All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the ... more With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C 3 Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C 3 Arg/85 infection. Infection of BHK-Rb cells with C 3 Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C 3 -Rb. The selected C 3 -Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A compari...
The selective pressures acting on viruses that replicate under enhanced mutation rates are largel... more 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...
The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human a... more The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report ...
Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a pos... more Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5'-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different su...
Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by... more Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by the fact of sharing a phylogenetic position, need not be associated with similar diseases, reflecting that the nature of the interactions with their host organisms may depend in a subtle manner on minimal genetic change of the virus. Picornaviruses have served to establish core concepts in the understanding of viruses as mutated collectivities and in establishing the relevance of quasispecies for viral pathogenesis. The adaptive potential of RNA viruses is also manifested in the response to selective agents administered to inhibit their replication. High mutation rates result in the almost-systematic selection of viral mutants resistant to antiviral inhibitors, either because resistant mutants are present in mutant spectra or because they are rapidly generated during viral replication. The participation of interfering genomes in virus extinction constitutes the basis of the lethal defection model of virus extinction by enhanced mutagenesis. The initial experiments to test the validity for RNA viruses of the error threshold concept consisted of documenting an adverse effect on viral infectivity as a result of increasing the mutation rate of poliovirus (PV) and vesicular stomatitis virus by chemical mutagens and base and nucleoside analogues added during viral RNA replication. Genetic modifications upon extensive passage of FMDV in BHK- 21 cells included genomes with internal in-frame deletions that were infectious by complementation in the absence of standard, wildtype genomes.
Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R... more 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)-RN...
The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resoluti... more The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 β-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.
All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the ... more With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C 3 Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C 3 Arg/85 infection. Infection of BHK-Rb cells with C 3 Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C 3 -Rb. The selected C 3 -Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A compari...
The selective pressures acting on viruses that replicate under enhanced mutation rates are largel... more 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...
The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human a... more The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report ...
Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a pos... more Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5'-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different su...
Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by... more Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by the fact of sharing a phylogenetic position, need not be associated with similar diseases, reflecting that the nature of the interactions with their host organisms may depend in a subtle manner on minimal genetic change of the virus. Picornaviruses have served to establish core concepts in the understanding of viruses as mutated collectivities and in establishing the relevance of quasispecies for viral pathogenesis. The adaptive potential of RNA viruses is also manifested in the response to selective agents administered to inhibit their replication. High mutation rates result in the almost-systematic selection of viral mutants resistant to antiviral inhibitors, either because resistant mutants are present in mutant spectra or because they are rapidly generated during viral replication. The participation of interfering genomes in virus extinction constitutes the basis of the lethal defection model of virus extinction by enhanced mutagenesis. The initial experiments to test the validity for RNA viruses of the error threshold concept consisted of documenting an adverse effect on viral infectivity as a result of increasing the mutation rate of poliovirus (PV) and vesicular stomatitis virus by chemical mutagens and base and nucleoside analogues added during viral RNA replication. Genetic modifications upon extensive passage of FMDV in BHK- 21 cells included genomes with internal in-frame deletions that were infectious by complementation in the absence of standard, wildtype genomes.
Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R... more 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)-RN...
The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resoluti... more The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 β-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.
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Papers by Núria Verdaguer