Monitoring sequence space as a test for the target of selection in viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.     

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.     

Beneficial effects of population bottlenecks in an RNA virus evolving at increased error rate

RNA viruses replicate their genomes with a very high error rate and constitute highly heterogeneous mutant distributions similar to the molecular quasispecies introduced to explain the evolution of prebiotic replicators. The genetic information included in a quasispecies can only be faithfully transmitted below a critical error rate. When the error threshold is crossed, the population structure disorganizes, and it is substituted by a randomly distributed mutant spectrum. For viral quasispecies, the increase in error rate is associated with a decrease in specific infectivity that can lead to the extinction of the population. In contrast, a strong resistance to extinction has been observed in populations subjected to bottleneck events despite the increased accumulation of mutations. In the present study, we show that the mutagenic nucleoside analogue 5-azacytidine (AZC) is a potent mutagen for bacteriophage Qβ. We have evaluated the effect of the increase in the replication error rate in populations of the bacteriophage Qβ evolving either in liquid medium or during development of clonal populations in semisolid agar. Populations evolving in liquid medium in the presence of AZC were extinguished, while during plaque development in the presence of AZC, the virus experienced a significant increase in the replicative ability. Individual viruses isolated from preextinction populations could withstand high error rates during a number of plaque-to-plaque transfers. The response to mutagenesis is interpreted in the light of features of plaque development versus infections by free-moving virus particles and the distance to a mutation-selection equilibrium. The results suggest that clonal bacteriophage populations away from equilibrium derive replicative benefits from increased mutation rates. This is relevant to the application of lethal mutagenesis in vivo, in the case of viruses that encounter changing environments and are transmitted from cell to cell under conditions of limited diffusion that mimic the events taking place during plaque development.     

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 μM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.     

Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10(6)-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.     

An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.     

Influence of mutagenesis and viral load on the sustained low-level replication of an RNA virus

Lethal mutagenesis is an antiviral strategy that aims to extinguish viruses as a consequence of enhanced mutation rates during virus replication. The molecular mechanisms that underlie virus extinction by mutagenic nucleoside analogues are not well understood. When mutagenic agents and antiviral inhibitors are administered sequentially or in combination, interconnected and often conflicting selective constraints can influence the fate of the virus either towards survival through selection of mutagen-escape or inhibitor-escape mutants or towards extinction. Here we report a study involving the mutagenesis of foot-and-mouth disease virus (FMDV) by the nucleoside analogue ribavirin (R) and the effect of R-mediated mutagenesis on the selection of FMDV mutants resistant to the inhibitor of RNA replication, guanidine hydrochloride (GU). The results show that under comparable (and low) viral load, an inhibitory activity by GU could not substitute for an equivalent inhibitory activity by R in driving FMDV to extinction. Both the prior history of R mutagenesis and the viral population size influenced the selection of GU-escape mutants. A sufficiently low viral load allowed continued viral replication without selection of inhibitor-escape mutants, irrespective of the history of mutagenesis. These observations imply that reductions of viral load as a result of a mutagenic treatment may provide an opportunity either for immune-mediated clearing of a virus or for an alternative antiviral intervention, even if extinction is not initially achieved.     

Fitness alteration of foot-and-mouth disease virus mutants: measurement of adaptability of viral quasispecies.

We document the rapid alteration of fitness of two foot-and-mouth disease virus (FMDV) mutants resistant to a neutralizing monoclonal antibody. Both mutants showed a selective disadvantage in BHK-21 cells when passaged in competition with their parental FMDV. Upon repeated replication of the mutants alone, they acquired a selective advantage over the parental FMDV and fixed additional genomic substitutions without reversion of the monoclonal antibody-resistant phenotype. Thus, variants that were previously kept at low frequency in the mutant spectrum of a viral quasispecies rapidly became the master sequence of a new genomic distribution and dominated the viral population.     

Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin

A mutant poliovirus (PV) encoding a change in its polymerase (3Dpol) at a site remote from the catalytic center (G64S) confers reduced sensitivity to ribavirin and forms a restricted quasispecies, because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouth disease virus (FMDV) mutant that encodes a change in the polymerase catalytic site (M296I) exhibits reduced sensitivity to ribavirin without restricting the viral quasispecies. In order to resolve this apparent paradox, we have established a minimal kinetic mechanism for nucleotide addition by wild-type (WT) FMDV 3Dpol that permits a direct comparison to PV 3Dpol as well as to FMDV 3Dpol derivatives. Rate constants for correct nucleotide addition were on par with those of PV 3Dpol, but apparent binding constants for correct nucleotides were higher than those observed for PV 3Dpol. The A-to-G transition frequency was calculated to be 1/20,000, which is quite similar to that calculated for PV 3Dpol. The analysis of FMDV M296I 3Dpol revealed a decrease in the calculated ribavirin incorporation frequency (1/8,000) relative to that (1/4,000) observed for the WT enzyme. Unexpectedly, the A-to-G transition frequency was higher (1/8,000) than that observed for the WT enzyme. Therefore, FMDV selected a polymerase that increases the frequency of the misincorporation of natural nucleotides while specifically decreasing the frequency of the incorporation of ribavirin nucleotide. These studies provide a mechanistic framework for understanding FMDV 3Dpol structure-function relationships, provide the first direct analysis of the fidelity of FMDV 3Dpol in vitro, identify the β9-α11 loop as a (in)fidelity determinant, and demonstrate that not all ribavirin-resistant mutants will encode high-fidelity polymerases.     

A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.     

Analysis of ribavirin mutagenicity in human hepatitis C virus infection

The addition of ribavirin to alpha interferon therapy significantly increases response rates for patients with chronic hepatitis C virus (HCV) infection, but ribavirin's antiviral mechanisms are unknown. Ribavirin has been suggested to have mutagenic potential in vitro that would lead to "error catastrophe," i.e., the generation of nonviable viral quasispecies due to the increment in the number of mutant genomes, which prevents the transmission of meaningful genetic information. We used extensive sequence-based analysis of two independent genomic regions in order to test in vivo the hypothesis that ribavirin administration accelerates the accumulation of mutations in the viral genome and that this acceleration occurs only when HCV replication is profoundly inhibited by coadministered alpha interferon. The rate of variation of the consensus sequence, the frequency of mutation, the error generation rate, and the between-sample genetic distance were measured for patients receiving ribavirin monotherapy, a combination of alpha interferon three times per week plus ribavirin, or a combination of alpha interferon daily plus ribavirin. Ribavirin monotherapy did not increase the rate of variation of the consensus sequence, the mutation frequency, the error generation rate, or the between-sample genetic distance. The accumulation of nucleotide substitutions did not accelerate, relative to the pretreatment period, during combination therapy with ribavirin and alpha interferon, even when viral replication was profoundly inhibited by alpha interferon. This study strongly undermines the hypothesis whereby ribavirin acts as an HCV mutagen in vivo.     

Influence of the mutant spectrum in viral evolution: focused selection of antigenic variants in a reconstructed viral quasispecies

RNA viruses replicate as complex mutant distributions termed viral quasispecies. Despite this, studies on virus populations subjected to positive selection have generally been performed and analyzed as if the viral population consisted of a defined genomic nucleotide sequence; such a simplification may not reflect accurately the molecular events underlying the selection process. In the present study, we have reconstructed a foot-and-mouth disease virus quasispecies with multiple, low-frequency, genetically distinguishable mutants that can escape neutralization by a monoclonal antibody. Some of the mutants included an amino acid substitution that affected an integrin recognition motif that overlaps with the antibody-binding site, whereas other mutants included an amino acid substitution that affected antibody binding but not integrin recognition. We have monitored consensus and clonal nucleotide sequences of populations passaged either in the absence or the presence of the neutralizing antibody. In both cases, the populations focused toward a specific mutant that was surrounded by a cloud of mutants with different antigenic and cell recognition specificities. In the absence of antibody selection, an antigenic variant that maintained integrin recognition became dominant, but the mutant cloud included as one of its minority components a variant with altered integrin recognition. Conversely, in the presence of antibody selection, a variant with altered integrin recognition motif became dominant, but it was surrounded by a cloud of antigenic variants that maintained integrin recognition. The results have documented that a mutant spectrum can exert an influence on a viral population subjected to a sustained positive selection pressure and have unveiled a mechanism of antigenic flexibility in viral populations, consisting in the presence in the selected quasispecies of mutants with different antigenic and cell recognition specificities.     

Insights into RNA virus mutant spectrum and lethal mutagenesis events: replicative interference and complementation by multiple point mutants

RNA virus behavior can be influenced by interactions among viral genomes and their expression products within the mutant spectra of replicating viral quasispecies. Here, we report the extent of interference of specific capsid and polymerase mutants of foot-and-mouth disease virus (FMDV) on replication of wild-type (wt) RNA. The capsid and polymerase mutants chosen for this analysis had been characterized biochemically and structurally. Upon co-electroporation of BHK-21 cells with wt RNA and a tenfold excess of mutant RNA, some mutants displayed strong interference (<10% of progeny production by wt RNA alone), while other mutants did not show detectable interference. The capacity to interfere required an excess of mutant RNA and was associated with intracellular replication, irrespective of the formation of infectious particles by the mutant virus. The extent of interference did not correlate with the known types and number of interactions involving the amino acid residue affected in each mutant. Synergistic interference was observed upon co-electroporation of wt RNA and mixtures of capsid and polymerase mutants. Interference was specific, in that the mutants did not affect expression of encephalomyocarditis virus RNA, and that a two nucleotide insertion mutant of FMDV expressing a truncated polymerase did not exert any detectable interference. The results support the lethal defection model for viral extinction by enhanced mutagenesis, and provide further evidence that the population behavior of highly variable viruses can be influenced strongly by the composition of the quasispecies mutant spectrum as a whole.     

Mutagenesis versus inhibition in the efficiency of extinction of foot-and-mouth disease virus

RNA viruses replicate near the error threshold for maintenance of genetic information, and an increase in mutation frequency during replication may drive RNA viruses to extinction in a process termed lethal mutagenesis. This report addresses the efficiency of extinction (versus escape from extinction) of foot-and-mouth disease virus (FMDV) by combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H). Selection of G- or H-resistant, extinction-escape mutants occurred with low-fitness virus only in the absence of FU and with high-fitness virus with some mutagen-inhibitor combinations tested. The combination of FU, G, and H prevented selection of extinction-escape mutants in all cases examined, and extinction of high-fitness FMDV could not be achieved by equivalent inhibitory activity exerted by the nonmutagenic agents. The G-resistant phenotype was mapped in nonstructural protein 2C by introducing the relevant mutations in infectious cDNA clones. Decreases in FMDV infectivity were accompanied by modest decreases in the intracellular and extracellular levels of FMDV RNA, maximal intracellular concentrations of FU triphosphate, and a decrease in the intracellular concentrations of UTP. In addition to indicating a key participation of mutagenesis in virus extinction, the results suggest that picornaviruses provide versatile experimental systems to approach the problem of extinction failure associated with inhibitor-escape mutants during treatments based on enhanced mutagenesis.     

Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe

The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease virus (FMDV). Passage of FMDV in the presence of increasing concentrations of R resulted in the selection of FMDV with the amino acid substitution M296I in the viral polymerase (3D). Measurements of progeny production and viral fitness with chimeric viruses in the presence and absence of R documented that the 3D substitution M296I conferred on FMDV a selective replicative advantage in the presence of R but not in the absence of R. In polymerization assays, a purified mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate as a substrate in the place of GTP and ATP, compared with the wild-type enzyme. The results suggest that M296I has been selected because it attenuates the mutagenic activity of R with FMDV. Replacement M296I is located within a highly conserved stretch in picornaviral polymerases which includes residues that interact with the template-primer complex and probably also with the incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given that a 3D substitution, distant from M296I, was associated with resistance to R in poliovirus, the results indicate that picornaviral polymerases include different domains that can alter the interaction of the enzyme with mutagenic nucleoside analogues. Implications for lethal mutagenesis are discussed.     

Potential Benefits of Sequential Inhibitor-Mutagen Treatments of RNA Virus Infections

Lethal mutagenesis is an antiviral strategy consisting of virus extinction associated with enhanced mutagenesis. The use of non-mutagenic antiviral inhibitors has faced the problem of selection of inhibitor-resistant virus mutants. Quasispecies dynamics predicts, and clinical results have confirmed, that combination therapy has an advantage over monotherapy to delay or prevent selection of inhibitor-escape mutants. Using ribavirin-mediated mutagenesis of foot-and-mouth disease virus (FMDV), here we show that, contrary to expectations, sequential administration of the antiviral inhibitor guanidine (GU) first, followed by ribavirin, is more effective than combination therapy with the two drugs, or than either drug used individually. Coelectroporation experiments suggest that limited inhibition of replication of interfering mutants by GU may contribute to the benefits of the sequential treatment. In lethal mutagenesis, a sequential inhibitor-mutagen treatment can be more effective than the corresponding combination treatment to drive a virus towards extinction. Such an advantage is also supported by a theoretical model for the evolution of a viral population under the action of increased mutagenesis in the presence of an inhibitor of viral replication. The model suggests that benefits of the sequential treatment are due to the involvement of a mutagenic agent, and to competition for susceptible cells exerted by the mutant spectrum. The results may impact lethal mutagenesis-based protocols, as well as current antiviral therapies involving ribavirin.     

Topology of evolving,mutagenized viral populations: quasispecies expansion,compression, and operation of negative selection

Background  

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues.     

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection

Background

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.

Results

The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.

Conclusions

(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.     

Preextinction viral RNA can interfere with infectivity

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.