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. 1978 Dec;28(3):905–916. doi: 10.1128/jvi.28.3.905-916.1978

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

W Bossart, E Paoletti, D L Nuss
PMCID: PMC525815  PMID: 215785

Abstract

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.

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Selected References

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  1. Aloni Y., Dhar R., Laub O., Horowitz M., Khoury G. Novel mechanism for RNA maturation: the leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3686–3690. doi: 10.1073/pnas.74.9.3686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Berget S. M., Moore C., Sharp P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171–3175. doi: 10.1073/pnas.74.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Boone R. F., Moss B. Methylated 5'-terminal sequences of vaccinia virus mRNA species made in vivo at early and late times after infection. Virology. 1977 Jun 1;79(1):67–80. doi: 10.1016/0042-6822(77)90335-x. [DOI] [PubMed] [Google Scholar]
  6. Boone R. F., Moss B. Sequence complexity and relative abundance of vaccinia virus mRNA's synthesized in vivo and in vitro. J Virol. 1978 Jun;26(3):554–569. doi: 10.1128/jvi.26.3.554-569.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bossart W., Nuss D. L., Paoletti E. Effect of UV irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation. J Virol. 1978 Jun;26(3):673–680. doi: 10.1128/jvi.26.3.673-680.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chow L. T., Gelinas R. E., Broker T. R., Roberts R. J. An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell. 1977 Sep;12(1):1–8. doi: 10.1016/0092-8674(77)90180-5. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Geshelin P., Berns K. I. Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J Mol Biol. 1974 Oct 5;88(4):785–796. doi: 10.1016/0022-2836(74)90399-4. [DOI] [PubMed] [Google Scholar]
  11. Hickey E. D., Weber L. A., Baglioni C. Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Proc Natl Acad Sci U S A. 1976 Jan;73(1):19–23. doi: 10.1073/pnas.73.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hickey E. D., Weber L. A., Baglioni C. Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability. Biochem Biophys Res Commun. 1978 Jan 30;80(2):377–383. doi: 10.1016/0006-291x(78)90687-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kaempfer R., Rosen H., Israeli R. Translational control: recognition of the methylated 5' end and an internal sequence in eukaryotic mRNA by the initiation factor that binds methionyl-tRNAfMet. Proc Natl Acad Sci U S A. 1978 Feb;75(2):650–654. doi: 10.1073/pnas.75.2.650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kates J., Beeson J. Ribonucleic acid synthesis in vaccinia virus. II. Synthesis of polyriboadenylic acid. J Mol Biol. 1970 May 28;50(1):19–33. doi: 10.1016/0022-2836(70)90101-4. [DOI] [PubMed] [Google Scholar]
  17. Klessig D. F. Two adenovirus mRNAs have a common 5' terminal leader sequence encoded at least 10 kb upstream from their main coding regions. Cell. 1977 Sep;12(1):9–21. doi: 10.1016/0092-8674(77)90181-7. [DOI] [PubMed] [Google Scholar]
  18. Lodish H. F. Translational control of protein synthesis. Annu Rev Biochem. 1976;45:39–72. doi: 10.1146/annurev.bi.45.070176.000351. [DOI] [PubMed] [Google Scholar]
  19. Lowery C., Richardson J. P. Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesi termination factor p. I. Enzymatic properties and effects of inhibitors. J Biol Chem. 1977 Feb 25;252(4):1375–1380. [PubMed] [Google Scholar]
  20. Lowery C., Richardson J. P. Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesis termination factor p. II. Influence of synthetic RNA homopolymers and random copolymers on the reaction. J Biol Chem. 1977 Feb 25;252(4):1381–1385. [PubMed] [Google Scholar]
  21. Martin S. A., Paoletti E., Moss B. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9322–9329. [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Nuss D. L., Paoletti E. Methyl group analysis of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus. J Virol. 1977 Jul;23(1):110–116. doi: 10.1128/jvi.23.1.110-116.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paoletti E., Grady L. J. Transcriptional complexity of vaccinia virus in vivo and in vitro. J Virol. 1977 Sep;23(3):608–615. doi: 10.1128/jvi.23.3.608-615.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Paoletti E., Lipinskas B. R. The role of ATP in the biogenesis of vaccinia virus mRNA in vitro. Virology. 1978 Jun 15;87(2):317–325. doi: 10.1016/0042-6822(78)90137-x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Roberts B. E., Paterson B. M. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2330–2334. doi: 10.1073/pnas.70.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shafritz D. A., Weinstein J. A., Safer B., Merrick W. C., Weber L. A., Hickey E. D., Baglioni C. Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3. Nature. 1976 May 27;261(5558):291–294. doi: 10.1038/261291a0. [DOI] [PubMed] [Google Scholar]
  34. Weber L. A., Hickey E. D., Nuss D. L., Baglioni C. 5'-Terminal 7-methylguanosine and mRNA function: influence of potassium concentration on translation in vitro. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3254–3258. doi: 10.1073/pnas.74.8.3254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Wei C. M., Moss B. Methylation of newly synthesized viral messenger RNA by an enzyme in vaccinia virus. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3014–3018. doi: 10.1073/pnas.71.8.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]

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