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IFITM3 directly engages and shuttles incoming virus particles to lysosomes


Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR–Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.

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The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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E.T. acknowledges support a Marie Skłodowska-Curie postdoctoral fellowship. T.D. is supported by the Tri-Institutional Program in Chemical Biology at The Rockefeller University. H.-H.H. and C.M.R. acknowledge support from NIH R01AI091707. We thank W. Wei (Peking University) for sharing pCAS9 plasmid and gRNA vector (pGL3-U6). We thank Y.-C. Wang (The Rockefeller University) for the synthesis of dfTAT. We thank J. Yount and members of the Hang laboratory for helpful comments and discussion of the paper. T.P. acknowledges support from the National Natural Science Foundation of China (No. 21778010), the Shenzhen Science and Technology Innovation Committee (JCYJ20170412150832022), and Shenzhen Peacock Plan (KQTD2015032709315529). K.C. acknowledges grant support from NIH-NIAID R56AI088027. H.C.H. acknowledges grant support from NIH-NIGMS R01GM087544.

Author information

J.S.S., R.H., T.P., K.C. and H.C.H. conceived the study. J.S.S., R.H., H.-H.H., E.T., T.D., C.M.R., T.P., K.C. and H.C.H. planned the experiments. R.H. generated IFITM1, IFITM2 and IFITM3 knockout mammalian cell lines and performed cell biology studies as well as IAV infection experiments. J.S.S. performed live-cell imaging studies of viruses and IFITM3. H.-H.H. performed other virus infection experiments. T.D. performed S-fatty-acylation experiments, TfR turnover and additional IFITM3 imaging experiments. E.T. performed protease activity studies. T.P. generated reagents for site-specific labeling and live-cell imaging of IFITM3. J.S.S., R.H., H.-H.H., E.T., T.D., C.M.R., T.P., K.C. and H.C.H. interpreted the data. C.M.R., K.C. and H.C.H. supervised the study. J.S.S. and H.C.H. wrote the manuscript with input from other co-authors.

Competing interests

The authors declare no competing interests.

Correspondence to Tao Peng or Kartik Chandran or Howard C. Hang.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2, Supplementary Figures 1–18

Reporting Summary

Supplementary Video 1

Example of DiD-IAV dequenching in a LAMP-GFP-expressing HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.

Supplementary Video 2

Example of DiD-IAV dequenching following colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.

Supplementary Video 3

Example of DiD-IAV dequenching prior to colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.

Supplementary Video 4

Example of LASV GPC-pseudotyped DiD-VSV dequenching following colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.

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Fig. 1: Expression and antiviral activity of IFITM1/2/3 KO cell lines.
Fig. 2: Analysis of IFITM3 in IFN-α stimulated A549 WT and IFITM2/3-KO cells.
Fig. 3: IAV-DID imaging in HeLa WT and IFITM2/3-KO cells.
Fig. 4: Trafficking of DiD-IAV and IFITM3 in IFITM2/3-KO cells.
Fig. 5: Analysis of the IFITM3-N21Δ loss-of-function mutant in HeLa IFITM2/3-KO cells.
Fig. 6: Site-specific S-palmitoylation regulates IFITM3 trafficking to IAV particles.