Zeng C Q, Estes M K, Charpilienne A, Cohen J
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Virol. 1998 Jan;72(1):201-8. doi: 10.1128/JVI.72.1.201-208.1998.
The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.
轮状病毒的最核心部分由VP2组成，它形成一层蛋白质层，围绕着两种次要蛋白质VP1和VP3以及11段双链RNA基因组。这个由主要衣壳蛋白VP6包围的内核层构成了具有转录活性的双层颗粒。每个编码双层颗粒结构蛋白的基因已被克隆到杆状病毒重组体中，并在昆虫细胞中表达。此前，我们发现轮状病毒双层颗粒结构蛋白不同组合的共表达会导致病毒样颗粒（VLP）的形成，并且每个含有推测的RNA依赖性RNA聚合酶VP1的VLP在体外模板依赖性检测系统中检测时具有复制酶活性（C.Q.-Y.曾、M.J.温茨、J.科恩、M.E.埃斯蒂斯和R.F.拉米格，《病毒学杂志》70:2736 - 2742，1996年）。这项工作报道了含有截短VP2（VPdelta2，含氨基酸[aa]Met - 93至880）的VLP的构建和特性。单独表达VPdelta2导致形成单层delta2 - VLP。VPdelta2与VP6共表达产生双层delta2/6 - VLP。VPdelta2与VP1或VP3或VP1和VP3两者共表达形成的VLP导致形成同时缺乏VP1和VP3的VLP。VP6与VPdelta2的存在并未导致VP1和VP3的衣壳化。为了确定VP2结合VP1所需的结构域，使用一系列截短的VP2构建体进行了Far - Western印迹分析，以测试它们结合VP1的能力。这些分析表明：（i）全长VP2（aa 1至880）与VP1结合；（ii）任何缺少aa 1至25的N端截短都不能与VP1结合；（iii）一个C端截短296个氨基酸的VP2构建体（aa 1至583）保持与VP1结合的能力。这些分析表明轮状病毒VP2的N端对于VP1和VP3的衣壳化是必需的。
