Neutralizing the human Norovirus Histo-Blood Group Antigen Binding Pocket

杯状病毒科有5个属，分别是诺如病毒、萨波病毒、Lagovirus、Vesivirus和Nebovirus。迄今为止，组织血型抗原（HBGA）结合位点仅为诺如病毒和lagovirus所知。早期对诺如病毒突出(P)结构域的结构研究表明，基因组I和II （GI和GII）诺如病毒每个P二聚体具有两个等效的HBGA结合袋。类似地，lagovirus发现每个P二聚体结合两个HBGAs。我们最近发现，GII诺如病毒有两个额外的HBGA口袋，使每个P二聚体总共有四个HBGA结合位点。为了更好地描述杯状病毒与HBGAs的相互作用，我们将实现三个主要目标。在第一个目标中，我们将使用x射线晶体学确定HBGAs是否与vesivirus， sapovirus和nebovirus的P结构域结合。我们已经证明，x射线晶体学提供了一种确定这些结合相互作用的可靠方法。我们的团队已经解决了30多个不同的P域HBGA复杂结构，包括一些以前未知的结构。我们将扩展这一目标，包括诺如病毒样颗粒（vlp）和HBGA复合物，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，我们将使用x射线晶体学，核磁共振和质谱的组合分析HBGA的剂量依赖性和结合方式。我们现在知道GII诺如病毒每个P二聚体有四个HBGA袋，但对GI诺如病毒和其他杯状病毒的多个结合位点知之甚少。在第三个目标中，我们进一步分析并开发了一种结合GII诺如病毒HBGA结合袋的单克隆。目前，该抗体仅结合几种GII诺如病毒株，但通过进一步修饰，我们期望产生一种广泛反应性的抗体分子，可以结合大多数GII诺如病毒。我们将研究该抗体阻断HBGA附着于诺如病毒的能力，并可能作为诺如病毒感染的治疗方法。总的来说，该提案产生的数据将提供关于杯状病毒和HBGA相互作用的新信息，并包括可以阻止HBGA与GII诺如病毒结合的抗体的开发。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.