A 3′-end structure in RNA2 of a crinivirus is essential for viral RNA synthesis and contributes to replication-associated translation activity

The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3′ terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5′ and YSS-containing 3′ terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons.