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Mapping the Mutation Site of an Autographa californica Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Abstract:
A polyhedron morphology mutant of Autographa californica nuclear polyhedrosis virus, designated M5, was compared with wild-type virus by genotypic analysis with EcoRI, BamHI, HindIII, SstI, and SmaI restriction endonucleases. M5 DNA revealed several alterations relative to the wild-type pattern: (i) EcoRI fragment I was 400 base pairs larger; (ii) BamHI fragment F was missing; (iii) HindIII fragment F was 400 base pairs larger; (iv) an extra restriction fragment was obtained with both HindIII and SmaI; and (v) SstI fragment G was 400 base pairs larger. M5 virions contained two size classes of circular DNA, one of 100% of the wild type and one of about 58% of the wild-type molecule. A revertant of M5, designated M5R, was isolated on the basis of polyhedron morphology. The genome of M5R contained the insertion of DNA in EcoRI fragment I and in HindIII fragment F, but was similar to the wild type in its other restriction fragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptide smaller in size than the wild type or M5R.
Insights
A mutant Autographa californica nuclear polyhedrosis virus (AcNPV) showed significant genomic alterations, including DNA insertions and deletions, affecting polyhedrin production. A revertant virus restored normal polyhedrin synthesis, indicating genetic changes responsible for the mutant phenotype.
Area of Science:
- Molecular virology
- Insect pathology
- Genomics
Background:
- Autographa californica nuclear polyhedrosis virus (AcNPV) is a baculovirus used in molecular biology.
- Viral mutants are essential for understanding gene function and viral replication.
- Polyhedrin is a major structural protein of AcNPV, crucial for virion assembly and occlusion.
Purpose of the Study:
- To characterize the genomic alterations in an AcNPV polyhedron morphology mutant (M5).
- To investigate the genetic basis of altered polyhedrin synthesis in M5.
- To analyze the genetic makeup of a revertant (M5R) to confirm the role of specific mutations.
Main Methods:
- Genotypic analysis of wild-type AcNPV, M5 mutant, and M5 revertant using restriction endonucleases (EcoRI, BamHI, HindIII, SstI, SmaI).
- Analysis of viral DNA size classes in M5 virions.
- Comparison of polyhedrin polypeptide synthesis in infected cell cultures.
Main Results:
- AcNPV M5 mutant exhibited multiple DNA alterations, including fragment size changes and a missing fragment, compared to wild-type.
- M5 virions contained two distinct circular DNA populations: wild-type size and approximately 58% of wild-type size.
- The M5 revertant (M5R) showed DNA insertions in specific fragments, restoring normal polyhedrin production.
- M5-infected cells produced a smaller polyhedrin polypeptide than wild-type or M5R-infected cells.
Conclusions:
- The polyhedron morphology defect in AcNPV M5 is associated with specific genomic alterations, including DNA insertions and deletions.
- These genetic changes impact viral DNA structure and polyhedrin polypeptide synthesis.
- Reversion to normal morphology correlates with the restoration of specific genomic patterns and polyhedrin production.
