Evans J T, Leisy D J, Rohrmann G F
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301, USA.
J Virol. 1997 Apr;71(4):3114-9. doi: 10.1128/JVI.71.4.3114-3119.1997.
The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.
苜蓿银纹夜蛾多核衣壳核型多角体病毒有六个必需基因和三个对瞬时DNA复制有刺激作用的基因。我们证明，其中两个基因LEF - 1和LEF - 2的产物，在使用酿酒酵母的双杂交试验和谷胱甘肽S - 转移酶融合亲和试验中均有相互作用。通过酵母双杂交试验，我们将LEF - 2的相互作用结构域定位到第20至60位之间的氨基酸。对LEF - 1进行广泛的缺失分析未能揭示一个明确的相互作用结构域，这表明可能存在一些基本的二级结构元件，这些元件因这些缺失而失活。所有表达不能相互作用的LEF - 1和LEF - 2的克隆也不能支持显著水平的瞬时DNA复制，这表明这种相互作用是DNA复制所必需的。对LEF - 1的序列分析揭示了一个类似引发酶的基序WVVDAD。当这个基序突变为WVVQAD时，LEF - 1不再支持瞬时DNA复制。
