Abstract

Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease1. The host factors required for alphavirus entry remain poorly characterized2. Here we use a genome-wide CRISPR–Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O’nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8–Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8–Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O’nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.

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Acknowledgements

This study was supported by NIH grants R01AI114816, R01AI123348, and R01AI095436, contract HHSN272201400058C, and the Defense Reduction Threat Agency HDTRA1-15-1-0013. We thank M. Heise, T. Morrison, M. Farzan, J. Miner and T. Dermody for discussions, reagents and comments on the manuscript.

Reviewer information

Nature thanks S. Mahalingam, C. Richardson and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA

    • Rong Zhang
    • , Arthur S. Kim
    • , Julie M. Fox
    • , Sharmila Nair
    • , Hueylie Lin
    • , Subhajit Poddar
    •  & Michael S. Diamond
  2. Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO, USA

    • Arthur S. Kim
    •  & Michael S. Diamond
  3. Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO, USA

    • Katherine Basore
    • , Daved H. Fremont
    •  & Michael S. Diamond
  4. Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA

    • William B. Klimstra
  5. Integral Molecular Inc., Philadelphia, PA, USA

    • Rebecca Rimkunas
    • , Rachel H. Fong
    •  & Benjamin J. Doranz
  6. Vanderbilt Vaccine Center, Department of Pediatrics and Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    • James E. Crowe Jr
  7. Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, Saint Louis, MO, USA

    • Michael S. Diamond

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Contributions

R.Z. performed CRISPR–Cas9 screening and infection studies with J.M.F., S.N., H.L. and S.P. A.S.K. generated Mxra8–Fc and performed ELISAs. K.B. performed SPR experiments. R.R. and R.H.F. performed mapping experiments. R.Z., A.S.K., J.M.F., B.J.D., D.H.F. and M.S.D. designed experiments. W.B.K. and J.E.C. provided key reagents. R.Z., A.S.K., J.M.F., K.B., R.H.F. and B.J.D. performed data analysis. R.Z. and M.S.D. wrote the initial manuscript draft.

Competing interests

M.S.D. is a consultant for Inbios and Sanofi-Pasteur and on the Scientific Advisory Board of Moderna. J.E.C. is on the Scientific Advisory Boards of GigaGen, Meissa Vaccines, and PaxVax, and is a Founder of IDBiologics. B.J.D. is a shareholder of Integral Molecular.

Corresponding author

Correspondence to Michael S. Diamond.

Extended data figures and tables

  1. Extended Data Fig. 1 CRISPR–Cas9-based gene editing screen.

    a, Mouse 3T3 cells were transduced separately with two half libraries (A + B) comprising 130,209 sgRNAs, selected with puromycin and then inoculated with CHIKV-181/25-mKate2 (MOI of 1). One day later, mKate2-negative cells were sorted and then expanded in the presence of 2 μg ml−1 each of CHK-152 and CHK-166 neutralizing mAbs. Several days later, cells were re-inoculated with CHIKV-181/25-mKate2 without neutralizing mAbs and re-sorted for mKate2-negative cells. This procedure was repeated one additional time. Afterwards, genomic DNA was collected for sgRNA sequencing and compared to the parent library for abundance (see Supplementary Tables 1, 2). b, Diagram of the mouse Mxra8 and human MXRA8 orthologues. The transcript identification numbers and length of proteins are indicated to the right. Partial deletions in the isoforms 3 and 4 are shown as dashed lines. c, Phylogenetic tree of Mxra8 indicating genetic relationships. The neighbour-joining tree was constructed using MEGA 7 (https://www.megasoftware.net/). Scale bar shows the branch length. Right, identity (red) and similarity (yellow) matrix indicating the conservation of Mxra8 between species. The matrix was generated using MagGat 1.8 (http://iubio.bio.indiana.edu/soft/molbio/evolve/).

  2. Extended Data Fig. 2 Efficiency of targeting Mxra8 expression by CRISPR–Cas9 gene editing.

    a, 3T3 cells were edited with a control or three different Mxra8 sgRNAs. After puromycin selection, bulk cells were inoculated with CHIKV-181/25-mKate2 and processed for marker gene expression by flow cytometry. Data are pooled from three experiments and expressed as mean ± s.d. (n = 6, one-way ANOVA with a Dunnett’s multiple comparison test compared to control). b, Western blotting of Mxra8 in control and ΔMxra8 3T3 or MEF cells using hamster mAb 3G2.F5. One representative of two is shown. c, 3T3 and MEF cells (parent or ΔMxra8) were tested for Mxra8 surface expression by flow cytometry using anti-Mxra8 antibody (4E7.D10) and an isotype control mAb. One representative experiment of two is shown. d, Sanger sequencing of Mxra8 in control and ΔMxra8 3T3 or MEF cells. Sequencing data shows an alignment and individual out-of-frame deletions. e, Viability of control and ΔMxra8 3T3 and MEF cells. Equal numbers of cells were plated and viability was assessed over a 24-h period using the Cell-Titer Glo assay. The results were normalized to control cells and pooled from two experiments (n = 6). Error bars indicate s.d. ****P < 0.0001.

  3. Extended Data Fig. 3 CHIKV infectivity in CHO-K1 and CHO-745 cells in the presence or absence of ectopic Mxra8 expression.

    a, Surface expression of Mxra8 on CHO-K1 (wild type) and CHO-745 (glycosaminoglycan deficient15) cells stably transduced with control (vector-only) or mouse Mxra8 as judged by flow cytometry. b, Confirmation of heparan sulfate expression on the surface of CHO-K1 (wild type) and CHO-745 cells. Surface expression of heparan sulfate was evaluated using the R17 protein of rodent herpesvirus Peru, which binds to glycosaminoglycans on the surface of cells39. R17GAG2 is a mutant form of the protein that lacks binding to glycosaminoglycans and served as a negative control. In a and b, data are representative of two experiments. c, CHO-K1 (wild type) and CHO-745 cells were transduced stably with control (vector) or mouse Mxra8 and inoculated with CHIKV (strains 181/25, AF15561 or LR-2006) and processed for intracellular E2 protein staining by flow cytometry. Data are from three experiments: mean ± s.d. (n = 6, one-way ANOVA with a Dunnett’s multiple comparison test). ****P < 0.001.

  4. Extended Data Fig. 4 Growth curves of related alphaviruses in ΔMxra8 3T3 cells.

    Control and ΔMxra8 3T3 cells were inoculated with BEBV, BFV, GETV, UNAV, MIDV or SFV at an MOI of 0.01 (except for BEBV, which was at 0.001), and supernatants were collected at the indicated times for focus forming assay. Data are pooled from two (BEBV) or three (all others) experiments and expressed as mean ± s.d. (n = 6, BEBV; n = 9, BFV, GETV, UNAV and MIDV; n = 12, SFV; two-way ANOVA with Sidak’s multiple comparisons test). ***P < 0.001; ****P < 0.0001.

  5. Extended Data Fig. 5 Surface expression of MXRA8 in different human cell lines.

    Human cell lines were tested for MXRA8 surface expression by flow cytometry: 293T (embryonic kidney), A549 (lung adenocarcinoma), JEG3 (placental choriocarcinoma), U2OS (osteosarcoma), HFF-1 (foreskin fibroblasts), HeLa (cervical carcinoma), Huh7 (hepatocarcinoma), HTR8/SVneo (trophoblast progenitor), MRC-5 (lung carcinoma), hCMEC/D3 (cerebral microvascular endothelial cells), RPE (retinal pigment epithelial cell), Jurkat (T cell lymphoma), Raji (B cell lymphoma), K562 (eryrtholeukaemia), HT1080 (fibrosarcoma) and Hs 633T (fibrosarcoma) cells. Representative data are shown of two independent experiments. Grey histograms, isotype control mAb; red histograms, anti-MXRA8 mAb.

  6. Extended Data Fig. 6 MXRA8 supports enhanced infection of different CHIKV strains.

    a, Transduction and expression of different MXRA8 (MXRA8-1, MXRA8-2, MXRA8-3 and MXRA8-4) isoforms in HeLa cells. Representative data are shown from two experiments. Grey histograms, isotype control mAb; red histograms, anti-MXRA8 mAb. b, Effect of ectopic expression of MXRA8-2 on CHIKV (strains 181/25, AF15561, and LR-2006) infection of A549, HeLa or 293T cells. Cells were collected and stained for CHIKV antigen with an anti-E2 antibody. Data are pooled from three experiments and expressed as mean ± s.d. (n = 6; two-tailed t-test with Holm–Sidak multiple comparison correction). c. Transduction and expression of MXRA8-2 in 293T, A549, and HeLa cells. Representative data are shown from two experiments. ***P < 0.001; ****P < 0.0001.

  7. Extended Data Fig. 7 Gene-editing of MXRA8 in human cell lines.

    a, Flow cytometry analysis of MXRA8 expression in human MRC-5, HFF-1, RPE and Hs 633T cells expressing control or two different MXRA8 sgRNAs. Data are representative of two experiments. b, Gene-edited cells were inoculated with CHIKV (strains 181/25, AF15561 or LR-2006) in HFF-1, RPE, and Hs 633T cells. Cells were stained for viral antigen with an anti-E2 antibody. Data are pooled from two (HFF-1 and Hs 633T) or three (RPE) independent experiments and expressed as mean values ± s.d. (n = 6; one-way ANOVA with a Dunnett’s multiple comparison test compared to the control). ****P < 0.0001.

  8. Extended Data Fig. 8 Mxra8–Fc and anti-Mxra8 generation and function.

    a, Diagram of Mxra8–Fc (left) and SDS–PAGE (non-reducing (NR) and reducing (R) conditions) of purified material (right). Data are representative of three experiments. b, Scheme of anti-Mxra8 generation in Armenian hamsters. c, ELISA reactivity of anti-Mxra8 mAbs against Mxra8–Fc, MXRA8-2–Fc or OPG–Fc. Purified proteins (50 μl, 5 μg ml−1) were immobilized overnight at 4 °C on ELISA plates. Anti-Mxra8 and isotype control mAbs were incubated for 1 h at room temperature. Signal was detected at 450 nm after incubation with horseradish peroxide conjugated goat anti-Armenian hamster IgG (H + L) and development with 3,3′-5,5′ tetramethylbenzidine substrate. d, Blockade of CHIKV-181/25 infection in MRC-5 cells with seven different hamster anti-Mxra8 or isotype control mAbs. mAbs were pre-incubated with cells for 1 h at 37 °C before addition of virus. After infection, cells were processed for E2 staining by flow cytometry. Relative infection was compared to a no mAb condition using flow cytometry and anti-E2 staining. Data in c and d are pooled from two experiments (n = 6) and expressed as mean ± s.d. e, Anti-Mxra8 mAbs (1G11 + 7F1) or isotype control hamster mAbs (300 μg total) were administered via intraperitoneal route 8 or 24 h after inoculation of CHIKV-AF15561 in the footpad. Left, at 72 h after initial infection, CHIKV titres were measured in the ipsilateral and contralateral gastrocnemius (calf) muscles. Right, at 72 h, ipsilateral foot swelling was measured and compared to measurements taken immediately before infection. Data are pooled from two experiments (n = 8; two-tailed Mann–Whitney test) and expressed as median values. *P < 0.05, n.s., not significant.

  9. Extended Data Fig. 9 Expression of truncated forms of Mxra8 mutants.

    a, Cell-surface expression of Mxra8 in ΔMxra8 3T3 cells trans-complemented with vector, Mxra8, Mxra8 ΔC-tail, Mxra8 with GPI anchors (derived from PLAP or RHVP open reading frame R1) or MXRA8-2. Data are representative of two independent experiments. b, Effect of PI-PLC treatment on expression of different GPI-anchored forms or Mxra8. Data are representative of two independent experiments.

  10. Extended Data Fig. 10 Binding of Mxra8–Fc to surface-displayed E2 protein in virus-infected cells and mapping of Mxra8 binding site on E2.

    a, Diagram of the cell-based binding assay. After infection, viral structural proteins (for example, E2) traffic to the cell plasma membrane where progeny virion assembles and buds. E2 protein is displayed on the cell surface and is accessible to the binding of Mxra8–Fc and detection with a goat anti-mouse IgG secondary antibody by flow cytometry. b, Binding of Mxra8–Fc to virus-infected wild-type 3T3 cells. Cell were infected with the indicated viruses and processed for Mxra8–Fc binding by flow cytometry. Virus-specific anti-E2 antibodies were used as positive controls. Data are representative of two independent experiments. c, d, Mapped residues are shown as magenta spheres (c) or sticks (d) on the CHIKV p62–E1 structure (c, trimer of dimers, top view; d, heterodimer, side view) using PyMOL (Protein Data Bank code: 3N41). The E1 and E2 proteins are coloured in grey and cyan, respectively.

Supplementary information

  1. Supplementary Figure 1

    This file contains the uncropped gels.

  2. Reporting Summary

  3. Supplementary Table 1

    Frequency of sgRNA deep-sequencing reads (see attached Excel file). Data was obtained by deep-sequencing of sgRNAs from uninfected or sorted CHIKV-181/25-mKate2-negative cells. The two half libraries (A + B) were used for independent screens.

  4. Supplementary Table 2

    List of genes and scores after MaGeck analysis (see Excel file).

  5. Supplementary Table 3

    Mxra8-Fc binding to 293T cells transfected with alanine scanning mutants of C-E3-E2-6K-E1. Alanine mutations of E2 are indicated in the first column, and binding to Mxra-Fc and several CHIKV mAbs (84, 88, IM-CKV063, IM-CKV065, and C9) was tested in 293T cells and processed by flow cytometry. Percent binding of Mxra-Fc or mAbs to the respective mutant was normalized to cells transfected with wild-type C-E3-E2-6K-E1 plasmid. Data are representative of two independent experiments and parentheses indicate range. Yellow highlighting indicates mutants with loss-of-binding to Mxra-Fc that retain expression and folding. Green highlighting indicates mutations that affect overall domain/protein folding and expression, and cannot be evaluated with certainty.

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