Biological Effect of Muller's Ratchet: Distant Capsid Site Can Affect Picornavirus Protein Processing

Abstract

Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.As a consequence of the quasispecies population structure, when a virus is subjected to an extreme bottleneck regime, such as successive plaque-to-plaque transfers, it accumulates deleterious mutations that result in fitness loss (reviewed in references 15, 21, and 33). These observations constitute experimental support for the Muller's ratchet hypothesis, which states that asexual populations of organisms tend to acquire deleterious mutations unless compensatory mechanisms (such as sex or recombination) intervene (39,41). Several lines of evidence indicate that population bottlenecks are abundant in the life cycle of viruses, both during host-to-host transmission and during intrahost replication (2, 8, 10, 22, 26, 32, 42, 45-48, 52, 53). Most studies have addressed the effects of bottlenecks on the reduction of intramutant spectrum diversity in relation to virus survival and persistence, effects on fitness, or as promoters of stochastic processes and drift in viral evolution. Yet the possible biological effects of specific mutations fixed as a result of bottleneck events remain largely unexplored.Experimental designs consisting of many successive plaqueto-plaque transfers, without intervening large-population passages, are ideal for obtaining viral clones that are debilitated by the occurrence of mutations because negative selection is highly attenuated (15,21,33). The deleterious nature of some mutations that become fixed in viral genomes subjected to repeated bottlenecks can be inferred from their position in the viral genome and then confirmed experimentally. For example, an internal tract of four oligoadenylate residues that precede the second functional AUG initiation codon of foot-and-mouth disease virus (FMDV) was invariant among natural isolates of the virus or among populations subject...

Lethal Mutagenesis of Foot-and-Mouth Disease Virus Involves Shifts in Sequence Space

“…The present comparison has involved FMDV populations passaged in BHK-21 cells in the absence of ribavirin, that are capable of maintaining the viral genetic information for hundreds of passages (23,28,51), and populations passaged in the presence of ribavirin, which are doomed to extinction in a few passages (1,56). Contrary to expectations, replication in the presence of ribavirin did not result in an overall broader mutant repertoire than that in the absence of ribavirin (compare Fig.…”

Virus population dynamics examined with experimental model systems

“…(Decreases of specific infectivity are mentioned in Section 4.3 of Chapter 4 as one of the consequences of suboptimal codon usage, and are revisited in Chapter 9 as a diagnostic parameter of lethal mutagenesis). A proof that pursuing molecular analysis of clones subjected to many bottleneck transfers may provide new information was documented by the discovery of an amino acid substitution in capsid protein VP1 that produced virus thermosensitivity, and exerted an effect at a distance in the processing of the FMDV polyprotein (Escarmís et al, 2009), unveiling a new feature of picornavirus protein processing (Martínez-Salas and Ryan, 2010). Unusual FMDVs isolated after plaque transfers are summarized in Box 6.2.…”

Virus Population Dynamics Examined with Experimental Model Systems

“…(Decreases of specific infectivity is mentioned in Section 4.3 of Chapter 4 as one of the consequences of suboptimal codon usage, and are revisited in Chapter 9 as a diagnostic parameter of lethal mutagenesis.) A proof that pursuing molecular analysis of clones subjected to many bottleneck transfers may provide new information was documented by the discovery of an amino acid substitution in capsid protein VP1 that produced virus thermosensitivity, and exerted an effect at a distance in the processing of the FMDV polyprotein (Escarmís et al, 2009), unveiling a new feature of picornavirus protein processing (Martínez-Salas and Ryan, 2010). Unusual FMDVs isolated after plaque transfers are summarized in Box 6.2.…”