Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Phylogenetic tree of mammarenavirus GPC amino acid sequences. Tree generated via MUSCLE alignment, Gblock curation, PhyML phylogeny, and rendered with TreeDyn using phylogeny.fr. (B) Enrichment of guide RNAs from a CRISPR-Cas9 loss-of-function genome-wide screen after the second round of infection with chimeric VSV-eGFP-PICV (CoAN3739). Genes arranged by rank on the x-axis; significance (positive score) on the y-axis. (n=1, experimental replicate). (C) WT A549, CD164 KO , and CD164 KO +CD164 cells were infected with WT or chimeric VSV-eGFP expressing indicated glycoproteins (MACV, LCMV, PICV CoAN4763, PICV CoAN3739, PARV, FLEXV) at MOI=3. Six hours post-infection, % GFP-positive cells determined by flow cytometry (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005. (D) WT A549, CD164 KO , and CD164 KO +CD164 cells were used for plaque assays with WT LCMV (Armstrong), PICV (CoAN4763), and PARV. Dotted line indicates limit of detection (LOD). (n=3, independent experiments in triplicate). One-way ANOVA with Tukey’s multiple comparisons: *p< 0.05, **p< 0.005, ***p< 0.0005.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Generated, CRISPR, Genome Wide, Infection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Left: domain architecture of CD164 and deletion mutants; predicted N-linked glycosylation sites (purple), O-linked sites (grey). Right: schematic comparing CD164 and CRD-LAMP1 chimeric protein (CD164 CRD: blue; LAMP1 proximal domain: red). (B) CD164 KO HeLa cells expressing empty vector, CD164, CD164 deletion mutants (ΔMD1, ΔCRD, ΔMD2), or CRD-LAMP1 were inoculated with chimeric VSV-eGFP expressing indicated glycoproteins (MOI=2). % GFP-positive cells determined 6 hpi by flow cytometry; normalized to WT HeLa cells (Relative Infection) (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Glycoproteomics, Expressing, Plasmid Preparation, Flow Cytometry, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Acid by-pass assay in CD164 KO HeLa cells expressing plasma membrane-targeted CD164 (WT-CD164 PM ). VSV-eGFP-PICV3739 was bound to Bafilomycin A1-pretreated cells (MOI=3, 4°C, 1 hr) then fusion was triggered at indicated pH, media replaced, and infection proceeded for 5 hr in Bafilomycin A1. % GFP-positive cells normalized to WT HeLa at pH 5.0 (n=3, independent experiments in triplicate and technical replicates in duplicate). (B) Same assay as in (A) performed with WT, CD164 KO cells expressing empty vector, or WT-CD164 PM cells, with fusion triggered at pH 5.2. % GFP-positive cells normalized to WT HeLa (dotted grey line) (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005. (C) WT or CD164 KO A549 cells pretreated with cycloheximide were inoculated with VSV-P/eGFP-PICV3739 (MOI=400, 3 hr). Cells were fixed, stained with DAPI, WGA, anti-VSV-M, and imaged by Airyscan confocal microscopy. Representative cell shown per condition. VSV-M: red; P/eGFP: green; colocalization: yellow; WGA: white; DAPI; blue. Scale bar, 10 µm. (D) Quantification of P/eGFP and VSV-M colocalization from (C) (20 cells per condition). One-Way ANOVA (left) or Kruskal-Wallis ANOVA (right), * p <0.05, ** p <0.005, *** p <0.0005..

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Expressing, Clinical Proteomics, Membrane, Infection, Plasmid Preparation, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: Additional representative images from . WT or CD164 KO A549 cells pretreated with cycloheximide or DMSO, inoculated with VSV-P/eGFP-PICV3739 (A, 3 hr) or VSV-P/eGFP-G (B, 3 h). Cells were fixed, stained with DAPI (blue), WGA (Alexa 647, white), anti-VSV-M (Alexa 594, red), imaged by Airyscan confocal microscopy. Scale bar, 10 µm. (C) Widefield microscopy of WT or CD164 KO A549 pretreated with cycloheximide, inoculated with VSV-P/eGFP-PICV3739, fixed, and stained at 2 hours post-infection.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Staining, Confocal Microscopy, Microscopy, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Binding of 2.5 µM PICV sGP1 to 100 nM immobilized sCD164 as a function of pH; baseline corrected to binding of MACV sGP1 at same pH (n=3, independent experiments in triplicate). (B) Final binding response of 2.5 µM PICV, PARV, FLEXV, and MACV sGP1 to sCD164 at pH 5.0 (blue) or 7.4 (grey) (n=3, independent experiments in triplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.00005. (C) pH 5.0 binding curve of PICV sGP1 to sCD164 (100 nM). Left: 1:1 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinity (K D ) calculated from 1:1 Langmuir fit, K D = 0.259 µM ± 0.001 µM. (n=3, independent experiments in triplicate). (D) pH 5.0 binding of PICV sGP1 to soluble sCRD (200 nM). Left: 1:2 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinities (K D ) calculated from 1:2 Langmuir fit K D1 = 1.144 μM ± 0.019 µM, and K D2 = 31.86 μM ± 0.555 µM. (n=3, independent experiments in triplicate) (E) Mean fluorescence intensity (MFI/10,000) of AF647-PICV sGP1 binding to CD164 KO +WT-CD164 PM or CD164 KO cells at pH 6.0 or 7.4 (n=3, independent experiments in triplicate). One-Way ANOVA of AUC: *p< 0.05, **p< 0.005, ***p< 0.0005, ****p< 0.00005.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Coomassie-stained SDS-PAGE of soluble proteins used in . 2.5 µg of PNGaseF treated, nondenatured, or denatured purified protein. (B) Nonlinear fit of binding curves at pH 5.0 (blue) and 7.4 (grey) for PICV sGP1, PARV sGP1, FLEXV sGP1, and MACV sGP1 to 100 nM of immobilized sCD164. Curves represent the averaged final response at the end of association (n=3, independent experiments in triplicate). (C) Binding of 5 µM MACV sGP1 (maroon triangle) or 1 µg mouse anti-human TfR1 (CD71) antibody (cyan diamond) to 100 nM of immobilized hTfR1-Fc as a function of pH. Normalized to max binding at pH 8.0 (n=2, independent experiments in duplicate). (D) pH 7.4 binding curve of MACV sGP1 to hTfR1 (100 nM). Left: 1:1 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinity (K D ) calculated from 1:1 Langmuir fit, K D = 2.52 µM ± 0.006 µM. (n=2, independent experiments in duplicate). (E) Representative histogram of mean fluorescence intensity (MFI) from . Cells bound with 2.5 µM AF647-labeled PICV sGP1 at pH=6.0 (orange: CD164 KO +WT-CD164 PM ; purple: CD164 KO ) or pH 7.4 (red: CD164 KO +CD164 PM ; blue: CD164 KO ) (n=3, independent experiments in triplicate) (F) MFI (/10,000) of CD164 KO +WT-CD164 PM or CD164 KO cells from and S4E, labeled with 5 µM AF647-PICV sGP1 at pH 6.0 and then washed at pH 7.4 (hashed) or 6.0 (solid). (n=3, independent experiments in triplicate).

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Staining, SDS Page, Purification, Binding Assay, Fluorescence, Labeling

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Alphafold3 predicted structure of PICV sGP1 (yellow) interacting with the CRD of CD164 (blue). Left: full predicted structure; right: zoom on predicted interaction region. Interface Predicted Template Modeling (ipTM) score=0.81. (B) DMS of CRD identifies key determinants for the acid-dependent GP1 interaction. DMS selection for loss of GP1 binding at pH 6.0 (GP1-sorted population S5A). Top: heat map of mutation differential scores (dms_tools2); blue: enriched mutations (>0), red: de-enriched (<0). Bottom: total enriched positive changes per amino acid site (score>0). (n=1, experimental replicate). (C) Averaged final association response of PICV sGP1 binding to sCD164 containing indicated mutations in CRD. Blue: response >WT sCD164 (0.066, white); red: response WT (white, 1.0); red:

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Selection, Binding Assay, Mutagenesis, Expressing, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Reducing/denaturing or non-denaturing Coomassie of sCD164 mutant proteins . Asterisk (*) denotes denaturing. (B) Final binding response of 1 µg N6B6 anti-human CD164 antibody to 100nM immobilized sCD164 mutants. (C) Inhibition of chimeric VSV expressing indicated glycoproteins by anti-CD164 monoclonal antibody, N6B6. Cells were pretreated with N6B6 for 1 hour on ice, virus was then added at MOI=1. At 6 hours post-infection, cells were fixed and % GFP-positive cells was determined by flow cytometry. % GPF+ was normalized to HeLa cells infected with no inhibition (n=2). (D) Relative expression of CD164 in CD164 mutant addback cells. Top: area under the curve (CD164/actin) compared to WT; bottom: representative Western blot. (E) Representative histograms of total CD164 expression in CD164 mutant addback cells.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Mutagenesis, Binding Assay, Inhibition, Expressing, Virus, Infection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Impact of alanine mutations within the predicted ß-sheet of PICV sGP1 on binding to CD164. Binding of 2.5 µM WT or mutant PICV sGP1 to 100 nM of immobilized sCD164. Averaged response over association and dissociation measured by BLI. (n=3, independent experiments in triplicate). (B) Plaque assays in WT and CD164 KO A549 infected with VSV expressing WT PICV GPC (yellow), K213A mutant (orange), F215A-N216A mutant (pink). Dotted line: LOD. (n=3, independent experiments in triplicate). Two-tailed unpaired t-test, of CD164 KO compared to WT infected with indicated virus: *p< 0.05, **p< 0.005, ***p< 0.0005. (C) Impact of stabilizing ß-sheet mutations (predicted by Protein MPNN) on CD164 binding, measured by BLI as in (A) (n=3, independent experiments in triplicate). (D) Impact of new consensus sequence generated by Protein MPNN analysis (RCTRSC, orange) on GPC-mediated infection by plaque assay in WT and CD164 KO A549 cells as in (B) (n=3, independent experiments in triplicate). Two-tailed unpaired t-test, of CD164 KO compared to WT infected with indicated virus: *p< 0.05, **p< 0.005, ***p< 0.0005

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Binding Assay, Mutagenesis, Infection, Expressing, Two Tailed Test, Virus, Sequencing, Generated, Plaque Assay