The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA∶polymerase complexes, providing a mechanism for the genesis of viral genome∶polymerase complexes observed in vivo.
Vyšlo v časopise: The N-Terminus of the RNA Polymerase from Infectious Pancreatic Necrosis Virus Is the Determinant of Genome Attachment. PLoS Pathog 7(6): e32767. doi:10.1371/journal.ppat.1002085 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002085
1. BruennJA 2003 A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases. Nucleic Acids Res 31 1821 1829
2. DelarueMPochOTordoNMorasDArgosP 1990 An attempt to unify the structure of polymerases. Protein Eng 3 461 467
3. GorbalenyaAEPringleFMZeddamJLLukeBTCameronCE 2002 The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage. J Mol Biol 324 47 62
4. CoulibalyFChevalierCGutscheIPousJNavazaJ 2005 The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120 761 772
5. MullerHIslamMRRaueR 2003 Research on infectious bursal disease–the past, the present and the future. Vet Microbiol 97 153 165
6. Rodriguez Saint-JeanSBorregoJJPerez-PrietoSI 2003 Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods. Adv Virus Res 62 113 165
7. PanJVakhariaVNTaoYJ 2007 The structure of a birnavirus polymerase reveals a distinct active site topology. Proc Natl Acad Sci U S A 104 7385 7390
8. DobosP 1993 In vitro guanylylation of infectious pancreatic necrosis virus polypeptide VP1. Virology 193 403 413
9. PanJLinLTaoYJ 2009 Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site. Virology 395 87 96
10. XuHTSiWDDobosP 2004 Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis. Virology 322 199 210
11. DobosP 1995 Protein-primed RNA synthesis in vitro by the virion-associated RNA polymerase of infectious pancreatic necrosis virus. Virology 208 19 25
12. CalvertJGNagyESolerMDobosP 1991 Characterization of the VPg-dsRNA linkage of infectious pancreatic necrosis virus. J Gen Virol 72 2563 2567
13. GarrigaDNavarroAQuerol-AudiJAbaituaFRodriguezJF 2007 Activation mechanism of a noncanonical RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A 104 20540 20545
14. HardingMM 2002 Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr D Biol Crystallogr 58 872 874
15. von EinemUIGorbalenyaAESchirrmeierHBehrensSELetzelT 2004 VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase. J Gen Virol 85 2221 2229
16. ButcherSJGrimesJMMakeyevEVBamfordDHStuartDI 2001 A mechanism for initiating RNA-dependent RNA polymerization. Nature 410 235 240
17. HuangHChopraRVerdineGLHarrisonSC 1998 Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282 1669 1675
18. TaoYFarsettaDLNibertMLHarrisonSC 2002 RNA synthesis in a cage–structural studies of reovirus polymerase λ3. Cell 111 733 745
19. ZamyatkinDFParraFAlonsoJMHarkiDAPetersonBR 2008 Structural insights into mechanisms of catalysis and inhibition in Norwalk virus polymerase. J Biol Chem 283 7705 7712
20. PoranenMMSalgadoPSKoivunenMRWrightSBamfordDH 2008 Structural explanation for the role of Mn2+ in the activity of Φ6 RNA-dependent RNA polymerase. Nucleic Acids Res 36 6633 6644
21. GaoGOrlovaMGeorgiadisMMHendricksonWAGoffSP 1997 Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection. Proc Natl Acad Sci U S A 94 407 411
22. BressanelliSTomeiLRousselAIncittiIVitaleRL 1999 Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci U S A 96 13034 13039
23. GoharaDWCrottySArnoldJJYoderJDAndinoR 2000 Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B. J Biol Chem 275 25523 25532
24. KorneevaVSCameronCE 2007 Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket. J Biol Chem 282 16135 16145
25. LeslieAGW 1992 Mosflm - a programme for processing X-ray diffraction data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 26
26. EvansP 2006 Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62 72 82
27. WinterG 2010 xia2: an expert system for macromolecular crystallography data reduction. Journal of Applied Crystallography 43 186 190
28. McCoyAJGrosse-KunstleveRWAdamsPDWinnMDStoroniLC 2007 Phaser crystallographic software. J Appl Crystallogr 40 658 674
29. EmsleyPCowtanK 2004 Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60 2126 2132
30. BlancERoversiPVonrheinCFlensburgCLeaSM 2004 Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr D Biol Crystallogr 60 2210 2221
31. DavisIWLeaver-FayAChenVBBlockJNKapralGJ 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35 W375 383
32. SmartOSBrandlMFlensburgCKellerPPaciorekW 2008 Annual Meeting of the American Crystallographic Association, Knoxville, TN. Abstract TP139
33. KleywegtGJBrungerAT 1996 Checking your imagination: applications of the free R value. Structure 4 897 904
34. KrissinelEHenrickK 2004 Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr 60 2256 2268
35. TheobaldDLWuttkeDS 2006 THESEUS: maximum likelihood superpositioning and analysis of macromolecular structures. Bioinformatics 22 2171 2172
36. BondCSSchuttelkopfAW 2009 ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr D Biol Crystallogr 65 510 512
37. MakeyevEVBamfordDH 2000 The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J 19 6275 6284
38. LaurilaMRMakeyevEVBamfordDH 2002 Bacteriophage Φ6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer. J Biol Chem 277 17117 17124
39. PagratisNRevelHR 1990 Minus-strand RNA synthesis by the segmented double-stranded RNA bacteriophage Φ6 requires continuous protein synthesis. Virology 177 281 288
40. SambrookJRussellD 2001 Molecular cloning: a laboratory manual (3rd edition) Cold Spring Harbor, New York Cold Spring Harbor Laboratory Press
41. SarinLPPoranenMMLehtiNMRavanttiJJKoivunenMRL 2009 Insights into the pre-initiation events of bacteriophage Φ6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex. Nucleic Acids Res 37 1182 1192
42. ShwedPSDobosPCameronLAVakhariaVNDuncanR 2002 Birnavirus VP1 proteins form a distinct subgroup of RNA-dependent RNA polymerases lacking a GDD motif. Virology 296 241 250
43. PetkovDLinnemannEKapczynskiDRSellersHS 2007 Full-length sequence analysis of four IBDV strains with different pathogenicities. Virus Genes 34 315 326
Zvýšte si kvalifikáciu online z pohodlia domova
Nemáte účet? Registrujte sa
Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.
