The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization.
Vyšlo v časopise: A Freeze Frame View of Vesicular Stomatitis Virus Transcription Defines a Minimal Length of RNA for 5′ Processing. PLoS Pathog 7(6): e32767. doi:10.1371/journal.ppat.1002073 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002073
1. SleatDEBanerjeeAK 1993 Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase. J Virol 67 1334 1339
2. EmersonSUWagnerRR 1973 L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. J Virol 12 1325 1335
3. EmersonSUYuY 1975 Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. J Virol 15 1348 1356
4. GrdzelishviliVZSmallwoodSTowerDHallRLHuntDM 2005 A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J Virol 79 7327 7337
5. HercykNHorikamiSMMoyerSA 1988 The vesicular stomatitis virus L protein possesses the mRNA methyltransferase activities. Virology 163 222 225
6. LiJFontaine-RodriguezECWhelanSP 2005 Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J Virol 79 13373 13384
7. LiJRahmehAMorelliMWhelanSP 2008 A conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping. J Virol 82 775 784
8. LiJWangJTWhelanSP 2006 A unique strategy for mRNA cap methylation used by vesicular stomatitis virus. Proc Natl Acad Sci U S A 103 8493 8498
9. OginoTBanerjeeAK 2007 Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. Mol Cell 25 85 97
10. HuntDMMehtaRHutchinsonKL 1988 The L protein of vesicular stomatitis virus modulates the response of the polyadenylic acid polymerase to S-adenosylhomocysteine. J Gen Virol 69 Pt 10 2555 2561
11. StillmanEAWhittMA 1999 Transcript initiation and 5′-end modifications are separable events during vesicular stomatitis virus transcription. J Virol 73 7199 7209
12. WangJTMcElvainLEWhelanSP 2007 Vesicular stomatitis virus mRNA capping machinery requires specific cis-acting signals in the RNA. J Virol 81 11499 11506
13. LiJRahmehABrusicVWhelanSP 2009 Opposing effects of inhibiting cap addition and cap methylation on polyadenylation during vesicular stomatitis virus mRNA synthesis. J Virol 83 1930 1940
14. RoseJKLodishHFBrockML 1977 Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine. J Virol 21 683 693
15. GallowaySEWertzGW 2008 S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein. J Virol 82 12280 12290
16. PochOBlumbergBMBougueleretLTordoN 1990 Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains. J Gen Virol 71 Pt 5 1153 1162
17. OginoTYadavSPBanerjeeAK 2010 Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase. Proc Natl Acad Sci U S A 107 3463 3468
18. FuruichiYShatkinAJ 2000 Viral and cellular mRNA capping: past and prospects. Adv Virus Res 55 135 184
19. KeeneJDLazzariniRA 1976 A comparison of the extents of methylation of vesicular stomatitis virus messenger RNA. Virology 69 364 367
20. MoyerSAAbrahamGAdlerRBanerjeeAK 1975 Methylated and blocked 5′ termini in vesicular stomatitis virus in vivo mRNAs. Cell 5 59 67
21. MoyerSABanerjeeAK 1976 In vivo methylation of vesicular stomatitis virus and its host-cell messenger RNA species. Virology 70 339 351
22. RhodesDPBanerjeeAK 1975 5′-terminal sequence of vesicular stomatitis virus mRNA's synthesized in vitro. J Virol 17 33 42
23. RhodesDPMoyerSABanerjeeAK 1974 In vitro synthesis of methylated messenger RNA by the virion-associated RNA polymerase of vesicular stomatitis virus. Cell 3 327 333
24. RoseJK 1975 Heterogneeous 5′-terminal structures occur on vesicular stomatitis virus mRNAs. J Biol Chem 250 8098 8104
25. TestaDBanerjeeAK 1977 Two methyltransferase activities in the purified virions of vesicular stomatitis virus. J Virol 24 786 793
26. RahmehAALiJKranzuschPJWhelanSP 2009 Ribose 2′-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein. J Virol 83 11043 11050
27. BujnickiJMRychlewskiL 2002 In silico identification, structure prediction and phylogenetic analysis of the 2′-O-ribose (cap 1) methyltransferase domain in the large structural protein of ssRNA negative-strand viruses. Protein Eng 15 101 108
28. FerronFLonghiSHenrissatBCanardB 2002 Viral RNA-polymerases – a predicted 2′-O-ribose methyltransferase domain shared by all Mononegavirales. Trends Biochem Sci 27 222 224
29. DaffisSSzretterKJSchriewerJLiJYounS 2010 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468 452 456
30. RahmehAASchenkADDanekEIKranzuschPJLiangB 2010 Molecular architecture of the vesicular stomatitis virus RNA polymerase. Proc Natl Acad Sci U S A 107 20075 20080
31. BaltimoreDHuangASStampferM 1970 Ribonucleic acid synthesis of vesicular stomatitis virus, II. An RNA polymerase in the virion. Proc Natl Acad Sci U S A 66 572 576
32. EmersonSUWagnerRR 1972 Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J Virol 10 297 309
33. BanerjeeAKRhodesDP 1973 In vitro synthesis of RNA that contains polyadenylate by virion-associated RNA polymerase of vesicular stomatitis virus. Proc Natl Acad Sci U S A 70 3566 3570
34. Piwnica-WormsHKeeneJD 1983 Sequential synthesis of small capped RNA transcripts in vitro by vesicular stomatitis virus. Virology 125 206 218
35. LiTPattnaikAK 1999 Overlapping signals for transcription and replication at the 3′ terminus of the vesicular stomatitis virus genome. J Virol 73 444 452
36. WertzGWWhelanSLeGroneABallLA 1994 Extent of terminal complementarity modulates the balance between transcription and replication of vesicular stomatitis virus RNA. Proc Natl Acad Sci U S A 91 8587 8591
37. WhelanSPWertzGW 1999 Regulation of RNA synthesis by the genomic termini of vesicular stomatitis virus: identification of distinct sequences essential for transcription but not replication. J Virol 73 297 306
38. WhelanSPWertzGW 2002 Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome. Proc Natl Acad Sci U S A 99 9178 9183
39. StillmanEAWhittMA 1997 Mutational analyses of the intergenic dinucleotide and the transcriptional start sequence of vesicular stomatitis virus (VSV) define sequences required for efficient termination and initiation of VSV transcripts. J Virol 71 2127 2137
40. ColonnoRJBanerjeeAK 1978 Complete nucleotide sequence of the leader RNA synthesized in vitro by vesicular stomatitis virus. Cell 15 93 101
41. WhelanSPBarrJNWertzGW 2000 Identification of a minimal size requirement for termination of vesicular stomatitis virus mRNA: implications for the mechanism of transcription. J Virol 74 8268 8276
42. HammondDCLesnawJA 1987 The fates of undermethylated mRNA cap structures of vesicular stomatitis virus (New Jersey) during in vitro transcription. Virology 159 229 236
43. HaglerJShumanS 1992 A freeze-frame view of eukaryotic transcription during elongation and capping of nascent mRNA. Science 255 983 986
44. ZhangXWalkerSBChipmanPRNibertMLBakerTS 2003 Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A. Nat Struct Biol 10 1011 1018
45. SimsRJ3rdBelotserkovskayaRReinbergD 2004 Elongation by RNA polymerase II: the short and long of it. Genes Dev 18 2437 2468
46. BarrJNWhelanSPWertzGW 2002 Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus. Biochim Biophys Acta 1577 337 353
47. FuerstTRNilesEGStudierFWMossB 1986 Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83 8122 8126
48. WhelanSPBallLABarrJNWertzGT 1995 Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci U S A 92 8388 8392
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