Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1978 Oct;28(1):75–83. doi: 10.1128/jvi.28.1.75-83.1978

Absence of Detectable Capping and Methylating Enzymes in Influenza Virions

Stephen J Plotch 1, Jeno Tomasz 2, Robert M Krug 1
PMCID: PMC354249  PMID: 702657

Abstract

In the presence of Mg2+ and a specific dinucleotide primer (ApG or GpG), the influenza virion transcriptase synthesizes the eight discrete segments of complementary RNA (cRNA) containing polyadenylic acid (Plotch and Krug, J. Virol. 21:24-34, 1977). Virions were examined for their ability to cap and methylate cRNA containing di- or triphosphorylated 5′ termini. By using the primers ppApG, pppApG, or ppGpG, viral cRNA was synthesized in vitro with [α-32P]-GTP and S-[methyl-3H]adenosylmethionine as labeled precursors. DEAE-Sephadex chromatography of the RNase T2 digest of the cRNA product demonstrated no 3H incorporation at all and the absence of a 32P-labeled cap structure. The 5′ terminus of ppApG-primed cRNA could be capped and methylated by enzymes from vaccinia virus, indicating that the two 5′-terminal phosphates derived from the primer were preserved in the product cRNA. The cap structure formed by the vaccinia enzymes and released by RNase T2 digestion as m7GpppAmpGp was radioactively labeled at its 3′-terminal phosphate only when [α-32P]CTP was used as the labeled precursor during transcription. This indicates that the 5′-terminal sequence of the cRNA is ppApGpC and that, therefore, ppApG most probably initiates transcription exactly at the 3′ GpCpUOH terminus of the virion RNA templates. Virions were also tested for their ability to cap and methylate ppApG in the absence of transcription. No such activities were detected, whereas under the same conditions the vaccinia virus enzymes successfully capped and methylated this compound. Consequently, these experiments, together with those reported earlier, have not detected in influenza virions any capping and methylating enzymes active on the 5′-initiated termini of viral cRNA chains synthesized in vitro, whether these termini possess one, two, or three phosphates. Some mechanism for capping and methylation of viral cRNA must, however, exist, because the viral mRNA (cRNA) synthesized in the infected cell contains 5′-terminal methylated cap structures (Krug et al., J. Virol. 20:45-53, 1976). Possible mechanisms are discussed.

Full text

PDF
75

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abraham G., Banerjee A. K. Sequential transcription of the genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976 May;73(5):1504–1508. doi: 10.1073/pnas.73.5.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abraham G., Rhodes D. P., Banerjee A. K. Novel initiation of RNA synthesis in vitro by vesicular stomatitis virus. Nature. 1975 May 1;255(5503):37–40. doi: 10.1038/255037a0. [DOI] [PubMed] [Google Scholar]
  3. Abraham G., Rhodes D. P., Banerjee A. K. The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus. Cell. 1975 May;5(1):51–58. doi: 10.1016/0092-8674(75)90091-4. [DOI] [PubMed] [Google Scholar]
  4. Ball L. A., White C. N. Order of transcription of genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976 Feb;73(2):442–446. doi: 10.1073/pnas.73.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baltimore D., Huang A. S., Stampfer M. Ribonucleic acid synthesis of vesicular stomatitis virus, II. An RNA polymerase in the virion. Proc Natl Acad Sci U S A. 1970 Jun;66(2):572–576. doi: 10.1073/pnas.66.2.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bishop D. H., Obijeski J. F., Simpson R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. I. Optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. J Virol. 1971 Jul;8(1):66–73. doi: 10.1128/jvi.8.1.66-73.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter C. A. Methylation of reovirus oligonucleotides in vivo and in vitro. Virology. 1977 Jul 15;80(2):249–259. doi: 10.1016/s0042-6822(77)80002-0. [DOI] [PubMed] [Google Scholar]
  8. Chow N. L., Simpson R. W. RNA-dependent RNA polymerase activity associated with virions and subviral particles of myxoviruses. Proc Natl Acad Sci U S A. 1971 Apr;68(4):752–756. doi: 10.1073/pnas.68.4.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colonno R. J., Banerjee A. K. A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro. Cell. 1976 Jun;8(2):197–204. doi: 10.1016/0092-8674(76)90003-9. [DOI] [PubMed] [Google Scholar]
  10. Colonno R. J., Stone H. O. Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2611–2615. doi: 10.1073/pnas.72.7.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Desrosiers R. C., Friderici K. H., Rottman F. M. Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus. Biochemistry. 1975 Oct 7;14(20):4367–4374. doi: 10.1021/bi00691a004. [DOI] [PubMed] [Google Scholar]
  12. Ensinger M. J., Martin S. A., Paoletti E., Moss B. Modification of the 5'-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2525–2529. doi: 10.1073/pnas.72.7.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Furuichi Y., Morgan M., Muthukrishnan S., Shatkin A. J. Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-. Proc Natl Acad Sci U S A. 1975 Jan;72(1):362–366. doi: 10.1073/pnas.72.1.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Furuichi Y., Morgan M., Shatkin A. J., Jelinek W., Salditt-Georgieff M., Darnell J. E. Methylated, blocked 5 termini in HeLa cell mRNA. Proc Natl Acad Sci U S A. 1975 May;72(5):1904–1908. doi: 10.1073/pnas.72.5.1904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Furuichi Y., Muthukrishnan S., Tomasz J., Shatkin A. J. Mechanism of formation of reovirus mRNA 5'-terminal blocked and methylated sequence, m7GpppGmpC. J Biol Chem. 1976 Aug 25;251(16):5043–5053. [PubMed] [Google Scholar]
  16. Furuichi Y., Shatkin A. J. 5'-termini of reovirus mRNA: ability of viral cores to form caps post-transcriptionally. Virology. 1977 Apr;77(2):566–578. doi: 10.1016/0042-6822(77)90482-2. [DOI] [PubMed] [Google Scholar]
  17. Furuichi Y., Shatkin A. J. Differential synthesis of blocked and unblocked 5'-termini in reovirus mRNA: effect of pyrophosphate and pyrophosphatase. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3448–3452. doi: 10.1073/pnas.73.10.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Groner Y., Hurwitz J. Synthesis of RNA containing a methylated blocked 5' terminus by HeLa nuclear homogenates. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2930–2934. doi: 10.1073/pnas.72.8.2930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huang A. S., Baltimore D., Bratt M. A. Ribonucleic acid polymerase in virions of Newcastle disease virus: comparison with the vesicular stomatitis virus polymerase. J Virol. 1971 Mar;7(3):389–394. doi: 10.1128/jvi.7.3.389-394.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kates J. R., McAuslan B. R. Poxvirus DNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1967 Jul;58(1):134–141. doi: 10.1073/pnas.58.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krug R. M. Cytoplasmic and nucleoplasmic viral RNPs in influenza virus-infected MDCK cells. Virology. 1972 Oct;50(1):103–113. doi: 10.1016/0042-6822(72)90350-9. [DOI] [PubMed] [Google Scholar]
  22. Krug R. M. Influenza viral RNPs newly synthesized during the latent period of viral growth in MDCK cells. Virology. 1971 Apr;44(1):125–136. doi: 10.1016/0042-6822(71)90159-0. [DOI] [PubMed] [Google Scholar]
  23. Krug R. M., Morgan M. A., Shatkin A. J. Influenza viral mRNA contains internal N6-methyladenosine and 5'-terminal 7-methylguanosine in cap structures. J Virol. 1976 Oct;20(1):45–53. doi: 10.1128/jvi.20.1.45-53.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lavi S., Shatkin A. J. Methylated simian virus 40-specific RNA from nuclei and cytoplasm of infected BSC-1 cells. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2012–2016. doi: 10.1073/pnas.72.6.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin S. A., Moss B. Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9330–9335. [PubMed] [Google Scholar]
  26. Martin S. A., Moss B. mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites. J Biol Chem. 1976 Dec 10;251(23):7313–7321. [PubMed] [Google Scholar]
  27. Moss B., Gershowitz A., Wei C. M., Boone R. Formation of the guanylylated and methylated 5'-terminus of vaccinia virus mRNA. Virology. 1976 Jul 15;72(2):341–351. doi: 10.1016/0042-6822(76)90163-x. [DOI] [PubMed] [Google Scholar]
  28. Moss B., Keith J. M., Gershowitz A., Ritchey M. B., Palese P. Common sequence at the 5' ends of the segmented RNA genomes of influenza A and B viruses. J Virol. 1978 Jan;25(1):312–318. doi: 10.1128/jvi.25.1.312-318.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moss B., Koczot F. Sequence of methylated nucleotides at the 5'-terminus of adenovirus-specific RNA. J Virol. 1976 Feb;17(2):385–392. doi: 10.1128/jvi.17.2.385-392.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moss B. Utilization of the guanylyltransferase and methyltransferases of vaccinia virus to modify and identify the 5'-terminals of heterologous RNA species. Biochem Biophys Res Commun. 1977 Jan 24;74(2):374–383. doi: 10.1016/0006-291x(77)90314-x. [DOI] [PubMed] [Google Scholar]
  31. Penhoet E., Miller H., Doyle M., Blatti S. RNA-dependent RNA polymerase activity in influenza virions. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1369–1371. doi: 10.1073/pnas.68.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perry R. P., Kelley D. E., Friderici K., Rottman F. The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5' terminus. Cell. 1975 Apr;4(4):387–394. doi: 10.1016/0092-8674(75)90159-2. [DOI] [PubMed] [Google Scholar]
  33. Plotch S. J., Krug R. M. Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J Virol. 1977 Jan;21(1):24–34. doi: 10.1128/jvi.21.1.24-34.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Plotch S. J., Krug R. M. Segments of influenza virus complementary RNA synthesized in vitro. J Virol. 1978 Feb;25(2):579–586. doi: 10.1128/jvi.25.2.579-586.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  36. Shatkin A. J., Sipe J. D. RNA polymerase activity in purified reoviruses. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1462–1469. doi: 10.1073/pnas.61.4.1462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Simoncsits A., Tomasz J., Allende J. E. Synthesis of pppGpN type dinucleotide derivatives: the 5' end sequence of some RNAs. Nucleic Acids Res. 1975 Feb;2(2):257–263. doi: 10.1093/nar/2.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Skehel J. J. RNA-dependent RNA polymerase activity of the influenza virus. Virology. 1971 Sep;45(3):793–796. doi: 10.1016/0042-6822(71)90197-8. [DOI] [PubMed] [Google Scholar]
  39. Taylor J. M., Illmensee R., Litwin S., Herring L., Broni B., Krug R. M. Use of specific radioactive probes to study transcription and replication of the influenza virus genome. J Virol. 1977 Feb;21(2):530–540. doi: 10.1128/jvi.21.2.530-540.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wei C. M., Gershowitz A., Moss B. 5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. Biochemistry. 1976 Jan 27;15(2):397–401. doi: 10.1021/bi00647a024. [DOI] [PubMed] [Google Scholar]
  41. Wei C. M., Gershowitz A., Moss B. Methylated nucleotides block 5' terminus of HeLa cell messenger RNA. Cell. 1975 Apr;4(4):379–386. doi: 10.1016/0092-8674(75)90158-0. [DOI] [PubMed] [Google Scholar]
  42. Wei C. M., Moss B. Methylated nucleotides block 5'-terminus of vaccinia virus messenger RNA. Proc Natl Acad Sci U S A. 1975 Jan;72(1):318–322. doi: 10.1073/pnas.72.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wei C., Moss B. 5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3758–3761. doi: 10.1073/pnas.74.9.3758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winicov I., Perry R. P. Synthesis methylation, and capping of nuclear RNA by a subcellular system. Biochemistry. 1976 Nov 16;15(23):5039–5046. doi: 10.1021/bi00668a014. [DOI] [PubMed] [Google Scholar]
  45. Young R. J., Content J. 5'-terminus of influenza virus RNA. Nat New Biol. 1971 Mar 31;230(13):140–142. doi: 10.1038/newbio230140a0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES