STRUCTURE AND FUNCTION OF THE ALEUTIAN MINK DISEASE VIRUS GENOME

本项目的目的是讲述ADV的结构特点 基因组对各种病毒分离株在培养中的复制能力的影响 并引起水貂的疾病。 我们一直在继续研究嵌合的基因组分子克隆 非致病性ADV-G株和致病性ADV株之间的片段。 通过进行片段交换和进行聚合酶链式反应突变，我们已经 发现将单个甘氨酸残基突变为天冬氨酸 衣壳蛋白基因丧失感染性AdV-G克隆的能力 在细胞培养中复制和生产传染性病毒。通过比较 用全长克隆转染后，我们发现阻断了对 复制似乎不能产生单链后代 DNA从全长克隆中救出的病毒没有一种会引起疾病 在成年水貂中，表明ADV的致病性及其对 在CRFK细胞中允许复制是以负方式连接的。 已获得额外的Adv-Utah 1序列信息， 表示以前没有发现的实质性序列变异。 利用原核表达系统，我们证明了 ADV的非结构蛋白-2(NS-2)，但未能鉴定出 Adv RX基因的预测产物(NS-3)。 同时表达VP1和VP2的重组痘苗病毒或 VP2单独合成大小适中的蛋白质；这些蛋白质是 被运送到细胞核并形成空的病毒颗粒。因此，VP1 不是粒子集合所必需的。VP1/VP2衣壳，但不是 VP2是唯一的一种，可以像真核一样与Adv DNA复制形式结合 高级病毒粒子。重组病毒能刺激小鼠体内产生抗ADV抗体 小鼠，但水貂的免疫原性似乎很差。类似的VP1和 VP2片段已导入重组杆状病毒， 表示为AcADV，它也表达蛋白质和蛋白质 自我组装成衣壳。

The purpose of this project is to relate structural features of the ADV genome to the ability of various virus isolates to replicate in culture and to cause disease in mink. We have continued to study molecular clones chimeric for genomic segments between the nonpathogenic ADV-G and pathogenic ADV strains. By performing segmental exchanges and doing PCR mutagenesis, we have found that mutating a single glycine residue to aspartic acid in the capsid proteins gene abrogates the ability of an infectious ADV-G clone to replicate and produce infectious virus in cell culture. By comparing transfections with full length clones, we have found that the block to replication appears to be a failure to generate single stranded progeny DNA. None of the viruses rescued from full length clones cause disease in adult mink, suggesting that pathogenicity of ADV and its ability to replicate permissively in CRFK cells are linked in a negative fashion. Additional ADV-Utah 1 sequence information has been obtained which indicates substantial sequence variation not previously recognized. Using prokaryotic expression systems, we demonstrated the existence of a nonstructural protein-2 (NS-2) for ADV, but have failed to identify a predicted gene product of the ADV RX mRNA (NS-3). Recombinant vaccinia viruses expressing either both VP1 and VP2 or VP2 singly synthesize appropriately sized proteins; the proteins are transported to the nucleus and form empty virus particles. Thus VP1 is not required for particle assembly. The VP1/VP2 capsids, but not the VP2 only ones, can bind to ADV DNA replicative forms like bona fide ADV virions. The recombinant viruses stimulate anti-ADV antibody in mice, but appear to be poorly immunogenic in mink. A similar VP1 and VP2 segment has been introduced into a recombinant baculovirus, denoted AcADV, which also expresses both proteins and the proteins self-assemble into capsids.