Conformational states and immunological targeting of the arenaviral glycoprotein complex: implications for therapeutic design
Keywords: x-ray crystallography
cryo-electron microscopy
rabies virus
arenavirus
viral glycoproteins

Envelope-displayed viral glycoproteins mediate host cell entry and are a key determinant of species tropism. As the glycoproteins are the sole antigens exposed on the mature virion surface, they are also primary targets of the host humoral immune response that arises during infection and immunisation. Macromolecular structure determination of these therapeutically important targets constitutes an important approach to obtain molecular-level insights into viral protein structure and function.
The emergence and re-emergence of Old World (OW) and New World (NW) mammarenaviruses (family Arenaviridae) from rodent reservoirs persistently threatens human health. The GP1 subunit of the envelope glycoprotein spike complex (GP) is responsible for receptor attachment during host cell entry. Here, I report the GP1 structure of OW Loei River virus (LORV) and the GP1 structures of NW Junín virus (JUNV) and NW Machupo virus (MACV) in complex with neutralising antibodies (nAbs). Comparative analyses of the GP1 glycoprotein architectures of LORV and previously characterised mammarenaviruses expands our understanding of the structural classes sampled by the GP1 glycoproteins and provide molecular blueprint for immunogen design that is capable of mimicking the mature OW mammarenaviral GP. Glycan mapping analysis further revealed a pronounced glycan-mediated masking of the LORV GP, rationalising the observed immune-evasive properties of OW mammarenaviruses. Furthermore, structural characterisation of the nAb epitopes on JUNV and MACV GP1s provides a template for understanding the antigenic surface of the NW GP1 and augments efforts to develop nAb-based prophylactics that target the NW arenavirus surface.
Rabies lyssavirus (RABV; family Rhabdoviridae) is the causative agent of rabies, which causes approximately 60,000 deaths annually. NAb-based therapeutics constitute an important part of the rabies treatment. Structure elucidation of the higher-order assembly of RABV glycoprotein (RV-G) allows identification and characterisation of the full antigenic surface of the spike, including the quaternary nAb epitopes. Here, I characterised the structure of RV-G in complex with two nAbs. The structural data revealed the architecture of the first full-length lyssavirus glycoprotein structure in prefusion state and defined nAb-targeting epitopes in the context of a trimeric spike. This work serves as a template for structure-guided engineering of next generation RABV vaccines. The structural insights on the nAb epitopes may also augment the optimisation of pre-existing antibody-based post-exposure prophylactics.
Through crystallographic and cryogenic electron microscopy analysis, the work included in this thesis expand our appreciation of the conformational landscape covered by the glycoproteins of mammarenaviruses and lyssaviruses. The data presented will empower strategies focused on the development of immunogens that resemble the glycoprotein spike presented on the mature virion and capable of eliciting protective immune response.