Development of Molecular Tools for the Investigation of Arenavirus Spike Architecture

The Arenaviridae family of segmented RNA viruses includes several important human pathogens, some of which are the causative agents of haemorrhagic fever, a disease with severe morbidity and high mortality. Currently, effective therapeutic and preventative measures for such pathogens are limited. The prototypical arenavirus is lymphocytic choriomeningitis virus (LCMV), which on account of its low pathogenicity in humans provides the opportunity to understand more about arenavirus infections under bio-safety level 2 containment. The work described here sought to develop and exploit molecular tools for arenavirus study. Firstly, a high-affinity antibody reactive against LCMV nucleocapsid protein (NP) was generated. This LCMV NP antibody was subsequently used to investigate the intracellular localisation of the NP during the viral lifecycle and to develop a method for the titration of non-cytolytic LCMV, allowing the quantification of infectious virus. Secondly, the LCMV NP antibody and the titration method were subsequently used to optimise a reverse genetics system, designed to recover infectious recombinant LCMV entirely from cDNAs. This system was further engineered to introduce the eGFP gene into the S segment of LCMV, permitting simple and rapid monitoring of LCMV gene expression and infection through fluorescent live cell analysis. The reverse genetics system was further engineered to successfully generate infectious LCMV carrying a C-terminal-6xHis-tagged NP, which could be utilised to purify native viral ribonucleoprotein complexes from infected cells. The successful production of both of these tagged replication-competent recombinant LCMV variants are valuable tools for the study of arenaviruses. Using the molecular tools generated here, LCMV was highly concentrated and purified for its examination by cryo-electron tomography, alongside another arenavirus, Pichindé virus (PICV), providing a direct comparison between the two arenaviruses. Sub-tomogram averaging was then used to resolve the structures of the native glycoprotein complex (GPC) entry spikes, present on the viral surface. These results indicated structural differences of the GPC between multiple arenaviruses, which could provide information on different entry requirements and their pathogenicity