Purification and Characterization of OF-1, a Host Factor Implicated in Herpes Simplex Replication

• Published in: The Journal of biological chemistry 2000-09, Vol.275 (39), p.30050-30057
• Main Authors: Baker, Robert O., Murata, Lauren B., Dodson, Mark S., Hall, Jennifer D.
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Binding, Competitive; DNA Replication; DNA, Single-Stranded - metabolism; DNA, Viral - metabolism; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Herpes simplex virus 1; Herpesvirus 1, Human - growth & development; Humans; Molecular Sequence Data; OF-1; oriS gene; Protein Binding; Protein Structure, Quaternary; Replication Origin; UL9 protein; Viral Proteins - metabolism; Virus Replication
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002154200

A human cellular factor (OF-1) has been previously implicated in replication of herpes simplex virus, type 1. This protein binds to three conserved regions (Boxes I, II, and III) in the viral replication origin and appears to be required for viral DNA synthesis (Dabrowski, C. C., Carmillo, P. J., and Schaffer, P. A. (1994) Mol. Cell. Biol. 14, 2545–2555). In the present study, we have partially purified and characterized OF-1 from human cells. This protein appears to consist of a tetramer composed of two heterodimers with subunits of 73 and 90 kDa. The smaller subunit contains the DNA binding activity. We have investigated the binding specificity of OF-1 using a mobility shift analysis. These studies reveal that binding is specific for both duplex and single-stranded Box I sequences and that the strongest preference is for the bottom strand of Box I. We present evidence suggesting that the binding of OF-1 to Box I DNA is enhanced in the presence of the herpes simplex-encoded UL9 protein, which also binds to Box I in oriS and is required for viral replication. Implications of these findings for the initiation step in viral replication are discussed.