Koromyslova Anna D, Hansman Grant S
Schaller Research Group at the University of Heidelberg and the DKFZ, Germany, Heidelberg, Germany, and Department of Infectious Diseases, Virology, University of Heidelberg, Germany, Heidelberg, Germany.
Schaller Research Group at the University of Heidelberg and the DKFZ, Germany, Heidelberg, Germany, and Department of Infectious Diseases, Virology, University of Heidelberg, Germany, Heidelberg, Germany
J Virol. 2015 Mar;89(5):2718-30. doi: 10.1128/JVI.03176-14. Epub 2014 Dec 17.
Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ~10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability.
We characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.
人诺如病毒是二十面体单链RNA病毒。衣壳蛋白分为外壳（S）和突出（P）结构域，它们通过一个灵活的铰链区相连。存在众多基因和抗原性不同的诺如病毒，且优势毒株每隔一年就会发生演变。在细胞培养中培养人诺如病毒存在困难以及毒株不断演变，这阻碍了疫苗和抗病毒药物的研发。在此，我们展示了人诺如病毒P结构域与两种不同纳米抗体结合的X射线晶体结构。一种纳米抗体Nano - 85具有广泛的反应性，而另一种纳米抗体Nano - 25具有毒株特异性。我们发现这两种纳米抗体都与P结构域的下部区域结合，且具有纳摩尔亲和力。Nano - 85的结合位点主要由基因不同的II组诺如病毒中高度保守的氨基酸组成。几个保守残基也被一种广泛反应性的单克隆抗体识别，这表明该区域包含一个主要表位。将P结构域纳米抗体复合物结构叠加到冷冻电子显微镜颗粒结构中发现，两种纳米抗体都结合在颗粒上的封闭位点。包含约10至12个氨基酸的灵活铰链区可能允许P结构域在颗粒上有一定程度的移动，以容纳纳米抗体。有趣的是，Nano - 85与完整颗粒的结合相互作用导致颗粒在体外解体。总之，这些结果表明高度保守 的Nano - 85结合表位包含颗粒解体的触发机制。从根本上说，这个表位代表了诺如病毒的一个潜在脆弱位点。
我们对两种与人诺如病毒结合的不同纳米抗体（Nano - 85和Nano - 25）进行了表征。两种纳米抗体都以高亲和力结合到P结构域的下部区域，该区域在完整颗粒上是封闭的。Nano - 25对GII.10具有特异性，而Nano - 85结合几种不同的GII基因型，包括GII.4、GII.10和GII.12。我们表明，Nano - 85能够使用夹心酶联免疫吸附测定法检测临床粪便标本中的诺如病毒病毒粒子。重要的是，我们发现Nano - 85与完整颗粒的结合导致颗粒解体。我们相信，经过进一步测试，Nano - 85不仅将作为诺如病毒检测系统中的诊断试剂发挥作用，还可能作为一种广泛反应性的II组诺如病毒抗病毒药物发挥作用。
