Restoration of Type I Interferon Expression by Heme and Related Tetrapyrroles Through Inhibition of NS3/4A Protease

Zhaowen Zhu 0 1 2 M. Meleah Mathahs 1 2 Warren N. Schmidt () 0 1 2 0 Department of Internal Medicine , Roy G. and Lucille A. Carver College of Medicine, University of Iowa , Iowa City 1 Received 1 November 2012; accepted 26 April 2013; electronically published 29 July 2013. Presented in part: 52nd Annual Meeting of the American Association for the Study of Liver Diseases , San Francisco, CA, 5 November 2011. Abstract 365. cine , University of Iowa Hospitals and Clinics , 200 Hawkins Dr, 4553 JCP, Iowa City, IA 52242 2 Department of Internal Medicine and Research Service, Veterans Affairs Medical Center Background. Tetrapyrrole substrates and products of heme oxygenase are potent inhibitors of hepatitis C virus (HCV) replication. It is not clear whether this occurs through primary induction of type I interferon (IFN), inhibition of viral NS3/4A protease, or a combination of these mechanisms. We studied the antiviral actions of tetrapyrroles and their potential influence on type I IFN induction. Methods. The effects of tetrapyrrole on NS3/4A protease activity and type I IFN induction were assessed in HCV-permissive cells, replicons, or human embryonic kidney (HEK) 293 cells transfected with NS3/4A protease. Activation of innate immune signaling was determined after transfection of double-strand surrogate nucleic acid antigens or infection with defined sequence HCV cell culture (HCVcc) RNA. Results. Tetrapyrroles failed to directly induce IFN expression at concentrations that inhibited HCV replication and NS3/4A protease activity. However, they potently restored IFN induction after attenuation with NS3/4A protease, a process accompanied by preservation of the adapter protein, mitochondrial antiviral signaling protein, nuclear localization of IFN regulatory factor 3, and augmentation of IFN-stimulated gene products. Conclusions. Tetrapyrroles do not directly induce IFN, but they dramatically restore type I IFN signaling pathway after attenuation with NS3/4A protease. They show immunomodulatory as well as antiprotease activity and may be useful for treatment of HCV infection. - hepatitis C virus; HCV protease inhibitors; type I interferon; heme metalloporphyrins. Hepatitis C (HCV) is a major cause of cirrhosis and end-stage liver disease worldwide. Since discovery of the virus, interferon (IFN) has been the cornerstone for successful antiviral therapy. The early addition of ribavirin and now the recent approval of first-generation protease inhibitors (PIs) have greatly improved treatment outcomes [1]. Nevertheless, in a sizeable percentage of patients, clinical cure is still not achieved because of weak IFN response and low tolerability of drug combinations [2, 3]. Consequently, intense research efforts have focused on development of new directacting anti-HCV drugs. The HCV NS3/4A protease-helicase protein complex is a useful target for drug development [4]. The enzyme performs crucial proteolytic steps in the viral life cycle and also has multiple extraviral interactions within the host cell that enable the virus to evade host innate immune defenses and increase viral efficiency [5, 6]. NS3/4A cleaves and inactivates adapter proteins, such as mitochondrial antiviral signaling protein (MAVS) or Toll/interleukin 1 receptor domaincontaining adapterinducing IFN- (TRIF), which allow host pattern recognition receptors (PRRs) to initiate signaling and induction of IFN- and IFN- responses [5, 79]. It is not known whether PIs significantly alter the success of antiviral therapy through counterinhibition of NS3/4A at the level of PRR adapter proteins, but further study of these interactions is important. Recent work has shown that new PIs require higher intracellular concentrations (>100 fold) in vitro to restore IFN signaling than is necessary for inhibition of polyprotein processing and viral replication [6, 10]. Heme oxygenase 1 (HO-1) is an inducible enzyme that oxidizes the metalloporphyrin, heme, to the linear tetrapyrrole, biliverdin (BV), which is then rapidly reduced to bilirubin (BR). Past work has demonstrated that precursors and products of the heme oxygenase system, including heme and BV have antiviral activities that target not only HCV but also human immunodeficiency virus (HIV) and hepatitis B virus [11]. In model systems, HCV replication and infection is inhibited with induction of HO-1 [12, 13] or incubation with HO-1related tetrapyrroles, such as BV and heme [14]. The antiviral behavior of BV has been attributed to direct binding and inhibition of the NS3/ 4A protease in our work [14] and direct induction of type I IFN in another study [15]. Although heme and its metabolites have extensive interactions with inflammatory mediators and stressresponse signaling pathways [1620], it is not clear how or where these agents might influence IFN induction. Except in a single report [15], tetrapyrroles have not been reported to directly stimulate IFN pathways. Consequently, we were prompted to investigate further their potential interactions with IFN induction further. In the present study, we investigated the effects of several tetrapyrroles on type I IFN induction, both directly and in relation to their intracellular antiprotease activities. Although these agents do not directly induce type I IFN, they do restore innate immune system signaling and IFN expression after attenuation with NS3/ 4A protease. These findings expand the known antiviral capabilities of natural tetrapyrroles and suggest that they represent a novel class of PIs that should be further investigated in vivo. MATERIALS AND METHODS See the Supplementary Material for a detailed description of our methods. We have shown elsewhere that the tetrapyrroles BV and heme inhibit NS3/4A protease and severely attenuate HCV replication in replicons and infected hepatocytes [14]. To investigate whether tetrapyrrole antiviral activity might also be accompanied by IFN induction in replicons, we assayed IFN- and IFN- messenger RNA (mRNA) levels after incubation with various concentrations of BV, heme, and zinc protoporphyrin (ZnPP) (Figure 1). Although all the tetrapyrroles potently inhibited HCV replication as well as NS3/4A protease activity, they did not significantly elevate type I IFN mRNA levels (Figure 1AD), which did not change even when >90% of viral RNA was eliminated. Similar results were obtained with fulllength Con 1 replicons [21], indicating that antiviral activity was not selective for NS replicons (not shown). We also considered the possibility that these replicons might be refractory to IFN. However, addition of exogenous IFN rapidly reduced HCV replication (Figure 1E), and we have shown elsewhere that tetrapyrroles such as BV actually increase the antiviral activity of exogenous IFN- [14]. It is known that cell lines permissive for HCV, such as Huh 7.5, generally have poor type I IFN induction responses [22, 23]. Consequently, it was important to test whether cells with intact IFN induction (...truncated)