Replicase of L-A virus-like particles of Saccharomyces cerevisiae. In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form.

Virus-like particles that contain L-A double-stranded RNA are known to have transcriptase activity whose product is L-A single-stranded plus RNA. In low salt conditions, these particles release their double-stranded RNA and can then use added plus L-A or plus M1 single-stranded RNAs as templates to synthesize their respective double-stranded RNAs. The reaction requires dialyzed L-A virus-like particles as the source of the enzyme, a partially purified cell extract (host factor(s)), added single-stranded RNA as a template, and polyethylene glycol 6000, along with four NTPs. Crude host factor extracts prepared from mak3 or mak10ta mutants also support the reaction as effectively as that from a wild type strain, while a crude extract prepared from a pet18 mutant grown under the nonpermissive conditions is less effective. Template specificity of the in vitro reaction is the same as that expected for the enzyme reaction in vivo. Plus L-A and plus M1 single-stranded RNAs, but not 18 S rRNA, are converted to their respective double-stranded RNAs with net RNA synthesis. The newly synthesized strand of M1 double-stranded RNA is a full-length minus strand. This demonstration of replicase activity in the mature L-A virus-like particles which contain L-A double-stranded RNA is consistent with our previous L-A double-stranded RNA replication model; the difference between the mature L-A virus-like particles and L-A double-stranded RNA-synthesizing particles (expected to be replication intermediates in vivo) is just that the former contain L-A double-stranded RNA, while the latter contain L-A plus single-stranded RNA.