Agnitha Xavier, University of Ottawa

Agnitha Xavier 1, 2, 3; Marceline Côté 1, 2, 3 (1 Department of Biochemistry, Microbiology, and Immunology, University of Ottawa; 2 Ottawa Institute of Systems Biology, University of Ottawa; 3 Centre for Infection, Immunity, and Inflammation, University of Ottawa)

Breaking & Entering

Ebolavirus (EBOV), a member of the Filoviridae family which causes severe haemorrhagic fever in humans, is known to infect cells by hijacking the endosomal trafficking network. Viral membrane fusion requires the interaction of the viral glycoprotein with the late endosome/lysosome (LE/LY)-resident protein Niemann Pick C-1 (NPC1), but how virions are trafficked to NPC1 compartments following internalization is not clear. We and others have shown that the activity of the PIKfyve-VAC14-FIG4 complex – which controls the turnover of the endosomal phosphoinositide PI(3, 5)P2 – is required for EBOV glycoprotein (GP)-mediated entry, although few details are known about how the complex is recruited and regulated. The best characterized effectors of PI(3,5)P2 are the LE/LY calcium channels TRPML1 and TPC1/2 – also thought to play roles in EBOV entry – which suggests that trafficking of EBOV to NPC1 LE/LY via PIKfyve requires calcium signalling. Here, we show that calmodulin, a calcium-binding protein essential to signal transduction, is involved in the recruitment of the PIKfyve-VAC14-FIG4 complex. We show through co-immunoprecipitation studies that calmodulin binding in complex with PIKfyve-VAC14-FIG4 is highly dynamic, may be required for the production of PI(3,5)P2, and is potentially further regulated by ubiquitination. Following this, we found that both small-molecule inhibition and siRNA knockdown of calmodulin reduces EBOV GP-mediated entry, which was not seen in VSV-G mediated entry. Taken together, our findings strongly suggest that EBOV hijacks PI(3, 5)P2 production via calmodulin, and further elucidate signalling mechanisms during EBOV entry.