Summary
Two types of particles were isolated during purification of rotavirus. Dense (D) particles have a density of 1.38 in CsCl and exhibit spontaneously a fully active endogenous transcriptase. Light (L) particles (density 1.36 in CsCl) need to be treated with chelating agents to show a polymerase activity. The activation process of L particles was studied under strictly controlled monovalent, divalent, and hydrogen ion concentrations. These experiments demonstrate that i) activation is not affected by the ionic strength ii) activation occurs only at a pH higher than 7.1 iii) a low concentration of chelating agent (40 µm EDTA) is sufficient to activate the enzyme. Treatment of particles with EGTA, which chelates selectively Ca2+, leads to unmasking even in the presence of magnesium, indicating that the concentration of free calcium ions plays a major role in the activation process. Various glycosidases, detergents, and chelating agents were tested in respect to unmasking properties. Of these compound only chelating agents turned out to be efficient. Following activation, two glycopeptides were solubilized. These glycopeptides have an apparent molecular weight of 34,000 and 31,000 daltons and react with concanavalin A.
The role of Ca2+ upon the stability of virus particles, and the activation of the endogenous transcriptasein vitro and in the infected cells is discussed.
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Baker, P. F., Hodgkins, A. L., Ridgeway, E. B.: Depolarization and calcium entry in squid axons. J. Physiol. (London)218, 709–755 (1971).
Black, D. R., Knight, C. A.: Ribonucleic acid transcriptase activity in purified wound tumor virus. J. Virol.6, 194–198 (1970).
Borsa, J., Long, D. G., Sargent, M. D., Copps, T. P., Chapman, J. D.: Reovirus transcriptase activationin vitro: involvement of an endogenous uncoating activity in the second stage of the process. Intervirology4, 171–188 (1974).
Borsa, J., Sargent, M. D., Long, D. G., Chapman, J. D.: Extraordinary effect of specific monovalent cation on activation of reovirus transcriptase by chymotrypsinin vitro. J. Virol.11, 207–217 (1973).
Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem.72, 248–254 (1976).
Brady, J. N., Winston, V. D., Consigli, R. A.: Dissociation of Polyoma virus by the chelation of calcium ions found associated with purifed virus. J. Virol.23, 717–724 (1977).
Bridger, J. C., Woode, G. N.: Characterization of two particle types of calf rotavirus. J. gen. Virol.31, 245–250 (1976).
Carafoli, E., Crompton, M.: The regulation of intracellular calcium. Current Topics in Membrane and Transport10, 151–216 (1978).
Chasey, D.: Different particle types in tissue culture and intestinal epithelium infected with Rotavirus. J. gen. Virol.37, 443–451 (1977).
Chater, K. F., Morgan, D. H.: Ribonucleic acid synthesis by isolated viruses ofPenicillium stoloniferum. J. gen. Virol.24, 307–317 (1974).
Cohen, J.: Ribonucleic acid polymerase activity associated with purified calf rotavirus. J. gen. Virol.36, 395–402 (1977).
Cohen, J., Maget-Dana, R., Roche, A. C., Monsigny, M.: Calf rotavirus: detection of outer capsid glycoprotein by lectins. FEBS Letters87, 26–30 (1978).
Durham, A. C., Hendry, D. A.: Cation binding by tobacco mosaic virus. Virology77, 510–519 (1977).
Durham, A. C., Hendry, D. A., von Wechmar, M. B.: Does calcium ion binding control plant virus disassembly? Virology77, 524–533 (1977).
Durham, A. C., Mounir Abou Haidar: Cation binding by tobacco rattle virus. Virology77, 520–523 (1977).
Elias, M. M.: Separation an infectivity of two particle types of human rotavirus. J. gen. Virol.37, 191–194 (1977).
Flewett, T. H., Bryden, A. S., Davies, H.: Virus particles in gastroenteritis. Lancetii, 1497 (1973).
Holmes, I. H., Ruck, B. J., Bishop, R. F., Davidson, G. P.: Infantile enteritis viruses: morphogenesis and morphology. J. Virol.16, 937–943 (1975).
Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)227, 680–685 (1970).
Lapierre, H., Astier-Manifacier, S., Cornuet, P.: Activité RNA polymérase associée á des préparation purifiée du virus dePenicillium stoloniferum. Compterendus de l'Académie des Sciences de ParisD 273, 992–994 (1971).
Maher, P., Molday, R. S.: Binding of concanavalin A toRicinus communis agglutinin and its implication in cell surface labelling studies. FEBS letters84, 391–394 (1977).
Palmer, E. L., Martin, M. L., Murphy, F. A.: Morphology and stability of infantile gastroenteritis virus: comparison with reovirus and Blue Tongue Virus. J. gen. Virol.35, 403–414 (1977).
Portzehl, H., Caldwell, P. C., Ruegg, J. C.: The dependence of contraction on relaxation of muscle fibres from the crabMaia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta79, 581–591 (1964).
Rodger, S. M., Schnagl, R. D., Holmes, I. H.: Biochemical and biophysical characteristics of diarrhea viruses of human and calf origin. J. Virol.16, 1229 to 1235 (1975).
Rodger, S. M., Schnagl, R. D., Holmes, I. H.: Further biochemical characterization, including the detection of surface glycoproteins of human, calf and simian viruses. J. Virol.24, 91–98 (1977).
Skehel, J. J., Joklik, W. K.: Studies on thein vitro transcription of reovirus RNA catalysed by reovirus cores. Virology39, 822–831 (1969).
Todd, D., McNulty, M. S.: Biochemical studies on a reovirus like agent (Rotavirus) from lamb. J. Virol.21, 1215–1218 (1977).
Verwoerd, D. W., Huismans, H.: Studies on thein vitro andin vivo transcription of the BTV genome. Onderstepoort. J. vet. Res.39, 185–192 (1972).
Williams, R. J. P.: Calcium chemistry and its relation to biological function. Symp. Soc. exp. Biol.30, 1–17 (1976).
Wood, H. A., Viruses with double stranded genomes. J. gen. Virol.20, 61–85 (1973).
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Cohen, J., Laporte, J., Charpilienne, A. et al. Activation of rotavirus RNA polymerase by calcium chelation. Archives of Virology 60, 177–186 (1979). https://doi.org/10.1007/BF01317489
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DOI: https://doi.org/10.1007/BF01317489