Passage of foot-and-mouth disease virus (FMDV) in cell culture resulted in the generation of defective RNAs that were infectious by complementation. Deletions (of nucleotides 417, 999, and 1017) mapped in the L proteinase and capsid protein-coding regions. Cell killing followed two-hit kinetics, defective genomes were encapsidated into separate viral particles, and individual viral plaques contained defective genomes with no detectable standard FMDV RNA. Infection in the absence of standard FMDV RNA was achieved by cotransfection of susceptible cells with transcripts produced in vitro from plasmids encoding the defective genomes. These results document the first step of an evolutionary transition toward genome segmentation of an unsegmented RNA virus and provide an experimental system to compare rates of RNA progeny production and resistance to enhanced mutagenesis of a segmented genome versus its unsegmented counterpart.Mutation, recombination, and ensuing phenotypic modifications in response to selective pressures can be readily observed and quantitated with RNA viruses both in cell culture and in vivo within modest time periods. This has rendered viruses suitable experimental systems for studies of basic Darwinian processes of genetic modification, competition, and selection or random drift as agents of genome diversification and biological adaptation (reviewed in references 1, 8, and 12). However, some major evolutionary transitions such as RNA genome segmentation have never been observed in the laboratory, yet they have probably occurred, given the hundreds of animal, plant, bacterial, and fungal viruses with segmented RNA genomes that have been described, including picornavirus-like plant RNA viruses (43). We have carried out extensive studies on the population dynamics of the important animal picornavirus foot-and-mouth disease virus (FMDV), including that of phenotypic evolution upon long-term serial cytopathic infections of BHK-21 cells (2). Unexpectedly, after more than 200 serial infections of viral population C-S8c1 of FMDV (a clone of serotype C [reviewed in reference 33]), defective genomes became dominant in the population. Defective interfering (DI) particles are frequently produced upon passage of RNA viruses at a high multiplicity of infection (MOI). DI particles are deletion mutants that require the presence of helper virus for replication and can interfere with the replication of the standard infectious virus (14-16, 29, 32, 35). The results described here show that defective genomes which individually could not cause cytopathology could nevertheless complement each other to produce progeny and kill cells in the absence of standard virus. The results provide, to our knowledge, the first description of defective RNA genomes occurring during viral replication that can be stably maintained by complementation upon passage at a high MOI in the absence of standard infectious virus. Therefore, the results provide experimental evidence of the initial step of an evolutionary transition towards genome ...

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Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the 'relevant' stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the 'relevant' and 'nonrelevant' terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented. STABILISATION OF PROTEINS WITH A TWO-STATE EQUILIBRIUMThe conformational stability of a protein is the free energy difference of the native/denatured equilibrium.Where no intermediates complicate this equilibrium, the stability can be easily measured from thermal or chemical denaturation [1]. Fuelled by the interest in protein stability, small model proteins have been used to investigate both the principles and practical strategies of protein stabilisation [2,3]. Although some basic questions regarding what stabilises proteins may not be settled [4,5] there are now various ways to attempt, with a reasonable probability of success, the increase of protein stability from a judicious analysis of protein structure [6]. The question is, Are these strategies similarly useful to stabilise proteins with more complex equilibria? THE 'RELEVANT' CONFORMATIONAL STABILITY OF PROTEINS WITH COMPLEX EQUILIBRIA: THE THREE-STATE CASELet us consider a simple three-state folding equilibrium with a single intermediate conformation appearing at mildly denaturing conditions (e.g., moderate urea concentration or moderately high temperatures) before the full denaturation takes place.For proteins of this kind, the conformational stability is made of two terms that, respectively, represent the stability of the native conformation relative to the intermediate (∆G NI ) and that of the intermediate relative to the denatured state (∆G ID ). However, provided the intermediate is no longer active (and the odds are it will not be), the 'relevant' conformational stability is given by just the first term, ∆G NI . The point is that the energetics of NI equilibria are so poorly understood that it is not clear whether the strategies found to stabilise two-state proteins will work well for proteins with equilibrium intermediates. If the energetics of protein intermediates are close to those of denatured states, there is no problem; but if, energetically, intermediates are not very different from the native state ...