Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin

We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic poly-hedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C a and C 2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains Ca and C ~ were compared with that of strain H. Strains Ca 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, C 1 and C 2 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 C a or C 2 polyhedrin genes, but amorphous tructures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxy-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest hat 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