Browsing by Author "Verdaguer, Nuria"

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.

RNA-dependent RNA polymerases (RdRPs) play central roles in both transcription and viral genome replication. In picornaviruses, these functions are catalyzed by the virally encoded RdRP, termed 3D. Polymerase 3D also catalyzes the covalent linkage of UMP to a tyrosine on the small protein VPg. Uridylylated VPg then serves as a protein primer for the initiation of RNA synthesis. Seven different crystal structures of foot-and-mouth disease virus (FMDV) 3D catalytic complexes have enhanced our understanding of template and primer recognition, VPg uridylylation, and rNTP binding and catalysis. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds.

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 a8 of the fingers domain and helix a13 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.

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 9- 11 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 9- 11 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.