Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1978 Aug;27(2):399–408. doi: 10.1128/jvi.27.2.399-408.1978

Multiple Roles for ATP in the Synthesis and Processing of mRNA by Vaccinia Virus: Specific Inhibitory Effects of Adenosine (β,γ-Imido) Triphosphate

Alan Gershowitz 1, Robert F Boone 1, Bernard Moss 1
PMCID: PMC354178  PMID: 691115

Abstract

Adenosine (β,γ-imido)triphosphate (AMP-PNP) and guanosine (β,γ-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable γ-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.

Full text

PDF
399

Images in this article

405Image
on p.405

Selected References

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

  1. Fournier F., Tovell D. R., Esteban M., Metz D. H., Ball L. A., Kerr I. M. The translation of vaccinia virus messenger RNA in animal cell-free systems. FEBS Lett. 1973 Mar 15;30(3):268–272. doi: 10.1016/0014-5793(73)80667-2. [DOI] [PubMed] [Google Scholar]
  2. Gold P. H., Dales S. Localization of nucleotide phosphohydrolase activity within vaccinia. Proc Natl Acad Sci U S A. 1968 Jul;60(3):845–852. doi: 10.1073/pnas.60.3.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Howard B. H., de Crombrugghe B. ATPase activity required for termination of transcription by the Escherichia coli protein factor rho. J Biol Chem. 1976 Apr 25;251(8):2520–2524. [PubMed] [Google Scholar]
  4. JOKLIK W. K. The preparation and characteristics of highly purified radioactively labelled poxvirus. Biochim Biophys Acta. 1962 Aug 20;61:290–301. doi: 10.1016/0926-6550(62)90091-9. [DOI] [PubMed] [Google Scholar]
  5. Jaureguiberry G., Ben-Hamida F., Chapeville F., Beaud G. Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores. J Virol. 1975 Jun;15(6):1467–1474. doi: 10.1128/jvi.15.6.1467-1474.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Kates J., Beeson J. Ribonucleic acid synthesis in vaccinia virus. I. The mechanism of synthesis and release of RNA in vaccinia cores. J Mol Biol. 1970 May 28;50(1):1–18. doi: 10.1016/0022-2836(70)90100-2. [DOI] [PubMed] [Google Scholar]
  8. Kleiman J. H., Moss B. Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions. J Biol Chem. 1975 Apr 10;250(7):2430–2437. [PubMed] [Google Scholar]
  9. Kleiman J. H., Moss B. Purification of a protein kinase and two phosphate acceptor proteins from vaccinia virions. J Biol Chem. 1975 Apr 10;250(7):2420–2429. [PubMed] [Google Scholar]
  10. Lowery-Goldhammer C., Richardson J. P. An RNA-dependent nucleoside triphosphate phosphohydrolase (ATPase) associated with rho termination factor. Proc Natl Acad Sci U S A. 1974 May;71(5):2003–2007. doi: 10.1073/pnas.71.5.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Moss B., Rosenblum E. N., Gershowitz A. Characterization of a polyriboadenylate polymerase from vaccinia virions. J Biol Chem. 1975 Jun 25;250(12):4722–4729. [PubMed] [Google Scholar]
  15. Munyon W., Paoletti E., Grace J. T., Jr RNA polymerase activity in purified infectious vaccinia virus. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2280–2287. doi: 10.1073/pnas.58.6.2280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Munyon W., Paoletti E., Ospina J., Grace J. T., Jr Nucleotide phosphohydrolase in purified vaccinia virus. J Virol. 1968 Mar;2(3):167–172. doi: 10.1128/jvi.2.3.167-172.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nevins J. R., Joklik W. K. Poly (A) sequences of vaccinia virus messenger RNA: nature, mode of addition and function during translation in vitra and in vivo. Virology. 1975 Jan;63(1):1–14. doi: 10.1016/0042-6822(75)90365-7. [DOI] [PubMed] [Google Scholar]
  18. Paolette E., Rosemond-Hornbeak H., Moss B. Two nucleid acid-dependent nucleoside triphosphate phosphohydrolases from vaccinia virus. Purification and characterization. J Biol Chem. 1974 May 25;249(10):3273–3280. [PubMed] [Google Scholar]
  19. Paoletti E., Cooper N., Moss B. Regulation of synthesis of two immunologically distinct nucleic acid-dependent nucleoside triphosphate phosphohydrolases in vaccinia virus-infected HeLa cells. J Virol. 1974 Sep;14(3):578–586. doi: 10.1128/jvi.14.3.578-586.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Paoletti E. High molecular weight virion-associated RNA of vaccinia. A possible precursor to 8 to 12 S mRNA. J Biol Chem. 1977 Feb 10;252(3):872–877. [PubMed] [Google Scholar]
  21. Paoletti E. In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia. J Biol Chem. 1977 Feb 10;252(3):866–871. [PubMed] [Google Scholar]
  22. Paoletti E., Lipinskas B. R. Soluble endoribonuclease activity from vaccinia virus: specific cleavage of virion-associated high-molecular-weight RNA. J Virol. 1978 Jun;26(3):822–824. doi: 10.1128/jvi.26.3.822-824.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paoletti E., Moss B. Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores. J Virol. 1972 Sep;10(3):417–424. doi: 10.1128/jvi.10.3.417-424.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paoletti E., Moss B. Two nucleic acid-dependent nucleoside triphosphate phosphohydrolases from vaccinia virus. Nucleotide substrate and polynucleotide cofactor specificities. J Biol Chem. 1974 May 25;249(10):3281–3286. [PubMed] [Google Scholar]
  25. Pelham H. R., Sykes J. M., Hunt T. Characteristics of a coupled cell-free transcription and translation system directed by vaccinia cores. Eur J Biochem. 1978 Jan 2;82(1):199–209. doi: 10.1111/j.1432-1033.1978.tb12012.x. [DOI] [PubMed] [Google Scholar]
  26. Pelham H. R. Use of coupled transcription and translation to study mRNA production by vaccinia cores. Nature. 1977 Oct 6;269(5628):532–534. doi: 10.1038/269532a0. [DOI] [PubMed] [Google Scholar]
  27. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  28. Watanabe Y., Sakuma S., Tanaka S. A possible biological function of the protein kinase associated with vaccinia and vesicular stomatitis virions. FEBS Lett. 1974 May 1;41(2):331–334. doi: 10.1016/0014-5793(74)81241-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Wittek R., Menna A., Schümperli D., Stoffel S., Müller H. K., Wyler R. HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA. J Virol. 1977 Sep;23(3):669–678. doi: 10.1128/jvi.23.3.669-678.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yount R. G., Babcock D., Ballantyne W., Ojala D. Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P--N--P linkage. Biochemistry. 1971 Jun 22;10(13):2484–2489. doi: 10.1021/bi00789a009. [DOI] [PubMed] [Google Scholar]

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

RESOURCES