Kumar Shantanu, Ochoa Wendy, Kobayashi Shinichi, Reddy Vijay S
Department of Molecular Biology, TPC-6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Virol. 2007 Feb;81(3):1119-28. doi: 10.1128/JVI.01909-06. Epub 2006 Nov 1.
Noroviruses (NoVs) are the causative agents of nonbacterial acute gastroenteritis in humans. NoVs that belong to genogroup II (GII) are quite prevalent and prone to undergo recombination, and their three-dimensional structure is not yet known. Protein homology modeling of Sinsiro virus (SV), a member of the GII.3 NoVs, revealed the presence of a surface-exposed 20-amino-acid (aa) insertion in the P2 domain of the capsid protein (CP) relative to the Norwalk virus (NV) CP, which is a well known hot spot for mutations to counter the host immunological response. To further characterize the role of the long insertion in SV, the capsid protein gene was expressed using the recombinant baculovirus system. Trypsinization of the resultant virus-like particles yielded two predominant bands (31.7 and 26.1 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. N-terminal sequencing and analysis of the mass spectroscopic data indicated that these fragments correspond to residues 1 to 292 (26.1 kDa) and 307 to 544 (31.7 kDa). In addition, the above data taken together with the comparative modeling studies indicated that the trypsin cleavage sites of the Sinsiro virus CP, Arg292 and Arg307, are located at the beginning of and within the 20-aa insertion in the P2 domain, respectively. This study demonstrates that the presence of the surface-exposed loop in the GII.3 NoVs facilitates the trypsinization of the capsid protein in the assembled form. The SV particles remain intact even after trypsin digestion and retain the suggested receptor binding linear epitope of residues 325 to 334. The above results are distinct from those obtained from the trypsinization studies performed earlier on the NV (GI) and VA387 (GII) viruses, both of which lack the large surface insertion and associated basic residues. These new observations may have implications for host receptor binding, cell entry, and norovirus infection in general.
诺如病毒（NoVs）是人类非细菌性急性胃肠炎的病原体。属于基因组II（GII）的诺如病毒相当普遍且易于发生重组，其三维结构尚不清楚。GII.3诺如病毒成员辛西罗病毒（SV）的蛋白质同源性建模显示，与诺沃克病毒（NV）衣壳蛋白（CP）相比，衣壳蛋白（CP）的P2结构域存在一个表面暴露的20个氨基酸（aa）插入，这是对抗宿主免疫反应的突变热点。为了进一步表征SV中长插入的作用，使用重组杆状病毒系统表达衣壳蛋白基因。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹分析中，所得病毒样颗粒经胰蛋白酶处理产生两条主要条带（31.7和26.1 kDa）。N端测序和质谱数据分析表明，这些片段对应于残基1至292（26.1 kDa）和307至544（31.7 kDa）。此外，上述数据与比较建模研究一起表明，辛西罗病毒CP的胰蛋白酶切割位点Arg292和Arg307分别位于P2结构域20-aa插入的起始处和内部。这项研究表明，GII.3诺如病毒中表面暴露环的存在促进了组装形式的衣壳蛋白的胰蛋白酶处理。即使经过胰蛋白酶消化，SV颗粒仍保持完整，并保留了残基325至334的推测受体结合线性表位。上述结果与早期对NV（GI）和VA387（GII）病毒进行胰蛋白酶处理研究所得结果不同，这两种病毒都缺乏大的表面插入和相关碱性残基。这些新观察结果可能对宿主受体结合、细胞进入以及一般的诺如病毒感染具有重要意义。
