Capturing HSV entry glycoprotein complexes

PROJECT SUMMARY/ABSTRACT Herpes simplex viruses (HSV-1 and HSV-2) infect most humans for life and cause skin sores, blindness, encephalitis, developmental abnormalities, and disseminated disease. No vaccines are yet available, and current therapeutics are non-curative. Therefore, there is an urgent need to develop new prophylactic and therapeutic interventions. Being enveloped, HSV-1 and HSV-2 penetrate target host cells by fusing their lipid envelopes with cellular membranes. This process requires several viral proteins – gD, gH, gL, and gB – each of which performs distinct functions. This contrasts with most enveloped viruses, which combine all entry functions in one protein. Such distribution of functions raises the question of how these proteins act, how they coordinate their activities to ensure efficient membrane fusion and viral entry, and how best to target them for vaccine development. High- resolution structures of the individual glycoproteins and stable complexes, such as gH/gL, have been indispensable for guiding functional, mechanistic, and immunological studies. But little is known about how these glycoproteins interact. High-resolution structural “snapshots” of glycoprotein complexes relevant to entry would help answer many outstanding mechanistic questions, but complexes have been difficult to isolate, so no structures of complexes (beyond that of gH/gL) are yet available. The current application proposes to apply new methods to isolate HSV glycoprotein complexes and characterize their structures by cryoEM. Aim 1 will use antibodies to stabilize gD/gH/gL complexes and characterize their structures. Aim 2 will use non-detergent extraction methods to isolate HSV gH/gL/gB complexes and characterize their structures. The proposal will leverage extensive experience in biochemical and structural studies of HSV glycoproteins as well as a newly established cryoEM pipeline. A detailed structural and biochemical knowledge of how HSV glycoproteins interact to facilitate cell entry will open new opportunities for rational vaccine and therapeutics design.