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Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for
Abstract:
We generated four point mutations in the DNA-binding protein (DBP) gene of adenovirus type 5 by oligonucleotide-directed site-specific mutagenesis. The sites mutated were in the three conserved regions (CR; amino acids 178-186 [CR1], 322-330 [CR2], and 464-475 [CR3]) identified previously by comparative sequence analysis (G. R. Kitchingman, Virology 146:90-101, 1985). The mutations resulted in changes in amino acids 181 (Trp to Leu), 323 (Arg to Leu), 324 (Trp to Leu), and 469 (Phe to Ile). The mutated DBP genes were put under the control of the simian virus 40 early promoter and analyzed by transfection for their ability to help adeno-associated virus replicate its DNA in COS-1 monkey cells. Mutations in the aromatic amino acids 324 and 469 reduced the amount of AAV DNA replication approximately 10-fold, while the mutation in Arg 323 produced a reduction of approximately fourfold. The Trp-to-Leu mutation in amino acid 181 had no effect on AAV DNA replication. The decreased helper activity of the 323, 324, and 469 mutations was not caused by any effect of the mutation on the stability of the DBP. These results suggest that CR2 and CR3 are involved in AAV helper activity, specifically in AAV DNA replication. The relevance of these findings to the identification of residues important for the functions of DBP in adenovirus infection is discussed.
Insights
Mutations in conserved regions 2 and 3 of adenovirus type 5 DNA-binding protein (DBP) impair adeno-associated virus DNA replication. These findings highlight key residues for DBP function in viral replication.
Area of Science:
- Molecular Biology
- Virology
Background:
- Adenovirus type 5 DNA-binding protein (DBP) is crucial for viral DNA replication.
- Conserved regions (CR) in DBP have been identified through comparative sequence analysis.
- Understanding DBP's role in replication is essential for viral pathogenesis studies.
Purpose of the Study:
- To investigate the role of specific conserved regions within the adenovirus type 5 DNA-binding protein (DBP) in supporting adeno-associated virus (AAV) DNA replication.
- To identify key amino acid residues essential for DBP's helper function in AAV replication.
Main Methods:
- Oligonucleotide-directed site-specific mutagenesis was used to generate four point mutations in the DBP gene of adenovirus type 5.
- Mutated DBP genes were analyzed for their ability to facilitate AAV DNA replication in COS-1 monkey cells following transfection.
- The stability of the mutated DBP proteins was assessed to rule out protein degradation as a cause for reduced helper activity.
Main Results:
- Mutations in aromatic amino acids at positions 324 and 469 (CR2 and CR3) reduced AAV DNA replication approximately 10-fold.
- A mutation at position 323 (CR2) resulted in a fourfold reduction in AAV DNA replication.
- A mutation at position 181 (CR1) showed no significant effect on AAV DNA replication, and protein stability was not compromised by the mutations.
Conclusions:
- Conserved regions 2 and 3 of adenovirus type 5 DBP are critical for its helper activity in adeno-associated virus DNA replication.
- Specific amino acid residues within CR2 and CR3 play important roles in facilitating AAV DNA replication.
- These findings contribute to understanding the functional significance of DBP residues in adenovirus infection and replication.