Abstract
In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.
Highlights
Sendai virus (SeV),1 a member of the Paramyxovirus family in the order Mononegavirales, contains a monopartite negative strand RNA genome, which consists of six genes encoding the viral proteins, nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinneuraminidase glycoprotein (HN), and large protein (L)
Little is known about the mechanism of mode switching of the RNA polymerase from transcription to replication, it has been shown that three viral proteins, the NP, P, and L proteins are required for transcription and replication of the genome RNA (4 – 6) and that the L protein interacts with the P protein to form the polymerase complex for replication and transcription [4, 7]
We have shown that phosphoglycerate kinase (PGK) stimulates viral mRNA synthesis at the elongation step, probably through its interaction with tubulin integrated into the initiation complex
Summary
SeV, Sendai virus; PGK, phosphoglycerate kinase; RNP, ribonucleoprotein; VSV, vesicular stomatitis virus; HPIV, human parainfluenza virus; DTT, dithiothreitol; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TPCK, N-tosyl-L-phenylalanyl chloromethyl ketone. We purified the host factor activity complementary to tubulin, and showed it has at least two components, and we identified one of them as phosphoglycerate kinase (PGK), a glycolytic enzyme This is the first example showing the involvement of a glycolytic enzyme in the transcription of paramyxoviruses. We have shown that PGK stimulates viral mRNA synthesis at the elongation step, probably through its interaction with tubulin integrated into the initiation complex Tubulin stimulated both mRNA synthesis and leader RNA synthesis of SeV, while PGK failed to stimulate leader RNA synthesis, suggesting that mRNA and leader RNA synthesis may be regulated by different sets of host proteins
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