key: cord-1026404-ljz7rfny
authors: Kim, Hyejeong; Ellis, Victor D.; Woodman, Andrew; Zhao, Yan; Arnold, Jamie J.; Cameron, Craig E.
title: RNA-dependent RNA polymerase speed and fidelity are not the only determinants of the mechanism or efficiency of recombination
date: 2019-09-14
journal: bioRxiv
DOI: 10.1101/769224
sha: ec223563f366aa8ed21ec84d1b0d21a911362cfa
doc_id: 1026404
cord_uid: ljz7rfny

Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity. The availability of a “universal” method to create attenuated viruses for use as vaccine strains would permit a rapid response to outbreaks of newly emerging viruses. Targeting RdRp fidelity has emerged as such a universal approach. However, because polymerase fidelity and speed are inextricably linked, the effort to attribute the attenuated phenotype to a single biochemical property of the RdRp may be futile. Here, we show that this circumstance is even more complex. We provide evidence for the existence of a biochemical parameter that combines with fidelity and speed to govern the mechanism and/or efficiency of recombination. We conclude that the field will be served best by continued emphasis on discovery of manipulatable functions of the RdRp instead of debating the importance of individual properties.

In spite of the substantial resources that have been allocated by the National Institute of Allergy and 54 Infectious Diseases and the Centers for Disease Control and Prevention to support prediction of 55 emerging viral pathogens (https://www.niaid.nih.gov/research/emerging-infectious-diseases-7 plaque formation relative to KH PV (Fig. 2B) . Indeed, the specific infectivity of IF-KH PV was 141 equivalent to WT. The addition of PS to IF-KH reduced the efficiency of plaque formation; the 142 specific infectivity of IF-PS-KH PV was reduced by twofold relative to IF-KH PV (Fig. 2B) .

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

The one caveat of the recombination assay is that the recombinant viruses produced must be 187 able to form plaques, which may mean that the recombinant virus must spread by a lytic mechanism. It 188 was possible that PS-KH PV was defective for virus assembly and/or spread, at least lytic spread ( Fig.   189 2B). In order to score for recombinant viruses that spread by either a lytic or non-lytic mechanism, we 190 engineered the acceptor template to encode the UnaG (35) green fluorescent protein (Fig. 3C) yielding products that are much greater than the average length of template used in the reaction (36).

The observation that only a subset of the KH mutants were competent for recombination, 205 presumably by a copy-choice mechanism (37), provided the opportunity to determine the extent to 206 which the poly(rU) polymerase activity can predict biological phenotypes. The congruence between 207 the two experiments was remarkable (Fig. 4B) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint polymerase activity (Fig. 4B) . These results further confirm template switching as the primary 210 mechanism of poly(rU) polymerase activity and validate this assay as a screen for identification of 211 RdRps with deficits in template switching. be identified as the one-nucleotide-extended product (n+1). Addition of the remaining nucleotides in 220 the absence or presence of the acceptor template will yield a strong-stop RNA product. In the presence 221 of a complementary acceptor RNA, the strong-strop RNA product (donor) will be extended, creating a 222 transfer product. 223 We evaluated each KH-containing RdRp derivative in this assay. Substrate and product 224 analysis are presented in Fig. 5C , with the quantitation of the transfer product relative to WT presented 225 in Fig. 5D . KH RdRp was defective in this assay as well, as transfer product was not detected (Fig.   226 5C). Importantly, the failure to transfer was not a reflection of the inability to assemble or produce 227 strong-stop donor RNA (Fig. 5C) . In this assay, PS-KH RdRp outperformed IF-KH RdRp, and PS-KH 228 and IF-PS-KH RdRp were essentially identical in activity. 229 We conclude that copy-choice and forced-copy-choice recombination use distinct mechanisms, The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint

For more than a decade now, our laboratory and others have published studies asserting 234 connections between viral RdRp fidelity and viral fitness (10-15). However, in the few instances in 235 which RdRp derivatives exhibiting perturbed fidelity have been characterized, these derivatives also 236 exhibit changes to the rate of nucleotide addition (6, 10, 17, 22, 33). Relative to wild-type polymerase, 237 a higher fidelity polymerase is a slower enzyme, and a lower fidelity polymerase is a faster enzyme (6, 238 10, 17, 22, 33). Polymerase speed will clearly determine replication kinetics and therefore can 239 contribute to viral fitness, as has been suggested recently (17). The most extensively characterized 240 fidelity mutants of PV harbor an RdRp with a substitution located at a remote site that likely causes 241 substantial collateral damage, further confounding the fidelity-versus-speed debate (4, 16). This study 242 was motivated by the need to understand better the relationship between the biochemical properties of 243 the viral RdRp and viral fitness, virulence, and pathogenesis. We have had in hand for a long time a 244 PV mutant whose speed and fidelity were perturbed by changing an RdRp active-site residue, K359H 245 (24). We did not publish this PV mutant until now because of its genetic instability (Fig. 1A) . We 246 realized that this genetic instability might actually represent an opportunity, as the biochemical 247 phenotypes reverted by the second-site suppressors might highlight the biochemical properties driving 248 viral fitness.

Lys-359 protonates the pyrophosphate leaving group during nucleotidyl transfer (24, 42). The 250 K359H substitution will change the efficiency of that protonation event for two reasons. First, the pKa 251 value of histidine is substantially lower than lysine and will therefore not be protonated to the same 252 extent. Second, the distance between the nucleotide phosphates and the imidazole ring of the histidine 253 will be greater than that of the amino group of lysine. It is likely that the distance is more of an issue, 254 because K359R PV is stable and K359R RdRp exhibits the same reduction in catalytic efficiency as 255 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint recombination phenotype (Fig. 3B ) may suggest that forced-copy-choice recombination contributes to 302 recombination in cells as well. 303 The current narrative of the characterized PV mutants impaired for recombination is that 304 recombination acts as a mechanism to purge deleterious mutations in the viral population (45-47). Our conserved residues capable of perturbing conserved, biochemical function (6, 8, 10). As a field, we 316 have focused on RdRp fidelity (6) but have come to the realization that RdRp speed should not be 317 ignored (17). This study shows that speed, fidelity, and undefined biochemical properties of the RdRp 318 exist and contribute to both the mechanism and efficiency of recombination (Fig. 6) . This inextricable 319 connection of the myriad biochemical properties of the RdRp precludes attribution of a single 320 biochemical property to viral fitness, virulence, and pathogenesis (Fig. 6) . The field will be served best The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint and treated with 2U DNAse Turbo (ThermoFisher) to remove residual DNA template. The RNA 348 transcripts were purified using RNeasy Mini Kit (Qiagen) before spectrophotometric quantification.

Purified RNA in RNase-free H 2 O was transfected into either HeLa or L929 fibroblasts using 350 TransMessenger (Qiagen). Virus yield was quantified by plaque assay. Briefly, cells and media were 351 harvested at 2-3 days post-transfection, subjected to three freeze-thaw cycles, and clarified. Supernatant 352 was then used on fresh HeLa cells in 6-well plates; virus infection was allowed to continue for 30 min. Luciferase assays. Subgenomic luciferase assays were performed as described previously (32). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint was isolated 24 h post-infection and used for qRT-PCR to determine genomes/mL or plaque assay to 579 determine pfu/mL, with the quotient yielding specific infectivity, genomes/pfu. (C) Ribavirin 580 sensitivity. HeLa cells were infected at a MOI 0.1 with each PV in the presence of 600 µM ribavirin.

After a 24-h incubation at 37 °C, virus was isolated and used for plaque assay. Indicated is the titer of 582 virus recovered in the presence of ribavirin normalized to that recovered in the absence of ribavirin. The heteropolymeric, symmetrical, primed-template substrate (sym/sub) has been used to establish an assay for forced-copy-choice recombination in vitro (22, 38). RdRp assembles on sym/sub. ATP is added and incorporated to yield a stable elongation complex. It is this elongation complex that is monitored for extension and transfer. Transfer is strictly dependent on the presence of an acceptor RNA with 5 complementarity to the 3'-end of donor RNA. (B) Schematic of the experimental design is indicated. Products in boxes are those observed and monitored by denaturing polyacrylamide gel electrophoresis. (C) Reaction products were resolved by electrophoresis and detected by phosphorimaging. The only regions of the gel with bands are shown; these correspond to the sym/sub primer, one-nucleotide-extended primer (n+1), four-nucleotides-extended product (strong stop) and non-templated addition of nucleotides to that product, and the transfer product. (D) Transfer products were quantified and are expressed as

Undefined Recombination Fidelity Speed Figure 6 : The biochemical properties of the RdRp are inextricably linked and collectively determine biological outcomes. PV RdRp is among the most extensively studied enzyme both in cells and in test tubes (4). A vast majority of these studies have emphasized elaboration of mechanisms governing efficient incorporation of nucleotides (speed) with high specificity (fidelity). Whether or not speed is a consequence of fidelity, vice versa, or completely separable is not clear (17, 33, 52-54). However, these two parameters are linked to recombination efficiency. Moreover, the studies reported 5 herein point to the existence of a biochemical property other than speed and fidelity, referred to here as undefined, that contributes to recombination efficiency. Together, the observations reported herein demonstrate that the current state of the art precludes association of a single biochemical property to viral fitness, virulence, and/or pathogenesis.

. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint

The cost of replication fidelity in an RNA virus