Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Viral titer of rVSVs expressing G, ANDV Gn/Gc, or SNV Gn/Gc in human or mouse primary lung endothelial cells. Means ± SD: n = 6 wells of infected cells examined over three independent experiments. b Surface expression of endogenous PCDH1 in primary mouse lung microvascular endothelial cells (MLMECs) used in ( a ). Cells were immunostained with PCDH1-specific monoclonal antibody (mAb) 3305 or a negative control mAb (Ctrl.) Scale bar, 20 µm. c Infectivity of rVSVs bearing HTNV, ANDV, or SNV Gn/Gc in primary MLMECs expressing flag-tagged, wild-type (WT) or mouse-variant (F83L) human PCDH1. rVSV infectivities are expressed as fold change relative to that in non-complemented cells (set to one). Means ± SD: n = 35 wells of infected cells examined over three independent experiments. d Cells described in ( c ) were immunostained with an anti-flag antibody to detect total human PCDH1 expression in transfected MLMECs. Scale bar, 20 µm. e Alignment of human PCDH1-EC1 amino acid sequences with a selection of rodent and primate species. All of the residues within EC1 which deviate from the consensus are shown and highlighted. Residues that specifically deviate from human EC1 are indicated [green, experimentally tested SNV-susceptible hosts; purple, experimentally unknown; orange, residues in EC1 that are different between human and other species with no known link to susceptibility]. Alignments generated by Clustal Omega. f The capacity of U2OS PCDH1 -KO cells expressing the indicated PCDH1 variants to support hantavirus Gn/Gc-dependent entry. Cells were exposed to rVSVs bearing the indicated Gn/Gc proteins or authentic ANDV, SNV, or HTNV. “None” indicates no PCDH1 expression. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. rVSV means ± SD: n = 8 wells of infected cells examined over three independent experiments. ANDV/SNV/HTNV means ± SD: one experiment examining n = 3 (ANDV and SNV) or n = 10 (HTNV) wells of infected cells. Infectious units ( a ) were compared by unpaired, two-tailed t test with Welch’s correction. Infectivities ( c , f ) were compared by one-way ANOVA with Dunnett’s correction for multiple comparisons; ns > 0.05, ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Expressing, Infection, Negative Control, Variant Assay, Transfection, Selection, Generated, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Diagram of direct binding ELISA comparing sEC1-2(WT) and sEC1-2(F83L) capture of rVSVs. b Direct binding ELISA. rVSVs expressing ANDV, SNV, or HTNV Gn/Gc were added to sEC1-2(WT) or sEC1-2(F83L) coated ELISA plates. Means ± SD: n = 4 wells examined over two independent experiments. A, absorbance. c Diagram depicting competition ELISA comparing sEC1-2(WT) and sEC1-2(F83L) as competitive reagents. d Competition ELISA. rVSVs expressing ANDV or SNV Gn/Gc were pre-incubated with sEC1-2(WT) or sEC1-2(F83L) before added to sEC1-2(WT) coated ELISA plates. The ELISA signal was normalized to that obtained without competing sEC1-2. Means ± SEM: n = 7 wells examined over three independent experiments (ANDV), n = 4 wells examined over two independent experiments (SNV). e Diagram depicting infection-inhibition assay comparing sEC1-2(WT) and sEC1-2(F83L) as inhibiting reagents. f Infection-inhibition assay using sEC1-2(WT) and sEC1-2(F83L) to block infection (MOI of 0.1) of rVSVs bearing ANDV or SNV Gn/Gc on primary human endothelial cells (HUVECs). The infectivity of each virus was normalized to that obtained without sEC1-2. Averages ± SD: n = 6 wells examined over two independent experiments. (sEC1-2, soluble extracellular cadherin domains 1 and 2). Figures ( a , c , e ) were created with BioRender.com. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Infection, Inhibition, Blocking Assay, Virus

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Relative infectivity of rVSVs bearing ANDV, SNV, or HTNV Gn/Gc on U2OS PCDH1- KO cells complemented with WT or mutant PCDH1. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 wells of infected cells examined over three independent experiments (rVSV-ANDV-Gn/Gc infection on U2OS PCDH1- KO cells complemented with V86A had n = 8). Infectivities were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. b Relative infectivity of authentic ANDV, SNV, or HTNV on the cell lines described in ( a ). The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 infected wells were examined over three independent experiments (HTNV infection on U2OS PCDH1- KO cells complemented with D85A and F83A/D85A had n = 6 wells of infected cells examined over two independent experiments). For ANDV and SNV infection on the control cell line, U2OS PCDH1- KO cells complemented with WT, n = 12 (SNV) and n = 18 (ANDV) wells of infected cells were examined over four independent experiments. Infectivities were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Infection, Mutagenesis, Virus, Control

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Crystal structure of the proposed anti-parallel EC1-4 trans-dimer. Structure is in the “open conformation” displaying residues representing three degrees of binding strength to ANDV Gn/Gc. Residues that when mutated bind similarly to WT (green); those that display a mild reduction in binding (orange); and those that display a strong reduction in binding (purple). The Gn/Gc binding site relative to the EC1:EC4 adhesive interface (dark blue) is indicated. An alternative view of the EC1:EC4 binding interface is shown to the right. Structure adapted from PDB 6MGA. b Competition ELISA using WT and mutant sEC1-2 as competitive reagents to the binding of rVSV-ANDV-Gn/Gc to WT sEC1-2 coated wells. Averages ± SD: n = 4 wells of each sEC1-2 dilution examined over two independent experiments [ n = 3 for one of the dilutions of sEC1-2(E137R)]. c Relative infectivity of rVSVs bearing ANDV, SNV, or HTNV Gn/Gc on U2OS PCDH1- KO cells complemented with WT or ΔEC4 PCDH1. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 wells of infected cells examined over three experiments. d Schematic representation of WT or mutant sEC1-4 proteins forming monomers or dimers. e Non-reduced and reduced purified WT and mutant sEC1-4 were separated on an SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue staining. kDa, kilodalton. f Non-reduced samples in ( e ) were run on a native-polyacrylamide gel and visualized as in ( e ). A representative gel from one experiment of two independent experiments is shown for ( e ) and ( f ). g ELISA detecting WT and mutant sEC1-4 coated plates, using an anti-Flag-HRP antibody. Mean ± SD: n = 4 wells examined over two independent experiments. ( h ) Capacity of rVSV-ANDV-Gn/Gc to bind to WT or mutant sEC1-4 coated plates. Done in parallel with ( g ). Mean ± SD: n = 4 wells of each viral particle dilution examined over two independent experiments. Infectivities ( c ) and ELISA signal ( g ) were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. (sEC1-4, soluble extracellular cadherin domains 1–4). Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Binding Assay, Adhesive, Enzyme-linked Immunosorbent Assay, Mutagenesis, Infection, Virus, Purification, Staining

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Reference nucleotide and amino acid sequence of PCDH1-EC1 Syrian hamster (WT, above) and representative sequences and trace files of Syrian hamsters after CRISPR-Cas9 genome editing [PCDH1(F83A/D85R), lower left] and [PCDH1(10a.a.) lower right]. The nucleotides encoding the corresponding human PCDH1-EC1 Gn/Gc-interacting residues, are highlighted: F83 in purple and D85 in green along with the location of the single guide RNAs (KI, knock-in; sgRNA, single guide RNA). b Immunoblot detecting PCDH1 in lung tissue lysates from WT or CRISPR knock-in mutant Syrian hamsters. Antibody targets PCDH1’s cytoplasmic tail. kDa, kilodalton. A representative blot from a single experiment of two independent experiments is shown. Uncropped blots in Source Data. c Syrian hamster ANDV challenge. Groups of WT, PCDH1(F83A/D85R), and PCDH1(10a.a.) CRISPR knock-in mutant hamsters were inoculated intranasally with ANDV (2,000 PFU). Mortality was monitored and hamsters were euthanized on day 35 post-exposure. One experiment was performed, with n = 8 hamsters for each group. Data was analyzed using two-sided, log-rank Mantel–Cox test. d Lung sections from WT and PCDH1(F83A/D85R) hamsters were collected 15 days post ANDV exposure. Representative histochemical images indicate inflammation in pulmonary tissue (left), ANDV nucleoprotein (N) (middle, tan staining), and ANDV RNA (right, red staining, detected by in situ hybridization). Representative images from one experiment from one out of three hamsters from each group are shown. Scale bars represent 100 µm. Figure ( a ) includes an image from Flaticon.com. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Sequencing, CRISPR, Knock-In, Western Blot, Mutagenesis, Staining, In Situ Hybridization

Journal: PLoS ONE

Article Title: Functional Characterization of a CRH Missense Mutation Identified in an ADNFLE Family

doi: 10.1371/journal.pone.0061306

Figure Lengend Snippet: To investigate intracellular distribution of CRH, cell were fixed in PFA and probed with mouse polyclonal anti-GM130 (red) for Golgi visualization and rabbit polyclonal anti-CRH (green) antibodies.

Article Snippet: After washing twice with PBS buffer, wells were probed overnight in PBS containing 5% (w/v) BSA with anti-CRH rabbit polyclonal antibody (1∶1000) (Source Biosciences).

Techniques:

Journal: Nature

Article Title: Protocadherin-1 is essential for cell entry by New World hantaviruses

doi: 10.1038/s41586-018-0702-1

Figure Lengend Snippet: a, b, Relative infectivity of rVSVs bearing the indicated viral glycoproteins. Wild-type (WT) and PCDH1-knockout (KO) HAP1 cell lines lacking (-cDNA) or expressing (+cDNA) WT human PCDH1 cDNA were exposed to rVSVs expressing hantavirus glycoproteins (rVSV-GPs) (a) or to hantaviruses (HVs) (b).a, Infected cells positive for enhanced green fluorescent protein (eGFP; pseudocoloured green) were detected by fluorescence microscopy. Representative images are shown. Scale bar, 100 μm. b, Hantavirus-infected cells were detected and enumerated by immunofluorescence microscopy. Averages ± s.d. from three experiments are shown in b; n = 6 (ANDV); n = 5 (SNV); WT versus PCDH1-KO cells, two-way ANOVA with Tukey’s test, ***P< 0.001 (n indicates the number of biologically independent samples). c, Expression of PCDH1 in HUVECs and HPMECs was detected by immunostaining with PCDHl-specific monoclonal antibody (mAb) 3305 or negative control antibody (see Extended Data Fig. 4d) and visualized by immunofluorescence microscopy. Scale bar, 20 μm. Experiments were performed three times with similar results. d, HPMECs transduced to co-express the endonuclease Cas9 and control or single-guide RNAs (sgRNAs) targeting PCDH1 were exposed to rVSVs. The results are averages ± s.d. from five experiments; n = 16 for ANDV; n = 18 for SNV; n = 14 for HTNV. PCDH1 sgRNA versus control sgRNA, two-way ANOVA with Sidak’s test; NS, P > 0.05; ****P < 0.0001.

Article Snippet: In brief, HAP1 and U2OS cells were exposed to virus at a MOI of 0.5–3 PFU per cell (ANDV and HTNV) or fluorescent focus-forming units (FFU) per cell (SNV) (see below for specific MOIs), and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection (see below for specific times) with rabbit polyclonal sera specific for ANDV, HTNV or SNV nucleoproteins (NR-9673, NR-9674 and NR-12152 (all from BEI Resources), respectively).

Techniques: Infection, Knock-Out, Expressing, Fluorescence, Microscopy, Immunofluorescence, Immunostaining, Negative Control, Control

Journal: Nature

Article Title: Protocadherin-1 is essential for cell entry by New World hantaviruses

doi: 10.1038/s41586-018-0702-1

Figure Lengend Snippet: a, Organization of PCDH1. b, U2OS PCDH1-KO cell lines complemented with the indicated PCDH1 proteins were exposed to rVSVs or hantaviruses. Left, averages ± s.e.m.: five experiments, n = 25 for ANDV and HTNV; five experiments, n = 15 for SNV except full-length PCDH1 (four experiments, n = 12)); four experiments, n = 24 for MPRLV and SEOV; three experiments, n = 10 for PHV. Right, averages ± s.d.: three experiments, n = 5 for ANDV and SNV, except SNV ΔEC1 and ΔEC2 (two experiments, n = 3)); two experiments, n = 4 for HTNV. c, Capacity of sEC1–2 (0–2.2 μM) to block authentic hantavirus infection. Viruses were preincubated with sEC1–2, and then allowed to infect WT U2OS cells. Averages ± s.d.: two experiments, n = 4 or 5 for ANDV; two experiments, n = 4 for HTNV. Untreated versus sEC1–2-treated, two-way ANOVA with Dunnett’s test; NS, P > 0.05; ****P < 0.0001. d, e, Capacity of soluble, truncated PCDH1 (sEC1–2, 0–5 μM) to block viral entry. rVSVs were preincubated with sEC1–2, and then allowed to infect HPMECs (d) and HUVECs (e). d, Averages ± s.d.: three experiments, n = 8 for ANDV and HTNV; n = 6 for SNV; n = 4 for MPRLV. e, Averages ± s.e.m.: six experiments, n = 15 or 16 for ANDV and HTNV; three experiments, n = 5 or 6 for SNV; four experiments, n = 8 for PHV. f, Capacity of PCDH1 EC1-specific mAb 3305 to block viral entry. HUVECs were preincubated with mAb 3305 (0–680 nM), and then exposed to rVSVs. Averages ± s.d.: three experiments, n = 4 for ANDV, SNV and HTNV); four experiments, n = 9 for PHV. Untreated versus antibody-treated by two-way ANOVA with Dunnett’s test: NS, P > 0.05; ****P < 0.0001.

Article Snippet: In brief, HAP1 and U2OS cells were exposed to virus at a MOI of 0.5–3 PFU per cell (ANDV and HTNV) or fluorescent focus-forming units (FFU) per cell (SNV) (see below for specific MOIs), and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection (see below for specific times) with rabbit polyclonal sera specific for ANDV, HTNV or SNV nucleoproteins (NR-9673, NR-9674 and NR-12152 (all from BEI Resources), respectively).

Techniques: Blocking Assay, Infection