HASTIE, KATHRYN MARIE
Project Summary/AbstractThere are around 50 known viruses in the Arenavirus family. These include Lassa (LASV), Lujo(LUJV), Machupo (MACV), and Junín (JUNV), which cause hemorrhagic fever in Africa and SouthAmerica. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), also causesencephalitis and birth defects in humans worldwide. Arenaviruses express one glycoprotein on theirsurface, termed GPC, which is responsible for receptor engagement, cell tropism, and entry.Understanding the structure of GPC, particularly in its trimeric, prefusion conformation, is key tounderstanding why the different arenaviruses recognize distinct receptors and in designingtherapeutics and vaccines against them. However, the GPC is metastable: it easily disassembles intoits component subunits and then springs into its more stable, post-fusion conformation. Thismetastability hindered structural biology efforts for years. Indeed, there has been no structure of anytrimeric, prefusion arenavirus GP until this year. A ten-year protein engineering effort in the labrecently culminated in the crystal structure of LASV GP in its trimeric, prefusion complex, the firstsuch structure for any arenavirus. This landmark structure illuminated new findings: that the properlyassembled prefusion trimer is essential for recognition by the most effective neutralizing antibodiesand for recognition by the LASV and LCMV cell surface receptor matriglycan. In the absence of stablyengineered prefusion GP, such potently protective antibodies would have been difficult to identify andcharacterize. There remains, however, no trimeric prefusion GP structure for any other arenavirus.Fortunately, the model built for LASV GP provides the blueprints we need to propel this effort forward.The premise of this proposal is that the correct quaternary assembly of GP can be engineeredfor other arenaviruses using the LASV GP structure as a template and the foundation ofknowledge laid by this structure. We will combine strong preliminary results and state-of the-artbiophysical techniques, with bio-layer interferometry and ELISA to analyze the interaction ofengineered MACV, JUNV, LCMV and LUJV GPs with their distinct receptors and with unique panelsof antibodies from human survivors (JUNV and LUJV) and mice (MACV and LCMV). In aim 1 we willdevelop the stable prefusion GPs necessary to determine if MACV and JUNV, like LASV, also elicitquaternary-epitope antibodies and the stoichiometry by which these viruses bind their TfR1 receptor.In aim 2 we will develop the GPs necessary to solve an LCMV-matriglycan complex, map theepitopes of LCMV-binding antibodies and identify and characterize LUJV GP antibodies and theLUJV GP structure involved in its atypical mechanism of entry. Multiple lines of inquiry will belaunched by the innovative research effort proposed here.