Interaction between the Herpes Simplex Virus Type 1 Origin-Binding and DNA Polymerase Accessory Proteins

Abstract

Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complexin vitroas determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathioneS-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9–UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 μM bound increasing amounts of UL9 expressed byin vitrotranscription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.