title: Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance.
abstract: RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3D(pol), as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3D(pol) may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3D(pol)-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3D(pol) decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3D(pol) increased the frequency of recombination. The 3D(pol) Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3D(pol) Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance.
  
  
  
  Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.
authors.affiliation: Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Kempf
authors.affiliation: Department of Biochemistry, Colorado State University, Fort Collins, Colorado, USA.
authors.forename: Olve B
authors.initials: OB
authors.lastname: Peersen
authors.affiliation: Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA david.barton@ucdenver.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Barton
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D024882:Drug Resistance, Viral; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D017955:Poliovirus / Q000187:drug effects* / Q000201:enzymology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012254:Ribavirin / Q000494:pharmacology*
chemical_list: D000998:Antiviral Agents; D012254:Ribavirin; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D007930:Leucine