Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors

Abstract

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step that is required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes, depending on the cell or tissue type. The virus spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abelson (Abl) kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titres and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which makes experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titres by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying the host cell pathways required for coronavirus infection. This will provide an insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

Keywords: Abl kinase; Abl1; Abl2; GNF2; GNF5; IBV; MERS-CoV; SARS-CoV; cell-cell fusion; coronavirus; imatinib; virus-cell fusion.

Fig. 1.

IBV infection is suppressed by Abl kinase inhibitors. Vero cells were pre-treated for 1 h with dimethyl sulphoxide (DMSO) or 10 µM imatinib, GNF2 or GNF5 at 37 °C. Cells were adsorbed at 37 °C for 1 h with IBV at an m.o.i. of 0.1 in the presence of DMSO or drug. Cells were washed and fresh medium containing DMSO or drug was added and infection was allowed to proceed for 18 h at 37 °C. (a) Supernatants were harvested and used for plaque assays to determine the viral titre. (b) Immunofluorescence staining of the IBV S protein was used to count the number of infected cells in the presence of DMSO or drug. In excess of 1000 cells were analysed per experimental condition in 3 independent experiments, and the % of cells infected was calculated by comparing the number of infected cells to the total number of cells analysed. The error bars represent the standard error and P values were calculated using a paired t-test. **P<0.01, ***P<0.001.

Fig. 2.

Abl kinase inhibitors block the first round of IBV infection. Vero cells were pre-treated for 1 h with DMSO, 10 µM imatinib, GNF2 or GNF5. Cells were adsorbed with IBV at an m.o.i of 2 in the presence of DMSO or drug. Adsorption was performed at 4 °C to allow the synchronization of virus-receptor binding. The cells were washed, fresh medium containing DMSO or drug was added and infection was allowed to proceed for 8 h at 37 °C. (a) Supernatants were harvested and used for plaque assays to determine the viral titre. (b) Indirect immunofluorescence staining of the IBV S protein was used to count the number of infected cells in the presence of DMSO or drug. In excess of 500 cells were analysed per experimental condition in 3 independent experiments, and the % of cells infected was calculated by comparing the number of infected cells to the total number of cells analysed. The error bars represent the standard error and P values were calculated using a paired t-test. *P<0.05, **P<0.01.

Fig. 3.

Imatinib interferes with the entry step of IBV infection. Vero cells were pre-treated for 1 h at 37 °C with DMSO or 10 uM imatinib. Cells were adsorbed with IBV for 1 h at m.o.i. 2 in the presence of DMSO or imatinib. Adsorption was performed at 4 °C to allow synchronization of virus receptor-binding. The cells were washed, fresh medium containing DMSO or imatinib was added and infection was allowed to proceed for 8 h at 37 °C. (a) Schematic showing duration of imatinib treatment. (b) Supernatants were harvested and used for plaque assays to determine the viral titre. The results shown were calculated from three independent experiments. The error bars represent the standard error and P values were calculated using a paired t-test. **P<0.01. (c) Indirect immunofluorescence staining of the IBV S protein was used to visualize infected cells in the presence of DMSO or imatinib.

Fig. 4.

Abl kinase inhibitors prevent virus-induced syncytia formation in IBV-infected cells. Vero cells were pre-treated for 1 h at 37 °C with DMSO, 10 µM imatinib, GNF2 or GNF5. Cells were adsorbed for 1 h with IBV at an m.o.i of 0.1 in the presence of DMSO or drug. The cells were washed, fresh medium containing DMSO or drug was added and infection was allowed to proceed for 18 h at 37 °C. (a) Indirect immunofluorescence assay detecting the S protein in infected cells. (b) Quantification of nuclei per syncytium in the presence or absence of Abl kinase inhibitors. In excess of 50 syncytia were analysed in 3 independent experiments. The error bars represent the standard deviation and P values were calculated using a paired t-test. *P<0.05.

Fig. 5.

Abl kinase inhibitors prevent syncytia formation in cells expressing coronavirus S proteins exogenously. Vero cells were transfected with a plasmid encoding IBV S (a, b) or SARS-CoV S (c, d) and DMSO or 10 µM imatinib and GNF2 or GNF5 were added to the medium 3 h post-transfection. The cells were examined 48 h post-transfection. The top images show an indirect immunofluorescence assay detecting IBV S (a) or SARS-CoV S (c). The graphs show the quantification of nuclei per syncytium for IBV S (b) or SARS-CoV S (d) in the presence of DMSO or drug. (b, d) In excess of 50 syncytia were analysed in three independent experiments. The error bars represent standard deviation and P values were calculated using a paired t-test. *P<0.05, **P<0.01.

Fig. 6.

Abl kinase inhibitors block cell–cell hemifusion. HeLa cells were transfected with plasmids encoding IBV S and YFP-GPI. Vero cells were transfected with a plasmid encoding dsRed. Cells were co-cultured 24 h post-transfection, −/+10 µM imatinib, GNF2 or GNF5. IBV S, YFP-GPI and dsRed were detected 24 h later. Surface IBV S (not shown) was labelled with mouse anti-IBV S and Cy5 anti-mouse IgG. Surface YFP-GPI was labelled with rabbit anti-GFP and Alexa 488 anti-rabbit IgG. (a) Fusion events were analysed and categorized as fusion (top panels) or hemifusion (bottom panels). (b) Quantification of hemifusion and full fusion events from two independent experiments. Total fusion events in control and drug-treated cells were normalized to 100 % and the percentage of hemifusion events was calculated based on comparison to total events for each treatment condition. Black and white indicates % of full fusion and hemifusion events, respectively.

Fig. 7.

Abl kinase inhibitors do not affect IBV S processing. Vero cells were pre-treated for 1 h with DMSO or 10 µM imatinib, GNF2 or GNF5 at 37 °C. Cells were adsorbed at 37 °C for 1 h with IBV at an m.o.i. of 0.1 in the presence of DMSO or drug. The cells were washed, fresh medium containing DMSO or drug was added and infection was allowed to proceed for 18 h at 37 °C. (a) A sucrose cushion was used to concentrate virus from supernatants of control and drug-treated IBV-infected cells. Concentrated virus was analysed by SDS-PAGE and Western blot using rabbit anti-IBV S and Licor anti-rabbit IgG 680. (b) The amount of S0, S2 and S2′ was calculated by normalizing the signal to the total amount of S within each sample. The results were confirmed in two independent experiments.