Foot-and-mouth disease virus low-fidelity polymerase mutants are attenuated

Abstract

Previous studies have shown that RNA viruses can be attenuated by either increased or decreased viral polymerase replication fidelity. Although foot-and-mouth disease virus (FMDV) high-fidelity RNA-dependent RNA polymerase (RdRp) variants with an attenuated phenotype have been isolated using mutagens, no FMDV mutant with a low-fidelity polymerase has been documented to date. Here, we describe the generation of several FMDV RdRp mutants using site-directed mutagenesis via a reverse genetic system. Mutation frequency assays confirmed that five rescued FMDV RdRp mutant populations had lower replication fidelity than the wild-type virus population, which allowed us to assess the effects of the change in replication fidelity on the virus phenotype. These low-fidelity FMDV RdRp mutants showed increased sensitivity to ribavirin or 5-fluorouracil (5-FU) treatment without a loss of growth capacity in cell cultures. In addition, decreased fitness and attenuated virulence were observed for the RdRp mutants with lower fidelity. Importantly, based on a quantitative analysis for fidelity and virulence, we concluded that lower replication fidelity is associated with a more attenuated virus phenotype. These results further contribute to our understanding of the replication fidelity of polymerases of RNA viruses and its relationship to virulence attenuation.

Coronavirus Host Range Expansion and Middle East Respiratory Syndrome Coronavirus Emergence: Biochemical Mechanisms and Evolutionary Perspectives

“…Mutator phenotypes have been isolated from natural populations of HIV-1 (120) and influenza A virus (121). Additionally, targeted mutations have resulted in mutator strains for poliovirus (114,122), coxsackievirus B3 (123), chikungunya virus (124), foot and mouth disease virus (125), and coronaviruses (10)(11)(12). However, coronaviruses are the only RNA viruses capable of proofreading their genomes, in a process mediated by the nsp10-nsp14 complex (8); nsp14 contains a conserved DEDD superfamily motif that facilitates exoribonuclease activity (9).…”

Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis

“…D5N at 3D was selected upon passage of FMDV Asia 1 in BHK-21c2 cells in the presence of ribavirin, and the mutant virus exhibited high polymerase fidelity according to the mutation frequency of the population (55). Modeled on the structural elements of the polymerase of FMDV of serotype C (36), at least 18 different amino acid replacements at 9 separate structural elements in 3D of FMDV serotype C, A, or Asia 1 have been involved in significant alterations of copying fidelity: D5N, K18E, and K20E (at the N-terminal region of the enzyme, with K20 within ␤1); A38V (immediately following ␤2); P44S (loop ␤2-␣2); G62S (loop ␣2-␣3); R84H (within ␣3); D165E (loop ␤5-␤6); P169S (within ␤6), K172R, and V173I (within ␣6) (the three substitutions in motif F); M194I (within ␣7); M296V and M296I (loop ␤9-␣11); W237F, W237I, and W237L (within ␤8, in motif A); G361S (loop ␣12-␤12) (29)(30)(31)(32)(33)(55)(56)(57)(58) (Fig. 6).…”

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 7 - Pathogenesis and Molecular Biology

“…Alongside host determinants of the virulence of FMDV infections (see (Arzt et al., ), under Pathogenesis), molecular characteristics of the virus can have profound impacts on the virulence of the strain. Attenuation has been linked with both low‐ and high‐fidelity variants of the viral RNA‐dependent RNA polymerase, the latter of which may act by limiting the potential for adaptive quasispecies emergence (Xie et al., ; Zeng et al., ). In addition, an elegant series of in vitro experiments tracked the differential virulence of two A serotype field strains isolated during the Argentine outbreaks of 2000/1 to differences in the structure of the IRES (internal ribosome entry site) of the viral genome (Garcia‐Nunez et al., ).…”