Coxsackieviruses and quasispecies theory: evolution of enteroviruses.

Enterovirus populations display quasispecies dynamics, characterized by high rates of mutation and recombination, followed by competition, selection, and random drift acting on heterogeneous mutant spectra. Direct experimental evidence indicates that high mutation rates and complex mutant spectra can serve for the adaptation of enteroviruses to complex environments. Studies with the RNA-dependent RNA polymerase of picornaviruses suggest that multiple enzyme sites may influence the template-copying fidelity (incorporation of incorrect vs correct nucleotide) during RNA replication. Mutation and recombination are an unavoidable consequence of the molecular mechanisms inherent to the process of viral genome replication and underlie the diversification of enterovirus genomes as they multiply in human and animal hosts. The diversity of disease manifestations associated with closely related enteroviruses is probably attributable to profound biological effects of some mutations that, because of their limited number, do not necessarily affect the phylogenetic position of the virus. The combination of highly dynamic mutant spectra with unpredictable alterations of biological behavior by minimal genetic change defies classical classification schemes. The result is the need to update the grouping of enteroviruses quite frequently into genetic and serological types and subtypes. The tolerance of enterovirus genomes to remain replication-competent despite multiple mutation and recombination events encourages the engineering of live-attenuated vaccines. Also, the application of quasispecies theory to an understanding of the limits of viral genomes to accept mutations, together with an increasingly deeper understanding of the mechanisms of mutagenesis by nucleoside analogs, has paved the way for the application of lethal mutagenesis as a new antiviral strategy.

Population bottlenecks in quasispecies dynamics.

The characteristics of natural populations result from different stochastic and deterministic processes that include reproduction with error, selection, and genetic drift. In particular, population fluctuations constitute a stochastic process that may play a very relevant role in shaping the structure of populations. For example, it is expected that small asexual populations will accumulate mutations at a higher rate than larger ones. As a consequence, in any population the fixation of mutations is accelerated when environmental conditions cause population bottlenecks. Bottlenecks have been relatively frequent in the history of life and it is generally accepted that they are highly relevant for speciation. Although population bottlenecks can occur in any species, their effects are more noticeable in organisms that form large and heterogeneous populations, such as RNA viral quasispecies. Bottlenecks can also positively select and isolate particles that still keep the ability to infect cells from a disorganized population created by crossing the error threshold.

Foot-and-mouth disease virus evolution: exploring pathways towards virus extinction.

Foot-and-mouth disease virus (FMDV) is genetically and phenotypically variable. As a typical RNA virus, FMDV follows a quasispecies dynamics, with the many biological implications of such a dynamics. Mutant spectra provide a reservoir of FMDV variants, and minority subpopulations may become dominant in response to environmental demands or as a result of statistical fluctuations in population size. Accumulation of mutations in the FMDV genome occurs upon subjecting viral populations to repeated bottleneck events and upon viral replication in the presence of mutagenic base or nucleoside analogs. During serial bottleneck passages, FMDV survive during extended rounds of replication maintaining low average relative fitness, despite linear accumulation of mutations in the consensus genomic sequence. The critical event is the occurrence of a low frequency of compensatory mutations. In contrast, upon replication in the presence of mutagens, the complexity of mutant spectra increases, apparently no compensatory mutations can express their fitness-enhancing potential, and the virus can cross an error threshold for maintenance of genetic information, resulting in virus extinction. Low relative fitness and low viral load favor FMDV extinction in cell culture. The comparison of the molecular basis of resistance to extinction upon bottleneck passage and extinction by enhanced mutagenesis is providing new insights in the understanding of quasispecies dynamics. Such a comparison is contributing to the development of new antiviral strategies based on the transition of viral replication into error catastrophe.

Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA.

The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15 degrees C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37 degrees C. This difference is responsible for the apparent inhibition of a21 dna transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enxyme is three times greater than that for the complex with the holoenzyme.

Effects and molecular action of ribosome-inactivating proteins on ribosomes from Streptomyces lividans.

The effects of 29 type 1 and 2 type 2 ribosome-inactivating proteins (RIPs) from plants on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from Streptomyces lividans were studied. Only dianthin 32, saporins R1 and R3, momordin I, trichokirin, Hura crepitans RIP 5 from latex, crotins 2 and 3, and PAPs C, R, and S, inhibited polyphenylalanine synthesis. Both the type 2 RIPs ricin and volkensin were ineffective on translation. The magnesium concentration affected the inhibition of translation to a considerable extent. Upon treatment with inhibitory RIPs, extraction of rRNA and further treatment with acid aniline, S. lividans ribosomes released an RNA fragment of about 130 nucleotides. The 5' terminal sequence of this rRNA fragment was 5'-GAGGACCGGGACGGACGAACCUCUGGUGUGCCAGUUGU-3', similar to the sequence obtained in Escherichia coli. This indicates that the most probable molecular action of these RIPs on S. lividans and E. coli ribosomes is the same: depurination of the rRNA at a site relevant to the translation mechanism and that has been highly conserved throughout evolution.

Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller's ratchet.

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular events underlying fitness recovery of a highly debilitated foot- and-mouth disease virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutations scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal polyadenylate was the first event in the process of fitness recovery. Further increases of fitness were associated with very few true reversions and with the accumulation of additional mutations affecting non-coding and coding regions. Remarkably, four biological subclones of the same debilitated FMDV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield the wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA virus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape.

Genetic lesions associated with Muller's ratchet in an RNA virus.

The molecular basis of Muller's ratchet has been investigated using the important animal pathogen foot-and-mouth disease virus (FMDV). Clones from two FMDV populations were subjected to serial plaque transfers (repeated bottleneck events) on host BHK-21 cells. Relative fitness losses were documented in 11 out of 19 clones tested. Small fitness gains were observed in three clones. One viral clone attained an extremely low plating efficiency, suggesting that accumulation of deleterious mutations had driven the virus near extinction. Nucleotide sequence analysis revealed unique genetic lesions in multiply transferred clones that had never been seen in FMDVs isolated in nature or subjected to massive infections in cell culture. In particular, a frequent internal polyadenylate extension has identified a mutational hot spot on the FMDV genome. Furthermore, amino acid residue substitutions in internal capsid sites which are severely restricted during FMDV evolution, amounted to half of capsid replacements in the transferred clones. In addition, a striking dominance of non-synonymous replacements fixed upon large population infections of FMDV was not observed upon serial plaque transfers. The nucleotide sequence of the entire genome of a severely debilitated clone suggests that very few mutations may be sufficient to drive FMDV near extinction. The results provide an account of the molecular basis of Muller's ratchet for an RNA virus, and insight into the types of genetic variants which populate the mutant spectra of FMDV quasispecies.

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

Population dynamics of RNA viruses: the essential contribution of mutant spectra.

Cells and their viral and cellular parasites are genetically highly diverse, and their genomes contain signs of past and present variation and mobility. The great adaptive potential of viruses, conferred on them by high mutation rates and quasispecies dynamics, demands new strategies for viral disease prevention and control. This necessitates a more detailed knowledge of viral population structure and dynamics. Here we review studies with the important animal pathogen Foot-and-mouth disease virus (FMDV) that document modulating effects of the mutant spectra that compose viral populations. As a consequence of interactions within mutant spectra, enhanced mutagenesis may lead to viral extinction, and this is currently investigated as a new antiviral strategy, termed virus entry into error catastrophe.

Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages.

The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29.

Fusidic acid-dependent ribosomal complexes protect Escherichia coli ribosomes from the action of the type 1 ribosome-inactivating protein crotin 2.

The type 1 ribosome-inactivating protein crotin 2 depurinated Escherichia coli ribosomes which, upon treatment of the isolated rRNA with acid aniline, released a fragment of around 240 nucleotides whose 5'-end sequence was 5'-GAGGACCGGAGUGGAC-3'. The formation of fusidic acid-dependent ribosomal complexes completely prevented release of the fragment. Ribosomes from crotin 2-pretreated fusidic acid complexes were insensitive to acid aniline. They released the RNA fragment only after a second treatment with crotin 2 and acid aniline whereas unprotected ribosomes released the fragment directly after acid aniline.

Molecular action of the type 1 ribosome-inactivating protein saporin 5 on Vicia sativa ribosomes.

The type 1 ribosome-inactivating protein (RIP) saporin 5 isolated from seeds of Saponaria officinalis L. strongly inhibited translation carried out by Vicia sativa L. purified ribosomes. The toxin multidepurinated V. sativa rRNA, which upon treatment with acid aniline releases several RNA fragments including an RNA fragment of approximately 370 nucleotides the 5'-end sequence of which was 5'-GAGGAACG-3'.

Genomic nucleotide sequence of a foot-and-mouth disease virus clone and its persistent derivatives. Implications for the evolution of viral quasispecies during a persistent infection.

The consensus nucleotide sequence of the entire genome of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) has been completed, and compared with that of two persistent derivatives R99 and R146, rescued after 99 and 146 passages of the carrier BHK-21 cells. Consensus sequences were determined directly from supernatants of persistently infected cells, without intervening cytolytic amplification of the viruses. These genomic sequences have also been compared with that of FMDV R100, a virus that was also rescued from persistently infected cells, but that was subjected to cytolytic amplification prior to sequencing. Mutation frequencies for R99 and R146 relative to C-S8c1 were in the range of 2.8x10(-3) to 7.7x10(-3) substitutions per nucleotide for the 5'-UTR and the L-, P1-, P2- and P3-coding regions. No mutations were fixed in the polymerase (3D)-coding region. Striking contrasts were noted regarding the distribution of mutation types along the persistent genomes, notably the complete absence of transversion mutations within the 5'-UTR, compared with 53% transversions in the L- and P1-coding regions. The sequencing results presented here, combined with previous sequences of FMDV C-S8c1 genomes at the onset of persistence, provide evidence of sequence fluctuations with a non-linear accumulation of mutations during prolonged persistence, a hallmark of quasispecies dynamics.

Modifications of the 5' untranslated region of foot-and-mouth disease virus after prolonged persistence in cell culture.

The nucleotide sequence of the 5'-untranslated region (5'UTR) of the genome of foot-and-mouth disease virus (FMDV) R100, rescued after 100 passages of persistently infected BHK-21 cells, has been compared with that of the parental FMDV C-S8c1. The nucleotide sequence divergence between the two viruses in heteropolymeric regions is 1%. The few mutations located at the 5'-most terminal region (S fragment) and at the internal ribosome entry site (IRES) do not appear to affect significantly the tight secondary structure predicted for these RNA segments. Comparison of the 5'UTR of C-S8c1 or R100 RNA with that of other FMDV serotypes and subtypes indicates the presence of block deletions (or insertions) which do not correlate with the serological classification of FMDV. Remarkably, FMDV R100, a virus highly attenuated for mice and cattle, contains a polyribocytidylate (poly C) tract of about 420 nucleotides, 145 residues longer than its parental, virulent FMDV C-S8c1. This long poly C of R100 RNA includes a few uridine residues interspersed at fairly regular intervals. This is the longest highly homopolymeric tract described in a viral genome and, to our knowledge, in any informational biomolecule.

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

Large deletions in the 5'-untranslated region of foot-and-mouth disease virus of serotype C.

Nucleotide sequences of the 5'-untranslated region (5'-UTR), at the 3'-side of the poly C tract, have been compared for 21 isolates of foot-and-mouth disease virus (FMDV) of serotype C from Europe, South America and The Philippines. A deletion of 43 nucleotides is present in the European isolates as compared with most American isolates. A larger deletion of 86 nucleotides is present in some viruses from South America and The Philippines. These deletions include the loss of one or two pseudoknot structures predicted in this region of the 5'-UTR. In addition, multiple point mutations have allowed the derivation of a phylogenetic tree which defines a grouping of isolates very similar to that derived from the capsid gene sequences of the same viruses. The study provides evidence that deletion (or addition) events must be very frequent during evolution of FMDV type C, since viruses which are phylogenetically very closely related (they belong to the same tree branch) may differ in the presence or absence of these deletions. Implications for FMDV evolution are discussed.

Population dynamics in the evolution of RNA viruses.

RNA virus quasispecies are subjected to processes of positive Darwinian selection, to a very active and continuous negative selection and to random genetic drift. The course of RNA virus evolution is often unpredictable, and recent results suggest that even highly conserved motifs, once regarded as essential for infectivity, may be rendered dispensable by singular evolutionary events. An immediate consequence of the quasispecies genetic organization of RNA viruses is a surprising ability to gain fitness once a minimal replication ability is established in a biological environment. The unique features of RNA genetics should not be underestimated since they are at the basis of the emergence of new viral diseases and of the current difficulties to control many diseases associated variable viruses.

[Variability and development of viral populations: assessment and implications]. / Variabilité et evolution des populations virales: bilan et implications.

RNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases.