Biomedical subjects

Cristina Escarmís

Publications and source records attributed to Cristina Escarmís.

At least 19 recordsLinked to original sources

PubMed2006

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes.

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.

Animals↗

PubMed2006

The structure of a protein primer-polymerase complex in the initiation of genome replication.

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

Crystallography, X-Ray↗

PubMed2005

A comparison of viral RNA-dependent RNA polymerases.

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg, to initiate RNA replication. The recent explosion of structural information on picornaviral 3D polymerases has provided insights into the initiation of RNA synthesis and chain elongation. Comparing these data with results from previous structural analyses of viral RNA-dependent RNA polymerases that catalyze de novo RNA synthesis sheds light on the different strategies that these viruses use to initiate replication.

Picornaviridae↗

PubMed2005

Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding.

A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)15 and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D). DMD and the G118 mutants showed impaired binding to template-primer RNA whereas the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that maintained only substitutions V239M and G373D. Consistently, when infectious transcripts encoded 3D with either G118D, G118A or D338A, viruses with reversions to the wild-type sequence were isolated. The implication of G118 in template-primer binding is supported by the location of this residue in the template-binding groove of the FMDV polymerase. In addition to identifying an amino acid residue that is critical for the binding of polymerase to RNA, the results document the presence of defective genomes in the transition of virus to error catastrophe.

Binding Sites↗

PubMed2005

A segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness.

Serial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome.

Animals↗

PubMed2005

High mutation rates, bottlenecks, and robustness of RNA viral quasispecies.

Population bottlenecks are stochastic events that strongly condition the structure and evolution of natural populations. Their effects are readily observable in highly heterogeneous populations, such as RNA viruses, since bottlenecks cause a fast accumulation of mutations. Considering that most mutations are deleterious, it was predicted that the frequent application of bottlenecks would yield a population unable to replicate. However, in vitro as well as in vivo systems evolving through bottlenecks present a remarkable resistance to extinction. This observation reveals the robustness of RNA viruses and points to the existence of internal mechanisms which must confer a high degree of adaptability to fast mutating populations. In this contribution, we review experimental observations regarding the survival of RNA viruses, both in laboratory experiments and in natural populations. By means of a simple theoretical model of evolution which incorporates strong reductions of the population size, we explore the relationship between the number of replication rounds that a single founder particle undergoes before the next bottleneck is applied, and the mutation rate in a particular environment. Our numerical results reveal that the mutation rate has evolved in a concerted way with the degree of optimization achieved by the population originated from the founder particle. We hypothesize that this mechanism generates a mutation-selection equilibrium in natural populations that maximizes adaptability while maintaining their structure.

Base Sequence↗

PubMed2005

Invariant aphthovirus consensus nucleotide sequence in the transition to error catastrophe.

RNA viruses replicate as complex distributions of non-identical but closely related variant genomes termed viral quasispecies. When the error rate during genome replication exceeds a threshold value, the genetic information cannot be maintained and the system enters error catastrophe. This violation of the error threshold results in virus extinction and it is currently being investigated as a new antiviral strategy, based on antiviral activity of some mutagenic agents. Previous studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have shown that FMDV entry into error catastrophe is associated with an increase of complexity (mutation frequency and Shannon entropy) of the mutant spectrum of the quasispecies and that mutated, pre-extinction RNA interferes with the infectivity of standard RNA. Here, we report that despite the increase of complexity, the genomic consensus nucleotide sequence of pre-extinction FMDV RNA remains invariant, and that the fitness of pre-extinction FMDV is at least six-fold lower than the fitness of the parental viral clone, prior to mutagenic treatments. Thus, a low fitness genome ensemble can suppress replication of high fitness virus. Furthermore, the results show that profound genetic modifications associated with fitness decrease of a virus population can take place without any manifestation in the consensus genomic sequence. Thus, increase in mutant spectrum complexity and invariance of the consensus sequence characterizes FMDV extinction through error catastrophe.

Animals↗

PubMed2005

Quasispecies dynamics and RNA virus extinction.

The extinction of foot-and-mouth disease virus (FMDV) is strongly influenced by mutation rates, types of mutations, relative viral fitness and virus population regimens during infection. Here we review experimental results and theoretical models that describe a contrast between the effective extinction of FMDV subjected to increased mutagenesis, and the remarkable resistance to extinction of the same and related FMDV clones subjected to serial bottleneck events. The results suggest procedures to master key parameters to develop effective antiviral strategies based on virus entry into error catastrophe.

Animals↗

PubMed2004

Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA.

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.

Amino Acid Motifs↗

PubMed2004

Fitness increase of memory genomes in a viral quasispecies.

Viral quasispecies may contain a subset of minority genomes that reflect those genomic sequences that were dominant at an early phase of quasispecies evolution. Such minority genomes are referred to as memory in viral quasispecies. A memory marker previously characterized in foot-and-mouth disease virus (FMDV) is an internal oligoadenylate tract of variable length that became dominant upon serial plaque-to-plaque transfers of FMDV clones. During large population passages, genomes with internal oligoadenylate were outcompeted by wild-type revertants but remained in the mutant spectra as memory genomes. Here, we report a quantification of relative fitness of several FMDV clones, harboring internal oligoadenylate tracts of different length, and that were retrieved at early or late times (passage number) after implementation of memory. The results show that for any given length range of the oligoadenylate, maintenance in memory resulted in an increase in relative fitness, comparable to the increase undergone by the entire population. The fitness increase is in agreement with the Red Queen hypothesis, and implies a replicative memory mechanism. Thus, permanence of memory genomes may be a source of high fitness variants despite their initial low fitness, and despite having remained hidden in mutant spectra. This reinforces the interest of diagnosing minority genomes during chronic human and animal viral infections.

Animals↗

PubMed2004

Evolutionary transition toward defective RNAs that are infectious by complementation.

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.

Animals↗

PubMed2003

Resistance of virus to extinction on bottleneck passages: study of a decaying and fluctuating pattern of fitness loss.

RNA viruses display high mutation rates and their populations replicate as dynamic and complex mutant distributions, termed viral quasispecies. Repeated genetic bottlenecks, which experimentally are carried out through serial plaque-to-plaque transfers of the virus, lead to fitness decrease (measured here as diminished capacity to produce infectious progeny). Here we report an analysis of fitness evolution of several low fitness foot-and-mouth disease virus clones subjected to 50 plaque-to-plaque transfers. Unexpectedly, fitness decrease, rather than being continuous and monotonic, displayed a fluctuating pattern, which was influenced by both the virus and the state of the host cell as shown by effects of recent cell passage history. The amplitude of the fluctuations increased as fitness decreased, resulting in a remarkable resistance of virus to extinction. Whereas the frequency distribution of fitness in control (independent) experiments follows a log-normal distribution, the probability of fitness values in the evolving bottlenecked populations fitted a Weibull distribution. We suggest that multiple functions of viral genomic RNA and its encoded proteins, subjected to high mutational pressure, interact with cellular components to produce this nontrivial, fluctuating pattern.

Animals↗

PubMed2003

Fitness distributions in exponentially growing asexual populations.

We explore a mean-field model for the evolution of exponentially growing populations of mutating replicators. Motivated by recent in vitro experiments devised to analyze phenotypic properties of bacterial and viral populations subjected to serial population transfers, we allow our in silico individuals to undergo unrestricted growth before applying bottleneck events. Different dynamical regimes of our model can be mapped to different experimental situations. Numerical and analytical results for fitness distributions calculated at the statistically stationary states of the dynamics compare favorably with available experimental data. Our model and results provide a common framework to better understand populations evolving under different selection pressures.

Biological Evolution↗

PubMed2003

Evolution of foot-and-mouth disease virus.

Foot-and-mouth disease virus evolution is strongly influenced by high mutation rates and a quasispecies dynamics. Mutant swarms are subjected to positive selection, negative selection and random drift of genomes. Adaptation is the result of selective amplification of subpopulations of genomes. The extent of adaptation to a given environment is quantified by a relative fitness value. Fitness values depend on the virus and its physical and biological environment. Generally, infections involving large population passages result in fitness gain and population bottlenecks lead to fitness loss. Very different types of mutations tend to accumulate in the foot-and-mouth disease virus (FMDV) genome depending on the virus population size during replication. Quasispecies dynamics predict higher probability of success of antiviral strategies based on multivalent vaccines and combination therapy, and this has been supported by clinical and veterinary practice. Quasispecies suggest also new antiviral strategies based on virus entry into error catastrophe, and such procedures are under investigation. Studies with FMDV have contributed to the understanding of quasispecies dynamics and some of its biological implications.

Amino Acid Sequence↗

PubMed2002

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.

Animals↗

PubMed2002

Duration and fitness dependence of quasispecies memory.

The duration and fitness dependence of memory in viral quasispecies evolving in cell culture have been investigated using two genetic markers of foot-and-mouth disease virus (FMDV). In lineages of antigenic variant FMDV RED, which reverted to FMDV RGD, memory FMDV RED genomes were detected after 50 infectious cycles, and memory level was fitness dependent. In growth-competition experiments between a reference FMDV RGD and two different FMDV RED populations, a 7.6-fold higher fitness of the initial FMDV RED population resulted in 30 to 100-fold higher memory level. In lineages of low-fitness clones containing an elongated internal polyadenylate tract, revertants lacking excess adenylate residues became dominant by passage 20. However, genomes including a larger number of adenylate residues were detected as memory genomes after at least 150 infectious cycles. Thus, quasispecies memory can be durable and is fitness dependent, as predicted from the growth competition of two mutant forms of a genome. An understanding of factors influencing quasispecies memory levels and duration may have implications for the extended diagnosis of viruses based on the quantification of minority genomes.

Animals↗

PubMed2002

Foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.

Animals↗

PubMed2002

Modeling viral genome fitness evolution associated with serial bottleneck events: evidence of stationary states of fitness.

Evolution of fitness values upon replication of viral populations is strongly influenced by the size of the virus population that participates in the infections. While large population passages often result in fitness gains, repeated plaque-to-plaque transfers result in average fitness losses. Here we develop a numerical model that describes fitness evolution of viral clones subjected to serial bottleneck events. The model predicts a biphasic evolution of fitness values in that a period of exponential decrease is followed by a stationary state in which fitness values display large fluctuations around an average constant value. This biphasic evolution is in agreement with experimental results of serial plaque-to-plaque transfers carried out with foot-and-mouth disease virus (FMDV) in cell culture. The existence of a stationary phase of fitness values has been further documented by serial plaque-to-plaque transfers of FMDV clones that had reached very low relative fitness values. The statistical properties of the stationary state depend on several parameters of the model, such as the probability of advantageous versus deleterious mutations, initial fitness, and the number of replication rounds. In particular, the size of the bottleneck is critical for determining the trend of fitness evolution.

Animals↗