Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down-regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB-DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB-DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half-life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3 0 untranslated region sequence is the element responsible for the effect of IMB-DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70-related HCV replication. Treatment of the HCV-infected hepatocytes with IMB-DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB-DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;52:845-853) H epatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family. 1 Current standard therapy for hepatitis C in the clinic is the combination of pegylated-interferon with ribavirin. 2,3 The regimen is effective in 40%-50% of patients infected with HCV genotype 1 and is associated with significant side effects. 3,4 Recently, telaprevir and boceprevir, two peptidomimetic inhibitors of the HCV nonstructural protein 3/4A (NS3/4A) protease, have shown great promise in clinical patients. [5][6][7][8] However, antiviral therapy targeting specific HCV proteases has caused emergence of drug-resistant mutations, 9-12 and thus, new mechanisms for anti-HCV drugs are highly desirable.Human heat shock cognate 70 (Hsc70, or heat shock protein A8) is a cytoplasm adenosine triphosphate-binding protein with 646 amino acids. 13 It is a member of the heat shock protein 70 (Hsp70) family with the gene located in chromosome 11. 14 The functions of Hsc70 protein in normal cells are complicated and remain to be clarified. 15 Virology research has shown that Hsc70 might play a role in regulating virion capsid assembly. 16 A recent study by Parent et al. demonstrated that host Hsc70 is part of the HCV particle. 17 They showed Hsc70 interaction HPD (HisPro-Asp) motif present on the E2 envelope of HCV (the J6/JFH [Japanese fulminant hepatitis] strain) and coexistence of Hsc70 with the HCV core and E2 proteins around lipid droplets in the infected cells.

Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(1) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. Conclusion: hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery.

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Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity purification. Herein, we propose a new approach, termed as the four-dimensional orthogonal electrophoresis (4-DE) system, to find and analyze the cytoplasmic protein complexes. 4-DE system is composed of two parts: nondenaturing part (Part I) and denaturing part (Part II). Through Part I and decision procedure separations, six protein complex candidates 20S core particle of proteasome (CP), hemoglobin (Hb, α2β2), Hb (α2δ2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat shock protein 60 (HSP60) were separated. CP, Hb (α2β2), PRDX2, and HSP60 with different MWs and pI's were chosen for Part II proteomic analysis. The results indicate that 4-DE is not only suitable for studying protein complexes and protein-protein interactions as well as structural proteomics from complex biological samples, but can also be easy to separate and concentrate intact protein complexes from dilute complex samples.

The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (alpha7beta7beta7alpha7) forming a symmetrical barrel-shaped structure. Typically, in previous reports, the purification of CP mainly relied on the antibody, liquid chromatography and affinity tag-based strategies. In this study, we report a relatively simple and economical protocol for proteomic analyses of CP, which combines differential centrifugations with native-PAGE. Furthermore, it is compatible with both scaled-up purification from erythrocytes and scaled-down purification from low to around 3.0 x 10(7) pancreatic cancer cells, SW1990. In addition, a direct three-dimensional gel electrophoresis approach that omits the interval procedure between native-PAGE and IEF/SDS-PAGE is introduced. The results obtained in this study show that this protocol has a valuable potential for the studies of CP and other protein complexes.