Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1990 May;64(5):2280–2289. doi: 10.1128/jvi.64.5.2280-2289.1990

Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression.

R F Massung 1, R W Moyer 1
PMCID: PMC249389  PMID: 2157885

Abstract

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.

Full text

PDF
2280

Images in this article

Selected References

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

  1. Bloom D. C., Massung R., Savage L., Morrison D. K., Moyer R. W. Recruitment to the cytoplasm of a cellular lamin-like protein from the nucleus during a poxvirus infection. Virology. 1989 Mar;169(1):115–126. doi: 10.1016/0042-6822(89)90047-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Broyles S. S., Yuen L., Shuman S., Moss B. Purification of a factor required for transcription of vaccinia virus early genes. J Biol Chem. 1988 Aug 5;263(22):10754–10760. [PubMed] [Google Scholar]
  4. Chang A., Metz D. H. Further investigations on the mode of entry of vaccinia virus into cells. J Gen Virol. 1976 Aug;32(2):275–282. doi: 10.1099/0022-1317-32-2-275. [DOI] [PubMed] [Google Scholar]
  5. Cochran M. A., Mackett M., Moss B. Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. Proc Natl Acad Sci U S A. 1985 Jan;82(1):19–23. doi: 10.1073/pnas.82.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DALES S., KAJIOKA R. THE CYCLE OF MULTIPLICATION OF VACCINIA VIRUS IN EARLE'S STRAIN L CELLS. I. UPTAKE AND PENETRATION. Virology. 1964 Nov;24:278–294. doi: 10.1016/0042-6822(64)90167-9. [DOI] [PubMed] [Google Scholar]
  7. Dales S. Early events in cell-animal virus interactions. Bacteriol Rev. 1973 Jun;37(2):103–135. doi: 10.1128/br.37.2.103-135.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dales S., Pogo B. G. Biology of poxviruses. Virol Monogr. 1981;18:1–109. doi: 10.1007/978-3-7091-8625-1. [DOI] [PubMed] [Google Scholar]
  9. Easterbrook K. B. Controlled degradation of vaccinia virions in vitro: an electron microscopic study. J Ultrastruct Res. 1966 Mar;14(5):484–496. doi: 10.1016/s0022-5320(66)80077-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Feldherr C. M., Richmond P. A. Manual enucleation of Xenopus oocytes. Methods Cell Biol. 1978;17:75–79. doi: 10.1016/s0091-679x(08)61135-8. [DOI] [PubMed] [Google Scholar]
  12. Golini F., Kates J. R. A soluble transcription system derived from purified vaccinia virions. J Virol. 1985 Jan;53(1):205–213. doi: 10.1128/jvi.53.1.205-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Katz E., Moss B. Formation of a vaccinia virus structural polypeptide from a higher molecular weight precursor: inhibition by rifampicin. Proc Natl Acad Sci U S A. 1970 Jul;66(3):677–684. doi: 10.1073/pnas.66.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katz E., Moss B. Vaccinia virus structural polypeptide derived from a high-molecular-weight precursor: formation and integration into virus particles. J Virol. 1970 Dec;6(6):717–726. doi: 10.1128/jvi.6.6.717-726.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Morrison D. K., Carter J. K., Moyer R. W. Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase. J Virol. 1985 Sep;55(3):670–680. doi: 10.1128/jvi.55.3.670-680.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morrison D. K., Moyer R. W. Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions. Cell. 1986 Feb 28;44(4):587–596. doi: 10.1016/0092-8674(86)90268-0. [DOI] [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. 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]
  24. Moss B., Rosenblum E. N. Letter: Protein cleavage and poxvirus morphogenesis: tryptic peptide analysis of core precursors accumulated by blocking assembly with rifampicin. J Mol Biol. 1973 Dec 5;81(2):267–269. doi: 10.1016/0022-2836(73)90195-2. [DOI] [PubMed] [Google Scholar]
  25. Moss B., Rosenblum E. N., Paoletti E. Polyadenylate polymerase from vaccinia virions. Nat New Biol. 1973 Sep 12;245(141):59–63. doi: 10.1038/newbio245059a0. [DOI] [PubMed] [Google Scholar]
  26. Moss B., Rosenblum E. N. Vaccinia virus polyriboadenylate polymerase: convalent linkage of the product with polyribonucleotide and polydeoxyribonucleotide primers. J Virol. 1974 Jul;14(1):86–98. doi: 10.1128/jvi.14.1.86-98.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moyer R. W., Rothe C. T. The white pock mutants of rabbit poxvirus. I. Spontaneous host range mutants contain deletions. Virology. 1980 Apr 15;102(1):119–132. doi: 10.1016/0042-6822(80)90075-6. [DOI] [PubMed] [Google Scholar]
  28. Moyer R. W. The role of the host cell nucleus in vaccinia virus morphogenesis. Virus Res. 1987 Sep;8(3):173–191. doi: 10.1016/0168-1702(87)90014-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Oie M., Ichihashi Y. Characterization of vaccinia polypeptides. Virology. 1981 Aug;113(1):263–276. doi: 10.1016/0042-6822(81)90153-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Pennington T. H. Vaccinia virus morphogenesis: a comparison of virus-induced antigens and polypeptides. J Gen Virol. 1973 Apr;19(1):65–79. doi: 10.1099/0022-1317-19-1-65. [DOI] [PubMed] [Google Scholar]
  33. Puckett C., Moss B. Selective transcription of vaccinia virus genes in template dependent soluble extracts of infected cells. Cell. 1983 Dec;35(2 Pt 1):441–448. doi: 10.1016/0092-8674(83)90177-0. [DOI] [PubMed] [Google Scholar]
  34. Rohrmann G., Moss B. Transcription of vaccinia virus early genes by a template-dependent soluble extract of purified virions. J Virol. 1985 Nov;56(2):349–355. doi: 10.1128/jvi.56.2.349-355.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rohrmann G., Yuen L., Moss B. Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell. 1986 Sep 26;46(7):1029–1035. doi: 10.1016/0092-8674(86)90702-6. [DOI] [PubMed] [Google Scholar]
  36. Shuman S., Surks M., Furneaux H., Hurwitz J. Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme). J Biol Chem. 1980 Dec 10;255(23):11588–11598. [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Wilton S., Dales S. Influence of RNA polymerase II upon vaccinia virus-related translation examined by means of alpha-amanitin. Virus Res. 1986 Sep;5(4):323–341. doi: 10.1016/0168-1702(86)90027-4. [DOI] [PubMed] [Google Scholar]
  40. Wilton S., Dales S. Relationship between RNA polymerase II and efficiency of vaccinia virus replication. J Virol. 1989 Apr;63(4):1540–1548. doi: 10.1128/jvi.63.4.1540-1548.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES