CHARACTERIZING THE MECHANISM OF VIRAL ENTRY INITIATION IN HSV - PROJECT SUMMARY/ABSTRACT HERPESVIRUSES HAVE UNUSUAL VIRAL ENTRY MECHANISMS. WHILE MOST ENVELOPED VIRUSES TYPICALLY HAVE ONE SURFACE GLYCOPROTEIN THAT MEDIATES VIRAL ENTRY, HERPESVIRUSES REQUIRE MULTIPLE GLYCOPROTEINS WORKING TOGETHER. FOR EXAMPLE, ALPHA-HERPESVIRUSES HSV-1 AND HSV-2 EMPLOY 4 SURFACE GLYCOPROTEINS TO MEDIATE VIRAL ENTRY: GD, GH, GL, AND GB. WHILE THE GENERAL ROLES OF THESE ENTRY GLYCOPROTEINS HAVE BEEN CHARACTERIZED, THE MECHANISM OF HOW THESE GLYCOPROTEINS INTERACT TO PROMOTE THE MEMBRANE FUSION NECESSARY FOR VIRAL ENTRY ARE LESS CLEAR. THE OBJECTIVE OF THIS PROPOSAL IS TO ELUCIDATE THE MECHANISM OF THE EARLY STEPS OF VIRAL ENTRY IN HERPES SIMPLEX VIRUS 2 (HSV-2), SPECIFICALLY, THE INTERACTIONS BETWEEN THE RECEPTOR-BINDING PROTEIN GD AND ITS PARTNER GH/GL. PRIOR STUDIES HAVE SHOWN THAT THESE PROTEINS INTERACT PRIOR TO AND DURING MEMBRANE FUSION. THEREFORE, THE CENTRAL HYPOTHESIS IS THAT THE GD-GHGL COMPLEX CAN ADOPT TWO MAIN STATES: AN "OFF" STATE, WHICH REPRESENTS THE COMPLEX PRIOR TO RECEPTOR BINDING, AND AN "ON" STATE THAT IS INDUCED BY RECEPTOR BINDING. HERE, HIGH-RESOLUTION STRUCTURES OF THESE STATES WILL BE DETERMINED BY CRYOEM AND VALIDATED BY USING BIOCHEMICAL AND FUNCTIONAL ASSAYS. AIM 1 WILL CHARACTERIZE THE STRUCTURE OF THE "OFF" STATE OF THE GD-GHGL COMPLEX WHEREAS AIM 2 WILL CHARACTERIZE THE STRUCTURE OF THE "ON" STATE OF THE ENTRY COMPLEX. THE TWO STATES WILL BE COMPARED TO IDENTIFY CONFORMATIONAL CHANGES IN GD AND GH/GL AND THEIR INTERACTIONS DURING TRANSITION BETWEEN THESE TWO STATES. THIS PROPOSAL IS INNOVATIVE AS IT WILL USE CUTTING-EDGE APPROACHES TO YIELD NEW MECHANISTIC INSIGHTS INTO HSV ENTRY. IT IS SIGNIFICANT BECAUSE IT WILL IDENTIFY KEY FUNCTIONAL REGIONS WITHIN THE ENTRY COMPLEX TO INFORM THE DEVELOPMENT OF ANTIVIRAL THERAPIES DESIGNED TO BLOCK VIRAL ENTRY. THIS PROPOSAL HAS THE POTENTIAL TO MAKE MAJOR CONTRIBUTIONS FOR THE DEVELOPMENT OF THERAPEUTICS AGAINST HSV AND ITS RELATED DISEASES. THE SPONSOR OF THIS PROPOSAL, DR. EKATERINA HELDWEIN, WILL ENSURE THAT THE APPLICANT ACQUIRES THE TECHNICAL AND CONCEPTUAL SKILLS NECESSARY FOR THE SUCCESSFUL COMPLETION OF THIS PROJECT AND FOR THEIR DEVELOPMENT INTO A PREMIER SCIENTIST SO THAT THEY MAY ESTABLISH A SUCCESSFUL CAREER AS AN INDEPENDENT INVESTIGATOR.