Nakazawa H, Kendirgi F, Belloncik S, Ito R, Takagi S, Minobe Y, Higo K, Sumida M, Matsubara F, Mori H
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
J Gen Virol. 1996 Jan;77 ( Pt 1):147-53. doi: 10.1099/0022-1317-77-1-147.
We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C1 and C2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains C1 and C2 were compared with that of strain H. Strains C1 and C2 contained two and three sites of mutation in their polyhedrin genes, respectively. Four amino acids (249RLLV) were added at the carboxy terminus of the polyhedrin of strain A, C1 and C2 and the corresponding polyhedrin genes were introduced into a baculovirus expression vector. Intracellular localization of expressed polyhedrin as well as the morphology and localization of polyhedra were investigated by Western blot and microscopy analysis. Recombinant baculovirus containing the polyhedrin gene of strain H produced hexahedral polyhedra in both the cytoplasm and the nucleus. However, the hexahedral polyhedra of strain A were localized only in the nucleus. Normal polyhedra were not observed in cells infected with recombinant baculoviruses expressing strain C1 or C2 polyhedrin genes, but amorphous structures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxyl-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest that a mutation at the carboxy terminus of BmCPV polyhedrin led to nuclear localization of polyhedrin and that several other mutations were responsible for modification of the crystallization pattern of polyhedrin.
我们已经克隆了家蚕细胞质多角体病毒（BmCPV）野生型菌株H及其突变体菌株A的多角体蛋白基因。在这项工作中，克隆了突变型BmCPV菌株C1和C2的多角体蛋白基因，并测定了它们的核苷酸序列。将菌株C1和C2的多角体蛋白氨基酸序列与菌株H的进行了比较。菌株C1和C2的多角体蛋白基因分别含有两个和三个突变位点。在菌株A、C1和C2的多角体蛋白的羧基末端添加了四个氨基酸（249RLLV），并将相应的多角体蛋白基因导入杆状病毒表达载体。通过蛋白质免疫印迹和显微镜分析研究了表达的多角体蛋白的细胞内定位以及多角体的形态和定位。含有菌株H多角体蛋白基因的重组杆状病毒在细胞质和细胞核中均产生六面体多角体。然而，菌株A的六面体多角体仅定位在细胞核中。在感染表达菌株C1或C2多角体蛋白基因的重组杆状病毒的细胞中未观察到正常多角体，但在感染细胞中发现了无定形结构。含有菌株A羧基末端序列的嵌合荧光素酶表达结果表明，该序列负责核定位。我们认为，BmCPV多角体蛋白羧基末端的突变导致多角体蛋白的核定位，而其他几个突变则负责多角体蛋白结晶模式的改变。
