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 (, & ); 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 (); 3) proteasome inhibitors, epoxomicin and MG132, block the degradation of NS5A (), 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 (); 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 ( & ). 6) There is no evidence that ZnMP affects HCV core protein levels in CNS3 cells (), in Huh-7/T7 cells transfected with pFK-Con1/GND ( & ), a plasmid encoding a replication deficient variant of Con1 (), 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 () and Huh-7/T7 cells transfected with pFK-Con1/GND ( & ). 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
, 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.