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: 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

Journal: IUCrJ

Article Title: Structural insights into the antiviral efficacy of AG7404 against human rhinovirus 3C proteases

doi: 10.1107/S2052252525008929

Figure Lengend Snippet: Anti-rhinoviral efficacy and enzymatic inhibition of AG7404. ( A ) Dose-response curve analysis of AG7404 against hRV-B14, -A16 and -A21. HeLa cells were mock-infected (blue triangle) or infected with hRV-B14 (red circle), hRV-A16 (yellow square) and hRV-A21 (green rhombus) with various concentrations of AG7404. After 72 h, cell viability was measured using the MTT assay. Data represent means (±SD) from at least two independent experiments performed in duplicate. ( B ) Inhibitory activity of AG7404 against purified hRV-B14 3C protease. The enzymatic assay was performed using a colorimetric peptide substrate (EALFQ-pNA), and the release of pNA was measured at 405 nm to calculate the IC 50 value. Data represent means (±SD) from triplicate experiments.

Article Snippet: Then, hRV B14 (ATCC VR-284), A16 (ATCC VR-283), and A21 (ATCC VR-496) were propagated in the H1HeLa cells and stored at −70°C until further use.

Techniques: Inhibition, Infection, MTT Assay, Activity Assay, Purification, Enzymatic Assay

Journal: IUCrJ

Article Title: Structural insights into the antiviral efficacy of AG7404 against human rhinovirus 3C proteases

doi: 10.1107/S2052252525008929

Figure Lengend Snippet: Overall structure of the hRV-B14 3C protease bound to the AG7404 inhibitor. ( A ) Orthogonal views of the hRV-B14 3C protease–AG7404 complex structure with domain I in brown, domain II in green, and the linker region connecting the two domains in violet. The catalytic triad residues (Cys146, His40 and Glu71) are labeled and secondary structural elements (α-helices and β-strands) are annotated. AG7404 is depicted as a cyan stick model positioned within the active site. The blue box indicates a region showing minor conformational differences among protomers in the asymmetric unit. ( B ) Close-up view of the active site showing AG7404 covalently bonded to Cys146. A polder map was generated using phenix.polder after omitting Cys146 and AG7404, with the resulting electron density shown as a yellow mesh contoured at 3.0σ. The residues of the catalytic triad are in stick representation. ( C ) The superposition of the four protomers in the asymmetric unit, presenting structural conservation and minor conformational variability in the loop between αC and βaII (blue box). The AG7404 molecules are shown as stick models bound at identical positions across all protomers. ( D ) Sequence alignment of hRV-B14, hRV-A16 and hRV-A21 3C proteases with secondary structural annotations derived from the hRV-B14 structure. Conserved residues are shaded in gray and black. The flexible region in the superposition of the four protomers is indicated by a blue box. The variable regions (VR1: residues 91–97; VR2: residues 107–110) derived from sequence alignments between hRV serotypes are underlined in orange. Catalytic residues (red circles) and AG7404-interacting residues (green diamonds) are highlighted.

Article Snippet: Then, hRV B14 (ATCC VR-284), A16 (ATCC VR-283), and A21 (ATCC VR-496) were propagated in the H1HeLa cells and stored at −70°C until further use.

Techniques: Labeling, Generated, Sequencing, Derivative Assay

Journal: IUCrJ

Article Title: Structural insights into the antiviral efficacy of AG7404 against human rhinovirus 3C proteases

doi: 10.1107/S2052252525008929

Figure Lengend Snippet: Sequence and structural comparison of hRV-B14, hRV-A16 and hRV-A21 3C proteases. ( A ) Sequence alignment of hRV-B14, hRV-A16 and hRV-A21 3C proteases. Conserved residues are shaded in gray and black, and variable regions (VR1: residues 91–97; VR2: residues 97–110) are highlighted with blue boxes. Red arrows indicate interacting residues. ( B ) Structural superposition of hRV-B14 (green), hRV-A16 (pink; AlphaFold -predicted model) and hRV-A21 (light blue; AlphaFold -predicted model) 3C proteases. Variable region 1 (blue box) is positioned opposite the active site (green circle) and variable region 2 (orange box) is located near the substrate-binding cleft. ( C ) A close-up view of variable region 2 (orange box) and the catalytic loop (red arrow) of hRV-B14 (green) and hRV-A21 (light blue) 3C proteases. The key residues of hRV-B14 3C protease are denoted without parentheses, whereas hRV-A21 3C protease residues are shown in parentheses.

Article Snippet: Then, hRV B14 (ATCC VR-284), A16 (ATCC VR-283), and A21 (ATCC VR-496) were propagated in the H1HeLa cells and stored at −70°C until further use.

Techniques: Sequencing, Comparison, Binding Assay

Journal: IUCrJ

Article Title: Structural insights into the antiviral efficacy of AG7404 against human rhinovirus 3C proteases

doi: 10.1107/S2052252525008929

Figure Lengend Snippet: MD simulations of AG7404-bound hRV-A16 and hRV-A21 3C proteases. ( A ) RMSD plots of AG7404-bound hRV-A16 (left) and hRV-A21 (right) 3C proteases for 300 ns of MD simulations. The protein RMSD is depicted in black and the ligand RMSD is displayed in cyan. ( B ) AG7404-bound hRV-A16 (left) and hRV-A21 (right) 3C protease structures at the end of the 300 ns MD simulation. AG7404 molecules are depicted as a cyan ball-and-stick model. Key residues and the water molecules (w) interacting with AG7404 are labeled.

Article Snippet: Then, hRV B14 (ATCC VR-284), A16 (ATCC VR-283), and A21 (ATCC VR-496) were propagated in the H1HeLa cells and stored at −70°C until further use.

Techniques: Labeling