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The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV), plays a critical role in HCV replication and is an attractive target for the therapy of HCV infection. So far, little is known about the post-translational regulation of NS5A protein and its precise role in HCV RNA replication. Our objectives were to elucidate the down-regulation of NS5A protein and HCV RNA replication by zinc mesoporphyrin (ZnMP), and the mechanism by which this process occurs.
Human hepatoma cells expressing HCV proteins were used to investigate the post-translational regulation of ZnMP on NS5A protein by Western blots (WB) and immunoprecipitation (IP). Quantitative RT-PCR (qRT-PCR) was used to determine the effects of ZnMP on HCV RNA replication.
ZnMP selectively and markedly down-regulated NS5A protein levels by increasing degradation of NS5A protein [half life fell from 18.7 h to 2.7 h]. The proteasome inhibitors, epoxomicin and MG132, significantly abrogated degradation of NS5A protein by ZnMP without affecting levels of NS5A in the absence of ZnMP. Analysis of immunoprecipitates with an anti-ubiquitin antibody revealed polyubiquitination of NS5A, suggesting that ZnMP induces ubiquitination of NS5A protein. In addition, 10 μM of ZnMP reduced HCV replication by ~63% in the Con1 replicon cells, ~70% in J6/JFH1 HCV transfected cells, and ~90% in J6/JFH1 HCV infected cells without affecting cell viability.
ZnMP produces a rapid and profound down-regulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent catabolism. Zinc mesoporphyrin may hold promise as a novel agent to treat HCV infection.
Hepatitis C virus (HCV) is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer1. Current treatments for HCV infection using pegylated interferon (Peg-IFN) and ribavirin (RBV) fail to produce sustained virological responses in fewer than 50% of adequately treated patients infected with HCV genotype 1, and have numerous unpleasant side effects. These side-effects and the rather high costs, and required duration of such treatment limit its use. Thus, it is too costly for most patients in developing countries and many in developed countries to afford. Clearly, more effective strategies to treat HCV infection are urgently needed.
The hepatitis C virion contains a 9.6-kb positive single-strand RNA genome which encodes a single polyprotein of approximately 3010 amino acids, that is then processed into structural (C, E1, E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins2–5. The nonstructural 5A (NS5A) protein, a major component of HCV proteins, is a 447-amino-acid phosphorylated zinc-metalloprotein with largely unknown functions6, 7. Recent evidence has shown that NS5A plays a critical role in the replication of HCV, both directly, with regard to viral RNA replication, and indirectly, by modulating the host cell environment to favor the virus6–8. NS5A has been shown to interact with nonstructural 5B (NS5B) protein, the viral RNA-dependent RNA polymerase, and this interaction is essential for maintenance of subgenomic replications in Huh-7 replicon cells9, 10. NS5A binds to the 3′-ends of HCV positive strand RNA with high affinity, and this binding is essential for genome replication and infection11. A recent study revealed that a cellular microRNA-196 (miR-196) directly targets NS5A and is able to substantially attenuate viral replication in JFH1 replicon Huh-7 cells12. The most recent study from Hughes et al reported that domain III of NS5A is implicated in both RNA replication and assembly of hepatitis C virus particles in JFH1-infected cells13. Although precise functions of NS5A in both HCV genomic RNA replication and modulation of physiology of the host cell remain unclear, targeting NS5A is an attractive emerging strategy for the therapy of HCV infection8, 14.
Zinc mesoporphyrin (ZnMP) is a non-heme metalloporphyrin and a synthetic heme analogue with a central zinc of the mesoporphyin macrocycle. ZnMP has been demonstrated as a potent inhibitor of heme oxygenase (HO) and proposed as a therapeutic agent for severe unconjugated hyperbilirubinemia (e.g., Crigler-Najjar syndrome type II) and for prolonging effects of heme in therapy of acute porphyric syndromes15, 16. Recent studies from our and other laboratories have shown that ZnMP17, tin mesoporphyrin (SnMP)18 and heme19 induce ubiquitination and proteasomal degradation of Bach1, a transcriptional repressor of the heme oxygenase 1 (HO-1) gene, in human hepatoma cells and NIH3T3 cells as one of the mechanisms by which the HO-1 gene is up-regulated. In addition, we have recently reported that cobalt protoporphyrin (CoPP) down-regulates HCV core protein and suppresses HCV replication in human hepatoma cells stably expressing HCV proteins20. So far, little is known about the ubiquitin-proteasome degradation of NS5A protein and its role in HCV RNA replication. In this study, we show down-regulation of NS5A protein by ZnMP and the molecular mechanism involved in this process. We demonstrate that ZnMP decreases the level of NS5A protein at the post-translational level by modulating protein degradation through a proteasome-ubiquitin degradation pathway. In addition, ZnMP suppress HCV RNA replication. We propose that zinc porphyrin-mediated ubiquitin-proteasome degradation pathway may play a role in affecting the HCV RNA replication through controlling the amount of NS5A protein. The observations presented herein suggest that zinc porphyrins may be of value in treatment of HCV infection.
See Supplemental Materials and Methods for more details.
The 9–13, CNS3 and Huh-7/T7 cell lines were kindly provided by R. Bartenschlager (University of Heidelberg, Heidelberg, Germany). 9–13 subgenomic replicon cells, containing a replicating HCV nonstructural region of the Con1 isolate, stably express HCV NS3 to NS5B21. CNS3 cells stably express HCV core to NS3. Huh-7/T7 cells constitutively express the bacteriophage T7 RNA polymerase22. Cell line Huh-7.5, a highly permissive, alpha interferon-cured Huh-7 derivative was kindly provided by C. M. Rice (The Rockefeller University, New York, NY). The Con1 genotype 1b HCV replicon cell line was from Apath LLC (St, Louis, MO). The Con1 cell line is a Huh-7.5 cell population containing the full-length HCV genotype 1b replicon 23, 24.
The pFK-Con1/GDD and pFK-Con1/GND constructs (genotype 1b) were kind gifts of Dr. R. Bartenschlager (University of Heidelberg, Heidelberg, Germany). The pFK-Con1/GDD construct containing the wild type Con1 genome was described21 and pFK-Con1/GND construct was a replication-deficient variant of pFK-Con1/GDD with a single amino substitution, which changed the GDD motif of the NS5B polymerase active site to GND25. Transfection of pFK-Con1/GDD or pFK-Con1/GND was performed as described in Suppl. Materials and Methods.
The HCV infectious clone pJ6/JFH1 was generously provided by Dr. C. Rice (the Rockefelller University, New York, NY). The full-length chimeric genome was constructed with the use of the core-NS2 gene regions from the infectious J6 (genotype 2a) and NS3-NS5B gene regions from the infectious JFH1 (genotype 2a) as described by Lindenbach et al23. To generate HCV J6/JFH1 RNA, the pJ6/JFH1 plasmid was linearized with XbaI, and purified by ethanol precipitation, digestion with proteinase K, and phenol-chloroform extraction. The linearized plasmid was used as a template for in vitro transcription using the MEGAscript T7 kit (Ambion, Austin, TX). For HCV RNA transfection, Huh-7.5 cells were plated in 24-well plates one day prior to transfection and transfected at 70~80% confluence. Cells were transfected at an RNA/lipofectamine ratio of 1:2 by using 2 μg/well of HCV RNA and 4 uL/well Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 48 h. For HCV infection, cell culture supernatants from the cells transfected with HCV RNA for 48 h were collected and filtered through a 0.20 μm filter, and infected naïve Huh-7.5 cells in 24-well plates for 72 h. Total RNA and protein were extracted for subsequent RT-PCR and Western blot measurements.
Western blot analysis and quantitative RT-PCR were performed using the standard protocols of our laboratory as described previously17. Immunoprecipitation was carried out according to the manufacturer’s protocol from Santa Cruz (Santa Cruz, CA). Antibodies used are described in Supplemental Material and Methods.
9–13 cells were incubated with 100 ug/mL cycloheximide (CHX) in the presence or absence of 10 uM ZnMP. Western blots were performed using anti-HCV NS5A and anti-GAPDH antibodies. Band intensities of Western blots were measured by densitometric analysis. GAPDH bands were used as internal controls to correct for protein loadings.
Earlier studies from our and other laboratories have shown that heme or cobalt protoporphyrin (CoPP) significantly reduced HCV20 or HBV26 core proteins. However, little attention has been given to the effect of ZnMP on the HCV proteins. To investigate whether ZnMP down-regulates the HCV proteins, we measured NS5A protein levels in 9–13 and Con1 cells, and core protein levels in CNS3 cells exposed to different concentrations of ZnMP (0, 1, 5, 10 μM) for 4 h, ZnMP led to a marked decrease of NS5A protein levels in 9–13 and Con1 cells in a dose-dependent fashion (Fig. 1A & B).
The effect was selective and specific: there were no detectable effects of ZnMP on HCV core protein levels in CNS3 cells and in Huh-7/T7 cells transfected with pFK-Con1/GND, a plasmid encoding a replication deficient variant of Con1 (Fig. 1C & D). We also observed that protein levels were not affected by 10 μM free mesoporphyrin or ZnCl2 (Fig. 2A). Tin mesoporphyrin (SnMP), another competitive HO inhibitor, has been reported recently to down-regulate Bach1 protein levels and induce the HO-1 gene expression in NIH 3T3 cells. We thus performed comparative studies on ZnMP vs SnMP down-regulation of NS5A in 9–13 cells. As shown in Fig. 2B, ZnMP markedly and rapidly decreased NS5A protein levels after exposure to ZnMP for as little as 2 h. In contrast, no detectable effects of SnMP on NS5A protein levels were observed. In addition, the potent zinc chelator, N, N, N, N-tetrakis-(2-pyridylmethyl) ethylenediamine (TEPN) did not affect ZnMP-mediated profound down-regulation of NS5A protein levels in 9–13 cells (Fig. 3).
Next we examined whether down-regulation of NS5A protein by ZnMP occurs at a post-translational level. As shown in Fig. 4, NS5A protein levels in 9–13 cells treated with ZnMP and cycloheximide (CHX) were greatly and rapidly reduced. NS5A protein levels in 9–13 cells that were not treated with ZnMP were also decreased by CHX, but to a much lesser extent. Specifically, ZnMP at a concentration of 10 μM decreased the NS5A protein half life (t1/2) from 18.7 h to 2.7 h (Fig. 4B & C).
To understand whether degradation of NS5A protein by ZnMP is proteasome dependent, 9–13 cells were treated with ZnMP (5, 10 μM) and selected proteasome inhibitors, epoxomicin (5, 10 μM) and MG132 (10, 20 μM). We found that epoxomicin (5, 10 μM) and MG132 (10, 20 μM) completely abrogated the degradation of NS5A in cells exposed to a lower concentration of ZnMP (5 μM), and cells treated with ZnMP (10 μM) and epoxomicin (5, 10 μM) or MG132 (10, 20 μM) displayed significant, although less than complete, diminution of the degradation of NS5A by ZnMP (Fig. 5A & B), however, epoxomicin or MG132 did not affect NS5A protein levels in the absence of ZnMP in 9–13 cells (Fig. 5C), suggesting that the proteasome-dependent degradation pathway is involved in ZnMP-mediated NS5A breakdown. The highest concentration of epoxomicin used in these experiments was 10 μM, because cells exposed to 20 μM epoxomicin failed to grow well, indicating that this concentration of epoxomicin was toxic to the cells.
To gain insight into the mechanism by which ZnMP mediates degradation of NS5A protein, we examined whether ZnMP induce polyubiquitination of NS5A [(Ub)n-NS5A]. After treating cells with ZnMP, or with vehicle (DMSO) alone as control, cell lysates were prepared and were subjected to immunoprecipitation (IP) of NS5A with anti-NS5A monoclonal antibody. An immunoblotting (IB) analysis of the precipitates with an anti-ubiquitin antibody revealed polyubiquitination of NS5A [(Ub)n-NS5A] following ZnMP treatment (Fig. 6B). In contrast, it did not show a significant level of ubiquitination under control condition (Fig. 6B). Immunoblot analysis with anti-NS5A antibody confirmed that NS5A proteins were immunoprecipitated (Fig. 6C) and that higher-molecular-weight bands may represent ubiquitylated forms of the NS5A protein (Fig. 6C). The results suggest that ZnMP induce polyubiquitination of NS5A which contributes to the degradation of NS5A by ZnMP.
To evaluate whether ZnMP-mediated degradation of NS5A may play a role in inhibiting HCV replication, Con1 full-length HCV replicon cells were treated with different concentrations of ZnMP for 24 h. As expected, vehicle (DMSO) alone did not alter the amounts of HCV replicon RNA, whereas treatment with ZnMP resulted in a dose-dependent reduction in viral RNA (Fig. 7A), and protein levels (Fig. 7B), suggesting that ZnMP-mediated rapid degradation of NS5A may lead to reduction of HCV RNA replication, and subsequent decrease in HCV protein expression. And then we asked if NS5A is an actual target of ZnMP and the effects of ZnMP on HCV RNA replication and core protein levels are secondary to ZnMP-mediated rapid degradation of NS5A. To this end, we performed parallel experiments with HCV proteins expressed from a DNA plasmid pFK-Con1/GND in Huh-7/T7 cells, where their expression would not be linked to viral RNA polymerase but only to T7 RNA polymerase. ZnMP markedly decreased NS5A protein levels in a dose-dependent fashion, whereas HCV core protein levels remained unaffected after 24 h of ZnMP treatment (Fig. 7C). We further observed that ZnMP resulted in reduction of core in the system that HCV proteins were expressed from pFK-Con1/GDD in Huh-7/T7 cells, where their expression would be partly linked to viral RNA polymerase (Fig. 7D), however, the reduction of core was much less than the effect in Con1 replicon system, where expression of HCV proteins were linked to viral RNA polymerase.
Next, we used the JFH1-based HCV cell culture system to investigate whether ZnMP down-regulates NS5A protein and displays anti-viral activity in this novel JFH1-based (genotype 2a) HCVcc system. We measured NS5A and core protein levels in J6/JFH1 transfected Huh-7.5 cells treated with indicated concentrations of ZnMP for 4 or 24 h. ZnMP led to a rapid and profound decrease of NS5A protein levels, while core protein levels were not affected after 4 h of ZnMP treatment and showed a decrease after 24 h exposure to ZnMP (Fig. 8A-D). To further examine whether ZnMP inhibits HCV RNA replication/infection in J6/JFH1 transfected and infected cell culture system, we analyzed HCV RNA expression after ZnMP treatment. 10 μM of ZnMP markedly decreased HCV RNA levels by ~70% in HCV-transfected cells and ~90% in HCV-infected cells (Fig. 8E & F).
To exclude the possibility that the reduction in HCV viral replication could be a non-specific effect of ZnMP to cause cytotoxicity of treated cells, cells were exposed to ZnMP (0, 1, 5, 10 μM) for 2, 6, or 24 h. As shown in Suppl. Fig. 2, ZnMP treatments did not significantly affect cell viability, suggesting that the effects of ZnMP on HCV viral RNA and protein expression were not due to cytotoxic effects.
The major findings of this paper are as follows: 1) ZnMP, but not the free mesoporphyrin nor ZnCI2 down-regulates NS5A protein levels in a dose–dependent fashion in human hepatoma cells stably expressing HCV proteins (Fig. 1, ,22 & 3); 2) ZnMP reduces the stability of NS5A protein by decreasing its half life from 18.7 h to 2.7 h, suggesting that the effect of ZnMP on NS5A occurs at a post-translational level (Fig. 4); 3) proteasome inhibitors, epoxomicin and MG132, block the degradation of NS5A (Fig. 5), indicating that down-regulation of NS5A by ZnMP is proteasome dependent; 4) ZnMP significantly induces polyubiquitination of NS5A, supporting the hypothesis that the proteasome-ubiquitin degradation pathway is involved in down-regulation of NS5A (Fig. 6); 5) ZnMP significantly reduces HCV RNA replication in Con1 replicon cells, JFH1-based HCV transfected cells, and JFH1-based HCV infected cells, suggesting that ZnMP-mediated degradation of NS5A may play a part in inhibiting HCV replication/infection (Fig. 7 & 8). 6) There is no evidence that ZnMP affects HCV core protein levels in CNS3 cells (Fig. 1C), in Huh-7/T7 cells transfected with pFK-Con1/GND (Fig. 1D & 7C), a plasmid encoding a replication deficient variant of Con1 (Fig. 1D), indicating that the effect of ZnMP on NS5A is selective and specific.
In this study, three different cell lines that stably express HCV proteins and JFH1-based HCV cell culture system were used (Suppl. Fig. 1). ZnMP decreased NS5A protein levels in Con1-based replicon cells and JFH1-based HCV cell culture system but did not show significant down-regulation of core protein in CNS3 (Fig. 1) and Huh-7/T7 cells transfected with pFK-Con1/GND (Fig.1D & 7C). ZnMP at concentrations of 10 μM was not toxic (Suppl. Fig. 2) and the maximal concentration of ZnMP used in this study was at 10 μM. Therefore it is clear that the down-regulation of ZnMP on NS5A and HCV RNA repliction are not due to a general, non-specific toxic effect, but due to its ability to activate the ubiquitin-proteasomal pathway of NS5A protein catabolism.
In recent years, it has become increasingly clear that that NS5A plays a key role in HCV RNA replication by participating in polyprotein cleavage, interferon response and cellular signaling pathways8, 14. HCV NS5A includes domain I (amino acids 1–213), domain II (amino acids 250–342), and domain III (amino acids 355–447)8, 27. The N-terminal domain (domain I) contains a zinc binding motif, which is a vital component of the HCV replication7. Domain II, containing an interferon sensitivity-determining region (amino acids 237–276), is associated with IFN resistance and lack of response to treatment in genotype1a-infected patients28. In this report, we observed that ZnMP induces markedly down-regulation of NS5A without affecting core protein and significantly reduces HCV RNA replication. We thus propose that the reduction of HCV RNA replication occurs primarily through controlling the amount of HCV NS5A protein by ZnMP. These results also provide strong evidence to support the novel strategy for treatment of HCV infection by targeting NS5A protein levels.
Ubiquitin (Ub) was first identified as a highly-conserved small protein in eukaryotic cells that is composed of 76 amino acids with a predicted molecular weight of 8.5 kDa29, 30. The ubiquitin-proteasome degradation pathway has been well accepted as an important regulatory system in many cellular processes such as cell cycle, DNA repair, embryogenesis, the regulation of transcription and apoptosis29–31. In the ubiquitin-proteasome pathway, protein substrates are first marked for degradation by covalent linkage to multiple molecules of ubiquitin (polyubiquitination) and then are hydrolyzed by the 26 S proteasome, a 2000 kDa ATP-dependent proteolytic complex. We previously reported that ZnMP produces profound degradation of Bach1 protein in a proteasome-dependent manner as one of molecular mechanisms by which ZnMP up-regulates the HO-1 gene expression17. Others have reported that HCV proteins, such as NS5B32, core33, 34, the unglycosylated cytosolic form of E235, NS236, and F protein37 are degraded through the ubiquitin-proteasome pathway. To our knowledge, the present study is the first report showing the degradation of NS5A protein and reduction of HCV RNA replication by degradation of NS5A. Taken together, these studies suggest that the ubiquitin-proteasome pathway plays a role in the HCV life cycle or viral pathogenesis. In addition, our data provide a rationale to develop antiviral drugs that increases ubiquitination of NS5A.
ZnMP induces polyubiquitination of NS5A and display anti-viral activity, we found ZnMP an attractive choice, and we chose to use mainly ZnMP because of the profound effects on Bach117, because mesoporphyrin derivatives lack reactive vinyl groups and are therefore more chemically stable than protoporphyrin derivatives38, and because it is taken up by intact liver cells far better than other zinc derivatives such as bisglycol-deuteroporphyrin congener39. Zinc porphyrins, under some circumstances, may be toxic to hematopoietic cells in vitro40. There was nothing to suggest cytotoxicity to liver cells in this work (Suppl. Fig.2). In addition, earlier work showed no evidence of toxicity of intravenously administrated ZnMP in intact rats. The ZnMP in these animals partitioned mainly to liver and spleen and inhibited heme oxygenase activity particularly in these organs. In this study, we chose to administer ZnMP bound to human serum albumin (HSA), because like other metalloporphyrins, ZnMP has limited solubility in physiologic buffer, but its solubility and uptake into liver cells are greatly enhanced by albumin binding41, 42 and because ZnMP, when administered as an albumin complex, is nontoxic and is taken up preferentially by the liver and spleen43.
In conclusion, we have found that ZnMP, but not the free mesoporphyrin nor the metallic ion itself induces post-translational down-regulation of HCV NS5A protein in an ubiquitin-proteasome degradation pathway. Our results indicate that ZnMP significantly suppresses HCV viral replication in the Con1 full length replicon Huh-7.5 cells and JFH1-based HCV cell culture system, suggesting that ZnMP may hold promise as a novel agent to treat HCV infection. We propose that ZnMP-mediated ubiquitin-proteasome degradation pathway may play a role in affecting the HCV RNA replication through controlling the amount of NS5A protein. Our studies also raise many important new questions that need to be answered in future. Further studies are underway in our laboratory, designed to unravel the molecular mechanism that underlies the effect of ZnMP to trigger ubiquitination of Bach1 or HCV NS5A, as well as to search for other zinc derivatives, which might display greater effects on NS5A degradation and HCV RNA replication.
We thank Dr. Ralf Bartenschlager for kindly providing the 9–13, CNS3 and Huh-7/T7 cells, pFK-Con1/GDD and pFK-Con1/GND constructs, and his helpful suggestions. We thank Dr. Charles M. Rice for generously providing the J6/JFH1 molecular clone.
Grant Support: This work was supported by United States Public Health Service Grants RO1-DK38825, contracts NO-1 DK92326 and UO-1 DK065193, and Carolinas Medical Center Grant CMC 08-35
Author Contributions: Hou W was responsible for the conception, design, experimentation, and drafting of this paper; Tian Q and Zheng J each contributed to experiments as well as data analysis. Bonkovsky HL was instrumental in the conception and in revising this paper critically.
Disclosure: All authors have no conflicts of interest to disclose
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