The Interaction of Herpes Simplex Type 1 Virus Origin-binding Protein (UL9 Protein) with Box I, the High Affinity Element of the Viral Origin of DNA Replication

Abstract

The herpes simplex type 1 (HSV-1) origin binding protein, the UL9 protein, exists in solution as a homodimer of 94-kDa monomers. It binds to Box I, the high affinity element of the HSV-1 origin, Ori s , as a dimer. The UL9 protein also binds the HSV-1 single strand DNA-binding protein, ICP8. Photocross-linking studies have shown that although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I. It is this form of the UL9 homodimer that upon interaction with ICP8, promotes the unwinding of Box I coupled to the hydrolysis of ATP to ADP and P i . Photocross-linking studies have also shown that the amount of UL9 protein that interacts with Box I is reduced by its interaction with ICP8.Antibody directed against the C-terminal ten amino acids of the UL9 protein inhibits its Box I unwinding activity, consistent with the requirement for interaction of the C terminus of the UL9 protein with ICP8. Inhibition by the antibody is enhanced when the UL9 protein is first bound to Box I, suggesting that the C terminus of the UL9 protein undergoes a conformational change upon binding Box I.

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

“…In Lee and Lehman (10,37), it was found that ICP8 would promote the unwinding of a short oriS-containing duplex if it either contained a ss tail or if the A͞T-rich spacer was unpaired. In He and Lehman (39), the 18-bp A͞T-rich spacer between boxes I and II was shown by hyperchromicity assays to be bound and opened by the addition of ICP8 and UL9 in an ATP-independent manner.…”

“…When UL9 protein and ICP8 are incubated with linear, nonorigin-containing DNA possessing a 3Ј ss tail, UL9 is loaded onto the tails and, in the presence of ATP, translocates into the duplex segment unwinding it and providing a template for ICP8 binding (22,28). UL9 protein is a homodimer in solution, and several studies have shown that two dimers are bound to oriS containing all three binding boxes (9,10). Electron microscopy (EM) studies from this laboratory revealed a particle with a mass consistent with a double dimer assembled at oriS (29).…”

“…Some origin-binding proteins also exhibit helicase activity, which facilitates origin unwinding, and several form hexamers or double hexamers on the DNA, a structure typical of many helicases; examples include simian virus 40 T antigen (3)(4)(5) and the E1 protein of the papillomaviruses (6, 7). Herpes simplex virus 1 (HSV-1) UL9 protein provides origin recognition function but binds as a double dimer to the HSV-1 origins rather than as a hexamer (8)(9)(10).…”

Replication of Herpes Simplex Virus DNA

“…Expression of the 37-kDa C-terminal DNA-binding domain has a dominant negative effect on viral replication, presumably because of its ability to occupy the origin nonproductively (16,17). Although the UL9 protein can bind to its recognition sequence as a homodimer, only one of the two monomers contacts the DNA (18). The dimerization of UL9 protein is mediated through the N-terminal part of the protein, presumably through a leucine zipper motif encompassing residues 150 -171 (19,20).…”

Functional Properties of the Herpes Simplex Virus Type I Origin-binding Protein Are Controlled by Precise Interactions with the Activated Form of the Origin of DNA Replication

“…It is a stable dimer that binds specifically and cooperatively to oriS (17). The C-terminal domain of OBP binds as a monomer to the recognition sequence GTTCGCAC referred to above as box I (18,19). Cooperative binding to boxes I and II is dependent on the N-terminal helicase domains of the protein (20).…”