1887

Abstract

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be adapted by the incorporation of commonly used epitope tags, facilitating their detection with commercially available reagents. In this study, we used random transposon-mediated mutagenesis to produce a library of 15 nt insertions in the FMDV non-structural polyprotein. Using a replicon-based assay, we isolated multiple replication-competent as well as replication-defective insertions. We adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins for use in a variety of downstream assays. Additionally, we showed that replication of some of the replication-defective insertion mutants could be rescued by co-transfection of a ‘helper’ replicon, demonstrating a novel use of random mutagenesis to identify intergenomic -complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornavirus replication complexes.

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2015-12-01
2020-01-18
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References

  1. Arias A. , Perales C. , Escarmís C. , Domingo E. . ( 2010;). Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus. PLoS One 5: e10735 [CrossRef] [PubMed].
    [Google Scholar]
  2. Beard C. W. , Mason P. W. . ( 2000;). Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus. J Virol 74: 987–991 [CrossRef] [PubMed].
    [Google Scholar]
  3. Belsham G. J. , Brangwyn J. K. . ( 1990;). A region of the 5′ noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control. J Virol 64: 5389–5395 [PubMed].
    [Google Scholar]
  4. Birtley J. R. , Knox S. R. , Jaulent A. M. , Brick P. , Leatherbarrow R. J. , Curry S. . ( 2005;). Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity. J Biol Chem 280: 11520–11527 [CrossRef] [PubMed].
    [Google Scholar]
  5. Brune W. , Ménard C. , Hobom U. , Odenbreit S. , Messerle M. , Koszinowski U. H. . ( 1999;). Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis. Nat Biotechnol 17: 360–364 [CrossRef] [PubMed].
    [Google Scholar]
  6. Carrillo C. , Tulman E. R. , Delhon G. , Lu Z. , Carreno A. , Vagnozzi A. , Kutish G. F. , Rock D. L. . ( 2005;). Comparative genomics of foot-and-mouth disease virus. J Virol 79: 6487–6504 [CrossRef] [PubMed].
    [Google Scholar]
  7. Clarke B. E. , Brown A. L. , Currey K. M. , Newton S. E. , Rowlands D. J. , Carroll A. R. . ( 1987;). Potential secondary and tertiary structure in the genomic RNA of foot and mouth disease virus. Nucleic Acids Res 15: 7067–7079 [CrossRef] [PubMed].
    [Google Scholar]
  8. de Felipe P. , Hughes L. E. , Ryan M. D. , Brown J. D. . ( 2003;). Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide. J Biol Chem 278: 11441–11448 [CrossRef] [PubMed].
    [Google Scholar]
  9. Doedens J. R. , Kirkegaard K. . ( 1995;). Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. EMBO J 14: 894–907 [PubMed].
    [Google Scholar]
  10. Donnelly M. L. , Hughes L. E. , Luke G. , Mendoza H. , ten Dam E. , Gani D. , Ryan M. D. . ( 2001;). The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J Gen Virol 82: 1027–1041 [CrossRef] [PubMed].
    [Google Scholar]
  11. Escarmís C. , Dopazo J. , Dávila M. , Palma E. L. , Domingo E. . ( 1995;). Large deletions in the 5′-untranslated region of foot-and-mouth disease virus of serotype C. Virus Res 35: 155–167 [CrossRef] [PubMed].
    [Google Scholar]
  12. Falk M. M. , Sobrino F. , Beck E. . ( 1992;). VPg gene amplification correlates with infective particle formation in foot-and-mouth disease virus. J Virol 66: 2251–2260 [PubMed].
    [Google Scholar]
  13. Ferrer-Orta C. , Arias A. , Perez-Luque R. , Escarmís C. , Domingo E. , Verdaguer N. . ( 2004;). Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA. J Biol Chem 279: 47212–47221 [CrossRef] [PubMed].
    [Google Scholar]
  14. Ferrer-Orta C. , Arias A. , Agudo R. , Pérez-Luque R. , Escarmís C. , Domingo E. , Verdaguer N. . ( 2006;). The structure of a protein primer-polymerase complex in the initiation of genome replication. EMBO J 25: 880–888 [CrossRef] [PubMed].
    [Google Scholar]
  15. Ferrer-Orta C. , Agudo R. , Domingo E. , Verdaguer N. . ( 2009;). Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr Opin Struct Biol 19: 752–758 [CrossRef] [PubMed].
    [Google Scholar]
  16. Forrest S. , Lear Z. , Herod M. R. , Ryan M. , Rowlands D. J. , Stonehouse N. J. . ( 2014;). Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers. J Gen Virol 95: 2649–2657 [CrossRef] [PubMed].
    [Google Scholar]
  17. Forss S. , Schaller H. . ( 1982;). A tandem repeat gene in a picornavirus. Nucleic Acids Res 10: 6441–6450 [CrossRef] [PubMed].
    [Google Scholar]
  18. García-Arriaza J. , Domingo E. , Escarmís C. . ( 2005;). A segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness. Virology 335: 155–164 [CrossRef] [PubMed].
    [Google Scholar]
  19. García-Briones M. , Rosas M. F. , González-Magaldi M. , Martín-Acebes M. A. , Sobrino F. , Armas-Portela R. . ( 2006;). Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells. Virology 349: 409–421 [CrossRef] [PubMed].
    [Google Scholar]
  20. Giachetti C. , Hwang S. S. , Semler B. L. . ( 1992;). cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication. J Virol 66: 6045–6057 [PubMed].
    [Google Scholar]
  21. Gladue D. P. , O'Donnell V. , Baker-Bransetter R. , Pacheco J. M. , Holinka L. G. , Arzt J. , Pauszek S. , Fernandez-Sainz I. , Fletcher P. , other authors . ( 2014;). Interaction of foot-and-mouth disease virus nonstructural protein 3A with host protein DCTN3 is important for viral virulence in cattle. J Virol 88: 2737–2747 [CrossRef] [PubMed].
    [Google Scholar]
  22. González-Magaldi M. , Postigo R. , de la Torre B. G. , Vieira Y. A. , Rodríguez-Pulido M. , López-Viñas E. , Gómez-Puertas P. , Andreu D. , Kremer L. , other authors . ( 2012;). Mutations that hamper dimerization of foot-and-mouth disease virus 3A protein are detrimental for infectivity. J Virol 86: 11013–11023 [CrossRef] [PubMed].
    [Google Scholar]
  23. González-Magaldi M. , Martín-Acebes M. A. , Kremer L. , Sobrino F. . ( 2014;). Membrane topology and cellular dynamics of foot-and-mouth disease virus 3A protein. PLoS One 9: e106685 [CrossRef] [PubMed].
    [Google Scholar]
  24. Grubman M. J. , Zellner M. , Bablanian G. , Mason P. W. , Piccone M. E. . ( 1995;). Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus. Virology 213: 581–589 [CrossRef] [PubMed].
    [Google Scholar]
  25. King A. M. , Sangar D. V. , Harris T. J. , Brown F. . ( 1980;). Heterogeneity of the genome-linked protein of foot-and-mouth disease virus. J Virol 34: 627–634 [PubMed].
    [Google Scholar]
  26. Knowles N. J. , Davies P. R. , Henry T. , O'Donnell V. , Pacheco J. M. , Mason P. W. . ( 2001;). Emergence in Asia of foot-and-mouth disease viruses with altered host range: characterization of alterations in the 3A protein. J Virol 75: 1551–1556 [CrossRef] [PubMed].
    [Google Scholar]
  27. Li S. , Gao M. , Zhang R. , Song G. , Song J. , Liu D. , Cao Y. , Li T. , Ma B. , other authors . ( 2010;). A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein. Virus Genes 41: 406–413 [CrossRef] [PubMed].
    [Google Scholar]
  28. Li S. , Gao M. , Zhang R. , Song G. , Song J. , Liu D. , Cao Y. , Li T. , Ma B. , other authors . ( 2011;). A mutant of Asia 1 serotype of Foot-and-mouth disease virus with the deletion of an important antigenic epitope in the 3A protein. Can J Microbiol 57: 169–176 [CrossRef] [PubMed].
    [Google Scholar]
  29. Li P. , Bai X. , Cao Y. , Han C. , Lu Z. , Sun P. , Yin H. , Liu Z. . ( 2012;). Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus. PLoS One 7: e41486 [CrossRef] [PubMed].
    [Google Scholar]
  30. López de Quinto S. , Martínez-Salas E. . ( 1997;). Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation. J Virol 71: 4171–4175 [PubMed].
    [Google Scholar]
  31. López de Quinto S. , Sáiz M. , de la Morena D. , Sobrino F. , Martínez-Salas E. . ( 2002;). IRES-driven translation is stimulated separately by the FMDV 3′-NCR and poly(A) sequences. Nucleic Acids Res 30: 4398–4405 [CrossRef] [PubMed].
    [Google Scholar]
  32. Ma X. , Li P. , Sun P. , Bai X. , Bao H. , Lu Z. , Fu Y. , Cao Y. , Li D. , Chen Y. , Qiao Z. , Liu Z. . ( 2015;). Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus. Infect Genet Evol 31: 17–24.[CrossRef]
    [Google Scholar]
  33. Mason P. W. , Bezborodova S. V. , Henry T. M. . ( 2002;). Identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus. J Virol 76: 9686–9694 [CrossRef] [PubMed].
    [Google Scholar]
  34. Mason P. W. , Grubman M. J. , Baxt B. . ( 2003;). Molecular basis of pathogenesis of FMDV. Virus Res 91: 9–32 [CrossRef] [PubMed].
    [Google Scholar]
  35. McMahon C. W. , Traxler B. , Grigg M. E. , Pullen A. M. . ( 1998;). Transposon-mediated random insertions and site-directed mutagenesis prevent the trafficking of a mouse mammary tumor virus superantigen. Virology 243: 354–365 [CrossRef] [PubMed].
    [Google Scholar]
  36. Moffat K. , Howell G. , Knox C. , Belsham G. J. , Monaghan P. , Ryan M. D. , Wileman T. . ( 2005;). Effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2BC but not 3A blocks endoplasmic reticulum-to-Golgi transport. J Virol 79: 4382–4395 [CrossRef] [PubMed].
    [Google Scholar]
  37. Moffat K. , Knox C. , Howell G. , Clark S. J. , Yang H. , Belsham G. J. , Ryan M. , Wileman T. . ( 2007;). Inhibition of the secretory pathway by foot-and-mouth disease virus 2BC protein is reproduced by coexpression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C. J Virol 81: 1129–1139 [CrossRef] [PubMed].
    [Google Scholar]
  38. Möhl B. S. , Böttcher S. , Granzow H. , Fuchs W. , Klupp B. G. , Mettenleiter T. C. . ( 2010;). Random transposon-mediated mutagenesis of the essential large tegument protein pUL36 of pseudorabies virus. J Virol 84: 8153–8162 [CrossRef] [PubMed].
    [Google Scholar]
  39. Nayak A. , Goodfellow I. G. , Belsham G. J. . ( 2005;). Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J Virol 79: 7698–7706 [CrossRef] [PubMed].
    [Google Scholar]
  40. O'Donnell V. K. , Pacheco J. M. , Henry T. M. , Mason P. W. . ( 2001;). Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A. Virology 287: 151–162 [CrossRef] [PubMed].
    [Google Scholar]
  41. Pacheco J. M. , Henry T. M. , O'Donnell V. K. , Gregory J. B. , Mason P. W. . ( 2003;). Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus. J Virol 77: 13017–13027 [CrossRef] [PubMed].
    [Google Scholar]
  42. Pacheco J. M. , Gladue D. P. , Holinka L. G. , Arzt J. , Bishop E. , Smoliga G. , Pauszek S. J. , Bracht A. J. , O'Donnell V. , other authors . ( 2013;). A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle. Virology 446: 260–267 [CrossRef] [PubMed].
    [Google Scholar]
  43. Paul A. V. , van Boom J. H. , Filippov D. , Wimmer E. , Protein-primed R. N. A. . ( 1998;). synthesis by purified poliovirus RNA polymerase. Nature 393: 280–284 [CrossRef] [PubMed].
    [Google Scholar]
  44. Paul A. V. , Yin J. , Mugavero J. , Rieder E. , Liu Y. , Wimmer E. . ( 2003;). A “slide-back” mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus. J Biol Chem 278: 43951–43960 [CrossRef] [PubMed].
    [Google Scholar]
  45. Remenyi R. , Qi H. , Su S. Y. , Chen Z. , Wu N. C. , Arumugaswami V. , Truong S. , Chu V. , Stokelman T. , other authors . ( 2014;). A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins. MBio 5: e01469–e01414 [CrossRef] [PubMed].
    [Google Scholar]
  46. Rodríguez P. L. , Carrasco L. . ( 1993;). Poliovirus protein 2C has ATPase and GTPase activities. J Biol Chem 268: 8105–8110 [PubMed].
    [Google Scholar]
  47. Rodríguez Pulido M. , Sobrino F. , Borrego B. , Sáiz M. . ( 2009;). Attenuated foot-and-mouth disease virus RNA carrying a deletion in the 3′ noncoding region can elicit immunity in swine. J Virol 83: 3475–3485 [CrossRef] [PubMed].
    [Google Scholar]
  48. Rowlands D. J. , Harris T. J. , Brown F. . ( 1978;). More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA. J Virol 26: 335–343 [PubMed].
    [Google Scholar]
  49. Ryan M. D. , Drew J. . ( 1994;). Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 13: 928–933 [PubMed].
    [Google Scholar]
  50. Ryan M. D. , Flint M. . ( 1997;). Virus-encoded proteinases of the picornavirus super-group. J Gen Virol 78: 699–723 [CrossRef] [PubMed].
    [Google Scholar]
  51. Sáiz M. , Gómez S. , Martínez-Salas E. , Sobrino F. . ( 2001;). Deletion or substitution of the aphthovirus 3′ NCR abrogates infectivity and virus replication. J Gen Virol 82: 93–101 [CrossRef] [PubMed].
    [Google Scholar]
  52. Teterina N. L. , Zhou W. D. , Cho M. W. , Ehrenfeld E. . ( 1995;). Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations. J Virol 69: 4245–4254 [PubMed].
    [Google Scholar]
  53. Teterina N. L. , Levenson E. A. , Ehrenfeld E. . ( 2010;). Viable polioviruses that encode 2A proteins with fluorescent protein tags. J Virol 84: 1477–1488 [CrossRef] [PubMed].
    [Google Scholar]
  54. Teterina N. L. , Lauber C. , Jensen K. S. , Levenson E. A. , Gorbalenya A. E. , Ehrenfeld E. . ( 2011a;). Identification of tolerated insertion sites in poliovirus non-structural proteins. Virology 409: 1–11 [CrossRef] [PubMed].
    [Google Scholar]
  55. Teterina N. L. , Pinto Y. , Weaver J. D. , Jensen K. S. , Ehrenfeld E. . ( 2011b;). Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells. J Virol 85: 4284–4296 [CrossRef] [PubMed].
    [Google Scholar]
  56. Thorne L. , Bailey D. , Goodfellow I. . ( 2012;). High-resolution functional profiling of the norovirus genome. J Virol 86: 11441–11456 [CrossRef] [PubMed].
    [Google Scholar]
  57. Tiley L. , King A. M. , Belsham G. J. . ( 2003;). The foot-and-mouth disease virus cis-acting replication element (cre) can be complemented in trans within infected cells. J Virol 77: 2243–2246 [CrossRef] [PubMed].
    [Google Scholar]
  58. Towner J. S. , Mazanet M. M. , Semler B. L. . ( 1998;). Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins. J Virol 72: 7191–7200 [PubMed].
    [Google Scholar]
  59. Tulloch F. , Pathania U. , Luke G. A. , Nicholson J. , Stonehouse N. J. , Rowlands D. J. , Jackson T. , Tuthill T. , Haas J. , other authors . ( 2014;). FMDV replicons encoding green fluorescent protein are replication competent. J Virol Methods 209: 35–40 [CrossRef] [PubMed].
    [Google Scholar]
  60. Xia H. , Wang P. , Wang G. C. , Yang J. , Sun X. , Wu W. , Qiu Y. , Shu T. , Zhao X. , other authors . ( 2015;). Human enterovirus nonstructural protein 2CATPase functions as both an RNA helicase and ATP-independent RNA chaperone. PLoS Pathog 11: e1005067 [CrossRef] [PubMed].
    [Google Scholar]
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