The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Abstract

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Figure 1. Validation of interactions detected by Y2H hybrid screening in LUMIER assays.

Z-scores were calculated as described from duplicate experiments for 86 interactions observed in Y2H screens. 44 of the reproducible and specific interactions (category A) were tested. In addition, 42 interactions which were observed only once in a screen were tested (category B). These are compared to a negative reference set of non-interacting proteins. Shown in the Y-axis is the fraction of protein pairs above a threshold value (X-axis). The SARS interactions depicted here are listed in Table S1.

Figure 2. Localization of SARS-CoV ORFs and interaction network of virus host protein interactions.

Figure 2A shows an overview of the SARS-CoV ORFs used as the basis for the construction of the viral ORFeome . Individual ORFs were PCR amplified by primers specific for the predicted N- and C- terminal ends including sequences of the GATEWAY cassette. Additionally, hydrophobic sequences were deleted from ORFs containing transmembrane regions. Amino acid positions of these fragments (small bars, not drawn to scale) are given behind the respective ORF name and refer to the starting position of each individual ORF. Hypothetical ORF14 was also subcloned. Figure 2B shows highly confident interaction partners of SARS-CoV ORF as identified by ORFeome-wide Y2H screen. Viral proteins are shown in turquoise, and are connected to direct cellular interaction partners shown in orange.

Figure 3. Validation of SARS-CoV Nsp1 interaction with immunophilins (cyclophilins PPIA, PPIB, PPIG, PPIH and FK506-binding protein FKBP1A) and calcipressin (RCAN3) by modified Lumier assay.

Three versions of Nsp1 (Nsp1fl  =  aa 1–180, Nsp1N-terminus  =  aa 1–93 and Nsp1 C-terminus  =  aa119–180) and human cDNAs were cloned into protein A and Renilla Luciferase fusion vectors. Renilla-Nsp1 (A) or protein A-Nsp1 (B) was cotransfected with each respective cDNA into HEK293 cells. Complexes were purified via IgG-coated magnetic beads and Luciferase activity was determined as a measure for binding activity. As a positive control the very strongly interacting jun and fos genes were used. On the y-axis normalized signal to background ratios are shown.

Figure 4. SARS-CoV Nsp1 full length (Nsp1fl) induces NFAT-regulated gene expression in vitro independently of the NFAT molecular species, and the calcipressin RCAN3 extenuates the effect.

HEK293 cells were transiently cotransfected with NFAT reporter plasmid (NFAT luc) and expression plasmids encoding NFAT3, Calcineurin (CnA) and SARS-CoV Nsp1fl (A). RCAN3 was additionally expressed in (B). In (C) and (D), NFAT1 and NFAT2 species were expressed instead of NFAT3, respectively. The respective empty plasmid vector DNA was added to each individual transfection setup in order to obtain identical DNA concentrations. After transfection cells were cultured in absence or presence of the calcineurin stimulators PMA and ionomycin (PMA/Io.) and the NFAT-pathway inhibitor Cyclosporin A (CspA). ** P<0.01.

Figure 5. SARS-CoV isolate “Hongkong” induces NFAT-regulated gene expression.

HEK 293lp cells were transiently transfected with NFAT reporter plasmid (NFATluc). 24 h post transfection cells were infected with SARS-CoV isolate “Hongkong” (SARS-CoV HK) and the medium was supplemented with the calcineurin stimulators PMA and ionomycin (PMA/Io.). 17 h post infection the luciferase readout was carried out. * P<0.05.

Figure 6. Influence of Nsp1 on Interleukin promoters.

HEK293 cells were transiently cotransfected with interleukin reporter plasmids IL2 luc (A), IL4 luc (B), IL8 luc (C) and expression plasmids encoding NFAT3, CnA and either SARS-CoV Nsp1fl or the empty plasmid vector. All experiments were also done with an additional overexpression of the Calcipressin RCAN3. After transfection cells were cultured in absence or with the calcineurin stimulators PMA/Io. and the inhibitor CspA. * P<0.05; ** P<0.01.

Figure 7. Influence SARS-CoV Nsp1fl on transcription factors NF-κB and AP-1.

HEK 293 and Jurkat cells were transiently co-transfected with NFκB-luc (A,C) or AP-1luc (B,D) and SARS-CoV Nsp1fl or an empty vector. Induction of the cells was carried out with PMA/Io. * P<0.05; ** P<0.01, ***P<0.005.

Figure 8. Effect of Cyclosporin A on human (SARS-CoV, HCoV-229E-luc and HCoV-NL63), animal CoV (FCoV, TGEV, IBV) and control virus (HIV-1/EMCV) replication.

SARS-CoV, and EMCV were plaque-titrated on VeroE6, IBV-Beaudette in Vero cells, HCoV-NL63 on CaCo-2, TGEV PUR46 on St-cells, FCoV Black and FCoV 791146 on FCWF cells. HCoV-229E-luc was titrated on Huh-7 Lunet and HIV-1 on C8166 SEAP cells. Data shown are mean values of at least three experiments. HIV-1 data show one representative experiment out of three, values are averages of triplicates. Left and right Y-axes represent the percentage of virus replication and cell viability with the mock-treated cells set as 100%, respectively. CspA concentrations used for each virus are given on the x-axis. The graphs were plotted using the Fit Spline algorithm of Prism Software 4.0 (for Mac) of Graphpad Software Inc. The 50% effective dose (EC50) was calculated by regression analysis of the respective virus CPE.

Figure 9. Effect of Cyclosporin A on human SARS-CoV replicon.

A) Schematic drawing of replicon structure. B) Inhibition assay: BHK cells were electroporated in six-well plates with in vitro transcribed replicon RNA containing the Metridia luciferase gene and N RNA. After 16 hours supernatant was removed and cells were washed twice with PBS. After addition of fresh medium cells were incubated for another 24 hours. Second wash PBS and supernatant taken after 24 hours (50 µl each) were analysed for Luciferase activity. Values are expressed as relative light units (RLU). * P<0.05; ** P<0.01, *** P<0.005. C) HEK293 cells were transiently cotransfected with NFAT or IL-2 reporter plasmid (NFAT luc, IL-2 luc) and expression plasmids encoding NFAT3, Calcineurin (CnA) and SARS-CoV ORF N. l After transfection cells were cultured in absence or presence of the calcineurin stimulators PMA and ionomycin (PMA/Io.)