STRUCTURE AND FUNCTION OF THE ADV GENOME

这个项目的目的是研究基因组结构的各个方面。 和阿留申水貂病细小病毒(ADV)的功能。 在过去的一年里，我们开始了对全长嵌合克隆的研究 ADV.策略一直是替换传染性病毒的片段 非致病性ADV-G株及其相应毒株的分子克隆 直接来源于致病性ADV的分子克隆片段 被感染的动物器官。到目前为止，检测的3个结构中有两个未能 细胞内连续传代后产生感染性Adv 文化；这些克隆是15-88或43-65地图的嵌合体 单位段。65-73地图单元片段的克隆嵌合体能够 复制类似于亲本Adv-G。这些结果表明，在 细胞培养中至少有一个允许复制的决定因素映射到 基因组中包含氨基末端的43-65个图谱单位部分 在病毒粒子蛋白中，非结构蛋白的羧基末端 以及与其中一个剪接相关联的序列。 我们还开始研究adv蛋白在牛痘中的表达。 基于真核表达系统。代表拼接的R3的数据段 编码这两种ADV结构蛋白的mRNA已重组为 一种传染性牛痘病毒。该重组痘苗病毒(VV-11L1)定向 VP1(P85)和VP2(P75)的合成。病毒抗原被定位 到原子核。此外，VV-IL1感染细胞的裂解物也可以使用 在对流免疫电泳法中作为ADV试验抗原。最后，细小病毒 用电子显微镜观察颗粒的形态。因此，所有必要的信号 用于生产和核运输VPL/VP2以及 颗粒组装所需的物质都存在。

The purpose of this project is to study various aspects of genome structure and function of the Aleutian mink disease parvovirus (ADV). In the past year we have initiated studies on full-length chimeric clones of ADV. The strategy has been to replace segments of an infectious molecular clone of the nonpathogenic ADV-G strain with corresponding fragments of molecular clones from pathogenic ADVs derived directly from infected animal organs. Two of the 3 constructs assayed to date failed to produce infectious ADV after transfection and serial passage in cell culture; these were clones chimeric for either the 15-88 or the 43-65 map unit segments. A clone chimeric for the 65-73 map unit segment was able to replicate similar to the parent ADV-G. These results suggested that at least one determinant for permissive replication in cell culture maps to the 43-65 map unit portion of the genome, which contains the amino terminus of the virion proteins, the carboxy terminus of the non-structural proteins and sequences associated with one of the splices. We have also begun to study the expression of ADV proteins in a vaccinia based eukaryotic expression system. A segment representing the spliced R3 mRNA that codes for both ADV structural proteins has been recombined into an infectious vaccinia virus. This recombinant vaccinia (VV-lL1) directed synthesis of both VP1 (p85) and VP2 (p75). The viral antigen was localized to the nucleus. Furthermore, lysates of VV-IL1 infected cells could be used as an ADV test antigen in counterimmunoelectrophoresis. Finally, parvovirus particles were observed by electron microscopy. Thus, all signals necessary for production and nuclear transport of both VPl/VP2 as well as those required for particle assembly were present.