ADENOVIRUS-RECEPTOR INTERACTION--STRUCTURE, FUNCTION

我们的腺病毒-12纤维结节结构域与CAR的可溶性片段形成复合体，CAR是B组柯萨奇病毒和许多腺病毒的细胞受体，挑战了已建立的病毒与受体结合机制的模型。旋钮上的CAR结合位点是由表面环形成的，表面环是旋钮蛋白中变化最大的部分，可以解释血清学差异，而不是来自更高度保守的区域，这些区域是基于Ad5旋钮的早期晶体结构被认为是候选结合位点的。具有保守特异性和抗原性可变区的受体结合部位的重叠强烈表明，病毒与受体结合的分子机制可能是独一无二的，在非病毒系统中是前所未有的。旋钮-CAR和HIV gp120-CD4界面都含有不同寻常的巨大的充满水的空腔，除了通过界面与氨基酸直接接触外，还允许通过水间接结合。这一特征可能是缓冲结合位点特异性以防止重叠抗原变异的机制的一部分。我们将利用X射线结晶学、诱变和生物化学相结合的方法，详细分析纽结-CAR结合的机制。将不同血清型的腺病毒纤维结节的结构单独和与CAR结合研究抗原变异对受体结合部位结构和活性的影响。将通过诱变和定量结合分析来评估通过空腔结合水分子直接或间接接触CAR的单个节状氨基酸的贡献。干扰纽结结合的纽结特异性单抗将被分离出来，以表征受体结合部位的抗原性结构。从我们的研究中获得的知识可能广泛适用于理解包括HIV在内的其他病毒系统中受体结合的机制。我们的结果也可能应用于疫苗和抗病毒药物的开发，并将影响重定向腺病毒载体用于基因治疗的努力。

Our structure of the adenovirus-12 fiber knob domain in complex with a soluble fragment of CAR, the cellular receptor for group B coxsackieviruses and many adenoviruses, challenges established models of the mechanism of virus-receptor binding. The CAR binding site on knob is formed from surface loops, which are the most variable parts of the knob protein and account for serological differences, rather than from more highly conserved regions that were proposed as candidate binding sites based on an earlier crystal structure of the Ad5 knob. The overlap of a receptor binding site of conserved specificity and antigenically variable regions strongly suggests that aspects of the molecular mechanism of virus-receptor binding may be unique and without precedent in non-viral systems. Both the knob-CAR and HIV gp120-CD4 interfaces contain unusual large water-filled cavities, allowing for indirect water-mediated binding in addition to direct contact of amino acids across the interface. This feature may be part of a mechanism to buffer binding site specificity against overlapping antigenic variation. We will analyze the mechanism of knob-CAR binding in detail, using the combined approaches of x-ray crystallography, mutagenesis and biochemistry. The structures of fiber knobs from different adenovirus serotypes will be solved alone and in complex with CAR to study the impact of antigenic variation on the structure and activity of the receptor binding site. The contribution of individual knob amino acids that contact CAR directly or indirectly through cavity-bound water molecules will be assessed by mutagenesis and quantitative binding assays. Knob-specific monoclonal antibodies which interfere with knob-CAR binding will be isolated to characterize the antigenic structure of the receptor binding sites. Knowledge gained from our studies may be broadly applicable to understanding mechanisms of receptor binding in other virus systems, including HIV. Our results also may have application to the development of vaccines and anti- viral drugs, and they will impact efforts to retarget the tropism of adenovirus-based vectors for gene therapy.