STRUCTURE AND FUNCTION OF THE ALEUTIAN MINK DISEASE VIRUS GENOME

本项目的目的是将ADV的结构特征 不同病毒分离株在培养物中复制的能力 并导致水貂患病。 我们继续研究基因组嵌合的分子克隆， 非致病性ADV-G和致病性ADV毒株之间的片段。 通过进行片段交换和PCR诱变， 发现将单个甘氨酸残基突变为天冬氨酸， 衣壳蛋白基因消除感染性ADV-G克隆的能力 在细胞培养中复制并产生感染性病毒。通过比较 用全长克隆进行转染，我们发现， 复制似乎不能产生单链子代 DNA. 从全长克隆中拯救出来的病毒都不会引起疾病 表明ADV对成年水貂具有致病性， 在CRFK细胞中允许复制的基因以阴性方式连接。 已经获得了额外的ADV-Utah 1序列信息， 表明先前未识别的大量序列变异。 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 mRNA的预测基因产物（NS-3）。 表达VP 1和VP 2的重组牛痘病毒，或 VP 2单独合成适当大小的蛋白质;这些蛋白质是 运输到细胞核并形成空病毒颗粒。 因此，VP 1 并不是颗粒组装所必需的。VP 1/VP 2衣壳，而不是 只有VP 2能与ADV DNA复制形式结合， ADV病毒粒子。 重组病毒在小鼠体内刺激抗ADV抗体， 小鼠，但似乎是弱免疫原性水貂。类似的VP 1和 将VP 2片段导入重组杆状病毒， 表示为AcADV，其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.