Innate immune control of influenza virus interspecies adaptation via IFITM3

Abstract

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigate whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We find that IFITM3-deficient mice and human cells can be infected with low doses of avian influenza viruses that fail to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious virus dose threshold. Remarkably, influenza viruses passaged through Ifitm3^-/- mice exhibit enhanced host adaptation, a result that is distinct from viruses passaged in mice deficient for interferon signaling, which exhibit attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates potentially zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

Fig. 1. IFITM3 deficiency lowers the minimum infectious dose threshold for avian influenza viruses in vivo.

WT and Ifitm3^−/− mice were intranasally infected with (a–c) 1, 10, or 50 TCID50 of H5N1 avian influenza strain (2 independent experiments for doses 1 and 10 (n = 10 mice) and 1 experiment for dose of 50 (n = 5 mice)) or with (d–f) 1 or 10 TCID50 of H7N3 avian influenza strain (n = 5 mice). a, d Viral titers from lung homogenates at day 3 post infection. b, e ELISA quantification of IL-6 levels in lung homogenates at day 3 post infection. c, f ELISA quantification of IFNβ levels in lung homogenates at day 3 post infection. All error bars represent SEM. Comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between WT and Ifitm3^−/− mice for each dose are shown. a–f Each data point represents an individual mouse. The numbers shown above the graph represent exact p-values. Source data are provided as a Source Data file.

Fig. 2. IFITM3 limits animal-origin influenza virus infection of human cells.

a Schematic of in vitro infection with animal-origin influenza viruses and representative example flow cytometry dot plots from infected A549 human lung cells. b, e The indicated A549 cells or THP-1 differentiated macrophages were treated +/- IFNβ for 18 h followed by infection with the indicated viruses (MOI 1) for 24 h. Percent infection was determined by flow cytometry and normalized to respective control cells without IFNβ pre-treatment. Error bars represent SEM. Only statistical comparisons between shControl versus shIFITM3 and WT versus IFITM3^−/− are shown, determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data are representative of 3 independent experiments, each performed in triplicate (n = 9). c, d Western blots of cell lysates at 18 h +/− IFNβ treatment. Note that commercial IFITM3 antibodies weakly detect IFITM2 in addition to IFITM3. The numbers shown above the graph represent exact p values. Source data are provided as a Source Data file. a Created in BioRender. Denz, P. (2024) BioRender.com/z17v580. Gating strategies for flow cytometry are depicted in Supplementary Fig. 2a. A549 cells and Supplementary Fig. 2b. THP-1 cells.

Fig. 3. IFITM3 deficiency lowers the minimum infectious dose threshold for avian influenza viruses in vivo.

a, b The indicated A549 cells were infected with the indicated avian-origin influenza viruses at a range of 0.001 to 10 multiplicity of infection (MOI) for 24 h. Percent infection was determined by flow cytometry. Data are representative of 3 independent experiments, each performed in triplicate (n = 9). Only comparisons between control and knockdown cells are shown at each dose. c A549 cells were infected with the indicated influenza viruses at an MOI of 1 and incubated in media containing TPCK-Trypsin for 48 h to allow for multi-cycle replication. The supernatants were collected to determine viral titer. Data are representative of 2 independent experiments, each performed in triplicate (n = 6). Only comparisons between control and knockdown cells are shown for each virus. All error bars represent SEM. The numbers shown above the graph represent exact p-values. Comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Fig. 4. Human-origin H3N2 influenza virus adapts to a new species more readily in the absence of IFITM3.

a Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1000 TCID50 of parental viruses. b Schematic of WT mouse challenge with parental A/Victoria/361/2011 (H3N2) or passaged viruses. c–h Groups of WT mice (n = 5 per group) were challenged with 1000 TCID₅₀ of A/Victoria/361/2011 (H3N2) virus passaged 1, 5, or 10 times through WT or Ifitm3^−/− mice and compared to the parent virus (passage 0). c, f Viral titers from lung homogenates collected at day 7 (c represents 2 independent infections). d, g ELISA quantification of IL-6. e, h ELISA quantification of IFNβ. c–h Error bars represent SEM and comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Each dot represents an individual mouse. The numbers above the graph represent exact p-values. Source data are provided as a Source Data file. a, b Created in BioRender. Denz, P. (2024) BioRender.com/h97v852.

Fig. 5. Human-origin H1N1 influenza virus adapts to a new species more readily in the absence of IFITM3.

Influenza virus A/California/04/2009 (H1N1) was passaged through mice as described in Fig. 4a. Groups of WT mice (n = 5 per group) were challenged with 1000 TCID₅₀ of A/California/04/2009 (H1N1) virus passaged 1, 5, or 10 times through WT or Ifitm3^−/− mice and compared to the parent virus (passage 0). a, e Viral titers from lung homogenates collected at day 7 (a) or day 6 (e) post infection. Error bars represent SEM, comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. b Weight loss for the H1N1 series 1 challenge. Error bars represent SEM, comparisons were made using the Mann-Whitney test. ELISA quantification of IL-6 (c, g) and IFNβ (d, h) levels in lung homogenates of WT and IFITM3 KO mice at day 7 (c, d) or day 6 (g, h) post infection. Error bars represent SEM and comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. f Weight loss for the H1N1 series 2 challenge. Skull and crossbones indicate humane euthanasia of all animals infected with KO passage 10. Error bars represent SEM, comparisons were made using the Mann-Whitney test. (a, c, d, e, g, h) Each dot represents an individual mouse. b, f dots represent averages of individual mice (n = 5 per group). All numbers above the graphs represent exact p-values. Source data are provided as a Source Data file.