PROTEIN FACTORS REQUIRED FOR IN-VITRO TRANSCRIPTION OF SENDAI-VIRUS GENOME

To elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription s ystem using purified virus particles. The in vitro RNA synthesis was a lmost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissu es, and even from plant cells. The RNA products were almost identical to authentic mRNA species synthesized in the infected cells, in their size distribution, the presence of 3'-poly(A) tail and the presence of methylated 5'-cap structure (m(7)GpppAm). Ribonuclease protection exp eriments after annealing the in vitro RNA with viral genomic RNA (vRNA ) indicated that the virion-associated RNA-dependent RNA polymerase tr anscribes correct regions of the RNA genome in vitro. The active compo nent(s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two comple mentary fractions, one of which could be replaced by highly purified c ellular tubulin. When viral ribonucleoprotein complexes were used inst ead of virus particles in the in, vitro transcription, only Sendai vir us-infected cell extracts supported mRNA synthesis, and extracts from uninfected cells or cells infected with other viruses were found to be inert. These results suggest that, in addition to the general factors which are present ubiquitously in eukaryotic cells, a factor(s) speci fic to Sendai virus-infection is required for Sendai virus transcripti on.