The molecular mechanisms involved in poxvirus entry into cells remain perplexing. Our goal is to dissect these mechanisms by focusing on the envelope proteins involved in entry of three orthopoxviruses: vaccinia (VACV), variola (VARV) and monkeypox (MPXV). The accidental or intentional release of VARV, coupled with the potential of MPXV to become a more efficient human pathogen, underscore the need for further investigation of these viruses in terms of their biodefense importance and as emerging infectious agents. The cell receptor(s) and the viral receptor-binding protein(s) for poxviruses are unknown. Our preliminary data suggest that the MV protein L1 is a receptor binding protein that may trigger fusion that is carried out by the virus fusion complex. We found that a soluble form of L1 binds to cells independently of glycosaminoglycans and blocks virus entry, suggesting it competes with virion associated L1 for a cell receptor. In Aim 1, we will map regions on L1 critical for its function in entry and identify the cellular receptor(s) to which it binds. We will determine which residues of L1 are involved in its ability to block virus entry using structure based mutagenesis. Similar studies will be done in collaboration with colleagues at CDC for MPXV and VARV. We will use two approaches to identify the cell receptor(s) for L1. First, we will use L1 itself to try to fish out the receptor from susceptible cells. Second, we will take a more unbiased approach using RNAi to screen for proteins that are important in VACV entry, focusing particularly on ones that may target L1. In Aim 2 we will study how the MV envelope proteins function in poxvirus entry. We will use assays to follow events that occur after receptor binding using a novel confocal-FACS technology. We will employ bimolecular fluorescence complementation to study interactions between VACV envelope proteins in intact cells with the goal of relating them to VACV entry/fusion. Our studies should provide the molecular details of both the viral and cellular factors involved in poxvirus entry. Additionally, we may identify novel targets for developing small molecule inhibitors of poxvirus entry.