title: Structure of foot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin.
abstract: Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop beta9-alpha11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 A, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop beta9-alpha11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.
authors.affiliation: Institut de Biología Molecular de Barcelona (CSIC), Parc Cientific de Barcelona, Baldiri i Reixac 10, E-08028 Barcelona, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ferrer-Orta
authors.forename: Macarena
authors.initials: M
authors.lastname: Sierra
authors.forename: Rubén
authors.initials: R
authors.lastname: Agudo
authors.forename: Ignacio
authors.initials: I
authors.lastname: de la Higuera
authors.forename: Armando
authors.initials: A
authors.lastname: Arias
authors.forename: Rosa
authors.initials: R
authors.lastname: Pérez-Luque
authors.forename: Cristina
authors.initials: C
authors.lastname: Escarmís
authors.forename: Esteban
authors.initials: E
authors.lastname: Domingo
authors.forename: Nuria
authors.initials: N
authors.lastname: Verdaguer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005536:Foot-and-Mouth Disease / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012254:Ribavirin / Q000494:pharmacology*; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D014764:Viral Proteins; D012254:Ribavirin; D012321:DNA-Directed RNA Polymerases
references.citation: Biochemistry. 2004 May 11;43(18):5149-58
references.pmid: 15122880
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7289-94
references.pmid: 12754380
references.citation: Nature. 2006 Jan 19;439(7074):344-8
references.pmid: 16327776
references.citation: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9463-8
references.pmid: 17517631
references.citation: Biochemistry. 2003 Sep 9;42(35):10462-71
references.pmid: 12950173
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10027-32
references.pmid: 10468556
references.citation: J Biol Chem. 2000 Feb 25;275(8):5329-36
references.pmid: 10681506
references.citation: J Virol. 2007 Feb;81(4):2012-24
references.pmid: 17151116
references.citation: Virology. 1983 Jul 30;128(2):310-8
references.pmid: 6310859
references.citation: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6895-900
references.pmid: 11371613
references.citation: PLoS Pathog. 2005 Oct;1(2):e11
references.pmid: 16220146
references.citation: PLoS One. 2009;4(5):e5554
references.pmid: 19436746
references.citation: Curr Top Microbiol Immunol. 2008;320:137-56
references.pmid: 18268843
references.citation: J Virol. 2000 Sep;74(18):8316-23
references.pmid: 10954530
references.citation: J Virol. 2004 Nov;78(21):11678-85
references.pmid: 15479809
references.citation: Virus Res. 2005 Feb;107(2):129-39
references.pmid: 15649559
references.citation: Virus Res. 2005 Feb;107(2):165-71
references.pmid: 15649562
references.citation: J Mol Biol. 1996 Nov 29;264(2):255-67
references.pmid: 8951375
references.citation: Virology. 2003 Jul 5;311(2):339-49
references.pmid: 12842623
references.citation: J Virol. 2007 Apr;81(7):3583-96
references.pmid: 17251299
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: J Mol Biol. 2005 Nov 11;353(5):1021-32
references.pmid: 16216271
references.citation: Biochemistry. 2004 May 11;43(18):5126-37
references.pmid: 15122878
references.citation: Nat Med. 2000 Dec;6(12):1375-9
references.pmid: 11100123
references.citation: Gastroenterology. 2007 May;132(5):2050-2
references.pmid: 17484896
references.citation: J Biol Chem. 2004 Nov 5;279(45):47212-21
references.pmid: 15294895
references.citation: Rev Med Virol. 2006 Jan-Feb;16(1):37-48
references.pmid: 16287208
references.citation: J Virol. 2001 May;75(10):4771-9
references.pmid: 11312349
references.citation: EMBO J. 2004 Sep 1;23(17):3462-71
references.pmid: 15306852
references.citation: J Virol. 2008 Dec;82(24):12346-55
references.pmid: 18829745