Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2
Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2
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DOI：
10.1099/vir.0.80902-0
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发表时间：
2005-10-01
影响因子：
3.8
通讯作者：
Nakai, T
中科院分区：
文献类型：
--
作者：
Iwamoto, T;Mise, K;Nakai, T
条纹杰克神经坏死病毒（SJNNV），感染鱼类，是贝塔诺达病毒属的模式种。该病毒具有正链RNA的二分基因组，命名为RNA 1和RNA 2。一个小的RNA（CA。0（.）个字符。从SJNNV感染的细胞中检测到一个新合成的RNA片段（约4kb），它对应于RNA 1的3 ′-末端区域。cDNA末端快速扩增分析表明，该小RNA（命名为RNA 3）的5'端起始于相应RNA 1序列的2730 nt，含有5'端帽结构。RNA 1内的亚基因组RNA序列的第一个核苷酸的取代选择性地抑制正链RNA 3的产生，但不抑制负链RNA 3的产生，这表明RNA 3可能通过提前终止模型合成。在大肠杆菌中表达单个RNA 3编码的蛋白（命名为蛋白B2），纯化并用于免疫兔以获得抗蛋白B2的多克隆抗体。免疫学试验表明，该抗原特异性地存在于感染条纹杰克鱼幼虫（Pseudocaranxdentex）的中枢神经系统和视网膜中，并存在于感染培养的E-11细胞的细胞质中。这些结果表明，SJNNV从RNA 1产生亚基因组RNA 3，并在病毒增殖过程中合成蛋白B2，如对嗜铬细胞瘤病毒所报道的。此外，在表达绿色荧光蛋白的转基因植物中建立的农杆菌共浸润测定表明，SJNNV蛋白B2具有有效的RNA沉默抑制活性，如对感染昆虫的烟草赤星病毒的蛋白B2所发现的。
Striped jack nervous necrosis virus (SJNNV), which infects fish, is the type species of the genus Betanodavirus. This virus has a bipartite genome of positive-strand RNAs, designated RNAs 1 and 2. A small RNA (ca. 0(.)4 kb) has been detected from SJNNV-infected cells, which was newly synthesized and corresponded to the 3'-terminal region of RNA1. Rapid amplification of cDNA ends analysis showed that the 5' end of this small RNA (designated RNA3) initiated at nt 2730 of the corresponding RNA1 sequence and contained a 5' cap structure. Substitution of the first nucleotide of the subgenomic RNA sequence within RNA1 selectively inhibited production of the positive-strand RNA3 but not of the negative-strand RNA3, which suggests that RNA3 may be synthesized via a premature termination model. The single RNA3-encoded protein (designated protein B2) was expressed in Escherichia coli, purified and used to immunize a rabbit to obtain an anti-protein B2 polyclonal antibody. An immunological test showed that the antigen was specifically detected in the central nervous system and retina of infected striped jack larvae (Pseudocaranx dentex), and in the cytoplasm of infected cultured E-11 cells. These results indicate that SJNNV produces subgenomic RNA3 from RNA1 and synthesizes protein B2 during virus multiplication, as reported for alphanodaviruses. In addition, an Agrobacterium co-infiltration assay established in transgenic plants that express green fluorescent protein showed that SJNNV protein B2 has a potent RNA silencing-suppression activity, as discovered for the protein B2 of insect-infecting alphanodaviruses.