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Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin
H Nakazawa1, F Kendirgi, S Belloncik
1Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
Abstract:
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.
Insights
Mutations in Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) polyhedrin genes affect polyhedron formation and localization. Carboxy-terminal changes drive nuclear localization, while other mutations alter crystallization patterns.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- The polyhedrin gene is crucial for Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) polyhedron formation.
- Previous studies cloned polyhedrin genes from wild-type BmCPV (strain H) and mutant strain A.
- Understanding sequence variations in BmCPV polyhedrin genes is key to deciphering polyhedron assembly and localization.
Purpose of the Study:
- To clone and determine the nucleotide sequences of polyhedrin genes from BmCPV mutant strains C1 and C2.
- To compare the polyhedrin amino acid sequences of strains C1 and C2 with strain H.
- To investigate the impact of specific mutations on polyhedrin localization and polyhedron morphology.
Main Methods:
- Cloning of polyhedrin genes from BmCPV strains C1 and C2.
- Nucleotide sequencing and amino acid sequence comparison.
- Introduction of polyhedrin genes into a baculovirus expression vector.
- Western blot and microscopy for analyzing intracellular polyhedrin localization and polyhedron morphology.
Main Results:
- Strains C1 and C2 exhibited two and three mutations in their polyhedrin genes, respectively.
- A four-amino acid addition (249RLLV) was observed at the carboxy terminus of polyhedrin in strains A, C1, and C2.
- Recombinant baculovirus expressing strain H polyhedrin produced hexahedral polyhedra in cytoplasm and nucleus, while strain A showed nuclear localization only.
- Cells infected with recombinant viruses for strains C1 and C2 showed amorphous structures instead of normal polyhedra.
Conclusions:
- Mutations at the carboxy terminus of BmCPV polyhedrin are responsible for its nuclear localization.
- Additional mutations within the polyhedrin gene contribute to the altered crystallization patterns observed in mutant strains.
- The carboxy-terminal sequence of BmCPV polyhedrin plays a critical role in directing its subcellular localization and influencing polyhedron formation.