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.