Okano K, Mikhailov V S, Maeda S
Laboratory of Molecular Entomology and Baculovirology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, Japan.
J Virol. 1999 Jan;73(1):110-9. doi: 10.1128/JVI.73.1.110-119.1999.
We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107-3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.
我们最近从家蚕核型多角体病毒（BmNPV）中鉴定出一种DNA结合蛋白（DBP），它能使双链DNA不稳定（V.S.米哈伊洛夫、A.L.米哈伊洛娃、M.岩永、S.古见和S.前田，《病毒学杂志》72:3107 - 3116，1998年）。发现DBP是一种早期基因产物，在出芽病毒粒子或包涵体衍生病毒粒子中不存在。为了表征病毒复制过程中DBP的定位，用DBP抗体通过免疫染色和共聚焦显微镜检查了BmNPV感染的BmN细胞。DBP在感染后（p.i.）4至6小时首次表现为弥漫性核染色，然后在感染后6至8小时定位于细胞核内的几个特定病灶。在感染后约8小时病毒DNA复制开始后，这些病灶开始扩大，到感染后14小时最终占据细胞核的一半以上。在感染后约20小时病毒DNA复制终止后，DBP染色区域似乎分解成细胞核内约100个小病灶。在感染后8小时，DBP以及IE - 1或LEF - 3（两种参与杆状病毒DNA复制的蛋白质）的分布与用溴脱氧尿苷掺入标记的DNA复制位点的分布很好地重叠。用IE - 1和DBP或IE - 1和LEF - 3进行的双重染色实验进一步证实，在感染后8至14小时之间，IE - 1和LEF - 3的分布与DBP的分布重叠。然而，在感染后4小时，IE - 1在DBP或LEF - 3之前定位于特定病灶。在DNA合成抑制剂阿非科林存在的情况下，到感染后8小时观察到含有IE - 1、LEF - 3和DBP的未成熟病灶。然而，这些病灶随后的扩大被完全抑制，这表明扩大依赖于病毒DNA复制。在感染后4小时，IE - 1病灶的数量与感染复数（MOI）在0.4至10之间相关。在较高的感染复数（例如50）下，病灶数量在约15处趋于平稳。这些结果表明，每个细胞核中大约有15个预先存在的位点，它们与病毒DNA复制的起始和病毒DNA复制工厂的组装有关。
