5′-coterminal subgenomic RNAs in Citrus tristeza virus-infected cells

Three unusual 5 ' coterminal positive-stranded subgenomic (sg) RNAs, two of about 0.8 kb and one of 10 kb (designated LMT1, LMT2, and LaMT, respectively), from Citrus spp, plants and Nicotiana benthamiana protoplasts infected with Citrus tristeza virus (CN) were characterized. The 5 ' termini of the LMT RNAs were mapped by runoff reverse transcription and found to correspond with the 5 ' terminus of the genomic RNA. The LMT 5 ' -coterminal sgRNAs consisted of two modal lengths of 744-746 and 842-854 nts. The 3 ' of the LaMT RNAs terminated near the junction of ORF Ib and ORF 2 (p33). None of the 5 ' sgRNAs had detectable amounts of corresponding negative-sense RNAs, as occurs with the genomic and 3 ' coterminal subgenomic RNAs of CTV. The abundance of the short and long 5 ' sgRNAs differed considerably in infected cells. The LMT RNAs were considerably more abundant than the genomic RNAs, while the larger LaMT RNA accumulated to much lower levels. The kinetics of accumulation of LMT1 and LMT2 in synchronously infected protoplasts differed. The larger RNA. LMT1, accumulated earlier with a strong hybridization signal at 2 days postinfection, a time when only traces of genomic and 3 ' sgRNAs were detected. The lack of corresponding RNAs, that could be 3 ' cleavage products corresponding to the 5 ' coterminal sgRNAs and the lack of complementary negative strands, suggest that these sgRNAs were produced by termination during the synthesis of the genomic positive strands. (C) 2001 Academic Press.