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.     

Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mutations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5A markedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5A mutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, suggesting that all three domains of NS5A are important for HCV morphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation and mammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific protein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles.     

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...     

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.     

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.     

Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-alpha) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.     

Enhancement of hepatitis C virus replication by Epstein-Barr virus-encoded nuclear antigen 1.

Based on our recent observation that Epstein-Barr virus (EBV) is detected in 37% of the tissues of hepatocellular carcinoma, and especially frequently in cases with hepatitis C virus (HCV), the effect of EBV infection on the replication of HCV was investigated. EBV-infected cell clones and their EBV-uninfected counterparts in cell lines MT-2 (a human T-lymphotropic virus type I-infected T-cell line), HepG2 (a hepatoblastoma cell line) and Akata (a Burkitt's lymphoma cell line) were compared in terms of their permissiveness for HCV replication following inoculation of HCV derived from patients who were HCV carriers. The results indicated that EBV-infected cell clones, but not their EBV-uninfected counterparts, promoted HCV replication. EBV-encoded nuclear antigen 1 (EBNA1), which is invariably expressed in EBV-infected cells, supported HCV replication. Deletion analysis of the EBNA1 gene showed good correlation between transactivation activity and the activity supporting HCV replication. The present findings suggest that EBV acts as a helper virus for 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.     

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.     

Viral RNA mutations are region specific and increased by ribavirin in a full-length hepatitis C virus replication system

High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid "error catastrophe." Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5' untranslated region (5' UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3' cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5' UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.     

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.     

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.     

Modulation of the type I interferon pathways by culture-adaptive hepatitis C virus core mutants

Hepatitis C virus (HCV) often establishes a persistent infection that leads to chronic liver diseases. The viral core protein modulates various cellular activities involved in this process. We found two mutations, K23E and V31A, in the core gene of the transfected HCV JFH-1 genome, which had been replicated for a prolonged period. The mutant viruses escaped immunochemical detection by a core-specific antibody and demonstrated enhanced RNA replication and protein expression, compared to the parental virus. The mutant core proteins bound less tightly than the parental type core to the DEAD-box RNA helicase DDX3 and attenuated the TBK1-mediated activation of interferon-related promoters. These results suggest a mechanism by which the viruses adapt to attenuate cellular antiviral activity and to establish persistent infection.     

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.     

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.     

Inhibition of hepatitis C virus replication by single and dual small interfering RNA using an HCV-infected cell model

Dual siRNA against different regions of gene in hepatitis C virus (HCV) synergistically inhibited replication of HCV RNA. An HCV-infected cell model was established, and HCV RNA and core protein were detected by RT-PCR and Western blot, respectively. Four HCV-specific siRNAs (siCore, siNS3, siNS4B, siNS5B) were designed and transfected into HCV-infected Huh7.5.1 cells. The antiviral efficacies of the siRNAs were compared using real time PCR and agarose gel electrophoresis. HCV replication in infected cells was inhibited by IFNα-2b in a dose-dependent manner. Synergistic inhibition effects were achieved with combination treatment of any two of the siRNAs (siCore, siNS3 and siNS5B) at low doses (0.1 and 10 nM), as compared to single siRNA treatment (P < 0.05). Furthermore, CCK-8 assay showed no toxicity of the siRNAs to Huh7.5.1 cells. These findings indicate a promising new therapeutic approach for treatment of HCV.     

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.     

Synthesis and SAR of piperazinyl-N-phenylbenzamides as inhibitors of hepatitis C virus RNA replication in cell culture

The RNA replication machinery of HCV is a multi-subunit membrane–associated complex. NS5A has emerged as an active component of HCV replicase, possibly involved in regulation of viral replication and resistance to the antiviral effect of interferon. We report here substituted piperazinyl-N-(aryl)benzamides as potent inhibitors of HCV replication exerted via modulation of the dimerization of NS5A.