1887

Journal of General Virology header logo

Volume 71, Issue 11

Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors Free

Abstract

The baculovirus expression system has been used to produce non-structural proteins encoded by bottom- component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of nuclear polyhedrosis virus (/lcNPV). Upon infection of cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In cells infected by recombinant NPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology 1990
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-11-2509
1990-11-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/11/JV0710112509.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-11-2509&mimeType=html&fmt=ahah

References

  1. Argos P., Kamer G., Nicklin M. J. H., Wimmer E. 1984; Similarity in gene organization and homology between proteins of animal picornaviruses and plant comovirus suggest a common ancestry of these virus families. Nucleic Acids Research 12:7251–7276
     [Google Scholar]
  2. Blake M. S., Johnston K. H., Russel-Jones G. J., Gotschlich E. C. 1984; A rapid, sensitive method for detection of alkaline phosphatase-conjugated antibody on Western blots. Analytical Biochemistry 136:175–179
     [Google Scholar]
  3. Bustos M. M., Luckow V. A., Griffing L. R., Summers M. D., Hall T. C. 1987; Expression, glycosylation and secretion of phaseolin in a baculovirus system. Plant Molecular Biology 10:475–488
     [Google Scholar]
  4. De Vries S., Hoge H., Bisseling T. 1988; Isolation of total and polysomal RNA from plant tissues. Plant Molecular Biology Manual B6:1–13 Dordrecht: Kluwer Academic Publishers;
     [Google Scholar]
  5. Dorssers L., Van Der Meer J., Van Kammen A., Zabel P. 1983; The cowpea mosaic virus RNA replication complex and the host- encoded RNA-dependent RNA polymerase-template complex are functionally different. Virology 125:155–174
     [Google Scholar]
  6. Dorssers L., Van Der Krol S., Van Der Meer J., Van Kammen A., Zabel P. 1984; Purification of cowpea mosaic virus RNA replication complex: identification of a 110,000 dalton polypeptide responsible for RNA chain elongation. Proceedings of the National Academy of Sciences, U.S.A 81:1951–1955
     [Google Scholar]
  7. Eggen R., Van Kammen A. 1988; RNA replication in comoviruses. In RNA Genetics 1 pp. 49–69 Ahlquist P., Holland J., Domingo E. Edited by Boca Raton: CRC Press;
     [Google Scholar]
  8. Eggen R., Kaan A., Goldbach R., Van Kammen A. 1988; Cowpea mosaic virus RNA replication in crude membrane fractions from infected cowpea and Chenopodium amaranticolor . Journal of General Virology 69:2711–2720
     [Google Scholar]
  9. Eggen R., Verver J., Wellink J., De Jong A., Goldbach R., Van Kammen A. 1989; Improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cDNA: impact of terminal nucleotide sequences. Virology 173:447–455
     [Google Scholar]
  10. Franssen H., Leunissen J., Goldbach R., Lomonossoff G. P., Zimmern D. 1984a; Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. EMBO Journal 3:855–861
     [Google Scholar]
  11. Franssen H., Moerman M., Rezelman G., Goldbach R. 1984b; Evidence that the 32,000-dalton protein encoded by bottom- component RNA of cowpea mosaic virus is a proteolytic processing enzyme. Journal of Virology 50:183–189
     [Google Scholar]
  12. Goldbach R.W. 1986; Molecular evolution of plant RNA viruses. Annual Review of Phytopathology 24:289–310
     [Google Scholar]
  13. Goldbach R. 1987; Genome similarities between plant and animal RNA viruses. Microbiological Sciences 4:197–202
     [Google Scholar]
  14. Goldbach R., Vank Kammen A. 1985; Structure, replication and expression of the bipartite genome of cowpea mosaic virus. In Molecular Plant Virology 2 pp. 83–120 Davies J. W. Edited by Boca Raton: CRC Press;
     [Google Scholar]
  15. Goldbach R. W., Rezelman G., Van Kammen A. 1980; Independent replication and expression of B-component RNA of cowpea mosaic virus. Nature; London: 286297–300
     [Google Scholar]
  16. Hink W. F. 1970; Established insect cell line from the cabbage looper, Trichoplusia ni . Nature; London: 226466–467
     [Google Scholar]
  17. Hooft Van Iddekinge B. J. L., Smith G. E., Summers M. D. 1983; Nucleotide sequence of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. Virology 131:561–565
     [Google Scholar]
  18. Korneluk R. G., Quan F., Gravel R. A. 1985; Rapid and reliable dideoxy sequencing of double-stranded DNA. Gene 40:317–323
     [Google Scholar]
  19. Kozak M. 1986; Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
     [Google Scholar]
  20. Kunkel T. A. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences, U.S.A 82:488–492
     [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
     [Google Scholar]
  22. Lomonossoff G. P., Shanks M. 1983; The nucleotide sequence of cowpea mosaic virus B-RNA. EMBO Journal 2:2253–2258
     [Google Scholar]
  23. Luckow V. A., Summers M. D. 1988a; Trends in the development of baculovirus expression vectors. Bio/Technology 6:47–55
     [Google Scholar]
  24. Luckow V. A., Summers M. D. 1988b; Signals important for high- level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167:56–71
     [Google Scholar]
  25. McMaster G. K., Carmichael G. G. 1977; Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proceedings of the National Academy of Sciences, U.S.A 74:4835–4838
     [Google Scholar]
  26. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. L. 1987; Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. Journal of General Virology 68:1233–1250
     [Google Scholar]
  28. Miller L. K. 1988; Baculoviruses as gene expression vectors. Annual Review of Microbiology 42:177–199
     [Google Scholar]
  29. Possee R. D., Howard S. C. 1987; Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 15:10233–10248
     [Google Scholar]
  30. Richards O. C., Eggen R., Goldbach R., Van Kammen A. 1989; High-level synthesis of cowpea mosaic virus RNA polymerase and protease in Escherichia coli . Gene 78:135–146
     [Google Scholar]
  31. Rohrmann G. F. 1986; Polyhedrin structure. Journal of General Virology 67:1499–1513
     [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A 74:5463–5467
     [Google Scholar]
  33. Summers M. D., Smith G. E. 1987; A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin1555 Texas Agricultural Experiment Station, Texas A & M University, College Station, Tx., U.S.A;
     [Google Scholar]
  34. Van Der Meer J., Dorssers L., Van Kammen A., Zabel P. 1984; The RNA-dependent RNA polymerase of cowpea is not involved in cowpea mosaic virus RNA replication: immunological evidence. Virology 132:413–425
     [Google Scholar]
  35. Van Wezenbeek P., Verver J., Harmsen J., Vos P., Van Kammen A. 1983; Primary structure and gene organization of the middle component RNA of cowpea mosaic virus. EMBO Journal 2:941–946
     [Google Scholar]
  36. Vaughn J. L., Goodwin R. H., Thompkins G. J., McCawley P. 1977; The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13:213–217
     [Google Scholar]
  37. Wellink J., Van Kammen A. 1989; Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins. Journal of General Virology 70:2279–2286
     [Google Scholar]
  38. Wellink J., Rezelman G., Goldbach R., Beyreuther K. 1986; Determination of the proteolytic processing role in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus. Journal of Virology 59:50–58
     [Google Scholar]
  39. Wellink J., Jaegle M., Goldbach R. 1987; Detection of a novel protein encoded by the bottom-component RNA of cowpea mosaic virus, using antibodies raised against a synthetic peptide. Journal ofVirology 61:236–238
     [Google Scholar]
  40. Whitford M., Stewart S., Kuzio J., Faulkner P. 1989; Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. Journal of Virology 63:1393–1399
     [Google Scholar]
  41. Zabel P., Dorssers L., Wernars K., Van Kammen A. 1981; Terminal uridylyl transferase of Vigna unguiculata : purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3′-end of a RNA primer. Nucleic Acids Research 9:2433–2453
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-11-2509
Loading
Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors
J. Gen. Virol. 71, 2509 (1990); https://doi.org/10.1099/0022-1317-71-11-2509
/content/journal/jgv/10.1099/0022-1317-71-11-2509
/content/journal/jgv/10.1099/0022-1317-71-11-2509
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error