ASSEMBLY OF THE T=4 NUDAURELIA CAPENSIS OMEGA VIRUS CAPSID PROTEIN, POSTTRANSLATIONAL CLEAVAGE, AND SPECIFIC ENCAPSIDATION OF ITS MESSENGER-RNA IN A BACULOVIRUS EXPRESSION SYSTEM
ASSEMBLY OF THE T=4 NUDAURELIA CAPENSIS OMEGA VIRUS CAPSID PROTEIN, POSTTRANSLATIONAL CLEAVAGE, AND SPECIFIC ENCAPSIDATION OF ITS MESSENGER-RNA IN A BACULOVIRUS EXPRESSION SYSTEM
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DOI：
10.1006/viro.1995.1054
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发表时间：
1995-02-20
期刊：
影响因子：
3.7
通讯作者：
JOHNSON, JE
中科院分区：
文献类型：
--
作者：
AGRAWAL, DK;JOHNSON, JE
我们用杆状病毒载体在昆虫细胞中表达了冠状新城疫病毒衣壳蛋白(CP)基因。CP的表达导致病毒样颗粒(VLP)的形成，其大小与天然病毒粒子的T=4准对称性一致。这是第一次展示T=4粒子的组装，通过异源表达，在四个不同的环境中存在化学上相同的亚基。VLP的初始产率很低，开发了一种高效的一步非变性过程，涉及到不连续的甘油梯度分离。利用这种方法，得到了足够数量的VLP，以供进一步表征。电子显微镜观察发现，病毒颗粒大小为40 nm，形态与天然病毒相似。SDS-PAGE显示，这些颗粒由一个62 kDa的主要蛋白和一个70 kDa的次要蛋白组成。脉冲追逐实验表明，在感染过程中，较大的物种被处理成较小的物种的过程非常缓慢。II还确定，这种切割显然依赖于这些颗粒从细胞中释放。此外，这些颗粒被发现包裹了多角体蛋白启动子引导的CP mRNA，具有明显的特异性和选择性。这项研究证实，CP编码序列中存在特定的包膜信号，重建这种病毒形态发生的大部分步骤(如果不是全部步骤)所需的成分可以在杆状病毒感染细胞中完成。这一结果与T=3和T=4四病毒在颗粒组装中使用相似的生物学策略的信念是一致的。(C)1995年学术出版社。
We have expressed the gene encoding the coat protein (CP) of Nudaurelia capensis omega Virus in insect cells with a baculovirus vector. Expression of CP resulted in formation of virus-like particles (VLPs) having a size consistent with the T = 4 quasi-symmetry observed for native virions. This is the first demonstration of assembly for a T = 4 particle, with chemically identical subunits present in four distinct environments, by heterologous expression. Initial yields of VLPs were low, and an efficient one step nondenaturing procedure involving separation on discontinuous glycerol gradients was developed. Using this method, VLPs were obtained in quantities sufficient for further characterization. Electron microscopic observation revealed 40-nm particles that were morphologically similar to native virus. SDS-PAGE revealed that these particles were composed of a 62-kDa major protein and a minor 70-kDa protein. Pulse-chase experiments revealed that the larger species was processed into the smaller one very slowly over the course of an infection. II was also determined that this cleavage was apparently dependent on release of these particles from the cell. Furthermore, these particles were found to encapsidate the polyhedrin promoter-directed CP mRNA with an apparently striking degree of specificity and selectivity. This investigation establishes that a specific encapsidation signal exists within the CP coding sequences and that the components required for reconstructing most, if not all, steps in the morphogenesis of this virus can be accomplished in baculovirus-infected cells. The results presented here are consistent with the belief that similar biological strategies are utilized by T = 3 nodaviruses and T = 4 tetraviruses in particle assembly. (C) 1995 Academic Press, Inc.