Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5 nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.The translation of eukaryotic mRNAs occurs either by capdependent scanning or by direct binding of a ribosome to a specialized RNA element called an internal ribosomal entry site (IRES) (12). Although the canonical translation initiation factors function in translation through IRES elements, many RNA-binding proteins have also been shown to play important roles (12,14). For instance, host cellular proteins such as polypyrimidine tract-binding protein (PTB), the La autoantigen, poly(rC)-binding proteins, and upstream of N-ras (Unr) bind directly to IRESs and enhance the translation of picornaviral mRNAs (12,14).Hepatitis C virus (HCV), the major causative agent of virusrelated liver cirrhosis and hepatocellular carcinoma in humans, is a positive-sense RNA virus. The HCV RNA contains a 341-nucleotide (nt) 5Ј nontranslated region (5ЈNTR) and a very long open reading frame encoding a polyprotein that is proteolytically processed into 10 or more viral proteins (9, 15). Translation of the HCV polyprotein is directed by an IRES element spanning the 5ЈNTR (29).Curiously, while most IRESs require only the 5ЈNTR for full activity, the HCV IRES depends on the presence of a proteincoding sequence downstream of the initiating AUG (19,26). An HCV RNA encoding the N-terminal part of the HCV polyprotein was shown to be required for full HCV IRES activity when heterologous reporter genes (influenza NSЈ and secreted alkaline phosphatase) were connected to the HCV 5ЈNTR (26). Moreover, the same region was absolutely required for the generation of a viable chimeric poliovirus that used the HCV IRES element for translation (19). However, several other reports have suggested that the core-coding sequence is not strictly essential for HCV IRES activity (27,29,30). Thus, the true molecular basis of translational activation by the HCV core-coding sequence is still a subject of some debate.Here we investigated the HCV IRES translational initiation mechanism by analyzing proteins that were found to specifically interact with the IRES element....

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), one of the major causative agents of virus-related liver cirrhosis and hepatocellular carcinoma in humans, contains a single-stranded RNA genome of positive polarity. HCV RNA contains nontranslated regions (NTRs) at the 5Ј and 3Ј ends (18,23) and a long open reading frame encoding polyprotein that is synthesized through a single translational initiation event directed by an RNA element designated the internal ribosomal entry site (IRES) (21, 38) at the 5Ј NTR (46). The polyprotein is proteolytically processed into 10 or more viral proteins.In general, IRES elements need several canonical translation factors (except eukaryotic initiation factor 4E [eIF4E]) for their activities (40,42). However, HCV IRES-dependent translation requires only a few canonical factors (eIF2, eIF3, eIF5, and eIF5B) for function (30,39,41). Additionally, cellular proteins known as IRES-specific cellular transacting factors (ITAFs) are required for the efficient translation of HCV mRNA. For instance, polypyrimidine tract-binding protein (PTB) interacting with HCV IRES is required for IRES function (2, 3). La antigen interacting with the GCAC site near the initiator AUG is necessary for the optimal function of the HCV IRES (1, 10, 43). Recent studies have shown that NSAP1 interacting with the adenosine-rich core-coding region of HCV mRNA augments HCV IRES-dependent translation (27).Heterogeneous ribonucleoprotein D (hnRNP D), also known as AU-rich element RNA-binding protein 1 (AUF1), is an hnRNP family member that shuttles between the nucleus and cytoplasm (44, 48). hnRNP D was initially identified owing to its ability to bind and destabilize c-myc mRNA in a crude in vitro decay system (6). The protein has four isoforms of different molecular weights (p37, p40, p42, and p45), all of which are produced by alternate splicing of a single transcript (11,47). The hnRNP D protein has various functions, including mRNA decay (6), telomere maintenance (12) MATERIALS AND METHODSPlasmid construction and small interfering RNA (siRNA). Dual reporters harboring the HCV IRES, encephalomyocarditis virus (EMCV) IRES, and cmyc IRES were constructed as described previously (27). The monocistronic reporter containing the HCV IRES and EMCV IRES, followed by firefly luciferase used for in vitro translation, were prepared according to methods described in a previous report (28). Plasmids expressing hnRNP D, pFLAG-CMV2 p37, pFLAG-CMV2 p40, pFLAG-CMV2 p42, and pFLAG-CMV2 p45 were kindly provided by R. J. Schneider at New York University School of Medicine (44). To generate pRSET A-hnRNP D for recombinant hnRNP D purification, pFLAG-CMV2 p45 was treated with HindIII-Klenow-EcoRI, and the resulting DNA fragment was cloned into NcoI-Klenow-EcoRI-treated pRSET A (Invitrogen).To construct pH(130-228)CAT and pH(229-402)CAT used for generating an RNA probe (see Fig. 2B, below), HCV IRES corresponding to positions 130 to 228 and 229 to 402 were amplified from pH(18-402)CAT (27) using the following primer pairs: 5Ј-CTAGGTACCGGGAGAGCCATAG-3Ј and 5...

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.