The subcellular localization of the nonstructural protein C of Sendai virus was investigated by means of indirect immunofluorescence microscopy of Sendai virus-infected cells, using an antiserum specific for C protein. In infected cells, C protein was detected exclusively in the cytoplasm as granular fluorescence, which coincided very well with the distribution of nucleocapsid protein NP and phosphoprotein P, which were also detected with specific antisera. This suggested that these proteins are present together in inclusions, probably forming nucleocapsids. In contrast, when the NP and C proteins were individually expressed in COS cells by transfection with expression plasmids containing cDNA for these proteins, their distribution patterns in the cytoplasm were found to be quite different from each other. Protein-blot analyses of purified virions revealed the presence of a significant amount of the C protein in virions, which indicated that C protein is integrated into virions. Under conditions in which most of the envelope-associated proteins, such as HN, F, and M, were removed from the virions by a detergent, the C protein remained tightly associated with the nucleocapsids--about 40 molecules per nucleocapsid.
To elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription system using purified virus particles. The in vitro RNA synthesis was almost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissues, 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 (m7GpppAm). Ribonuclease protection experiments after annealing the in vitro RNA with viral genomic RNA (vRNA) indicated that the virion-associated RNA-dependent RNA polymerase transcribes correct regions of the RNA genome in vitro. The active component(s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two complementary fractions, one of which could be replaced by highly purified cellular tubulin. When viral ribonucleoprotein complexes were used instead of virus particles in the in vitro transcription, only Sendai virus-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) specific to Sendai virus-infection is required for Sendai virus transcription.
We have generated two serum-and anchorage-dependent revertants from NIH 3T3 cells transformed with multiple copies of the human c-H-ras oncogene. In both revertants, the c-H-ras oncogene was fully expressed. Fusion of either revertant with untransformed cells or of the two revertants with one another resulted in transformed progeny. These results indicated that the two revertants were recessive and in different complementation groups. We believe that in our two revertants some of the genes mediating the transforming activity of the c-H-ras oncogene are defective; we are attempting to identify these mediator genes.We wish to identify genes that mediate the transforming activity of the c-H-ras oncogene (1). The approach chosen requires the isolation of recessive revertants from cells transformed with the oncogene (3,9,19). In the revertants sought, the oncogene is fully expressed, yet the transformed state is not manifested in consequence of a defect in a gene (mediator gene) required for mediating the transforming activity. We plan to retransform the revertants by transfection with DNA from normal cells and to identify the retransforming gene.For this purpose, we have transfected (7) mouse NIH 3T3 cells with a plasmid carrying an activated human c-H-ras oncogene from the T24 bladder carcinoma cell line (13) and, as a selectable marker, the neomycin (G418) resistance gene (12). From the resulting G418-resistant clones, we picked one (FT9) carrying five to seven copies of the integrated ras oncogene. Revertants (serum dependent) were obtained from FT9 (serum independent) by incubation in serum-free medium with bromodeoxyuridine and irradiation (17). The procedure was repeated with the survivors except that the incubation with bromodeoxyuridine was in the presence of 1% fetal calf serum (FCS) to eliminate cells with a low serum requirement. After screening of 162 revertant clones, 2 with flat morphology (R116 and R260) were chosen for further studies. R116 cells were similar in size to NIH 3T3 cells; R260 cells were smaller (Fig. 1A). Both revertants were serum dependent: in the absence of serum, they formed only a few small colonies in conditions in which FT9 cells reached confluency ( Fig. 2A). The efficiency of growth of the two revertants in 1% FCS was similar to that of NIH 3T3 cells and clearly lower than that of FT9 cells (Table 1)
An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter of E. coli expression plasmid ptac12-Bam. A new protein produced in E. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion. Antiserum raised against the purified protein specifically reacted with the C protein in infected cells. Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.
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