Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir

Abstract

Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Müller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schöpf, J., Kögl, M., Friedel, C., Müller, M.A., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Züst, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Weßels, C., Pöhlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.

Keywords: Cyclophilin A; Cyclosporine/FK506-non-immunosuppressive derivatives; FKBP; HCoV-NL63; Inhibition of viral replication.

Fig. 1

Chemical structures of novel CsA derivatives (compounds 1–5) and the FK506 derivative (compound 6) used in this work. The position where the R substituents are attached to the CsA ring core at position 1 is marked with an asterisk. Parent FK506 is substituted at position C-21 (dotted circle).

Fig. 2

Effect of CsA, FK506 and derivatives on NFAT-GFP nuclear translocation as a measure of immunosuppressive activity. NFAT3-GFP expression plasmid was transfected into HeLa cells. Subsequently, inhibitor compounds (comp.) were added to the medium at a final concentration of 40 nM. 19 h later, ionomycin was added at a final concentration of 2 μM to induce NFAT3-GFP translocation. Pictures were taken 10 min after Ionomycin (Iono.) induction. CsD (not depicted here) showed the same behavior as CsA.

Fig. 3

Effect of CsA and FK506 derivatives on HCoV-NL63 replication in Caco-2 cells. (A) and >(B) show independent sets of experiments using compounds CsA, 3, ALV, NIM811 and CsD, CsA, 1, 2, 4, 5, and FK506, 6, respectively. CsA represents an internal control included in each set. Genome equivalents were determined by qPCR and cell viabilities by Cell Titer Glow kit (Promega). Data shown are mean values of a representative experiment performed in at least triplicates. Left Y-axes represent the percentage of reduction of virus replication in linear scale (left panel column) and in log scale (right panel column). Right Y-axes indicate the percentage of the cell viabilities. X-axes indicate increasing inhibitor concentrations at which virus replication was determined. Closed/open squares and closed/open circles represent the reduction of genome equivalents and cell viability at the indicated inhibitor concentrations (X-axes), respectively. The graphs were plotted using Prism 5 (GraphPad Software, Inc.) and by a non-linear regression with a variable slope algorithm, the curve was fitted for each respective inhibitor and the EC₅₀ calculated.

Fig. 4

Western blot analysis of N protein expression in HCoV-NL63- infected CaCo-2 cells upon treatment with increasing concentrations of CsA, ALV, NIM811 and compound 3. N- Protein was detected with a mouse mab against N-protein. A rabbit anti-Lamin A antibody was used to detect Lamin A as a loading control.

Fig. 5

Characterization of CypA and CypB in Caco-2 knockdown cells and growth analysis of HCoV-NL63. CypA and CypB expression was determined by Western blot using an anti-CypA and anti-CypB (A) antibody and by qPCR (B). Lamin B was detected with an anti-lamin B antibody as a loading control. hTOP1 was used in qPCR to standardize cyclophilin expression. Growth of HCoV-NL63 on Caco-2 wt, Caco-2 shCtr non-target cells, Caco-2 ΔCypA (CypA KD) and Caco-2 ΔCypB (CypB KD) knockdown mutants was analyzed by plaque titration assay. Virus titers are depicted in (C).