly, we observed that the inactivation rate constants for these three unheated laboratory populations were significantly higher than those obtained for natural FMDV populations (i.e. field isolates subjected to limited amplification in cultured cells) (Table 1). This indicates that some reduction in thermostability may occur during adaptation of FMDV to cell culture conditions, perhaps because of the absence, in those conditions, of the selective pressure exerted in the field by occasional heat extremes. However, comparison of the results of thermal inactivation assays with the unheated and heated populations showed no significant differences between them regarding the inactivation rate constant (Table 1), as observed for the populations subjected to few passages.
When the conditions involve infection of the same cell by more than one viral particle from a quasispecies, one can always have some proportion of particles in which the genotype and the phenotype do not match. Thus, in principle, some thermoresistant particles could encapsidate a non-mutated genome, and some thermosensitive particles could carry a genome with mutations potentially conferring thermoresistance. However, we believe that such mixing of genotype and phenotype would not affect the interpretation of the results described here. If mutant genomes leading to thermostable particles were minimally abundant, and had no preference for encapsidation in a thermosensitive or a thermoresistant capsid, thermostable particles should have been observed irrespective of some of those genomes being encapsidated in thermosensitive capsids. Regarding the procedures that we used and the inactivation temperature, they were very similar or nearly identical to those described by Bachrach et al. (1960) and Pringle (1964) in their early studies, and cannot possibly account for the different results obtained. In fact, two aspects of their work that may be highly significant are: (i) the thermoresistant population reverted to a thermosensitive phenotype if the selective pressure by heat was relaxed; this genetic instability indicates a reduced fitness of the thermoresistant variants. (ii) No thermoresistant variants could be obtained in one of the four populations analysed by Pringle (1964). Also, even though some thermostable poliovirus variants have been isolated, variants able to withstand moderate temperatures over long periods of time could not be obtained (Shiomi et al., 2004). The results of our study, with both uncloned and cloned populations of FMDV from three different serotypes recovered from cytolytic or persistent infections and subjected to either very few passages or extensive passaging in cell culture, do not rule out the possibility that thermostable variants could be present in some specific FMDV populations. However, they clearly indicate that the presence of thermostable virus variants, even in small proportions, is not a general feature of FMDV quasispecies.
A way to reconcile the presence of thermostable variants in some FMDV populations, but not in others, may be to contemplate two aspects: (i) the number, type and location of mutations needed to confer thermostability and (ii) the effect of those mutations on the biological fitness. Shiomi et al. (2004) showed that a single mutation in a poliovirus capsid may be enough to confer thermostability with no apparent effect on the genetic stability of the virus. However, the possibility that these variants could have a reduced fitness (for example, because of a slower uncoating) and would be outcompeted by other, less stable variants cannot be excluded. Different studies with small, non-enveloped viruses, including FMDV, have shown that a large majority of mutations in a virus capsid may have a negative effect on infectivity and/or fitness. This is particularly true regarding the interfaces between the pentameric subunits that form the FMDV capsid, where mutations that could increase the thermostability through the introduction of further intersubunit interactions would occur (Mateo et al., 2003). Accordingly, most mutations aimed at increasing the stability of FMDV through the rational introduction of intersubunit salt bridges or disulfide bonds proved lethal. Most of the non-lethal mutants showed no increase in stability; only one mutant appeared to lose infectivity at a slower rate, compared with the non-mutated control. This mutant is under study (unpublished results). Further, although different FMDVs may show a somewhat different thermostability (Doel & Baccarini, 1981; Nettleton et al., 1982; this study), all FMDV isolates are surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot-climate regions where FMD is prevalent. Thus, a likely scenario is that the vast majority of single mutations potentially able to increase the stability of the FMDV capsid may have a negative effect on fitness, are strongly selected against, and are heavily under-represented, or not represented, in FMDV quasispecies.
On the other hand, compensatory mutations able to counteract the deleterious effect of some mutations in the FMDV capsid, including those located at the interpentamer interfaces, have been detected (Mateo et al., 2003; unpublished results). Thus, there is the possibility that particular second-site mutations could compensate for the disadvantage tentatively associated with single mutations that increase thermostability, restoring fitness. However, the variant genomes carrying particular combinations of mutations would be probabilistically present in the mutant spectrum at much lower frequencies than single mutants. While some FMDV populations facing a strong selective pressure by heat could easily adapt, many others would face extinction, depending on the stochastic presence or absence of the right combination of mutations in one or a few variant genomes in the mutant spectrum. The observations above provide an example for a different, non-deterministic adaptability of different FMDV quasispecies in response to a single selective agent.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 August 2006;
accepted 6 November 2006.
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