BTP2 restricts Tulane virus and human norovirus replication independent of store-operated calcium entry

Figures

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  Fig 1. Orai channel inhibitors block SOCE in MA104 cells but variably affect TV replication. (A) Representative traces of GCaMP6s fluorescence following 1 µM thapsigargin treatment (black arrow) and 2 mM calcium perfusion (red arrow) in MA104G6s cells pretreated with DMSO control (gray), 5 µM Ro2959 (purple), or 10 µM BTP2 (blue), Synta66 (pink), or GSK (orange) for 20 min. (B) Maximum fold change in GCaMP6s fluorescence per imaging field-of-view after 2 mM calcium perfusion relative to DMSO control. All experiments were performed with a minimum of three biological repeats. (C) Lactate dehydrogenase (LDH)-based cytotoxicity in MA104G6s cells after 24 h treatment with media alone (black), DMSO (gray), 10 µM BTP2, Synta66, GSK, or 5 µM Ro2959. All experiments were performed with a minimum of three biological repeats of six technical replicates. (D) TV yield in PFU/mL 24 hours post-infection (hpi) at multiplicity of infection (MOI) 1 with BTP2, Synta66, GSK (10 µM), and Ro2959 (5 µM). All compounds were added 1 hpi. Data are shown as an average of technical duplicates across at least three biological replicates. (E) Representative traces of GCaMP6s fluorescence following thapsigargin…

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  Fig 2. BTP2 inhibits both early- and late-stage TV replication. (A) Percent inhibition of monolayer clearance in crystal violet-based assay in TV-infected monolayers (MOI 10) treated with 0.001, 0.01, 0.1, 1, 10, or 100 µM BTP2. The dose curve was fit with a non-linear regression (dotted line) to estimate the inhibitory concentration 50 (IC50). (B) Time course assay measuring TV yield in PFU/mL at 1, 6, 12, and 24 hpi with DMSO (black) or BTP2 (purple) treatment at 1 hpi with an MOI of 1 infection. (C) Schematic of BTP2 time-of-treatment. A total of 10 µM BTP2 stock was incubated with virus or cell monolayers for 1 h prior to infection, or with cell monolayers during the 1 h inoculum incubation, or 1, 6, or 10 h post-inoculation. All infections were harvested at 24 hpi. (D–G) Plaque assay titrations of TV yield following MOI 1 infection and the corresponding pre-treatment scheme of TV (D) or pre- and post-treatment schemes of cell monolayers (E–G). (H) Plaque assay titration of TV yield after MOI 10 infection and treatment with BTP2 at 1, 6, or 10 hpi. All data are shown as an average of technical duplicates across at least three biological replicates. For all experiments, normality was…

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  Fig 3. Early and late stage BTP2 addition reduces TV protein and RNA synthesis. (A) TV VP1 or NS1-2 protein expression detected by western blot at 1, 6, 10, or 24 hpi. (B) TV VP1 and NS1-2 expression at 24 h following DMSO (−) or 10 µM BTP2 (+) addition 1 h before infection (pre), during viral inoculation (inoc), or 1, 6, or 10 hpi at an MOI of 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the loading control. For quantitation, band intensity was plotted relative to GAPDH. Each point represents an independent biological replicate. (C) TV genome equivalents were determined through quantitative reverse transcription PCR (RT-qPCR) of infected cells treated with DMSO (−) or BTP2 (+) at the indicated time points. RNA was harvested at 2 and 24 hpi, and genome equivalents were determined based on a standard curve from in vitro transcribed TV RNA. (D) Fold change in TV genome equivalents between 2 and 24 h with DMSO (−) or BTP2 (+) treatment at the indicated time points. Data are shown from four biological replicates, and the RT-qPCR data represent an average of technical duplicates for each biological replicate. ****P < 0.0001 by one-way analysis of variance (ANOVA) with…

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  Fig 4. TV overcomes BTP2 susceptibility with passage through the emergence of resistant quasi-species. (A) Representative images of crystal-violet-stained MA104 cell monolayers after infection with DMSO-passaged TV (A, passage [P] 1–4) or BTP2-passaged TV (B, passage [P] 1–4) in the presence of DMSO or 10 µM BTP2. (C) TV yield in PFU/mL from passage 4 DMSO or BTP2-passaged virus, challenged with DMSO (white, gray) or 10 µM BTP2 (purple, light blue). Data are shown as an average of technical duplicates across at least three biological replicates. (D) Representative images of crystal violet-stained plaques following DMSO or 5 µM BTP2 addition to the plaque assay overlay (left panel) and quantitation of plaque diameter (millimeters) across three independent infections (right panel, n > 62 plaques). (E and F) TV yield in PFU/mL from DMSO (E) or BTP2 (F) solid overlay plaque picks, challenged with DMSO (white) or 10 µM BTP2 (purple). Data are shown from at least three biological replicates. For all experiments, normality was assessed by the Shapiro-Wilk test. TV yield assay data are plotted as geometric mean ± SD. **P < 0.01 by Mann-Whitney or unpaired T test. Plaque diameter data were…

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  Fig 5. Amino acid changes in TV VP1 and VP2 are associated with BTP2 resistance. (A) Percent inhibition of monolayer clearance in crystal violet-based assay with three serial plaque-isolated susceptible viruses (gray) and three serial isolated resistant viruses (blue) (MOI 3) treated with a dose range of BTP2. (B) TV yield assay with one serial plaque-isolated susceptible virus (clone 1) and one serially isolated resistant virus (clone 1) challenged with DMSO or 10 µM BTP2. (C) Time course assay measuring yield at 1, 6, 12, and 24 hpi with one serial plaque-isolated susceptible virus (clone 1, gray) and one serially isolated resistant virus (clone 1, blue). (D) Schematic of amino acid differences identified between BTP2-susceptible and resistant clones. (E) Representative images of mean fluorescence intensity of cells transduced with adeno-associated viral (AAV) vectors expressing VP2 or mScarlet alone. Images were taken at 48 hours post-transduction (hpt). Scale bar = 100 µM. (F) Quantitation of mean fluorescence intensity across four biological replicates. (G) Western blot demonstrating expression of FLAG-tagged susceptible or resistant VP2 following AAV transduction. Cell lysates…

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  Fig 6. TV structural proteins mediate BTP2 susceptibility. (A) Schematic of susceptible or resistance-associated amino acids in the four recombinant Tulane virus variants. (B) Recombinant TV yield at 24 hpi following an MOI 1 infection with DMSO or 10 µM BTP2 treatment at 1 hpi. (C) Percent inhibition of monolayer clearance with recombinant TV (MOI 3) infection and treatment with BTP2 at 1 hpi at the indicated dose range. (D) Recombinant TV yield at 24 hpi following an MOI 1 infection with DMSO or 10 µM BTP2 treatment at 1 or 10 hpi. For yield experiments, normality was assessed by the Shapiro-Wilk test, and data are plotted as geometric mean ± SD. ***P < 0.001 by unpaired T test.

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  Fig 7. BTP2 inhibits HuNoV replication. (A) Quantitation of HuNoV genome equivalents in jHIOs inoculated with 100 TCID50s of GII.4 virus at 2 and 24 hpi after treatment with a dose range of BTP2 or 50 µM 2-CMC. Both compounds were incubated with enteroid monolayers for 1 h before infection and maintained in the maintenance media until harvest (24 h). (B) Quantitation of HuNoV genome equivalents in jHIOs inoculated with 100 TCID50s of GII.3 (P21) virus at 2 and 24 hpi. jHIOs were treated with BTP2 or 2-CMC as described above at the indicated concentrations. (C and D) Percent inhibition of GII.4 (C) or GII.3 (D) replication across the BTP2 dose range for three independent experiments. EC50 and CC50 values were calculated using non-linear regression, and the selective indices were calculated by dividing the CC50 by the EC50. (E) Percent viability as measured by LDH assay over a dose range of BTP2. Data are pooled from five independent experiments. For HuNoV replication data, ****P < 0.0001 by two-way ANOVA with Dunnett’s multiple comparisons test.