The devastating impact on public health, global economy and social stability incurred by the COVID-19 pandemic in the last two years has highlighted the importance of basic research into zoonotic pathogens. This application describes structural and functional studies into the rodent-borne human pathogen lymphocytic choriomeningitis virus (LCMV), a member of the Arenaviridae family in the Bunyavirales order. Like other members of the same family, LCMV has a negative sense, bi-segmented genome consisting of a large (L) and a small (S) segment. The L segment encodes the RNA-dependent RNA polymerase (L RdRp) protein and the multi-functional matrix protein (Z). The S segment encodes the viral nucleoprotein (NP) and the glycoprotein (GP) precursor of the glycoprotein complex (GPC) that is later cleaved into a stable signal peptide (SSP), GP1, and GP2. In the virion, nucleocapsids of NP coated L and S segments associated with the L protein are copackaged through interactions with membrane-associated Z proteins, which also interact with GPs embedded in the membrane envelope. Although structures of individual proteins from AVs have been solved by x-ray crystallography or cryo electron microscopy (cryoEM), the architectural organization of these proteins in the virion and the assembly mechanism of NP and RNA into the nucleocapsid are poorly understood. We hypothesize that NP interacts with genomic RNA segments and L RdRp to form a nucleocapsid, which is recruited to GP-decorated membrane patches through Z for budding of virions. The proposed structural and functional studies aim to test this hypothesis of LCMV virion and nucleocapsid assembly with techniques just established by our team in the collaborative studies of vesicular stomatitis virus (VSV), another negative sense RNA virus. Specifically, cryo electron tomography (cryoET) will be used to reconstruct the first 3D model of the LCMV virion at molecular resolution and atomic models of individual proteins will be fitted into the virion tomogram to establish the architectural framework of the virion and to unveil molecular interactions among GP, Z, NP and L proteins (Aim 1). Near-atomic resolution with novel sub-particle reconstruction method will be used to image fully assembled nucleocapsids consisting of NP protein and genomic RNA segment to define the protein-RNA interactions at atomic details. The nucleocapsid structure will be used to guide sub-particle reconstruction workflow and be complemented by in situ structures of nucleocapsids from virions (Aim 2). In both Aims, structure-guided functional studies will be performed to test hypotheses of assembly mechanisms of LCMV nucleocapsid and virion. Structure-function relationship relevant to viral RNA synthesis will also be explored. Overall, the anticipated results will provide new insights into the mechanism of virion assembly and viral RNA synthesis, not only for LCMV but also for Arenaviruses in general. The proposed studies harness cutting-edg...