RNA-dependent RNA polymerase speed and fidelity are not the only determinants of the mechanism or efficiency of recombination

Hyejeong Kim; Victor D. Ellis; Andrew Woodman; Yan Zhao; Jamie J. Arnold; Craig E. Cameron

Therefore, the exaggerated behavior of PS-KH PV in the PFU-based growth assay relative to the 144 replicon assay likely reflects an additional defect to virus assembly and/or spread. based on the 2-log difference in sensitivity to ribavirin (Fig. 2C) . IF-KH and PS-KH PVs also 152 exhibited a higher fidelity than WT PV (Fig. 2C) . Importantly, both mutants exhibited essentially 153 equivalent fidelity phenotypes (Fig. 2C) , consistent with the suggestion above that the reduced 154 efficiency of plaque formation observed for PS-KH PV is likely related to impairment of virus 155 assembly and/or spread. Finally, evaluation of IF-PS-KH PV revealed equivalent sensitivity of this 156 mutant to ribavirin as observed for WT PV (Fig. 2C) , suggesting that fidelity had been returned to Based on the myriad RdRp fidelity mutants that have been reported to date, there appears to be 164 a direct correlation between the rate of nucleotide addition (speed) and the fidelity of nucleotide 165 addition (6, 10, 17, 22, 33). This correlation also extends to recombination efficiency (22) . If this is 166 the case, then the ongoing debate of speed versus fidelity as the key determinant of viral fitness will 167 become even more complicated to resolve (17). 168 The current state of the art for evaluation of recombination in cell culture is based on the co-169 transfection of two viral (sub)genomic RNAs incapable of producing infectious virus (34). The donor 170 RNA is a replication-competent, subgenomic RNA that encodes a luciferase reporter instead of the 171 viral capsid (Fig. 3A) . The acceptor RNA is a replication-incompetent, genomic RNA that has a 172 defective cis-acting replication element, termed oriI ( Fig. 3A) (34). Initiation of replication on the 173 donor followed by a switch to the acceptor at a site after the oriI locus will yield an infectious genome 174 that can be scored by plaque assay (Fig. 3A) (34). 175 We evaluated our panel of KH-containing PV mutants using this assay, and the outcomes were, 176 in most cases, quite unexpected (Fig. 3B) . KH PV was unable to produce viable recombinants, as 177 expected for a high-fidelity RdRp (22). The first surprise was that IF-KH and PS-KH PVs did not 178 exhibit the same phenotypes (Fig. 3B) . While IF-KH PV produced viable recombinant virus, PS-KH 9