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 2-4kb 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 20kb 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.

Involvement of a subgenomic mRNA in the generation of a variable population of defective citrus tristeza virus molecules

“…These results imply that viral sgRNA messengers also function as building components for genomic rearrangement and exchange of complete viral genes. a Primers dT14V, where V represents either A, C, or G, and P-dT were used for first-strand cDNA synthesis from the polyadenylated templates as described elsewhere (15).…”

“…Infected plants contain the large replicative-form (RF) RNA molecule and a nested set of at least nine smaller species, 3Ј-coterminal subgenomic RNAs (sgRNAs) corresponding to the 3Ј-terminal ORFs 2 to 11. Each sgRNA is present as a single-stranded RNA (ssRNA) molecule and as a corresponding double-stranded RNA (dsRNA) species (9,15). Recombination of CTV molecules, resulting in different sizes of defective RNA molecules (D-RNAs) and different junction sites, has been demonstrated to occur in both the VT isolate of CTV (CTV-VT) and other CTV isolates (16,18).…”

“…The considerable homogeneity of the 3Ј termini and close resemblance to the ORF11 subgenomic RNA of 0.9 kb suggested the identity of these molecules. In order to test this possibility, we have isolated the 0.9-kbp dsRNA fraction from total dsRNA extracted from CTV-VT-infected bark tissue (6,15). The dsRNA was denatured with methylmercury and the [␥-32 P]ATP-labeled P2 primer was used for cDNA extension, according to the method described in reference 14.…”

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“…The hybridization probes consisted of a 611-bp and a 762-bp cDNA fragment from the 3 H and 5 H ends of CTV-VT genome, respectively (15). The DNA probes were either non-radioactively labeled using the Gene Images Random Prime Labeling Module Kit from Amersham or radioactively labeled with 32 P according to Mawassi et al (17). RNA probes labeled with 32 32P-UTP were synthesized, with the Riboprobe System-T7 kit (Promega) according to the manufacturer's instructions, from cDNA fragments of 611 bp and 762 bp of the CTV-VT 3 H and 5 H ends, respectively, cloned in pGEM (Promega).…”

“…In addition to the large replicative form (RF) RNA molecule, the infected plants contain a nested set of at least nine smaller species of 3 H -co-terminal single-and double-stranded subgenomic RNAs (sgRNAs). These sgRNAs correspond to the 3 H -terminal ORFs (16,17). Cloning of the VT strain of CTV revealed the presence of several defective (D) RNAs of various sizes, composed of the 5 H and 3 H termini of the genomic RNA with extensive internal deletions, along with the full-length virus.…”