Agranovsky A A, Koenig R, Maiss E, Boyko V P, Casper R, Atabekov J G
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
J Gen Virol. 1994 Jun;75 ( Pt 6):1431-9. doi: 10.1099/0022-1317-75-6-1431.
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.
甜菜黄化线形病毒（BYV）的正义RNA基因组包含病毒衣壳蛋白（CP，开放阅读框6）和CP同源物（p24，开放阅读框5）的开放阅读框。将开放阅读框5和6的序列插入受噬菌体T5启动子控制的大肠杆菌表达载体pQE - 9中。这些蛋白质在细菌中表达、纯化，并用于在兔体内产生抗血清。当在蛋白质免疫印迹中用每种抗血清检测时，重组BYV CP和p24显示出血清学交叉反应。抗p24血清与CP以及抗CP血清与p24之间的交叉反应通过用异源抗原预吸附而消除，这表明CP和p24共享一个对SDS变性有抗性的共同表位。在间接平板捕获抗原ELISA中也观察到可溶性CP和p24的交叉反应，而在双抗体夹心ELISA中几乎没有观察到。使用用重组CP预吸附的多克隆抗p24血清，在BYV感染的植物中检测到了p24。对BYV感染的番杏亚细胞组分的分析表明，这两种蛋白质主要位于宿主细胞的可溶性组分中。对携带CP和p24基因的亚基因组RNA的各个双链形式进行引物延伸分析，使它们能够被定位，并且它们的5'起始位点分别位于完整基因组序列中的核苷酸位置13588和12815处。这分别对应于CP和p24亚基因组RNA中52和105个核苷酸的5'非翻译区。所获得的数据表明，两种亚基因组RNA的合成均起始于负链RNA上保守序列5' CCAUUUPyA（以正义链显示）内的一个腺苷残基，因此该序列可能代表亚基因组启动子的核心元件。这个保守序列也类似于烟草花叶病毒和雀麦花叶病毒科成员（与BYV进化关系最密切的病毒）CP亚基因组RNA 5'端的序列。
