The Bipartite 3′-cis-Acting Signal for Replication Is Required for Formation of a Ribonucleoprotein Complex in Vivo between the Viral Genome and Its RNA Polymerase in Yeast 23 S RNA Virus

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.9 kb) encodes only its RNA-dependent RNA polymerase, p104, and forms a ribonucleoprotein complex with p104 in vivo. Previously we succeeded in generating 23 S RNA virus in yeast from an expression vector containing the entire viral cDNA sequence. Using this system, we have recently identified a bipartite 3' cis-acting signal for replication. The signal consists of a stretch of four cytidines (Cs) at the 3' end and a mismatched pair of purines in a stem-loop structure that partially overlaps the terminal four Cs. Although the 3' terminal and penultimate Cs are not essential for virus launching, the generated viruses efficiently recovered these terminal nucleotides. In this work, we expressed RNA transcripts containing the entire 23 S RNA genome but incapable of generating the virus because of the presence of non-viral extra sequences at the 3' ends. These transcripts could form complexes with p104 in vivo, and a detailed analysis indicated that the mismatched pair of purines as well as the third and fourth Cs from the viral 3' end was essential for this complex-forming activity. Given that 23 S RNA virus does not have genes for capsid proteins, the binding of p104 to the viral 3' end, in addition to the efficient 3' terminal repair, may play a crucial role in virus persistence by protecting and maintaining the correct viral 3' end in vivo.