In Vitro mRNA Synthesis by Sendai Virus: Isolation and Characterization of the Transcription Initiation Complex

We have developed an in vitro Transcription system using purified Sendai virus particles in which viral mRNA synthesis is almost entirely dependent on the addition of cellular proteins (host factors), one of which can be replaced by highly purified cellular tubulin (Mizumoto et al. (1995) J.Biochem. 117, 527–534). In this study, to elucidate the function of host factors in transcription, we islated an active initiation complex as aviral ribonucleoprotein, by incubating virus particles with bovine brain extract in the absence of nucleoside triphosphates, followed by ultracentrifugation.RNA products from the isolated initiation complex contained six mRNA species corresponding to all the virus-encoded genes, and most of them had a 5'-cap structure as well as a3'-poly(A) tail. Immunob lotting showed that tubulin was sepecifically associated in the active complex. These data sugest that cellular tubulin, one of the host factor essential for Sendai virus transcription, interacts with the viral ribonucleoprotein to form an active complex at the initiation step.