20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome only encodes its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing from a vector genomic strands fused at the 3’ ends to the hepatitis delta virus anti-genomic ribozyme. Using this launching system we analyzed 3’ cis signals for replication present on the genomic strand. The viral genome has 5 nt inverted repeats at both termini (5’ GGGGC…GCCCC-OH). The fifth G from the 3’ end was dispensable for replication, while the third and fourth Cs were essential. The 3’ terminal and penultimate Cs could be eliminated or modified to other nucleotides, however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3’ end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the viral 3’ end correct in the size and sequence. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from anti-genomic strands, provided an active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3’ end in the anti-genomic strand was essential for replication. In this work we also present the first conclusive evidence that 20 S RNA and 23 S RNA viruses are independent replicons.