Summary
Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsids precursors of either of the viruses.
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Cleveland, D. W., Fischer, S. G., Kirschner, M. W., Laemmli, U. K.: Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem.252, 1102–1106 (1977).
Kitamura, N., Semler, B. L., Rothberg, P. G., Larsen, G. R., Adler, C. J., Dorner, A. J., Emini, E. A., Hanecak, R., Lee, J. J., van der Werf, S., Anderson, C. W., Wimmer, E.: Primary sturcture gene organization and polypeptide, expression of poliovirus RNA. Nature (London)291, 547–553 (1981).
Korant, B. D.: Cleavage of viral precursor proteinsin vivo andin vitro. J. Virol.10, 751–759 (1972).
Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)227, 680–685 (1970).
Larsen, G. R., Anderson, C. W., Dorner, A. J., Semler, B. L., Wimmer, E.: Cleavage sites within the poliovirus capsid protein precursors. J. Virol.41, 340–344 (1982).
Laskey, R. Mills, A.: Quantitative film detection of3H and14C in polyacrylamide gels by fluorography. Eur. J. Biochem.56, 335–341 (1975).
Matthews, R. E. F.: Fowel report of the International Committee on Taxonomy of Viruses. Intervirol.17, 1–200 (1982).
Moore, N. F., Kearns, A., Pullin, J. S. K.: Characterization of Cricket paralysis. virus-induced polypeptides inDrosophila cells. J. Virol.33, 1–9 (1980).
Moore, N. F., Pullin, J. S. K.: Plaque purification of Cricket paralysis virus using an agar overlay onDrosophila cells. J. Invertebr. Pathol.39, 10–14 (1982).
Moore, N. F., Pullin, J. S. K., Reavy, B.: The intracellular proteins induced by Cricket paralysis virus inDrosophila cells: the effect of protease inhibitors and amino acid analogues. Arch. Virol.70, 1–9 (1981).
Moore, N. F., Reavy, B., Pullin, J. S. K.: Processing of Cricket paralysis virus induced polypeptides inDrosophila cells: production of high molecular weight polypeptides by treatment with iodoacetamide. Arch. Virol.68, 1–8 (1981).
Moore, N. F., Reavy, B., Pullin, J. S. K., Plus, N.: The polypeptides induced inDrosophila cells byDrosophila C virus (strain Ouarzazate). Virology112, 411–416 (1981).
Palmenberg, A. C., Rueckert, R. R.: Evidence for intramolecular self-cleavage of picornaviral replicase precursors. J. Virol.41, 244–249 (1982).
Pelham, H. R. B.: Synthesis and proteolytic processing of cowpea mosaic virus proteins in reticulocyte lysates. Virology96, 463–477 (1979).
Pelham, H. R. B., Jackson, R. J.: An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem.67, 247–256 (1976).
Plus, N., Croizier, G., Reinganum, C., Scotti, P. D.: Cricket paralysis virus andDrosophila C virus: serological analysis and comparison of capsid polypeptides and host range. J. Invertebr. Pathol.31, 296–302 (1978).
Reavy, B., Moore, N. F.:In vitro translation of Cricket paralysis virus RNA. Arch. Virol.67, 175–180 (1981).
Reavy, B., Moore, N. F.: Cell-free translation of Cricket paralysis virus RNA: analysis of the synthesis and processing of virus-specified proteins. J. gen. Virol.55, 429–438 (1981).
Reavy, B., Moore, N. F.: The replication of small RNA-containing viruses of insects. Microbiologica5, 63–84 (1982).
Rueckert, R. R.: On the structure and morphogenesis of picornaviruses. In:Fraenkel-Conrat, H., Wagner, R. R. (eds.), Comprehensive Virology, Vol. 6, 131–213. New York: Plenum Publishing Corp. 1976.
Scotti, P. D.: Cricket paralysis virus replicates in culturedDrosophila cells. Intervirol.6, 333–342 (1976).
Scotti, P. D.: End-point dilution and plaque-assay methods for titration of Cricket paralysis virus in culturedDrosophila cells. J. gen. Virol.35, 393–396 (1977).
Semler, B. L., Hanecak, R., Anderson, C. W., Wimmer, E.: Cleavage sites in the polypeptide precursors of poliovirus protein P2-X. Virology114, 589–594 (1981).
Shih, D. S., Shih, C. T., Kew, O., Pallansch, M., Rueckert, R. R., Kaesberg, P.: Cell-free synthesis and processing of the proteins of poliovirus. Proc. Nat. Acad. Sci. U.S.A.75, 5807–5811 (1978).
Svitkin, Y. V., Gorbalenya, A. E., Kazachkov, Y. A., Agol, V. I.: Encephalomyocarditis virus-specific polypeptide p22 possessing a proteolytic activity. Preliminary mapping on the viral genome. FEBS Letts.108, 6–9 (1979).
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Reavy, B., Moore, N.F. Cell-free translation ofDrosophila C virus RNA: Identification of a virus protease activity involved in capsid protein synthesis and further studies onin vitro processing of Cricket paralysis virus specified proteins. Archives of Virology 76, 101–115 (1983). https://doi.org/10.1007/BF01311694
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DOI: https://doi.org/10.1007/BF01311694