Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A) Schematic of CRISPR-based KO screen done in A549 lung epithelial cells for the identification of LCMV host factors. (B) Gene enrichment for CRISPR screen of rLCMV-mCherry infection. Enrichment scores were determined by MaGECK analysis and genes were colored by biological function. Dotted line indicates −log10(Enrichment Score) = 4. All genes and their enrichment scores can be found in Table S1 . (C) Percentage of infected cells as determined by flow cytometry following infection of A549 homozygous knockouts (CD164, SPR14, IL2RA, KHNYN) or heterozygous knockouts (ARFRP1, YKT6, ACKR4, RAB10, EMC1, SYS1) with rLCMV-mCherry. Wildtype cells were used as normalization controls. Cells were infected at MOI 1 and harvested at 24 hpi. Error bars indicate standard error of three independent experiments. (D) Quantification of viral infection in WT, Δ CD164 , Δ CD164 complemented with human CD164 (Δ CD164 + hCD164 ), and Δ CD164 complemented with mouse Cd164 (Δ CD164 + mCd164 ) in A549, 293T, and 3T6 cell type backgrounds. Cells were infected with rLCMV-mCherry at MOI 1 and harvested at 24 hpi. Error bars indicate standard error of three independent experiments.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: CRISPR, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A) Log fold changes (LFC) of individual sgRNA of the top 10 scoring genes and CD164 (red) when comparing the infected and sorted cell population versus the uninfected cell population. Overall sgRNA distribution is shown at the bottom of the graph and dotted line indicates mean LFC of all sgRNAs. (B-D) Dose-response curve of v-ATPase inhibitors on rLCMV-mCherry infection at MOI 1 in A549 cells at 24 hpi, yielding (B) Bafilomycin A 1 IC50 = 2.96 nM, (C) Bafilomycin B 1 IC50 = 4.97 nM, and (D) Concanamycin A IC50 = 0.83 nM. Error bars indicate standard error of three independent experiments. (E) Genotyping of clonal A549 where the target loci were PCR-amplified, Sanger-sequenced, and aligned to WT reference sequence. (F) Analysis of cell proliferation of WT and clonal A549 KO cells. Cells were plated in 96-well and proliferation was measured daily using Cell Titer Glo. Error bars indicate standard error from three separate well per cell line per time point. (G-I) Western blot analysis of WT, Δ CD164 , Δ CD164 + hCD164 , and Δ CD164 + mCd164 for A549, 293T, and 3T6 cell lines. Human cell lines (A549 and 293T) were probed with anti-hCD164 antibody except for the mCd164 addback which was probed with anti-mCd164 antibody. Mouse cell line 3T6 was probed with anti-mCd164 antibody except for the hCD164 addback, which was probed with anti-hCD164 antibody. GAPDH was used as loading control.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Infection, Amplification, Sequencing, Western Blot

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A-B) Western blot analysis of Δ CD164 , Δ DAG1 , and Δ CD164 /Δ DAG1 double KO cells in (A) A549 or (B) 293T cell backgrounds. GAPDH was used as a loading control.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A-E) Percent infection of Δ CD164 , Δ DAG1 , and Δ CD164 /Δ DAG1 double-KO cells relative to WT in either A549 or 293T cell type backgrounds following inoculation with low DAG1 affinity LCMV strains (A) Armstrong 53b-GP or (B) WE2.2-GP, and high DAG1 affinity strains (C) Armstrong Clone 13-GP, (D) W54-GP, or (E) WE-GP pseudotyped virus as determined by flow cytometry for GFP positivity. Cells were infected at MOI 1 and measured 24 hpi. Error bars indicate standard error of three independent experiments. (F-H) Percent infection of ΔCD164, ΔDAG1, and ΔCD164/ΔDAG1 double-KO cells relative to WT in either A549 or 293T cell type backgrounds following inoculation with (D) LASV-GP, (E) GTOV-GP, or (F) MACV-GP pseudotyped virus as determined by flow cytometry for GFP positivity. Cells were infected at MOI 1 and measured 24 hpi. Error bars indicate standard error of three independent experiments.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A) Schematic (left) of wildtype, Δ CD164 , Δ CD164 + hCD164 (ΔE1), Δ CD164 + hCD164 (ΔE1-2), Δ CD164 + hCD164 (ΔE1-3), Δ CD164 + hCD164 (ΔE1-4), Δ CD164 + hCD164 (ΔE1-5), and Δ CD164 + hCD164 (ΔE2-6). Complemented A549 and 293T cells were challenged with rLCMV-mCherry (MOI 1) and infection was measured by flow cytometry at 24 hpi (right). Percent infection was normalized to wildtype. Error bars represent standard error of three independent experiments. (B) Schematic (left) of wildtype, ΔCD164 KO + hCD164(N72A), ΔCD164 + hCD164(N77A), ΔCD164 + hCD164(N94A), and ΔCD164 + hCD164(N104A). Complemented A549 and 293T cells were challenged with rLCMV-mCherry (MOI 1) and infection was measured by flow cytometry at 24 hpi (right). Percent infection was normalized to wildtype. Error bars represent standard error of three independent experiments. (C) Amino acid similarities of the cysteine-rich region in human CD164 and mouse Cd164 determined using the ClustalW program on SnapGene. Yellow circles indicate cysteine residues, red N symbolizes N-linked glycosylation sites, and identical amino acids are highlighted in green. (D) Blockade of LCMV infection with serial dilutions of anti-human CD164 monoclonal mouse antibody clone N6B6 or mouse IgG2a-κ isotope control in wild type A549 cells. Cells were infected at MOI 1 and infection measured at 24 hpi. Error bars indicate standard error of three independent experiments.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A-B) Western blot analysis of deletion domain addbacks in (A) A549 or (B) 293T cell backgrounds. (C-D) Western blot analysis of alanine mutagenesis addbacks in (C) A549 or (D) 293T cell backgrounds. All CD164 addbacks were probed with anti-FLAG antibody. GAPDH was used as a loading control. (E) AlphaFold prediction of CD164 protein structure. Prediction had low position error for the signal peptide, the cysteine-rich region, the transmembrane domain, and the cytoplasmic tail and high position error for the two mucin domains. Location of residue 104 is noted with an arrow.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A) Double immunofluorescence staining for CD164 and CK7 or isotypes staining followed by counterstaining with DAPI in villous trophoblastic tissue. Original images were taken by confocal microscopy at 100x magnification. Scale bar represents 20 μm. (B) Immunofluorescence imaging of JEG-3 placenta cells pre-incubated with various concentrations of anti-CD164 mAb N6B6 and infected with r3LCMV-mCherry at MOI 0.5. Cells were fixed and imaged at 10x magnification 24 hpi. Scale bar represents 20 μm. (C) Quantification of percent infection of JEG-3 placenta cells pre-incubated with various concentrations of anti-CD164 mAb N6B6 and infected with r3LCMV-mCherry at MOI 0.5. Analysis was done on 4 FOV in 2 independent infections and normalized to infection control.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Double Immunofluorescence Staining, Staining, Confocal Microscopy, Immunofluorescence, Imaging, Incubation, Infection

Journal: bioRxiv

Article Title: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

doi: 10.1101/2022.01.24.477570

Figure Lengend Snippet: (A) Immunofluorescence staining of CD164 or isotype control followed by counterstaining with DAPI on placenta tissue at the maternal decidua and fetal villi. Original images taken at 40x magnification. Scale bar represents 50 μm. (B) Immunofluorescence imaging of CD164 or isotype control followed by counterstaining with DAPI on JEG-3 placenta cell line. Original images taken at 10x magnification. Scale bar represents 100 μm. (C) Immunofluorescence imaging JEG-3 placenta cell line with and without infection by r3LCMV-mCherry at MOI 1 and imaged at 24 hpi. Original images taken at 10x magnification. Scale bar represents 100 μm.

Article Snippet: Human CD164 (Origene, #RC202234) and mouse Cd164 (Origene, #MR201951) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene #17452, gift from Eric Campeau & Paul Kaufman)( ) using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Techniques: Immunofluorescence, Staining, Imaging, Infection