STRUCTURE AND FUNCTION OF THE ALEUTIAN MINK DISEASE PARVOVIRUS GENOME

这个项目正在进行的目的是研究人类的基因组 阿留申水貂病细小病毒及其相关结构特征 基因组与功能相关，如抗原表位， 致病决定因素、菌株变异和寄主范围。 通过构建额外的全长嵌合分子克隆 细胞培养适应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，它能够复制 细胞培养，对水貂也有传染性。给动物注射这种物质 病毒感染后出现典型的AD病理改变。XXX-J-8-10为 除部分衣壳蛋白序列(73-88)外，全部为Adv-G 地图单位)，这是ADV-犹他州。这种致病和非致病的病毒 分离株在这一区域只有4个氨基酸残基不同。这 衣壳蛋白序列的一部分包含表面类似物 犬细小病毒(CPV)第4环，与宿主范围有关的区域和 这种病毒的致病性。其他嵌合体是在 XXX-J-8-10的开发表明，至少有两种不同的 衣壳蛋白序列中控制复制的决定因素 在体外的能力。 ADV衣壳蛋白基因在10个非重叠组织中表达 原核表达载体pMAL-c2中的片段。受感染的血清 水貂在选定的区域表现出优先的反应性。这些地区 对应于VP1唯一序列和CPV表面环3和4 始终如一的反应。衣壳的高变区 与CPV表面环2相对应的蛋白质被发现含有一种类型 异种抗血清识别的特异性线性表位 抗体和水貂血清。制备了异种抗血清。 CPV表面环和VP1独特区域的类似物。虽然 它们都与真正的ADV衣壳蛋白发生免疫印迹反应(S)， 只有环3和环4的抗血清能够中和ADV 体外感染性及其与纯化衣壳结合的免疫电子研究 显微镜。这些发现定义了表面环路的关键作用 ADV衣壳蛋白。计算机分析表明，环路3和 4在病毒上形成位于3个折叠对称轴的棘突 粒子。 用冷电子推导AdV病毒粒子结构的研究 显微镜和x射线结晶学已经开始使用Empty 在重组杆状病毒中表达衣壳蛋白。

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.