In many ways, viruses are the ultimate molecular hijackers. Over millions of years of coevolution with their hosts, they have found ways to exploit cellular machinery and processes to facilitate infection, replication, and immune evasion. Ebola virus (EBOV) entry is no exception, relying on the intricately regulated endosomal trafficking system—normally used to transport cellular cargo—to instead traffic virions to their receptor, NPC1.
How EBOV manages to navigate the complex endosomal labyrinth is largely uncharacterized, however, in our study, we found that it requires the activity of a host enzyme complex, the PIKfyve-ArPIKfyve-Sac3 (PAS) complex. This complex is responsible for the production and turnover of the lipids phosphatidylinositol-3-phosphate (PtdIns3P) and phosphatidylinositol(3,5)bisphosphate (PtdIns(3,5)P2), which are important for transport of cargos towards the late endosomal/lysosomal compartment—where NPC1 resides.
EBOV induces relocalization of a PtdIns(3,5)P2 probe to intracellular vesicles. Serum-starved Vero cells transfected with FYVE-RFP and 2XML1N-GFP probes were seeded in 8 well chamber slides and treated with EGF (100 ng/mL), EGF+ apilimod (50 nM), VLPs harbouring EBOV ΔM GP (MOI~100), or VLPs + apilimod for 30 min. Live cells were imaged on an LSM800 confocal microscope (Zeiss). Bar = 10 µm.
While initial experiments using small molecular inhibitors of PIKfyve activity suggested that its enzymatic activity was important for viral entry, it was really our experiments using CRISPR to delete each member of the complex from cells that demonstrated simply and elegantly how important these proteins are for viral infection. One surprise was that Sac3 expression, but not its enzymatic activity, is important for EBOV entry. It turned out that Sac3 is required to enable full PIKfyve enzymatic activity, highlighting PIKfyve and PtdIns(3,5)P2 production as the key players for EBOV entry. This was further confirmed using fluorescent PtdIns(3,5)P2 binding probes that showed that the virus not only depends on production of this lipid, but actually is able to stimulate its production.
The PAS complex is vital for development and cell survival, however, there are still many unanswered questions about how it functions within cells. Evidence that viruses exploit this complex to facilitate infection raises the exciting possibility that we can use viruses as tools to understand how basic cellular machinery functions, thus learning two sides of the same story: pathogen and host.
Introducing the author
Shirley Qiu, University of Ottawa
About the research
Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry
Virology, Volume 513, 1 January 2018, Pages 17-28