IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

Abstract

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.

Figure 1. IFITM3 inhibits infection after viral binding but before viral transcription.

A) A549 cell lines were incubated on ice with H1N1 WSN/33 to permit viral-host binding. Cells were washed, fixed and immunostained for surface-bound HA protein, then analyzed by flow cytometry. Values given are percentage of cells staining for surface HA. Values are representative of three independent experiments. B) MDCK cells transduced with the empty vector control (Vector) or IFITM3 were incubated with A/Puerto Rico/8/34 H1N1 (PR8) on ice. Warm media was added at time zero. Cells were then fixed at the indicated time points and hybridized with RNA probes against the viral NP mRNA (red) and stained for DNA (blue), then imaged by confocal microscopy. Images are representative of three independent experiments. (Scale bar: 20 µm).

Figure 2. IFN prevents vRNP nuclear entry, and IFITM3 is necessary and sufficient for this action.

A) Normal diploid human fibroblasts (WI-38 cells) were stably transduced with retroviruses containing either IFITM3 (IFITM3), a shRNA against IFITM3 (shIFITM3), an empty viral vector alone (Vector), or a non-targeting control shRNA (shScramble, Fig. S2). Cells were incubated with PR8 on ice, and then warm media was added at time zero. Cells were fixed at the indicated times p.i. and stained for NP (green) and DNA and analyzed by confocal microscopy. Image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on DIC images and DNA staining, respectively). Red arrows: cytosolic compartments containing NP. Images are representative of four independent experiments. (Scale bar: 15 µm). B) As in (A) except that cells were treated with IFN-α prior to infection.

Figure 3. IFITM3 overexpression leads to both a retention of viral genomes in the cytosol, and a decrease in viral genomes entering the nucleus.

MDCK cells transduced with the empty vector control (A) or IFITM3 (B) were incubated with PR8 on ice. Warm media containing lysotracker red dye (LTRed, red) was added at time zero. Cells were then fixed at the indicated time points and hybridized with RNA probes against the viral NP genome (NP vRNA, green) and stained for DNA, then imaged by confocal microscopy. Image analysis software was used to define the nuclear boundaries (blue lines) based on DNA staining. Images are representative of four independent experiments. (Scale bar: 20 µM). C) Quantitation of nuclear vRNA particles. The number of viral RNA particles per nucleus of the MDCK-Vector and IFITM3 cells at the indicated time points are shown. Values represent the mean +/− the SD of three independent experiments. D) Percent colocalization of vRNA and LTRed-containing compartments for MDCK-Vector and IFITM3 cells lines treated as in A and B, at the indicated time points.

Figure 4. Ifitm knockout cells are more vulnerable to vRNP nuclear entry and are rescued by the restoration of Ifitm3 expression.

MEFs, either A) wild type (WT) or B) IfitmDel−/−, which are missing all five of the mouse Ifitm proteins, were either left untreated (left panels, Buffer), or treated (right panels) with IFN-γ. The following day cells were incubated with PR8 on ice. Cells were next incubated in warm media containing LTRed. Cells were then fixed at the indicated times and immunostained with anti-NP antibodies (green), stained for DNA (blue), and imaged by confocal microscopy. Image analysis software was used to define the nuclear boundaries (blue lines). Images are representative of three independent experiments. (Scale bar: 12 µm).

Figure 5. HA or VSV-G-mediated fusion is inhibited by IFN or IFITM3.

A) Schematic model of the established viral fusion assay , comprised of lentiviral pseudoparticles (pps) containing the β-lactamase protein fused to the HIV-1 accessory protein Vpr (BLAM-Vpr, shown in orange/red) and expressing HA and NA (WSN/33) on their surfaces. The H1N1pps were incubated for 2 h with cells, which were subsequently loaded with the β-lactamase flourogenic substrate, CCF2. Upon viral fusion, BLAM-Vpr enters the cytosol and can cleave CCF2, producing a wavelength shift from green to blue in emitted light when analyzed by flow cytometry ([37]). B) MDCK cells stably overexpressing IFITM3 (MDCK-IFITM3) or empty vector control cells (MDCK-Vector) were exposed for 2 h to viral pseudoparticles containing a BLAM-Vpr and expressing either the HA and NA envelope proteins of Influenza A virus (WSN/33, H1N1pp) or the VSV-G envelope protein (VSV-Gpp), then loaded with CCF2. After incubation with the indicated pseudoparticles, the cells were fixed and assayed for cleavage of CCF2 by determining the conversion of the fluorescence emission from 520 nm (uncleaved CCF2) to 447 nm (cleaved CCF2) using flow cytometry. Fusion of the pseudoparticles was inhibited by bafilomycin A1 (Baf). These results are representative of six independent experiments. C) IFITM3 inhibits fusion of H1N1pps in normal diploid fibroblasts. WI-38 fibroblasts stably transduced with IFITM3 (WI-38 M3) or the empty vector (WI-38 V) were exposed for 2 h to serial dilutions of H1N1pps containing BLAM-Vpr, with or without Baf. These results are representative of four independent experiments. D) Fusion of H1N1pps increases after IFITM3 knockdown. WI-38 fibroblasts stably transduced with a shRNA against IFITM3 (WI-38 shM3), a shRNA control with a scrambled sequence (WI-38 shScr), or the IFITM3 cDNA (WI-38 M3) were exposed to either no virus, H1N1pps or VSV-Gpps containing BLAM-Vpr. These results are representative of two independent experiments. E) Fusion of H1N1pps is inhibited by IFN-γ. WI-38 fibroblasts were treated with IFN-γ for 24 h or buffer alone prior to incubation with H1N1pps containing BLAM-Vpr. These results are representative of three independent experiments.

Figure 6. IFITM3 partially colocalizes with Rab7 and LAMP1, and compartments containing these proteins are amplified with IFITM3 overexpression.

A) A549 cells stably transduced with either IFITM3 or with the empty vector alone, were incubated with LTRed (red) at 37°C, then fixed and immunostained for confocal imaging of IFITM3 (endogenous and overexpressed, gold), and LAMP1 (endogenous, green). DNA = blue. (Scale bars: 20 µM throughout). B) Percent colocalization of IFITM3, LTRed and LAMP in A549-Vector (blue) or IFITM3 (red) cells in (A). C) A549 cells stably transduced with either IFITM3 or with the empty vector alone were immunostained for confocal visualization of IFITM3 (endogenous and overexpressed, red) and Rab7 (endogenous, green). DNA = blue. D) Percent colocalization of IFITM3 and Rab7 in either the A549-Vector or A549-IFITM3 cells in (C). E) MDCK-Vector or MDCK-IFITM3 cells stained for exogenous IFITM3 (overexpressed, red) and LAMP1 (endogenous, green). DNA = blue. F) Percent colocalization of IFITM3 and LAMP1 in MDCK-Vector or MDCK-IFITM3 cells in (E). G) MDCK cells stably overexpressing Rab7-YFP and either IFITM3 (MDCK-IFITM3) or the empty vector control (MDCK-Vector) were immunostained and confocally imaged for IFITM3 (overexpressed, red) and Rab7-YFP (fluorescent signal from exogenous protein, green). Nuclear peripheries are represented by blue lines. H) Percent colocalization of Rab7-YFP and IFITM3 in either the MDCK-Vector or IFITM3 cells in (G). I) Enlarged view of images outlined by white boxes shown in (G), with MDCK-IFITM3 cells stably overexpressing both IFITM3 (red) and Rab7-YFP (green).

Figure 7. IFN treatment or IFITM3 overexpression expands late endosomes and lysosomes.

A549 cells stably expressing IFITM3 (IFITM3) or empty vector (Vector) were (A) left untreated (Buffer) or (B) treated with IFN-α, then fixed, permeabilized and immunostained for IFITM3 (endogenous and overexpressed, red), Rab7 (endogenous, gold), LAMP1 (endogenous, green), and for DNA (blue, merged image). Images were obtained using a confocal microscope. Similar results were observed with IFN-γ (data not shown). (Scale bars: 20 µm throughout). C) Whole-cell lysates from A549-IFITM3 or A549-Vector cells in (A) and (B) treated or untreated with IFN-α or γ were subjected to immunoblotting against the proteins indicated. GAPDH levels are provided to demonstrate comparable protein loading. Molecular weights in kDa are provided to the left. These images are representative of three independent experiments. D) A549 cells stably expressing Rab7-YFP (fluorescent signal from exogenous protein, green) were left untreated (Buffer) or treated with IFN-α, then fixed, permeabilized and immunostained for IFITM3 (endogenous, red) and imaged confocally. DNA = Blue. Similar results were obtained for IFN-γ (data not shown). E) Percent colocalization of IFITM3 and Rab7-YFP in the A549 cells in (D), with or without IFN-α treatment.

Figure 8. IFITM3 overexpression results in the expansion of acidified organelles.

A) MDCK or (B) A549 cell lines, stably overexpressing IFITM3 or the empty vector alone, were incubated with either the acidophilic dye acridine orange (AO), LTRed, or a flourogenic cathepsin-L substrate (Cath-L). All cells were also stained for DNA (blue). After incubation cells were imaged on a confocal microscope. Middle panels show enlarged images of the IFITM3 cells. (Scale bars: 20 µm throughout). C) Vector (blue) or IFITM3 (red) transduced cell lines, either MDCK (left) or A549 (right), were incubated with LTRed then analyzed by flow cytometry. D) A549 cells stably transduced with IFITM3 or with the vector alone were incubated with LTRed (red), then immunostained for confocal imaging of LC3 (endogenous, green). DNA = blue. E) MDCK cells stably transduced with IFITM3 or with the vector alone were immunostained for confocal imaging of LC3 (endogenous, red) and CD63 (endogenous, green). DNA = blue. F) Confocal images of MDCK cells overexpressing IFITM3 or the empty vector alone showing the distribution and fluorescence intensities of a stably expressed mCherry-EGFP-LC3B fusion protein using fluorescence channels that detect light emitted from the mCherry protein, EGFP or both (merge). DNA = blue. G) Model of IFITM3-mediated restriction of virus replication. Endocytosed viruses enter late endosomes where IFITM3 is present. IFITM3 prevents viral fusion within the endosomes and likely lysosomes via an unknown mechanism, perhaps by altering pH, membrane characteristics, lipid composition, transport speed or destination. Trapped viruses are trafficked to lysosomes and/or autolysosomes where they undergo degradation.