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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058809-03
Application #
6386389
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Wolfe, Paul B
Project Start
1999-08-01
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
3
Fiscal Year
2001
Total Cost
$276,208
Indirect Cost
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210