Investigating the assembly and egress of arenaviruses

There are around 70 segmented RNA viruses contained within the Arenaviridae family, and multiple members are associated with fatal haemorrhagic fevers, including Lassa and Junín viruses. Established LCMV strains are biosafety level (BSL) 2 pathogens, and can model for these serious pathogens since fatality is only common within immunocompromised populations. Here, the project details three aims which collectively investigate the later stages of arenavirus multiplication. Firstly, an siRNA library, which targeted cellular trafficking components, identified coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes as important during LCMV infection. Immunofluorescence (IF) was performed for cells infected with an infectious recombinant LCMV harbouring a FLAG tag within GP-1, which identified close co-localisation of COPA, AP4E1 and viral proteins. Additionally, the inhibitor Brefeldin A (BFA) was applied to LCMV infection, which suggests that COPI and AP-4 complexes are critical for efficient virion production.
Secondly, it was noted during live-cell imaging that cells initially infected with LCMV result in infection of neighbouring cells, with discrete foci formed. IF analysis revealed LCMV structural proteins co-localise within cell-cell connections resembling tunnelling nanotubes (TNT-like). Fluorescent in situ hybridisation was applied and identified that TNT-like connections also contain genomic sense RNA. Strikingly, blocking the extracellular route of infection (post initial entry) through application of a potent neutralising antibody did not abolish progression of LCMV infection, and thus implied intracellular connections could account for around half of infection events.
Finally, we investigated the phosphorylation of LCMV NP during infection. We noted that CK-869 caused an increased post-translational modification of NP. Through investigations, we reveal that S343 is phosphorylated, and we established infectious NP phospho-ablantant (S343A) and phospho-mimetic (S343E) mutants. Here, we identified that S343 phosphorylation of NP is not a viral-dependent process, and acts in an anti-viral manner within the mouse macrophage RAW264.7 cell line. Mechanistically, we suggest that phosphorylation of NP may trigger lysosomal degradation, reporting co-localisation of NP and LAMP-1 during S343E infected cultures. Additionally, we reveal that S343 NP phosphorylation is LCMV strain specific, which may contribute to difference in pathogenicity.
Taken together, these results increase our knowledge surrounding the later stages of arenavirus multiplication, which may contribute to the development of effective preventatives or therapeutics.