Figure 4. IFITM3 blocks fusion pore formation between single influenza viruses and endosomes.

Pseudoviruses bearing WSN HA and NA glycoproteins were co-labeled with HIV-1 Gag-iCherry (viral content marker, red) and YFP-Vpr (viral core marker, green). Viruses were pre-bound in the cold to A549-Vector cells (A, B) or MDCK-Vector cells (C, D) and their entry was initiated by raising the temperature. (A, C) Images of IAVpp are extended projections of 3 Z-stacks illustrating the loss of the mCherry signal (arrow) upon virus-endosome fusion. A schematic illustration between image panels A and C illustrates fusion between the YFP-Vpr (green) and Gag-iCherry (red) labeled IAVpp and an endosome (gray). (B, D) Mean mCherry and YFP fluorescence intensities obtained by tracking the particles shown in panels A and C. (E) Normalized efficiencies of IAVpp fusion (content release) with A549 and MDCK cells transduced with an empty vector or with IFITM3. The middle bar shows the lack of mCherry release in A549-Vector cells in the presence of NH₄Cl. ***, P<0.001. See movies S4 and S5.