Lamb J W, Duncan G H, Reavy B, Gildow F E, Mayo M A, Hay R T
School of Biological and Medical Sciences, University of St Andrews, UK.
J Gen Virol. 1996 Jul;77 ( Pt 7):1349-58. doi: 10.1099/0022-1317-77-7-1349.
DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
将编码马铃薯卷叶病毒（PLRV）苏格兰分离株外壳蛋白（P3）的DNA插入苜蓿银纹夜蛾核多角体病毒（AcNPV）的基因组中，使得外壳蛋白以未修饰的形式表达，或者在N端添加氨基酸序列MHHHHHHGDDDDKDAMG（P3-6H）。感染这些重组杆状病毒的昆虫细胞积累了大量的P3和P3-6H。除非在SDS中变性，否则无法从细胞提取物中回收P3，但一部分P3-6H在非变性条件下可以以可溶形式回收。对感染细胞切片的免疫金标记显示，P3在细胞核中以大的无定形物体形式积累。相比之下，尽管大部分P3-6H也在细胞核中积累，但它形成了病毒样颗粒（VLP），这些颗粒通常紧密堆积成几乎结晶的阵列。当涂有抗PLRV抗体的电子显微镜网格漂浮在含有P3-6H的细胞提取物上时，捕获的VLP与从PLRV感染植物提取物中捕获的PLRV颗粒无法区分。VLP在蔗糖梯度中与PLRV颗粒共沉降，这表明VLP含有RNA。从蔗糖密度梯度级分中收集的VLP含有与螯合到次氮基三乙酸的镍反应的蛋白质，次氮基三乙酸是一种组氨酸特异性试剂。感染任何一种重组杆状病毒的细胞还合成了一种分子量约为17000的蛋白质，该蛋白质被证明是外壳蛋白基因中+1阅读框内P4基因的翻译产物。这种蛋白质也存在于感染细胞的核部分中，但比P3更易溶解。
