SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

HCV reoccurs after liver transplantation and increases mortality. Cyclosporine, but not tacrolimus, has potent antiviral effects against HCV replication in cell culture. To determine the conditions, if any, under which HCV is susceptible to cyclosporine in vivo, we selected for cyclosporine-resistant mutant HCV in vitro. The resulting mutations were mapped to x-ray crystallographic structures and sequence databases. Mutations selected by cyclosporine were clustered in the nonstructural (NS) proteins NS5A and NS5B. Different sets of mutations in NS5A, paired with the same 2 NS5B mutations, conferred different levels of cyclosporine resistance when engineered back into the HCV replicon. Mutations in NS5B are structurally consistent with a proposed model of regulation of RNA binding by cyclophilin B (CyPB). These mutations also highlight a natural polymorphism between different HCV genotypes that correlates with the variation in response to cyclosporine A (CsA) noted in some clinical trials. Replicons engineered to have mutations in only NS5A (P ≤ 0.0001) or only NS5B (P = 0.002) suggest that while both NS5A or NS5B variants alter cyclosporine susceptibility, NS5A has the largest effect. Conclusion: Preexisting sequence variation could alter the effect of cyclosporine on HCV in vivo. (HEPATOLOGY 2007.)

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide.1 It is the principal indication for liver transplantation throughout the world. Infection of the new liver graft is universal. The problem is compounded by the fact that the immunosuppressants needed to prevent organ rejection worsen HCV-mediated disease. Consequently, while HCV takes decades to cause significant liver damage in an immunocompetent patient, end-stage liver disease develops in 5 to 7 years in HCV-infected liver transplant patients.2 Initially, recurrent HCV posttransplantation was thought to be fairly benign.3 However, more recent studies have demonstrated that HCV-infected liver transplant patients have significantly higher mortality and morbidity than patients transplanted for cholestatic liver disease.4 This difference may be more pronounced in recent transplants compared to those prior to 1990.5 It remains controversial whether differences between immunosuppressant regimens play a role.

Cyclosporine A (CsA) has recently been demonstrated to have potent anti-HCV activity both in the HCV replicon system and in the recently described JFH-1 2a cell culture system.6–9 CsA binds molecular chaperones called cyclophilins (CyPs) in cells and inhibits their peptidyl-proline isomerase activity.10 The CsA-CyP complex also binds calcineurin and inhibits its activation of T cells.11 FK506 binds FK506-binding proteins and inhibits calcineurin activation independent of CyPs. However, FK506 lacks antiviral activity in the HCV replicon system.7, 12 Certain CsA analogs, such as NIM811 and DEBIO-025, have been shown to have potent anti-HCV activity.9, 13 These CsA analogs retain their ability to bind CyPs, but are no longer recognized by calcineurin. These findings point toward CyP inhibition, not calcineurin inhibition, as being critical for the anti-HCV activity of CsA. Although CsA has been shown to inhibit all the HCV replicons tested thus far, the level of inhibition varies between the different HCV genotypes. The JFH-1 2a replicon has been demonstrated to have lower CsA susceptibility compared to a genotype 1b replicon.8 This, together with clinical data, suggests that different HCV genotypes may have a variable response to immunosuppressants. Thus, treatment of HCV liver transplant patients may need to be tailored based on sequence diversity.

Potentially conflicting models have been proposed to explain the inhibition of the HCV replicon by CsA. Cyclophilin B (CyPB) has been shown to bind the HCV polymerase and regulate the binding of RNA template to the polymerase.14 Another model argues that replication of the HCV replicon depends upon CyPs A, B, and C.6 Both models were based on experiments that used RNA silencing to target specific host CyPs that are known targets of CsA. We undertook a forward genetic approach in which no assumptions were made about the mechanism of CsA inhibition of HCV. We exposed HCV replicons to CsA and selected for HCV replicons with decreased susceptibility to CsA. We identified mutations in the selected replicon pool that mapped to the nonstructural proteins (NS) NS5A and NS5B. These mutations conferred CsA resistance. Mutations in NS5B, the viral RNA-dependent RNA polymerase, fit with a model of CyPB as a functional regulator of the viral polymerase.14 Our study is the first to demonstrate a role of NS5A in the CsA susceptibility of HCV. We show that NS5A has a larger effect than NS5B by separating the NS5A and NS5B mutations. Additionally, our data suggests that naturally occurring HCV polymorphisms may be critical factors in determining whether CsA inhibits HCV in individual patients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cells and Plasmids.

Huh7 and EN5.3 cells were kindly provided by Dr. Lemon (University of Texas Medical branch, Galveston, TX). Yi et al.15 and Ikeda et al.16 describe the production of EN5.3 cells and the pNtat2ANeo/SI. The plasmids pNtat2ANeo/SI and the pNNeo/3-5b/SI replicon containing the HCV 1bN consensus sequence (GenBank accession no. AF139594) with an adaptive mutation S232I in NS5A were also kindly provided by Dr. Lemon.15, 16 After transfection with replicon RNA, we selected the EN5.3 cells and we maintained them in the growth media containing 2.0 μg/mL blasticidin (Invitrogen) and 0.5 to 1.0 mg/mL G418 (Geneticin, Invitrogen).

CsA Passage Experiments.

We passaged Huh7 cells that were stably transfected with the pNNeo/3-5B/SI replicon in the presence of 1 mg/mL of G418 for 3 weeks both with and without 10 μg/mL of CsA (Sigma). We pooled the replicon cells and split them every 3 to 5 days to maintain a cell confluency of approximately 30% and we replaced the media with new CsA and G418.

Amplification of Replicon RNA Pool by Reverse Transcription–PCR and Cloning.

At the end of 3 weeks, we isolated total RNA using TRIZOL (Invitrogen) from the CsA passaged pool and control replicon cells. We used a SuperScript III One-Step reverse transcription polymerase chain reaction (PCR) mix (Invitrogen) to generate 7 independent PCR amplifications that covered the entire replicon from the HCV internal ribosome entry sequence to the 3′ untranslated region. We cloned the resulting PCR products into TOPO TA vectors (Invitrogen). We sequenced 5 to 16 TOPO TA clones containing PCR products from an individual PCR amplification. We repeated the process for RNA isolated from the control replicon cells. After we used the initial round of sequencing to determine the region with the highest frequency of mutations, 2 independent cDNA reactions were done using SuperScript II Reverse Transcriptase (Invitrogen) followed by PCR amplifications with a high-fidelity polymerase to generate NS5A-NS5B fragments from amino acid 214 of NS5A to amino acid 591 of NS5B. We generated 7 TOPO TA clones from the 2 cDNA amplifications. We then digested these clones with BlpI and ClaI and cloned them into the pNtat2ANeo/SI construct to replace the original sequence and generate mutant replicons 1 to 7. We transfected the original pNtat2ANeo/SI and the mutant replicon RNAs in parallel into EN5.3 cells and passaged them for several weeks in the presence of 0.5 mg/mL G418 and 2 μg/mL blasticidin. A total of 3 of the 7 mutant replicons succeeded in establishing stable replicon cell lines, which we called the CsA-1s, CsA-2s, and CsA-3s (GenBank accession numbers 855173, 860232, and 855179, respectively). For construct CsA-1s 5A, we cut CsA-1s with Blp1 and BstXI and inserted it into pNtat2ANeo/SI. For the CsA-1s 5B construct, we inserted the BstXI-ClaI portion of CsA-1s back into pNtat2ANeo/SI.

RNA Transcription and Transfection.

We linearized replicon DNA with XbaI and transcribed it using a T7 Ampliscribe Flash transcription kit (Epicentre Biotechnology). We transfected RNA into EN5.3 cells with a Transit mRNA transfection kit (Mirus). At 4 to 8 weeks after the establishment of stable replicon cell lines using G418 and blasticidin, we seeded an equal number of cells into 6-well plates with or without CsA, in the absence of G418. On day 6, we removed the media, washed it extensively with phosphate-buffered saline (PBS), and replaced it with new media and CsA. We used the secreted alkaline phosphatase (SEAP) assay to quantitate replicon RNA as described.15

Recombinant CyPB.

We obtained mature CyPB cDNA by RT-PCR from Huh7 cDNA using primers containing BamHI and EcoRI sites. We generated the glutathione S transferase (GST)-CyPB construct by inserting the CyPB PCR product into pGEX-2T using BamHI and EcoRI restriction sites contained in the primers. We transformed the pGEX-2T and GST-CyPB plasmids into Rosetta bacterial cells (Novagen). We purified the recombinant protein as in Cheng et al.17

In Vitro Translation and Pull-Down Assays.

We generated all NS5B constructs by PCR with forward and reverse primers containing XhoI and PacI, respectively. We inserted PCR products into a modified p-internal ribosome entry sequence vector (Clontech) through the XhoI-PacI restriction sites. We used quick-change mutagenesis to make the P540A mutant of NS5B and to truncate 1bN and 1a NS5B constructs with a stop codon. We generated the NS5B protein using a TNT Quick Coupled Transcription/Translation System (Promega) with the incorporation of [35S]methionine (GE Healthcare) following the manufacturer's protocol. We performed the pull-down assay as described in Cheng et al.17

Statistical Analyses.

The SEAP values of the pNtat2ANeo/SI, CsA-1s 5A, and CsA-1s 5B (3 experiments), each measured at 4 different CsA levels, were normalized through division by the corresponding SEAP value at the CsA level of 0 μg/mL within the respective experiment for each replicon. Using SAS 9.1 PROC MIXED, we constructed a fixed-effects linear regression model with repeated measures having the log10-transformed normalized SEAP values as outcomes. Its main effects were the 3 replicons, CsA level (0, 1, 2, or 2.5 μg/mL), and SEAP value replicate number (1, 2, or 3). To model the correlation of the three log10-transformed normalized SEAP values per experimental unit, we chose a compound symmetry covariance structure.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Selection and Cloning of HCV Replicons with Decreased Susceptibility to CsA.

We selected CsA-resistant HCV replicons to uncover more about the antiviral mechanism(s) of CsA. We exposed Huh7 cells bearing the pNNeo/3-5b/SI replicon to 10 μg/mL CsA and 1 mg/mL G418. We used a high concentration of G418 to maintain the replicon despite the presence of CsA. The presence of both G418 and CsA concentration in the media led to cell death after each split, but this lessened over time. We passaged cells continuously in the presence of both drugs. After 3 weeks, we seeded an equal number of CsA-exposed cells and control cells and treated them with different concentrations of CsA to detect resistance via a real-time PCR assay. The CsA-passaged pool demonstrated considerable resistance to CsA compared to control cells (data not shown). We then isolated RNA from stably transfected Huh7/pNNeo/SI cells that were exposed to CsA and control cells. We performed multiple independent reverse-transcriptions and PCR amplifications and we sequenced the products. We obtained several synonymous mutations in both the control replicon and in the CsA-passaged replicon cells. We primarily found nonsynonymous mutations in the CsA-passaged pool. The nonsynonymous mutations that occurred frequently (>80%) were limited to the latter half of NS5A and the carboxy terminus of NS5B (Table 1). In particular, 5 mutations in NS5A and 2 mutations in NS5B showed up repeatedly from independent RT-PCRs. The only other consistent nonsynonymous mutation with a frequency higher than 20% was a glutamic acid to glycine mutation at amino acid 175 of NS3. Since this was genetically distant from the more frequently occurring mutations, we did not further investigate it. We then obtained PCR fragments that covered amino acids 214 of NS5A to 591 of NS5B with fidelity polymerase from cDNA amplified by Superscript II Reverse Transcriptase. We then cloned these PCR fragments into the TOPO TA vector to generate 7 independent amplicons. The same 7 mutations found in the initial round of sequencing occurred repeatedly in almost all 7 amplicons with some individual nonsynonymous mutations (Fig. 1A; Table 1). With the exception of amplicon 4 (Fig. 1A), all other amplicons contained the same 5 NS5A mutations and the 2 NS5B mutations. We cloned the 7 amplicons back into a cell culture–adapted replicon to determine if this locus could alter CsA susceptibility. The recipient replicon, pNtat2ANeo/SI, expressed not only neomycin resistance, but also the HIV trans-activating Tat protein that induces SEAP in EN5.3 cells.15 SEAP levels correlate with viral RNA, which allows replicon RNA levels to be monitored without cell lysis.15 Only 3 of the 7 replicon RNAs replicated efficiently enough to isolate stable cell lines (Fig. 1A). The 3 replicons, referred to as CsA-1s, CsA-2s, and CsA-3s, were then compared to the original pNtat2ANeo/SI replicon-containing cells to measure CsA susceptibility. The IC90 of the replicon-containing mutations selected by CsA exposure increased from 0.5 to 1.3 (1s), 1.2 (2s), and 2.2 (3s) μg/mL CsA (Fig. 1B). All 3 replicons replicated to the same levels as the pNtat2ANeo/SI. We did not observe a difference in kinetics between the pNtat2ANeo/SI and the mutants over a period of 7 days (data not shown). We confirmed by sequencing that the original mutations were still present and that we had found no further nonsynonymous mutations. We performed a second round of isolating stable replicon cell lines for replicon RNAs pNtat2ANeo/SI, CsA-1s, and CsA-3s, to reconfirm the decreased CsA susceptibility. The 2 mutants showed the same response to CsA as the first CsA-1s and CsA-3s replicon cell lines (data not shown).

Table 1. Frequency of Mutations in NS5A and NS5B from Sequencing of Independent PCR Fragments
MutationsFrequency of Mutations
NS5A 
 E260G15/16
 F288S5/16
 V284A16/16
 L307Q15/16
 R360G15/16
 V448A15/16
NS5B 
 P538T14/16
 S556G14/15
thumbnail image

Figure 1. Mutations in both NS5A and NS5B confer decreased CsA susceptibility. (A) Summary of mutations obtained on passaging the pNNeo/3-5b/SI replicon in the presence of CsA for 3 weeks. Column 2 and 3 indicate mutations found in NS5A and NS5B amplified from the selected pool and cloned back into the pNtat2ANeo/SI replicon. The wild-type amino acid and residue number are indicated on the top while the mutated residues found in CsA selected replicons is indicated below. The CsA IC90 value for each replicon is shown in column 4. (B) Comparison of the pNtat2ANeo/SI replicon with mutants CsA-1s, CsA-2s, and CsA-3s. We stably transfected EN5.3 cells with the pNtat2ANeo/SI, CsA-1s, CsA-2s, and CsA-3s replicons using G418 selection. We seeded equal numbers of each replicon cell and we treated them with noncytotoxic concentrations of CsA for 7 days in the absence of neomycin. On day 6, we washed the cells extensively and we replaced the media with new media and CsA. We collected media aliquots on day 7 and assayed them for SEAP activity. Data are representative of 3 separate experiments.

Download figure to PowerPoint

Sequence Variation in NS5A Has a Greater Effect on CsA Susceptibility than NS5B.

Since mutants with different NS5As and the same NS5Bs showed different levels of CsA susceptibility (Fig. 1B), our data implied that NS5A itself altered CsA susceptibility. We made 2 additional constructs, CsA-1s 5A and CsA-1s 5B, to determine if the mutations in NS5A or NS5B alone were sufficient to confer CsA resistance. We obtained stable replicon cell lines for both these constructs, together with the CsA-1s mutant and the pNtat2ANeo/SI replicons. The CsA-1s NS5A mutant conferred CsA resistance to the replicon without concomitant mutations in NS5B (Fig. 2) and, in fact, was not significantly different in CsA susceptibility than CsA-1s. The NS5B mutations alone also conferred a level of CsA resistance (P = 0.002), though the change in CsA susceptibility was relatively small compared to only the NS5A mutations (P ≤ 0.0001) (described in Table 2).

thumbnail image

Figure 2. NS5A has a larger effect on CsA susceptibility than NS5B. (A) Summary of mutations as indicated in Fig. 1A. (B) Comparison of the pNtat2ANeo/SI replicon with mutants CsA-1s, CsA-1s 5A, and CsA-1s 5B. We individually cloned NS5A and NS5B from the CsA-1s mutant into the pNtat2ANeo/SI replicon. We called these replicons CsA-1s 5A and Csa-1s 5B, respectively. Comparison of these replicons to CsA is described in Fig. 1B.

Download figure to PowerPoint

Table 2. A Fixed-Effects Regression Model for Repeated Log10-Normalized SEAP Scores
EffectEstimateStandard ErrorDFt valueP > |t|
  1. *Ref denotes the reference group for each replicon or CsA concentration or replicate number (Each experiment was performed in triplicate). Abbreviation: DF, degrees of freedom.

Intercept−2.720.086−36.05<0.0001
CsA-1s NS5A0.830.07611.74<0.0001
CsA-1s NS5B0.370.0765.260.0019
pNtat2ANeo/SIRefRefRefRefRef
0 μg/mL CsA2.320.072432.53<0.0001
1 μg/mL CsA0.870.072412.15<0.0001
2 μg/mL CsA0.200.07242.770.0107
2.5 μg/mL CsARefRefRefRefRef
Experiment 10.000.06160.010.9905
Experiment 2−0.000.0616−0.050.9574
Experiment 3RefRefRefRefRef

Structural Differences in NS5B that Correlate with Genotype and CsA Resistance.

We initially sought to identify what role the NS5B mutations played in conferring resistance to CsA, as our NS5B mutations mapped to a previously identified region of the polymerase that bound CyPB. One proposed mechanism for the antiviral effect of CsA is that it inhibits either a CyPB-NS5B interaction or alters the interaction between NS5B and RNA, which is regulated by CyPB.14 We found 2 mutated NS5B residues in our CsA selection. One of these residues is a proline to threonine change at residue 538. This residue is in close proximity to the proline residue at position 540. A proline to alanine mutation at position 540 had been shown to disrupt the CyPB binding to the polymerase.14 Both the 538 and 540 residues are surface-exposed prolines that likely play important structural roles in the conformation of NS5B (Fig. 3A, dark blue). Our second mutation is a serine to glycine change at position 556. The 556 residue is a naturally occurring glycine in the relatively CsA resistant strain JFH-1 (genotype 2a)8 and in all genotype 2s (Table 3). The wild-type serine residue at position 556 directly interacts with RNA according to the x-ray crystal structure of the polymerase cocrystallized with a RNA oligomer (Fig. 3B, light blue).18 To examine the role of residue 556 in CsA resistance, we compared the 1b NS5B crystal structure (Fig. 3B, green and pink,18 to the YUY1 genotype 2a structure (Fig. 3B, orange),19 which has a naturally occurring glycine at residue 556. The overall root mean squared deviation between the 2 genotype 1b NS5Bs in the NS5B:RNA structure, and between the genotype 1b and 2a NS5Bs is relatively small, as has been reported.19 However, when comparing the carboxyl terminus of both structures, we noticed a large shift in one region of the thumb domain between the genotype 2a structure and two 1b structures. We then calculated the root mean squared deviation20 for the carboxy terminal residues 538-563 (previously linked to CyPB binding), and found it to be much larger between the 2a structure and the two 1b structures (2.494 Å) than between the two 1b structures alone (0.859 Å) suggesting significant structural differences (Fig. 3B). Thus it is possible that the decrease in CsA susceptibility of the CsA-1s NS5B replicon (Fig. 2B) might arise from a change in conformation of the mutant polymerase.

thumbnail image

Figure 3. Mapping CsA-adapted mutations on the HCV 1b polymerase. (A) X-ray crystallographic structure of the 1b polymerase with the RNA primer (in light blue) bound to the catalytic site. Residues in dark blue indicate the consensus sequence of 2 prolines separated by a hydrophobic residue (PXP). CsA-selected residues from Fig. 1 (538 and 556) are indicated by arrows. (B) Stereo picture of the shift in α carbon backbone for the carboxy terminus for 1bN7 molecule 1 (green), molecule 2 (purple), and 1YUY (orange, glycine at 556). Structures were superimposed by Dali20 and root mean squared deviation was calculated. While both of the 2 genotype 1b structures reveal serine 556 as within hydrogen bonding range with the RNA (light blue), no such side-chain interaction occurs with a genotype 2a glycine structure and the whole α carbon chain is shifted from the genotype 1b configuration.

Download figure to PowerPoint

Table 3. Alignment of Genotypes 1b, 1a, 2a, 2b, and 3 Polymerase Sequence from Residues 538 to 558 in the Los Alamos Database
SubtypesSequenceSequence Variability at Residue 556
1BPIPAASXLDLSXWFVAGYSGG102/115 S
1APIAAAGRLDLSGWFTAGYSGG14/14 S
2APLPEAXXLDLSSWFTVGAGGG20/20 G
2BPLPEAXRLDLSGWFTVGAGGG23/23 G
3PLPXXGQLDLSXWFTVGVGGN7/7 G

Binding of NS5B to CyPB in the Absence of NS5A.

It was previously demonstrated that residues 521-570 of the polymerase were important for CyPB binding. To determine if changes at position 538 and 556 of the polymerase NS5B alter CyPB binding, we performed a GST-CyPB pull-down assay with the CsA-1s NS5B. We also included a mutated 1bN NS5B with a proline to alanine change at position 540 that was demonstrated to disrupt GST-CyPB binding.14 The 1bN served as a positive control. The H77 1a NS5B was also included in the pull-down because position 540 is an alanine in all 14 sequences from genotype 1a (Table 3) and thus might not be expected to bind CyPB based on previous data. The 1bN, 1a, CsA-1s, and P540A NS5B all interacted with GST-CyPB (Fig. 4A). We observed no interaction of NS5B with GST unless it was fused to CyPB. Further truncations of both 1bN and H771a localized the NS5B region required for the CyPB interaction site to amino acids 398-469 of the NS5B polymerase in the absence of NS5A (Fig. 4B).

thumbnail image

Figure 4. Cyclophilin B interacts with both 1A and mutant CsA-1s NS5B polymerases. (A) A GST pull-down assay was performed as follows: The 1bN NS5B, 1a NS5B, CsA-1s NS5B, and a 1bN NS5B with a proline to alanine change at position 540 were in vitro translated and [35S] labeled. We incubated the translated products with 0.5 μM GST alone (GST) or recombinant GST-CyPB (CYPB) fusion proteins. After extensive washing, we loaded the samples onto an SDS-PAGE gel. Input represents 1/10th of the amount used in the GST/GST-CyPB pull-down assay. (B) Mapping the CyPB binding region of NS5B. We made mutant truncates of the 1bN and 1a NS5B proteins by inserting stop codons at position 469 and 398. Together, we compared the truncated mutant NS5Bs with the wild-type 1bN and 1a NS5Bs in the GST pull-down assay, as described above. Deletions and point mutations carboxy terminal to 469 were not disruptive of the CyB:NS5B interaction (for 1b and 1a; data not shown), while deletions at 398 were disruptive for both 1a NS5B and 1b NS5B.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Several different HCV replicons (H771a, JFH-1 2a, and 3 different 1bs) are all sensitive to CsA to different degrees in vitro.6, 8, 13 It is unclear if CsA has an antiviral effect in patients. This is an area of high scrutiny because recently, 2 trends have been observed simultaneously in patients. First, there has been a shift from CsA-based regimens to tacrolimus-based regimens.21 Second, while likely multifactorial, recurrent liver disease in HCV-infected patients may be worsening.22 Landmark clinical trials showed no difference between CsA and tacrolimus in liver transplants23, 24 and a recent meta-analysis was also unable to demonstrate differential outcome in CsA-managed compared to tacrolimus-managed HCV-infected liver transplant recipients.25 Studies that have demonstrated a beneficial effect of CsA on HCV have only been able to do so in genotype 1, generally in conjunction with interferon.26–30

We used a forward genetics approach to investigate the mechanism of CsA inhibition of the HCV replicon. CsA selected for mutations in NS5A and NS5B. This is unusual because most antivirals select for relatively uniform, single-point mutations and have a low genetic barrier to resistance.31 Three replicons, each with an NS5A that varied in its carboxy-terminal sequence, and the same 2 mutations in the carboxy-terminus of NS5B, were shown to have reduced CsA susceptibility. Interestingly, CsA-3s was more resistant than the other 2 replicons even though all 3 shared essentially the same NS5B sequence. NS5B mutations fit with a model of CyPB modulating NS5B activity.14 However, at least 1 mutant NS5A was able to confer decreased CsA susceptibility without NS5B mutations. The magnitude of this change in CsA resistance was large compared to that of the NS5B mutations alone. All of the mutant replicons replicated to same levels as the control and showed similar growth kinetics (data not shown). Additionally, all of the mutant replicons contained their original mutations and did not revert back to the wild-type sequence despite the fact that stable cell lines were obtained in the absence of CsA.

A mutant replicon containing NS5B mutations alone conferred a small, but significant amount of resistance compared to wild type. Although our data confirmed a CyPB:NS5B interaction, our attempts with in vitro assays to measure a CyPB enhancement of NS5B:RNA interactions found the background NS5B:RNA interaction to be too high to reliably detect a CyPB enhancement (data not shown). Our mapping of the CyPB:NS5B interaction in the absence of NS5A and RNA implicated a region between 398 and 469 to be important for this interaction. Genotypic and subgenotypic structural differences in the thumb may be important since it is a site for several nonnucleoside allosteric inhibitors in preclinical and clinical trials.19, 32, 33 Our data suggest not only genotype 1b NS5B, but also genotype 1a NS5B maintains the CyPB interaction despite genotype 1a having an alanine at position 540 (Fig. 4; Table 3). Although our mutant NS5B did not abrogate CyPB binding, it remains consistent with a model previously suggested,14 in which NS5B has a significant but not major role in CsA inhibition of the replicon.

The carboxy-terminus of NS5A is polymorphic, thought to be unstructured, and remains functional in the replicon despite insertions and deletions. Nevertheless, chimpanzee experiments have shown this region to be important for in vivo replication and possibly for interferon resistance (Enomoto et al.34 and reviewed in Appel et al.35). Additionally, this region of NS5A binds NS5B.36 It is possible that CsA may be acting by 2 separate mechanisms on NS5A and NS5B or through a single antiviral effect on the NS5A:NS5B replicase complex. Our data does not distinguish between these possibilities, because even though our NS5A and NS5B mutations were selected for and conferred CsA resistance together, they could also do so alone. The NS5B serine/glycine polymorphism at position 556 or NS5A polymorphisms that distinguish genotype 2/3 from genotype 1 could potentially be responsible for the differences in CsA susceptibility.27, 28 Nevertheless, the data presented here points to a polymorphic region of NS5A as having the largest effect on CsA susceptibility.

Nonimmunosuppressive analogs of CsA with even more potent antiviral activity against both HCV and HIV than CsA in cell culture are in early clinical trials.9 These drugs may have an important role to play in therapy, especially in HIV/HCV coinfected patients. Additional studies are needed to determine if the antiviral effect of CsA is clinically relevant, and what resistance mutations, if any, arise in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Jim Keck for help analyzing crystal structures and Michael Lucey for helpful discussion and comments on this work. We also thank Richard Yang and Dipankar Bhattacharya for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Wasley A, Alter MJ. Epidemiology of hepatitis C: geographic differences and temporal trends. Semin Liver Dis 2000; 20: 116.
  • 2
    Terrault NA. Treatment of recurrent hepatitis C in liver transplant recipients. Clin Gastroenterol Hepatol 2005; 3: S125131.
  • 3
    Gane EJ, Portmann BC, Naoumov NV, Smith HM, Underhill JA, Donaldson PT, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med 1996; 334: 815820.
  • 4
    Forman LM, Lewis JD, Berlin JA, Feldman HI, Lucey MR. The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 2002; 122: 889896.
  • 5
    Berenguer M, Ferrell L, Watson J, Prieto M, Kim M, Rayon M, et al. HCV-related fibrosis progression following liver transplantation: increase in recent years. J Hepatol 2000; 32: 673684.
  • 6
    Nakagawa M, Sakamoto N, Tanabe Y, Koyama T, Itsui Y, Takeda Y, et al. Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins. Gastroenterology 2005; 129: 10311041.
  • 7
    Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. HEPATOLOGY 2003; 38: 12821288.
  • 8
    Ishii N, Watashi K, Hishiki T, Goto K, Inoue D, Hijikata M, et al. Diverse effects of cyclosporine on hepatitis C virus strain replication. J Virol 2006; 80: 45104520.
  • 9
    Paeshuyse J, Kaul A, De Clercq E, Rosenwirth B, Dumont JM, Scalfaro P, et al. The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro. HEPATOLOGY 2006; 43: 761770.
  • 10
    Takahashi N, Hayano T, Suzuki M. Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature 1989; 337: 473475.
  • 11
    Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991; 66: 807815.
  • 12
    Nakagawa M, Sakamoto N, Enomoto N, Tanabe Y, Kanazawa N, Koyama T, et al. Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem Biophys Res Commun 2004; 313: 4247.
  • 13
    Ma S, Boerner JE, TiongYip C, Weidmann B, Ryder NS, Cooreman MP, et al. NIM811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis C virus alone or in combination with alpha interferon. Antimicrob Agents Chemother 2006; 50: 29762982.
  • 14
    Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005; 19: 111122.
  • 15
    Yi M, Bodola F, Lemon SM. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 2002; 304: 197210.
  • 16
    Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 2002; 76: 29973006.
  • 17
    Cheng W, Altafaj X, Ronjat M, Coronado R. Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling. Proc Natl Acad Sci U S A 2005; 102: 1922519230.
  • 18
    O'Farrell DTR, Rowlands D, Jager J. Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): structural evidence for nucleotide import and de-novo initiation. J Mol Biol 2003; 326: 10251035.
  • 19
    Biswal BK, Cherney MM, Wang M, Chan L, Yannopoulos CG, Bilimoria D, et al. Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J Biol Chem 2005; 280: 1820218210.
  • 20
    Holm L, Sander C. Protein structure comparison by alignment of distance matrices. J Mol Biol 1993; 233: 123138.
  • 21
    Kaufman DB, Shapiro R, Lucey MR, Cherikh WS, T Bustami R, Dyke DB. Immunosuppression: practice and trends. Am J Transplant 2004; 4(Suppl 9): 3853.
  • 22
    Berenguer M. Natural history of recurrent hepatitis C. Liver Transpl 2002; 8(10 Suppl 1 ): S14S18.
  • 23
    Randomised trial comparing tacrolimus (FK506) and cyclosporin in prevention of liver allograft rejection. European FK506 Multicentre Liver Study Group. Lancet 1994; 344: 423428.
  • 24
    A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. The U.S. Multicenter FK506 Liver Study Group. N Engl J Med 1994; 331: 11101115.
  • 25
    Berenguer M, Royuela A, Zamora J. Immunosuppression with calcineurin inhibitors with respect to the outcome of HCV recurrence after liver transplantation: results of a meta-analysis. Liver Transpl 2007; 13: 2129.
  • 26
    Lorho R, Turlin B, de Lajarte-Thirouard AS, Camus C, Lakehal M, Compagnon P, et al. Improved liver function and decreased hepatitis C viral load after tacrolimus was replaced by cyclosporine. Transplant Proc 2005; 37: 28712872.
  • 27
    Yvon Calmus DS, Georges Pageaux, Michel Messner, Phillippe Wolf, Lionel Rostaing, Claire Vanlemmens, et al. Multicenter randomized trial in HCV-infected patients treated with peginterferon alfa-2A and ribavirin alone after liver transplantation: 18-month report. In: The Liver Meeting 2006; Boston, MA: Schering-Plough; 2006. p. 47.
  • 28
    Inoue K, Sekiyama K, Yamada M, Watanabe T, Yasuda H, Yoshiba M. Combined interferon alpha2b and cyclosporin A in the treatment of chronic hepatitis C: controlled trial. J Gastroenterol 2003; 38: 567572.
  • 29
    Rayhill SC, Barbeito R, Katz D, Voigt M, Labrecque D, Kirby P, et al. A cyclosporine-based immunosuppressive regimen may be better than tacrolimus for long-term liver allograft survival in recipients transplanted for hepatitis C. Transplant Proc 2006; 38: 36253628.
  • 30
    Firpi RJ, Zhu H, Morelli G, Abdelmalek MF, Soldevila-Pico C, Machicao VI, et al. Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation. Liver Transpl 2006; 12: 5157.
  • 31
    Richman DD. Antiviral drug resistance. Antiviral Res 2006; 71: 117121.
  • 32
    Love RA, Parge HE, Yu X, Hickey MJ, Diehl W, Gao J, et al. Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme. J Virol 2003; 77: 75757581.
  • 33
    Wang X, Krupczak-Hollis K, Tan Y, Dennewitz MB, Adami GR, Costa RH. Increased hepatic Forkhead Box M1B (FoxM1B) levels in old-aged mice stimulated liver regeneration through diminished p27Kip1 protein levels and increased Cdc25B expression. J Biol Chem 2002; 277: 4431044316.
  • 34
    Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996; 334: 7781.
  • 35
    Appel N, Schaller T, Penin F, Bartenschlager R. From structure to function: new insights into hepatitis C virus RNA replication. J Biol Chem 2006; 281: 98339836.
  • 36
    Qin W, Yamashita T, Shirota Y, Lin Y, Wei W, Murakami S. Mutational analysis of the structure and functions of hepatitis C virus RNA-dependent RNA polymerase. HEPATOLOGY 2001; 33: 728737.