The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication

Abstract

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

Figures

Fig 1

CoPP induces HO-1 expression without affecting cell viability. (A) Representative Western blot demonstrating HO-1 induction in Vero VP30, Huh 7.0 VP30, and 293T cells after treatment with 10 μM CoPP from 0 to 24 h. IB, immunoblot. (B to D) Percent viability (compared to vehicle-treated cells) of Vero VP30 or Huh 7.0 VP30 cells on days 3, 5, and 7 or 293T cells on days 1, 2, and 3 posttreatment with the indicated concentrations of CoPP. The data are presented as means ± standard deviations (SD) and are representative of at least three independent experiments.

Fig 2

CoPP attenuates EBOV growth in Vero VP30 and Huh 7.0 VP30 cells. (A and B) Growth kinetics (FFU/ml) of EbolaΔVP30 virus in Vero VP30 cells (A) and Huh 7.0 VP30 cells (B) after treatment with the indicated concentrations of CoPP 8 h prior to infection (pretreatment) or immediately following infection (posttreatment) with EbolaΔVP30 virus at an MOI of 0.001. All values for CoPP-treated cells (pre- and postinfection) are statistically significant (P ≤ 0.05) compared to the values for vehicle-treated cells on the same day, except the 10 μM posttreatment in Vero VP30 cells on day 3 at an MOI of 0.001. (C) Growth kinetics of EbolaΔVP30 in Vero VP30 cells following direct incubation with vehicle or 15 μM CoPP for 1 h. The data are presented as means ± SD (for small SD, the error bars may not be clearly visible) and are representative of at least three independent experiments.

Fig 3

CoPP-induced attenuation of EBOV is HO-1 dependent. siRNAs specific to HO-1 were transfected into Vero VP30 or Huh 7.0 VP30 cells, which were then treated with vehicle or CoPP (10 μM and 15 μM, respectively) for 24 h. Then, the cells were infected with EbolaΔVP30 virus at an MOI of 0.001 for 4 days. (A) Western blots demonstrating the downregulation of CoPP-induced HO-1 by siRNAs on day 4 postinfection. (B) Growth of EbolaΔVP30 virus in Vero VP30 or Huh 7.0 VP30 cells on day 4 postinfection. The data are presented as means ± SD and are representative of at least three independent experiments.

Fig 4

Stable expression of HO-1 attenuates EBOV growth kinetics. Shown are the growth kinetics of EbolaΔVP30 virus in HEK 293 VP30 and HEK 293 VP30/HO-1 cells infected at an MOI of 0.001. The numbers of focus-forming units on days 3, 5, and 7 postinfection are presented as means ± SD and are representative of two independent experiments. The asterisks indicate statistical significance compared to the control (P ≤ 0.05).

Fig 5

The competitive HO-1 inhibitor SnPP does not attenuate EBOV growth. (A) Western blot demonstrating the induction of HO-1 in Vero VP30, Huh 7.0 VP30, and 293T cells by the selective HO-1 enzymatic inhibitor SnPP. (B) Growth of EbolaΔVP30 virus in Vero VP30 or Huh 7.0 VP30 cells 5 days posttreatment with CoPP or SnPP (10 μM in Vero VP30 and 15 μM in Huh 7.0 VP30 cells) and infection at an MOI of 0.01. All data are presented as means and SD and are representative of at least three independent experiments. The asterisks indicate statistical significance compared to the control (P ≤ 0.05).

Fig 6

CoPP treatment does not affect EBOV entry or budding. (A and B) Relative luciferase activity in Vero VP30 cells (A) or Huh 7.0 VP30 cells (B) after treatment with 10 μM or 15 μM CoPP for 16 h and infection with VSVΔG EbGP Luc at MOIs of 0.1, 0.01, and 0.001. (C) Representative Western blot demonstrating VP40 expression in VLPs and cell lysate from 293T cells treated with 10 μM CoPP for 16 h and transfected with a VP40 protein expression plasmid for 2 days. The error bars indicate SD.

Fig 7

HO-1 expression attenuates EBOV transcription/replication. (A) Relative polymerase activity (compared to vehicle-treated cells) from an EBOV minireplicon assay in 293T cells after treatment with 10 μM CoPP or SnPP for 2 days. (B) Relative polymerase activity (compared to empty vector) from an EBOV minireplicon assay in 293T cells that were transfected with a HO-1 overexpression plasmid for 2 days. (C) Relative polymerase activity (compared to HEK 293 VP30 cells) 2 days posttransfection with EBOV minireplicon plasmids in HEK 293 VP30/HO-1 cells. The data are presented as means ± SD and are representative of at least three independent experiments. The asterisks indicate statistical significance compared to the control (P ≤ 0.05).