Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy

Abstract

The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter. We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2. ORF1 expression, like a host ski- mutation, can suppress the usual requirement of M1 for the MAK11, MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cell a ski- phenocopy. Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein.