Neutralizing the human Norovirus Histo-Blood Group Antigen Binding Pocket

杯状病毒科有5个属，即诺如病毒属、沙普病毒属、拉格氏病毒属、新城疫病毒属和新病毒属。到目前为止，组织血型抗原(HBGA)结合位点只对诺沃克病毒和拉格病毒已知。早期对诺如病毒突起(P)结构域的结构研究表明，基因I和II(GI和GII)诺如病毒每个P二聚体有两个等价的HBGA结合口袋。类似地，滞后病毒被发现在每个P二聚体上结合两个HBGA。我们最近发现GII诺如病毒有两个额外的HBGA口袋，使得每个P二聚体总共有四个HBGA结合位点。在这项提案中，我们将实现三个主要目标，以更好地描述杯状病毒与HBGA的相互作用。在第一个目标中，我们将使用X射线结晶学来确定HBGA是否与VsiVirus、Sap Virus和NeboVirus P结构域结合。我们已经证明，X射线结晶学提供了一种确定这些结合作用的可靠方法。我们的团队已经解决了30多个不同的P结构域HBGA复杂结构，其中包括几个以前未知的结构。我们将把这一目标扩展到包括诺沃克病毒样颗粒(VLP)和HBGA复合体，以便更好地了解完整颗粒背景下的结合机制。在第二个目标中，我们将结合X射线结晶学、核磁共振和MS分析HBGA结合的剂量依赖关系和方式。我们现在知道GII诺如病毒每个P二聚体有四个HBGA口袋，但对GI诺如病毒和其他杯状病毒中的多个结合部位知之甚少。在第三个目标中，我们进一步分析和开发了与GII诺如病毒HBGA结合口袋结合的单抗。目前，这种抗体只与几个GII诺如病毒株结合，但经过进一步的修改，我们预计会产生一种能与大多数GII诺如病毒结合的广谱抗体分子。我们将研究抗体阻断HBGA与诺沃克病毒附着的能力，并可能作为诺沃克病毒感染的治疗手段。总之，这项提案产生的数据将提供有关杯状病毒和HBGA相互作用的新信息，并包括开发能够阻止HBGA与GII诺如病毒结合的抗体，GII诺如病毒是临床上最重要的人类诺如病毒。

There are five genera in the Caliciviridae family, Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. To date, histo-blood group antigen (HBGA) binding sites are only known for norovirus and lagovirus. Earlier structural studies with norovirus protruding (P) domains showed that genogroup I and II (GI and GII) noroviruses had two equivalent HBGA binding pockets per P dimer. Similarly, lagovirus was found to bind two HBGAs per P dimer. We recently discovered that GII noroviruses had two additional HBGA pockets, making a total of four HBGA binding sites per P dimer. In this proposal, we will perform three main aims in order to better describe calicivirus interactions with HBGAs. In the first aim, we will determine if HBGAs bind to vesivirus, sapovirus, and nebovirus P domains using X-ray crystallography. We have already shown that X-ray crystallography provides a reliable method of determining these binding interactions. Our group has solved over 30 different P domain HBGA complex structures, including several that were previously unknown. We will extend this aim to include norovirus virus-like particles (VLPs) and HBGA complexes in order to gain a better understanding of the binding mechanisms in the context of the intact particles. In the second aim, we will analyze the dose-dependence and manner of HBGA binding using a combination of X-ray crystallography, NMR, and MS. We now know that GII noroviruses have four HBGA pockets per P dimer, but little is known about the multiple binding sites in GI noroviruses and other caliciviruses. In the third aim, we further analyze and develop a monoclonal that binds at the GII norovirus HBGA binding pocket. Currently, this antibody only binds several GII norovirus strains, but with further modifications we expect to produce a broadly-reactive antibody molecule that can bind most GII noroviruses. We will study the ability of the antibody to block HBGA attachment to norovirus and possibly function as a treatment for norovirus infections. Altogether, the data generated from this proposal will provide new information on calicivirus and HBGA interactions and include the development of antibody that can block HBGA from binding to the GII noroviruses - the most clinically important human noroviruses.