Cell lines and culture conditions
The cell lines used in these studies have been previously described. RP7 was derived by transfection of Huh7 cells (ATCC) with a sub-genomic HCV replicon (genotype 1b, CON1)(Elazar et al., 2003
). Two cell lines that confer resistance of HCV against 10uMnitazoxanide (NTZ) or tizoxanide (TIZ), NTZ-11 and TIZ-9, were derived by serial passage of RP7 in increasing concentrations of either NTZ or TIZ(Korba et al., 2008a
). Stock cultures were maintained in DMEM with 250ug/ml G418 as previously described(Korba et al., 2008a
). Medium for the two resistant cell lines also contained 10uM NTZ. Stock solutions of NTZ or TIZ (supplied by Romark Laboratories, LC, Tampa, FL) were 10mM in 100% tissue culture grade DMSO (Sigma-Aldrich, Inc., St Louis, MO), and were stored for up to two weeks at 4°C.
All three cell lines were ‘cured’ of resident HCV replicons by eight serial passages with 1000IU/ml recombinant human interferon alfa-2b (PBL Laboratories, Inc, Piscataway, NJ). Medium for the two resistant cell lines also contained 10uM NTZ during curing. Loss of HCV RNA was confirmed by polymerase chain reaction (PCR) analysis (data not shown). In addition, cultures of all three cured cell lines in 6-well culture plates failed to produce colonies when cultured in the presence of 500 ug/ml G418 (initial population of 1–2 × 106 cells, data not shown).
Transfection with HCV RNA
Cultures were transfected in 6-well culture dishes (Nunc, Inc) using the Lipofectamine2000TM
reagent (Gibco, Inc., Gaithersburg, MD) following the Manufacturer’s instructions and previously described procedures (Korba et al., 2008a
). In brief, 24 hours post-transfection, cultures were exposed to 500μg/ml G418 and antiviral compounds for an additional 14–17 days. Media was replaced three times a week. Resultant macroscopic colonies were fixed in 7% formaldehyde/water and stained with 1% crystal violet dye (1:1 EtOH:water) and counted manually. Duplicate wells were used for each experimental condition in each experiment. The number of colonies in each drug-treated well was compared with the average number of colonies produced by each source RNA in the absence of drug treatment to express colony formation as a percent of untreated controls. Two to four independent experiments were conducted with two different preparations of each RNA source, resulting in 4–8 replicates for each treatment and source RNA. Whole cell RNA used for transfections was purified using Maxi or Midi columns (Qiagen, Inc., Germantown, MD) according to the Manufacturer’s instructions. HCV replicon RNA from individual cloned genomes as prepared as described below.
Construction of HCV replicons containing point mutations
Three specific point mutations in the 5’-UTR were created in an HCV CON1 sub-genomic replicon background using site-directed mutagenesis (QuikChange II, Stratagene, Inc., La Jolla, California): G17A, G18A, C20T (C20U in HCV RNA). Sequencing of the complete 5’-UTR region in the resultant clones was performed to confirm that only a single mutation was present. HCV replicon RNA was transcribed and purified from ScaI linearized replicon source plasmid DNA using the MegaScriptkit (Ambion, Inc., Austin, TX),and purified using MEGAclear kit (Ambion, Inc., Austin, TX).
Cloning and analysis of HCV sequences
Full length cDNAcomplementary to the HCVreplicon genome was created with the SuperScriptOne-Step R/T-PCR for Long Template Kit(Invitrogen, Inc., Carlsbad, CA)following the Manufacturer’s instructions using the corresponding primer pair:R/T-PCR P02 (5’-ACT TGA TCT GCA GAG AGG CCA GTA TC-3’) and PCR P01(5’-GCA GCT GAG TGA TGG TAA GAC TAG AGA GG-3’).
Producing full genome length HCV replicon cDNA proved to be highly inefficient (approximately one of every 50–75 clones). As such, only a limited number of full length clones were produced. For efficiency, the 5’-UTR region was cloned separately and the HCV NS gene sequence region and 3’-UTR were cloned in two portions that overlapped by approximately 150 bases. The first sub-length HCV clones contained the NS3, NS4Aand most of the NS4B coding regions, and the second sub-length clones contained the remainder of NS4B, the NS5A and NS5B coding regions, and the 3’UTR. Analysis of HCV sequences from the RP7 parental cell line did not reveal any significant sequence variances between the full length and sub-length clones. The intent of this practical approach was that, if any common mutations were observed to occur for both cell lines in each of the two sub-length clones, then full length sequences would be created to determine if these existed in the same HCV replicons.
The primer pair of R/T-PCR P01 (5’-GCA GCT GAG TGA TGG TAA GAC TAG AGA GG-3’) and PCR P01 (5’-GCC AGC CCC CGA TTG-3’) was used for reactions with the one-step R/T-PCR kit, to make the first sub-length DNAs. The primer pair of R/T-PCR P02 with PCR P02 (5’-GGATGAACCGGCTGATAGCGTTCG-3’) was used to make the second sub-length DNAs. The DNA made by the R/T-PCR reactions was inserted into vectors using either the TOPO TA Cloning Kit (Invitrogen, Inc.) or the pGEM-T Vector System (Promega, Inc., Madison, WI), and then transformed into E. coli Top 10 or E. coli Top 10F’ chemically competent cells (Invitrogen, Inc.). Positive clones were selected by colony PCR of the single colonies from the culture plates with same primers used to make the cDNAs. Plasmid DNA from the selected colonies was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Inc.) and checked by restriction enzyme digestion as indicated below:Full length clones: EcoRI + Hind III; firstsub-length clones: EcoRI or Hind III + Sac II; second sub-length-clones:Nde I + Xho I or EcoRI + Kpn I. Clones containing the proper restriction enzyme fragments were subjected to DNA sequencing (Macrogen, Inc., Rockville, MD).
The primers utilized for the sequencing were as follows. SP6 promoter: 5’-ATTTAGGTGACACTATAG-3’, T7 Promoter: 5’-TAATACGACTCACTATAGGG-3’, 1stsub-clone sequencing primers: SP P03: 5’-GGAATGCAAGGTCTGTTGAATGTCGTGAAGG-3’, SP P04: 5’-CATCATCACTAGCCTCACAGGCC-3’, SP P05: 5’-CGTATGCAGCCCAAGGGTATAAGG-3’, SP P06: 5’-GCCATTCCAAGAAGAAATGTGATGAGCTCG-3’, SP P07: 5’-GTGTCTCATACGGCTAAAGCC-3’, SP P08: 5’-CAGGCTTGTCCACTCTGCCTG-3’; 2ndsub-clone sequencing primers: SP P10: 5’-CGTAAAGTGCCCGTGTCAGG-3’, SP P11: 5’-CTCCAAGCGGAGGAGGATGAG-3’, SP P12: 5’-CAGGCGCCCTGATCACGCCATG-3’, SP P13: 5’-GGCCCTTTACGATGTGGTCTC-3’, SP P14: 5’-GACAGCTAGACACACTCCAGTCAATTCCTGG-3’, SP P15: 5’-GGAGACATATATCACAGCCTGTCTCG-3’.
Uptake of 14C-TIZ
Cultures were grown to approximately 80% confluence on 12-well culture plates. 14C-labelled TIZ (14.20 mCi/mmol, ABC Laboratories, Inc., Madison, FL) was added to the culture medium (c.a. 5 × 105 CPM/ml in a final TIZ concentration of1.0uM, 0.1% DMSO, 0.5ml/well). Triplicate cultures were harvested at various time points post-exposure. For harvesting, culture medium was removed and the cultures were rinsed three times with PBS. Cells were then removed by trypsinization, centrifuged, the pellets lysed with RLT buffer (Qiagen, Inc), and the lysates analyzed by scintillation counting.
In vitro analysis of HCV enzymatic activities.HCV NS3/4A Protease
Full length NS3 protease/helicase and NS4A cofactor genes from HCV (CON1) were amplified using the primers NS3FLHis (5’-CATATGCATCACCATCACCATCACGCGCCTATTACGGCCTACTCC-3’) with an Nde I restriction site, a hexahistidine tag, and NS4AFLR (5’-AAGCTTTCAGCACTCTTCCATCTCATCG-3’), with a stop codon and Hind III restriction site at the end. Both the pET32a vector and the full length NS3/4A amplicon were processed with Nde I and Hind III enzymes and ligated to obtain the pET32NS3/4AFL expression construct.
E. Coli BL21 Star (DE3) cells, transformed with pET32NS3/4AFL were grown at 37°C in Luria broth containing 100 mg/mL ampicillin. Protein expression was induced with 0.5 mM IPTG at an OD600 of 0.6, and incubated overnight at 16°C. The cells were harvested; lyzed by sonication in lysis buffer (50 mM HEPES; pH7.3, 20% glycerol, 0.5M NaCl, 1 mMb-mercaptoethnol, 1% NP-40). The lysate was centrifuged at 18,000g for 1hr and the supernatant was incubated for 1 hr with Talon metal affinity resin (Clontech Laboratories, Inc., Mountain View, CA) that was pre-equilibrated in the lysis buffer. After the incubation, the resin was washed with 20 column volumes of wash buffer (50mM HEPES; pH7.3, 20% glycerol, 0.5M NaCl, 20 mM imidazole, 1mM b-mercaptoehanol, 0.1% NP-40). The fusion protein was then eluted from the resin using the elution buffer (50 mM HEPES; pH7.3, 20% glycerol, 0.5M NaCl, 500 mM imidazole, 1mM β-mercaptoehanol, 0.1% NP-40). The eluted fractions were checked for purity by SDS-PAGE and quantified spectrophotometrically. Fractions of highest concentrations were pooled and dialysed against buffer containing 50 mM HEPES; pH7.3, 40 % glycerol, 5mM MgCl2,50mM NaCl, 5mM DTT and aliquots stored at −70°C.
Purified full length recombinant NS3/4A protease was used in conjunction with the SensoLyte™ 490 HCV Protease Assaybuffer and fluorogenic substrate components (Anaspec, Inc., San Francisco). The use of full length NS3/4A protein in the assay is prompted by the increasing evidence that the activities of NS3 protease and helicase domains are modulated by each other (Beran et al., 2007
; Du et al., 2002
; Lam et al., 2003
; Kuang et al., 2004
) and most likely more accurately models in vivo
reactions. The reaction consisted of 20nM purified enzyme and 1× HCV NS3/4A protease substrate, both diluted in 1× SensoLyte 490 HCV assay buffer. The HCV NS3/4A protease substrate was an EDANS/DABCYL FRET peptide which upon cleavage has excitation and emission maxima at 340nm and 490nm respectively. Fluorescence was measured after 30 minutes of incubation at room temperature. Analysis of inhibitors was performed by incubating test compounds with the enzyme for 15 minutes followed by 30 minutes incubation with the substrate. An established, clinically relevant, HCV protease inhibitor, VX-950 (telaprevir) (Perni et al., 2006
) (purchased from Acme Bioscience, Inc., Palo Alto, CA) was used as an assay control.
HCV NS3 Helicase
A double-stranded nucleic acid unwinding assay previously described (Kuang et al., 2004
; Lam et al., 2003
) was adopted with minor modifications. Full length NS3/NS4A protein was used to assay helicase activity. The helicase assay is dependent on the unwinding of a synthetic duplex DNA or RNA oligonucleotide substrate in which one of the strand is 32
P-labeled to enable radioactive detection after electrophoretic separation of the completed reactions.
The substrate DNA was generated by annealing an 18-base 32
P-labeled and an unlabelled 28-base oligonucleotide, creating a duplex substrate with a 3’-ssDNA overhang (required for helicase recognition and activity (Kuang et al., 2004
; Lam et al., 2003
; Rosales-Leon et al., 2007
). The reaction consisted of 1nM dsDNA substrate and 20nM HCV helicase incubated in reaction buffer (25mM MOPS, pH6.5, 3mM MgCl2
, 0.1% Tween 20) for 30 min at 37°C. Unlabelled shorter oligonucleotides (250nM) were used as trap DNA to prevent re-annealing of the unwound oligonucleotides (Kuang et al., 2004
). The 10 μl reactions were initiated by the addition of 5mM ATP and terminated by 2.5μL of 5× stop buffer (250nM Tris-HCl, pH 7.5, 20mM EDTA, 0.5% SDS, 0.1% Nonidet p-40, 0.1% bromophenol blue, 0.1% Xylene cyanole FF, 50% glycerol). The reaction products were analyzed by non-denaturing 14% polyacrylamide gel electrophoresis. Helicase was measured by the relative quantification of the radioactivity in the single-stranded reaction product and the duplex substrate using an InstantImagerTM
Beta Scanner (PerkinElmer, Inc., Shelton, CT).
For the testing of inhibitors of HCV helicase, the compounds were diluted in dimethyl sulfoxide (DMSO), and pre-incubated with the enzyme for 15 minutes at 37°C in the assay buffer. Activation of assay was initiated by the addition of substrate and ATP, followed by further incubation at 37°C for 30 minutes. DMSO (0.1%) was added to the enzyme assay as a blank. Thioflavine S (Sigma-Aldrich, Inc.) was used as an inhibitor control. This dye is a non-proprietary compound that is reported to inhibit HCV helicase in a dose-response manner (Frick, 2010
HCV NS5B polymerase
Several enzymatic assays for the HCV NS5B polymerase (RdRP)have been described (Behrens et al., 1996
; Heck et al., 2008
; Lohmann et al., 1997
; Ludmerer et al., 2005
). Most of these assays utilize either a non-specific RNA template, or a short template containing the 3’-UTR. The 5′-UTR also plays a pivotal role in the initiation of translation of the viral polyprotein(Brown et al., 1992
; Bukh et al., 1992
; Kolykhalov et al., 2000
). A unique template for the HCV RdRP assay was created based on studies with in vitro
Dengue Virus polymerase assays (You and Padmanabhan, 1999
) that is believed to more closely model viral genome interactions between the 5’ and 3’ UTRs. This mini-genome (674 nucleotides) is comprised of the HCV 3’ and 5’-UTR’s with small portions of the core (45 bp) and NS5B (28 bp) coding sequences as a linker. Particular advantages of this template scheme are that it allows the use of RNA templates with either genomic polarity, allows for genotype-specific matching of template and polymerase, and allows for the use of 32
P-labelled nucleotides other than UTP. NS5B was prepared and purified essentially as previously described (Behrens et al., 1996
; Lohmann et al., 1997
The in vitro
RdRP assay was performed in 50-μl reaction mixtures using incorporation of [α-32
P]-UTP for 1 hr. at 30°C. into the RNA products. Reactions contained300ng of HCV RNA templates, 100ngof NS5B proteins, 500uM of each ATP, CTP and GTP, 10uM of UTP, and 100unit of RNaseOut (Invitrogen, Inc., Carlsbad, CA) in 50mM HEPES buffer (pH 7.3). The RNA products were isolated using the RNeasy Mini Kit (Qiagen Inc.), then ethanol precipitated, separated on 1.5% formaldehyde agarose gels (3 hrs. at 100V), and dried. Radioactivity was quantitated using an InstantImagerTM
Beta Scanner. An established non-nucleoside NS5B inhibitor, HCV-796 (Howe et al., 2008
) was used as an assay inhibitor control.