Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species

Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, has a single-stranded positive-sense genomic RNA (gRNA) of 3.9 kb. The 5′ half of the gRNA encodes two proteins involved in replication, the p27 and its readthrough product, p86 (the viral RNA dependent-RNA polymerase, RdRp), and the 3′ half encodes two small movement proteins, p7 and p12, and the coat protein (CP). As other members of the family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs (sgRNAs). Analysis of double-stranded RNAs extracted from PFBV-infected leaves and Northern blot hybridizations of total RNA from infected plants or protoplasts revealed than PFBV produces four 3′-coterminal sgRNAs of 3.2, 2.9, 1.7 and 1.4 kb, respectively. The 5′ termini of the 1.7 and 1.4 kb sgRNAs mapped 26 and 143 nt upstream of the initiation codons of the p7 and CP genes, respectively, whereas the 5′-ends of the 3.2 and 2.9 kb sgRNAs were located within the readthrough portion of the RdRp gene. The PFBV sgRNAs begin with a motif which is also present at the 5′ terminus of the gRNA and the minus polarity of the regions preceding their corresponding start sites (in the gRNA) may be folded into hairpin structures resembling those found for the sgRNA promoters of other carmoviruses. The results indicate that, besides the sgRNAs involved in the translation of the movement proteins and the CP identified in most carmoviral infections, PFBV produces two additional sgRNAs whose biological significance is currently unknown. The possible participation of the 3.2 and 2.9 kb PFBV sgRNAs in the expression of readthrough portions of the RdRp is discussed.