human rhinovirus 16 strain 11757  (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) Workflow for the identification of RV-A16 2C interactors and their functional validation. HeLa-H1 cells were infected in triplicate with RV-A16 (MOI 20) for 4.5 h, 6 h or were left uninfected. Protein-protein interactions were then cross-linked with formaldehyde and cell lysates were immunoprecipitated using RV-A16 2C-specific or control antisera (2C-IP or control-IP). Eluates were TMT-labelled, multiplexed and analysed by LC-MS/MS. Differences between infected and uninfected conditions were assessed using two-sample Student’s t -test. Cellular proteins significantly enriched in infected 2C-IP fractions were further investigated for their role in RV-A16 replication through siRNA screening. The two most significant non-cytotoxic hits from the siRNA screen, RUVBL1 and RUVBL2, were further validated using a small molecule inhibitor. (B-C) Proteomics analysis of 2C-IPs. RV-A16 2C was immunoprecipitated from infected cells and the associated proteins were analysed as described in A. (B) Volcano plots showing in red the cellular proteins significantly enriched in 2C-IPs at 4.5 hpi (top) and 6 hpi (bottom), compared to uninfected conditions. (C) Corresponding heatmap, also showing infected control-IPs. (D-E) siRNA screen of the 2C-IP hits. HeLa-H1 cells were transfected with siRNA pools targeting the 2C-IPs hits or a non-targeting (NT) control siRNA pool. (D) At 72 h post-transfection, cells were infected with RV-A16 (MOI 20), and viral titres were quantified at 6 hpi. Data are presented as a percentage of the mean NT control (dashed grey line). (E) Viability of uninfected cells at 72 h post-transfection, for siRNA significantly reducing viral replication, presented as percentage of the mean NT control (dashed grey line). Statistical tests: one-way ANOVA with Dunnett’s post-hoc test, comparing to the NT control pool. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (F-G) Validation using CB-6644, a small molecule inhibitor of the ATPase activity of the RUVBL1/2 complex. (F) HeLa-H1 cells were infected with RV-A16 (MOI 20) and treated at 1 hpi with DMSO or increasing concentrations of CB-6644. Viral titres were quantified at 0 hpi and 6 hpi. Viral titres in CB-6644-treated cells at 6 hpi are shown as individual points with means connected by a line. Mean viral titres of untreated cells at 0 hpi (input) and of DMSO-treated cells at 6 hpi are represented by dashed lines. (G) Cell viability of uninfected CB-6644-treated cells measured in parallel of the infection, presented as a percentage of the DMSO control. (H) RUVBL1 and RUVBL2 co-immunoprecipitate with RV-A16 2C or 2BC in the absence of other viral components. HeLa-H1 cells were transfected with constructs encoding FLAG-RUVBL1, HA-RUVBL2, and Myc-tagged RV NSPs (2C or 2BC) or Myc-GFP. Myc-tagged proteins were immunoprecipitated from cell lysates. Cell lysates (input) and immunoprecipitated fractions (α-Myc IP) were analysed by western blotting for Myc, FLAG, and HA. (*) HA-RUVBL2 overlaps with IgG heavy chain. FLAG-RUVBL1 and HA-RUVBL2 with Myc-2C, N=4; FLAG-RUVBL1 with Myc-2BC, N=3; HA-RUVBL2 with Myc-2BC, N=2. For all graph panels (D-G), data from 3-4 independent experiments are shown as individual points coded by shape, according to experimental replicate, together with means (connected by lines in F-G). Non-graph panels (H) show representative images. See also Figure S1.

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: Functional Assay, Biomarker Discovery, Infection, Protein-Protein interactions, Immunoprecipitation, Control, Liquid Chromatography with Mass Spectroscopy, Transfection, Activity Assay, Construct, Western Blot

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: 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) Inhibitors of cellular transcription or of JAK1/2 do not abrogate the antiviral effect of CB-6644. HeLa-H1 cells were pre-treated for 1 h with actinomycin D (AMD), triptolide (TPL), ruxolitinib (RUX), or DMSO, and subsequently infected with RV-A16 (MOI 20) in the presence of the corresponding drug and DMSO or 500 nM CB-6644. Viral RNA was quantified by RT-qPCR at 6 hpi (N=3). (B) Generation of CB-6644-resistant RV-A16. HeLa-H1 cells were infected with RV-A16 (MOI 0.1) in the presence of 15 nM CB-6644 or without treatment and incubated until 100% cytopathic effect was observed. Passage 1 virus was harvested and used to infect fresh HeLa-H1 cells under the same conditions. This process was repeated until passage 22 (P22), with CB-6644 concentrations being increased stepwise (30 nM at P3, 60 nM at P16 and 100 nM at P19). (C) CB-6644-passaged RV-A16 exhibits reduced sensitivity to CB-6644. HeLa-H1 cells were infected with passaged RV-A16 (P22, from untreated [black] or CB-6644-treated [red] passages, MOI 1) and were treated with DMSO or increasing concentrations of CB-6644. Viral titres were quantified at 0 hpi and 16 hpi (N=3). Viral titres in CB-6644-treated cells at 16 hpi are shown as individual points with means connected by lines. Mean viral titres of untreated cells at 0 hpi (input) and of DMSO-treated cells at 16 hpi are represented by dashed lines. (D) Mutations in RV-A16 2C reduce sensitivity to CB-6644. HeLa-H1 cells were infected with wild-type RV-A16, CB-6644-passaged virus (P22, from B), or with recombinant RV-A16 viruses carrying a single mutation (VP1 V285A, 2A Y92H, 2C M121V, or 2C T284S), two mutations (VP1 V285A + 2A Y92H [VP1-2A DM], or 2C M121V + T284S [2C DM]), or all four mutations in combination (quadruple mutant [QM]) (MOI 1). Infected cells were treated with DMSO or the indicated concentrations of CB-6644, and viral titres were quantified at 16 hpi (N=4). Dashed lines indicate mean titres of wild-type RV-A16 under each treatment. For all panels, N=number of independent experiments. For all graph panels (A, C-D), data are shown as individual points, coded by shape according to experimental replicate, with means (connected by lines in C). Statistical tests: one-way ANOVA with Dunnett’s post-hoc test (A), two-way ANOVA with Holm-Sidak’s post-hoc test (D). For D, only statistically significant comparisons are shown; all other comparisons within each treatment group were non-significant. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See also Figure S4.

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, Incubation, Virus, Recombinant, Mutagenesis

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: bioRxiv

Article Title: Identification of ICAM-1–targeting DNA aptamers as a host-directed strategy to inhibit Human Rhinovirus infection

doi: 10.64898/2026.04.20.717810

Figure Lengend Snippet: A. Schematic representation of the infection experiment to assess the ability of aptamers to block rhinovirus infection. Aptamers (I4, I5 and I8) were tested at doses of 1 and 0.1 µM. As positive control, for assessing viral inhibition, a VP1 inhibitor (Pleconaril, pleco) was also included. H1-HeLa cells were pre-treated with aptamers for 7 minutes and next the infection with HRV-A16 (MOI 0.1) was performed. After 1 hours, the inoculum was removed and infection was allowed to proceed for 72 hours at 33°C. Infectious virus released into the supernatant was quantified by TCID₅₀ assay (B), and viral RNA levels were measured (C). B. Normalized viral titers (%), expressed relative to vehicle (100%) and mock (0%) (mean ± SEM). C. Viral RNA levels assessed by RT-PCR using HRV-specific primers. Data are shown as 2 -ΔCt . Statistical significance was determined by ordinary one-way ANOVA followed by Bonferroni’s multiple comparison correction vs vehicle.

Article Snippet: HRV-A16 (Human Rhinovirus A 16) strain 11757 was purchased from ATCC (LGC, Italy, cat. n°: VR-283).

Techniques: Infection, Blocking Assay, Positive Control, Inhibition, Virus, Reverse Transcription Polymerase Chain Reaction, Comparison