Zinc Mesoporphyrin Induces Rapid Proteasomal Degradation of Hepatitis C Nonstructural 5A Protein in Human Hepatoma Cells

Abstract

Background & Aims 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. Methods 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. Results 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. Conclusions 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.

Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A

“…Interestingly, the ubiquitinated form of NS5A was also detected in the absence of MG132, suggesting that HCV-NS5A protein is a heavily ubiquitinated protein. It is noteworthy that zinc mesoporphyrin, which suppresses HCV replication, exhibited enhanced ubiquitination of HCV-NS5A (39). To determine whether ISGylation of NS5A affects ubiquitination of NS5A, which leads to the protein degradation via 26S proteasome, we examined ubiquitination of HCV-NS5A (K379R) mutant protein.…”

Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation

“…Positive-stranded RNA viruses adopt cellular degradation pathways to maintain appropriate viral protein levels that avoid inhibitory effects of high-level RNA-dependent RNA polymerases (RdRps) on virus assembly, or premature apoptosis induced by excess viral proteases, thus supporting productive viral propagation (80). Accordingly, the protein levels of HCV core, NS2, NS5B, and NS5A are all tightly regulated by cellular protein degradation pathways (24,80). Moreover, studies of positive-stranded RNA viruses, including encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV), have implied that the degradation-mediated regulation is fine-tuned dynamically at different viral life cycle stages (80).…”

“…On the other hand, NS5A has been reported to be regulated by ubiquitin-proteasome degradation (24). Administration of zinc mesoporphyrin (ZnMP), a synthetic nonheme metalloporphyrin, induces NS5A ubiquitination and proteasome degradation and, hence, inhibition of HCV RNA replication (24).…”

Roles of the two distinct proteasome pathways in hepatitis C virus infection

“…Zinc mesoporphyrin (ZnMP) is a non-heme metalloporphyrin and a synthetic heme analog of the central zinc in the mesoporphyrin macrocycle. ZnMP enhances the polyubiquitylation and proteasomal degradation of NS5A and suppresses HCV RNA replication [49] . The physiological role of the ubiquitin-dependent proteasomal degradation of NS5A protein is still unclear and the ubiquitin ligase that targets NS5A also remains to be identified.…”