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Bombyx mori nucleopolyhedrovirus encodes a DNA-binding protein capable of destabilizing duplex DNA
V S Mikhailov1, A L Mikhailova, M Iwanaga
1N. K. Koltzov Institute of Developmental Biology, Moscow, Russia. vsmikha@postman.riken.go.jp
Abstract:
A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3'-->5' exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.
Insights
A novel DNA-binding protein (DBP) from Bombyx mori nucleopolyhedrovirus (BmNPV) was identified and characterized. This protein preferentially binds single-stranded DNA and can unwind DNA duplexes, suggesting a role in viral replication.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant insect pathogen.
- Understanding viral protein functions is crucial for insect pest control strategies.
Purpose of the Study:
- To purify and characterize a novel DNA-binding protein (DBP) from BmNPV.
- To elucidate the DNA-binding properties and potential function of BmNPV DBP.
Main Methods:
- Protein purification from infected BmN cells.
- Peptide sequencing and homology analysis to identify the gene encoding DBP.
- In vitro DNA binding and DNA unwinding assays.
Main Results:
- A 38 kDa DNA-binding protein (DBP) was purified from BmNPV-infected cells.
- DBP is encoded by ORF16 (dbp) and is a homolog of AcMNPV ORF25.
- DBP binds preferentially to single-stranded DNA, protects it from exonuclease digestion, and exhibits DNA helix-destabilizing activity.
Conclusions:
- BmNPV DBP is a single-stranded DNA-binding protein with DNA unwinding capabilities.
- DBP likely plays a role in viral DNA replication and/or other DNA metabolism processes during BmNPV infection.