Mutations in the hepatitis C virus polymerase that increase RNA binding can confer resistance to cyclosporine A

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with ≈8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. Conclusion: An increased level of CsA resistance is associated with distinct mutations in the NS5B gene that increase RNA binding in the presence of CsA, and the intramolecular communications between residues of the thumb and the C-terminal domains are important for HCV replicase function. (HEPATOLOGY 2009.)

Cyclosporine A (CsA), a commonly used immunosuppressant for transplant patients, has recently emerged as a potential new anti-hepatitis C virus (HCV) therapeutic. CsA and its derivatives potently inhibit HCV replication both in cell-culture systems and in mice with transplanted human liver,1–3 although no consensus has emerged on the in vivo benefits of using CsA over Tacrolimus (FK506), a compound that lacks anti-HCV effect in vitro, for HCV-infected liver-transplant patients.4–9 More recent clinical trials with a CsA derivative, DEBIO-025, yield promising results in human immunodeficiency virus (HIV) and HCV-coinfected patients, but drug resistance in vivo has not been studied.10

Studies of the antiviral effect of CsA on HCV replicons has led to the identification of cyclophilin (CyP) as an essential cofactor for HCV replication11–13; point mutations in nonstructural protein 5B (NS5B) and NS5A are associated with CsA resistance in vitro.14, 15 In addition, NS5B interacts with both CyPA and CyPB both in vitro and in vivo.12, 13 These data establish NS5B as an indirect viral target for the CsA-mediated inhibition of HCV replication. The CyPA-NS5B interaction has also been shown to be the principal mediator of cyclosporine resistance in vitro.12

How CyPA affects NS5B function is unclear. The crystal structure of HCV NS5B lacking the C-terminal transmembrane domain has been solved. It adopts a typical right-handed shape that contains finger, palm, and thumb domains surrounding the active site.16–18 Characterization of allosteric, nonnucleoside inhibitors revealed extensive communication between the different domains.19 Here, we identify a series of mutations in NS5B that conferred resistance to CsA and show that the mutations can affect RNA binding and synthesis by the HCV polymerase.

Abbreviations

CsA, cyclosporine A; CyP, cyclophilin; HCV, hepatitis C virus; GT, genotype; NS5B: nonstructural protein 5B; RdRp, RNA-dependent RNA polymerase; wt, wild-type.

Materials and Methods

Compounds and Cell Lines.

Cyclosporine A was purchased from Alexis (San Diego, CA). GS5- and CsA-resistant replicons have been described.14, 20 All replicon cells are routinely cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 500 μg/mL of G418.

Replicon Sequencing, Site-Directed Mutagenesis, and Replication Assays.

These experiments were performed using published procedures.14, 20 Detailed information on these methods can be found in the Supporting Methods. Primer sequences used in the cloning and sequencing are shown in Supporting Tables 1 and 2.

Molecular Biology Assays.

Transient replication was measured by isolation of total cellular RNAs from cells 7 hours, 4 days, and 10 days after electroporation of the Rep1b or JFH-1 RNA. We performed real-time reverse-transcriptase polymerase chain reaction (RT-PCR) with a SYBR Green PCR kit (Applied Biosystems) to detect HCV IRES and GAPDH expression. Sequences of the qRT-PCR primers are shown in Supporting Table 3.

In vitro transcription, electroporation, colony formation, and Poly-U RNA binding assays were performed as described.12, 14

Infections and Fluorescence Staining.

Infection of Huh-7.5 cells by cell culture-derived virus was performed as described.21 Immunostaining of infected Huh-7.5 cells was carried out according to standard methods with a mouse monoclonal antibody against the core protein (Affinity Bioreagents, Golden, CO) and an Fluoresbrite Carboxylate YG (FITC)-conjugated goat antimouse secondary antibody for detection (Sigma, St. Louis, MO).

Protein Purification and RNA-Dependent RNA Polymerase (RdRp) Assays.

Wild-type (wt) and mutant versions of the NS5B Δ21 were expressed in E. coli as C-terminal 6xHis tagged proteins and purified first by a metal ion chromatography and then with polyuridylate resins. Standard RdRp assays were performed as described.22 Protein concentrations were determined by comparison with known concentrations of bovine serum albumin (BSA) in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Differential Scanning Fluorimetry.

Differential scanning fluorimetry was performed in a Stratagene MX3005P machine with an excitation of 492 nm and emission of 610 nm. Each sample was prepared in a total volume of 50 μL containing solutions of protein at 2.5 μM final concentration, SYPRO orange (Molecular Probes) at 2.5 × final concentration. The samples were heated at a rate of 0.5°C/minute, from 25°C to 95°C, and the Tm values were calculated from the maximum of the first derivative with KaleidaGraph software (Synergy Software, Reading, PA).

Fluorescence Anisotropy Assay.

Measurements were made with an LS55 spectrometer (Perkin-Elmer) and cuvettes with an optical pathlength of 0.4 cm at 22°C. The fluorescein-labeled RNA SL-UC was at 0.2 μM in 50 mM Tris-Cl (pH 7.5) and 25 mM NaCl. The final volume of protein added did not exceed 5% of the initial sample volume. The excitation wavelength was 495 nm, and each emission was scanned from 510 to 560 nm. The average of 10 scans was taken per data point and the data were analyzed by nonlinear least-square fitting using KaleidaGraph.

Results

Isolation of Replicon RNA with Distinct Levels of CsA Resistance.

We have recently isolated CsA-resistant replicons using a combination of antibiotic selection and cell sorting.14 When single-cell clones of the resistant population were analyzed, various levels of CsA resistance were observed (Fig. 1A). The three single-cell clones with the highest levels of resistance were RS1-2, RS1-8, and RS1-9 (Fig. 1A); other clones had lower levels of resistance, similar to that of RS1-10 (Supporting Fig. 1A). To confirm that the CsA resistance of these single-cell clones was indeed conferred by the replicon RNA, we introduced total RNAs isolated from these cells, which contain the replicon RNAs, into naïve Huh-7.5 cells. The new replicons are designated RS1-2.2, RS1-8.2, RS1-9.2, and RS1-10.2, respectively. The replicon RNAs from all these clones phenocopied the resistant phenotype to the new replicon cells (Supporting Fig. 1B). Importantly, the relative level of resistance was maintained in the progeny replicons, demonstrating that the replicon RNAs in these clones contain distinct mutations that are responsible for the resistant phenotype.

Figure 1.

NS5B mutations that confer CsA-resistance. (A) Single-cell clones exhibit distinct levels of CsA resistance. The replicon cells were treated with increasing concentrations of CsA for 4 days before being analyzed by western blotting to detect NS5A expression. (B) Point mutations in NS5B confer resistance to a Con1 replicon without GFP. The wt replicon (Rep1b) and its derivatives containing the indicated NS5B mutations were treated with CsA for 4 days. The cells were then lysed and analyzed by SDS-PAGE and western blotting for NS5A and a cellular protein, Ku80. (C) Colony formation assay. One μg of in vitro transcribed replicon RNAs was electroporated into Huh-7.5 cells. The transfected cells were then selected for 3 weeks with 500 μg/mL of G418 in the presence or absence of 0.375 μg/mL of CsA. Statistical analysis was performed using Student's t test to compare the number of colonies formed by each mutant with that by wt replicon in the presence or absence of CsA.

Identification of Point Mutations in NS5B that Confer Resistance.

The HCV NS5B gene, which encodes the viral RdRp, has been implicated in the CsA-mediated inhibition of HCV and CsA resistance in vitro.12–15 We sequenced the NS5B region of the single cell clones and compared them to the wt sequence represented by GS5 replicon. Despite having a similar level of resistance, RS1-8, RS1-9, and RS1-2 contained different mutations in the NS5B coding region. A single common I432V mutation was found in RS1-2 NS5B, whereas combinations of two mutations, I11V/I454V and Q438R/E440G, were found in RS1-8 and RS1-9, respectively. To determine whether these mutations could confer CsA resistance, we engineered them into a wt genotype 1b replicon, Rep1b, and assayed the resultant constructs for CsA resistance. Expression of the NS5A protein of Rep1b was below the detectable level when the replicon was treated with 0.375 μg/mL of CsA. In contrast, NS5A could be detected when the mutant replicons were treated with the same concentration of CsA (Fig. 1B). This result was confirmed with a colony-formation assay. In the absence of CsA treatment, electroporated replicon RNAs resulted in formation of G418-resistant colonies as a result of the expression of the neomycin phosphotransferase gene carried by these replicons. Inclusion of 0.375 μg/mL of CsA abolished the ability of the wt replicon (Rep1b) to form colonies, whereas the three mutant replicon RNAs retained colony formation (Fig. 1C). No significant differences were observed between the mutant and wt replicons' ability to form colonies in the absence of CsA. These results demonstrate that these NS5B mutations are sufficient to confer increased CsA resistance without enhancing replication. However, the full level of CsA resistance of the original resistant isolates may require mutations in other genes (compare the CsA dosages in Fig. 1A and Fig. 1B and see Supporting Table 4).

CsA-Resistance Mutations Locate to Two Functionally Related Regions in NS5B Structure.

All of the mutations lie within two regions of the NS5B structure (Fig. 2A). First, mutations I432V, I11V, and V405L are within the template channel or the Δ1 loop that extends from the finger subdomain to the thumb subdomain to form a latch over the template channel. This locking mechanism is primarily due to hydrophobic interactions between the Δ1 loop and the residues on the outer surface of the template channel.22 The second group of mutations, Q438R, E440G, I454V, and W550R, lie along one side of a concave surface at the back of the template channel, along a groove. Mutation R556V could not be mapped because it was not present in the crystal structure, but the trajectory of the C-terminal tail is projected to be near the template channel. To extend this analysis, we also examined the locations of the two mutations in NS5B identified by Fernandes et al.,15 S556G and P538T. S556G is located within the template channel and P538T is within the same side of the concave surface of NS5B, lining the aforementioned groove. A compilation of all nine mutations in the NS5B structure is shown in Fig. 2B, including a cut-away view that better exposes the locations of the mutations within the template channel.

Figure 2.

Locations of the mutations in NS5B that conferred resistance to CsA. (A) Models of the mutations in NS5B from RS1-2, RS1-8, RS1-9, and RS1-10. The structure of the NS5B protein is from Ago et al.16 (pdb: 1QUV). Several features in the structures serve as landmarks. The Δ1 loop that extends from the thumb to the finger subdomains is colored cyan. The GDD residues that coordinate divalent metals for catalysis are shown in yellow. Note that the active site within the enclosed template channel was obscured in some views. The back of the template channel includes a concave surface, the location of several of the mutations. (B) CsA mutations exist in two regions within the NS5B protein. The mutations include those identified in this study and the two from Fernandes et al.,15 which also conferred resistance to CsA, and the mutation N142S from Wiedmann et al.34 The first region is in or near the template channel (I11, N142, V405, I432, and S556) and the second is within one face of the concave surface (Q438, E440, I454, P538, and W550). A cut-away view of the molecule is shown to allow visualization of the mutations in the template channel.

RNA Binding by HCV Replication Proteins in the Presence of CsA.

On the basis of their locations in the NS5B structure, we hypothesize that the mutations could affect RNA binding by NS5B. To test this hypothesis, we assayed whether NS5B produced in cells could bind poly-U (pU) RNA in vitro in a CsA-sensitive manner.13 Cell lysates were incubated with pU resin, then washed and subjected to western blot analysis with antiserum to NS5B. Binding to protein G Sepharose served as a negative control. All NS5B variants were able to bind pU RNA in the absence of CsA (Fig. 3A). The interaction between the wt NS5B and pU RNA was reduced by more than 90% upon CsA treatment. In contrast, all three of the NS5B proteins produced by mutant replicons retained pU RNA binding when treated with CsA (Fig. 3A). The reductions in RNA binding by mutant proteins containing I432V, Q438R/E440G, and I11V/I454V were 0%, 5%-10%, and 40%-50%, respectively (Fig. 3B). Note that these cells were treated with CsA for only 22 hours, a time at which total NS5B protein level has not yet decreased. Longer treatment with CsA (>24 hours) would have resulted in a reduction in total NS5B.

Figure 3.

CsA-resistant RNA binding of the mutant NS5B proteins. (A) An in vitro poly-U RNA binding by NS5B. Cell lysates of CsA-treated (8 μg/mL) or untreated Rep1b or mutant replicon cells were incubated with either poly-U RNA-conjugated Sepharose or protein G Sepharose beads in the presence or absence of 32 μg/mL of CsA. NS5B bound to poly-U RNA was detected with anti-NS5B antibody. The amount of input lysate used for binding was three times that of the “total.” “U”: poly U RNA; “G”: protein G. (B) The percentage of the NS5B bound to RNA was calculated by quantification of the intensities of the western blot bands. The ratio of “bound” to “total” accounted for the 3-fold difference in loading. The error bars represent the standard derivation of three experiments.

To further determine if the mutations in NS5B that confer resistance to CsA affect NS5B activity, we expressed several of the mutants in E. coli in a form that lacked the C-terminal transmembrane domain (NS5B Δ21). The mutant proteins include I432V, I11V/I454V, Q438R/E440G, and a mutant that contained all five mutations (I11V, I432V, I454V, Q438R, and E440G), named Mut5 (Fig. 4A).

Figure 4.

Activity of recombinant NS5B proteins containing CsA-resistant mutations. (A) Coomassie blue-stained SDS-PAGE of the recombinant proteins purified for the experiments presented here and in Fig. 6. The purified proteins have been adjusted to be of comparable concentration. (B) Sequence and secondary structures of LE19, the template for the RdRp assay. The monomeric structure of LE19 can be used for de novo initiation, whereas the partially annealed dimer can direct primer extension. (C) Products of RNA synthesis by the Δ21 (wild-type for this study) and mutant RdRps. Each set of reactions was performed with increasing concentrations of LE19 in the reaction. The identities of the 19-nt de novo initiated the 32-nt primer-extended and the template-switch products are shown. Most proteins were named according to the mutated residues they contain. Mut5: the mutant that contained five mutations: I11V, I432V, Q438R, E440G, and I454V. (D) The amounts of products were determined by phosphorimage analysis and the ratios adjusted to the comparable reaction performed with Δ21. Results with Δ21, I432V and Q438R/E440G were confirmed in two additional experiments.

Figure 6.

CsA-resistance mutations alleviate a lethal mutation at the C-terminal domain of NS5B. (A) Transient replication of Rep1b, P540A, and the derivatives. One μg of in vitro transcribed replicon RNAs was electroporated into Huh-7.5 cells and total RNA was isolated 7 hours, 4 days, and 10 days after electroporation. The ratio of HCV RNA to the GAPDH RNA was plotted for each mutant at each timepoint. The mock-transfected cells were electroporated with buffer. P: P540A; PI: P540A/I432V; PII: P540A/I11V/I454V; P11: P540A/I11V; P454: P540A/I454V; PQE: P540A/Q438R/E440G; PQ: P540A/Q438R; PE: P540A/E440G. (B) Colony formation assay for the same set of mutants. The cells were selected with 500 μg/mL of G418 for 3 weeks to allow the formation of the colonies. The results shown are representative of three to four independent experiments.

The HCV RdRp can initiate RNA synthesis de novo from the 3′ end of the viral genome23, 24 or by extension from a primed template.25 Both activities could be measured in the same reaction with RNA LE19 that exists in equilibrium between a monomer and a dimer. The monomer forms an intramolecular hairpin that directs de novo initiation to produce a 19-nucleotide (nt) product. A dimer can serve as the template-primer complex for an extension reaction 32 nt in length22 (Fig. 4B). In addition, template-switch products that are multimers of the 19-nt RNA are also produced from some of the de novo initiated 19-mers will.26 All mutant proteins retained the ability to synthesize the de novo transcript, the primer-extended products, and the template switch products (Fig. 4C), indicating that the mutations that conferred resistance to CsA did not affect residues of the polymerase that are essential for catalytic activity.

The mutant proteins had more subtle differences with regard to RNA synthesis. First, RNA synthesis by I432V was increased several-fold (Fig. 4C). Higher template concentrations were previously reported to inhibit RNA synthesis by Δ21 protein.21 In contrast, I432V was not inhibited in RNA synthesis at higher ligand concentrations (Supporting Fig. 2). Second, mutants Q438R/E440G, I11V/I454V, and Mut5 produced up to 4.6-fold more of the 19-nt de novo initiated RNA than of the 32-nt primer-extension product (Fig. 4D). Notably, I432V produced a ratio of the de novo-initiated and the primer-extended products similar to that of Δ21, suggesting that I432V is functionally distinct from the other mutants. Because Mut5 also contained the I432V mutation, the effects of the other mutations on the ratio of RNA synthesis appear to dominate that of I432V.

Spectroscopic Analysis of Select Mutant RdRps.

The conformation of the HCV RdRp is linked to the mode of RNA synthesis. To examine the effects of the mutations on NS5B conformation, we selected mutant proteins I432V (near the template channel) and Q438R/E440G (in the concave surface) as representing the two classes of mutant proteins. Differential scanning fluorimetry27 was used to measure proteins' unfolding as a function of temperature. The assay uses SYPRO orange, which fluoresces when it binds to hydrophobic regions of proteins that are exposed upon thermal denaturation. Both I432V and Q438R/E440G differed distinctly in thermal denaturation profiles from Δ21 (Fig. 5A) and from each other, in agreement with the observed differences in RNA synthesis (Fig. 4C). The functional and spectroscopic analyses support the model that the CsA-resistant mutations affect the conformation of the NS5B protein.

Figure 5.

Spectroscopic characterizations of Δ21 and mutant proteins I432V and Q438R/E440G. (A) Differential scanning fluorimetry used to compare protein unfolding rates. Temperature increase was regulated by a Stratagene Mx3005P real-time PCR machine. The first derivative of the change in fluorescence is plotted. (B) Fluorescence anisotropy results revealing the affinities of the proteins for the RNA SL-UC (inserted in the graph to the left). The symbol attached to the 5′ terminus of the RNA represents fluorescein. Purified proteins were titrated into a cuvette fitted with a magnetic stir bar. Background fluorescence of the buffer and protein were small at the wavelengths used (emission: 520 nm; excitation: 560 nm) but were subtracted from the data. The fit to the binding isotherms was performed with a hyperbola equation (y − m1*x/[m2+x]; m2 = kD) and plotted with KaleidaGraph. The quality of the fit is shown by the R2 value.

RNA Binding by Recombinant HCV RdRps.

A fluorescence anisotropy assay was used to determine the affinity of RdRp-RNA binding. The RNA used was chemically synthesized to contain 5′ fluorescein (Fig. 5B) and a stable hairpin needed for efficient de novo initiation of RNA synthesis.28, 29 The binding isotherms for the three proteins were complex to fit, perhaps reflecting that the proteins may exist in equilibrium of two or more conformers, but when fitted to the same hyperbola equation the affinities for SL-UC binding were 16 μM for Δ21 and less than 2 μM for I432V and Q438R/E440G (Fig. 5B). The apparent kD values were reproducible within 10% in two independent experiments. These results show that mutations that conferred CsA resistance can increase RNA binding by NS5B in the absence of other proteins. Strikingly, both I432V (near the template channel) and Q438R/E440G (the concave surface of the RdRp) conferred increased RNA binding by the HCV RdRp.

Rescue of P540A Lethal Phenotype by NS5B Mutations.

We sought additional evidence for the functional relationship between the template channel and the concave surface of the HCV RdRp. A point mutation, P540A, that resides in the concave surface of the HCV RdRp abolished the replication of a genotype 1b replicon13 and I432V rescued replication when engineered into the P540A background,14 demonstrating communication between the mutations at the two patches. We tested all the CsA-resistance mutations for their ability to alleviate the lethal effect of P540A in a transient replication assay. Replicon RNAs carrying P540A or P540A plus the CsA-resistance mutations were delivered into Huh-7.5 cells by electroporation. Total RNAs were extracted and analyzed by real-time RT-PCR. The input RNAs were at comparable levels in all the replicon derivatives at 7 hours. After 4 days in culture, however, the P540A RNA had decreased to that of the negative control, whereas the replicon derivatives had levels close to that of the wt (Fig. 6). At day 10, the RNA levels of P540A/Q438R and P540A/E440G were lower than that of the wt but three orders higher than that of P540A (Fig. 6A). The colony-formation assay results confirmed these observations (Fig. 6B). These results provide additional evidence that the two patches in the NS5B protein affected by CsA are functionally related.

A Naturally Occurring I454V Variation Averts the Lethal Effect of P540A Mutation.

The NS5B gene of GT2a carries a valine at position 454 instead of an isoleucine in the wt sequence. Importantly, the JFH-1 isolate of the GT2a genome can efficiently produce infectious particles in cell culture.30 To examine if Val454 could counteract the lethal phenotype of P540A in this genetic background, we engineered the P540A mutation into the JFH-1 genome and examined its effect on viral replication and infection of this isolate. In the transient replication assay, the P540A mutant was impaired in its ability to replicate but accumulated to a level higher than a nonviable control virus (GDD changed to GND in the catalytic core of NS5B) (Fig. 7A). In contrast, the double mutant of V454I and P540A (PAVI) was not viable, whereas the V454I single mutant replicated close to the wt level (Fig. 7A). There is a significant decrease in RNA level from 7 hours to day 4 for all the samples, likely a result of the higher amount of input RNA (10 μg as opposed to 1 μg used for the replicons in Fig. 6A) needed to produce infectious viruses. Nevertheless, for the wt V454I, and P540A, the RNA level stabilized from day 4 through day 10, indicating sustained replication. Furthermore, when the supernatants from these cells were tested for their abilities to infect naïve Huh-7.5 cells, similar results were obtained (Fig. 7B). These results strongly suggest that the presence of a naturally occurring valine at position 454 could prevent the lethal effect of P540A in an experimental setting that recapitulates the full cycle of HCV infection.

Figure 7.

Intramolecular compensation for P540A by valine at position 454 in an infection system. (A) Ten μg of in vitro transcribed JFH-1 and its derivative RNAs were electroporated into Huh-7.5 cells. Transient replication assay was performed as described in Fig. 6A using JFH-1-specific primers for RT-PCR. (B) Supernatant collected 19 days postelectroporation in (A) was used to infect naïve Huh-7.5 cells, which were then stained for the expression of core protein 7 days after infection. WT: wild type; PAVI: P540A/V454I. The results shown are representative of those from three experiments.

Discussion

Here, we report that mutations in the HCV polymerase that confer CsA-resistant replication increased RNA binding by NS5B, which is an indirect viral target of CsA's inhibitory effect on HCV replication. Together with our previous report that CyPA interacts with NS5B and regulates HCV replication,12 these results suggest a model in which CyPA assists NS5B in binding RNA for HCV RNA synthesis. When mutations arise within NS5B that improve RNA binding, CyPA is not as necessary for NS5B function. Structural analysis revealed that the mutations associated with CsA resistance and a lethal mutation for which they can compensate are located in two distinct patches in NS5B, one near the template channel and one on the back of the template channel. The two patches both affected the same activity, perhaps RNA binding with or without CyPA. Although the template channel certainly contacts the RNA, the patch behind the template channel may serve to stabilize RNA binding either directly or with assistance from CyPA.

NS5B exists in a closed conformation that is correlated with de novo initiation, which, on the basis of the ternary structure of the phage f6 RdRp, is likely to be the form that binds single-stranded RNA for de novo initiation.31 The template channel is too narrow, however, to accommodate the double-stranded RNA intermediate that must form during RNA synthesis; the RdRp must assume a more open conformation during elongative RNA synthesis.22 The open and closed conformations probably exist in equilibrium and we propose that CyP and/or mutations within the RdRp could affect the equilibrium. With the CsA mutations, the increased de novo initiation versus primer extension by several of the mutations led us to propose that at least some of the mutations affect the conformation of the RdRp, especially mutations I432V and I11A, which could contact the Δ1 loop.

The NS5B mutations did not completely restore CsA resistance when engineered into a Con1 replicon background. Mutations elsewhere in the genome, particularly in the NS5A region, can probably augment the CsA resistance in combination with the NS5B mutations.14, 15 NS5A has been proposed to serve as a cofactor for NS5B, and interactions between the two have been reported.32, 33 The NS5A-NS5B interaction may therefore modulate the conformation of NS5B and NS5A mutations may synergize with the NS5B mutations to increase CyP or RNA-binding. Interestingly, although the CsA-resistant replicons contained mutations in NS5A and NS3 (Supporting Table 4), these mutations were also found in GS5, which is a CsA-sensitive replicon.12, 14 It is possible that mutations in other NS proteins, although unable to confer resistance by themselves, can augment CsA-resistance when combined the NS5B mutations.

Polymorphism of the HCV NS genes may affect HCV replication fitness and drug sensitivity in vivo. Although P540A is clearly lethal to genotype 1b replicon, an alanine at position 540 exists in many naturally occurring isolates. Interestingly, all these isolates also contain at least one of the rescuing mutations that we identified as natural variations.14 Taking advantage of the naturally occurring I454V variation in the JFH-1 isolate, we confirmed the intramolecular compensation using a full-length infection system. These results demonstrate that the mutations we found to confer CsA resistance in cultured cells could be found in natural HCV isolates. Also of note, the concentrations of CsA that we used here to select resistant replicons are in the same range of the blood concentrations of CsA and DEBIO-025 in liver transplant and HCV-infected patients, respectively. These data suggest that CsA-based therapies may have varying effects based on the patient population.

Acknowledgements

We thank Drs. Takaji Wakita and Charlie Rice for reagents, Ruth Didier for assistance with flow cytometry, and Dr. Anne B. Thistle for proofreading.

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