RIPK1 Is Cleaved by 3C Protease of Rhinovirus A and B Strains and Minor and Major Groups

Figures

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  Figure 1. Infection with different RVs results in RIPK1 cleavage. O-HeLa cells were infected with the RV strains indicated at an M.O.I. of 3, for 1 h of adsorption, then cultured for a further 24 h post-infection (hpi). Protein lysates were collected at the indicated times and subject to SDS-PAGE (12.5%), transferred to nitrocellulose membrane and blocked. (A) Blots were probed with anti-RIPK1 antibodies. Tubulin was used as a loading control for all blots and is included below the corresponding RIPK1 blots. The position of bands correlating to the indicated full-length protein, or the cleavage products are indicated to the right of the blot. The relevant molecular weights are indicated to the left of the blots. The blots shown are representative of three repeats. (B) ImageJ was used to plot the densitometric profile of each of the lanes in the blot; each peak corresponds to a band in the blot. Profile of the molecular weight marker lane and the size corresponding to each peak is shown at the top. Coloured boxes indicate peaks corresponding to RIPK1 bands. (C,D) Intensity of RIPK1 bands was measured with ImageJ and normalized to the corresponding tubulin band. Data shown are mean ± SEM of…

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  Figure 2. RV protease activity correlates with RIPK1 cleavage. O-HeLa cells were infected with the RV strains as per Figure 1. Protein lysates were collected at the indicated times and subject to SDS-PAGE, transferred to nitrocellulose membrane and blocked. Blots were probed with (A) anti-PABP antibodies, or (B) anti-eIF4G antibodies. Tubulin was used as a loading control for all blots and is included below the corresponding blots. The position of bands correlating to the indicated full-length protein, or the cleavage products are indicated to the right of the blot. The relevant molecular weights are indicated to the left of the blots. Representative western blots of three independent experiments have been shown. Please note that proteins were separated on 12.5% (A) or 8% (B) gels.

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  Figure 3. Inhibition of 3Cpro abrogates cleavage of RIPK1. O-HeLa cells were infected with the RV strain indicated at an M.O.I. of 3, for 1 h of adsorption and then a further 12 hpi. At 3 hpi, cells were treated with ActoD (5 µg/mL) or remained untreated, and at 9 hpi, cells were treated with Rupintrivir (1 µM) or remained untreated. Protein lysates were subject to SDS-PAGE, transferred to nitrocellulose membrane and blocked. Blots were probed with (A) anti-RIPK1 antibodies (upper blot), anti-caspase 3 antibodies (middle blot), (B) anti-eIF4G antibodies (upper blot), anti-PABP antibodies (lower blot). Tubulin was used as a loading control for all blots, and is included below the corresponding blots in (A, B). The position of bands correlating to the indicated full-length protein, or the cleavage products are indicated to the right of the blot. The relevant molecular weights are indicated to the left of the blots. Representative western blots of three independent experiments have been shown. Please note that proteins were separated on 12.5% (RIPK1, PABP blots) or 8% (eIF4G blot) gels.

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  Figure 4. RVA but not RVB strains modulate apoptotic pathways. O-HeLa cells were infected with the RV strain indicated at an M.O.I. of 3, for 1 h of adsorption. (A) Cells were treated at 3 hpi with ActoD (5 µg/mL) or remained untreated followed by caspase 8 inhibitor (4 µM) at 4 hpi, or remained untreated. At 24 hpi, virus cultures were frozen and thawed. The virus was clarified and titrated. Results are expressed as the mean of 3 independent experiments and error bars are the SEM. * p < 0.05. (B) At 3 hpi, cells were treated with ActoD (5 µg/mL) or remained untreated. Both adherent and non-adherent cells were collected at 12 hpi and stained with Annexin-V (FITC) antibodies and Propidium Iodide and processed for FACS analysis. Live cells were subject to doublet discrimination before quadratic gating to determine viable (AnV − Pi−), apoptotic (AnV + Pi−), late apoptotic (AnV + Pi+), or necrotic (AnV − Pi+) populations. Representative FACS plots are shown. (C) Quantitation as % of single cells (all cells) of data represented in (B). Data are representative of two experiments performed in triplicate and columns represent the mean with the two data points shown on each.