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. 2007 Jun;81(11):5829-40.
doi: 10.1128/JVI.02524-06. Epub 2007 Mar 21.

Characterization of hepatitis C virus subgenomic replicon resistance to cyclosporine in vitro

John M Robida  1 , Heather B Nelson, Zhe Liu, Hengli Tang
Affiliations

Characterization of hepatitis C virus subgenomic replicon resistance to cyclosporine in vitro

John M Robida et al. J Virol. 2007 Jun.

Abstract

Treatment of hepatitis C virus (HCV) infection has been met with less than satisfactory results due primarily to its resistance to and significant side effects from alpha interferon (IFN-alpha). New classes of safe and broadly acting treatments are urgently needed. Cyclosporine (CsA), an immunosuppressive and anti-inflammatory drug for organ transplant patients, has recently been shown to be highly effective in suppressing HCV replication through a mechanism that is distinct from the IFN pathway. Here we report the selection and characterization of HCV replicon cells that are resistant to CsA treatment in vitro, taking advantage of our ability to sort live cells that are actively replicating HCV RNA in the presence of drug treatments. This resistance is specific to CsA as the replicon cells most resistant to CsA were still sensitive to IFN-alpha and a polymerase inhibitor. We demonstrate that the resistant phenotype is not a result of general enhanced replication and, furthermore, that mutations in the coding region of HCV NS5B contribute to the resistance. Interestingly, a point mutation (I432V) isolated from the most resistant replicon was able to rescue a lethal mutation (P540A) in NS5B that disrupts its interaction with its cofactor, cyclophilin B (CypB), even though the I432V mutation is located outside of the reported CypB binding site (amino acids 520 to 591). Our results demonstrate that CsA exerts selective pressure on the HCV genome, leading to the emergence of resistance-conferring mutations in the viral genome despite acting upon a cellular protein.

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Figures

FIG. 1.
FIG. 1.
Selection and characterization of CsA-resistant HCV replicon cells in vitro. (A) Diagram of the selection scheme which combines antibiotic selection and live cell sorting. Note that the double selection with CsA and G418 was carried out for 3 weeks to obtain colonies, and the subsequent sorting of resistant cells was performed 3 days posttreatment. (B) CsA resistance profiles of the various replicon populations. GS5 cells, a population consisting of the top 15% of GFP-expressing GS4 cells sorted in the absence of CsA treatment, were used as the negative control. The CsA-resistant cells remained susceptible to IFN-α (C) and 2CMA (D). The GFP-based replicon cells were routinely treated with the indicated drugs for 4 days before being fixed for FACS analysis. The percentage of GFP-positive cells at the end of the treatments is normalized to the value of the untreated sample (set at 100%). (E) RS2 cells were resistant to long-term treatment with CsA. GS5 and RS2 cells were treated with 0.25 and 0.5 μg/ml CsA for 10 days, and cells were fixed for FACS analysis at days 3, 6, and 10 after the addition of the drug.
FIG. 2.
FIG. 2.
Quantification of CsA resistance at the RNA level. (A) A representative Northern blot. The GS5 and RS2 cells were treated with the indicated amounts of CsA for 48 h. Total RNA from the replicon cells was then extracted and subjected to Northern analysis with a radioactively labeled probe that corresponded to the NS5B sequence of the GT1b HCV (29). A photograph of the RNA electrophoresis gel before Northern transfer showed that equivalent amounts of RNA were loaded for each sample. (B) Quantification of the ratio of HCV RNA to 28S rRNA using band densities from panel A. The Northern blot bands were analyzed with a PhosphorImager (Molecular Dynamics; Storm 860 scanner), and the rRNA bands were analyzed with a Bio-Rad ChemiDoc system. The ratio (plotted) serves as an indicator of the HCV RNA steady-state levels in the various samples after normalization to total RNA loaded.
FIG. 3.
FIG. 3.
Replicon RNA, not the host cell, was responsible for the CsA resistance of RS2 cells. (A) Experimental design for mapping the resistance to the replicon RNA or the host cell. We separated the viral RNA and host cells for both GS5 and RS2 replicons and remixed them to generate all four possible combinations of new replicons (see Materials and Methods for details). (B) Only the RS2 RNA was able to confer CsA resistance to the new replicons. The new replicons containing GS5 RNA, regardless of the host cell origin, retained the same level of CsA sensitivity as did GS5 cells, while the RS2 RNA was able to confer resistance to host cells originated from the GS5 cells. (C) Western blot analysis results of an independent experiment confirming the FACS results in panel B. Concentrations of CsA are indicated above each lane.
FIG. 4.
FIG. 4.
A point mutation at amino acid 432 of NS5B contributes to the CsA resistance of RS2 cells. (A) Rep1b and a derivative replicon containing the I432V mutation were treated with increasing amounts of CsA for 4 days before being subjected to Western blotting with an anti-NS5A antibody. Concentrations of CsA are indicated above each lane. The detection of Ku80 served as a loading control. (B) Replication of I432V is resistant to CsA in a colony formation assay. In the CsA-treated group, 0.375 μg/ml CsA was included in the selection medium, which also contained 500 μg/ml of G418. −, not treated with CsA; +, treated with CsA.
FIG. 5.
FIG. 5.
I432V rescues replication of a GT1b replicon from the lethal effect of P540A mutation. (A) Colony formation assay for Rep1b and derivative replicons. One microgram of the replicon RNA generated by in vitro transcription was electroporated into either Huh-7.5 or GS5 IFN-cured cells. G418-resistant cells were selected with 500 μg/ml of G418 for 3 weeks. Cell colonies were stained with crystal violet at the end of the selection. (B) Transient replication assay. Equivalent amounts (1 μg) of the RNA of the three replicons generated by in vitro transcription (left) were used in the electroporation of Huh-7.5 cells. Total RNA was then extracted 4 and 8 days postelectroporation and subjected to RT-PCR for the detection of HCV RNA. Primers that amplify the NS5B coding region of GT1b were used to detect HCV RNA, and the cDNA for RNA helicase A (RHA), a cellular gene, was amplified as a control for the input of total cellular RNA.
FIG. 6.
FIG. 6.
The I432V/P540A replicon and the Rep1b replicon are equally sensitive to CsA. (A) We treated the indicated replicon cells with increasing amounts of CsA for 72 h and then detected HCV NS5A expression. (B) RNA analysis. The total RNA in the CsA-treated replicon cells was extracted 72 h posttreatment and then analyzed with real-time RT-PCR to detect HCV RNA and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) RNA simultaneously. The ratios of HCV RNA versus GAPDH RNA were calculated through the relative cycle threshold values. (C) RS1-2 is more resistant than the I432V replicon. The RS1-2 replicon, which contains a single I432V mutation in NS5B and many other mutations elsewhere in the genome, was treated and analyzed in the same manner as the Rep1b-based replicon cells shown in panel A.
FIG. 7.
FIG. 7.
RNA binding activity of NS5B is enhanced in CsA-resistant replicon cells. (A) An in vitro RNA-NS5B binding assay was conducted as described in Materials and Methods. Cell lysates of CsA-treated (8 μg/ml) or untreated GS5 or RS1-2 replicon cells were incubated with poly(U)-Sepharose or protein G-Sepharose beads in the presence or absence of 32 μg/ml of CsA. Precipitates were separated by SDS-PAGE and immunoblotted with anti-NS5B and anti-CypB antibodies. Twofold amounts of cell lysate were used for “input” in the RNA binding assay. “pU” represents poly(U), and “G” represents protein G-Sepharose beads. −, absence of; +, presence of. (B) The percentage of the NS5B bound to RNA was calculated by quantifying the band intensities. The ratio of “bound” versus “input” is plotted with the twofold difference in loading taken into consideration. (C) Direct comparison of the effect of CsA on the RNA binding activity of NS5B from GS5 and RS1-2 replicons. The percentage of bound RNA in the presence or absence of CsA is calculated and shown for both GS5 and RS1-2 cells. Error bars indicate standard deviations.
FIG. 8.
FIG. 8.
Cyclophilin B knockdown in replicon cells had no significant effect on NS5A expression. (A) Various wild-type and CsA-resistant replicon cells were transduced with lentiviral vectors expressing either a luciferase siRNA (si-Luc) or a CypB siRNA (si-CypB) for 7 days before being lysed for protein analysis. After SDS-PAGE separation, NS5A, CypB, and Ku80 were detected by sequential probing and stripping of these same membranes. (B) Quantitative analysis of the results shown in panel A. After normalization to the loading control Ku80, no specific effect on HCV expression was observed for the CypB knockdown. Error bars indicate standard deviations.
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