1887

Abstract

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-73-11-2775
1992-11-01
2021-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/73/11/JV0730112775.html?itemId=/content/journal/jgv/10.1099/0022-1317-73-11-2775&mimeType=html&fmt=ahah

References

  1. Assink A. M., Swaans H., van Kammen A. 1973; The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves. Virology 53:384–391
    [Google Scholar]
  2. Bienz K., Egger D., Rasser Y., Bossart W. 1983; Intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures. Virology 131:39–48
    [Google Scholar]
  3. Bienz K., Egger D., Pasamontes L. 1987; Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology 160:220–226
    [Google Scholar]
  4. Bienz K., Egger D., Troxler M., Pasamontes L. 1990; Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. Journal of Virology 64:1156–1163
    [Google Scholar]
  5. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. 1984; A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-body on Western blots. Analytical Biochemistry 136:175–179
    [Google Scholar]
  6. de Zoeten G. A., Assink A. M., van Kammen A. 1974; Association of cowpea mosaic virus-induced double-stranded RNA with a cytopathological structure in infected cells. Virology 59:341–355
    [Google Scholar]
  7. 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]
  8. 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]
  9. Eggen R., van Kammen A. 1988; RNA replication in como-viruses. In RNA Genetics. vol 1 pp. 49–69 Edited by Ahlquist P., Holland J., Domingo E. Boca Raton: CRC Press;
    [Google Scholar]
  10. 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]
  11. 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]
  12. Flanegan J. B., Baltimore D. 1977; A poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proceedings of the National Academy of Sciences, U. S. A. 74:3677–3680
    [Google Scholar]
  13. Franssen H., Leunissen J., Goldbach R., Lomonossoff G. P., Zimmern D. 1984a; Homologous sequences in non-structural proteins from cowpea mosaic virus and picomaviruses. EMBO Journal 3:855–861
    [Google Scholar]
  14. 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]
  15. Franssen H., Goldbach R., van Kammen A. 1984c; Translation of bottom component RNA of cowpea mosaic virus in reticulocyte lysate: faithful proteolytic processing of the primary translation product. Virus Research 1:39–49
    [Google Scholar]
  16. Goldbach R. W., Rezelman G., van Kammen A. 1980; Independent replication and expression of B-component RNA of cowpea mosaic virus. Nature, London 286:297–300
    [Google Scholar]
  17. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. 1988; A novel superfamily of nucleoside triphosphatebinding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination. FEBS Letters 235:16–24
    [Google Scholar]
  18. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. 1989; Two related superfamilies of putative helicases involved in replication, recombination, repair, and expression of DNA and RNA genomes. Nucleic Acids Research 17:4713–4730
    [Google Scholar]
  19. Groebe D. R., Chung A. E., Ho C. 1990; Cationic lipid-mediated co-transfection of insect cells. Nucleic Acids Research 18:4033
    [Google Scholar]
  20. Hink W. F. 1970; Established insect cell line from the cabbage looper, Trichoplusia ni. Nature, London 226:466–467
    [Google Scholar]
  21. Korneluk R. G., Quan F., Gravel R. A. 1985; Rapid and reliable dideoxy sequencing of double-stranded DNA. Gene 40:317–323
    [Google Scholar]
  22. Kunkel T. A. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences, U. S. A. 82:488–192
    [Google Scholar]
  23. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  24. Lomonossoff G. P., Shanks M. 1983; The nucleotide sequence of cowpea mosaic virus B RNA. EMBO Journal 2:2253–2258
    [Google Scholar]
  25. Luckow V. A., Summers M. D. 1988; Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167:56–71
    [Google Scholar]
  26. Peters S. A., Voorhorst W. G. B., Wery J., Wellink J., van Kammen A. 1992; A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins. Virology (in press)
    [Google Scholar]
  27. Poch O., Sauvaget I., Delarue M., Tordo N. 1989; Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO Journal 8:3867–3874
    [Google Scholar]
  28. 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]
  29. Rezelman G., Franssen H. J., Goldbach R. W., Ie T. S., van Kammen A. 1982; Limits to the independence of bottom component RNA of cowpea mosaic virus. Journal of General Virology 60:335–342
    [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. Roosien J., Belsham G. J., Ryan M. D., King A. M. Q., Vlak J. M. 1990; Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors. Journal of General Virology 71:1703–1711
    [Google Scholar]
  32. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: A Laboratory Manual. , 2nd edn.. New York: Cold Spring Harbor Laboratory;
    [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 Bulletin, no. 1555. College Station: Texas Agricultural Experiment Station;
    [Google Scholar]
  34. Tobias J. W., Shrader T. E., Rocap G., Varshavsky A. 1991; The N-end rule in bacteria. Science 254:1374–1377
    [Google Scholar]
  35. van Bokhoven H., Wellink J., Usmany M., Vlak J. M., Goldbach R., van Kammen A. 1990; Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors. Journal of General Virology 71:2509–2517
    [Google Scholar]
  36. van Bokhoven H., Mulders M., Wellink J., Vlak J. M., Goldbach R., van Kammen A. 1991; Evidence for dissimilar properties of comoviral and picomaviral RNA polymerases. Journal of General Virology 72:567–572
    [Google Scholar]
  37. van der wilk F., van lent J. W. M., Vlak J. M. 1987; Immunogold detection of polyhedrin, pl0 and virion antigens in Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells. Journal of General Virology 68:2615–2623
    [Google Scholar]
  38. van Lent J. W. M., Groenen J. T. M., Klinge-Roode E. C., Rohrmann G. F., Zuidema D., Vlak J. M. 1990; Localization of the 34 kDa polyhedron envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. Archives of Virology 111:103–114
    [Google Scholar]
  39. Vaughn J. L., Goodwin R. H., Tompkins 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]
  40. Verver J., Goldbach R., Garcia J. A., Vos P. 1987; In vitro expression of a full-length DNA copy of cowpea mosaic virus B-RNA: identification of the B RNA encoded 24-kd protein as viral protease. EMBO Journal 6:549–554
    [Google Scholar]
  41. Vos P., Verver J., Jaegle M., Wellink J., van Kammen A., Goldbach R. 1988a; Two viral proteins involved in the proteolytic processing of the cowpea mosaic virus polyproteins. Nucleic Acids Research 16:1967–1985
    [Google Scholar]
  42. Vos P., Jaegle M., Wellink J., Verver J., Eggen R., van Kammen A., Goldbach R. 1988b; Infectious RNA transcripts derived from full-length DNA copies of the genomic RNAs of cowpea mosaic virus. Virology 165:33–41
    [Google Scholar]
  43. 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]
  44. Wellink J., Rezelman G., Goldbach R., Beyreuther K. 1986; Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus. Journal of Virology 59:50–58
    [Google Scholar]
  45. 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 of Virology 61:236–238
    [Google Scholar]
  46. Wellink J., van Lent J., Goldbach R. 1988; Detection of viral proteins in cytopathic structures in cowpea protoplasts infected with cowpea mosaic virus. Journal of General Virology 69:751–755
    [Google Scholar]
  47. Zabel P., Weenen-Swaans H., van Kammen A. 1974; In vitro replication of cowpea mosaic virus RNA I. Isolation and properties of the membrane-bound replicase. Journal of Virology 14:1049–1055
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-73-11-2775
Loading
/content/journal/jgv/10.1099/0022-1317-73-11-2775
Loading

Data & Media loading...

Most cited this month 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