RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Abstract

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 KH 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.broad-spectrum therapeutics [2-4] and a target for function/mechanism-based strategies for viral attenuation [5-10]. One function/mechanism of the RdRp that has been targeted most is that required for faithful incorporation of nucleotides [5][6][7][8][9][10]. Changing RdRp fidelity decreases or increases the genetic variation of the viral population, which, in turn, decreases fitness and virulence of the viral population [11][12][13][14][15][16].Known RdRp variants exhibiting a high-fidelity phenotype can also exhibit a reduced speed of nucleotide incorporation, at least at the biochemical level [17]. Replication speed is also a determinant of viral fitness and virulence [18]. So, is it the increased fidelity or decreased speed of nucleotide addition that gives rise to the attenuated phenotype? Further complicating the fidelity-versus-speed question are the recent observations that changes to fidelity also have consequence for the efficiency of recombination [19][20][21][22][23][24]. Increased RdRp fidelity decreases recombination efficiency and vice versa [19][20][21][22][23][24]. It will likely be impossible to attribute a single biochemical property of the RdRp to biological outcome.The most extensively studied PV fidelity mutants encode RdRps with amino acid substitutions at sites remote from the active site. Constructing equivalent substitutions conferring equivalent phenotypes in RdRps other than PV is difficult if not impossible. Our laboratory has therefore pursued active-site-based strategies to manipulate the fidelity, speed, and/or recombination efficiency of the RdRp [25]. We have shown that a lysine (Lys-359 in PV) present in ...

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

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An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

“…Furthermore, when subjected to serial passage in ribavirin, the L420A mutation virus reverted in 3/3 lineages (Table 1), resulting in the classic ribavirin-resistant G64S poliovirus. While polymerase speed, fidelity, and efficiency of recombination are intricately intertwined (64,65), our data suggest that polymerase residue L420 is required for efficient viral RNA recombination and for counteracting ribavirin-induced error catastrophe (18). Furthermore, even though polymerase speed and fidelity are intricately intertwined, recent studies suggest that polymerase speed is more important than fidelity for virulence (66).…”

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“…There is some evidence that recombination events leading to D-RNA formation are largely, though not exclusively, polymerase driven (64)(65)(66) and are influenced by polymerase characteristics such as fidelity. For example, low-recombination variants of Senecavirus exhibited a high-fidelity polymerase phenotype (67), a low-fidelity SINV polymerase mutant showed increased rates of virion D-RNA production (34), and a high-fidelity poliovirus polymerase mutant exhibited decreased rates of recombination (68).…”