ABSTRACT
Hepatitis C virus (HCV) core protein has been shown to exhibit several biological properties which suggest an important role in liver pathogenesis and carcinogenesis. During a previous study, we showed that core mutants, isolated from tumour, could directly interact with PKR and maintain it in an activated form. In the present report, we have further investigated this interaction and mapped the core and PKR domains involved. Using glutathion S-transferase fusion protein harbouring the different domains of core or PKR, we determined that the N-terminal 1-58 amino acid (aa) of core protein and the N-terminal 1-180 aa of PKR are responsible for this direct interaction. Using this system we also confirmed that the core-PKR interaction induced PKR autophosphorylation. Furthermore, we found that core protein co-localized and co-immunoprecipitated with PKR in cells expressing a full-length HCV replicon, thus confirming that this interaction occurs when all HCV proteins are expressed. Considering that the activation of PKR has been observed in some cancer cell lines and tissues, it suggests that, depending on the cellular context, PKR may stimulate or inhibit cell proliferation. The precise mapping of core-PKR interaction provides new data to study the molecular mechanism underlying HCV pathogenesis.
Subject(s)
Hepacivirus/chemistry , RNA, Double-Stranded/metabolism , Viral Core Proteins/metabolism , eIF-2 Kinase/metabolism , Protein Structure, Tertiary , Viral Core Proteins/geneticsABSTRACT
Hepatitis C virus (HCV) is a major risk factor for human hepatocellular carcinoma (HCC) but the mechanisms underlying HCV-induced carcinogenesis are still poorly understood. We have hypothesized that viral variants, selected during long-term infection, might contribute to cellular transformation. To address this issue, we have investigated the effect of natural HCV core variants isolated from liver tumors (T), or their non-tumor (NT) counterparts, on the tumor growth factor-beta (TGF-beta) pathway, a major regulator of cellular proliferation, differentiation and apoptosis. We have found a significant reduction in TGF-beta reporter gene activity with the expression of core sequences isolated from liver tumors. In contrast, moderate or no effects were observed with non-tumor mutants or a core reference sequence. The molecular mechanisms have been characterized and involved the inhibition, by tumor-derived cores, of the DNA-binding activity of the Smad3/4 transcription factors complex. This inhibition occurs through a direct interaction between the central domain (amino acids 59-126) of tumor-derived core and the MH1 DNA-binding domain of Smad3, thus preventing its binding to DNA. We have therefore identified a new cell-signaling pathway targeted by HCV core and inhibited by tumor-derived core sequences. These results suggest that during chronic infection, there is selection of viral variants that may promote cell transformation by providing, to clonally expanding cells, resistance to TGF-beta antiproliferative effects.
Subject(s)
Carcinoma, Hepatocellular/virology , DNA-Binding Proteins/physiology , Hepacivirus/genetics , Liver Neoplasms/virology , Trans-Activators/physiology , Transforming Growth Factor beta/physiology , Viral Core Proteins/physiology , Amino Acid Sequence , Carcinoma, Hepatocellular/physiopathology , Cell Transformation, Neoplastic , Hepacivirus/physiology , Humans , Liver Neoplasms/physiopathology , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Signal Transduction , Smad3 Protein , Smad4 Protein , Tumor Cells, Cultured , Viral Core Proteins/geneticsABSTRACT
<p><b>OBJECTIVE</b>To study the role of different truncated core proteins (CORE) of hepatitis C virus (HCV) played in the pathogenesis of HCV persistent infection and hepatocellular carcinoma, and to construct seven different truncated prokaryotic expression plasmids of HCV CORE.</p><p><b>METHODS</b>The gene sequences of different truncated HCV genotype 1b CORE were amplified from plasmids containing CORE sequences derived from tumor and non-tumor tissues of a patient infected with HCV. Amino acid (aa) lengths of HCV BT (from tumor tissue, patient B) were: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; of BNT (non-tumor tissue, patient B) were: 1-172 aa and HCV C191 (HCV-J6): 1-172 aa. PCR products were cleaved with restriction enzymes BamH I and EcoR I and cloned into pGEX-4T-1. Positive clones were transformed into BL21 and glutathione S-transferase(GST)-CORE fusion proteins were expressed with isopropylthio-beta-D-galactoside (IPTG) induction, purified and verified by Western blot.</p><p><b>RESULTS</b>Different truncated GST-CORE fusion proteins were expressed with different quantities. Except the fragment of 59-126 aa, the longer the fragment, the less its expression. The levels of truncated expression of CORE of BT and BNT were higher than that of C191, even though they all contained 1-172 aa. Some of truncated CORE of HCV genotype 1b could form dimmers.</p><p><b>CONCLUSIONS</b>Successful construction of truncated GST-CORE expression plasmids lays a basis for future study of the function of different domains of CORE of different HCV strains; different expression levels of HCV COREs might be related to their different hydrophobicity, cytotoxicity and their functions in HCV structure and their roles played in the pathogenesis; the domain of 59-126 aa is responsible for the HCV genotype 1b CORE dimmer formation.</p>
