The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found
The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found
复制标题
DOI：
10.1128/jvi.79.1.140-149.2005
复制
发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Lo, CF
中科院分区：
文献类型：
--
作者：
Leu, JH;Tsai, JM;Lo, CF
对虾白斑综合征病毒(WSSV)基因组的一个独特特征是存在一个由18,234个核苷酸组成的巨大开放阅读框架(ORF)，编码一个由6,077个氨基酸组成的长多肽，其功能迄今尚不清楚。本研究应用蛋白质组学方法，对WSSV病毒粒子的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳谱进行了LC-MS/MS分析，发现VP664是WSSV病毒结构蛋白之一。对逆转录-聚合酶链式反应产物进行测序分析，证实了相应的18kb转录本的存在，这也表明vp664是无内含子的。时程分析表明，该转录物在后期转录活跃，提示该基因产物主要参与病毒粒子的组装和形态发生。制备了几种针对该巨蛋白的多克隆抗血清，并成功地将其中一种抗血清用于免疫电子显微镜分析以定位该蛋白在病毒粒子中的位置。免疫电子显微镜和金标记的二抗显示，金颗粒规则地分布在核衣壳的外围，其周期性与呈现为条纹的特征堆积的环亚单位相匹配。根据这一证据和其他证据，我们认为这个巨大的ORF实际上编码了WSSV的核衣壳蛋白。
One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.