STRUCTURE AND FUNCTION OF THE ALEUTIAN MINK DISEASE PARVOVIRUS GENOME

该项目的目的是研究人类的基因组。 水貂阿留申病细小病毒（ADV）及其相关结构特征 基因组的功能相关，如抗原表位， 致病性决定因素、菌株变异和宿主范围。 通过构建额外的全长嵌合分子克隆， 细胞培养适应性ADV-G和致病性ADV-Utah分离株，我们 已经开发出一种病毒，XXX-J-8-10，可以复制， 细胞培养，对水貂也有感染性。注射了这个的动物 病毒出现典型的AD病理改变。 XXX-J-8-10是 除了衣壳蛋白序列的一部分外，全部为ADV-G（73-88 地图单位），即ADV-Utah。这种致病性和非致病性病毒 分离株在该区域仅相差4个氨基酸残基。 这 衣壳蛋白序列的一部分含有表面蛋白的类似物 犬细小病毒（CPV）的环4，涉及宿主范围的区域， 这种病毒的致病性。 在本研究期间制备的其他嵌合体 XXX-J-8-10的发展表明，至少有两个不同的 衣壳蛋白序列中控制复制的决定簇 体外能力 ADV衣壳蛋白基因在10个非重叠克隆中得到表达， 片段在原核表达载体pMAL-c2中表达。感染者血清 水貂表现出对选定区域的优先反应性。 领域 对应于VP 1独特序列和CPV表面环3和4 一直都很积极 衣壳的高变区 蛋白，对应于CPV表面环2，被发现含有一种类型 异源抗血清识别的特异性线性表位， 抗体和水貂血清。 制备了针对该抗体的异源性抗血清。 CPV表面环和VP 1独特区域的类似物。 虽然 所有这些都在免疫印迹中与真正的ADV衣壳蛋白反应， 只有抗环3和4的抗血清能够中和ADV 体外感染性和免疫电子中与纯化衣壳结合 显微镜 这些发现定义了表面环的关键作用 ADV衣壳蛋白。计算机分析表明，循环3和 4形成位于病毒上的3重对称轴处的尖峰 粒子 ADV病毒粒子结构的低温电子学研究 显微镜和X射线晶体学已经开始使用空的 在重组杆状病毒中表达的衣壳。

The ongoing purpose of this project is to study the genome of the Aleutian mink disease parvovirus (ADV) and to relate structural features of the genome to functional correlates, such as antigenic epitopes, pathogenicity determinants, strain variation and host range. By constructing additional full-length chimeric molecular clones between the cell-culture adapted ADV-G and the pathogenic ADV-Utah isolates, we have developed a virus, XXX-J-8-10, that is replication competent for cell culture and also is infectious for mink. Animals injected with this virus developed typical pathological findings of AD. XXX-J-8-10 is entirely ADV-G except for a portion of the capsid protein sequence (73-88 map units) which is ADV-Utah. This pathogenic and nonpathogenic viral isolates differ only by 4 amino acid residues in this region. This portion of the capsid protein sequence contains the analogue to surface loop 4 of canine parvovirus (CPV), a region implicated in host range and pathogenicity of that virus. Other chimeras prepared during the development of XXX-J-8-10 revealed that there are at least 2 distinct determinants in the capsid protein sequence that govern replication competence in vitro. The capsid protein gene of ADV was expressed in 10 non-overlapping segments in a prokaryotic expression vector, pMAL-c2. Sera from infected mink exhibited preferential reactivity for selected regions. The areas corresponding to the VP1 unique sequence and CPV surface loops 3 and 4 were consistently reactive. The hypervariable region of the capsid protein, corresponding to CPV surface loop 2, was found to contain a type specific linear epitope recognized by heterologous antisera, a monoclonal antibody and mink sera. Heterologous antisera were prepared against the analogues of the CPV surface loops and the VP1 unique regions. Although all reacted in immunoblot against the bona fide ADV capsid proteins(s), only the antisera against loops 3 and 4 were able to neutralize ADV infectivity in vitro and bind to purified capsids in immune electron microscopy. These findings define a pivotal role for the surface loops of the ADV capsid proteins. Computer analysis suggests that loops 3 and 4 form the spike located at the 3 fold axes of symmetry on the viral particle. Studies to derive the structure of the ADV virion by cryo-electron microscopy and x-ray crystallography have been initiated using empty capsids expressed in a recombinant baculovirus.