Narnavirus 23S RNA is a persistent positive-stranded RNA virus found in yeast Saccharomyces cerevisiae. The viral genome (2.9 kb) only encodes its RNA-dependent RNA polymerase, p104. Here we report the generation of 23S RNA virus, with high frequency, from a vector containing the entire viral cDNA sequence. When the conserved GDD (Gly-Asp-Asp) motif of RNA-dependent RNA polymerase was modified, the vector failed to generate the virus, indicating that an active p104 is essential for replication. Successful launching required transcripts having the proper viral 3' terminus generated in vivo. This was accomplished through in vivo processing of the primary transcripts by the hepatitis delta virus antigenomic ribozyme directly fused to the 3' terminus of the 23S RNA genome. Although the primary transcripts also contained extra nucleotides at their 5' ends derived from the vector, the launched virus possessed the authentic 5' terminus of the viral genome without these extra nucleotides. Modifications of the genome sequence at the 5' and 3' termini abolished viral generation, indicating that the viral genome has cis-acting signals for replication at both termini. The great ease to generate the virus will facilitate the identification of these cis-acting signals. Furthermore, the virus, once generated, can be transmitted to daughter cells indefinitely without the vector or any selection, which makes the 23S RNA virus-launching system particularly useful for investigating the basis for RNA virus persistence.