ADENOVIRUS-RECEPTOR INTERACTION--STRUCTURE, FUNCTION

我们的腺病毒-12纤维球形结构域与B族柯萨奇病毒和许多腺病毒的细胞受体CAR的可溶性片段复合物的结构，挑战了病毒-受体结合机制的既定模型。旋钮上的CAR结合位点是由表面环形成的，这是旋钮蛋白中最易变化的部分，并解释了血清学差异，而不是由基于Ad5旋钮早期晶体结构提出的作为候选结合位点的更高度保守的区域形成的。保守特异性受体结合位点和抗原可变区域的重叠强烈表明，病毒-受体结合的分子机制可能是独特的，在非病毒系统中没有先例。旋钮- car和HIV gp120-CD4界面都含有不寻常的大充满水的空腔，除了允许氨基酸通过界面直接接触外，还允许间接的水介导结合。这一特征可能是缓冲结合位点特异性对抗重叠抗原变异机制的一部分。我们将利用x射线晶体学、诱变和生物化学的结合方法，详细分析旋钮- car结合的机制。不同腺病毒血清型的纤维旋钮的结构将被单独解决，并与CAR联合研究抗原变异对受体结合位点结构和活性的影响。通过空腔结合水分子直接或间接与CAR接触的单个球形氨基酸的作用将通过诱变和定量结合试验进行评估。将分离干扰旋钮- car结合的旋钮特异性单克隆抗体，以表征受体结合位点的抗原结构。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的受体结合机制。我们的结果也可能应用于疫苗和抗病毒药物的开发，它们将影响重新定位腺病毒载体的基因治疗倾向的努力。

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.