Merrington C L, Kitts P A, King L A, Possee R D
NERC Institute of Virology and Environmental Microbiology, Oxford, United Kingdom.
Virology. 1996 Mar 1;217(1):338-48. doi: 10.1006/viro.1996.0121.
In order to define factors involved in very late Autographa californica nucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5'-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of the EcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and in lef-2. Further mapping experiments demonstrated that only replacement of the point mutation in lef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that the lef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in the lef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed that lef-2 may play a dual role, both in DNA replication and very late gene expression.
为了确定与苜蓿银纹夜蛾核型多角体病毒（AcMNPV）极晚期基因功能相关的因素，通过5'-溴脱氧尿苷处理对杆状病毒重组体（AcUW1.lacZ）进行了随机诱变。选择了5种在极晚期基因表达方面存在缺陷的病毒。通过互补分析对它们进行了表征。发现一种突变病毒VLD1在极晚期基因功能方面完全缺失。这种病毒可以被辅助病毒互补以表达极晚期基因，这表明突变病毒在极晚期基因表达激活因子方面存在缺陷。进一步的研究表明，与野生型病毒相比，VLD1的复制在时间上有所延迟。VLD1中的突变被定位到AcMNPV基因组EcoRI-I区域0至5个图距单位之间的一个亚片段。序列分析显示在ORF2和lef-2中存在点突变。进一步的定位实验表明，只有用野生型序列替换lef-2中的点突变才能使VLD1恢复正常表型。先前的研究表明lef-2基因产物参与DNA复制。通过比较野生型和VLD1感染细胞中的DNA复制对此进行了研究。发现VLD1的lef-2基因中的突变对DNA复制没有影响。有人提出lef-2可能在DNA复制和极晚期基因表达中都发挥双重作用。
