1. Microbiology and Infectious Disease
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Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

  1. Maria Eugenia Dieterle
  2. Carles Solà-Riera
  3. Chunyan Ye
  4. Samuel M Goodfellow
  5. Eva Mittler
  6. Ezgi Kasikci
  7. Steven B Bradfute
  8. Jonas Klingström
  9. Rohit K Jangra  Is a corresponding author
  10. Kartik Chandran  Is a corresponding author
  1. Department of Microbiology and Immunology, Albert Einstein College of Medicine, United States
  2. Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Sweden
  3. University of New Mexico Health Science Center, Center for Global Health, Department of Internal Medicine, United States
Short Report
Cite this article as: eLife 2021;10:e69708 doi: 10.7554/eLife.69708
2 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Suitability of TIME cells as a model to study hantavirus entry and the generation of knockout cells.

(a) Upper panels, total flow cytometry plots of HUVEC and TIME cells stained for endothelial cell markers PECAM and von Willebrand factor (vWF). Medium and lower panels, surface flow cytometry plots of HUVEC and TIME cells stained for PCDH1, β3 integrin, DAF, β1 integrin. (b) Surface flow cytometry of wild-type (WT) and knockout (KO) TIME cells stained as above. Histograms of WT cells are shown in gray; single- and double-KO cells are shown in color. (c) Western blot analysis of WT TIME cells and KO cells ± cDNA. β-Actin was used as a loading control.

Figure 1—source data 1

Original blot of WT TIME cells and KO cells ± cDNA.

https://cdn.elifesciences.org/articles/69708/elife-69708-fig1-data1-v1.zip
Figure 1—figure supplement 1
Sanger sequences retrieved from the targeted genomic loci for each knockout cell population.

Sequences of interests were amplified by PCR and then TA-cloned into the pGEM-T vector. For each knockout cell population, 15–20 clones were subjected to Sanger sequencing. All sequenced clones showed an indel at the targeted site, resulting in a frameshift that brought one or more stop codons into frame.

Figure 2 with 1 supplement
Hantavirus receptor requirement in endothelial cells.

(a) Representative images of eGFP-positive rVSV-infected wild-type (WT) and PCDH1, ITGB3, and DAF knockout (KO) TIME cells. Nuclei were stained with Hoechst (blue). (b) WT and KO cells were exposed to the indicated rVSV-Gn/Gc. n=11 for each cell line from three independent experiments. WT versus KO cells, two-way ANOVA with Dunnett’s test; ***p<0.0001. Other comparisons were not statistically significant (p>0.05). (c) WT and KO cells lacking (–cDNA) or expressing the corresponding cDNA (+cDNA) were exposed to rVSVs bearing the indicated hantavirus glycoproteins. Viral infectivities are shown in the heatmap. Averages are from three independent experiments (d) WT and double-KO cells were exposed to rVSV-Gn/Gc. PCDH1/ITGB3 KO, n = 12; PCDH1/DAF KO, n = 12; and ITGB3/DAF KO, n=9 from three independent experiments. WT versus KO cells, two-way ANOVA with Dunnett’s test; ****p<0.0001. Other comparisons were not statistically significant. (e) Cells were exposed to authentic hantaviruses and infected cells were manually enumerated by immunofluorescence microscopy for ANDV, HTNV, and PUUV (each point represents infectivity of the average of positive cells per field relative to WT). Data are from two independent experiments. PHV- and SNV-infected cells were detected and enumerated by automated imaging following immunofluorescence staining. For PHV: WT and PCDH1 KO n=18, ITGB3 KO n = 16 from four independent experiments; DAF KO, n=10, ITGB1 KO n=8, from three independent experiments. For SNV: WT, ITGB3 KO, DAF KO n=6; PCDH1 KO n = 5 from three independent experiments. Averages ± SD are shown. WT versus KO cells, two-way ANOVA with Tukey’s test; ***p<0.0001. Other comparisons were not statistically significant (p>0.05).

Figure 2—figure supplement 1
Dispensability of β1 integrin in TIME cells.

(a) Representative images of eGFP-positive rVSV-infected wild-type (WT) and ITGB1 knockout (KO) TIME cells. Nuclei were stained with Hoechst (blue). (b) WT and KO cells were exposed to the rVSV bearing the ANDV, SNV, PHV, HTNV, and EBOV glycoproteins. n=12 for each cell line from three independent experiments. WT versus KO cells, two-way ANOVA. Comparisons were not statistically significant (p>0.05).

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)PCDH1GenBankGene ID: 5097
Gene (Homo sapiens)ITGB3GenBankGene ID: 3690
Gene (Homo sapiens)DAFGenBankGene ID: 1604
Gene (Homo sapiens)ITGB1GenBankGene ID: 3688
Strain, strain background (virus)rVSV eGFP ANDV Gn/GcKleinfelter et al., 2015
Strain, strain background (virus)rVSV eGFP SNV Gn/GcKleinfelter et al., 2015
Strain, strain background (virus)rVSV eGFP HTNV Gn/GcKleinfelter et al., 2015
Strain, strain background (virus)rVSV eGFP SEOV Gn/GcJangra et al., 2018
Strain, strain background (virus)rVSV eGFP DOBV Gn/GcSlough et al., 2019
Strain, strain background (virus)rVSV eGFP MPRLV Gn/GcJangra et al., 2018
Strain, strain background (virus)rVSV eGFP PHV Gn/GcJangra et al., 2018
Strain, strain background (virus)rVSV mNeongreen-P PUUV-Gn/GcKerkman et al., 2019, This studyLaboratory of K. Chandran
Strain, strain background (virus)rVSV mNeongreen-P CHOV Gn/GcThis studyGenBank # KT983772.1Laboratory of K. Chandran/VSV antigenome plasmid (Whelan et al., 1995), plasmids expressing T7
polymerase and VSV N, P, M, G, and L (Witko et al., 2006)
Strain, strain background (virus)rVSV-EBOV/Mayinga GP (EBOV/H.sap-tc/COD/76/
Yambuku-Mayinga)
Wong et al., 2010
Strain, strain background
(Hantavirus)
ANDV isolate Chile-9717869N/A
Strain, strain background (Hantavirus)HTNV isolate 76–118N/A
Strain, strain background (Hantavirus)PUUV isolate SotkamoN/A
Strain, strain background (Hantavirus)SNV isolate SN77734Botten et al., 2000
Strain, strain background (Hantavirus)PHVN/ALaboratory of K. Chandran
Cell line (H. sapiens)293TATCCCat. # CRL-3216
Cell line (H. sapiens)HUVECLonzaCat. # C2517APrimary cell
Cell line (H. sapiens)TIME (endothelial cell line)ATCCCat. # CRL-4025
Cell line (C. aethiops)VeroCCL-81Cat. # CCL-81
Genetic reagent (H. sapiens)TIME PCDH1 KO (endothelial cell line)This studyLaboratory of K. Chandran/Lentiviral transduction/lentiCRISPR v2-sgRNA PCDH1
Genetic reagent (H. sapiens)TIME ITGB3 KO (endothelial cell line)This studyLaboratory of K. Chandran/Lentiviral transduction/lentiCRISPR v2-sgRNA ITGB3
Genetic reagent (H. sapiens)TIME ITGB1 KO (endothelial cell line)This studyLaboratory of K. Chandran/Lentiviral transduction/lentiCRISPR v2-sgRNA ITGB3
Genetic reagent (H. sapiens)TIME DAF KO (endothelial cell line)This studyLaboratory of K. Chandran/Lentiviral transduction/lentiCRISPR v2-sgRNA DAF
Genetic reagent (H. sapiens)TIME PCDH1/ITGB3 KO (endothelial cell line)This studyLaboratory of K. Chandran/TIME PCDH1 KO + Lentiviral transduction/lentiCRISPR v2-sgRNA ITGB3
Genetic reagent (H. sapiens)TIME PCDH1/DAF KO (endothelial cell line)This studyLaboratory of K. Chandran / TIME PCDH1 KO + Lentiviral transduction/ lentiCRISPR v2-sgRNA DAF
Genetic reagent (H. sapiens)TIME ITGB3/DAF KO (endothelial cell line)This studyLaboratory of K. Chandran / TIME ITGB3 KO + Lentiviral transduction/ lentiCRISPR v2-sgRNA DAF
Genetic reagent (H. sapiens)TIME PCDH1 KO + PCDH1 (endothelial cell line)This studyCanonical isoform Q08174-1 (Uniprot)Laboratory of K. Chandran / Retroviral transduction/ pBabe-PCDH1
Genetic reagent (H. sapiens)TIME ITGB3 KO + ITGB3 (endothelial cell line)This studyCanonical isoform P05106-1 (Uniprot)Laboratory of K. Chandran / Retroviral transduction/ pBabe-ITGB3
Genetic reagent (H. sapiens)TIME DAF KO + DAF (endothelial cell line)This studyCanonical isoform P08174-1 (Uniprot)Laboratory of K. Chandran / Retroviral transduction/ pBabe-DAF
AntibodyAF480-α-Human-vWF (Rabbit monoclonal)AbcamCat. # ab195028Flow 1:250
AntibodyPeCy7-α-Human-PECAM (Mouse monoclonal)BDCat. # 563651Flow 1:1000
AntibodyAF 647-α-Human- β3-Integrin (Mouse monoclonal)BiolegendsCat. # 336407Flow 1:200
AntibodyPE-α-Human-DAF (Mouse monoclonal)BDCat. # 555694Flow 1:1600
AntibodyAPC-α-Human-β1-Integrin (Mouse monoclonal)BDCat. # 59883Flow 1:20
Antibodyα–Human-PCDH1 mAb 3305 (human monoclonal)Jangra et al., 2018Flow 1:200
AntibodyConvalescent serum (human polyclonal)Stoltz et al., 2007IF: 1:40
AntibodyAF488-α–-Human IgG (Goat polyclonal)InvitrogenCat. # A-11013Flow 1:200
AntibodyAF594-α–Human IgG (Goat polyclonal)InvitrogenCat. # A-11014IF 1:500
AntibodyAF594-α-Mouse IgG (Goat polyclonal)InvitrogenCat. # A32742IF 1:500
AntibodyAF488-α-Rabbit IgG (Goat polyclonal)InvitrogenCat. # A-11008IF 1:500
Antibodyα–Human PCDH1 (Mouse monoclonal)Santa CruzCat. # sc-81816WB 1:200
Antibodyα–Human β3 Integrin (Rabbit polyclonal)Cell SignalingCat. # 4702WB 1:300
Antibodyα–Human-DAF (Mouse monoclonal)Santa CruzCat. # NaM16-4D3WB 1:200
Antibodyα–Human β Actin (Mouse monoclonal)Santa CruzCat. # sc-47778WB 1:300
AntibodyIRDye 680LT α-Mouse (Goat polyclonal)LI-CORCat. # 926–68020WB 1:10,000
AntibodyIRDye 680LT Goat α-Rabbit IgG 680 (Goat polyclonal)LI-CORCat. # 926–68021WB 1:10,000
Antibodyα-hantavirus nucleocapsid B5D9 (Mouse monoclonal)ProgenCat. # B5D9-CIF 1:50
Antibodyα-HTNV nucleoprotein NR-12152 (Rabbit polyclonal)BEI resourcesCat. # NR-12152IF 1:500
Recombinant DNA reagentpBabe-puro (plasmid)Morgenstern and Land, 1990Addgene plasmid # 1764
Recombinant DNA reagentpBabe-PCDH1 (plasmid)Jangra et al., 2018
Recombinant DNA reagentpBabe-ITGB3 (plasmid)This studyLaboratory of K. Chandran
Recombinant DNA reagentpBabe-DAF
(plasmid)
This studyLaboratory of K. Chandran
Recombinant DNA reagentlentiCRISPR v2 (plasmid)Sanjana et al., 2014; Shalem et al., 2014
Recombinant DNA reagentlentiCRISPR v2-sgRNA PCDH1 (plasmid)Jangra et al., 2018Laboratory of K. Chandran/5′-GTTTGAGCGGCCCTCCTATGAGG-3′ /PAM sequence is underlined but not included in oligos
Recombinant DNA reagentlentiCRISPR v2-sgRNA DAF (plasmid)This studyLaboratory of K. Chandran / 5′-CCCCCAGATGTACCTAATGCCCA-3′ / PAM sequence is underlined but not included in oligos
Recombinant DNA reagentlentiCRISPR v2-sgRNA ITGB3 (plasmid)This studyLaboratory of K. Chandran / 5′-CCACGCGAGGTGTGAGCTCCTGC-3′ /PAM sequence is underlined but not included in oligos
Recombinant DNA reagentlentiCRISPR v2-sgRNA ITGB1 (plasmid)This studyLaboratory of K. Chandran/5′-AATGTAACCAACCGTAGCAAAGG-3′ /PAM sequence is underlined but not included in oligos
Sequence-based reagentSNV degenerate primersThis studyLaboratory of S. Bradfute/SNV F: CAgCTgTgTCTgCATTggAgAC
SNV R: TARAgYCCgATggATTTCCAATCA
Sequence-based reagentSNV probeThis studyLaboratory of S. Bradfute/ TMGB1: F-TCAAAACCTgTTgATCCA NFQ MGB
Commercial assay or kitpGEM-T VectorPromegaCat. # A3600
Commercial assay or kitVIC/TAMRA-dye probeApplied
Biosystems
Cat. # 4310881E
Commercial assay or kitTaqMan Fast Advanced Master MixApplied BiosystemsCat. # 4444556
Commercial assay or kitFoxp3/Transcription Factor StainingTonboCat. # TNB-0607-KIT
Commercial assay or kitZombie NIR Fixable Viability KitBioLegendCat. # 423105Flow 1:4000
Software, algorithmCytation 5 Cell Imaging Multi-Mode ReaderBiotek
Author response table 1
gRNA target site locationModified sequence in pBabe- PCDH1/
ITGB3/ DAF
PCDH1GTTTGAGCGGCCCTCCTATGAGGaTTcGAGaGaCCtagtTATGAGG
ITGB3CCACGCGAGGTGTGAGCTCCTGCCtACtaGAGGcGTatcaagCTGt
DAFCCCCCAGATGTACCTAATGCCCACCgCCtGAcGTcCCaAAcGCgCA

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