human ace2 gene  (Addgene inc)

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) Enrichment analysis of the expression levels of interferon gamma signaling genes in the indicated cell lines using publicly available RNA-seq data sets. (B) Comparison of the expression levels of ACE2 protein (x-axis) and mRNAs in the interferon gamma signaling pathway (y-axis) in the indicated cell lines. (C) UMAP analysis of cell surface protein expression in the indicated cell lines. (D) RT-qPCR analysis of ACE2 mRNA expression in the indicated cell lines. (E) Immunoblot analysis of ACE2 and β-actin expression in the indicated cell lines. (F) Cells were inoculated with SARS-CoV-2 (USA-WA1-2020) bearing WT S or S E484D or B.1.526 SARS-CoV-2 variant bearing S-E484K substitution at an MOI of 1 i.u./cell. Cell-associated viral RNA levels was analyzed at 4 h and 72 h post-infection by RT-qPCR from n=3 independent replicates. Data show the mean, error bars show the SD.

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Expressing, RNA Sequencing, Comparison, Quantitative RT-PCR, Western Blot, Variant Assay, Infection

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522-ACE2 KO, H661 and H661-ACE2 KO cells were inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. RT-qPCR analysis of cell-associated viral RNA at 4 and 72 hpi is shown. (B) WT H661 and four different H661 ACE2 KO clonal cells were inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. RT-qPCR for cell-associated viral RNA at 72 hpi is shown. (C) H522 and H661 WT cells pre-treated with 50 mM ammonium chloride for 2 h and inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. At 24 hpi, ammonium chloride was removed, and cells were replenished with fresh media. RT-qPCR analysis of cell-associated SARS-CoV-2 RNA at 48 hpi is shown. (D) H522 and H661 cells were pre-treated with a combination of heparinase I, II and III for 90 min. Cells were subsequently inoculated with SARS-CoV-2-mNG S E484D at an MOI of 0.1. RT-qPCR for cell-associated SARS-CoV-2 RNA at 72 hpi is shown. (E, F) H522 (E) and H661 cells (F) were infected with SARS-CoV-2-mNG S E484D (MOI of 0.1) in the presence of mAbs. Amount of cell-associated vRNA was quantified as above. All data show the mean from three independent biological replicates with error bars displaying the SEM. (nonsignificant (ns); ** P < 0.01; *** P < 0.001; **** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Quantitative RT-PCR, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 TMEM106B KO cells stably transduced with an empty vector (EV), full length TMEM106B (FL), TMEM106BΔCTD, TMEM106BΔNTD as well as H522 cells stably expressing ACE2 were inoculated with SARS-CoV-2-mNG S E484D or WT SARS-CoV-2-mNG at an MOI of 1 at 16°C. At 1 hpi, cells were fixed and analyzed for viral RNA (green) using RNAScope as detailed in Materials and Methods. Nuclear DAPI staining is shown in blue. (B) Quantification of the data shown in A. For S E484D infections, all data was compared to EV, for S-WT infections H522-ACE2 OE data was compared to H522-TMEM106BΔNTD (* P < 0.05; **** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons). Scale bar=10μM.

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, RNAscope, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 WT cells were inoculated with WT VSV-GFP-SARS-CoV-2 S (WT) or a derivative bearing the S E484D substitution at an MOI of 1. GFP positive cells were enumerated by flow cytometry at 48 hpi. (B) VSV-GFP-SARS-CoV-2 S E484D was serially passaged in H522 cells for 8 rounds as explained in Materials and Methods. Aliquots of virus from each passage was titered on H522 and H522-TMEM106B KO cells using 1, 5, 25 or 125 µl of the inoculum. Data show the percentage of GFP positive cells at the indicated inoculum for all 8 passages. (C) Aliquots of virus from (B) was titered on H661 cells. (D) Five different virus clones were plaque purified from Passage 7 and sequenced. Table shows the observed substitutions and their location within S. (E-G) H522-ACE2 ( E ), H522 ( F ), and H661 ( G ) cells were inoculated with parental VSV-GFP-SARS-CoV-2 S E484D or the five different plaque purified virus clones from (D) at an MOI of 0.1. H522-ACE2 cells were fixed at 16 hpi, and H522 and H661 were fixed at 48 hpi for enumeration of GFP positive cells by flow cytometry. Data show the mean from three independent experiments, error bars show the SEM. For D, E, and F, all five clones were compared to the parental virus (nonsignificant (ns), * P < 0.05 by one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Flow Cytometry, Virus, Clone Assay, Purification

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Diagram of the RBD substitutions that distinguish Omicron BA.2.86 from Wuhan-Hu-1 (top), Omicron KP.3.1.1 from BA.2.86 (middle), and Omicron LP.8.1 from BA.2.86 (bottom). Dashed lines show propagation of BA.2.86 changes to KP.3.1.1 and LP.8.1, and italicized mutation in LP.8.1 (H445R) indicates a secondary substitution at a site that previously changed from Wuhan-Hu-1 to BA.2.86. Wuhan-Hu-1 reference spike numbering is used throughout the manuscript. ( B-D ) Quality control of the KP.3.1.1 and LP.8.1 RBD site-saturation mutagenesis library as assessed by PacBio sequencing, illustrating the distribution of number of amino acid mutations per barcoded variant (B), the average number of mutations of each type across library variants (C), and the distribution of mutations across sites in the RBD over all variants (D). ( E, F ) Representative FACS gates used to sort RBD + singlet cells for ACE2 binding (E) and singlet cells for RBD expression (F), which is followed by high-throughput sequencing of cells sorted into each bin. ( G, H ) Correlation in per-mutant deep mutational scanning measurements between independently barcoded replicate libraries for ACE2-binding affinity (G) and RBD expression (H) experiments. Red dash indicates the 1:1 line.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Control, PacBio Sequencing, Variant Assay, Binding Assay, Expressing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on ACE2-binding affinity as determined from FACS-seq experiments with yeast-displayed RBD mutant libraries. ACE2 contact residues (black squares, bottom) defined as RBD residues with non-hydrogen atoms <5Å from ACE2 in the BA.2.86 RBD structure (PDB 8QSQ ). Raw data available in Supplemental Data 1. ( B ) Deep mutational scanning data from (A) mapped to the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ), illustrating the average effect of mutations at a site (left), and the maximal effect of any mutation at a site (right). Sites of interest are labeled. ACE2 (key motifs only) is shown as transparent gray cartoon. ( C ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on yeast-surface expression levels, a proxy for folding and expression efficiency. Raw data available in Supplemental Data 1. An interactive version of these heatmaps alongside previously assayed SARS-CoV-2 variant RBDs is available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/RBD-heatmaps/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Labeling, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Correlation between KP.3.1.1 mutant effects on ACE2 binding in the pseudovirus whole-spike DMS study of Dadonaite et al. (change in potency of pseudoviral neutralization via soluble ACE2 protein) versus the yeast-display RBD DMS study presented here (change in binding affinity from FACS-seq titration). Correlations are separated based on residue distance from the ACE2 interface in the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ). Mutations whose effect on spike-mediated pseudoviral entry was < -2 units per the assay of Dadonaite et al. were not included because it is difficult to reliably measure soluble-ACE2-inhibition of entry when entry is poor. ( B ) Structural context of residues F374 and H505 in the spike trimer (PDB 9ELH ). Center, overall top-down view of the spike trimer with two RBDs in the “down” and one in the “up” conformation. Left, zoom-in on the down-down interface of RBD packing; right, zoom-in on the up-down interface of RBD packing.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Binding Assay, Neutralization, Titration, Residue, Inhibition

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Epistatic shift in the effects of mutations on ACE2 binding at each RBD position as measured in the Wuhan-Hu-1 (previously reported in ), KP.3.1.1 or LP.8.1 background compared to those previously measured in Omicron BA.2.86 (previously reported in ). ( B ) Mutation-level plots of epistatic shifts between BA.2.86 and KP.3.1.1 or LP.8.1 at sites of interest. Each scatterplot shows the measured ACE2-binding affinity of each amino acid (plotting character, – indicates deletion character) in the BA.2.86 versus KP.3.1.1 or LP.8.1 backgrounds. Red dashed lines mark the respective wildtype RBD affinities on each axis, and the gray dashed line indicates the additive (non-epistatic) expectation. Interactive plots enabling the comparison of all SARS-CoV-2 variants and scatterplots for all RBD sites are available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/epistatic-shifts/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Comparison