Abstract
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.
Highlights
Double-stranded RNA1 viruses encapsidate their plus single-stranded genomic RNAs into viral particles or inner cores and subsequently convert them into double-stranded form by the particle-associated RNA-dependent RNA polymerase
The sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C4194 was directly involved in binding to empty particles rather than indirectly through its potential base pairing with G4190
It is reasonable to assume that most of the interactions exerted by empty particles can be ascribed to the Pol part of the fusion protein, since the packaging activity of RNA with the encapsidation signal resides in the N-terminal quarter of Pol [10] and neither Gag nor the Gag part of the fusion protein appears to be involved in the recognition of the encapsidation signal [21]
Summary
Double-stranded RNA (dsRNA)1 viruses encapsidate their plus single-stranded genomic RNAs into viral particles or inner cores and subsequently convert them into double-stranded form by the particle-associated RNA-dependent RNA polymerase. The sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C4194 was directly involved in binding to empty particles rather than indirectly through its potential base pairing with G4190.
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