Expression of the beet yellows closterovirus capsid protein and p24, a capsid protein homologue, in vitro and in vivo.

Abstract

The positive-sense RNA genome of beet yellows closterovirus (BYV) encompasses open reading frames (ORFs) for the viral capsid protein (CP, ORF 6) and for a CP homologue (p24, ORF 5). The sequences of the ORFs 5 and 6 were inserted into an Escherichia coli expression vector, pQE-9, under the control of the bacteriophage T5 promoter. The proteins were expressed in bacteria, purified, and used for antiserum production in rabbits. The recombinant BYV CP and p24 showed serological cross-reactions when probed with each antiserum on Western blots. The cross-reactions of the anti-p24 serum with the CP, and of the anti-CP serum with the p24, were abolished by preadsorption with the heterologous antigens, suggesting that CP and p24 share a common epitope(s) resistant to SDS denaturation. Cross-reactivity of the soluble CP and p24 was also observed in indirect plate-trapped antigen ELISA, whereas virtually none was encountered in double-antibody sandwich ELISA. Using a polyclonal anti-p24 serum preadsorbed with the recombinant CP, the p24 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected Tetragonia expansa indicated that both proteins are predominantly located in the soluble fraction of the host cells. Primer extension analysis of the individual double-stranded forms of the subgenomic RNAs bearing the CP and p24 genes allowed them to be mapped and their 5' start sites to be located at nucleotide positions 13,588 and 12,815, respectively, in the complete genome sequence. This corresponds to the 5' untranslated regions of 52 and 105 nucleotides in the subgenomic RNAs for CP and p24, respectively. The data obtained indicate that the synthesis of both subgenomic RNAs is initiated on a negative RNA strand at an adenosine residue found within the conserved sequence 5' CCAUUUPyA (shown as positive-sense), which may thus represent a core element of the subgenomic promoter. This conserved sequence also resembles the sequences at the 5' ends of the CP subgenomic RNAs of tobamoviruses and the Bromoviridae family members, the viruses evolutionarily most closely related to BYV.