Populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic RNA molecules

Abstract

Plants infected by citrus tristeza virus (CTV) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. The CTV particles and RNA extracts from purified CTV preparations were fractionated by sucrose gradient centrifugation and the RNA molecules from different fractions translated in a reticulocyte translation system. Fractions containing predominantly an RNA band of approximately 3.2 kb directed the synthesis of CTV coat protein (CP), which in SDS-PAGE had an estimated molecular mass of 28 kDa. Three additional polypeptides, with estimated sizes of 21 kDa, 23 kDa and 27 kDa, were translated from a range of RNA molecules smaller than 3.2 kb. Hybridization with cDNA to the CP gene (CTV-CPG) and with a 350 base clone complementary to the 3' and 5' termini of the genes for CTV p20 and p23.5, respectively, indicated that preparations of CTV particles contain, in addition to the genomic (20 kb) RNA, two sub-genomic RNA molecules of 3.2 kb and 2.4 kb and probably also two smaller molecules of 1.6 kb and 0.9 kb. Only the 3.2 kb RNA, its corresponding dsRNA molecule and populations of larger RNAs, including the 20 kb genomic RNA, hybridized with a CTV-CPG probe, thus conflicting with our previous assignment of the CTV-CPG to the 0.8 kbp dsRNA. Based on these results we propose that distinct populations of CTV particles encapsidate smaller RNAs which were formed as a nested set of subgenomic RNAs. Sequence analysis of 2540 nucleotides downstream to CTV-CPG of strain VT revealed four open reading frames (ORFs) potentially encoding in the 5' to 3' direction, 18 kDa (p18), 13 kDa (p13), 20 kDa (p20) and 23.5 kDa (p23.5) proteins. The CTV-VT ORFs showed variable but usually close levels of homology with the corresponding ORFs of CTV-T36 from Florida.