Vanarsdall Adam L, Mikhailov Victor S, Rohrmann George F
Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804, USA.
Virology. 2007 Aug 1;364(2):475-85. doi: 10.1016/j.virol.2007.03.024. Epub 2007 Apr 20.
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.
苜蓿银纹夜蛾多核多角体病毒（AcMNPV）编码两种具有单链DNA结合蛋白（SSB）典型特性的蛋白质，即晚期表达因子3（LEF-3）和一种被称为DNA结合蛋白（DBP）的蛋白质。虽然LEF-3是一种多功能蛋白，对病毒DNA复制、将解旋酶转运到细胞核中以及与杆状病毒碱性核酸酶形成稳定复合物至关重要，但DBP在杆状病毒复制中的作用仍不清楚。因此，为了更好地了解DBP在病毒复制中的功能作用，从AcMNPV杆粒中构建了一种DBP敲除病毒并进行了分析。生长曲线分析结果表明，dbp敲除构建体无法产生出芽病毒，这表明dbp是必不可少的。如用dbp敲除构建体转染的细胞中早期（LEF-3）、晚期（VP39）和极晚期（P10）蛋白的积累所显示的那样，DBP的缺失不会导致病毒基因表达的普遍关闭。为了研究DBP在DNA复制中的作用，采用了基于实时PCR的检测方法，结果表明，虽然在转染了dbp敲除构建体的细胞中发生了病毒DNA合成，但合成水平低于对照病毒，这表明DBP是正常水平的DNA合成或新生病毒DNA稳定性所必需的。此外，通过脉冲场凝胶电泳分析dbp敲除病毒复制的病毒DNA，未能检测到基因组长度DNA的存在。此外，对感染细胞中的DBP分析表明，与LEF-3相似，DBP与病毒染色质紧密结合。通过免疫电子显微镜评估DBP相对于复制的病毒DNA的细胞定位表明，在感染后24小时，DBP与新生DNA在细胞核内不同的电子致密区域共定位。最后，对用dbp敲除构建体转染的细胞进行免疫电子显微镜分析表明，DBP是产生外观正常的核衣壳和形成病毒发生基质所必需的。
