Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification

Area of Science:

• Molecular Virology
• Protein Biochemistry

Background:

• Adenovirus type 2 encodes a single-stranded DNA binding protein (DBP).
• DBP plays a crucial role in viral DNA replication and other processes.
• The post-translational modification status of DBP was not fully characterized.

Purpose of the Study:

• To investigate the phosphorylation status of adenovirus type 2 DBP.
• To characterize the different forms of DBP and their properties.
• To identify the specific amino acid residues involved in DBP phosphorylation.

Main Methods:

• Pulse-chase labeling experiments using [35S]methionine and 32PO4.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess molecular weight and forms.
• Single-stranded DNA-cellulose chromatography for DNA binding analysis.
• Immunoprecipitation using specific antisera.
• Paper electrophoresis of acid hydrolysates to identify phosphorylated amino acids.

Main Results:

• Adenovirus type 2 DBP is a phosphoprotein.
• DBP exists in at least two forms with different molecular weights (74,000 and 77,000 Da) observable by SDS-PAGE.
• Both forms of DBP bind to single-stranded DNA-cellulose and are immunoprecipitated by specific antisera.
• Phosphorylation of DBP involves phosphoserine, not phosphothreonine.
• The larger form of DBP (77,000 Da) likely results from post-translational modification during a chase period.

Conclusions:

• Adenovirus type 2 DBP undergoes phosphorylation, primarily on serine residues.
• The observed molecular weight heterogeneity of DBP is due to phosphorylation.
• Phosphorylated DBP retains its DNA-binding and antigenic properties.