There are five families of double-stranded RNA virus-like particles (L-A, L-BC, M, T, and W) and two distinct single-stranded RNA virus-like entities (20S RNA and 23S RNA) that replicate in cells of Saccharomyces cerevisiae. We have studied how these genomes replicate in yeast with emphasis on the role of the host. Highly purified virus-like particles (VLPs) carry our both (+) strand and (-) strand synthesis of L-A, L-BC, or M RNA in vitro in a conservative, sequential reaction. We developed an in vitro replication system ((-) strand synthesis on a viral (+) strand template producing dsRNA) for the L-A system. Using isolated opened empty viral particles, we demonstrated site- specific binding of viral (+) strands. The internal sequence 5' UUUGGCCAGG 3' determines this binding. In addition to this sequence, the replicase reaction requires the correct 3' end, but not the 5' end. The viral 180 kDa minor protein binds ssRNA and is encoded by L-A, having an N-terminal domain with immunologic relationship to the major coat protein and the C-terminal region responsible for ssRNA binding. We have cloned and sequenced the L-A genome and find that the 180 kDa protein is encoded by the fusion of two overlapping ORFs, ORFl and ORF2. ORFl encodes the major coat protein, and ORF2 encodes the ssRNA binding domain of the 180 kDa protein. ORF2 has substantial homology with viral RNA polymerases of picornaviruses and togaviruses. We have designed an RNA virus-based vector for yeast using our clone of X dsRNA, a deletion derivative of L-A dsRNA.
