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.

Replication of Lettuce chlorosis virus (LCV), a crinivirus in the family Closteroviridae, is accompanied by the production of LCV RNA 1-derived novel RNAs.

Cloned infectious complementary DNAs of the bipartite genomic RNAs of Lettuce chlorosis virus (LCV) were constructed. Inoculation of tobacco protoplasts with the in vitro produced RNAs 1 and 2 transcripts, or with RNA 1 transcript alone, resulted in viral replication accompanied by the production of novel LCV RNA 1-derived RNAs. They included the abundantly accumulating LM-LCVR1-1 (~0.38 kb) and LM-LCVR1-2 (~0.3 kb), and the lowly accumulating HM-LCVR1-1 (~8.0 kb) and HM-LCVR1-2 (~6.6 kb), all of which reacted with riboprobes specific to the 5' end of RNA 1 in Northern blot analysis. LM-LCVR1-1 and HM-LCVR1-2 accumulated as positive-stranded RNAs that lacked complementary negative strands, while HM-LCVR1-1 and LM-LCVR1-2 accumulated in both polarities. Additional Northern blot, reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed LM-LCVR1-2 to be an authentic RNA 1-derived defective (D)RNA, suggesting that its synthesis and maintenance are supported in trans by an RNA 1 encoded replication machinery.

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2.

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3' non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.