Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination

et al. 2019

Genes

Self Cite

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 ...

Section: Discussionmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Kim

Ellis

et al. 2019

Genes

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“…Over the past few years, a connection has been made between RdRp fidelity and recombination 14,32,39,41,42 . With more incorporation errors and/or consumption of nucleotide analogues by the RdRp comes an increase in the frequency of recombination both in cells and in test tubes 39 .…”

Section: Discussionmentioning

confidence: 99%

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

Janissen¹,

Lee

et al. 2020

Preprint

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The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazinecarboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra-and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

“…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 (18-23). Increased RdRp fidelity decreases recombination efficiency and vice versa (18-23).…”

Section: Introductionmentioning

confidence: 99%

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

Kim

Ellis

et al. 2019

Preprint

Self Cite

25Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we 26 have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl 27 transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the 28 context of the virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F 29 (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and 30 enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique 31 recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH 32 being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits 33 biochemical properties within twofold of wild type, the virus is impaired substantially for 34 recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to 35 speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven 36 nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible 37 to attribute a single property of the RdRp to fitness. However, the derivatives described here may 38 permit elucidation of the importance of recombination on the fitness of the viral population in a 39 background of constant polymerase speed and fidelity.40 41 Significance 42The availability of a "universal" method to create attenuated viruses for use as vaccine strains would 43 permit a rapid response to outbreaks of newly emerging viruses. Targeting RdRp fidelity has emerged 44 as such a universal approach. However, because polymerase fidelity and speed are inextricably linked, 45 the effort to attribute the attenuated phenotype to a single biochemical property of the RdRp may be 46 futile. Here, we show that this circumstance is even more complex. We provide evidence for the 47 existence of a biochemical parameter that combines with fidelity and speed to govern the mechanism 48 . 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 3 and/or efficiency of recombination. We conclude that the field will be served best by continued 49 emphasis on discovery of manipulatable functions of the RdRp instead of debating the importance of 50 individual properties. 51 52 56 pathogens), viral outbreaks over the past few decades have been caused by viruses for which 57 surveillance was not considered a priority. Rapid response to an outbreak caused by an unexpected 58 viral pathogen requires, minimally, the existence of broad-spectrum, antiviral therapeutics. Prevention 59 requires the availability of vaccines, development of which could take years. Indeed, approved 60 vaccines still do not exist to prevent infections by West Nile virus or severe acute respiratory syndrome 61 (SARS) coronavirus, and these ...