Inhibition of Calcineurin or IMP Dehydrogenase Exerts Moderate to Potent Antiviral Activity against Norovirus Replication

Abstract

Norovirus is a major cause of acute gastroenteritis worldwide and has emerged as an important issue of chronic infection in transplantation patients. Since no approved antiviral is available, we evaluated the effects of different immunosuppressants and ribavirin on norovirus and explored their mechanisms of action by using a human norovirus (HuNV) replicon-harboring model and a surrogate murine norovirus (MNV) infectious model. The roles of the corresponding drug targets were investigated by gain- or loss-of-function approaches. We found that the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506) moderately inhibited HuNV replication. Gene silencing of their cellular targets, cyclophilin A, FKBP12, and calcineurin, significantly inhibited HuNV replication. A low concentration, therapeutically speaking, of mycophenolic acid (MPA), an uncompetitive IMP dehydrogenase (IMPDH) inhibitor, potently and rapidly inhibited norovirus replication and ultimately cleared HuNV replicons without inducible resistance following long-term drug exposure. Knockdown of the MPA cellular targets IMPDH1 and IMPDH2 suppressed HuNV replication. Consistent with the nucleotide-synthesizing function of IMPDH, exogenous guanosine counteracted the antinorovirus effects of MPA. Furthermore, the competitive IMPDH inhibitor ribavirin efficiently inhibited norovirus and resulted in an additive effect when combined with immunosuppressants. The results from this study demonstrate that calcineurin phosphatase activity and IMPDH guanine synthase activity are crucial in sustaining norovirus infection; thus, they can be therapeutically targeted. Our results suggest that MPA shall be preferentially considered immunosuppressive medication for transplantation patients at risk of norovirus infection, whereas ribavirin represents as a potential antiviral for both immunocompromised and immunocompetent patients with norovirus gastroenteritis.

Keywords: calcineurin inhibitors; cell culture; cell culture model; mycophenolic acid; norovirus; noroviruses; ribavirin.

FIG 1

Antiviral activities of CsA and its derivatives against HuNV replication. CsA (A) and its immunosuppressive derivative voclosporin (B) dose-dependently inhibited HuNV replication without potent toxicity to host cells after 48 h of treatment. The level of HuNV RNA was quantified by qRT-PCR and was compared to that in cells treated with 0.5% DMSO (control) (n = 3 independent experiments with 2 replicates each). (C) Its nonimmunosuppressive derivatives 431-32 and 440-02 failed to inhibit HuNV replication after 48 h of treatment, even at the highest concentration tested (n = 2 independent experiments with 2 replicates each). (D and F) Clearance assay with CsA and voclosporin. HG23 cells were treated with CsA or voclosporin for 1, 2, 6, or 10 days. At the end of each treatment period, the level of HuNV RNA was determined by qRT-PCR and normalized to that of the control cells from the same treatment time (n = 3 independent experiments with 2 replicates each). (E and G) Rebound assay with CsA and voclosporin. After 10 days of treatment, drugs were omitted, and HG23 cells (48-well tissue culture plate with 2.5 × 10⁴ cells per well) were cultured under the selective pressure of G418 (1.5 mg/ml). With another 5 days of culture, the cell layers were stained with hematoxylin and visualized by an inverted light microscope. Images are representative of three independent experiments with 2 replicates each. Data are presented as the means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).

FIG 2

CyPA, but not CyPB, is involved in CsA-caused inhibition of HuNV replication. (A and B) Knockdown of CyPA and CyPB through lentiviral shRNA vectors. HG23 cells were transduced with shRNA vectors against CyPA (shCyPA), CyPB (shCyPB), or control (shCTR). At the indicated time points, the level of cyclophilin RNA was measured by qRT-PCR and compared to the control (n = 2 independent experiments with 2 replicates each). (C) Western blot analysis of CyPA and CyPB in HG23 cells transduced with shRNAs. Images are representative of three independent experiments. Transduction of CyPA and CyPB shRNAs resulted in a dramatic downregulation of CyPA and CyPB expression at day 10 (10 d) but not at day 6 (6 d) postransduction. (D) qRT-PCR analysis of HuNV RNA in HG23 cells transduced with shRNAs. Knockdown of CyPA but not CyPB resulted in a significant decrease in viral replication at day 10 but not at day 6 postransduction (n = 3 independent experiments with 2 to 3 replicates each). (E and F) Knockdown of CyPA abrogated the inhibition of HuNV replication by CsA and voclosporin. After successful knockdown of mRNA and protein of CyPA, the responsiveness of HuNV to CsA and voclosporin treatment was detected after 48 h of treatment. CsA and voclosporin possessed the antinorovirus activity against shCTR-treated HG23 cells but failed to suppress HuNV replication in shCyPA HG23 cells. Data presented as the means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

FK506 moderately inhibited HuNV replication through FKBP12 and calcineurin. (A) Concentration-dependent antiviral effects of FK506 on HuNV replication after 48 h of treatment (n = 3 independent experiments with 2 replicates each). (B) Clearance assay with FK506. Long-term exposure to FK506 resulted in a moderate reduction of HuNV replication (n = 3 independent experiments with 2 replicates each). (C) Rebound assay with FK506. After long-term clearance phase, HG23 cells were cultured in the presence of G418 for another 5 days. Reduced cell confluence was observed, indicating that FK506 partially cleared the HuNV replicons from host cells. (D) Western blot analysis of FKBP12 knockdown by lentiviral shRNA vectors. Images are representative of three independent experiments. (E) qRT-PCR analysis of HuNV RNA level in shCTR, shFKBP8, and shFKBP12 cells after transduction of lentiviral shRNA vectors for 10 days. FKBP12 but not FKBP8 knockdown inhibited HuNV replication (n = 2 independent experiments with 2 replicates each). (F) FKBP12 knockdown partially blocked FK506-induced inhibition of HuNV after 48 h of treatment (n = 3 independent experiments with 2 to 3 replicates each). (G) qRT-PCR analysis of calcineurin knockdown at the RNA level. The level of calcineurin subunit PPP3CA RNA was presented as the relative value to the shRNA control (n = 2 independent experiments with 2 replicates each). (H) PPP3CA knockdown inhibited HuNV replication, as determined by qRT-PCR (n = 2 independent experiments with 2 replicates each). Data are presented as the means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 4

MPA potently inhibited norovirus replication and completely eliminated HuNV replicons from host cells without concomitant drug resistance. (A) Treatment with MPA for 48 h potently inhibited HuNV replication, as determined by qRT-PCR (n = 3 independent experiments with 2 replicates). (B) Clearance assay with MPA (n = 3 independent experiments with 2 replicates). (C) Rebound assay with MPA. Long-term treatment with 0.5 μg/ml MPA completely cleared HuNV replicons from host cells, and no colony formation was observed. (D) Comparison of the antinorovirus effects of MPA and 4 IMPDH inhibitors on HuNV. In the HG23 cells, treatment with 4 IMPDH inhibitors (the same concentration of 1 μM as MPA) resulted in a reduction of HuNV replication (n = 3 independent experiments with 2 replicates). (E) The antinorovirus effects of MPA were validated on MNV-1 as quantified by means of an MTT-based CPE reduction assay and qRT-PCR (n = 2 independent experiments with 2 to 3 replicates). ND, not detected. (F) Same as panel E for detecting the cellular MNV-1 RNA level; the viral RNA copy numbers in the supernatant (secreted viruses) were also detected after 24 h of treatment with MPA. MPA potently inhibited MNV-1 virus particle production (n = 2 independent experiments with 2 or 3 replicates). (G) The inhibitory efficacy of MPA against MNV-1 following 20 passages (20P) exposed to MPA or vehicle control. In the selection process, MNV-1 was either directly cultured in the presence of a fixed MPA concentration (0.1 or 0.5 μg/ml) or in a lengthy stepwise selection concentration from 0.1 to 0.5 μg/ml or from 0.5 to 1 μg/ml. After selection, the antinorovirus effects of MPA on the 20th passage of MNV-1 were determined by qRT-PCR. Data are presented as the means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001). 10P, 10 passages.

FIG 5

Simultaneous knockdown of IMPDH1/2 reduced HuNV replication, which was reversed by exogenous guanosine. (A and B) Validation of IMPDH downregulation in shCTR cells and shIMPDH cells by Western blotting. Images are representative of three independent experiments. (C) qRT-PCR analysis of HuNV RNA level in IMPDH knockdown cells. Compared to control cells, knockdown of IMPDH1 or IMPDH2 alone had no significant effect on HuNV replication (n = 2 independent experiments with 2 replicates each). (D) Validation of IMPDH downregulation after simultaneous knockdown of IMPDH1/2 by Western blotting. Images are representative of three independent experiments. (E) Simultaneous silence of IMPDH1/2 decreased HuNV replication (n = 2 independent experiments with 2 replicates). (F and G) Guanosine restored norovirus replication in MPA-treated cells. MNV-1 and the HuNV replicon were treated with MPA alone or combined with guanosine (1, 10, or 100 μg/ml). After 24 h of incubation, norovirus replication was quantified by qRT-PCR. (H) IMPDH1/2 knockdown cells were cultured with medium or increasing concentrations of guanosine. After 24 h of incubation, the HuNV RNA level was analyzed by qRT-PCR and was compared to the shCTR cells (n = 2 independent experiments with 2 replicates). Data presented as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 6

Ribavirin effectively inhibited norovirus replication. (A) Treatment with ribavirin for 48 h dose-dependently decreased HuNV replication, as determined by qRT-PCR (n = 3 independent experiments with 2 replicates each). (B) Clearance assay with ribavirin (n = 3 independent experiments with 2 replicates each). (C) Rebound assay with ribavirin. (D) The antinorovirus activity of ribavirin was also confirmed on MNV-1. Ribavirin treatment decreased MNV-1-induced CPE and viral RNA replication in RAW 264.7 cells (n = 3 independent experiments with 2 to 3 replicates). (E) Ribavirin treatment also decreased MNV-1 virus particle production in supernatant as quantified by viral RNA copy numbers (n = 3 independent experiments with 2 replicates). Data are presented as the means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 7

The combinatory effects of ribavirin and immunosuppressants on norovirus replication. The combinatory effects of two drugs in the 48-h antiviral assay with HuNV were analyzed using the mathematical model MacSynergy. The three-dimensional surface plot represents the differences (within 95% confidence interval [95% CI]) between actual experimental effects and theoretical additive effects of the combination at various concentrations of the two compounds (n = 5). The antiviral effects of FK506 in combination with CsA (A) or MPA (B) as well as CsA in combination with MPA (C) was analyzed by MacSynergy model. Combinations of ribavirin with CsA (D), FK506 (E) or MPA (F) were analyzed by the relative HuNV RNA level compared to the control and MacSynergy model.