Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28.

The killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs from that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previously demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus-like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA-free recipient cell. We also demonstrated that L28, like the L-A-H species in K1 killers, has [HOK] activity required for maintenance of M1-dsRNA, and predicted that M28 would share with M1 dependence on L-A for replication. We now confirm this prediction by genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resembles M1 in all respects. We also show that L28 is an L-A-H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1-cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L-A-H encoded capsid and cap-pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, but not of K28 immunity, requires the Kex2 endoprotease.