Hong J S, Engler J A
Department of Biochemistry and Molecular Genetics, School of Medicine and Dentistry, University of Alabama at Birmingham.
J Virol. 1996 Oct;70(10):7071-8. doi: 10.1128/JVI.70.10.7071-7078.1996.
Entry of human adenovirus into cells is a two-step process, mediated in the first step by a specific interaction between the trimeric fiber protein and a specific receptor on the surface of susceptible cells. Because of the interest in human adenovirus as a vector for gene therapy, we have mapped domains in the fiber protein that are important for proper assembly of this trimeric structure and for proper addition of O-linked N-acetylglucosamine (0-GlcNAc). Mutants of adenovirus type 2 fiber in this study were expressed in human cells by use of a recombinant vaccinia virus expression system that yielded protein indistinguishable from the fiber produced during adenovirus infection. The N-terminal half of the protein did not appear to influence fiber trimer formation, since deletions up to 260 amino acids (aa) from the N-terminal end as well as in-frame deletions within the shaft of the molecule still allowed trimerization; internal deletions in the shaft between aa 61 and 260 appeared to alter addition of 0-GlcNAc, as judged by loss of reactivity to a monoclonal antibody specific for this carbohydrate addition. Deletions from the C terminus of the molecule (as small as 2 aa) appeared to prevent trimer formation. Additions of amino acids to the C-terminal end of the fiber showed variable results: a 6-aa addition allowed trimer formation, while a 27-aa addition did not. These trimer-defective mutants were also relatively less stable, as judged kV pulse-chase experiments. Taken together, our results indicate that trimerization of the fiber requires at least two domains, the entire head (aa 400 to 582), and at least the C-terminal-most 15 aa of the shaft.
人腺病毒进入细胞是一个两步过程，第一步由三聚体纤维蛋白与易感细胞表面的特定受体之间的特异性相互作用介导。由于对人腺病毒作为基因治疗载体的兴趣，我们已经绘制了纤维蛋白中对这种三聚体结构的正确组装以及O-连接的N-乙酰葡糖胺（O-GlcNAc）的正确添加很重要的结构域。本研究中2型腺病毒纤维的突变体通过重组痘苗病毒表达系统在人细胞中表达，该系统产生的蛋白质与腺病毒感染期间产生的纤维无法区分。该蛋白的N端一半似乎不影响纤维三聚体的形成，因为从N端删除多达260个氨基酸（aa）以及分子杆内的框内缺失仍允许三聚化；杆内61至260 aa之间的内部缺失似乎改变了O-GlcNAc的添加，这通过对这种碳水化合物添加特异性的单克隆抗体反应性的丧失来判断。从分子C端的缺失（小至2 aa）似乎阻止了三聚体的形成。在纤维C端添加氨基酸显示出不同的结果：添加6个氨基酸允许三聚体形成，而添加27个氨基酸则不允许。通过脉冲追踪实验判断，这些三聚体缺陷突变体也相对不太稳定。综上所述，我们的结果表明，纤维的三聚化至少需要两个结构域，即整个头部（aa 400至582）和杆的至少最C端的15个aa。
