Yeast double-stranded RNA virus L-A deliberately synthesizes RNA transcripts with 5′-diphosphate

Abstract

L-A is a persistent double-stranded RNA virus commonly found in the yeast Saccharomyces cerevisiae. Isolated L-A virus synthesizes positive strand transcripts in vitro. We found that the 5′ termini of the transcripts are diphosphorylated. The 5′-terminal nucleotide is G, and GDP was the best substrate among those examined to prime the reaction. When GTP was used, the triphosphate of GTP incorporated into the 5′-end was converted to diphosphate. This activity was not dependent on host CTL1RNAtriphosphatase. The 5′-end of the GMP-primed transcript also was converted to diphosphate, the β-phosphate of which was derived from the γ-phosphate of ATP present in the polymerization reaction. These results demonstrate that L-A virus commands elaborate enzymatic systems to ensure its transcript to be 5′-diphosphorylated. Transcripts of M1, a satellite RNA of L-A virus, also had diphosphate at the 5′ termini. Because viral transcripts are released from the virion into the cytoplasm to be translated and encapsidated into a new viral particle, a stage most vulnerable to degradation in the virus replication cycle, our results suggest that the 5′-diphosphate status is important for transcript stability. Consistent with this, L-A transcripts made in vitro are resistant to the affinity-purified Ski1p 5′-exonuclease. We also discuss the implication of these findings on translation of viral RNA. Because the viral transcript has no conventional 5′-cap structure, this work may shed light on the metabolism of non-self-RNA in yeast. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.