Replication and translation of cowpea mosaic virus RNAs are tightly linked

  • J. Wellink
  • H. van Bokhoven
  • O. Le Gall
  • J. Verver
  • A. van Kammen
Conference paper
Part of the Archives of Virology Supplementum book series (ARCHIVES SUPPL, volume 9)


The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts trans- fected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV- specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.


Replicative Protein Mutant RNAs Infectious cDNA Clone Soybean Kunitz Trypsin Inhibitor Cowpea Mosaic Virus 


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  1. 1.
    Peters SA, Voorhorst WGB, Wellink J, Van Kämmen A (1992) Processing of VPg-containing polyproteins encoded by the B RNA from cowpea mosaic virus. Virology 191: 90–97PubMedCrossRefGoogle Scholar
  2. 2.
    Peters SA, Voorhorst WGB, Wery J, Wellink J, Van Kämmen A (1992) A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins. Virology 191: 81–89PubMedCrossRefGoogle Scholar
  3. 3.
    Franssen H, Leunissen J, Goldbach R, Lomonossoff G, Zimmern D (1984) Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. EMBO J 3: 855–861Google Scholar
  4. 4.
    Argos P, Kamer G, Nickiin MJH, Wimmer W (1984) Similarity in gene organisation and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families. Nucleic Acids Res 12: 7251–7267PubMedCrossRefGoogle Scholar
  5. 5.
    Bernstein HD, Sarnow P, Baltimore D (1986) Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. J Virol 60: 1040–1049PubMedGoogle Scholar
  6. 6.
    Hagino-Yamayishi U, Nomoto A (1989) In vitro construction of poliovirus defective interfering particles. J Virol 63: 5386–5392Google Scholar
  7. 7.
    Van Bokhoven H, Le Gall O, Kasteel D, Verver J, Wellink J, Van Kämmen A (1993) Cis and trans acting elements in cowpea mosaic virus RNA replication. Virology 195: 377–386PubMedCrossRefGoogle Scholar
  8. 8.
    Wellink J, Van Kämmen A (1989) Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins. J Gen Virol 70: 2279–2286CrossRefGoogle Scholar
  9. 9.
    Holness CL, Lomonossoff GP, Evans D, Maule AJ (1989) Identification of the initiation codons for translation of cowpea mosaic virus middle component RNA using site-directed mutagenesis of an infectious cDNA clone. Virology 172: 311–320PubMedCrossRefGoogle Scholar
  10. 10.
    Chen X, Bruening G (1992) Nucleotide sequence and genetic map of cowpea severe mosaic virus RNA2 and comparisons with RNA2 of other comoviruses. Virology 187: 682–692PubMedCrossRefGoogle Scholar
  11. 11.
    Kenan DJ, Query CC, Keene JD (1991) RNA recognition: towards identifying determinants of specificity. Trends Biochem Sei 16: 214–220CrossRefGoogle Scholar
  12. 12.
    Rezelman G, Van Kämmen A, Wellink J (1989) Expression of cowpea mosaic virus M RNA in cowpea protoplasts. J Gen Virol 70: 3043–3050CrossRefGoogle Scholar
  13. 13.
    Jofuka KD, Schipper RD, Goldberg RG (1989) A frameshift mutation prevents Kunitz trypsine inhibitor mRNA accumulation in soybean embryos. Plant Cell 1: 427–435CrossRefGoogle Scholar
  14. 14.
    Voelker TA, Moreno J, Chrispeels MJ (1990) Expression analysis of a pseudogene in transgenic tobacco: a frameshift mutation prevents mRNA accumulation. Plant Cell 2: 255–261PubMedCrossRefGoogle Scholar
  15. 15.
    Vancanneyt G, Rosahl S, Wilmitzer L (1990) Translatability of a plant mRNA strongly influences its accumulation in transgenic plants. Nucleic Acids Res 18: 2917–2921PubMedCrossRefGoogle Scholar
  16. 16.
    Eggen R, Verver J, Wellink J, Pley K, Van Kämmen A, Goldbach R (1989) Analysis of sequences involved in cowpea mosaic virus RNA replication using site specific mutants. Virology 173: 456–464PubMedCrossRefGoogle Scholar
  17. 17.
    Richards OC, Eggen R, Goldbach R, Van Kämmen A (1989) High level synthesis of cowpea mosaic virus RNA polymerase and protease in Escherichia coli. Gene 78: 135–146PubMedCrossRefGoogle Scholar
  18. 18.
    Van Bokhoven H, Wellink J, Usmany M, Vlak JM, Goldbach R, Van Kämmen 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. J Gen Virol 71: 2509–2517PubMedCrossRefGoogle Scholar
  19. 19.
    Van Bokhoven H, Mulders M, Wellink J, Vlak JM, Goldbach R, Van Kammen A (1991) Evidence for dissimilar properties of comoviral and picornaviral RNA polymerases. J Gen Virol 72: 567–572PubMedCrossRefGoogle Scholar
  20. 20.
    Van Bokhoven H, Van Lent JWM, Custers R, Vlak JM, Wellink J, Van Kammen A (1992) Synthesis of the complete 200K polyprotein of cowpea mosaic virus B RNA in insect cells. J Gen Virol 73: 2775–2784PubMedCrossRefGoogle Scholar
  21. 21.
    Van Bokhoven H, Verver J, Wellink J, Van Kämmen A (1993) Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA. J Gen Virol 79 (in press)Google Scholar
  22. 22.
    Takeda N, Kuhn RJ, Yang C, Takegami T, Wimmer E (1986) Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. J Virol 60: 43–53PubMedGoogle Scholar
  23. 23.
    Molla A, Paul AV, Wimmer E (1991) Cell-free de novo synthesis of poliovirus. Science 254: 1647–1651PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • J. Wellink
    • 1
  • H. van Bokhoven
    • 1
  • O. Le Gall
    • 1
  • J. Verver
    • 1
  • A. van Kammen
    • 1
  1. 1.Department of Molecular BiologyAgricultural UniversityWageningenThe Netherlands

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