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Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells [1]

['Okikiola M. Olajide', 'Institute Of Virology', 'Medical Center', 'University Of Freiburg', 'Freiburg', 'Faculty Of Medicine', 'Spemann Graduate School Of Biology', 'Medicine', 'Faculty Of Biology', 'Maria Kaukab Osman']

Date: 2023-07

Abstract The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infection with the bat IAV H18N11. Yet, it has been difficult to biochemically determine the H18:MHC-II binding. Here, we followed a different approach and generated MHC-II chimeras from the human leukocyte antigen DR (HLA-DR), which supports H18-mediated entry, and the nonclassical MHC-II molecule HLA-DM, which does not. In this context, viral entry was supported only by a chimera containing the HLA-DR α1, α2, and β1 domains. Subsequent modeling of the H18:HLA-DR interaction identified the α2 domain as central for this interaction. Further mutational analyses revealed highly conserved amino acids within loop 4 (N149) and β-sheet 6 (V190) of the α2 domain as critical for virus entry. This suggests that conserved residues in the α1, α2, and β1 domains of MHC-II mediate H18-binding and virus propagation. The conservation of MHC-II amino acids, which are critical for H18N11 binding, may explain the broad species specificity of this virus.

Citation: Olajide OM, Osman MK, Robert J, Kessler S, Toews LK, Thamamongood T, et al. (2023) Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells. PLoS Biol 21(7): e3002182. https://doi.org/10.1371/journal.pbio.3002182 Academic Editor: Andrew Mehle, University of Wisconsin-Madison, UNITED STATES Received: November 24, 2022; Accepted: June 2, 2023; Published: July 6, 2023 Copyright: © 2023 Olajide et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the European Research Council (ERC) to M.S. (NUMBER 882631—Bat Flu) and in part by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School) and the Ministry for Science, Research and Arts of the State of Baden-Wuerttemberg. O.M.O, M.K.O and J.R. are members of the Spemann graduate school of biology and medicine (SGBM). A.G.W. is supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001078), the UK Medical Research Council (FC001078), and the Wellcome Trust (FC001078). P.R. and K.C. are supported by the Hans A. Krebs Medical Scientist Programme of the Medical Faculty of the University of Freiburg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; HEK293T, human embryonic kidney 293T; HLA-DR, human leukocyte antigen DR; IAV, influenza A virus; Ig, immunoglobulin; MHC-II, major histocompatibility complex class II

Introduction Zoonotic transmission of viruses represents a constant threat to global health. Bats play an important role as reservoir hosts for diverse, potentially deadly viral pathogens [1–4]. However, until recently, bats were not recognized as a reservoir for influenza A viruses (IAVs); rather, all IAV strains were believed to have originated from wild waterfowls [5]. This notion was challenged by the discovery of the genome sequences of 2 novel IAV strains, H17N10 and H18N11, in New World bats [6–8]. Although these bat IAVs essentially resemble conventional IAVs, their surface glycoproteins (H17/18 and N10/11) differ fundamentally in function from those of conventional IAVs despite structural similarity. While the hemagglutinins of conventional IAV (H1 to H16) mediate attachment and entry via binding to sialic acid residues, both H17 and H18 are unable to bind sialylated glycans [8–10] and instead utilize major histocompatibility complex class II (MHC-II) molecules for cell entry [11]. MHC-II molecules are heterodimeric transmembrane proteins essential for adaptive immune responses as they present antigenic peptides of extracellularly derived proteins on the surface of professional antigen presenting cells to CD4+ T cells [12–14]. They consist of alpha (α)- and beta (β)-chains made of membrane-proximal barrel-shaped, immunoglobulin (Ig)-like α2 and β2 domains and juxtaposed membrane-distal domains (α1 and β1), which contribute almost equally to the formation of the peptide-binding groove [13,15,16]. MHC-II molecules fold in the endoplasmic reticulum, from which they are transported with the help of the invariant chain to late endosomal compartments, where the invariant chain is degraded and MHC-II loaded with peptides [13,14]. This peptide loading is facilitated by a chaperone, the nonclassical MHC-II molecule DM (in human HLA-DM) [14,17]. HLA-DM resides in the late endosomal compartments and shares high structural similarity with classical MHC-II molecules but does not have a functional peptide-binding groove [14,18]. After binding a peptide, MHC-II molecules are trafficked to the plasma membrane [19]. The H18 binding site on MHC-II is unknown. However, classical MHC-II molecules from all vertebrate species tested to date enable H18-mediated infection [11], suggesting that highly conserved MHC-II residues facilitate viral entry. Here, we sought to define these residues through a mutational approach in which we generated chimeric MHC-II molecules from permissive classical HLA-DR and nonpermissive nonclassical HLA-DM molecules. We identified conserved residues within the α2 domain of HLA-DR as key for H18N11 infection.

Discussion We elucidated the central role of the α2 domain of the MHC-II molecule in H18-mediated cell entry by generating sets of chimeras between the classical human MHC-II, HLA-DR, and the nonclassical MHC-II molecule, HLA-DM. Within the α2 domain, we identified the highly conserved amino acid residues N149 and V190 as critical for both H18- and H17-mediated infection. Single substitutions of these amino acids in HLA-DR prevented viral entry but did not affect the structural integrity of the HLA-DR molecules as these mutants were still able to accommodate antigenic peptides and activate T cells. The fact that these HLA-DR mutants were able to activate T cells demonstrates their overall structural integrity and clustering ability. The latter, which is critical for T cell activation, may also provide the avidity required for virus attachment and entry [19,26,27]. The α2 and β2 domains of MHC-II molecules are C1-set Ig-like domains [28], which makes them well suited to act as platforms for interactions with other proteins [29]. Indeed, the α2 domain, which we found to be crucial for H18N11 entry, was previously shown to bind TIRC7, a negative regulator of T cell activity [30]. Furthermore, the HLA-DR:CD4 and HLA-DR:HLA-DM interactions consist of large multidomain interfaces in which the α2 domain is a major contributor [31–33]. Our in silico structural modeling suggests that the interaction with H18 occurs through an interface involving the α2 domain and parts of the α1 and β1 domains in HLA-DR. Despite the involvement of multiple domains, we hypothesize that the H18:HLA-DR interaction is of relatively low affinity as classical biochemical approaches have yet failed to detect direct binding [11]. However, according to our predicted model, 1 H18 homotrimer can bind to 3 MHC-II complexes (S4F and S4G Fig), which would allow clustering of MHC-II at the viral entry site and provide the avidity required for host cell binding and subsequent uptake. Similar clustering of entry factors is also required for the uptake of classical IAV due to the low affinity of individual HA–sialic acid interactions [34,35]. On a genetic level, the MHC-II α- and β-chains are markedly conserved among all mammalian species with the obvious exception of the polymorphic residues that mainly cluster in the β1 domain [16]. Usage of such a conserved receptor allows bat IAV to infect a wide range of New World bat species, including the phylogenetically only distantly related Neotropical fruit bats, Artibeus spp. (family Phyllostomidae), and Velvety free-tailed bat, Molossus molossus (family Molossidae) [8]. However, despite the ability to utilize MHC-II of diverse mammalian species and the wide geographical distribution of seropositive bats across Central and South America, there is as of yet no evidence for natural infection of non-bat species with bat IAVs. This might suggest that there are additional molecular and/or ecological hurdles, which so far have prevented a spill over to other mammals including humans.

Material and methods Cell lines HEK293T cells were obtained from the American Type Culture Collection (ATCC; CRL-3216). Baby Hamster Kidney Fibroblasts (BHK-21) cells were obtained from the German Cell Culture Collection (DSZM). MDCKII cells stably overexpressing human MHC-II (MDCK-MHC-II) were generated previously [11] and selected using 2.5 μg per ml puromycin and 300 μg per ml hygromycin. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Fischer Scientific) containing 10% fetal calf serum (FCS), 100 U per ml penicillin, and 100 mg per ml streptomycin at 37°C with 5% CO 2 . Viruses GFP-encoding vesicular stomatitis virus whose glycoprotein was replaced by H18 with a polybasic cleavage site (VSV-H18) was generated as previously described [20]. Cell culture-adapted H18N11 (rP11) was produced in MDCKII cells stably expressing HLA-DR as previously described [24]. MHC-II expression plasmids cDNA sequences encoding V5- or HA-tagged wild-type, and chimeric MHC-II α- and β-chains were synthesized (Genewiz) and cloned into the pCAGGS vector via NotI and XhoI restriction enzymes (S1A Fig). Reference encoding sequences used include: HLA-DRA (NM_019111.4), HLA-DRB1 (NM_001243965.1), HLA-DRB1*0101 (HM067843.1), HLA-DMA (NM_006120.4), HLA-DMB (NM_002118.5), Aj-DRA (XM_037146055.1), and Aj-DRB (XM_037162922.1). Mutations were introduced by PCR using overlapping primers (Sigma Aldrich). Truncated MHC-II β-chain was generated by introduction of the amber stop codon at respective amino acid positions. Amino acid differences in the signal peptide of V5- and HA-tagged wild-type HLA-DR are due to codon optimization. Plasmids encoding HLA-DRB1*01:01 with CLIP 87-101 (PVSKMRMATPLLMQA) or HA 307-319 (PKYVKQNTLKLAT) were generated by PCR using overlapping primers (Sigma Aldrich) and cloned into the pCAGGS vector via NotI and XhoI restriction enzymes. MHC-II surface expression HEK293T cells seeded to approximately 70% confluency in 24-well plates were transfected with 250 ng each of the respective MHC-II α- and β-chain using Lipofectamine 2000 (Thermo Fisher, Germany). The next day, cells were detached and washed by pipetting up and down gently with FACS buffer (PBS supplemented with 2% FCS). After centrifugation at 1,200 rpm for 5 min at 4°C, cells were stained primarily with anti-V5 rabbit antibody (Abcam, catalog no. ab9116, 1:500) and anti-HA mouse antibody (Sigma Aldrich, catalog no. H3663, 1:500) for 30 min on ice. Following another washing and centrifugation step, cells were secondarily stained with BV421 goat anti-rabbit antibody (BD Biosciences, catalog no. 565014, 1:200) and APC goat anti-mouse antibody (BD Biosciences, catalog no. 550826, 1:200) for 30 min on ice. Zombie NIR Fixable Viability Kit (BioLegend, catalog no. 423105, 1:1000) was used to assess live versus dead status of cells. After a final wash and centrifugation step, cells were resuspended in FACS buffer, transferred to a FACS tube, and surface expression of MHC-II heterodimer was analyzed with a BD FACS Canto II (BD Biosciences) flow cytometer. Virus infections HEK293T cells seeded to approximately 70% confluency in 24-well plates were transfected with 250 ng each of the respective MHC-II α- and β-chain using Lipofectamine 2000 (Thermo Fisher, Germany). For VSV-H18 infection, cells were infected 24 h posttransfection at an MOI of 0.05 in infection medium [11]. At 24 h postinfection, cells were detached, washed, centrifuged, and stained as described for MHC-II surface expression. After staining and washing, cells were fixed in 2% PFA in PBS for 20 min on ice, washed, and centrifuged at 1,500 rpm for 10 min at 4°C. After a final wash and centrifugation step, cells were resuspended in FACS buffer, transferred to a FACS tube, and MHCII surface expression as well as the frequency of infected GFP-positive cells were analyzed with a BD FACS Canto II (BD Biosciences) or BD LSRFortessa (BD Biosciences) flow cytometer. For H18N11 infection, cells were infected 24 h posttransfection at an MOI of 5 in infection medium supplemented with 0.2 μg/ml TPCK trypsin. At 24 h postinfection, cells were resuspended in infection medium, washed, and centrifuged as described for MHC-II surface expression. Subsequently, cells were primarily stained with a rabbit polyclonal anti-H18 serum (1:100) [11] for 30 min on ice. After washing in FACS buffer and centrifugation at 1,200 rpm for 5 min at 4°C, cells were stained with BV421 goat anti-rabbit antibody (BD Biosciences, catalog no. 565014, 1:200) and Alexa Fluor 488–conjugated HA tag monoclonal antibody (Thermo Fisher, catalog no. A-21287, 1:200) for 30 min on ice. After washing and centrifugation, cells were fixed in 2% PFA in PBS for 20 min on ice, washed, and centrifuged at 1,500 rpm for 10 min at 4°C. After a final wash step, cells were resuspended in FACS buffer, transferred to a FACS tube, and MHC-II surface expression as well as the frequency of H18N11-infected Alexa Fluor 488–positive cells were analyzed with BD LSRFortessa (BD Biosciences) flow cytometer. Fluorescent images of GFP-positive VSV-H18-infected cells were acquired on a Zeiss Observer.ZI inverted epifluorescence microscope (Carl Zeiss) equipped with an AxioCamMR3 camera using a 10× objective. Molecular docking The HDOCK server (http://hdock.phys.hust.edu.cn/) [36–38] was used to computationally construct the three-dimensional (3D) complex model of H18:MHC-II using crystal structures of MHC-II molecules and H18 HA deposited in the PDB and its default hybrid docking protocol. Focusing on the HA1 region that binds the sialic acid receptor of conventional IAVs [39,40] and acquired amino acid mutations that increase replication competence of H18N11 [24], the PDB entry 4K3X [8], (chains A, C, E) of H18 were designated as the “interactor” and MHCII (PDB ID 1DLH, chains A, B) [41] as the “ligand.” After a global sampling of putative binding orientations at 15° rotational intervals, HDOCK provided docking results, including 10 top models based on its scoring function. Among these top 10 docking models, the top 2 models with the lowest docking score (below −200; the most possible binding model) and confidence score greater than 0.7 (very likely to bind), in addition to the criteria of using the HA head domain, not engaging the HLA-DR peptide binding groove, and being in upright orientation, were selected. Pymol was used for 3D structure visualization of H18:MHC-II complex model and PISA was used to calculate buried surface areas. T cell activation Baby Hamster Kidney Fibroblasts (BHK-21) (8.4 × 105 cells per well in 6-well format) were transfected with 2 pCAGGS expression vectors (500 ng each) encoding the HLA-DRB1*01:01 β-chain and the respective HLA-DRA1 α-chain using Lipofectamine 2000 (Thermo Fisher, Germany). The next day, 5 × 104 transfected cells were transferred into a well of a 96-well plate and cultured over night at 37°C in DMEM (Gibco, USA) containing 1% FCS. For exogenous peptide loading, 50 μM HA 307-319 peptide (PKYVKQNTLKLAT; GenScript, USA) or EBV EBNA1 515-527 peptide (TSLYNLRRGTALA; GENAXXON bioscience, Germany) were added to the culture medium. Medium was removed and cells were cocultured with CH7C17 Jurkat T cells (105 cells per well in 96-well format) in RPMI 1640 (Gibco, USA) supplemented with 10% FCS and 5% HEPES for 6 h at 37°C. Subsequently, cells were stained with FITC-labeled anti-CD3 antibody (BioLegend, USA, 1:200) and APC-labeled anti-CD69 antibody (Life Technologies, USA, 1:200) and analyzed with a BD FACSCanto II (BD Biosciences) flow cytometer. Polykaryon formation assay Subconfluent HEK293T cells were cultured in 6-well plates and cotransfected with 2 μg of pCAGGS-GFP and either pCAGGS-EV (empty vector) or pCAGGS-H18. Similarly, subconfluent BHK-21 cells were cotransfected with 2 μg of pCAGGS-HLA-DRB1 and either pCAGGS-HLA-DRA or pCAGGS-HLA-DRA N149H+G150S or pCAGGS-HLA-DRA V190A . At 24 h posttransfection, cells were detached by trypsin treatment and 2 × 105 HEK293T and 2 × 105 BHK21 cells, respectively, were seeded in collagen coated 24-well plates containing growth medium (DMEM, 10% FCS, 100 U per ml penicillin, and 100 mg per ml streptomycin) and incubated at 37°C and 5% CO 2 . The following day, cells were treated with TPCK trypsin (10 μg/ml in Opti-MEM) for 30 min at 37°C. Cells were subsequently washed with PBS, exposed to pH 5 PBS for 20 min at 37°C and 5% CO 2 , and then incubated in growth medium for 2 h at 37°C and 5% CO 2 . Finally, cells were washed with PBS, fixed using 4% paraformaldehyde in PBS for 20 min, and nuclei were stained for 1 h using 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired using a Zeiss Observer.ZI inverted epifluorescence microscope (Carl Zeiss) equipped with an AxioCamMR3 camera using a 20× objective. Conservation analysis To determine the cross-species conservation of amino acids within the α- and β-chain of HLA-DR, we performed a ConSurf-DB analysis [21,22] for 1DLH chain A and B (PDB DOI: 10.2210/pdb1DLH/pdb) [41]. The calculation was conducted on 300 hits out of 1,872 (α-chain) and 5,558 (β-chain) homologs, which were CT-HIT unique at 95% threshold. Where necessary, resulting conservation scores were plotted for our regions of interest using GraphPad Prism.

Acknowledgments We thank Anne Halenius for her contribution to the in silico design of MHC-II chimeras and Wolfgang Schamel for providing the CH7C17 Jurkat T cells.

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