Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus.
The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5' side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five nucleotides of the loop sequence ((4190)GAUCC(4194)) were not protected. However, T1 RNase protection and in vitro mutagenesis experiments indicated that G(4190) is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C(4194) was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G(4190). These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.
Fujimura
T
T
Instituto de Microbiologia Bioquimica/Departamento de Microbiologia y Genética, Consejo Superior de Investigaciones Cientificas/Universidad de Salamanca, Salamanca 37007, Spain. tfujimura@ww-micro.usal.es
Esteban
R
R
D001483:Base Sequence; D002213:Capsid / Q000502:physiology*; D016384:Consensus Sequence; D015685:Fusion Proteins, gag-pol / Q000502:physiology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012260:Ribonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014158:Transcription, Genetic
D015685:Fusion Proteins, gag-pol; D013463:Sulfuric Acid Esters; D012260:Ribonucleases; C007482:dimethyl sulfate