Abstract
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.
Highlights
Members of the genus Crinivirus are whitefly-transmitted, emerging plant viruses and affiliates of the alphavirus-like supergroup of single-stranded, positive (+)-sense RNA viruses with large (15.3–17.6 kb) and complex genomes[1,2]
We have investigated the role of the Y-shaped structure (YSS) in supporting viral RNA synthesis by assessing the relative amounts of progeny RNA produced when 3′NCR/YSS mutants of Lettuce chlorosis virus (LCV) RNA2 are co-inoculated with WT LCV RNA1 to tobacco protoplasts
SL1 and SL2 of LCV RNA1 are not separated by any nucleotide, while both SLs of LCV RNA2 are separated by 3 nts, of which two are moderately reactive to benzoyl cyanide (BzCN) modification
Summary
Members of the genus Crinivirus (family Closteroviridae) are whitefly-transmitted, emerging plant viruses and affiliates of the alphavirus-like supergroup of single-stranded (ss), positive (+)-sense RNA viruses with large (15.3–17.6 kb) and complex genomes[1,2]. The 3′NCR of RNA1 is 226-nucleotide (nt) long and shares 74% sequence identity with its 226-nt counterpart in RNA2 In this 226-nt region of RNA2, the first 128 nts are part of the ORF encoding P4.8, while the downstream 98 nts constitute the 3′NCR of the RNA. In this study, using Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE)[16,17,18,19] analysis of full-length LCV RNAs 1 and 2, we have identified a Y-shaped structure (YSS) consisting of two stem-loops, SL1 and SL2, and a closing stem, S3, in the 3′terminal regions of both RNAs, and have investigated its role in translation by performing in vivo and in vitro translation assays using constructs containing the firefly luciferase (F-Luc) coding sequence flanked with the 5′NCR and the YSS-containing 3′NCR of LCV RNAs 1 or 2. The implications of our findings for conceptualizing the role of the YSS in viral RNA synthesis and translation for LCV and other criniviruses are discussed
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