Infectious pancreatic necrosis virus: Identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2
Infectious pancreatic necrosis virus: Identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2
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DOI：
10.1128/jvi.73.4.3484-3490.1999
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发表时间：
1999-04-01
影响因子：
5.4
通讯作者：
Everitt, E
中科院分区：
文献类型：
--
作者：
Hjalmarsson, A;Carlemalm, E;Everitt, E
感染性胰腺坏死病毒（IPNV）的病毒粒子在对无盐缓冲液透析后完全崩解。通过电子显微镜直接观察这些制剂，发现5.0至6.5 nm厚的缠结丝。通过使用特定的胶体金免疫标记技术，这些结构被证明含有病毒蛋白VP 3。通过蔗糖梯度离心分离丝状体，然后进行血清学分析，证明病毒体的全部VP 3含量与放射性标记的基因组物质一起回收，形成独特的线状核糖核蛋白复合物。在灵敏的印迹分析中，IPNV的外部衣壳组分，即，主要结构蛋白VP 2显示出特异性结合识别N-乙酰半乳糖胺、甘露糖和岩藻糖的糖部分的凝集素。三种已建立的N-连接糖基化代谢抑制剂不能阻止糖残基添加到病毒体中，并且使用N-糖苷酶对分离的病毒体进行酶促去糖基化未能去除凝集素识别的VP 2糖残基。然而，温和的碱性β消除明显降低了凝集素识别VP 2的能力。这些结果表明，当IPNV在RTG-2细胞中增殖时，VP 2的糖基化为O-连接型。
Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline beta elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.