Herpes simplex virus (HSV) provides a powerful genetic model for understanding eukaryotic DNA replication because of its relative simplicity compared to eukaryotic cells and because its haploid genome renders it more genetically tractable. HSV encodes 7 proteins directly involved in and required for viral DNA synthesis: a ss DNA binding protein (ICP8), a processive DNA polymerase (pol/UL42 heterodimer), a heterotrimeric helicase/primase complex (UL5, UL52, and UL8), and a protein mechanism of initiation of DNA replication of native viral DNA genomes in infected cells. Initiation of viral DNA synthesis is likely to be dependent upon the ability of UL9 to form an open complex at ori's present in the input viral DNA. A multiplicity of protein-protein interactions among the HSV DNA replication proteins has been described, through the functions of very few have been elucidated. UL9 most likely plays a central role in assembling the other proteins at ori's since UL9 interacts with a member of each of the complexes described above (ICP8, UL8, and UL42). We hypothesize that UL42 acts as an adapter protein which facilitates the entry of the processive pol (perhaps together with ICP8 and helicase/primase) into an activated ori occupied by UL9. We will combine biochemical and immunologic approaches to better understand the function of the UL9-UL42 interaction on the known UL9 activities.
Three specific aims are proposed: 1) To measure the affinity of UL9 for UL42, pol, and pol/UL42 complex using the BIAcore 2000 and solution competition experiments with fusion proteins. The ability of UL9 to displace pol when it is complexed to UL42 will be determined as well as the stoichiometry of proteins in stable complexes. 2) To determine the ability and affinity to bind to different DNA substrates which vary in their structure and ori content, and the effect of UL42 on these properties. 3) To determine the functional significance of UL9-UL42 interaction by examining enzymatic mechanisms using pre-steady state and steady-state kinetic analysis. The role of UL42 in enhancing ATPase and helicase activities will be studied to differentiate specific from non-specific mechanisms of action. Effects of UL42 on load, initial rate constants, and processivity of helicase activities will be distinguished. The knowledge we gain in understanding the importance of interactions among DNA replciation proteins will help in defining an in vitro ori-dependent DNA replication system and in defining targets for the development of novel anti-viral compounds designed to disrupt the viral DNA replication complex.
| Trego, Kelly S; Zhu, Yali; Parris, Deborah S (2005) The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA. Nucleic Acids Res 33:536-45 |
| Zhu, Yali; Trego, Kelly S; Song, Liping et al. (2003) 3' to 5' exonuclease activity of herpes simplex virus type 1 DNA polymerase modulates its strand displacement activity. J Virol 77:10147-53 |
| Trego, Kelly S; Parris, Deborah S (2003) Functional interaction between the herpes simplex virus type 1 polymerase processivity factor and origin-binding proteins: enhancement of UL9 helicase activity. J Virol 77:12646-59 |
