Human heme oxygenase 1 is a potential host cell factor against dengue virus replication

Chin-Kai Tseng, Chun-Kuang Lin, Yu-Hsuan Wu, Yen-Hsu Chen, Wei-Chun Chen, Kung-Chia Young, Jin-Ching Lee, Chin-Kai Tseng, Chun-Kuang Lin, Yu-Hsuan Wu, Yen-Hsu Chen, Wei-Chun Chen, Kung-Chia Young, Jin-Ching Lee

Abstract

Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 μM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice's brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection.

Figures

Figure 1. HO-1 inhibits DENV protein synthesis…
Figure 1. HO-1 inhibits DENV protein synthesis and RNA replication.
Inhibition of DENV protein synthesis by HO-1 inducer (A) CoPP, (B) hemin or (C) transient expression of exogenous HO-1 in a dose-dependent manner. DENV-infected Huh-7 cells were treated with CoPP and hemin or were transfected with a vector that transiently expressed exogenous HO-1 at indicated concentrations for 3 days. Western blotting was performed to determine DENV protein synthesis in the infected Huh-7 cells treated with CoPP or hemin or transiently expressing exogenous HO-1. GAPDH was used as an equal loading control. Inhibition of DENV RNA replication by HO-1 inducer (D) CoPP or (E) hemin or (F) by the transient expression of exogenous HO-1 in a dose-dependent manner. DENV RNA level was analyzed by performing RT-qPCR, were normalized against the mRNA level of cellular gapdh, and was presented as percentage change relative to that in DENV-infected Huh-7 cells (defined as 100%). (G) Representative immunofluorescence images showing CoPP-induced inhibition of DENV replication in DENV-infected Huh-7 cells. DENV-infected Huh-7 cells were treated 0–30 μM CoPP for 3 days, fixed, and examined by performing the immunofluorescence assay with anti-DENV prM antibody, and stained with DAPI. (H) CoPP inhibited the replication of DENV serotypes 1–4. Huh-7 cells were infected with the 4 DENV serotypes and were treated with CoPP at indicated concentration for 3 days. RNA level of DENV serotypes 1–4 were analyzed by performing RT-qPCR, were normalized against the mRNA level of cellular gapdh, and were presented as percentage change relative to that in DENV-infected Huh-7 cells (defined as 100%). Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 2. HO-1-specific inhibitor SnPP attenuates the…
Figure 2. HO-1-specific inhibitor SnPP attenuates the inhibitory effect of HO-1 induction on DENV replication.
SnPP attenuates the inhibitory effect of (A) HO-1 inducer and (B) transient expression of exogenous HO-1 on DENV protein synthesis. DENV-infected Huh-7 cells were treated with 30 μM CoPP or with 2 μg vector that transiently expressed exogenous HO-1, followed by treatment with 0–20 μM SnPP for 3 days. Total cell lysate was collected for performing western blotting to analyze DENV protein synthesis. Levels of GAPDH were used as equal loading control. SnPP attenuates the inhibitory effect of (C) HO-1 inducer and (D) transient expression of exogenous HO-1 on DENV RNA replication. Huh-7 cells were treated with 30 μM CoPP or with 2 μg of the vector that transiently expressed exogenous HO-1, followed by treatment with 0–20 μM SnPP for 3 days. Total cellular RNA was collected for performing RT-qPCR to analyze DENV RNA replication. The mRNA level of gapdh was used as the equal loading control. Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 3. Biliverdin but not CO and…
Figure 3. Biliverdin but not CO and Fe3+ inhibits DENV replication.
Effect of (A) CO, Fe3+, and (B) biliverdin on DENV protein synthesis. Western blotting was performed to determine DENV protein synthesis in DENV-infected Huh-7 cells treated with CO donor MC, Fe3+ donor FeCl3, or biliverdin at indicated concentrations for 3 days. GAPDH was used as the equal loading control. Effect of (C) CO, Fe3+, and (D) biliverdin on DENV RNA replication. DENV-infected Huh-7 cells were treated with CO donor MC, Fe3+ donor FeCl3, or biliverdin at indicated concentrations for 3 days. DENV RNA level was analyzed by performing RT-qPCR, were normalized using the mRNA level of cellular gapdh, and was presented as percentage change relative to that in DENV-infected Huh-7 cells (defined as 100%). Effects of silencing the gene encoding BVR on DENV (E) protein synthesis (F) RNA replication. (G) BVR activity. Huh-7 cells were transfected with shBVR or shEGFP (control). After 6 h, the transfected cells were infected with 0.2 MOI DENV-2 strain 16681 for 2 h at 37 °C and were incubated with medium with or without 50 μM biliverdin for 3 days. Total cell lysate was analyzed by performing western blotting with anti-NS2B, anti-BVR, and anti-GAPDH (loading control) antibodies. Total cellular RNA was extracted for performing RT-qPCR with specific primers against DENV NS5 gene and gapdh (loading control). BVR activity was measured using a BVR assay kit and was calculated from a standard curve generated using a biliverdin-positive control solution. BVR activity is presented as percentage relative to that in shEGFP-treated and biliverdin-untreated cells (defined as 100%). Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 4. Biliverdin inhibits DENV NS2B/NS3 protease…
Figure 4. Biliverdin inhibits DENV NS2B/NS3 protease activity.
(A) Inhibition of DENV protease activity by biliverdin. DENV protease activity was determined by incubating the indicated concentrations of biliverdin with recombinant DENV protease and Boc-GRR-AMC in a cleavage buffer for 30 min. Free AMC was measured using a spectrofluorometer and was expressed as percentage change relative to that in biliverdin-untreated controls (defined as 100%). (B,C) Determination of mechanisms underlying the effect of biliverdin on DENV NS2B/NS3 protease activity. The fluorogenic peptide at indicated concentrations was incubated with recombinant DENV protease in the presence of 5, 12.5 and 25 μM biliverdin for 30 min. The rate of release of AMC as a function of DENV protease activity was determined using a spectrofluorometer. Kinetic parameters and mode of inhibition were calculated from a standard curve generated using an AMC-positive control solution and were analyzed using Lineweaver–Burk plots of reciprocal velocity at the indicated concentrations of biliverdin. (D) Schematic diagram of the NS2B/NS3 protease vector with autocleavage activity (E) Inhibition of cis-acting activity of DENV protease by biliverdin in Huh-7 cells. The pNS2B(H)-NS3pro-transfected Huh-7 cells were treated with biliverdin at increasing concentrations for 3 days. The western blotting analysis were performed using anti-DENV NS2B specific antibody. (F) Schematic diagram of the protease reporter vector and the protease vector (G,H) Inhibition of trans-acting activity of DENV protease by biliverdin in Huh-7 and DENV-infected Huh-7 cells. The protease reporter vector and protease expression vector were co-transfected into Huh-7 cells or protease reporter vector alone was transfected into DENV-infected Huh-7 cells, followed by incubation of biliverdin at increasing concentrations for 3 days. Each transfection mixture contained 0.1 μg of firefly luciferase expression vector as a transfection control for normalization against the nano luciferase activity. Transfection of dysfunctional NS2B/NS3 protease expression vector or non-DENV-infected Huh-7 cells served as a negative control. The trans-acting activity of DENV protease is presented as percentage change relative to that in wild type protease-transfected or DENV-infected Huh-7 cells and biliverdin-untreated cells (defined as 100%). Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 5. HO-1-specific inducer CoPP rescues DENV…
Figure 5. HO-1-specific inducer CoPP rescues DENV protease-suppressed IFNα production in DENV-infected Huh-7 cells.
CoPP rescues DENV protease-suppressed transcription of genes encoding (A) IFN-α-2, (B) IFN-α-5, and (C) IFN-α-17 in DENV-infected Huh-7 cells. DENV-infected Huh-7 cells were treated with 7.5, 15, and 30 μM CoPP for 3 days. Total cellular RNA was extracted for performing RT-qPCR with specific primers against the genes encoding IFN-α-2, IFN-α-5, and IFN-α-17. The mRNA level of these genes were normalized against that of cellular gapdh and were presented as percentage change relative to that in parental Huh-7 cells (defined as 100%). (D) CoPP rescues DENV protease-suppressed IFN-α protein expression in DENV-infected Huh-7 cells. DENV-infected Huh-7 cells were treated with 7.5, 15, and 30 μM CoPP for 3 days. The culture fluid was examined by performing ELISA to determine IFN-α protein levels. (E) SnPP attenuates the CoPP-induced expression of IFN-α in DENV-infected Huh-7 cells. DENV-infected Huh-7 cells were coincubated with 30 μM CoPP and increasing concentrations of SnPP for 3 days. The culture fluid was examined by performing ELISA to determine IFN-α protein levels. Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 6. HO-1-specific inducer CoPP induces antiviral…
Figure 6. HO-1-specific inducer CoPP induces antiviral IFN responses in DENV-infected Huh-7 cells.
(A) CoPP induces the ISRE promoter activity in DENV-infected Huh-7 cells. Huh-7 cells were transfected with pISRE-Luc. After 6 h, the transfected cells were infected with 0.2 MOI DENV-2 strain 16681 for 2 h at 37 °C and were treated with 7.5, 15, and 30 μM CoPP for 3 days. Luciferase activity was measured to determine the ISRE promoter activity and was presented as fold activation relative to that in CoPP-untreated cells (defined as 1). (B) SnPP attenuates CoPP-induced stimulation of the ISRE promoter activity in DENV-infected Huh-7 cells. Huh-7 cells were transfected with pISRE-Luc plasmid. After 6 h, the transfected cells were infected with 0.2 MOI DENV-2 strain 16681 for 2 h at 37 °C and were treated with 30 μM CoPP and increasing concentrations of SnPP for 3 days. Luciferase activity was measured to determine the ISRE promoter activity and was presented as fold activation relative to that in CoPP-/SnPP-untreated cells (defined as 1). CoPP increases mRNA level of (C) OAS1, (D) OAS2, (E) OAS3 and (F) PKR, which are attenuated by SnPP in DENV-infected Huh-7 cells. DENV-infected Huh-7 cells were treated with 15 and 30 μM CoPP in the presence or absence of 20 μM SnPP for 3 days. Total cellular RNA was extracted for performing RT-qPCR with specific primers against genes encoding OAS1-3 and PKR. The mRNA level of OAS1-3 and PKR were normalized against that of cellular gapdh and were presented as fold change relative to that in parental Huh-7 cells (defined as 1). Results are expressed as the mean ± SD (error bar) of 3 independent experiments.
Figure 7. HO-1-specific inducer CoPP shows therapeutic…
Figure 7. HO-1-specific inducer CoPP shows therapeutic efficacy in DENV-infected ICR suckling mice.
Six-day-old ICR suckling mice were injected with 2.5 × 105 pfu DENV-2 intracerebrally and with CoPP (50 mg/kg) intraperitoneally at 1, 3 and 5 dpi. (A) Survival rate, (B) clinical score, and (C) body weight of the mice were daily measured to 6 dpi. Illness symptoms were scored as follow: 0, no symptom; 1, slight weight loss and ruffled hair; 2, slow activity; 3, asthenia and anorexia; 4, paralysis and fatal illness; and 5, death. Next, 0.1 g of brain tissue of these mice was incubated in RPMI medium for viral titration. (D) DENV titer was determined by performing plague assay. DENV-infected ICR suckling mice were intraperitoneally injected with CoPP (50 mg/kg) at 1, 3, 5, 7 and 9 dpi. (E) Survival rate and (F) body weight of the mice were daily measured to 12 dpi. Mice in the control group were treated with 60 °C heat-inactive DENV for 30 min. Each group included 8–10 mice. Error bars indicate the mean ± SD of 3 independent experiments; *P < 0.05, **P < 0.01.
Figure 8. Andrographolide shows therapeutic efficacy in…
Figure 8. Andrographolide shows therapeutic efficacy in DENV-infected ICR suckling mice.
Six-day-old ICR suckling mice were injected with 2.5 × 105 pfu DENV-2 intracerebrally and with andrographolide (10 mg/kg) intraperitoneally at 1, 3, and 5 dpi. (A) Survival rate, (B) clinical score, and (C) body weight of the mice were daily measured to 6 dpi. Illness symptoms were scored as follows: 0, no symptoms; 1, slight weight loss and ruffled hair; 2, slowing of activity; 3, asthenia and anorexia; 4, paralysis and fatal illness; and 5, death. Next, 0.1 g brain tissue of these mice was incubated in an RPMI medium for viral titration. (D) A DENV titer was determined by performing the plague assay. DENV-infected ICR suckling mice were intraperitoneally injected with andrographolide (10 mg/kg) at 1, 3, 5, 7 and 9 dpi. (E) Survival rate and (F) body weight of the mice were daily measured to 12 dpi. Mice in the control group were treated with 60 °C heat-inactive DENV for 30 min. Each group included 8–10 mice.

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