Ana Grande-Pérez

Mutagenesis-Induced, Large Fitness Variations with an Invariant Arenavirus Consensus Genomic Nucleotide Sequence

ABSTRACT Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.

High Variability and Rapid Evolution of a Nanovirus

ABSTRACT Nanoviruses are multipartite single-stranded DNA (ssDNA) plant viruses that cause important diseases of leguminous crops and banana. Little has been known about the variability and molecular evolution of these viruses. Here we report on the variability of faba bean necrotic stunt virus (FBNSV), a nanovirus from Ethiopia. We found mutation frequencies of 7.52 × 10−4 substitutions per nucleotide in a field population of the virus and 5.07 × 10−4 substitutions per nucleotide in a laboratory-maintained population derived thereof. Based on virus propagation for a period of more than 2 years, we determined a nucleotide substitution rate of 1.78 × 10−3 substitutions per nucleotide per year. This high molecular evolution rate places FBNSV, as a representative of the family Nanoviridae, among the fastest-evolving ssDNA viruses infecting plants or vertebrates.

Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs

ABSTRACT We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus Nanovirus and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of Agrobacterium tumefaciens into Vicia faba plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species, Aphis craccivora and Acyrthosiphon pisum. This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.

No evidence of selection for mutational robustness during lethal mutagenesis of lymphocytic choriomeningitis virus.

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication. Theoretical studies suggest that viruses can evolve towards regions of their fitness landscapes at which they display resistance to the deleterious effects of mutations. It has been suggested that such mutational robustness could jeopardize lethal mutagenesis. We have used the Arenavirus lymphocytic choriomeningitis virus (LCMV) to explore whether treatment with the mutagenic base analogue 5-fluorouracil (FU) selected for viral populations displaying resistance to lethal mutagenesis. Neither average LCMV populations with a history of FU mutagenesis, nor individual biological LCMV clones derived from those populations, displayed any resistance to lethal mutagenesis by FU. They were as sensitive to FU-induced extinction as LCMV populations and clones treated in parallel, but without a history of FU mutagenesis. Current evidence of the molecular events affecting quasispecies dynamics suggests that it is unlikely that a viral population can acquire mutational robustness under the increased mutation rates associated with mutagenic treatments. We consider mechanisms by which viruses could escape extinction by lethal mutagenesis, and provide evidence that mutational robustness is unlikely to be one of them.

Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.

Future prospects for the treatment of rapidly evolving viral pathogens: insights from evolutionary biology.

The evolutionary dynamics of viruses must be taken into consideration in designing preventive and therapeutic treatments. Here we review mechanisms by which viruses adapt in response to antiviral interventions. We propose combination therapy and multiepitopic vaccines as adequate to circumvent virus adaptability. An alternative design, termed lethal mutagenesis or virus entry into error catastrophe is presented. It exploits the high error rates inherent in RNA virus replication, to provoke virus extinction through excess of mutations.

Populations of Genomic RNAs Devoted to the Replication or Spread of a Bipartite Plant Virus Differ in Genetic Structure

ABSTRACT RNA viruses within a host exist as dynamic distributions of closely related mutants and recombinant genomes. These closely related mutants and recombinant genomes, which are subjected to a continuous process of genetic variation, competition, and selection, act as a unit of selection, termed viral quasispecies. Characterization of mutant spectra within hosts is essential for understanding viral evolution and pathogenesis resulting from the cooperative behavior of viral mutants within viral quasispecies. Furthermore, a detailed analysis of viral variability within hosts is needed to design control strategies, because viral quasispecies are reservoirs of viral variants that potentially can emerge with increased virulence or altered tropism. In this work, we report a detailed analysis of within-host viral populations in 13 field isolates of the bipartite Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). The intraisolate genetic structure was analyzed based on sequencing data for 755 molecular clones distributed in four genomic regions within the RNA-dependent RNA polymerase (RNA1) and Hsp70h, CP, and CPm (RNA2) open reading frames. Our results showed that populations of ToCV within a host plant have a heterogeneous and complex genetic structure similar to that described for animal and plant RNA viral quasispecies. Moreover, the structures of these populations clearly differ depending on the RNA segment considered, being more complex for RNA1 (encoding replication-associated proteins) than for RNA2 (encoding encapsidation-, systemic-movement-, and insect transmission-relevant proteins). These results support the idea that, in multicomponent RNA viruses, function can generate profound differences in the genetic structures of the different genomic segments.

Arenaviruses and Lethal Mutagenesis. Prospects for New Ribavirin-based Interventions

Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments.

Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

Abstract We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis.

A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules.