PROJECT SUMMARY When a mosquito-borne flavivirus encounters a cell, it must adhere to a receptor on the cell surface, endocytose, and finally fuse with the endosomal membrane. To accomplish this, flaviviruses use an exterior lattice of 180 E proteins arrayed in a herringbone arrangement of 90 antiparallel dimers at neutral pH. Acidification in the late endosome induces a widespread conformational rearrangement, resulting in 60 outward-facing trimeric E spikes that can embed into the host membrane and pull the membranes together, fusing them and releasing the viral RNA into the host cytoplasm. Although flavivirus lattices are canonically described as 90 dimers lying flat (mature state) or 60 trimers facing outward (immature and fusion states), up to 50% of Dengue virions have incomplete maturation that results in a mosaic pattern of E dimers and trimers with different orientations. It is unknown whether mosaic lattices are less functional than their perfect counterparts due to steric hindrance of the conformational rearrangements, or alternatively might have certain advantages that explain why evolution has conserved their heterogeneous arrangement. This has strong consequences for the design of therapeutics, as the mature and immature patches have different epitope exposure and possibly different binding affinities for antibodies that are not well understood. We will explore this question through a combination of structural and functional studies performed on mosaic Dengue and West Nile viruses, using cryo-electron tomography and subtomogram averaging to determine the position and orientation of individual E proteins within the viral lattices, and designing new analyses to describe the heterogeneity of the viral population. We will evaluate how the mosaic surface affects fusion and antibody binding, and directly visualize the structure-function relationship by imaging viruses interacting with target membranes and cells. This approach will allow us to identify which areas of a mosaic lattice participate in adhesion or fusion, and whether functional virions favor more heterogeneous or homogeneous surfaces. Current structural biology is usually performed on samples that contain a large number of inactive virions; by imaging functional states directly, inactive virions are eliminated from analysis to facilitate identification of the structural states of the virus that should be prioritized in structure-based therapeutic and vaccine design. While this work will focus on the flavivirus lattice, functional lattices are ubiquitous in all the domains of life and play integral roles in human health and disease. The methods and analyses we develop to study flaviviruses will directly apply to other viruses, but will also potentially aid in understanding processes as diverse as bacterial chemotaxis, carbon fixation, and human cardiac muscle contraction.