Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus

Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RP), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3'-coterminal and two 5'-coterminal subgenomic RNAs (sgRNAs). The 3'-coterminal sgRNAs contain the MP (3'MP sgRNA) and CP (3'CP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5' end. These sgRNAs start with a hexanucleotide which is also present at the 5' terminus of the gRNA. The 5'-coterminal sgRNAs have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5'RPMP sgRNA) and most ORF1 (5'RP sgRNA), respectively. Their 3' termini map 35 and 40 nt upstream of the transcription initiation of the 3'CP and 3'MP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3'-coterminal sgRNAs transcribed from the minus gRNA strand. The 5'-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis.