MHC class II proteins mediate cross-species entry of bat influenza viruses

Abstract

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR–Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

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Fig. 1: Identification of HLA-DR as an entry determinant for bat IAV.
Fig. 2: HLA-DR mediates the entry of bat IAV into U-87MG cells.
Fig. 3: Homologues of HLA-DR from different species confer susceptibility to bat IAV.
Fig. 4: MHC-II knockout mice are resistant to bat IAV infection.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. The associated raw data for Fig. 1a are provided in Supplementary Table 1, and raw data for Fig. 1c in Supplementary Table 2. Any further relevant data are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank C. Kastenholz, K. Flämig, J. Brandel, S. Schuparis, S. Sander and G. Czerwinski for assistance; A. Dudek, P. Staeheli, D. Schnepf and A. Trkola for discussions; and R. Zengerle, D. Szabó, P. Koltay, A. Karsai and S. Zimmermann for their support. This work was supported by grants from the Swiss National Science Foundation to S.S. (310030E-164065) and B.G.H. (31003A_182464), a grant from the German Research Foundation to M.S. (SCHW 632/17-1) and M.B. (BE 5187/4-1) and the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School) to T.T. This work was also partly supported by CRIP (Center for Research on Influenza Pathogenesis) and NIAID funded Center of Excellence in Influenza Research and Surveillance (CEIRS) to A.G.-S. (HHSN272201400008C). M.W.C. and C.B. were supported by NIAID grant U19AI135972.

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Nature thanks Michael Farzan, David Steinhauer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors

Contributions

K.C. and W.R. contributed equally to this work. U.K., T.T., K.C., W.R., S.C.G., M.O.P., D.E., C.J., D.H., S.R., J.S., R.U., J.W., M.G.B.H., M.W.C., A. Hunziker, E.Y. and A. Halenius performed experiments; U.K., T.T., K.C., M.S. and S.S. designed experiments; T.A., F.K., J.O., A.O. and G.Z. contributed reagents and mice; M.M., C.B., B.G.H., A.G.-S., M.B., M.S. and S.S. supervised work and acquired funding; U.K., M.S. and S.S. wrote the manuscript.

Corresponding authors

Correspondence to Martin Schwemmle or Silke Stertz.

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Competing interests

C.J. is the patentee of the patent covering the technology of emulsion coupling (WO2016083793) and a shareholder in Actome GmbH (Germany), which holds the patent.

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Extended data figures and tables

Extended Data Fig. 1 Analysis of transcriptional profiles from H18N11-susceptible and non-susceptible cell lines.

a, MDCKII clone no. 1 (here labelled MDCKII #1), MDCKII clone no. 2 (here labelled MDCKII #2), U-87MG, U-118MG, Calu-3 and A549 cells were infected for 90 min at 37 °C with luciferase-encoding VLPs pseudotyped with H18N11 or H1N1, or control VLPs that lacked any surface glycoprotein (empty vector). At 48 h p.i., luciferase signals were measured and normalized to control samples. Fold increase in luciferase signals relative to samples infected with empty vector (EV) is plotted. b, MDCKII clone no. 1 (here labelled MDCKII #1), MDCKII clone no. 2 (here labelled MDCKII #2), U-87MG, U-118MG and Calu-3 cells were infected with BlaM1 VLPs pseudotyped with H1N1 or H18, for 4 h at 37 °C. Entry-positive cells were measured with the fluorogenic β-lactamase substrate CCF2-AM, and quantified by flow cytometry. Relative numbers of entry-positive cells are shown. **, signals from A549 were below the detection limit, owing to inefficient loading with the fluorogenic β-lactamase substrate. a, b, Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes. ce, Transcriptional profiles of the indicated H18N11-susceptible versus non-susceptible cell lines are shown as volcano plots. The two-group comparison—each of which consists of n = 4 independent samples—was performed using the gene-wise negative binomial generalized linear model with quasi-likelihood tests, implemented in EdgeR. Adjustments for multiple comparisons were made by applying the Benjamini–Hochberg method. Significantly (P ≤ 0.01) upregulated (fold change (expressed as log2(transcript level susceptible cell line/non-susceptible cell line) ≥ 1) and downregulated (fold change (expressed as log2(transcript level susceptible cell line/non-susceptible cell line)≤ −1) transcripts are shown in red and blue, respectively. Transcripts expressed at similar levels (−1 ≤ fold change (expressed as log2(transcript level susceptible cell line/non-susceptible cell line) ≤ 1) are shown in grey. Black dots indicate significantly upregulated transcripts, filtered according to the Gene Ontology annotations GO:0016020 (membrane), GO:0016021 (integral component of membrane) and GO:0005886 (plasma membrane), that overlap in the H18N11-susceptible cell lines. f, HLA-DR surface staining of U-87MG, U-118MG, Calu-3 and A549 cells, and DLA surface staining of MDCKII clone no. 1 (here labelled MDCKII #1) and MDCKII clone no. 2 (here labelled MDCKII #2) cells, is shown in contour plots. Representative plots of n = 3 independent experiments are shown.

Extended Data Fig. 2 Genome-wide CRISPR–Cas9 screen on U-87MG cells to identify potential receptor candidates for bat influenza virus.

a, Schematic of the genome-wide CRISPR–Cas9 screen. U-87MG cells stably expressing Cas9 (U-87MG–Cas9) were transduced with lentiviruses encoding a human CRISPR-knockout pooled sgRNA library at an MOI of 0.3. Two days after transduction, cells (U-87MG–Cas9–sgRNA) were selected with puromycin (1 μg/ml) for ten days, and subsequently infected with VSV-H18 at an MOI of 10. Survivors were re-infected with VSV-H18 at an MOI of 10 for 4 additional rounds, and collected on day 27 p.i. Genomic DNA from cell pellets was isolated and analysed using next-generation sequencing. b, Susceptibility of the survivors and the wild-type U-87MG cells was monitored by flow cytometry measuring GFP signals from cells infected with H18-VSV reporter virus at different MOIs. Relative numbers of GFP-positive cells are shown.

Extended Data Fig. 3 HLA-DR is required for bat IAV infection in U-87MG and Calu-3 cells.

a) U-87MG control, U-87MG KO no. 1 (here labelled KO #1) and U-87MG KO no. 2 (here labelled KO #2) cells were infected with recombinant VSV-H18 (red), VSV (light grey) and H7N7 (dark grey) reporter viruses expressing GFP at an MOI of 1, 0.1 and 0.1, respectively, for at least 24 h. The frequency of GFP-positive cells was quantified by flow cytometry. b, U-87MG control, U-87MG KO no. 1 and U-87MG KO no. 2 (labelled ‘KO #1’ and ‘KO #2’) were infected with recombinant VSV-H17 expressing GFP at an MOI of 1 for 72 h. Representative images of n = 3 independent experiments are shown. Scale bar, 100 μm. c, HLA-DR surface staining of U-87MG control, U-87MG KO no. 1 and U-87MG KO no. 2 (labelled ‘KO #1’ and ‘KO #2’), stably transduced with either a control lentivirus (labelled ‘LV-ctrl’) or a lentivirus encoding HLA-DRA (labelled ‘LV-HLA-DRA’) from a representative of n = 3 independent experiments is shown. d, e, Indicated U-87MG cell lines were infected with VSV-H18 reporter virus expressing GFP at an MOI of 5 for 48 h. Representative images are shown in d. Frequency of GFP-positive cells was quantified by flow cytometry and is depicted in e. a, e, Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes. f, HLA-DR surface staining of Calu-3 HLA-DRA knockout cells (labelled ‘KO #1’ and ‘KO #2’) and Calu-3 cells (labelled ‘Ctrl’) is shown from a representative of n = 2 independent experiments. g, Calu-3 control, Calu-3 KO no. 1 and Calu-3 KO no. 2 cells (labelled ‘KO #1’ and ‘KO #2’) were infected with BlaM1 VLPs pseudotyped with H1N1 or H18 for 4 h at 37 °C. Entry-positive cells were measured with the fluorogenic β-lactamase substrate CCF2-AM, and quantified by flow cytometry. Relative numbers of entry-positive cells are shown. Data are mean from n = 2 independent experiments. Values below background levels of 1 are displayed on the x axis.

Extended Data Fig. 4 Alignment of protein sequences of haemagglutinins from different subtypes of IAV.

Residues conserved across all subtypes are highlighted in red. Residues known to be part of the receptor-binding site in conventional IAV are highlighted by light purple boxes, and the fusion peptide is marked by a yellow box.

Extended Data Fig. 5 Ectopic expression of HLA-DR renders non-susceptible cell lines susceptible.

a, HLA-DR surface staining of A549 LV control (LV-ctrl) and A549 LV HLA-DR (LV-HLA-DR) (left), MDCKII clone no. 2 LV control and MDCKII clone no. 2 LV HLA-DR (middle), U-118MG LV control and U-118MG LV HLA-DR (right) from a representative of one experiment is shown. b, Indicated cell lines were infected with luciferase-encoding VLPs pseudotyped with H18 or H1N1, or control VLPs that lack any surface glycoprotein (empty vector). Fold increase in luciferase signals relative to samples infected with empty vector is plotted. c, Indicated cell lines were infected with VSV-H18 at an MOI of 10 and GFP-positive cells were quantified by flow cytometry at 24 h p.i. for A549 LV control and A549 LV HLA-DR (left), at 24 h p.i. for MDCKII clone no. 2 LV control and MDCKII clone no. 2 LV HLA-DR (middle), and at 72 h p.i. for U-118MG LV control and U-118MG LV HLA-DR (right). b, c, Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes. d, MDCKII clone no. 2 LV control and MDCKII clone no. 2 LV HLA-DR were infected with IAV H18N11 at MOI = 0.001. At indicated time points p.i., supernatants were collected and viral titres were determined by immunofluorescence on subconfluent MDCKII clone no. 1 cells, using rabbit polyclonal anti-H18. Data are mean ± s.d. from n = 3 independent experiments.

Extended Data Fig. 6 Polykaryon formation assay and PLAs detect interaction of H18 with HLA-DR complexes.

a, HEK293T cells were co-transfected with plasmids encoding mCherry and H1. Transfected HEK293T cells were treated with TPCK–trypsin to cleave haemagglutinin and seeded on top of control MDCKII clone no. 2 cells (labelled as LV-ctrl) or MDCKII clone no. 2 cells that stably express HLA-DR (labelled as LV-HLA-DR), in the presence or absence of the indicated antibodies. Polykaryon formation was examined after exposure of cells to low pH. Representative images of n = 3 independent experiments are shown. Cell nuclei were stained by DAPI (blue). Scale bar, 25 μm. b, Visualization of H18 and HLA-DR complexes by PLA. MDCKII clone no. 2 cells (LV-ctrl) or MDCKII clone no. 2 cells that express HLA-DR (LV-HLA-DR) were infected with VSV-H18 at an MOI of 100 at 37 °C. After 1 h, cells were fixed and subjected to PLA using mouse anti-H18 and rabbit anti-MHC-II antibodies. Specific PLA dots obtained after amplification of the PLA fluorescence probe are indicated by red dots. Cell nuclei were stained by DAPI (blue colour). Representative images of three independent experiments are shown. Scale bars, 20 μm. c, Scatter plot of the PLA dot count per cell obtained from PLA images taken under the conditions given in c is shown from a representative of three independent experiments. The cell number, counted at 37 °C, for LV-ctrl cells infected with VSV-H18, LV-HLA-DR mock-infected cells or LV-HLA-DR cells infected with VSV-H18 was 9,254, 10,747 and 14,966, respectively. Statistical significance was assessed by Mann–Whitney U test (two-sided). ***P ≤ 2.2 × 10−16. d, MDCKII clone no. 2 cells (LV-ctrl) or MDCKII clone no. 2 cells that express HLA-DR (LV-HLA-DR–HA-tag) were infected with VSV-H18 at an MOI of 10. GFP-positive cells were quantified by flow cytometry at 24 h p.i. Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes. e, Cartoon highlighting the principal steps of the emulsion coupling assay. Emulsion coupling is a digital assay concept based on the detection of double-labelled, individual molecular complexes in emulsion, which are identified by ddPCR. In a first step, MDCKII clone no. 2 cells that express HLA-DR that contains a haemagglutinin tag (HLA-DR–HA tag) were incubated with VSV-H18 at an MOI of 100 for 15 min at room temperature (A). To stabilize and prevent the dissociation of complexes in the following steps, the interaction was fixed with a membrane-impermeable cross-linker. Cells were lysed and the cell lysate was incubated with single-stranded-DNA-labelled antibodies (B) that specifically recognize HLA-DR–HA or H18, to achieve equilibrium binding (C). Before emulsification, the reaction was highly diluted (~100,000 times) to achieve single-complex separation. ddPCR was carried out using the standard ddPCR protocol, and the reactions were measured using a QX200 Droplet Digital PCR instrument (D). The evaluation of the reaction was based on the partitioning of the labels in the ddPCR reactions using fluorescently tagged PCR products (FAM- or VIC-labelled), and indicated as RFU (see scatter plot). According to the ddPCR standard evaluation, the number of labelled antibodies was determined in each reaction (counting all label-positive droplets for a given label, and using the same definition of clusters for all reactions). Without complexes (interacting proteins with two antibodies bound), the partitioning of the labelled antibodies follows a Poisson distribution, and results in a calculable number of double-coloured droplets. In the case of complexes present in the reaction, the number of the detected double-coloured droplets is larger than would be expected by Poisson distribution. We developed a Python code to calculate the number of complexes, which explains this difference on the basis of Poisson-based modelling of the partitioning of the molecular species of the reaction; this results in the absolute counts for the detected complexes.

Extended Data Fig. 7 HLA-DR homologues from different species confer susceptibility to bat IAV.

a, HEK293T cells were transfected with plasmids encoding CIITA, HLA-DRA or HLA-DRB1, and co-transfected with HLA-DRA and HLA-DRB1 or empty vector. At 48 h after transfection, cells were infected with VSV-H18 at an MOI of 10. Fluorescent microscopy images were taken at 72 h p.i. Representative images of n = 3 independent experiments are shown. Scale bar, 100 μm. b, c, HEK293T cells were co-transfected with plasmids encoding the α- and β-chains of HLA-DR homologues from different species: S. scrofa (SLA-DR), G. gallus (B-L), E. fuscus (E. fuscus DR), M. lucifugus (M. lucifugus DR) and P. alecto (P. alecto DR). HLA-DR was included as a positive control; cells transfected with empty vector served as a negative control. At 48 h after transfection, cells were infected with GFP encoding VSV-H18 (b) or VSV-H17 (c) at an MOI of 1. At 48 h (b) or 72 h (c) p.i., the frequency of GFP-positive cells was quantified by flow cytometry. d, CIITA from P. alecto (PaCIITA) was stably expressed by lentiviral transduction in kidney-derived cell lines from bat species S. lilium and P. alecto. Cells were infected with VSV-H18 at an MOI of 10 for at least 24 h. The frequency of GFP-positive cells was quantified by flow cytometry. bd, Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes.

Extended Data Fig. 8 Alignment of protein sequences of surface MHC-II α-chains from different species.

Sequences included are the α-chains of MHC-II from Homo sapiens (HLA-DRA, HLA-DPA1 and HLA-DQA1), S. scrofa (SLA-DRA), G. gallus (B-LA), E. fuscus (EfDRA), M. lucifugus (MlDRA) and P. alecto (PaDRA). Residues conserved across all species are highlighted in red.

Extended Data Fig. 9 Alignment of protein sequences of surface MHC-II β-chains from different species.

Sequences included are the β-chains of MHC-II from H. sapiens (HLA-DRB1, HLA-DPB1 and HLA-DQB1), S. scrofa (SLA-DRB1), G. gallus (B-LB2), E. fuscus (EfDRB5), M. lucifugus (MlDRB5) and P. alecto (PaDRB). Residues conserved across all species are highlighted in red.

Extended Data Fig. 10 MHC-II knockout mice are resistant to bat IAV infection.

a, HEK293T cells were co-transfected with plasmids encoding the α- and β-chains of mouse MHC-II H-2A or H-2E or transfected with control plasmid. At 48 h after transfection, cells were infected with BlaM1 VLPs pseudotyped with H1N1 or H18 for 4 h at 37 °C. Entry-positive cells were measured with the fluorogenic β-lactamase substrate CCF2-AM and quantified by flow cytometry. b, HEK293T cells were co-transfected with plasmids encoding the α- and β-chains of mouse MHC-II H-2A or H-2E, or transfected with control plasmid. At 48 h p.i., cells were infected with VSV-H18 expressing GFP at an MOI of 10 for 48 h. Representative images are shown on the left, and a quantification of GFP-positive cells by flow cytometry is depicted on the right. a, b, Data are mean ± s.d. from n = 3 independent experiments. Values below background levels of 1 are displayed on the x axes. c, Detection of viral antigen in paraffin-embedded tissue from B6 mice intranasally infected with H18N11 (n = 4, 1 × 105 ffu in 40 μl, panels A and B) or H3N2 (n = 3, 1 × 103 pfu in 20 μl, panels C and D) or mock-infected (n = 2, panels E and F) at 4 days p.i. IAV matrix protein M1 immunoreactivity was observed in an oligofocal and laminar pattern in the apical ciliary border of the epithelium (arrow), and in epithelial cells (arrowhead), which displayed equivocal faint cytoplasmic and nuclear immunoreactivity (panel A). The inset (B) provides an overview of the epithelium (arrow) highlighted in panel A, and shows unspecific immunoreactivity in the lumen of a blood vessel in the lamina propria (arrowhead). Panel C shows intense matrix protein immunoreactivity in a multifocal-to-coalescing cytoplasmic pattern, occasionally with an apically increased intensity (arrow). Some nuclei within the immunoreactive cells are also labelled (arrowhead). The inset (D) provides an overview of the localization of the immunoreactive respiratory epithelium (arrow) on the cranial nasal septum in the area of the transition from transitional (left side) to respiratory (right side) epithelium. In mock-infected animals (panel E), respiratory epithelium never displayed immunoreactivity. Only faint unspecific immunoreactivity was observed in the lumen of a blood vessel in the lamina propria (arrow) in panel E. The inset (F) provides an overview of the epithelium highlighted in panel E. Scale bars, 10 μm (panels A, C, E), 50 μm (panels B, D, F). d, Detection of viral antigen and viral RNA in paraffin-embedded tissue of B6 mice intranasally infected with H3N2 (n = 3) for four days as described in c. A moderate amount of oligo- to multifocally distributed IAV matrix protein (panel A) but no H18-haemagglutinin-specific RNA (panel B) was detectable in the respiratory epithelium in following sections of the nasal cavities by using immunohistochemistry and in situ hybridization, respectively.

Supplementary information

Reporting Summary

Supplementary Table 1

Differentially expressed genes in bat IAV susceptible versus non-susceptible cell lines. Transcriptional profiles of MDCKII #1, Calu-3 and U-87MG cells susceptible to H18-pseudotyped VLP infection were compared to those of non-susceptible MDCKII #2, A549 and U-118MG cells, respectively. The two-groups comparison, each group consisting of n = 4 independent samples, was performed using the genewise negative binomial generalized linear model with quasi-likelihood tests (glmQLfit) implemented in EdgeR. Adjustments for multiple comparison were made by applying the Benjamini-Hochberg method. The indicated numbers represent genes in the H18N11-susceptible cell lines significantly (P ≤ 0.01) upregulated (log2 (fold change) ≥1) and filtered according to gene ontology annotations GO:0016020 (membrane), GO:0016021 (integral component of membrane) and GO:0005886 (plasma membrane). The 10 overlapping genes from MDCK #1, Calu-3 and U-87MG cells are listed.

Supplementary Table 2

Results of CRISPR screen. Cas9 expressing U-87MG cells transduced with a lentiviral library encoding genome-wide pools of single-guide RNAs (sgRNA) were infected with VSV-H18. sgRNA sequences from surviving cells were sequenced. Data analysis was performed by MAGeCK to identify enriched sgRNAs and genes were rank-ordered by robust rank aggregation (RRA) scores. Tab 1 contains positive selection summaries at the gene level. Tab 2 contains the sgRNA score summary. Tab 3 contains raw sgRNA counts. Tab 4 contains normalized sgRNA counts. The statistical significance of selection at the gene level was assessed using modified robust ranking aggregation (RRA), a permutation test implemented as part of the MAGeCK software suite. Corrections for multiple comparisons were made using the Benjamini-Hochberg method. The screen was performed once (n = 1).

Supplementary Table 3

siRNA sequences used to screen potential receptor candidates.

Supplementary Table 4

PCR primers used for Illumina sequencing.

Supplementary Table 5

Oligos used in Emulsion Coupling.

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Karakus, U., Thamamongood, T., Ciminski, K. et al. MHC class II proteins mediate cross-species entry of bat influenza viruses. Nature 567, 109–112 (2019). https://doi.org/10.1038/s41586-019-0955-3

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