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Structural basis for influenza virus NS1 protein block of mRNA nuclear export

Abstract

Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1,2,3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1–nuclear transport factor 2-related export protein 1 (NXF1–NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1–NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1–NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1–NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

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Data availability

The coordinates of the NS1–NXF1–NXT1 structure have been deposited in the Protein Data Bank under accession number 6E5U. The data for the transcriptome analysis has been deposited in the Gene Expression Omnibus under accession number GSE129318. All other data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank Z. Wawrzak at Advanced Photon Source (APS) beamline 21-ID-F for assistance with X-ray data collection; P. Jeffrey for suggestions for data processing; and Y. M. Chook for reagents and assistance with protein purification. Y.R. and Y.X. are supported by funds from Vanderbilt University School of Medicine. Funding was provided by NIH R01 GM113874 to B.M.A.F.; R01 AI125524 to B.M.A.F. and A.G.-S. This work is also partially funded by CRIP (Center for Research on Influenza Pathogenesis), an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract no. HHSN272201400008C) and by NIAID grant U19AI135972.

Author information

B.M.A.F. and Y.R. conceived the study. K.Z., Y.X., R.M.-M., J.W., L.Z., M.E. and Y.R. performed the experiments. K.Z., Y.X., R.M.-M., J.W., L.Z., M.E., A.G.-S., B.M.A.F. and Y.R. analysed the data. K.Z., Y.X., B.M.A.F. and Y.R. wrote the manuscript with input from all authors.

Correspondence to Beatriz M. A. Fontoura or Yi Ren.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Tables 1 and 2, raw image figures and raw image Supplementary Figures.

Reporting Summary

Supplementary Table 3

Transcriptome profiling of A549 cells infected with influenza virus shows a subset of cellular mRNAs blocked in the nucleus.

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Fig. 1: Influenza A virus NS1 protein binds the nucleoporin-binding domain of NXF1 to block host mRNA nuclear export.
Fig. 2: Structure of a 2:2:2 complex of *NS1–NXF1(117–619)–NXT1.
Fig. 3: NS1 blocks binding of the FG nucleoporin Nup98 to NXF1–NXT1 in vitro and in influenza-infected cells.
Fig. 4: Influenza virus mutant on the NXF1-binding site of NS1 allows nuclear export of host mRNAs and is attenuated.