Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment Free

Abstract

The genome of broad bean mottle bromovirus (BBMV) contains three positive-sense ssRNA segments, each capped with mGpppA. Full-length transcribable cDNA clones for four strains of BBMY were constructed by employing reverse transcriptase-PCR (RT-PCR) and a high fidelity Vent DNA polymerase. The transcribed BBMY RNAs contained a 5′ non-viral G residue and, although delayed, produced symptoms similar to those observed in plants infected with authentic virion RNAs. The transcripts replicated inefficiently in protoplasts. In contrast, transcript-derived progeny BBMV RNAs had the repaired termini, were as infectious as the authentic BBMV RNAs and replicated to high levels in protoplasts. translation of synthetic RNAs confirmed the previously proposed gene expression strategy for BBMV. Sequencing of virion RNAs from the Bawden strain revealed two forms of BBMV RNA 3 components, the longer form containing 21 5′ extra nucleotides derived by the duplication of two short 5′ leader regions. The relative concentration of the two RNA 3 forms was found to be host-dependent, with the longer form prevailing in broad bean and infections and the shorter form in bean infections.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-3-693
1994-03-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/3/JV0750030693.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-3-693&mimeType=html&fmt=ahah

References

  1. Ahlquist P., Dasgupta R., Kaesberg P. 1984a; Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication. Journal of Molecular Biology 172:369–383
    [Google Scholar]
  2. Ahlquist P., French R., Janda M., Loesch-Fries S. 1984b; Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proceedings of the National Academy of Sciences U.S.A: 817066–7070
    [Google Scholar]
  3. Allison R., Janda M., Ahlquist P. 1988; Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus. Journal of Virology 62:3581–3588
    [Google Scholar]
  4. Allison R., Janda M., Ahlquist P. 1989; Sequence of cowpea chlorotic mottle virus RNA2 and 3 and evidence of a recombination event during bromovirus evolution. Virology 172:321–330
    [Google Scholar]
  5. Angenent G. C., Posthumus E., Bol J. F. 1989; Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2. Virology 173:68–76
    [Google Scholar]
  6. Aoki S., Takebe I. 1969; Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39:439–148
    [Google Scholar]
  7. Bawden F. C., Chaudhuri R. P., Kassanis B. 1951; Some properties of broad bean mottle virus. Annals of Applied Biology 38:774–784
    [Google Scholar]
  8. Bujarski J. J., Miller W. A. 1992; Use of in vitro transcription to study gene expression and replication of spherical, positive sense RNA plant viruses. In Genetic Engineering with Plant Viruses pp 115–147 Wilson T. M. A., Davies J. W. Edited by Boca Raton: CRC Press;
    [Google Scholar]
  9. Domier L. L., Franklin K. M., Hunt A. G., Rhoads R. E., Shaw J. G. 1989; Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proceedings of the National Academy of Sciences U.S.A.: 863509–3513
    [Google Scholar]
  10. Dunn J. J., Studier F. W. 1983; Complete nucleotide sequence of bacteriophage T7 elements. Journal of Molecular Biology 166:435–177
    [Google Scholar]
  11. Dzianott M. A., Bujarski J. J. 1989; Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs. Proceedings of the National Academy of Sciences U.S.A.: 864823–1827
    [Google Scholar]
  12. Dzianott M. A., Bujarski J. J. 1991; Nucleotide sequence and genome organization of the RNA-1 segment in two bromoviruses: broad bean mottle virus and cowpea chlorotic mottle virus. Virology 185:553–562
    [Google Scholar]
  13. 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]
  14. Gilmer D., Allmang C., Ehresmann C., Guilley H., Richards K., Jonard G., Ehresmann D. 1993; The secondary structure of the 5′-noncoding region of beet necrotic yellow vein virus RNA3: evidence for a role in viral RNA replication. Nucleic Acids Research 21:1389–1395
    [Google Scholar]
  15. Hayes R. J., Buck K. W. 1990; Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction. Journal of General Virology 71:2503–2508
    [Google Scholar]
  16. Janda M., French R., Ahlquist P. 1987; High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cDNA and effects of 5′ extensions on transcript infectivity. Virology 158:259–262
    [Google Scholar]
  17. Jones R. W., Jackson A. O., Morris T. J. 1990; Defective- interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts. Virology 176:539–545
    [Google Scholar]
  18. Kaesberg P. 1987; Organization of tripartite plant virus genomes: the genome of brome mosaic virus. In The Molecular Biology of the Positive-strand RNA Viruses pp 219–236 Rowlands D. J., Mayo M. A., Mahy B. W. J. Edited by London: Academic Press;
    [Google Scholar]
  19. Kroner P., Richards D., Traynor P., Ahlquist P. 1989; Defined mutations in a small region of the brome mosaic virus 2a gene cause diverse temperature-sensitive RNA replication phenotypes. Journal of Virology 63:5302–5309
    [Google Scholar]
  20. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  21. Lai M. M. C. 1992; RNA recombination in animal and plant viruses. Microbiological Reviews 56:61–79
    [Google Scholar]
  22. Lane L. 1981; Bromoviruses. In Handbook of Plant Virus Infections and Comparative Diagnosis pp 333–376 Kurstak E. Edited by Amsterdam: Elsevier/North-Holland;
    [Google Scholar]
  23. Langereis K., Mugnier M., Cornelissen B. J. C., Pinck L., Bol J. F. 1986; Variable repeats and poly(A)-stretches in the leader sequence of alfalfa mosaic virus RNA3. Virology 154:409–414
    [Google Scholar]
  24. Lohff C. J., Cease K. B. 1992; PCR using a thermostable polymerase with 3′ to 5′ exonuclease activity generates blunt products suitable for direct cloning. Nucleic Acids Research 20:144
    [Google Scholar]
  25. Makkouk K. M., Bos L., Rizkallah A., Azzam O. I., Katul L. 1988; Broad bean mottle virus: identification, host range, serology, and occurrence on faba bean (Vicia faba) in West Asia and North Africa. Netherlands Journal of Plant Pathology 94:195–212
    [Google Scholar]
  26. Mattila P., Korpela J., Tenkanen T., Pitkanen K. 1991; Fidelity of DNA synthesis by the Thermococcus littoralis DNA 699 Short communication polymerase - an extremely heat stable enzyme with proofreading activity. Nucleic Acids Research 19:4967–4973
    [Google Scholar]
  27. Miller W. A., Bujarski J. J., Dreher T. W., Hall T. C. 1986; Minus-strand initiation by brome mosaic virus replicase within the 3′-tRNA-like structure of native and modified RNA templates. Journal of Molecular Biology 187:537–546
    [Google Scholar]
  28. Neeleman L., Van Der Kuyl A. C., Bol J. F. 1991; Role of alfalfa mosaic virus coat protein gene in symptom formation. Virology 181:687–693
    [Google Scholar]
  29. Pogue G. P., Hall T. C. 1992; The requirement for a 5′ stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation. Journal of Virology 66:674–684
    [Google Scholar]
  30. Pogue G. P., Marsh L. E., Hall T. C. 1990; Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate ( + )-strand replication. Virology 178:152–160
    [Google Scholar]
  31. Romero J., Dzianott M. A., Bujarski 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]
  32. Romero J., Huang Q., Pogany J., Bujarski J. J. 1993; Characterization of defective interfering RNA components that increase symptom severity of broad bean mottle virus infections. Virology 194:576–584
    [Google Scholar]
  33. Sambrook J., Fritch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn.. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Van Der Vossen E. A. G., Neeleman L., Bol J. F. 1993; Role of the 5′ end leader sequence of alfalfa mosaic virus RNA3 in replication and translation of the viral RNA. Nucleic Acids Research 21:1361–1367
    [Google Scholar]
  35. Xiong Z., Lommel S. A. 1991; Red clover necrotic mosaic virus infectious transcripts synthesized in vitro . Virology 182:388–392
    [Google Scholar]
  36. Zucker M., Stiegler P. 1981; Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Research 9:133–148
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-3-693
Loading
/content/journal/jgv/10.1099/0022-1317-75-3-693
Loading

Data & Media loading...

Most cited Most Cited RSS feed