Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.     

Nuclear factors are involved in hepatitis C virus RNA replication

Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 5' and 3' termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein. HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 5'-3' interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.     

Viral and cellular determinants of hepatitis C virus RNA replication in cell culture

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.     

Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.     

Host-based ribavirin resistance influences hepatitis C virus replication and treatment response

Many individuals infected with hepatitis C virus (HCV) develop a chronic infection, and of those who are treated with pegylated interferon and ribavirin (RBV), many do not respond. While the nucleoside analog RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV's mechanism is controversial. Most of RBV's proposed mechanisms require RBV import into cells. Therefore, we explored whether host-based RBV resistance develops through reduced cellular uptake, akin to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on HCV replication in cultured hepatoma Huh7.5 liver cells and whether RBV resistance develops in HCV patients. When Huh7.5 cells were exposed to RBV, resistance developed through reduced RBV uptake via the ENT1 nucleoside transporter and antiviral efficacy was reduced. The uptake defect in RBV-resistant cells was specific to RBV, since transport of another ENT1 substrate, cytidine, was unaffected. Importantly, RBV uptake significantly declined in HCV patient peripheral blood mononuclear cells (PBMCs) following 4 weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance and suggest that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.     

Assays for RNA synthesis and replication by the hepatitis C virus

At least six major genotypes of Hepatitis C virus (HCV) cause liver diseases worldwide.The efficacy rates with current standard of care are about 50％ against genotype 1,the most prevalent strain in the...     

Persistent and transient replication of full-length hepatitis C virus genomes in cell culture

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.     

Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication

The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3' NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3'-terminal sequence designated the X tail or 3'X. Conflicting data as to the role the sequences in the 3' NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3' NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3'X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3' NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3' NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication

The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.     

Effects of mutations of the initiation nucleotides on hepatitis C virus RNA replication in the cell

Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5' and 3' ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5' end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5' end dramatically reduced the efficiency of cell colony formation. Likewise, the 3'-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3' end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5'-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5' end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5'-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3'-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3' ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3' end. We also determined the 5'-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5' end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.     

Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase

Crotty et al. recently proposed the primary antiviral action of ribavirin to be that of a potent RNA mutagen [Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379]. Here we investigate the effect of ribavirin triphosphate (RTP) on RNA synthesis catalyzed by a full-length hepatitis C virus (HCV) RNA polymerase in vitro. HCV polymerase can use RTP as a nucleotide substrate in a template-dependent manner, incorporating it opposite a pyrimidine (C or U) template residue, but not a purine (A or G). Kinetic analysis revealed that incorporation of ribavirin monophosphate (RMP) across from C is 3 times more efficient catalytically than that across from U, as determined by the k(cat)/K(m) parameter. The efficiency of RMP incorporation, however, is 50-100 fold lower than that of the natural NMP. RMP incorporation does not lead to termination of RNA chain synthesis, as evidenced by the ability of the polymerase to extend its RNA product many nucleotides beyond the site of RMP incorporation. However, multiple-RMP incorporation at low GTP concentrations induced the formation of stalled elongation complexes, particularly at the template region containing consecutive C residues. Most, but not all, such elongation blocks can be relieved by the re-addition of GTP. When ribavirin is present in the RNA template, pyrimidine (but neither purine nor ribavirin) monophosphate is incorporated opposite ribavirin, but at an exceedingly low catalytic efficiency (200-3000-fold lower) compared to the efficiencies of those templated by A or G. Consequently, the level of RNA synthesis on a ribavirin-containing template is significantly reduced. These findings suggest that ribavirin not only is mutagenic but also interferes with HCV polymerase-mediated RNA synthesis.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression

Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. Here, we report on the inhibition, by baculovirus-based vector-derived shRNAs, of core-protein expression in full-length hepatitis C virus (HCV) replicon cells. shRNAs were designed to target the highly conserved core region of the HCV genome. In particular, the core-shRNA452 containing nucleotides 452-472, as the target in the HCV core gene, dramatically inhibited the expression of the HCV core protein in replicon cells. Furthermore, HCV core-protein expression was inhibited more strongly by the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped baculovirus vector than by the wild-type baculovirus vector.     

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.     

Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase

In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5BDeltaC55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.     

3' nontranslated RNA signals required for replication of hepatitis C virus RNA

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.     

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.     

DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication

DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.