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LETTER https://doi.org/10.1038/s41586-019-0955-3 MHC class II proteins mediate cross-species entry of bat influenza viruses Umut Karakus1,17, Thiprampai Thamamongood2,3,4,5,17, Kevin Ciminski2,3, Wei Ran2,3, Sira C. Günther1, Marie O. Pohl1, Davide Eletto1, Csaba Jeney6, Donata Hoffmann7, Sven Reiche8, Jan Schinköthe8, Reiner Ulrich8, Julius Wiener9, Michael G. B. Hayes10, Max W. Chang10, Annika Hunziker1, Emilio Yángüez1, Teresa Aydillo11,12, Florian Krammer11, Josua Oderbolz13, Matthias Meier9, Annette Oxenius13, Anne Halenius2,3, Gert Zimmer14,15, Christopher Benner10, Benjamin G. Hale1, Adolfo García-Sastre11,12,16, Martin Beer7, Martin Schwemmle2,3,18* & Silke Stertz1,18* Zoonotic influenza A viruses of avian origin can cause severe disease To identify receptor candidates for bat IAV, we performed tran- in individuals, or even global pandemics, and thus pose a threat to scriptional profiling on three cell lines that are susceptible to bat IAV human populations. Waterfowl and shorebirds are believed to be (Madin–Darby canine kidney II (MDCKII) clone no. 1, and human the reservoir for all influenza A viruses, but this has recently been glioblastoma (U-87MG) and lung cancer (Calu-3) cell lines), and on challenged by the identification of novel influenza A viruses in three cells lines that are not susceptible (MDCKII clone no. 2, and bats1,2. The major bat influenza A virus envelope glycoprotein, human adenocarcinomic alveolar basal epithelial (A549) and human haemagglutinin, does not bind the canonical influenza A virus glioblastoma (U-118MG) cell lines). The susceptibility of each cell receptor, sialic acid or any other glycan1,3,4, despite its high sequence line was characterized by two different H18- or H18N11-pseudotyped and structural homology with conventional haemagglutinins. virus-like particle (VLP) assays10,11 (Extended Data Fig. 1a, b). This functionally uncharacterized plasticity of the bat influenza A Enrichment filtering for transcripts that encode membrane-integral virus haemagglutinin means the tropism and zoonotic potential of or membrane-associated proteins identified ten genes that were highly these viruses has not been fully determined. Here we show, using expressed in all H18-susceptible cells (Fig. 1a, Extended Data Fig. 1c–e, transcriptomic profiling of susceptible versus non-susceptible cells Supplementary Table 1). Five of these ten candidates encode MHC-II- in combination with genome-wide CRISPR–Cas9 screening, that the associated proteins—namely, the α- and β-chains of both HLA-DR and major histocompatibility complex class II (MHC-II) human leukocyte HLA-DQ, and the MHC-II-associated invariant chain (CD74), which antigen DR isotype (HLA-DR) is an essential entry determinant for is required for HLA processing and transport12,13. We tested the effect bat influenza A viruses. Genetic ablation of the HLA-DR α-chain of downregulating expression of the candidates by RNA interference on rendered cells resistant to infection by bat influenza A virus, whereas H18-mediated VLP entry. We also included HLA-DP as an additional ectopic expression of the HLA-DR complex in non-susceptible MHC-II receptor. As functional cell-surface HLA-DR, HLA-DQ and cells conferred susceptibility. Expression of MHC-II from different HLA-DP complexes require both α- and β-chains to be expressed, we bat species, pigs, mice or chickens also conferred susceptibility to downregulated the α-chain of each complex. Knockdown of CD74 or infection. Notably, the infection of mice with bat influenza A virus HLA-DRA (the α-chain of dimeric HLA-DR)—but not of other HLA resulted in robust virus replication in the upper respiratory tract, genes or any other candidate—markedly reduced entry mediated by whereas mice deficient for MHC-II were resistant. Collectively, our H18 (Fig. 1b). In addition, cell-surface levels of HLA-DR correlated with data identify MHC-II as a crucial entry mediator for bat influenza A susceptibility (Extended Data Fig. 1f). To obtain independent evidence viruses in multiple species, which permits a broad vertebrate tropism. for genes involved in entry mediated by H18, we performed a genome- Influenza A viruses (IAVs) bind to host cells via haemagglutinin, a wide CRISPR–Cas9 screen (Fig. 1c). We introduced single-guide RNAs trimeric virion glycoprotein. Haemagglutinins of all non-bat-derived (sgRNAs) into U-87MG cells that express Cas9, and infected these cells IAVs recognize terminal sialic acids on glycoproteins or glycolipids, with vesicular stomatitis virus (VSV) that expresses H18 with a polyba- with no known exception. Avian IAVs bind sialic acids that display sic cleavage site (VSV-H18) in place of VSV-G (Extended Data Fig. 2). an α2,3-linkage to the penultimate sugar, whereas human IAVs pref- sgRNA sequences from surviving cells revealed several candidates, erentially recognize the α2,6 linkage5–7. Receptor binding is followed including four transcription factors that are known to regulate MHC-II by endocytic uptake and pH-mediated fusion of viral and endosomal expression (RFXANK, RFX5, CIITA and RFXAP) and HLA-DRB1 (the membranes8. The discovery of two novel IAV genomes (designated β-chain of HLA-DR) (Fig. 1c, Supplementary Table 2). These data sug- H17N10 and H18N11) in Central and South American bat species gest that HLA-DR is an entry determinant of H18N11 (Fig. 1d). expanded the known host range of IAVs, and raised questions about To specify the role of HLA-DR in bat IAV entry, we generated two their zoonotic potential and fundamental biology1,2. Although these clones of U-87MG HLA-DRA knockout cells (U-87MG KO no. 1 and bat-derived IAVs resemble classical IAVs in many ways, their haemag- no. 2). Upon knockout, HLA-DR surface expression was reduced to glutinins (H17 and H18) are unable to bind sialic acids, and thus chal- background (Fig. 2a). Notably, the HLA-DRA knockout cells were resist- lenge previous characterizations of this binding as central to IAVs1,3,4. ant to infection with H18- or H17-pseudotyped VLPs, VSV-H18, and The bat IAV receptor has remained unknown but is speculated to be VSV-H17 (Fig. 2b–d, Extended Data Fig. 3a, b), which suggests that the proteinaceous9. requirement for HLA-DR is shared between both subtypes of bat IAV, 1Institute of Medical Virology, University of Zurich, Zurich, Switzerland. 2Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany. 3Faculty of Medicine, University of Freiburg, Freiburg, Germany. 4Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany. 5Faculty of Biology, University of Freiburg, Freiburg, Germany. 6Department of Microsystems Engineering – IMTEK, University of Freiburg, Freiburg, Germany. 7Institute of Diagnostic Virology, Friedrich-Loeffler Institut, Greifswald-Insel Riems, Germany. 8Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler Institut, Greifswald-Insel Riems, Germany. 9Helmholtz Pioneer Campus, Helmholtz Zentrum Munich, Neuherberg, Germany. 10Department of Medicine, University of California, San Diego, CA, USA. 11Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 12Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 13Institute of Microbiology, ETH Zurich, Zurich, Switzerland. 14Division of Virology, Institute of Virology and Immunology, Mittelhäusern, Switzerland. 15Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. 16Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 17These authors contributed equally: Umut Karakus, Thiprampai Thamamongood. 18These authors jointly supervised this work: Martin Schwemmle, Silke Stertz. *e-mail: [email protected]; [email protected] 7 MARCH 2019 | VOL 567 | NATURE | 109 RESEARCH LETTER a Calu-3 b effect. To probe the interaction between HLA-DR and H18, we used a siRNA: polykaryon assay and observed that H18-mediated polykaryon forma- HLA-DRA CD74 HLA-DRAHLA-DPHLA-DQAA ITGA7 B3GALNT1B3GNT5GPRC5BSORBS2 MDCKII no. 1 1,093 HLA-DRB1 No. 1 HLA-DQA1 tion strictly depended on HLA-DR expression (Fig. 2h, i). Furthermore, 42 13 HLA-DQB1 CD74 No. 2 H18-dependent—but not H1-mediated—polykaryon formation could 8 10 B3GALNT1 206 B3GNT5 No. 3 be blocked by an MHC-II-specific antibody (Fig. 2h, i, Extended Data ITGA7 Fig. 6a), which strongly suggests that H18 interacts with HLA-DR. The 485 GPRC5B No. 4 SORBS2 interaction between H18 and HLA-DR was confirmed by proximity U-87MG % ligation assay (Extended Data Fig. 6b, c) and emulsion coupling (an c ޒ10 70 >130 10–15 RFXANK approach for protein–protein interactions based on digital-droplet RFX5 d CIITA PCR) (Fig. 2j, Extended Data Fig. 6d, e). 10–11 HLA-DRB1 RFXAP CIITA LAMTOR4 We aimed to render non-susceptible human HEK293T cells suscep- 10–7 EIF3H CBFB RFXANK NFYA tible to bat IAV by ectopic expression of HLA-DR. HEK293T cells were RRA score RPS11 PRR21 NFYB transfected with plasmids that encode the α- and β-chains of HLA-DR –3 10 RFX5 RFXAP CREB NFYC HLA-DRB1 HLA-DRA (individually or in combination), or the transcriptional regulator CIITA. MHC-II gene locus 0 5,000 10,000 15,000 As expected, surface expression of HLA-DR was detected upon trans- Genes fection of both HLA-DRA and HLA-DRB1, and in CIITA-transfected Fig. 1 | Identification of HLA-DR as an entry determinant for bat IAV. cells (Fig. 3a). Only HEK293T cells that express

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