Yeast double-stranded RNA virus L-A deliberately synthesizes RNA transcripts with 5'-diphosphate.
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 CTL1 RNA triphosphatase. The 5'-end of the GMP-primed transcript also was converted to diphosphate, the beta-phosphate of which was derived from the gamma-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.
Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Salamanca 37007, Spain. tfujimura@usal.es
Tsutomu
T
Fujimura
Rosa
R
Esteban
D001483:Base Sequence; D011756:Diphosphates; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D043583:Nucleoside-Triphosphatase / Q000378:metabolism; D043264:Phosphodiesterase I / Q000378:metabolism; D020871:RNA Stability; D012328:RNA Viruses / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000821:virology*; D014771:Virion / Q000378:metabolism
D011756:Diphosphates; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D043264:Phosphodiesterase I; D043583:Nucleoside-Triphosphatase