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 complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-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 microM bound increasing amounts of UL9 expressed by in vitro transcription/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.

Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: Visualization of a specific preunwinding complex

“…UL9 protein interacts with ICP8 (23), UL8 protein, a component of the trimeric helicase͞primase (46), and UL42 protein, the DNA polymerase accessory protein (47). The G͞C-rich flanking sequences contain multiple binding sites for transcriptional factors, and their presence has been shown to stimulate replication significantly (41,45).…”

DNA Binding Activity of the Herpes Simplex Virus Type 1 Origin Binding Protein, UL9, Can Be Modulated by Sequences in the N Terminus: Correlation between Transdominance and DNA Binding

“…The ability of UL9 to bind specifically to the origin has been localized to the C-terminal one-third (residues 564 to 832) (1,6). The N terminus of UL9 has been shown to interact with UL8 and UL42 (22,23). Residues in the N-terminal region are also required both for dimerization and cooperative binding of UL9 at the origins (7,11,14).…”

Herpes Virus Replication

“…Subsequently, UL9, through its interaction with the UL8 loading protein (67), may recruit the viral helicase-primase to initiate primer synthesis and to establish a bi-directional replication fork. The viral DNA polymerase may be recruited to the site of initiation either by the interaction of its catalytic subunit (UL30) with UL8 (68) or via an interaction between its processivity factor (UL42) and UL9 (69).…”