Alternate Capping Mechanisms for Transcription of Spring Viremia of Carp Virus: Evidence for Independent mRNA Initiation

Abstract

Two alternate mechanisms of mRNA capping for spring viremia of carp virus have been observed. Under normal reaction conditions, a ppG residue of the capping GTP is transferred to a pA moiety of the 5′ termini of mRNA transcripts. However, in reaction conditions where GppNHp is used instead of GTP, an alternate capping mechanism occurs whereby a pG residue of the capping GTP is transferred to a ppA moiety of the transcripts. The first mechanism is identical to that described previously for vesicular stomatitis virus (G. Abraham, D. P. Rhodes, and A. K. Banerjee, Nature [London] 255 :37-40, 1975; A. K. Banerjee, S. A. Moyer, and D. P. Rhodes, Virology 61 :547-558, 1974), and thus appears to be a conserved function during the evolution of rhabdoviruses. The alternate mechanism of capping indicates not only that capping can take place by two procedures, but also that the substrate termini have di- or triphosphate 5′ ends, indicating that they are probably independently initiated. An analog of ATP, AppNHp, has been found to completely inhibit the initiation of transcription by spring viremia of carp virus, suggesting that a cleavage between the β and γ phosphates of ATP is essential for the initiation of transcription. However, in the presence of GppNHp, uncapped (ppAp and pppAp), capped (GpppAp), and capped methylated (m7GpppAmpAp and GpppAmpAp) transcripts are detected. Size analyses of oligodeoxythymidylic acid-cellulose-bound transcripts resolved by formamide gel electrophoresis demonstrated that full-size mRNA transcripts are synthesized as well as larger RNA species. The presence of GppNHp and S -adenosylhomocysteine in reaction mixtures did not have any effect on the type of unmethylated transcription products. Our results favor a transcription model postulated previously (D. H. L. Bishop, in H. Fraenkel-Conrat and R. R. Wagner, ed., Comprehensive Virology , vol. 10, Plenum Press, New York, 1977; D. H. L. Bishop and A. Flamand, in D. C. Burke and W. C. Russell, ed., Control Processes in Virus Multiplication , Cambridge University Press, Cambridge, 1975; D. H. L. Bishop and M. S. Smith, in D. Nayak, ed., The Molecular Biology of Animal Viruses , Marcel Dekker, New York, 1977; P. Roy and D. H. L. Bishop, J. Virol. 11 :487-501, 1973) in which mRNA synthesis is initiated independently; they do not support a model for transcripts being synthesized by plus-strand cleavage (A. K. Banerjee, G. Abraham, and R. J. Colonno, J. Gen. Virol. 34 :1-8, 1977; A. K. Banerjee, R. J. Colonno, D. Testa, and M. T. Franze-Fernandez, in B. M. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cells , Academic Press, London, 1978).