Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

Abstract

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 M ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 M ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 M ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.

Amino acid residues Ala283 and His421 in the RNA-dependent RNA polymerase of porcine reproductive and respiratory syndrome virus play important roles in viral ribavirin sensitivity and quasispecies diversity

“…For example, by using different strains of FMDV, one study identified one substitution (M296I) while other independent studies identified three (P44S, P169S and M296I) or four (D5N, A38V, M194I and M296V) different substitutions that confer resistance to ribavirin and increased fidelity (Agudo et al, 2010;Sierra et al, 2007;Zeng et al, 2014). A couple of amino acid mutations were also identified in coxsackievirus B3 virus, human enterovirus 71 virus and bovine viral diarrhea virus (Curti & Jaeger, 2013;Gnädig et al, 2012;Sadeghipour et al, 2013). For the two amino acid substitutions identified in PRRSV in this study, when they were introduced into the PRRSV VR2385 infectious clone backbone, the rescued reconstituted virus vA283T showed a more ribavirin-resistant profile and fewer quasispecies variants than the other reconstituted virus vH421Y (Fig.…”

“…The results indicated that PRRSV is highly sensitive to ribavirin, since it accumulates more lethal mutations by mistakenly incorporating ribavirin. For other RNA viruses, it has been reported that poliovirus can tolerate consecutive in vitro passages at 600 mM ribavirin, foot-mouth disease virus (FMDV) at 800 mM, and human enterovirus 71 at 1600 mM (Sadeghipour et al, 2013;Sierra et al, 2007;Vignuzzi et al, 2006). The high sensitivity of PRRSV to ribavirin suggested a potential usage as an antiviral to inhibit PRRSV infection (Kim & Lee, 2013).…”

“…One crucial mechanism of the high mutation rate for RNA viruses is the low fidelity of RNA-dependent RNA polymerase (RdRp) due to its lack of proofreading ability during RNA replication (Domingo & Holland, 1997;Smith et al, 2013). Studies of poliovirus and other RNA viruses revealed that certain amino acid residues in RdRp are fidelity checkpoints which affect the fidelity of RdRp, and that the fidelity of RdRp can be modulated by change of these checkpoint residues (Gnädig et al, 2012;Rozen-Gagnon et al, 2014;Sadeghipour et al, 2013;Vignuzzi et al, 2008;Zeng et al, 2014). Mutants carrying a high fidelity RdRp showed a decreased population quasispecies diversity and attenuated pathogenicity to hosts (Van Slyke et al, 2015).…”

“…JX044140) and strain MN184B (GenBank accession no. DQ176020), were consecutively passaged each in MARC-145 cells under the selection of ribavirin, which has been successfully used to screen high fidelity virus mutants in vitro by increasing the mutation frequency of RNA viruses above the point of error catastrophe, ultimately driving quasispecies with low fidelity RdRp into extinction (Crotty et al, 2000;Pfeiffer & Kirkegaard, 2003;Sadeghipour et al, 2013;Zeng et al, 2014).…”

“…The viruses from passages P5, P10, P15 and P20 were used to monitor the frequency of ribavirin resistance in virus populations, using a ribavirin resistance assay which has been described elsewhere (Sadeghipour et al, 2013). MARC-145 cells were pretreated with ribavirin at final concentrations of 0, 200 or 400 mM, respectively.…”