Kraschnefski Mark J, Bugarcic Andrea, Fleming Fiona E, Yu Xing, von Itzstein Mark, Coulson Barbara S, Blanchard Helen
Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222, Australia.
Glycobiology. 2009 Mar;19(3):194-200. doi: 10.1093/glycob/cwn119. Epub 2008 Oct 30.
The rotavirus spike protein VP4 mediates attachment to host cells and subsequent membrane penetration. The VP8() domain of VP4 forms the spike tips and is proposed to recognize host-cell surface glycans. For sialidase-sensitive rotaviruses such as rhesus (RRV), this recognition involves terminal sialic acids. We show here that the RRV VP8()(64-224) protein competes with RRV infection of host cells, demonstrating its relevance to infection. In addition, we observe that the amino acids revealed by X-ray crystallography to be in direct contact with the bound sialic acid derivative methyl alpha-D-N-acetylneuraminide, and that are highly conserved amongst sialidase-sensitive rotaviruses, are residues that are also important in interactions with host-cell carbohydrates. Residues Arg101 and Ser190 of the RRV VP8() carbohydrate-binding site were mutated to assess their importance for binding to the sialic acid derivative and their competition with RRV infection of host cells. The crystallographic structure of the Arg(101)Ala mutant crystallized in the presence of the sialic acid derivative was determined at 295 K to a resolution of 1.9 A. Our multidisciplinary study using X-ray crystallography, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and competitive virus infectivity assays to investigate RRV wild-type and mutant VP8() proteins has provided the first evidence that the carbohydrate-binding cavity in RRV VP8(*) is used for host-cell recognition, and this interaction is not only with the sialic acid portion but also with other parts of the glycan structure.
轮状病毒刺突蛋白VP4介导病毒与宿主细胞的附着以及随后的膜穿透过程。VP4的VP8()结构域形成刺突顶端，被认为可识别宿主细胞表面聚糖。对于诸如恒河猴轮状病毒（RRV）这类对唾液酸酶敏感的轮状病毒，这种识别涉及末端唾液酸。我们在此表明，RRV VP8()(64 - 224)蛋白可与RRV对宿主细胞的感染竞争，证明了其与感染的相关性。此外，我们观察到，通过X射线晶体学显示与结合的唾液酸衍生物α-D-N-乙酰神经氨酸甲酯直接接触且在对唾液酸酶敏感的轮状病毒中高度保守的氨基酸，也是在与宿主细胞碳水化合物相互作用中重要的残基。对RRV VP8()碳水化合物结合位点的精氨酸101和丝氨酸190残基进行突变，以评估它们对与唾液酸衍生物结合以及与RRV感染宿主细胞竞争的重要性。在295 K下测定了在唾液酸衍生物存在下结晶的精氨酸(101)丙氨酸突变体的晶体结构，分辨率为1.9 Å。我们使用X射线晶体学、饱和转移差异核磁共振光谱、等温滴定量热法和竞争性病毒感染性测定等多学科研究方法来研究RRV野生型和突变型VP8()蛋白，首次证明了RRV VP8(*)中的碳水化合物结合腔用于宿主细胞识别，并且这种相互作用不仅与唾液酸部分有关，还与聚糖结构的其他部分有关。
