Protein Factors Required for In Vitro Transcription of Sendai Virus Genome1

Abstract

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.

Identification of Sendai Virus L Protein Amino Acid Residues Affecting Viral mRNA Cap Methylation

“…However, such an addition would be very undesirable for our experiments, as these extracts might contain trans-active cellular cap MTases, which could complement L protein defects in cap methylation and thus prevent discrimination between mutants based on their ability to methylate mRNA caps. Therefore, we optimized the SeV in vitro transcription conditions using purified tubulin, which has been shown to stimulate SeV virion transcription even when other cellular components are absent (35,39). Interestingly, our optimal reaction conditions, producing amounts of viral mRNA similar to those of reactions with cell lysate from Vero cells (data not shown), generated about 200-fold less viral mRNA than did VSV virions transcribed under the same conditions (Fig.…”

“…SeV in vitro transcription by detergent-activated purified virions was conducted essentially as described previously (35). For [␣-…”

“…In addition to recombinant SeV, we used wt rVSV and rVSV hr1 viruses as positive and negative controls for cap methylation throughout all these assays. It is important that in contrast to the VSV system, the reactions with detergent-activated purified SeV (and many other members of the Mononegavirales) virions require the addition of cytoplasmic extracts to each reaction mixture (11,35,38,39). However, such an addition would be very undesirable for our experiments, as these extracts might contain trans-active cellular cap MTases, which could complement L protein defects in cap methylation and thus prevent discrimination between mutants based on their ability to methylate mRNA caps.…”

Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA

“…Complexes-In vitro mRNA products synthesized in the presence of AdoMet from SeV particles or RNP contain methylated cap structures, m 7 GpppA and m 7 GpppAm (21,22). This suggests that the viral RNP contains two cap methyltransferases, G-7-MTase and mRNA cap-ribose MTase.…”

“…We established an accurate and efficient in vitro mRNA synthesizing system using purified SeV particles or RNP complexes (21)(22)(23). Polyadenylated mRNA products synthesized from the isolated RNP complex contained cap structures, GpppA-, m 7 GpppA-(cap 0), and m 7 GpppAm-(cap I), suggesting that the complex possesses a capping enzyme system including mRNA guanylyltransferase, G-7-MTase, and cap-ribose MTase (22).…”