Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued D5N, A38V, and DAMM mutants but not the M194I and M296V mutants are high-fidelity RdRp variants. Mutagen resistance assays revealed that the higher replication fidelity was associated with higher-level resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains. IMPORTANCEThe ribavirin-isolated poliovirus (PV) RdRp G64S variant, the polymerases of which were of high replication fidelity, was attenuated in vivo. It has been proposed (M. Vignuzzi, E. Wendt, and R. Andino, Nat. Med. 14:154 -161, http://dx.doi.org/10.1038/nm 1726) that modulation of replication fidelity is a promising approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high-fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via several attenuated FMDV high-fidelity RdRp variants. Our findings may aid in the rational design of a new type of vaccine strain.

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Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5′ untranslated region. Regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition is antagonized by the viral 3C protease through cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K1-364, retains partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and modulation of ITAFs, providing new insights into translational control during viral infection. IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1-364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus, and provide new insights into translational control during viral infection.