Heme and HO-1 Inhibition of HCV, HBV, and HIV

Abstract

Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system inhibit replication of all 3 viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

Keywords: HBV; HCV; HIV; Heme; biliverdin; metalloporphyrins; proteases; viruses.

Figures

Figure 1

Representative porphyrin structures. (A) pyrrole ring, (B) porphyrin ring, (C) protoporphyrin IX, and (D) Heme, iron protoporphyrin IX.

Figure 2

Heme oxygenase and biliverdin reductase enzymes.

Figure 3

Tetrapyrrole inhibition of HCV NS3/4A protease. (A,B) Protease activity was determined fluorometrically (FRET assay) using recombinant NS3/4A enzyme and various concentrations of inhibitors. (C) Endogenous NS3/4A protease activity in microsomes of replicons was measured using the same FRET assay but employing endogenous, partially purified NS3/4A protease from replicon cells (Zhu et al., 2010a). (D) Reciprocal (Lineweaver–Burk) plot of substrate concentration vs. enzyme activity. Recombinant protease activity was determined fluorometrically. Each point is the mean ± SEM of 3–5 determinations per point. Plot of [BV] vs. either 1/Vap or Km/V (not pictured) showed highly significant linearity (r = 0.975 and r = 0.979 respectively, p < 0.005) indicating mixed inhibition of NS3/4A protease by BV (K′i = 1.1  mM and Ki = 0.6 mM, respectively). 0 to the commercial Inhibitor = NS3/4A protease competitive inhibitor, AnaSpec #25346. Biliverdin = >99% Biliverdin IX-α. Bilirubin-mixed isomers (MI) = 93% Bilirubin IX-α, and 6% associated Bilirubin isomers. Bilirubin IX-α = >99% bilirubin IX-α. (With permission Zhu et al., see original manuscript for further details.)

Figure 4

Disappearance of HCV RNA and HCV NS3/4A inhibition with biliverdin, heme, or ZnPP. (A–C) HCV RNA was determined in total RNA extracted from replicon cells incubated with the indicated concentrations of tetrapyrrole for 48 h. Independent determinations of the inhibitory activity of each tetrapyrrole for recombinant HCV NS3/4A was determined by FRET analysis (Zhu et al., 2010a). Results are means ± SEM of six determinations per point. (With permission; Zhu et al., .)

Figure 5

Schematic of HCV NS3/4A protease inhibition of innate immune signaling pathways: TLR3 and RIG1. Double stranded RNA (dsRNA) viruses or viruses with dsRNA intermediate activate innate immune system signaling pathways for type I interferon induction through binding to Pattern Recognition Receptors (PRR). For HCV, PRR are associated with at least two major signaling pathways: TLR3 and RIG1. Upon intracellular binding of dsRNA to associated adapter proteins TRIF and CARDIFF, respectively, signaling is transmitted through complex intermediate steps of recognition, binding, and phosphorylation leading to activation of the transcriptional factors IRF3 and NFκB. The activated nuclear transcription factors bind to type I interferon promoters and induce α/β-interferon transcription. HCV NS3/4A protease is known to cleave both TRIF and CARDIFF adapters thus crippling innate immune antigen recognition and signaling for type I interferon induction. With permission (Bode et al., 2007), see original review for terminology and abbreviations.

Figure 6

Biliverdin and Heme can restore IFN promoter activation and Interferon Stimulated Response Genes (ISRG) after interference of IFN signaling with HCV NS3/4A. (A,B) HEK 293 cells were transfected with dsRNA and vectors containing Type I IFN promoter-luciferase construct and complete NS3/4A sequence plasmid construct or control plasmid. Cells were then incubated with heme (20 μM) or Biliverdin (50 μM) for 48 h. Cellular lysates were prepared and assayed for interferon promoter activation using luciferase assay (Zhu et al., 2010b). Each point is the mean of six determinations (two separate cell culture incubations, and then three replicates for luciferase assay per incubation). (C,D) Parallel experiments showing that heme and BV can restore interferon stimulated response genes (ISRG) after inhibition with NS3/4A protease. HEK 293 cells were transfected with dsRNA with or without HCV NS3/4A plasmid then incubated with heme or BV for same time and concentrations as (A,B) OAS-1 mRNA was measured using Real Time RT-PCR. Each point is the mean of six determinations as described for (A,B) **p < 0.01; *p < 0.05; NS, not significant. (With permission Zhu et al., .)