Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae

Abstract

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3' end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

FIG. 1

Genome organization of the ilarviruses. (A) Diagram of the three triplet codon phases of plus-strand RNA 2 of the four ilarviruses. The AUG triplet and the three stop codons are shown as short and long vertical lines, respectively. (B) SpLV genome organization. Proteins 1a, 2a, and 3a are translated from genomic RNAs 1, 2, and 3, while the CP and the 2b protein are translated via subgenomic RNAs 4 and 4A. The open box represents the highly conserved 3′-terminal sequence of 163 nucleotides (nt). The target positions of the two probes (a and b) used in Northern blot analysis, as well as the approximate binding position of primer Xin-44 (arrows), are indicated. K, 10³.

FIG. 2

Alignment of the amino acid (aa) sequences of the putative 2b proteins encoded by the four ilarviruses (10, 12, 22, 23). The alignment was obtained by using the Pileup program of the Wisconsin GCG package (6) implemented at the Bioinformatic Center of the National University of Singapore. Residues identical in three or more 2b proteins are highlighted. Notably, the VVSG motif is absolutely conserved in all four ilarvirus 2b proteins.

FIG. 3

Characterization of expression of the SpLV 2b gene. (A) SpLV RNA 4A detection. Total RNAs were extracted from SpLV virions (lane 1) or from mock-inoculated (lanes 2 and 4) or SpLV-infected (lanes 3 and 5) N. tabacum (cv. Xanthi) plants, fractionated, and blotted onto an Amersham Hybond-N+ membrane as previously described (8). SpLV virion RNAs were visualized by ethidium bromide staining (lane 1). Alternatively, duplicate membranes were hybridized with either probe a, which is specific for all SpLV RNAs (lanes 2 and 3), or probe b, which is specific for the ORF 2b coding sequence only (lanes 4 and 5). The positions of SpLV RNAs 1, 2, 3, 4, and 4A are indicated. (B) Mapping of the 5′ end of SpLV RNA 4A. Total RNAs from healthy plants (lane 6) or plants infected with SpLV (lane 7) were annealed with primer Xin-44, reverse transcribed by avian myeloblastosis virus reverse transcriptase, and analyzed on a sequencing gel. Primer Xin-44 was also used to sequence a full-length cDNA clone of SpLV RNA 2 by using the Sequenase kit, version 2.0 (U.S. Biochemicals), to provide a sequence ladder (lanes A, C, G, and T). (C) In vitro translation. SpLV virion RNAs were translated in vitro by using the Promega wheat germ extract in accordance with the manufacturer’s instructions. The translational products from BMV RNAs provided by the manufacturer were also analyzed, and their sizes (in kilodaltons) are in parentheses. The positions of the ¹⁴C-labeled low-range protein molecular weight standards from Gibco Bethesda Research Laboratories are on the left.