Elucidation of JUNV GPC Structure and Molecular Mechanisms of Escape from Humoral Immunity - Elucidation of JUNV GPC Structure and Molecular Mechanisms of Escape from Humoral Immunity Junin virus (JUNV) is a New World arenavirus and causes Argentine hemorrhagic fever. The JUNV glycoprotein complex (GPC) on the surface of virions mediates viral entry by binding to the host protein, transferrin receptor 1 (TfR1). GPC is synthesized as a single polypeptide precursor that undergoes cleavage by the cellular protease SKI-1/S1P to yield subunits GP1 and GP2, which function in receptor binding and membrane fusion, respectively. Unique to arenaviruses is the stable signal peptide (SSP) which is retained in the mature spike glycoprotein complex and is critical for GPC stability and post-translational processing. Currently, all characterized JUNV neutralizing antibodies (nAbs) target the receptor binding site (RBS) within the GP1 domain. However, the RBS of New World arenaviruses is highly diverse in sequence despite sharing a common binding mode to their receptor, TfR1. This suggests there may be a broad mutational landscape compatible with TfR1 utilization, potentiating escape from RBS-directed antibodies. Importantly, a recent study demonstrated that MACV GPC can readily escape RBS-directed nAbs isolated from vaccinated mice. Our lab previously characterized a panel of nAbs targeting the RBS of JUNV, with one nAb in particular showing significant therapeutic efficacy in mice and guinea pig models. However, the propensity of JUNV to escape from nAbs, including this particular nAb, has not been investigated. Therefore, this proposal aims to examine the molecular mechanisms and evolutionary dynamics of JUNV escape by passaging VSV pseudotyped with JUNV GPC in the presence of these RBS-directed antibodies. By using a similar approach with convalescent sera depleted of GP1-directed antibodies, I will map nAb epitopes outside of GP1. The role of epitopes outside of GP1 in sera neutralization has not been explored and experiments proposed herein will elucidate whether they represent additional targets for vaccine and therapeutic development. Epitope information obtained will enable to design of antigenic probes for sorting B-cells specific for these epitopes in future studies. Efforts to study nAbs targeting epitopes outside of GP1 are hindered by the lack of GPC reagents critical for their isolation and characterization. Efforts to develop these stable New World GPC antigens are complicated by the lack of high resolution structures for use in rational protein design. Therefore, another central aim of this proposal is to obtain the structure of JUNV GPC by cryo-EM. If successful, the resulting model will be used for structure-based stabilization of the GPC. A stabilized JUNV GPC trimer would not only serve as a valuable research tool and promising vaccine antigen but would also provide a blueprint for structural characterization of other New World arenaviruses. Taken together, the research in this proposal will identify pathways for antibody escape in JUNV GPC, define novel neutralizing antibody epitopes, and provide a high-resolution structure of JUNV GPC to help inform vaccine and therapeutic antibody design efforts.
