STRUCTURE AND FUNCTION OF THE ALEUTIAN MINK DISEASE PARVOVIRUS GENOME

阿留申水貂病细小病毒(ADV)持续感染水貂 导致进行性免疫紊乱，其特征是高水平的 抗病毒抗体、高丙种球蛋白血症、浆细胞增多症和免疫 复杂的疾病。病毒在体内不被中和，而ADV存在于 感染性免疫复合体。ADV的分离株在它们的 诱发AD的能力；一些像ADV-Utah和ADV-tr是高度 在体内致病，但对CRFK细胞是复制缺陷的。这个 细胞培养的Adv-G对CRFK具有复制能力，但可以复制 在水貂中很差，如果有的话，也不会导致进行性疾病。 该项目的一个主要目标是将特定的DNA序列与 ADV基因组与功能相关，如致病性 决定因素和应变变异。另一个主要目标是研究 ADV病毒粒子的实际结构。对结构的理解位于 高分辨率将使我们能够(A)绘制表位和致病性图 病毒颗粒上谨慎坐标的决定因素，(B)定义 病毒组装所需的序列。 过去一年取得的成果摘要包括： *ADV的结构研究。用低温电子显微镜观察病毒粒子 在重组杆状病毒的表达下，ADV的结构已被 解析为22埃。病毒粒子的表面有几个独特的 在其他细小病毒中没有观察到的特征。结晶研究 已经启动了。全长衣壳蛋白也被 在大肠杆菌和巴斯德毕赤酵母中表达并纯化至接近 同质性。将这些蛋白质的自组装驱动成 粒子正在研发中。 *对嵌合病毒结构的分析。附加全长 对ADV-G和ADV-Utah之间的嵌合克隆进行了分析。复合体 体外复制能力的决定因素映射到 衣壳蛋白。详细映射表明，非连续的 序列相互作用或“串扰”形成这些决定因素。二 能够复制CRFK的嵌合体被发现会导致病毒血症 成年水貂的抗腺病毒抗体。

Persistent infection of mink with Aleutian mink disease parvovirus (ADV) leads to progressive immune disorder characterized by high levels of antiviral antibodies, hypergammaglobulinemia, plasmacytosis, and immune complex disease. Virus is not neutralized in vivo and ADV exists in infectious immune complexes. Isolates of ADV differ markedly in their ability to induce AD; some like ADV-Utah and ADV-TR are highly pathogenic in vivo, but are replication defective for CRFK cells. The cell-culture ADV-G is replication competent for CRFK, but replicates poorly, if at all, in mink and does not cause progressive disease. A major goal of this project is to correlate specific DNA sequences of the ADV genome to functional correlates, such as pathogenicity determinants and strain variation. Another major goal is to study the actual structure of the ADV virion. Understanding of the structure at high resolution will enable us to (a) map epitopes and pathogenicity determinants to discreet coordinates on the viral particle, (b) define sequences necessary for virus assembly. A summary of the results obtained within the last year includes: * Structural studies of ADV. Using cryo-electron microscopy of virions expressed by recombinant baculoviruses, the structure of ADV has been resolved to 22 Angstroms. The surface of the virion has several unique features not observed in other parvoviruses. Crystallization studies have been initiated. Full-length capsid proteins have also been expressed in E. coli and Pichia pastoris and purified to near homogeneity. Methods to drive self-assembly of these proteins into particles are being developed. * Analysis of chimeric virus constructions. Additional full length clones chimeric between ADV-G and ADV-Utah have been analyzed. Complex determinants for in vitro replication competence map to sequences within the capsid proteins. Detailed mapping indicates that non-contiguous sequences interact or "cross-talk" to form these determinants. Two chimeras, replication competent for CRFK, were found to induce viremia and anti-ADV antibody in adult mink.