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.     

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.     

Lethal mutagenesis of foot-and-mouth disease virus involves shifts in sequence space

Lethal mutagenesis or virus transition into error catastrophe is an antiviral strategy that aims at extinguishing a virus by increasing the viral mutation rates during replication. The molecular basis of lethal mutagenesis is largely unknown. Previous studies showed that a critical substitution in the foot-and-mouth disease virus (FMDV) polymerase was sufficient to allow the virus to escape extinction through modulation of the transition types induced by the purine nucleoside analogue ribavirin. This substitution was not detected in mutant spectra of FMDV populations that had not replicated in the presence of ribavirin, using standard molecular cloning and nucleotide sequencing. Here we selectively amplify and analyze low-melting-temperature cDNA duplexes copied from FMDV genome populations passaged in the absence or presence of ribovirin Hypermutated genomes with high frequencies of A and U were present in both ribavirin -treated and untreated populations, but the major effect of ribavirin mutagenesis was to accelerate the occurrence of AU-rich mutant clouds during the early replication rounds of the virus. The standard FMDV quasispecies passaged in the absence of ribavirin included the salient transition-modulating, ribavirin resistance mutation, whose frequency increased in populations treated with ribavirin. Thus, even nonmutagenized FMDV quasispecies include a deep, mutationally biased portion of sequence space, in support of the view that the virus replicates close to the error threshold for maintenance of genetic information.     

Efficient virus extinction by combinations of a mutagen and antiviral inhibitors

The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 10(6)-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.     

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.     

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-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.     

7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.     

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.     

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.     

Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.     

Mutagenesis-induced, large fitness variations with an invariant arenavirus consensus genomic nucleotide sequence

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.     

Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches

Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3' end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.