Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5-nontranslated region (5 NTR) and the 3 NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.Approximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for most cases of chronic liver disease, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (45,70).HCV has a single-stranded RNA genome of positive polarity that contains a single long open reading frame flanked by nontranslated regions (NTRs) at its 5Ј and 3Ј ends. Nearly the entire 5Ј NTR is needed for efficient RNA amplification, although the minimal replication element is within the first 120 nucleotides (20,35,58). In addition, the 5Ј NTR has been found to contain an internal ribosomal entry site (IRES), which is required for the translation of polyprotein (29,67,75). The 3Ј NTR is composed of a nonessential variable region, a poly(U)/poly(U ⅐ C) tract, and a highly conserved and essential 3Ј X domain (19,40,74). The viral proteins are translated as a single large polyprotein of 3,010 to 3,040 amino acids, which is cotranslationally and/or posttranslationally processed by cellular and viral proteases into mature structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) viral proteins (57). NS5b is an RNA-dependent RNA polymerase that produces complementary RNAs from template RNAs (3, 66).Subgenomic HCV RNA replicons have been developed to mimic the replication of a viral RNA infecting a host cell. These replicons are composed of the HCV 5Ј NTR; a selection marker, such as neomycin phosphotransferase; the IRES of encephalomyocarditis virus; HCV nonstructural proteins NS3, -4a, -4b, -5a, and -5b; and the HCV 3Ј NTR. When introduced into human hepatoma (Huh 7) cells, the HCV replicon RNA replicates autonomously (47). Using this replicon system, several groups have reported that HCV replication occurs in a distinct replication complex, which comprises viral RNA and HCV proteins (1,12,23,54). The replication complex is formed on intracellular membranes with vesic...

Hepatitis C virus (HCV) is a positive-sense RNA virus ∼9600 bases long. An internal ribosomal entry site (IRES) spans the 5 nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.

Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and can be involved in very long chronic infections up to 30 years or more. Therefore, it has been speculated that HCV possesses mechanisms capable of modulating host defense systems such as innate and adaptive immunity. To investigate this virus-host interaction, we generated HCV replicons containing various HCV structural proteins and then analyzed the sensitivity of replicon-containing cells to the apoptosis-inducing agent, TRAIL. TRAIL-induced apoptosis was monitored by cleavage of procaspase-3 and procaspase-9 as well as that of their substrate poly(ADP-ribose) polymerase. TRAIL-induced apoptosis was inhibited in cells expressing HCV E2. Moreover, expression of HCV E2 enhanced the colony forming efficiency of replicon-containing cells by 25-fold. Blockage of apoptosis by E2 seems to be related to inhibition of TRAIL-induced cytochrome c release from the mitochondria. Based on these results, we propose that E2 augments persistent HCV infection by blocking host-induced apoptosis of infected cells.