Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes

Suddala, Krishna C.; Lee, Christine C.; Meraner, Paul; Marin, Mariana; Markosyan, Ruben M.; Desai, Tanay M.; Cohen, Fredric S.; Brass, Abraham L.; Melikyan, Gregory B.

To visualize single LASV entry and fusion, which is not restricted by IFITM3 in A549 cells [15] , we pseudotyped the HIV-1 core containing the bi-functional mCherry-2xCL-YFP-Vpr marker with the LASV GPc envelope glycoprotein complex to generate LASV pseudoparticles (LASVpp). LASVpp imaging in A549 cells confirmed the ability to track single particles and detect their fusion (release of mCherry) in late endosomal compartments (Fig 5B, S10 Movie) . Interestingly, LASVpp fusion exhibited a unique feature rarely seen for other viruses, including IAV. The YFP-Vpr fluorescence, which is markedly decreased at mildly acidic pH [51, 52] , was consistently quenched at some point prior to viral fusion, demonstrating acidification of intraviral pH [53, 54] (schematized in Fig 5A) . Single frame images show that YFP-Vpr signal quenched for~10 min before viral fusion, which is observed as the loss of mCherry signal (red) and concomitant reappearance of the YFP-Vpr signal (Fig 5B and 5C, arrow) . The dequenching of YFP fluorescence can be attributed to the re-neutralization of the virus' interior through a fusion pore connecting it to the cytoplasm [52, 54, 55] . Based on the differences in IAVpp and LASVpp fusion, we classified single virus fusion events into "Type I", in which mCherry signal is lost without acidification of the virus interior (YFP quenching), as observed in IAV fusion, and "Type II" events, in which acidification of the virus interior occurs prior to fusion (mCherry release), as observed in LASV fusion. In A549 cells, only 9% of LASVpp fusion events are Type I, while most particles-91%-undergo Type II fusion (Fig 5D) . Of note, YFP-Vpr quenching occurs for most particles not undergoing fusion at later times after infection due to a non-specific acidification of the viral interior in late acidic compartments. These events representing a non-productive entry of LASVpp were excluded from analysis. Additional experiments to confirm single LASVpp fusion in A549 cells were also performed. Control cells were treated with a broad-spectrum arenavirus entry inhibitor, ST-193 [56] , which abrogated single LASVpp fusion events (Fig 5D) . A total of 90 Type II events were observed in at least 24 independent experiments, with 6430 viral particles in Vector control cells and 5264 viral particles observed in cells treated with . LASVpp fusion with A549 cells measured by a bulk BlaM assay also demonstrated potent inhibition of viral fusion in the presence of 10 μM ST-193 or upon raising the endosomal pH by 40 mM NH 4 Cl (Fig 5E) . Thus, the observed changes in fluorescent signals faithfully represent single LASVpp fusion.