1887

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Volume 75, Issue 6

Expression of the beet yellows closterovirus capsid protein and p24, a capsid protein homologue, and Free

Abstract

The positive-sense RNA genome of beet yellows closterovirus (BYY) encompasses open reading frames (ORFs) for the viral capsid protein (CP, ORF 6) and for a CP homologue (p24, ORF 5). The sequences of the ORFs 5 and 6 were inserted into an expression vector, pQE-9, under the control of the bacteriophage T5 promoter. The proteins were expressed in bacteria, purified, and used for antiserum production in rabbits. The recombinant BYV CP and p24 showed serological cross-reactions when probed with each antiserum on Western blots. The crossreactions of the anti-p24 serum with the CP, and of the anti-CP serum with the p24, were abolished by preadsorption with the heterologous antigens, suggesting that CP and p24 share a common epitope(s) resistant to SDS denaturation. Cross-reactivity of the soluble CP and p24 was also observed in indirect plate-trapped antigen ELISA, whereas virtually none was encountered in double-antibody sandwich ELISA. Using a polyclonal anti-p24 serum preadsorbed with the recombinant CP, the p24 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected indicated that both proteins are predominantly located in the soluble fraction of the host cells. Primer extension analysis of the individual double-stranded forms of the subgenomic RNAs bearing the CP and p24 genes allowed them to be mapped and their 5′ start sites to be located at nucleotide positions 13588 and 12815, respectively, in the complete genome sequence. This corresponds to the 5′ untranslated regions of 52 and 105 nucleotides in the subgenomic RNAs for CP and p24, respectively. The data obtained indicate that the synthesis of both subgenomic RNAs is initiated on a negative RNA strand at an adenosine residue found within the conserved sequence 5′ CCAUUUPyA (shown as positive-sense), which may thus represent a core element of the subgenomic promoter. This conserved sequence also resembles the sequences at the 5′ ends of the CP subgenomic RNAs of tobamoviruses and the family members, the viruses evolutionarily most closely related to BYV.

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© Society for General Microbiology 1994
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1994-06-01
2024-05-09
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References

  1. Agranovsky A. A., Boyko V. P., Karasev A. V., Lunina N. A., Koonin E. V., Dolja V. V. 1991; Nucleotide sequence of the 3′-terminal half of beet yellows closterovirus RNA genome: unique arrangement of eight virus genes. Journal of General Virology 72:15–23
     [Google Scholar]
  2. Agranovsky A. A., Koonin E. V., Boyko V. P., Maiss E., Frotschl R., Lunina N. A., Atabekov J. G. 1994; Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease. Virology 198:311–324
     [Google Scholar]
  3. Ahlquist P., Luckov V., Kaesberg P. 1981; Complete nucleotide sequence of brome mosaic virus RNA3. Journal of Molecular Biology 153:23–38
     [Google Scholar]
  4. Banerjee A. K. 1980; 5′-Terminal cap structure in eucaryotic messenger ribonucleic acids. Microbiological Reviews 44:175–205
     [Google Scholar]
  5. Bar-Joseph M., Hull R. 1974; Purification and partial characterization of sugar beet yellows virus. Virology 62:552–562
     [Google Scholar]
  6. Bar-Joseph M., Murant A. F. 1982; Closterovirus group. MI/AAB Descriptions of Plant Viruses260
     [Google Scholar]
  7. Barker R. F., Jarvis N. P., Thompson D. V., Loesch-Fries L. S., Fiall T. C. 1983; Complete nucleotide sequence of alfalfa mosaic virus RNA3. Nucleic Acids Research 11:2881–2891
     [Google Scholar]
  8. Boccard F., Baulcombe D. 1983; Mutational analysis of cis-acting sequences and gene function in RNA3 of cucumber mosaic virus. Virology 193:563–578
     [Google Scholar]
  9. Boyko V. P., Karasev A. V., Agranovsky A. A., Koonin E. V., Dolja V. V. 1992; Coat protein gene duplication in a filamentous RNA virus of plants. Proceedings of the National Academy of Sciences U.S.A.: 899156–9160
     [Google Scholar]
  10. Brunstedt J., Moseley J., Hull R. 1991; Nucleotide sequence of cDNA encoding the coat protein of beet yellows virus. Virus Genes 5:267–272
     [Google Scholar]
  11. Carpenter J. M., Kassanis B., White R. F. 1977; The protein and nucleic acid of beet yellows virus. Virology 77:101–109
     [Google Scholar]
  12. Clark M. F., Adams A. N. 1977; Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475–483
     [Google Scholar]
  13. Coffin R. S., Coutts R. H. A. 1993; The closteroviruses, capilloviruses and other similar viruses: a short review. Journal of General Virology 74:1475–1483
     [Google Scholar]
  14. Davies C., Symons R. H. 1988; Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA3. Virology 165:216–224
     [Google Scholar]
  15. Dolja V. V., Karasev A. V., Agranovsky A. A. 1990; Organization of the beet yellows closterovirus genome. In New Aspects of Positive-Strand RNA Viruses pp 31–35 Rueckert R., Brinton M. Edited by Washington D.C: ASM Publications;
     [Google Scholar]
  16. Dolja V. V., Boyko V. P., Agranovsky A. A., Koonin E. V. 1991; Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: two families with distinct patterns of sequence and probably structure conservation. Virology 184:79–86
     [Google Scholar]
  17. Donald R. G. K., Zhou H., Jackson A. O. 1993; Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology 195:659–668
     [Google Scholar]
  18. Fichot O., Girard M. 1990; An improved method for sequencing of RNA templates. Nucleic Acids Research 18:61–62
     [Google Scholar]
  19. Godefroy-Colburn T., Gagey M.-J., Berna A., Stussi-Garaud C. 1986; A non-structural protein of alfalfa mosaic virus in the walls of infected tobacco cells. Journal of General Virology 67:2233–2239
     [Google Scholar]
  20. Goelet P., Lomonossoff G. P., Butler P. J. G., Akam M. E., Gait M. J., Karn J. 1982; Nucleotide sequence of tobacco mosaic virus RNA. Proceedings of the National Academy of Sciences U.S.A.: 795818–5822
     [Google Scholar]
  21. Harlow E., Lane D. 1988 Antibodies: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  22. Hayakawa T., Mizukami M., Nakajima M., Suzuki M. 1989; Complete nucleotide sequence of RNA3 from cucumber mosaic virus (CMV) strain O: comparative study of nucleotide sequences and amino acid sequences among CMV strains O, Q, D and Y. Journal of General Virology 70:499–504
     [Google Scholar]
  23. Hochuli E., Bannwath W., Döbeli H., Gentz R., Stüber D. 1988; Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio /Technology 6:1321–1325
     [Google Scholar]
  24. Karasawa A., Nakaho K., Kakutani T., Minobe Y., Ehara Y. 1991; Nucleotide sequence of RNA3 of peanut stunt cucumovirus. Virology 185:464–467
     [Google Scholar]
  25. Karasev A. V., Agranovsky A. A., Rogov V. V., Miroshnichenko N. A., Dolja V. V., Atabekov J. G. 1989; Virion RNA of beet yellows closterovirus: cell-free translation and some properties. Journal of General Virology 70:241–245
     [Google Scholar]
  26. Kaufmann A., Koenig R., Leseman D.-E. 1992; Tissue print immunoblotting reveals' an uneven distribution of beet necrotic yellow vein and beet soil-borne viruses in sugarbeets. Archives of Virology 126:329–335
     [Google Scholar]
  27. Koenig R. 1981; Indirect ELISA methods for the broad specificity detection of plant viruses. Journal of General Virology 55:53–62
     [Google Scholar]
  28. Koonin E. V., Dolja V. V. 1993; Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. CRC Critical Reviews in Biochemistry and Molecular Biology 28:375–430
     [Google Scholar]
  29. Kozak M. 1986; Bifunctional messenger RNAs in eucaryotes. Cell 47:481–183
     [Google Scholar]
  30. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 27680–685
     [Google Scholar]
  31. Morris T. J., Dodds J. A. 1979; Isolation and analysis of doublestranded RNA from virus-infected plant and fungal tissue. Phytopathology 69:854–858
     [Google Scholar]
  32. Owen J., Shintaku M., Aeschleman P., Bentahar S., Palukaitis P. 1990; Nucleotide sequence and evolutionary relationships of cucumber mosaic virus (CMV) strains: CMV RNA 3. Journal of General Virology 71:2243–2249
     [Google Scholar]
  33. Romero J., Dzianott A. M., Bujarsky J. J. 1992; The nucleotide sequence and genome organization of the RNA2 and RNA3 segments in broad bean mottle virus. Virology 187:671–681
     [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989; . Molecular Cloning: A Laboratory Manual, 2nd edn.. New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  35. Shelbourn S. L., Day P. R., Buck K. W. 1988; Relationships and functions of virus double-stranded RNA in a P4 killer strain of Ustilago maydis . Journal of General Virology 69:975–982
     [Google Scholar]
  36. Solis I., Garcia-Arenal F. 1990; The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus. Virology 177:553–558
     [Google Scholar]
  37. Richards K. E., Tamada T. 1992; Mapping functions on the multipartite genome of beet necrotic yellow vein virus. Annual Review of Phytopathology 30:291–313
     [Google Scholar]
  38. Van Regenmortel M. H. V., Burckard J. 1980; Detection of a wide spectrum of tobacco mosaic virus strains by indirect enzyme-linked immunosorbent assays (ELISA). Virology 106:327–334
     [Google Scholar]
  39. Verwoerd T. C., Dekker B. M., Hockema A. 1989; A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research 17:2362
     [Google Scholar]
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Expression of the beet yellows closterovirus capsid protein and p24, a capsid protein homologue, in vitro and in vivo
J. Gen. Virol. 75, 1431 (1994); https://doi.org/10.1099/0022-1317-75-6-1431
/content/journal/jgv/10.1099/0022-1317-75-6-1431
/content/journal/jgv/10.1099/0022-1317-75-6-1431
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