Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins

Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines—HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A—D2303H, S2362G, and E2414K—enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC₅₀ values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.     

Cyclosporine Inhibits a Direct Interaction between Cyclophilins and Hepatitis C NS5A

Background

Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.

Methodology/Principal Findings

We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.

Conclusions/Significance

Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.     

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (betaR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the betaR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the betaR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.     

DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.     

Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.     

Effect of alpha interferon on the hepatitis C virus replicon

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.     

Evasion of Superinfection Exclusion and Elimination of Primary Viral RNA by an Adapted Strain of Hepatitis C Virus

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3′ untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.     

Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication

The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.     

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.     

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.     

Hepatitis C virus nucleotide inhibitors PSI-352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.     

Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro

Cyclosporine (CsA) and its derivatives potently suppress hepatitis C virus (HCV) replication. Recently, CsA-resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA-resistant replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing of CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of an siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.     

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.     

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B

Background

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood.

Principal Findings

Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction.

Conclusions

We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.     

Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.