Pedersen Torunn, Skjesol Astrid, Jørgensen Jorunn B
Department of Marine Biotechnology, Norwegian College of Fishery Sciences, University of Tromsø, N-9037 Tromsø, Norway.
J Virol. 2007 Jun;81(12):6652-63. doi: 10.1128/JVI.02831-06. Epub 2007 Apr 11.
Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.
传染性胰腺坏死病毒（IPNV）是双RNA病毒科的一种双链RNA（dsRNA）病毒，由两个片段组成，可在鲑科鱼类中引发广泛疾病。它的两个基因组片段与病毒RNA依赖性RNA聚合酶VP1以及假定的内部蛋白VP3一起，被包裹在由VP2组成的单壳衣壳中。IPNV分子生物学的主要方面，如病毒粒子组装以及对宿主大分子的干扰，目前仍知之甚少。为了解感染过程，分析病毒蛋白之间的相互作用至关重要。在本研究中，我们聚焦于VP3的相互作用特性，VP3被认为是双RNA病毒中病毒粒子组装的关键组织者。通过应用酵母双杂交系统并结合免疫共沉淀技术，证实VP3能与VP1结合且能强烈自缔合。此外，体外下拉实验表明VP3能以不依赖序列的方式特异性结合dsRNA。VP3与VP1之间的结合不依赖于dsRNA的存在。缺失分析确定了VP3的101个N端氨基酸内的自相互作用结构域以及VP3的62个C端氨基酸内的VP1相互作用结构域。C端对于VP3的dsRNA结合能力也很关键，但并不充分。对于VP1而言，90个C端氨基酸是维持与VP3结合能力的唯一可缺失部分。动力学分析显示，在IPNV感染的CHSE - 214细胞中，成熟病毒粒子形成之前就存在VP1 - VP3复合物，这表明其在促进组装过程中发挥作用。
