Low basal expression and slow induction of IFITM3 puts immune cells at risk of influenza A infection

Dannielle Wellington; Zixi Yin; Liwei Zhang; Jessica Forbester; Kerry Kite; Henry Laurenson-Schafer; Shokouh Makvandi-Nejad; Boquan Jin; Emma Bowes; Krishnageetha Manoharan; David Maldonado-Perez; Clare Verill; Ian Humphreys; Tao Dong

To confirm that the level of expression of IFITM3 is an important determinant 397 of viral control, we stimulated HEK293 cells with IFN for 24 hours prior to 398 infection with influenza A pseudotyped virus (S-FLU PR8:H1N1). IFITM3 399 expression was measured prior to infection (Figure 4a) However, IFITM3 is not the only anti-viral mechanism induced by IFN 411 stimulation. We therefore investigated the infection rates of IFN-stimulated 412 HEK293-IFITM3 -/cells. As expected, the infection rate was substantially 413 higher in IFITM3 -/cells with no IFN stimulation, but following type I IFN 414 stimulation the infection rate was attenuated to levels comparable to or lower 415 than those seen in un-stimulated HEK293-WT but not as low as in IFN-treated 416 HEK293-WT cells (Figure 4b) . This data suggests that IFITM3 contributed to 417 type I IFN mediated control in cells where IFITM3 was substantially 418 upregulated upon IFN stimulation. 419 420 To investigate the association between IFITM3 expression levels and 421 infection rates, we re-introduced IFITM3 into HEK293-IFITM3 -/cells using 422 plasmid transfection at different concentrations prior to infection with S-FLU 423 PR8:H1N1 (Figure 4c) . As the percentage of IFITM3+ cells increased, the 424 level of GFP+ (i.e. cells with replicating virus) was reduced. In fact, when we 425 looked at the correlation between IFITM3+ and GFP+ cells we saw a 426 significant inverse correlation between these levels (R 2 =0.4730, p=0.0046) 427 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2019.12.20.885590 doi: bioRxiv preprint (Figure 4d) .