Subversion of the Heme Oxygenase-1 Antiviral Activity by Zika Virus

Chaker El Kalamouni, Etienne Frumence, Sandra Bos, Jonathan Turpin, Brice Nativel, Wissal Harrabi, David A Wilkinson, Olivier Meilhac, Gilles Gadea, Philippe Desprès, Pascale Krejbich-Trotot, Wildriss Viranaïcken, Chaker El Kalamouni, Etienne Frumence, Sandra Bos, Jonathan Turpin, Brice Nativel, Wissal Harrabi, David A Wilkinson, Olivier Meilhac, Gilles Gadea, Philippe Desprès, Pascale Krejbich-Trotot, Wildriss Viranaïcken

Abstract

Heme oxygenase-1 (HO-1), a rate-limiting enzyme involved in the degradation of heme, is induced in response to a wide range of stress conditions. HO-1 exerts antiviral activity against a broad range of viruses, including the Hepatitis C virus, the human immunodeficiency virus, and the dengue virus by inhibiting viral growth. It has been reported that HO-1 displays antiviral activity against the Zika virus (ZIKV) but the mechanisms of viral inhibition remain largely unknown. Using a ZIKV RNA replicon with the Green Fluorescent Protein (GFP) as a reporter protein, we were able to show that HO-1 expression resulted in the inhibition of viral RNA replication. Conversely, we observed a decrease in HO-1 expression in cells replicating the ZIKV RNA replicon. The study of human cells infected with ZIKV showed that the HO-1 expression level was significantly lower once viral replication was established, thereby limiting the antiviral effect of HO-1. Our work highlights the capacity of ZIKV to thwart the anti-replicative activity of HO-1 in human cells. Therefore, the modulation of HO-1 as a novel therapeutic strategy against ZIKV infection may display limited effect.

Keywords: Zika virus; antiviral; heme-oxygenase 1; viral replication.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Generation and validation of ZIKA Virus (ZIKV) replicon in HEK-293A cells. (A) Schematic representation of overlapping fragments Z1 to Z4 covering of ZIKV replicon. Below, the flow chart representing the design of the experiment. (B) GFP, ZIKV NS3, ZIKV NS1 and RNA pol-II mRNA expression assessed by RT-PCR in ZIKV-infected A549 cells, ZIKV replicon cells and HEK 293A cells. (C) Cytometry monitoring of GFP after ribavirin treatment. (D) Fluorescence microscopy images of ZIKV replicon cells (GFP positive) after immunostaining of dsRNA with J2 antibody (red). (E) ISRE/SEAP activity evaluated in HEK 293A cells and ZIKV replicon cells. As positive control, cells were treated for 24 h with recombinant IFN-β (10,000 UI·mL−1). ** p < 0.01.
Figure 2
Figure 2
ZIKV replicon expression is inhibited by HO-1 induction or overexpression. (A) HO-1 protein expression was assessed by Western blot in HEK-293A cells expressing ZIKV replicon after treatment with several doses of CoPP for 20 h. Antibody against α-tubulin served as the protein loading control. (B) The percentage of GFP-expressing cells was analyzed by flow cytometry assay in ZIKV replicon cells after treatment with different concentrations of CoPP for 20 h. As positive control, cells were treated for 24 h with 40 µg·mL−1 (164 µM) of ribavirin. *** p < 0.001. (C) Overexpression of HO-1 in ZIKV replicon cells transfected with pcDNA3.1-HO-1-FLAG-Neo was assessed by Western blot using the anti-FLAG M2 and antibody against HO-1. Antibody against β-tubulin served as protein loading control. (D) The percentage of GFP-expressing cells was analyzed by flow cytometry assay in ZIKV replicon cells after transfection with pcDNA3.1-HO-1-FLAG encoding the human HO-1 protein or with pcDNA3.1. ** p < 0.01. In (E) and (F), cell viability was observed by optical microscopy or crystal violet staining respectively in ZIKV replicon cells transfected with pcDNA3.1 or pcDNA3.1-HO-1-FLAG after 7 days of treatment with G418 and puromycin to respectively allow the expression of HO-1 and the expression of ZIKV replicon. *** p < 0.001.
Figure 3
Figure 3
ZIKV replication and ZIKV infection decrease HO-1 protein and mRNA levels. (A) Western blot analysis of HO-1 protein expression in HEK 293A cells and ZIKV replicon cells using anti-HO-1. (B) RT-PCR analysis of HO-1, ZIKV NS1 and GAPDH mRNA expression in HEK 293A cells and ZIKV replicon cells. (C) RT-qPCR analysis of HO-1 expression in HEK 293A cells and ZIKV replicon cells. A549 cells were infected with ZIKV-PF13 at multiplicity of infection (MOI) of 5. In (D), HO-1 and ZIKV-E protein expression were analyzed by Western blot using anti-HO-1 and anti-ZIKV-E 4G2 antibody. Antibody against β-tubulin served as protein loading control. In (E), HO-1, ZIKV NS1 and GAPDH mRNA expression were analyzed by RT-PCR 24-h post infection. In (F) RT-qPCR analysis of HO-1 expression in A549 cells infected with ZIKV-PF13 24-h post-infection. * p < 0.05.
Figure 4
Figure 4
ZIKV infection decreases HO-1 induction mediated by CoPP at the protein and the mRNA levels. A549 cells were infected with ZIKV-PF13 at several multiplicity of infection (MOI) and then treated 2 h post-infection with CoPP for 16 h. In (A), HO-1 and ZIKV-E protein expression were analyzed by Western blot using anti-HO-1 and anti-ZIKV-E 4G2 antibody. Antibody against α-tubulin served as protein loading control. Quantification was done with the ImageJ software. In (B), HO-1, ZIKV NS1 and GAPDH mRNA expression were analyzed by RT-PCR. In (C) RT-qPCR analysis of HO-1 expression. ns = not significant.
Figure 5
Figure 5
The model of the crosstalk between HO-1 and ZIKV. HO-1 induction inhibits ZIKV at the replication level. ZIKV growth downregulates HO-1 level through its own replication.

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