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. Viruses that have RNA as genetic material include many important human, animal and plant pathogens. A new strategy against RNA viruses consists in using mutagenic nucleotides. The objective is to provoke an excessive number of mutations, to deteriorate the viral functions to the point that the virus can not survive. One of the mutagens used in research on lethal mutagenesis is ribavirin, extensively employed in clinical practice. Unfortunately, viral mutants that are resistant to ribavirin have been selected, thus facilitating escape from lethal mutagenesis. Here we describe a new mechanism by which foot-and-mouth disease virus (FMDV) can become resistant to ribavirin. Amino acid changes in the viral polymerase, selected by ribavirin, are able to modify the types of mutations produced in the presence of ribavirin. Biochemical data indicate that the alteration of the enzyme changes the pairing behavior of ribavirin, avoiding the production of an excess of some types of mutations, supporting the hypothesis that an unbalanced mutation repertoire is detrimental to the virus. Thus, this new mechanism of resistance to ribavirin is based not as much in limiting the number of mutations in the virus genetic material but in ensuring an equilibrium among different types of mutations that favors viral survival.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R) selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop β9-α11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 Å, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop β9-α11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.

The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. IMPORTANCE The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms.

Viruses are the most abundant biological entities on Earth, and yet, they have not received enough consideration in astrobiology. Viruses are also extraordinarily diverse, which is evident in the types of relationships they establish with their host, their strategies to store and replicate their genetic information and the enormous diversity of genes they contain. A viral population, especially if it corresponds to a virus with an RNA genome, can contain an array of sequence variants that greatly exceeds what is present in most cell populations. The fact that viruses always need cellular resources to multiply means that they establish very close interactions with cells. Although in the short term these relationships may appear to be negative for life, it is evident that they can be beneficial in the long term. Viruses are one of the most powerful selective pressures that exist, accelerating the evolution of defense mechanisms in the cellular world. They can also exchange genetic material with the host during the infection process, providing organisms with capacities that favor the colonization of new ecological niches or confer an advantage over competitors, just to cite a few examples. In addition, viruses have a relevant participation in the biogeochemical cycles of our planet, contributing to the recycling of the matter necessary for the maintenance of life. Therefore, although viruses have traditionally been excluded from the tree of life, the structure of this tree is largely the result of the interactions that have been established throughout the intertwined history of the cellular and the viral worlds. We do not know how other possible biospheres outside our planet could be, but it is clear that viruses play an essential role in the terrestrial one. Therefore, they must be taken into account both to improve our understanding of life that we know, and to understand other possible lives that might exist in the cosmos.

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication.