The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3,4,5,6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1—a member of the cadherin superfamily7,8—to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.
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See Supplementary Information for grants supporting this work. We thank A. Beck and the Einstein Histopathology Core for histopathology support; S. Garforth and the Einstein Macromolecular Therapeutics Development Facility for assistance with SEC-MALS; G. Pehau-Arnaudet, Institut Pasteur Paris, for electron microscopy of MPRLV VLPs; H. Galeno, Instituto de Salud Pública de Chile, for convalescent sera from Chilean patients with hantavirus; and M. Evans for feedback and discussions. Opinions, conclusions, interpretations and recommendations are those of the authors and are not necessarily endorsed by the US Army. The mention of trade names or commercial products does not constitute endorsement or recommendation for use by Department of the Army or Department of Defense.
Nature thanks B. Lee, G. Schönrich and the other anonymous reviewer(s) for their contribution to the peer review of this work.
R.K.J., L.T.J., K.C. and T.R.B. are named inventors on US patent application US20170173141A1, covering methods to treat hantavirus infections by targeting PCDH1 (the entry factor described here), the application being assigned to Albert Einstein College of Medicine. T.R.B. is co-founder and Scientific Advisory Board member of Haplogen GmbH, and co-founder and managing director of Scenic Biotech BV. F.A.R. is a consultant for Flagship Pioneering.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 PCDH1 is required for entry of New World hantaviruses into HAP1 and U2OS cells.
a, Genes enriched for gene-trap insertions in the rVSV-ANDV Gn/Gc-selected population versus unselected control cells. The size of each bubble reflects the number of independent gene-trap insertions observed. Candidate genes are associated with cholesterol metabolism (yellow), the endoplasmic reticulum membrane complex network (green), and transcription (blue). PCDH1 (red) is a singleton hit. b, Single-cell HAP1 clones deficient for PCDH1 were generated by CRISPR–Cas9 genome engineering. The sequences of PCDH1-knockout alleles in clones 1 and 22 are shown. The sgRNA target sequence is highlighted in red. i1, insertion of a single nucleotide in PCDH1. c, WT and HAP1 PCDH1-KO cell lines were exposed to rVSVs bearing the indicated viral glycoproteins at a MOI of 0.02 IU per cell. ‘+cDNA’ indicates complementation of PCDH1-KO cells with PCDH1. Cells were scored for infection at 20 hpi. One hundred per cent relative infection corresponds to 20–30% infected cells. Averages ± s.d. are shown: HAP1-WT, three experiments, n = 6; PCDH1-KO#1-cDNA, two experiments, n = 5; PCDH1-KO#1+cDNA, three experiments, n = 8; PCDH1-KO#22-cDNA, three experiments, n = 8, except for HTNV Gn/Gc, for which two experiments, n = 4; PCDH1-KO#1+cDNA, three experiments, n = 8; PCDH1-KO#22+cDNA, three experiments, n = 8. d, Single-cell U2OS clones deficient for PCDH1 were generated by CRISPR–Cas9 genome engineering. The sequences of PCDH1-knockout alleles in clone 5 are shown. The sgRNA target sequence is highlighted in red. i1, insertion of a single nucleotide in PCDH1. e, f, WT and U2OS PCDH1-KO cell lines were exposed to the indicated rVSVs at a MOI of 0.02 IU per cell. Cells were scored for infection at 20 hpi. eGFP-positive infected cells (pseudocoloured green) were detected by fluorescence microscopy (e) and enumerated by automated counting (f). One hundred per cent relative infection corresponds to 10–20% infected cells. Averages ± s.e.m. are shown, three experiments, n = 13. In c, f, wild type versus PCDH1-KO by two-way ANOVA with Tukey’s (c) or Sidak’s (f) tests: NS, P > 0.05; ****, P < 0.0001.
Extended Data Fig. 2 Expression and plasma-membrane localization of PCDH1 variants lacking domains EC1 or EC2 of PCDH1.
a, U2OS PCDH1-KO cell lines complemented with the indicated PCDH1 proteins were immunostained with an anti-Flag antibody and visualized by fluorescence microscopy. EV, empty vector. Scale bar, 20 µm. b, c, Live cells from a were stained with the PCDH1-EC7-specific mAb 3677 at 4 °C to detect cell-surface PCDH1 and visualized by flow cytometry. Cells were gated on PCDH1 immunofluorescence intensity (dotted red lines in c) to determine the percentage of cells with surface expression of each PCDH1 protein. Averages ± s.d. are shown in b: two experiments, n = 4, except in the case of WT, for which n = 3. c, Representative flow plots from b. Experiments were performed three times with similar results.
a, Purified sEC1–2 bearing C-terminal Flag and decahistidine (His10) epitope tags was resolved on an SDS–polyacrylamide gel and visualized by Coomassie blue staining. Asterisk, minor component of unknown origin. Mr, relative molecular weight (K denotes ×1,000); M, monomer peak. The experiment was performed three times with similar results. b, SEC-MALS analysis of purified sEC1–2 from a. Absorbance (arbitrary units, au) was monitored at 280 nm; m, monomer peak. Calculated (MWcalc) and observed (MWobs) molecular-weight estimates from MALS are shown in the inset. The experiment was performed twice with similar results. c, Generation of purified sEC1, comprising the first extracellular cadherin domain of PCDH1, and bearing a C-terminal His10 epitope tag. sEC1–2 is shown for comparison. The experiment was performed three times with similar results. d, Capacity of sEC1 and sEC2 to block hantavirus glycoprotein-dependent entry. rVSVs bearing ANDV Gn/Gc were preincubated with sEC1 or sEC2 (0–5 µM, in serial threefold dilutions), and then allowed to infect HUVECs at a MOI of 0.2 IU per cell. Cells were scored for infection at 9 hpi. One hundred per cent relative infection corresponds to 10–20% infected cells. Averages ± s.d.: three experiments, n = 8 for sEC1; n = 7 for sEC2.
a, Flow chart showing isolation of PCDH1-specific mAbs from Library F by phage display. b, Capacity of selected mAb clones to recognize recombinant fusion proteins comprising PCDH1 ectodomains (containing or lacking EC1) and the Fc antibody domain. BSA, bovine serum albumin. Data from a representative ELISA experiment are shown. Experiments were performed three times with similar results. c, Kinetic binding analysis of selected Fabs to PCDH1(ectodomain)–Fc fusion proteins by surface plasmon resonance. nd, not determined; Kd, equilibrium dissociation constant. d, mAb 3305 recognizes the first extracellular cadherin (EC1) domain of PCDH1. U2OS PCDH1-KO cells, uncomplemented (KO) or complemented either with full-length PCDH1 (WT) or with a PCDH1 variant lacking the EC1 domain (∆EC1), were co-immunostained with anti-Flag (α-Flag) mAb and anti-PCDH1 mAb 3305, or with anti-Flag and negative control antibodies (hIgG). Cells were visualized by fluorescence microscopy. Scale bar, 20 µm. Data from a representative experiment are shown. Experiments were performed three times with similar results.
HPMECs were preincubated with mAb 3305 or with human IgG control (Ctrl) (0–100 µg ml−1, 0–680 nM, in serial threefold dilutions), and then exposed to the indicated rVSVs Gn/Gc glycoproteins at a MOI of 0.2 IU per cell. Infected cells were scored at 9 hpi. One hundred per cent relative infectivity corresponds to 15–20% infected cells. Averages ± s.d.: three experiments; no-mAb samples (‘0’), n = 10 for ANDV and HTNV, n = 3 for SNV; mAb-treated samples, n = 5 for ANDV and HTNV, n = 3 for SNV. mAb 3305 versus control mAb, two-way ANOVA with Dunnett’s test: ns, P > 0.05; ****P < 0.0001.
293FT cells transfected with plasmids encoding ANDV or HTNV Gn/Gc were analysed by flow cytometry using 1E11/D3 mAb for total (after permeabilization) and surface (without permeabilization) expression of Gn/Gc. Empty-vector-transfected cells were used as a negative control for gating. Results are from a representative experiment with n = 2. Experiments were performed five times for ANDV and twice for HTNV with similar results.
Capacity of hantavirus glycoproteins expressed at the cell surface to capture Flag-tagged sEC1–2 from solution. a, rVSVs bearing ANDV or HTNV Gn/Gc were allowed to infect U2OS cells, and cell-surface expression of Gn/Gc was detected by immunofluorescence microscopy using mAb 1E11/D3. b, Cells expressing Gn/Gc were then exposed to sEC1–2 (200 nM), and sEC1–2 binding to the cell surface was detected by immunofluorescence microscopy using an anti-Flag mAb. c, The capacity of PCDH1-specific mAb 3305 to block binding of sEC1–2 to Gn/Gc-expressing cells was determined as in b. sEC1–2 (50 nM) was preincubated with the indicated amounts of a control IgG or mAb 3305, and then exposed to Gn/Gc-expressing cells. In a–c, representative images are shown, and experiments were performed three times with similar results. Scale bars, 20 µm. d, Biotinylated rVSVs bearing ANDV or HTNV Gn/Gc were incubated with sEC1–2 and then captured with streptavidin magnetic beads. Co-precipitated viral particles and sEC1–2 (‘Virus IP’) and a fraction of the input material (‘Input’) were detected by immunoblotting with mAb 23H12, specific for the VSV M matrix protein, and an anti-Flag mAb, respectively. ‘None’ indicates control precipitation of sEC1-2 in the absence of viral particles. Representative images are shown. Experiments were performed three times with similar results.
Extended Data Fig. 8 Expression and purification of MPRLV VLPs and their interaction with soluble PCDH1.
a, Purified MPRLV VLPs (8 µg protein) bearing an N-terminal StrepTag were resolved on an SDS–polyacrylamide gel and visualized by Coomassie blue staining. R, reducing conditions; NR, nonreducing conditions. b, MPRLV VLPs were negatively stained with uranyl acetate and visualized by electron microscopy. c, Kinetic constants for MPRLV VLP-sEC1–2 interaction were determined by biolayer interferometry. kon, association-rate constant; koff, dissociation rate constant. Values ± 95% confidence intervals derived from curve fits are shown for two independent experiments. These experiments were performed three times with similar results.
Extended Data Fig. 9 PCDH1-EC1 is required for hantavirus Gn/Gc-dependent entry and infection in primary Syrian hamster lung endothelial cells.
a, Capacity of soluble PCDH1 (sPCDH1) variants (sEC1–2, sEC1 or sEC2) to block hantavirus glycoprotein-dependent entry. rVSVs bearing ANDV or HTNV Gn/Gc glycoproteins were preincubated with the indicated amounts of the indicated sPCDH1 variants (0–5 µM, in serial threefold dilutions) at room temperature for 1 h, and then allowed to infect Syrian hamster lung endothelial cells (ECs). One hundred per cent relative infectivity corresponds to 15–20% infected cells. Averages ± s.d.: two experiments, n = 4. b, Capacity of PCDH1-EC1-specific mAb 3305 to block hantavirus glycoprotein-dependent entry. Syrian hamster lung ECs were preincubated with mAb 3305 or control human IgG (0–100 µg ml−1, 0–680 nM, in serial threefold dilutions), and then exposed to rVSVs bearing ANDV or SNV Gn/Gc at an MOI of 0.2 IU per cell. Cells were scored for infection at 9 hpi. One hundred per cent relative infectivity corresponds to 15–20% infected cells. Two experiments, n = 2, except in the case of no mAb (‘0’) for which n = 4.
Extended Data Fig. 10 Generation of a PCDH1-KO knockout Syrian hamster by CRISPR–Cas9 genome engineering.
a, Organization of the PCDH1 gene of the Syrian hamster (M. auratus). The sequence in exon 2 of PCDH1 that was targeted by an sgRNA is shown in magenta. Knockout animals bear two PCDH1 alleles that have been edited to lack a single nucleotide, highlighted in green. The sgRNA PAM is highlighted in blue. b, A PCR–RFLP strategy, based on loss of digestion by the restriction endonuclease BanI, was used to detect genome editing and genotype animals. c, PCR–RFLP results were confirmed by Sanger DNA sequencing of PCR amplicons from WT and genome-edited animals. Sequencing traces are shown. Sequence features are highlighted as in a. Red arrows denote the site of gene editing in the PCDH1-KO allele. Experiments were performed twice with similar results. d, Lung tissue isolated from WT and PCDH1-KO hamsters was solubilized, normalized by protein content, and subjected to SDS–PAGE. PCDH1 was detected by immunoblotting with EC1-specific mAb 3305. Control, nonspecific loading control. Experiments were performed three times with similar results. e, Viral loads in the sera of WT and PCDH1-KO hamsters at 14 dpi. The limit of detection is shown as a dotted line. Experiments in b–e were performed twice with similar results.
This file contains Supplementary Figure 1: Uncropped scans with size markers.
This file contains Supplementary Notes.
This file contains Results of haploid genetic screen for ANDV entry. The number of mutations retrieved from HAP1 cells selected with rVSV-ANDV Gn/Gc was compared per gene to a dataset of unselected HAP1 cells. A one-sided Fisher's exact test was used to gauge the enrichment of mutations in a selected versus the unselected population. P-values are false discovery rate (FDR)-corrected. Please see associated Excel file.
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Jangra, R.K., Herbert, A.S., Li, R. et al. Protocadherin-1 is essential for cell entry by New World hantaviruses. Nature 563, 559–563 (2018). https://doi.org/10.1038/s41586-018-0702-1
- Protocadherin (PCDH1)
- Hantavirus (HTNV)
- World Hantaviruses
- Andes Virus (ANDV)
- Hantavirus Pulmonary Syndrome (HPS)
T-cell immunoglobulin and mucin (TIM) contributes to the infection of human airway epithelial cells by pseudotype viruses containing Hantaan virus glycoproteins
Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model
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