IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion

Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.
Author Summary Interferon-induced transmembrane proteins (IFITMs) block infection of many enveloped viruses, including the influenza A virus (IAV) that enters from late endosomes. IFITMs are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. Here we performed single IAV imaging and found that IFITM3 did not interfere with hemifusion, but prevented complete fusion. Also, contrary to a current view that excess cholesterol in late endosomes of IFITM3-expressing cells inhibits IAV entry, we show that cholesterol-laden endosomes are permissive for virus fusion. The ability of IFITM3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. IFITM3 may also redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane.