rv b14  (ATCC)

Journal: bioRxiv

Article Title: A host ATPase essential for rhinovirus replication is an antiviral target with a high barrier to resistance

doi: 10.64898/2026.05.13.723454

Figure Lengend Snippet: (A-C) CB-6644 antiviral assays in cell lines. (A) HeLa-H1 cells, (B) BEAS-2B cells or (C) HeLa-E8 cells were infected with the indicated RV types (MOI 20 for A , MOI 1 for B-C ). Cells were treated at 1 hpi with DMSO or the indicated concentrations of CB-6644. Viral titres were quantified at the indicated times post-infection. N=4 or 5 independent experiments. (D-G) CB-6644 antiviral assays in WD-PNECs. (D) Workflow for generation of WD-PNEC cultures. Primary nasal epithelial cells (PNECs) were sampled via nasal brushing from volunteers, expanded in monolayers, and seeded into Transwells. When 100% confluent, after 4-8 days of incubation, apical medium was removed to initiate air-liquid interface (ALI), which triggers cell differentiation and the formation of a pseudostratified epithelium containing ciliated epithelial cells, goblet cells and basal cells. After 28 days of incubation, high quality WD-PNEC cultures were infected apically with the indicated RV (MOI 0.01). CB-6644 or DMSO was added apically 16 h before (E) or at different time points after (F) infection, as indicated. Viral titres in apical washes collected at the indicated times were quantified. N= 3 (E, RV-A16 and RV-B14) or 2 (E, RV-C15 and F) independent donors. (G) Viability of WD-PNECs apically treated with 2 μM CB-6644 or DMSO for 192 h, or with 1% Triton X-100 (TX100) for 2 h, presented as percentage viability relative to DMSO-treated control. N= 3 independent donors. For panels A-C and G, data are shown as individual points, coded by shape according to experimental replicate, with means. For panels E-F, data are shown as means (± SD) connected by lines colour-coded by treatment. Statistical tests: two-tailed paired t -test (A-C), one-way ANOVA with Dunnett’s post-hoc test (G). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See also Figure S2.

Article Snippet: RV-A1b (VR-1645, ATCC), RV-A16 (VR-283, ATCC), RV-A29 (VR-1809, ATCC), and RV-B14 (VR-284, ATCC) were propagated in HeLa-H1 cells.

Techniques: Infection, Incubation, Cell Differentiation, Control, Two Tailed Test

Journal: bioRxiv

Article Title: A host ATPase essential for rhinovirus replication is an antiviral target with a high barrier to resistance

doi: 10.64898/2026.05.13.723454

Figure Lengend Snippet: (A–C) CB-6644 inhibits RV RNA replication and NSP production. HeLa-H1 cells were infected with RV-A16 (MOI 20) and treated at 1 hpi with DMSO or 500 nM CB-6644. (A) Viral RNA was quantified by RT-qPCR at 0 hpi and 6 hpi. (B) At 6 hpi, lysates were analysed by western blotting for RV-A16 3C, RUVBL1, and lamin-B1. (C) 3C signal was quantified and normalised to lamin-B1. (D) Time-of-addition assay. HeLa-H1 cells were infected as above and treated with DMSO or 500 nM CB-6644 immediately after virus adsorption (0 hpi) or at the indicated times post-infection. Viral titres were quantified at 6 hpi. (E–J) siRNA knockdown of RUVBL1 inhibits RV RNA replication and NSP production. HeLa-H1 cells were transfected with siRNA targeting RUVBL1 or firefly luciferase for 72 h and then infected with RV-A16 (MOI 20). (E) Viral RNA was quantified by RT-qPCR at 0 hpi and 6 hpi. (F) At 6 hpi, lysates were analysed by western blotting for RV-A16 3C, RUVBL1, and lamin-B1. (G–H) Quantification of 3C and RUVBL1 signal from F, normalised to lamin-B1. (I) Immunofluorescence staining for RV-A16 2C (red) at 6 hpi; nuclei were stained with DAPI (blue). (J) Quantification of 2C-positive cells from (I). (K–L) RUVBL1/2 is required after RV entry. (K) HeLa-H1 cells were transfected with RV-A16, RV-A1a, or RV-B14 RNA in the presence of DMSO or 500 nM CB-6644. Viral titres were quantified at 14 h post-transfection. (L) Cell viability assessed in parallel of K in untransfected cells treated for 14 h with DMSO or 500 nM CB-6644. (M) RUVBL1/2 is not required for IRES-dependent translation. HeLa-H1 cells were transfected with a luciferase reporter RNA under RV-A16 IRES-dependent translational control, in the presence of DMSO, 500 nM CB-6644, or cycloheximide (CHX). Luciferase activity was measured at the indicated times. Values were t=0-subtracted and normalised to the DMSO 24 h post-transfection value within each experiment. The 0 h baseline is shown as a dashed grey line. (N-O) RUVBL1/2 is not required for RV-A16 polyprotein cleavage. (N) Myc-GFP-2BC3ABCD construct used for polyprotein processing assays, with expression under the control of a CMV promoter. (O) HeLa-H1 cells were transfected or not with the Myc-GFP-2BC3ABCD plasmid for 21 h, in the presence of DMSO or 500 nM CB-6644. In parallel, HeLa-H1 cells were infected or not with RV-A16 for 8 h. Lysates were then analysed by western blotting for Myc-GFP and RV-A16 2C, 3A and 3C. (P) CB-6644 inhibits negative-strand RNA synthesis. HeLa-H1 cells were infected with RV-A16 (MOI 20) and treated with DMSO or 500 nM CB-6644 at 1 hpi. Negative-strand RNA was quantified at the indicated times by RT-qPCR, normalised to 0 hpi. For all graph panels (A, C-E, G, H, J-M, P), data from 3-4 independent experiments are shown as individual points, coded by shape according to experimental replicate, with means (connected by lines in M and P). Non-graph panels (B, F, I, O) show representative images from 3 independent experiments. Statistical tests: two-tailed paired t-test (A, C, E, G, H, J-L), one-way ANOVA with Dunnett’s post-hoc test (D), two-way ANOVA, comparing drug treatments to the DMSO control at each time point (M, P). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. See also Figure S2 and S3.

Article Snippet: RV-A1b (VR-1645, ATCC), RV-A16 (VR-283, ATCC), RV-A29 (VR-1809, ATCC), and RV-B14 (VR-284, ATCC) were propagated in HeLa-H1 cells.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Virus, Adsorption, Knockdown, Transfection, Luciferase, Immunofluorescence, Staining, Control, Activity Assay, Construct, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Genome Biology

Article Title: Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

doi: 10.1186/s13059-021-02513-w

Figure Lengend Snippet: Validation of the top 10 hits from surfaceome and genome-wide screens. a Cell viability assay for determination of the protective effects of identified gene knockout on RV-B14-induced cell death. The assay is performed at 24 h post infection of RV-B14 at an MOI of 2. b RT-qPCR quantification of viral loads in medium supernatant. The supernatant is harvested at 24 h post infection of RV-B14 at an MOI of 2. c Immunofluorescence (IF) staining of RV-B14 envelope protein for evaluation of infection rates at individual cell lines. IF staining is performed at 16 h post infection of RV-B14 at an MOI of 2. Each biological replicate contains the quantification results from 2000 cells. d , e RT-qPCR quantification of viral loads in medium supernatant ( d ) and the lysate ( e ) of mock and knockout single clones. The supernatant and cell lysate are harvested at 24 h post infection of RV-B14 at an MOI of 2. The significant difference between knockout cells and non-targeting sgRNA mock groups are determined using two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB).

Techniques: Genome Wide, Viability Assay, Gene Knockout, Infection, Quantitative RT-PCR, Immunofluorescence, Staining, Knock-Out, Clone Assay, Two Tailed Test

Journal: Genome Biology

Article Title: Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

doi: 10.1186/s13059-021-02513-w

Figure Lengend Snippet: Validation of the effects of ICAM-1, RAB5C, and OLFML3 on RV infection. a , b Time-dependent viral replication of RV-B14 in mock and knockout cells, as determined by viral loads in medium supernatant ( a ) and cell lysate ( b ). Cells are infected with RV-B14 at an MOI of 2. Viral RNA in cell lysates ( b ) is normalized to RPLP0 expression. Significant difference between test groups and non-targeting sgRNA group is determined using two-way ANOVA with Dunnett’s multiple comparisons test. c , d Rescued susceptibility of knockout cells to the infection of RV-B14 ( c ) and RV-A16 ( d ) by overexpression of RAB5C and OLFML3 respectively. e , f Rescued replication of RV-B14 ( e ) and RV-A16 ( f ) in knockout cells by overexpression of RAB5C and OLFML3 respectively, as determined by viral loads in medium supernatant. g , h Rescued replication of RV-B14 ( g ) and RV-A16 ( h ) in knockout cells by overexpression of RAB5C and OLFML3 respectively, as determined by viral loads in cell lysates. Viral RNA in cell lysates is normalized to RPLP0 expression. i Representative images of CPEs induced by clinical RV strain. Scale bar, 100 μm. j Cell viability of mock and knockout cells upon challenge of clinically isolated RV strain. k Phylogenetic analyses of clinical RV strain using MEGA X , with VP4 gene as the reference. l , m The effects of gene knockout on the replication of clinical RV strain, as determined by RT-qPCR quantification of viral loads in medium supernatant ( l ) or cell lysates ( m ). For m , viral RNA in cell lysates is normalized to RPLP0 expression. For c – j and l , m , analyses are performed at 24 h post infection of RV at an MOI of 2. Significant difference between test groups and non-targeting sgRNA group is determined using two-tailed Student’s t test and the P values are shown. Significant difference between knockout and overexpression rescue groups is determined using two-tailed Student’s t test and the P values are shown above the lines

Article Snippet: To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB).

Techniques: Infection, Knock-Out, Expressing, Over Expression, Isolation, Gene Knockout, Quantitative RT-PCR, Two Tailed Test

Journal: Genome Biology

Article Title: Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

doi: 10.1186/s13059-021-02513-w

Figure Lengend Snippet: Dissection of the functions of RAB5C and OLFML3 in RV infection. a , b Viral loads in medium supernatant ( a ) and cell lysates ( b ) at 24 h after transfection of RV-A16 genome RNA. c , d Viral RNA in cell lysates of mock and knockout cells at 1, 3, and 6 h after infection with RV-B14 ( c ) and RV-A16 ( d ) at an MOI of 20 in the presence of 2 mM GuHCl. For a , b , significant difference between mock and test groups is determined using two-tailed Student’s t test. For c , d , significant difference between mock and RAB5C groups is determined using two-tailed Student’s t test. e Volcano plot showing differentially expressed genes (DEGs). RV infection-induced gene upregulation and downregulation are first calculated and the differentially upregulated or downregulated genes in mock and knockout cells are defined as DEGs. Cells are harvested and analyzed at 24 h after infection of RV-B14 at an MOI of 2. f GO analyses of biological processes of DEGs identified in E. g , h Heat map showing ISG expression in mock and OLFML3 −/− cells at 24 h post infection of RV-B14 ( g ) and RV-A16 ( h ) at an MOI of 2. These results are derived from RT-qPCR quantification. Gene expression is normalized to RPLP0

Article Snippet: To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB).

Techniques: Dissection, Infection, Transfection, Knock-Out, Two Tailed Test, Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Genome Biology

Article Title: Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

doi: 10.1186/s13059-021-02513-w

Figure Lengend Snippet: OLFML3 is a RV-inducible suppressor of type I IFN signaling during RV infection. a Structural organization of OLFML proteins. b RT-qPCR quantification of OLFML3 expression levels in H1-Hela cells in the absence or presence of RV-B14 or RV-A16. Samples are collected at 24 h post infection with a RV MOI of 2. c RT-qPCR quantification of IFIT2, OAS2, and ISG15 expression in WT and OLFML3 −/− cells at 0, 12, and 24 h after RV-B14 infection at an MOI of 2. d RT-qPCR quantification of IFNB, STAT1, and STAT2 expression in WT and OLFML3 −/− cells at 0, 12, and 24 h after RV-B14 infection at an MOI of 2. For c and d , gene expression is normalized to RPLP0 and significant difference was determined using two-way ANOVA with Sidak’s multiple comparisons test. Gene abbreviations are as follows: IFNB, interferon β; ISG15, IFN-stimulating genes 15; IFIT2, interferon induced protein with tetratricopeptide repeats 2; OAS2, 2′-5′-oligoadenylate synthetase 1; SOCS3, suppressor of cytokine signaling 3

Article Snippet: To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB).

Techniques: Infection, Quantitative RT-PCR, Expressing

Journal: Genome Biology

Article Title: Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

doi: 10.1186/s13059-021-02513-w

Figure Lengend Snippet: OLFML3 antagonizes type I IFN signaling in an SOCS3-dependent mechanism. a mRNA expression of SOCS3 in response to RV-B14 infection. Samples are collected at 0, 12, and 24 h after infection at an MOI of 2 and the fold change of SOCS3 expression before and after RV infection is shown. b RT-qPCR quantification of SOCS3 expression in mock or OLFML3 −/− cells in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. c RT-qPCR quantification of viral RNA in cell lysate in mock or OLFML3 −/− cells in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. Significant difference between non-target and each other group is determined unless indicated otherwise. d RT-qPCR quantification of STAT1, STAT2, IFNB, OAS2, ISG15, and IFIT2 expression in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. Significant difference between OLFML −/− and each other group is determined unless indicated otherwise. For a – d , gene expression is normalized to RPLP0 and significant difference is determined using Student’s t test. NS, no significance. e Schematic diagram illustrating OLFML3 and SOCS3-mediated inhibition of type I IFN signaling during RV infection. Gene abbreviations are as follows: RVs, rhinoviruses; ICAM-1, intercellular adhesion molecule 1; OLFML3, olfactomedin-like 3; SOCS3, suppressor of cytokine signaling 3; IFNs, interferons; IFNAR1, type I interferon receptor α chain; ISREs, interferon-stimulated response elements; ISGs, interferon-stimulating genes

Article Snippet: To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB).

Techniques: Expressing, Infection, Quantitative RT-PCR, Inhibition