The herpes simplex virus type I origin binding protein. DNA-dependent nucleoside triphosphatase activity

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Herpes simplex virus type 1 (HSV-1) DNA replication relies on specific viral proteins.
• The UL9 gene encodes the origin binding protein, essential for initiating viral DNA synthesis.

Purpose of the Study:

• To construct and characterize a recombinant baculovirus expressing the HSV-1 origin binding protein (UL9).
• To investigate the enzymatic properties of the purified UL9 protein, specifically its DNA-dependent nucleoside triphosphatase activity.

Main Methods:

• Construction of a recombinant baculovirus for UL9 overexpression.
• Purification of the recombinant UL9 protein.
• Assays to determine the DNA-dependent nucleoside triphosphatase activity under various conditions.

Main Results:

• The recombinant UL9 protein demonstrated DNA-dependent nucleoside triphosphatase activity comparable to the native enzyme.
• Optimal activity was observed under specific conditions: low salt, Mg2+, alkaline pH, high temperature, and with unstructured single-stranded DNA.
• Activity was inhibited by products and showed a dependence on DNA cofactor length and structure.
• The HSV-1 single-stranded DNA-binding protein ICP8 significantly stimulated UL9's enzymatic activity.
• A lag period was observed in the hydrolysis kinetics, influenced by DNA structure and temperature.

Conclusions:

• The recombinant UL9 protein is enzymatically active and possesses characteristics similar to the native HSV-1 origin binding protein.
• UL9's nucleoside triphosphatase activity is modulated by DNA structure and is significantly enhanced by ICP8, highlighting their functional interaction in HSV-1 DNA replication.