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Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line
Journal of Virological Methods 189 (2013) 110–117
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line Daniela Cavallone a , Francesco Moriconi a , Piero Colombatto a , Filippo Oliveri a , Ferruccio Bonino b , Maurizia Rossana Brunetto a,∗ a b
Hepatology Unit and Liver Physiopathology Laboratory, University Hospital of Pisa, Pisa 56124, Italy General Medicine Unit 2, Department of Internal Medicine, University Hospital of Pisa, Pisa 56124, Italy
a b s t r a c t Article history: Received 13 August 2012 Received in revised form 4 January 2013 Accepted 28 January 2013 Available online 4 February 2013 Keywords: Hepatitis B virus In vitro cell culture Transfection system HBV surface antigen HBV replication cycle
The Gunther’s vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48 h) and in higher amounts (2.51 ± 0.32 and 6.43 ± 0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R = 0.696, p = 0.017 overall; R = 0.928, p = 0.008), but not in GM (R = −0.437, p = 0.179 overall; R = −0.395, p = 0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human hepatitis B virus (HBV) infection leads to a wide spectrum of clinical conditions ranging from inactive infection without liver disease to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) (Lee, 1997; Lok and McMahon, 2007). A better understanding of the molecular mechanisms of HBV replication and pathogenesis could improve the clinical management of HBV carriers, however the lack of appropriate cell culture systems supporting stable and efficient in vitro HBV infection (Chang et al., 1987) has been a major limitation. Only primary human hepatocytes (Tuttleman et al., 1986) and differentiated HepaRG cells can support the complete HBV life cycle (Gripon et al., 2002), but major drawbacks of primary hepatocytes are their limited availability and inherent variability of human material, whereas HBV replication levels in HepaRG are low (Lucifora et al., 2010). Vector-based models were developed using plasmids that encode “greater-thangenome” HBV sequences to overcome the breaking of the HBV genes due to the linearization of viral genome (Brunelle et al., 2005; Chin et al., 2001; Durantel et al., 2004; Jacquard et al., 2006;
Abbreviations: GM, Gunther’s vector-free method; MLSE, Monomer-LinearSticky-Ends-DNA; MCC, Monomer-Circular-Closed. ∗ Corresponding author. Tel.: +39 050996857; fax: +39 050995457. E-mail address: [email protected] (M.R. Brunetto). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.01.012
Junker et al., 1987; Seeger and Mason, 2000; Sells et al., 1987a,b; Summers and Mason, 1982; Warner and Locarnini, 2008; Yaginuma et al., 1987; Yang et al., 2004). Alternatively, recombinant adenovirus or baculovirus carrying the HBV genome could be used (Abdelhamed et al., 2002; Delaney and Isom, 1998; Delaney et al., 1999). However, vector-based methods and baculovirus transfer system, despite their utility to study specific steps of HBV cell cycle, are far away from reproducing the in vivo conditions adequately. In the attempt to develop a vector-free approach and to reproduce the conditions of in vivo HBV infection, Günther et al. (1995) described an original method using the polymerase chain reaction (PCR) amplification of full length HBV-DNA. By this approach not all HBV isolates result replication-competent and intracellular HBV-DNA synthesis is low (Durantel et al., 2004; Villeneuve et al., 2003; Zhu et al., 2007). To overcome the low replicative efficiency, it has been suggested to increase the sensitivity in detecting viral replication by assays such as real-time PCR (Zoulim, 2006). However, such an approach might be misleading by amplifying not only replicative intermediates but also input DNA, as a significant proportion of the input linear double-strand HBVDNA remains intact even after DNAse I digestion (Günther et al., 1995). The present study was aimed to analyze the factors influencing the replication efficiency of the Günther’s method and to develop a new approach able to increase the in vitro replication levels of HBV.
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2. Materials and methods 2.1. Patients A panel of highly viraemic sera (median HBV-DNA 2.43 × 109 cp/ml, range 1.00 × 106 cp/ml to 7.16 × 1011 cp/ml) was obtained from 12 HBeAg positive untreated acute (n = 4) or chronic (n = 8) hepatitis B patients (median age 37 years, range: 18–70; 10 males and 2 females) (Table 1). All the patients were negative for antibodies against hepatitis C virus (HCV), hepatitis D virus (HDV) and human immunodeficiency virus (HIV). 2.2. HBV-DNA isolation HBV-DNA was extracted from serum according to the proteinase K protocol. Briefly 200 l of serum were added to 450 l of mix containing 1 mg/ml proteinase K, 5 mM Tris–HCl (pH 8.5), 2.0% SDS and 25 mM EDTA and incubated at 37 ◦ C overnight. HBV-DNA was precipitated with 1 vol of absolute isopropanol in presence of 20 g of Dextran T 500 and 1/10 vol of 3 M NaAc (pH 4.7). DNA was recovered by centrifugation at 20,000 × g for 15 min; finally pellets were washed with ethanol 70%, dried and resuspended in 50 l of water. 2.3. In house Gunther’s method Full length HBV-DNA was amplified by High Fidelity PCR system (Roche, Mannhein, Germany) with the sense primer FL-S 5 -CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTAATCATC-3 and with antisense primer FL-AS 5 -CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGTG-3 (Parekh et al., 2003). The thermal profile of the amplification was: pre-heating 2.5 min at 94 ◦ C, 10 cycles including denaturation 20 s at 94 ◦ C, annealing 30 s at 55 ◦ C and extension 3 min at 68 ◦ C; 30 cycles including denaturation 20 s at 94 ◦ C, annealing 30 s at 55 ◦ C and extension 3 min (+0.05 ◦ C/cycle) at 68 ◦ C followed by 10 min at 68 ◦ C. After amplification, HBV full length was purified after gel electrophoresis analysis. For transfection of PCR products HBV-DNA was cleaved with 1 U of SapI (New England BioLabs, Ipswich, USA) per g of DNA at 37 ◦ C overnight. Digested DNA was purified by QIAquick PCR purification kit (Qiagen, Hilden, Germany) and then spectrophotometrically quantified. 2.3.1. Optimization of enzyme restriction digestion During the development of the project different digestion profiles of SapI or BspQI (New England BioLabs, Ipswich, USA) were tested: enzyme concentration ranging from 0.5 U/g to 4 U/g and digestion time ranging from 5 h to overnight. The digestion temperature was 37 ◦ C and 50 ◦ C for SapI and BspQI respectively. 2.4. Preparation of Monomer Linear Sticky Ends (MLSE) and Monomer Circular Closed (MCC) HBV-DNA Monomer Linear Sticky Ends DNA (MLSE-DNA) and linear undigested full length HBV-DNA, share the same molecular weight and can not be separated by gel electrophoresis analysis after SapI or BspQI digestion (Fig. 1A). To obtain only replication competent DNA, full length HBV-DNA was cut with HindIII (2 U/g) and SacI (2 U/g) (Fermentas, Erembodegem, Belgium) and cloned into pUC18 vector (Sambrook et al., 1989). pUC18-HBV-DNA was cleaved with 1 U of SapI or BspQI per g of DNA at 37 ◦ C or at 50 ◦ C overnight and then the 3.2 Kb HBV monomer was recovered by gel electrophoresis. To become competent for replication after cell transfection, MLSE DNA requires circularization by the host’s enzyme: this step is crucial for replication, but cannot be monitored in the available in vitro systems. To overcome this limitation and to transfect
Fig. 1. (A) Effect of SapI treatment on electrophoretic mobility of full length HBVDNA. Full length HBV-DNAs, either digested or undigested by SapI or BspQI, migrate at the same molecular weight (3.2 Kb). (B) In vitro MLSE circularization. MLSE was treated by T4 DNA ligase in highly diluted solution to avoid the polymerization and to promote the in vitro monomer circularization.
Monomer Circular Closed HBV-DNA only, MLSE was circularized by T4 DNA ligase (Promega, Madison, USA) at low DNA concentration (5 g/ml) (Cavallone et al., 2010; Hirschman et al., 1980; Sureau et al., 1986; Wang et al., 1982) to obtain monomer circular closed (MCC). Thereafter, MCC-DNA and MLSE-DNA were separated by gel electrophoresis (migration at molecular weight of 2 and 3.2 Kb, respectively, Fig. 1B) and used for transfection after purification and quantification by spectrophotometer analysis. 2.5. Analysis of full length HBV-DNA by sequencing All HBV-DNA forms, that were used in transfection experiment were purified by Exosap (GE Healthcare, Life Science, LittleChalfont, UK) and directly sequenced by chain terminator method using CEQ 8000 Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Krefeld, Germany). Briefly, 2 l of purified template were added to 8.0 l of DTCS Quick Start Master Mix, 4.0 l of
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Table 1 Clinical and virological features of patients. No.
Genotype
Viral load (cp/ml)
HBeAg
Diagnosis
Replication competent (Gunther Sapl phenotypic assay)
Mutations affecting replication
1 2 3 4 5 6 7 8 9 10 11 12
A B C D D F D D D A D D
1.10 × 109 1.22 × 1010 1.00 × 106 2.70 × 109 6.40 × 108 3.09 × 109 2.19 × 1010 7.16 × 1011 2.16 × 109 2.50 × 1010 6.40 × 108 1.86 × 109
POS POS POS POS POS POS POS POS POS POS POS POS
CHB CHB Acute Hep. Acute Hep. CHB CHB CHB Acute Hep. CHB CHB CHB Acute Hep.
Yes Yes Yes Yes Yes No No Yes No No Yes Yes
No No No No No No No No No No No No
1.6 M sequencing primers (Fig. 2) and 6 l H2 O. Thermal cycling profile was 30 cycles of 20 s at 96 ◦ C, 20 s at 50 ◦ C, 4 min at 60 ◦ C followed by holding at 4 ◦ C. Sequencing was carried out using a CEQ 8000 XL analysis System (Beckman Coulter, Krefeld, Germany) and analyzed with Chromas lite 2.01 software. 2.6. Cell culture Human hepatoma HuH7 cells were generously provided by Dr. Fabien Zoulim. Huh7 was grown in Dulbecco’s Modified Eagle Medium (DMEM)-F12 medium (1:1) (Euroclone, Milano, Italy) supplemented with 100 U/ml of penicillin, 100 g/ml of streptomycin, 2 mM L-glutamine, 10 mM Hepes buffer solution, 0.5 mM sodium pyruvate and 10% fetal bovine serum (Gibco, Monza, Italy). The HuH7 cell line was maintained at 37 ◦ C in a humidified incubator at 5% CO2 . HuH7 cell line was regularly passaged and tested with mycoplasma stain kit (Sigma, St. Louis, USA).
with a [32 P]-labeled full length HBV probe and exposed from 10 to 16 h for autoradiography as indicated in the figure legend. 2.9. HBsAg quantification The culture medium was tested for HBsAg using the Architect HBsAg assay (Abbott Laboratories, Abbott Park, IL; dynamic range, 0.05–250.0 IU/ml). Briefly, after incubation of the sample with anti-HBs coated paramagnetic microparticles and washing, an acridinium-labeled anti-HBs conjugate was added. The resulting chemiluminescent reaction was measured in the relative light units (RLUs) and converted in IU/ml of HBsAg. The correlation between HBsAg production and HBV-DNA replication was analyzed using SPSS19 software. 3. Results 3.1. In house performance of the Günther’s method
2.7. Transfection of HBV-DNA HuH7 cells were plated at a density of 1 × 106 cells per well in 60 mm diameter-petri dish 1 day before transfection. The cells were transfected with 3 g of DNA and the transfection was carried out using Fugene reagent (Roche, Mannhein, Germany) according to the manufacturer’s instructions. The medium was changed 1 day after transfection, and cells were harvested at the indicated time points after transfection (from 1 h to 120 h). Each experiment was run in triplicate. Transfection efficiency was measured by cotrasfection of 1 g of reporter plasmid expressing enhanced green fluorescence protein. 2.8. Purification of HBV-DNA from intracellular core particles and Southern blot analysis The cells were washed three times with phosphate-buffered saline (PBS) and lysed in 500 l of lysis buffer [50 mM Tris–HCl (pH 8), 1 mM EDTA, 1% Nonidet P-40] per 60 mm diameter petri dish. The lysed cells were transferred to Eppendorf tubes, vortexed, and maintained in ice for 10 min. Nuclei were pelleted by centrifugation for 1 min at 1000 × g. The supernatant was adjusted to 10 mM MgCl2 and treated with 100 g of DNase I per ml for 1 h at 37 ◦ C. The reaction was stopped by the addition of EDTA to a final concentration of 25 mM. Proteins were digested with 0.5 mg of proteinase K per ml and 1% SDS at 37 ◦ C overnight. Nucleic acids were purified by phenol-chloroform (1:1) extraction and ethanol precipitation after the addition of 20 g of Dextran T500 and 1/10 vol of 3 M NaAc (pH 4.7). Replicative intermediates were then analyzed by electrophoresis in 1.2% agarose gels followed by Southern blotting as described previously (Sells et al., 1987a). Filters were hybridized
Using the Günther’s (GM) method, 8 out of 12 (67%) HBV isolates were replication competent: one (no. 8) with high and seven with low efficiency (Table 1). Mutation, potentially responsible for defective replication were not detected in the full length sequence of the 4 isolates which did not replicate (data not shown). In all isolates direct sequencing (DS) of the catalytic site of SapI showed the presence of the heterologous primers, in spite of the treatment with restriction enzyme. Given the sensitivity limit of DS in detecting a minor viral population, the results indicate that more than 80% of the input DNA was undigested, therefore unable to replicate, because missing the Sticky Ends required for circularization. 3.1.1. Optimization of enzyme digestion The efficiency of digestion was not improved by changing SapI concentrations (from 0.5 U/g to 4 U/g) or digestion time (from 5 h to overnight, data not shown). On the contrary, replacing SapI with the isoenzyme BspQI, high levels of HBV replication were obtained in 1 additional isolate (no. 3). In both the isolates with high replication levels (nos. 3 and 8) DS showed that most of the input DNA was digested. Given the more efficient digestion, BspQI was therefore used in all the following experiments. 3.2. Günther method vs. MLSE-DNA vs. MCC-DNA The HBV-DNA extracted from the serum with the highest viremia (no. 8, Table 1) was used to transfect HuH7 cells to compare the replication performance of the GM, MLSE-DNA and MCC-DNA methods (Fig. 3A). In all the 3 approaches (Fig. 3B), Southern blotting analysis showed a smears (extending from the
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Fig. 2. Amplification and sequencing primers.
position of single-strand HBV-DNA [SS] up to the position of relaxed circular [RC] HBV-DNA) characteristic of HBV replicative intermediates. Kinetic analysis of the intracellular replicative intermediates showed significant differences among the 3 methods (Fig. 3): MCCDNA was not only more efficient than MLSE-DNA and GM methods, but also showed an earlier detection of single strand HBV-DNA in the cytoplasmic HBV core particles. As expected, the untreated BspQI HBV-DNA full length (NRC) was replication defective and Double-Strand Linear HBV-DNA (DSL) was the only detected band (Fig. 3A and B) that resulted from the input DNA. Accordingly, DSL signal decreased progressively during post transfection time and no other HBV replicative intermediates were observed. Extracellular HBsAg was quantified at different post transfection time points and showed increasing levels over time in the three replication competent systems (Fig. 3C), the highest HBsAg levels being produced by MLSE. The untreated BspQI, full length replication defective HBV-DNA, also produced HBsAg although at the lowest levels (Fig. 3C).
The kinetics of single strand (SS) production analyzed by densitometric quantification showed that the transfection by MCC-DNA was associated with an earlier appearance (24 vs. 48 h) and a higher production of the replicative intermediates. In 3 different experiments (each of them run in triplicate) in vitro circularized MCC-DNA increased by 2.51(±0.32) and 6.43 (±0.43) fold the production of SS DNA replicative intermediate, at the 120 h post trasfection time, as compared to the MLSE and Günther’s methods, respectively (Fig. 4). HBV-DNA and HBsAg kinetics correlated positively in all the three replication competent systems (R = 1; p = 0.01) (Fig. 5), but not in the replication defective system (R = 0.354; p = 0.559) (Fig. 5). 3.3. Replication fitness of 12 HBV isolates from HBeAg positive patients by Günther method and MCC-DNA approach The experiments were run in triplicate with comparable results. Eight of the 12 isolates (67%) showed all the HBV replicative
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Fig. 3. Kinetics analysis of HBV replicative intermediates and HBsAg expression. (A) Schematic representation of HBV genome used in the different transfection approaches: Untreated BspQI HBV-DNA full length (replicative defective, NRC) used as negative control; in house Gunther’s method (GM); Monomer Linear Sticky Ends (MLSE); Monomer Circular Closed (MCC). (B) Southern blotting analysis of intracellular replicative intermediates extracted from viral core particles produced in HuH7 cells transfected with different types of full length HBV-DNAs BspQI treated (GM, MLSE, MCC) or untreated (NRC). In the mock control lane (mc) the cells were not transfected. Filter was autoradiographed at −80 ◦ C for 16 h. The position of the relaxed circular (RC), double strand linear (DSL) and single stand (SS) forms of the HBV-DNA genome are indicated Lane M, DNA size markers (Kb). Data shown are representative experiments performed in triplicate. (C) Extracellular HBsAg quantified by Architect (Abbott) at different time points after transfection.
intermediates and were defined replication competent by in house GM method (Fig. 6, panel A): only 2 had high replicative efficiency (Fig. 6, panel A, lanes 3 and 8). The remaining 4 isolates showed only linear double-strand HBV-DNA. By the MCC-DNA approach 10 out of 12 (83%) isolates were replication competent: 7 showed all the replicative intermediates at very high levels (Fig. 6 panel B, lanes: 2, 3, 5, 7, 8, 11 and 12); 3 showed only linear double-strand HBVDNA (Fig. 6 panel B, lanes: 4, 9 and 10). Using the MCC approach the detection of linear double strand HBV-DNA (3.2 Kb) is the hallmark of viral replication, at variance with the GM method where traces of the input 3.2 Kb DSL-DNA can contaminate the results. On the contrary, in MCC the circularized input DNA migrates with a molecular weight of 2 Kb, which differs from that of DSL-DNA. The remaining 2 isolates did not show any band and were defined not replication competent (Fig. 6 panel B, lanes 1 and 6). The cloning step that is required to prepare MCC-DNA may select minor viral populations, therefore the input DNA of all the isolates that were used in the GM and MCC approaches were sequenced by DS and no mutations potentially affecting viral replication were identified (data not shown). When transfection was performed by GM method, HBsAg was produced by all the isolates, independent of the replication competence. In 2 cases (nos. 3 and 11) where HBsAg values were GM
SS DNAcp
1.00E+10
MLSE
MCC
4. Discussion
1.00E+09 1.00E+08 1.00E+07 1.00E+06 24
48
72
96
low in spite of medium–high replication levels, DS analysis of the input DNA showed the presence of mixed wild-type and mutant sequences in the small S gene. The 2 mutations (TGG to TAG switch at codon s201 and TTA to TAA switch at codon s216) created a stop codon in the S proteins, without affecting the overlapping polymerase gene. By MCC method HBsAg was not produced by 2 isolates (nos. 6 and 11): one (no. 6) was replicative defective, the other (no. 11) was replication competent, but DS of the input DNA showed the presence of the s216 stop codon, possibly selected by the cloning step. The SS DNA and HBsAg levels across the different isolates (excluding no. 11 for the reasons explained above) showed a positive correlation in the MCC method (Spearman correlation: R = 0.696; p = 0.017). On the contrary, a negative correlation was observed in the Günther method, where higher HBsAg levels were found in isolates with lower levels of replication (Spearman correlation: R = −0.437; p = 0.179). To avoid possible interference due to the variability in the ratio between viral replication and antigen expression in the different genotypes, a sub-analysis was run in genotype D isolates only. In genotype D isolates the correlation between HBsAg levels and viral replication was even higher in the MCC system (Spearman correlation: R = 0.928; p = 0.008), but not in GM method (Spearman correlation: R = −0.395; p = 0.439).
120
post transfection time (h)
Fig. 4. Kinetics of single strand HBV-DNA production by the 3 different approaches. The intensity of SS DNA band was measured by densitometric analysis and then a reference HBV marker was used to convert the intensity value in copies (cp).
The present study investigated the reasons for the low replication efficiency of the Günther’s method and found that a major limiting step is the low proportion (<20%) of SapI digested DNA in the DNA used for transfection. The DS showed that the majority of the input DNA is not replication competent, since the continuity of the Basic Core Promoter (BCP) and Pre-Core (PC) regions is disrupted by the presence of the primers tails, that prevent DNA circularization. In the attempt to optimize the digestion conditions, SapI was replaced by BspQI enzyme, but replication was improved only in 2 of the 8 replication competent isolates. DS analysis showed that the 2 genomes with high replication efficiency
D. Cavallone et al. / Journal of Virological Methods 189 (2013) 110–117
B 600 500
1.50E+09
400
1.00E+09
300 200
5.00E+08
0 1
24
48
96
600 500
1.50E+09
400
1.00E+09
300 200
5.00E+08
100
0.00E+00
700
2.00E+09
SS DNA cp
2.00E+09
HBsAg IU
100
0.00E+00
0
120
1
post transfection time (h)
48
96
120
post transfection time (h)
C
D 2.50E+09
700
2.50E+09
2.00E+09
500
1.50E+09
400
1.00E+09
300 200
5.00E+08 0.00E+00 1
24
48
96
SS DNA cp
600
HBsAg IU
SS DNA cp
24
600
2.00E+09
500
1.50E+09
400
1.00E+09
300
100
5.00E+08
0
0.00E+00
120
700
200 100 0 1
post transfection time (h)
24
48
96
120
post transfection time (h)
HBsAg IU DNA SS cp
Post Transfection time (h) 24 48 96 0.0 15.3 65.5 153.5 0.0E+00 0.0E+00 0.0E+00 0.0E+00
120 185.0 0.0E+00
HBsAg IU DNA SS cp
0.8 0.0E+00
34.9 1.3E+06
135.9 1.2E+07
334.9 1.6E+08
469.5 3.1E+08
GM
HBsAg IU DNA SS cp
1.0 0.0E+00
57.7 1.0E+07
266.5 2.8E+08
488.1 7.9E+08
584.0 8.0E+08
MLSE
HBsAg IU DNA SS cp
0.4 0.0E+00
46.2 1.8E+08
153.3 1.4E+09
281.9 1.9E+09
299.2 2.0E+09
MCC
E
HBsAg IU
SS DNA cp
2.50E+09
700
2.50E+09
HBsAg IU
A
115
1
Method NRC
Fig. 5. Correlation between HBsAg production (•) and intrahepatic HBV replicative intermediates (o) in the 4 transfection conditions: (A) NRC, (B) GM, (C) MLSE, and (D) MCC. A positive correlation between HBV RI and HBsAg production could be observed in panels B, C and D but not in the replication defective system (panel A). All the values showed in the graphics are reported in panel E.
(Fig. 6A, patients number 3 and 8) were those more efficiently digested by BspQI enzyme. On the contrary, heterologous primer sequences were detected as prevalent population in all the other genomes with lower or absent replication. The evidence that the input DNA in the Günther’s method results from a mixture of digested and undigested full length HBV-DNA identifies a major confounding factor for the in vitro study of viral replication, whose efficiency appears to be influenced not only by viral features but also by technical artifacts. Accordingly, sequencing the full length of all genomes excluded that mutations affecting viral replication could have been eventually introduced by PCR amplification. Probably the use of High Fidelity Taq DNA polymerase could explain our results: accordingly, Günther reported that the frequency of the mutations decreased significantly when Taq DNA polymerase without proofreading activity was substituted by an enzyme with proofreading activity (High Fidelity) (Günther et al., 1998). In the attempt to improve further in vitro HBV replication the method was modified by transfecting, after cloning, pure replicative competent DNA either as Monomer Linear Sticky Ends DNA
(MLSE method), which still requires intracellular circularization, or Monomer Circular Closed DNA (MCC method), which is competent for replication immediately after transfection (Cavallone et al., 2010). Accordingly, by the latter approach (MCC method) HBV replicative intermediates were detected earlier than with Günther’s or MLSE methods (24 vs. 48 h) (Fig. 3B). In addition, the transfection with MLSE DNA or MCC DNA was two and six times more efficient in terms of viral replication than the original Günther’s method (Fig. 4). Similar results were recently reported by Qin et al. (2011) who used a similar approach. Using the MCC method very high level of replicative intermediates were produced by 7 of 12 HBV isolates (Fig. 6B), whereas 3 isolates showed only linear double-strand HBV-DNA (Fig. 6B, nos. 4, 9 and 10). Nevertheless, in the setting of MCC method the detection of linear double strand HBV-DNA (3.2 Kb) has to be considered a true expression of viral replication. In fact, at variance with Günther’s method where traces of the input 3.2 Kb DSL-DNA can contaminate the results, in the case of the MCC approach the circularized input DNA migrates with a molecular weight of 2 Kb.
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Fig. 6. Analysis of the replication fitness of 12 isolates from HBeAg positive patients by the in house Gunther’s method (A) and MCC DNA method (B). HuH7 cells transfected with 3 g of HBV-DNA full length isolated from patients (referred as number 1–12) by GM (A) or of MCC method (B): lanes correspond to DNA extracted from viral core particles derived from cell lysates. The figure shows a representative result of experiments performed in triplicate. In the mock control lane (mc) the cells were not transfected. Lane M, DNA size markers (Kb) Filters were autoradiographed at −80 ◦ C for 10 h.
Accordingly, when an isolate is unable to replicate in vitro with the MCC method no background band was observed (Fig. 6B, patients 1 and 6). Furthermore, in all 3 isolates where only DSL-DNA was present at Southern blotting, HBsAg was detected in the medium (Fig. 6B, nos. 4, 9 and 10), proving the presence of viral replication because in this setting HBsAg production results exclusively from viral replication. At variance with Gunther’s method, where linear undigested full length HBV-DNA, that is not replication competent but represents a significant proportion of input DNA, may produce HBsAg independently from HBV replication (Fig. 3C). Indeed, the mixture of different (digested and undigested) DNA forms could be responsible for the asymmetry between viral replication and HBsAg production that leads in the GM method to the inverse correlation between HBsAg levels and replicative intermediates, when different isolates are compared. By MCC method the HBsAg secretion showed a significant correlation with HBV replication particularly when the analysis was restricted to genotype D isolates, to avoid possible interference due to the variability in the ratio between viral replication and antigen expression that was described in the different genotypes (Sugiyama et al., 2006). Overall these findings suggest that the MCC method consistently reproduces the in vivo conditions, representing a reliable approach for the in vitro study of HBV biology. Particularly, the MCC method appears adequate for a precise analysis of the correlation between viral replication and HBsAg production, because only circularized HBV-DNA is transfected. The in vivo complexity of the interplay between viral replication and HBsAg production was underlined in the recent years by several clinical studies, suggesting the possible interference of both viral (genotype) and host (immune competence) factors (Brunetto, 2010; Brunetto et al., 2010). The possibility to study specifically the biological features of each HBV isolate, without loosing the chance of the population approach by creating a clone pool could warrant a better understanding of the virologic
factors which influence the dynamic relationship between viral replication, HB-virions and antigen production. In conclusion the present study demonstrates that HBV-DNA circularization improves in vitro viral replication and viral replication competent HBsAg production, which mimics better the in vivo conditions, thus this method could be used to investigate further the characteristics of the HBV life-cycle. 5. Conflict of interest The authors declare no conflict of interest in relation to this study. Acknowledgments This work was supported by 2010 CUCCS Grant of the University of Pisa, Italy. References Abdelhamed, A.M., Kelley, C.M., Miller, T.G., Furman, P.A., Isom, H.C., 2002. Rebound of hepatitis B virus replication in HepG2 cells after cessation of antiviral treatment. J. Virol. 76, 8148–8160. Brunelle, M.N., Jacquard, A.C., Pichoud, C., Durantel, D., Carrouée-Durantel, S., Villeneuve, J.P., Trépo, C., Zoulim, F., 2005. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology 41, 1391–1398. Brunetto, M.R., 2010. A new role for an old marker, HBsAg. J. Hepatol. 52, 475–477. Brunetto, M.R., Oliveri, F., Colombatto, P., Moriconi, F., Ciccorossi, P., Coco, B., Romagnoli, V., Cherubini, B., Moscato, G., Maina, A.M., Cavallone, D., Bonino, F., 2010. Use of hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers. Gastroenterology 139, 483–490. Cavallone, D., Colombatto, P., Moriconi, F., Stabile, A., Maina, A.M., Bonino, F., Brunetto, M.R., 2010. Improved in vitro HBV replication in HuH7 cell line transfected with full length circular closed HBV DNA by modifications of the Günther’s method. Dig. Liver Dis. 42 (Suppl. 1), S2.
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Qin, Y., Zhang, J., Garcia, T., Ito, K., Gutelius, D., Li, J., Wands, J., Tong, S.J., 2011. Improved method for rapid and efficient determination of genome replication and protein expression of clinical hepatitis B virus isolates. J. Clin. Microbiol. 49, 1226–1233. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Analysis and cloning of eukaryotic genomic DNA. In: Ford, N., Nolan, C., Ferguson, M. (Eds.), Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 9.1–9.62. Seeger, C., Mason, W.S., 2000. Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. 64, 51–68, Review. Sells, M.A., Chen, M.L., Acs, G., 1987a. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. U.S.A. 84, 1005–1009. Sells, M.A., Zelent, A.Z., Shvartsman, M., Acs, G., 1987b. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J. Virol. 62, 2836–2844. Sugiyama, M., Tanaka, Y., Kato, T., Orito, E., Ito, K., Acharya, S.K., Gish, R.G., Kramvis, A., Shimada, T., Izumi, N., Kaito, M., Miyakawa, Y., Mizokami, M., 2006. Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 44, 915–924. Summers, J., Mason, W.S., 1982. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29, 403–415. Sureau, C., Romet-Lemonne, J.L., Mullins, J.I., Essex, M., 1986. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 47, 37–47. Tuttleman, J.S., Pugh, J.C., Summers, J.W., 1986. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J. Virol. 58, 17–25. Villeneuve, J.P., Durantel, D., Durantel, S., Westland, C., Xiong, S., Brosgart, C.L., Gibbs, C.S., Parvaz, P., Werle, B., Trépo, C., Zoulim, F., 2003. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J. Hepatol. 39, 1085–1089. Wang, Y., Schäfer-Ridder, M., Stratowa, C., Wong, T.K., Hofschneider, P.H., 1982. Expression of hepatitis B surface antigen in unselected cell culture transfected with recircularized. HBV DNA. EMBO J. 1, 1213–1216. Warner, N., Locarnini, S., 2008. The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound. Hepatology 48, 88–98. Yaginuma, K., Shirakata, Y., Kobayashi, M., Koike, K., 1987. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc. Natl. Acad. Sci. U.S.A. 84, 2678–2682. Yang, H., Westland, C., Xiong, S., Delaney 4th., W.E., 2004. In vitro antiviral susceptibility of full-length clinical hepatitis B virus isolates cloned with a novel expression vector. Antiviral Res. 61, 27–36. Zhu, Y., Curtis, M., Snow-Lampart, A., Yang, H., Delaney, W., Miller, M.D., BorrotoEsoda, K., 2007. In vitro drug susceptibility analysis of hepatitis B virus clinical quasispecies populations. J. Clin. Microbiol. 45, 3335–3341. Zoulim, F., 2006. In vitro models for studying hepatitis B virus drug resistance. Semin. Liver Dis. 26, 171–180.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line Daniela Cavallone a , Francesco Moriconi a , Piero Colombatto a , Filippo Oliveri a , Ferruccio Bonino b , Maurizia Rossana Brunetto a,∗ a b
Hepatology Unit and Liver Physiopathology Laboratory, University Hospital of Pisa, Pisa 56124, Italy General Medicine Unit 2, Department of Internal Medicine, University Hospital of Pisa, Pisa 56124, Italy
a b s t r a c t Article history: Received 13 August 2012 Received in revised form 4 January 2013 Accepted 28 January 2013 Available online 4 February 2013 Keywords: Hepatitis B virus In vitro cell culture Transfection system HBV surface antigen HBV replication cycle
The Gunther’s vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48 h) and in higher amounts (2.51 ± 0.32 and 6.43 ± 0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R = 0.696, p = 0.017 overall; R = 0.928, p = 0.008), but not in GM (R = −0.437, p = 0.179 overall; R = −0.395, p = 0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human hepatitis B virus (HBV) infection leads to a wide spectrum of clinical conditions ranging from inactive infection without liver disease to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) (Lee, 1997; Lok and McMahon, 2007). A better understanding of the molecular mechanisms of HBV replication and pathogenesis could improve the clinical management of HBV carriers, however the lack of appropriate cell culture systems supporting stable and efficient in vitro HBV infection (Chang et al., 1987) has been a major limitation. Only primary human hepatocytes (Tuttleman et al., 1986) and differentiated HepaRG cells can support the complete HBV life cycle (Gripon et al., 2002), but major drawbacks of primary hepatocytes are their limited availability and inherent variability of human material, whereas HBV replication levels in HepaRG are low (Lucifora et al., 2010). Vector-based models were developed using plasmids that encode “greater-thangenome” HBV sequences to overcome the breaking of the HBV genes due to the linearization of viral genome (Brunelle et al., 2005; Chin et al., 2001; Durantel et al., 2004; Jacquard et al., 2006;
Abbreviations: GM, Gunther’s vector-free method; MLSE, Monomer-LinearSticky-Ends-DNA; MCC, Monomer-Circular-Closed. ∗ Corresponding author. Tel.: +39 050996857; fax: +39 050995457. E-mail address: [email protected] (M.R. Brunetto). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.01.012
Junker et al., 1987; Seeger and Mason, 2000; Sells et al., 1987a,b; Summers and Mason, 1982; Warner and Locarnini, 2008; Yaginuma et al., 1987; Yang et al., 2004). Alternatively, recombinant adenovirus or baculovirus carrying the HBV genome could be used (Abdelhamed et al., 2002; Delaney and Isom, 1998; Delaney et al., 1999). However, vector-based methods and baculovirus transfer system, despite their utility to study specific steps of HBV cell cycle, are far away from reproducing the in vivo conditions adequately. In the attempt to develop a vector-free approach and to reproduce the conditions of in vivo HBV infection, Günther et al. (1995) described an original method using the polymerase chain reaction (PCR) amplification of full length HBV-DNA. By this approach not all HBV isolates result replication-competent and intracellular HBV-DNA synthesis is low (Durantel et al., 2004; Villeneuve et al., 2003; Zhu et al., 2007). To overcome the low replicative efficiency, it has been suggested to increase the sensitivity in detecting viral replication by assays such as real-time PCR (Zoulim, 2006). However, such an approach might be misleading by amplifying not only replicative intermediates but also input DNA, as a significant proportion of the input linear double-strand HBVDNA remains intact even after DNAse I digestion (Günther et al., 1995). The present study was aimed to analyze the factors influencing the replication efficiency of the Günther’s method and to develop a new approach able to increase the in vitro replication levels of HBV.
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2. Materials and methods 2.1. Patients A panel of highly viraemic sera (median HBV-DNA 2.43 × 109 cp/ml, range 1.00 × 106 cp/ml to 7.16 × 1011 cp/ml) was obtained from 12 HBeAg positive untreated acute (n = 4) or chronic (n = 8) hepatitis B patients (median age 37 years, range: 18–70; 10 males and 2 females) (Table 1). All the patients were negative for antibodies against hepatitis C virus (HCV), hepatitis D virus (HDV) and human immunodeficiency virus (HIV). 2.2. HBV-DNA isolation HBV-DNA was extracted from serum according to the proteinase K protocol. Briefly 200 l of serum were added to 450 l of mix containing 1 mg/ml proteinase K, 5 mM Tris–HCl (pH 8.5), 2.0% SDS and 25 mM EDTA and incubated at 37 ◦ C overnight. HBV-DNA was precipitated with 1 vol of absolute isopropanol in presence of 20 g of Dextran T 500 and 1/10 vol of 3 M NaAc (pH 4.7). DNA was recovered by centrifugation at 20,000 × g for 15 min; finally pellets were washed with ethanol 70%, dried and resuspended in 50 l of water. 2.3. In house Gunther’s method Full length HBV-DNA was amplified by High Fidelity PCR system (Roche, Mannhein, Germany) with the sense primer FL-S 5 -CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTAATCATC-3 and with antisense primer FL-AS 5 -CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGTG-3 (Parekh et al., 2003). The thermal profile of the amplification was: pre-heating 2.5 min at 94 ◦ C, 10 cycles including denaturation 20 s at 94 ◦ C, annealing 30 s at 55 ◦ C and extension 3 min at 68 ◦ C; 30 cycles including denaturation 20 s at 94 ◦ C, annealing 30 s at 55 ◦ C and extension 3 min (+0.05 ◦ C/cycle) at 68 ◦ C followed by 10 min at 68 ◦ C. After amplification, HBV full length was purified after gel electrophoresis analysis. For transfection of PCR products HBV-DNA was cleaved with 1 U of SapI (New England BioLabs, Ipswich, USA) per g of DNA at 37 ◦ C overnight. Digested DNA was purified by QIAquick PCR purification kit (Qiagen, Hilden, Germany) and then spectrophotometrically quantified. 2.3.1. Optimization of enzyme restriction digestion During the development of the project different digestion profiles of SapI or BspQI (New England BioLabs, Ipswich, USA) were tested: enzyme concentration ranging from 0.5 U/g to 4 U/g and digestion time ranging from 5 h to overnight. The digestion temperature was 37 ◦ C and 50 ◦ C for SapI and BspQI respectively. 2.4. Preparation of Monomer Linear Sticky Ends (MLSE) and Monomer Circular Closed (MCC) HBV-DNA Monomer Linear Sticky Ends DNA (MLSE-DNA) and linear undigested full length HBV-DNA, share the same molecular weight and can not be separated by gel electrophoresis analysis after SapI or BspQI digestion (Fig. 1A). To obtain only replication competent DNA, full length HBV-DNA was cut with HindIII (2 U/g) and SacI (2 U/g) (Fermentas, Erembodegem, Belgium) and cloned into pUC18 vector (Sambrook et al., 1989). pUC18-HBV-DNA was cleaved with 1 U of SapI or BspQI per g of DNA at 37 ◦ C or at 50 ◦ C overnight and then the 3.2 Kb HBV monomer was recovered by gel electrophoresis. To become competent for replication after cell transfection, MLSE DNA requires circularization by the host’s enzyme: this step is crucial for replication, but cannot be monitored in the available in vitro systems. To overcome this limitation and to transfect
Fig. 1. (A) Effect of SapI treatment on electrophoretic mobility of full length HBVDNA. Full length HBV-DNAs, either digested or undigested by SapI or BspQI, migrate at the same molecular weight (3.2 Kb). (B) In vitro MLSE circularization. MLSE was treated by T4 DNA ligase in highly diluted solution to avoid the polymerization and to promote the in vitro monomer circularization.
Monomer Circular Closed HBV-DNA only, MLSE was circularized by T4 DNA ligase (Promega, Madison, USA) at low DNA concentration (5 g/ml) (Cavallone et al., 2010; Hirschman et al., 1980; Sureau et al., 1986; Wang et al., 1982) to obtain monomer circular closed (MCC). Thereafter, MCC-DNA and MLSE-DNA were separated by gel electrophoresis (migration at molecular weight of 2 and 3.2 Kb, respectively, Fig. 1B) and used for transfection after purification and quantification by spectrophotometer analysis. 2.5. Analysis of full length HBV-DNA by sequencing All HBV-DNA forms, that were used in transfection experiment were purified by Exosap (GE Healthcare, Life Science, LittleChalfont, UK) and directly sequenced by chain terminator method using CEQ 8000 Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Krefeld, Germany). Briefly, 2 l of purified template were added to 8.0 l of DTCS Quick Start Master Mix, 4.0 l of
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Table 1 Clinical and virological features of patients. No.
Genotype
Viral load (cp/ml)
HBeAg
Diagnosis
Replication competent (Gunther Sapl phenotypic assay)
Mutations affecting replication
1 2 3 4 5 6 7 8 9 10 11 12
A B C D D F D D D A D D
1.10 × 109 1.22 × 1010 1.00 × 106 2.70 × 109 6.40 × 108 3.09 × 109 2.19 × 1010 7.16 × 1011 2.16 × 109 2.50 × 1010 6.40 × 108 1.86 × 109
POS POS POS POS POS POS POS POS POS POS POS POS
CHB CHB Acute Hep. Acute Hep. CHB CHB CHB Acute Hep. CHB CHB CHB Acute Hep.
Yes Yes Yes Yes Yes No No Yes No No Yes Yes
No No No No No No No No No No No No
1.6 M sequencing primers (Fig. 2) and 6 l H2 O. Thermal cycling profile was 30 cycles of 20 s at 96 ◦ C, 20 s at 50 ◦ C, 4 min at 60 ◦ C followed by holding at 4 ◦ C. Sequencing was carried out using a CEQ 8000 XL analysis System (Beckman Coulter, Krefeld, Germany) and analyzed with Chromas lite 2.01 software. 2.6. Cell culture Human hepatoma HuH7 cells were generously provided by Dr. Fabien Zoulim. Huh7 was grown in Dulbecco’s Modified Eagle Medium (DMEM)-F12 medium (1:1) (Euroclone, Milano, Italy) supplemented with 100 U/ml of penicillin, 100 g/ml of streptomycin, 2 mM L-glutamine, 10 mM Hepes buffer solution, 0.5 mM sodium pyruvate and 10% fetal bovine serum (Gibco, Monza, Italy). The HuH7 cell line was maintained at 37 ◦ C in a humidified incubator at 5% CO2 . HuH7 cell line was regularly passaged and tested with mycoplasma stain kit (Sigma, St. Louis, USA).
with a [32 P]-labeled full length HBV probe and exposed from 10 to 16 h for autoradiography as indicated in the figure legend. 2.9. HBsAg quantification The culture medium was tested for HBsAg using the Architect HBsAg assay (Abbott Laboratories, Abbott Park, IL; dynamic range, 0.05–250.0 IU/ml). Briefly, after incubation of the sample with anti-HBs coated paramagnetic microparticles and washing, an acridinium-labeled anti-HBs conjugate was added. The resulting chemiluminescent reaction was measured in the relative light units (RLUs) and converted in IU/ml of HBsAg. The correlation between HBsAg production and HBV-DNA replication was analyzed using SPSS19 software. 3. Results 3.1. In house performance of the Günther’s method
2.7. Transfection of HBV-DNA HuH7 cells were plated at a density of 1 × 106 cells per well in 60 mm diameter-petri dish 1 day before transfection. The cells were transfected with 3 g of DNA and the transfection was carried out using Fugene reagent (Roche, Mannhein, Germany) according to the manufacturer’s instructions. The medium was changed 1 day after transfection, and cells were harvested at the indicated time points after transfection (from 1 h to 120 h). Each experiment was run in triplicate. Transfection efficiency was measured by cotrasfection of 1 g of reporter plasmid expressing enhanced green fluorescence protein. 2.8. Purification of HBV-DNA from intracellular core particles and Southern blot analysis The cells were washed three times with phosphate-buffered saline (PBS) and lysed in 500 l of lysis buffer [50 mM Tris–HCl (pH 8), 1 mM EDTA, 1% Nonidet P-40] per 60 mm diameter petri dish. The lysed cells were transferred to Eppendorf tubes, vortexed, and maintained in ice for 10 min. Nuclei were pelleted by centrifugation for 1 min at 1000 × g. The supernatant was adjusted to 10 mM MgCl2 and treated with 100 g of DNase I per ml for 1 h at 37 ◦ C. The reaction was stopped by the addition of EDTA to a final concentration of 25 mM. Proteins were digested with 0.5 mg of proteinase K per ml and 1% SDS at 37 ◦ C overnight. Nucleic acids were purified by phenol-chloroform (1:1) extraction and ethanol precipitation after the addition of 20 g of Dextran T500 and 1/10 vol of 3 M NaAc (pH 4.7). Replicative intermediates were then analyzed by electrophoresis in 1.2% agarose gels followed by Southern blotting as described previously (Sells et al., 1987a). Filters were hybridized
Using the Günther’s (GM) method, 8 out of 12 (67%) HBV isolates were replication competent: one (no. 8) with high and seven with low efficiency (Table 1). Mutation, potentially responsible for defective replication were not detected in the full length sequence of the 4 isolates which did not replicate (data not shown). In all isolates direct sequencing (DS) of the catalytic site of SapI showed the presence of the heterologous primers, in spite of the treatment with restriction enzyme. Given the sensitivity limit of DS in detecting a minor viral population, the results indicate that more than 80% of the input DNA was undigested, therefore unable to replicate, because missing the Sticky Ends required for circularization. 3.1.1. Optimization of enzyme digestion The efficiency of digestion was not improved by changing SapI concentrations (from 0.5 U/g to 4 U/g) or digestion time (from 5 h to overnight, data not shown). On the contrary, replacing SapI with the isoenzyme BspQI, high levels of HBV replication were obtained in 1 additional isolate (no. 3). In both the isolates with high replication levels (nos. 3 and 8) DS showed that most of the input DNA was digested. Given the more efficient digestion, BspQI was therefore used in all the following experiments. 3.2. Günther method vs. MLSE-DNA vs. MCC-DNA The HBV-DNA extracted from the serum with the highest viremia (no. 8, Table 1) was used to transfect HuH7 cells to compare the replication performance of the GM, MLSE-DNA and MCC-DNA methods (Fig. 3A). In all the 3 approaches (Fig. 3B), Southern blotting analysis showed a smears (extending from the
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Fig. 2. Amplification and sequencing primers.
position of single-strand HBV-DNA [SS] up to the position of relaxed circular [RC] HBV-DNA) characteristic of HBV replicative intermediates. Kinetic analysis of the intracellular replicative intermediates showed significant differences among the 3 methods (Fig. 3): MCCDNA was not only more efficient than MLSE-DNA and GM methods, but also showed an earlier detection of single strand HBV-DNA in the cytoplasmic HBV core particles. As expected, the untreated BspQI HBV-DNA full length (NRC) was replication defective and Double-Strand Linear HBV-DNA (DSL) was the only detected band (Fig. 3A and B) that resulted from the input DNA. Accordingly, DSL signal decreased progressively during post transfection time and no other HBV replicative intermediates were observed. Extracellular HBsAg was quantified at different post transfection time points and showed increasing levels over time in the three replication competent systems (Fig. 3C), the highest HBsAg levels being produced by MLSE. The untreated BspQI, full length replication defective HBV-DNA, also produced HBsAg although at the lowest levels (Fig. 3C).
The kinetics of single strand (SS) production analyzed by densitometric quantification showed that the transfection by MCC-DNA was associated with an earlier appearance (24 vs. 48 h) and a higher production of the replicative intermediates. In 3 different experiments (each of them run in triplicate) in vitro circularized MCC-DNA increased by 2.51(±0.32) and 6.43 (±0.43) fold the production of SS DNA replicative intermediate, at the 120 h post trasfection time, as compared to the MLSE and Günther’s methods, respectively (Fig. 4). HBV-DNA and HBsAg kinetics correlated positively in all the three replication competent systems (R = 1; p = 0.01) (Fig. 5), but not in the replication defective system (R = 0.354; p = 0.559) (Fig. 5). 3.3. Replication fitness of 12 HBV isolates from HBeAg positive patients by Günther method and MCC-DNA approach The experiments were run in triplicate with comparable results. Eight of the 12 isolates (67%) showed all the HBV replicative
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Fig. 3. Kinetics analysis of HBV replicative intermediates and HBsAg expression. (A) Schematic representation of HBV genome used in the different transfection approaches: Untreated BspQI HBV-DNA full length (replicative defective, NRC) used as negative control; in house Gunther’s method (GM); Monomer Linear Sticky Ends (MLSE); Monomer Circular Closed (MCC). (B) Southern blotting analysis of intracellular replicative intermediates extracted from viral core particles produced in HuH7 cells transfected with different types of full length HBV-DNAs BspQI treated (GM, MLSE, MCC) or untreated (NRC). In the mock control lane (mc) the cells were not transfected. Filter was autoradiographed at −80 ◦ C for 16 h. The position of the relaxed circular (RC), double strand linear (DSL) and single stand (SS) forms of the HBV-DNA genome are indicated Lane M, DNA size markers (Kb). Data shown are representative experiments performed in triplicate. (C) Extracellular HBsAg quantified by Architect (Abbott) at different time points after transfection.
intermediates and were defined replication competent by in house GM method (Fig. 6, panel A): only 2 had high replicative efficiency (Fig. 6, panel A, lanes 3 and 8). The remaining 4 isolates showed only linear double-strand HBV-DNA. By the MCC-DNA approach 10 out of 12 (83%) isolates were replication competent: 7 showed all the replicative intermediates at very high levels (Fig. 6 panel B, lanes: 2, 3, 5, 7, 8, 11 and 12); 3 showed only linear double-strand HBVDNA (Fig. 6 panel B, lanes: 4, 9 and 10). Using the MCC approach the detection of linear double strand HBV-DNA (3.2 Kb) is the hallmark of viral replication, at variance with the GM method where traces of the input 3.2 Kb DSL-DNA can contaminate the results. On the contrary, in MCC the circularized input DNA migrates with a molecular weight of 2 Kb, which differs from that of DSL-DNA. The remaining 2 isolates did not show any band and were defined not replication competent (Fig. 6 panel B, lanes 1 and 6). The cloning step that is required to prepare MCC-DNA may select minor viral populations, therefore the input DNA of all the isolates that were used in the GM and MCC approaches were sequenced by DS and no mutations potentially affecting viral replication were identified (data not shown). When transfection was performed by GM method, HBsAg was produced by all the isolates, independent of the replication competence. In 2 cases (nos. 3 and 11) where HBsAg values were GM
SS DNAcp
1.00E+10
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4. Discussion
1.00E+09 1.00E+08 1.00E+07 1.00E+06 24
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96
low in spite of medium–high replication levels, DS analysis of the input DNA showed the presence of mixed wild-type and mutant sequences in the small S gene. The 2 mutations (TGG to TAG switch at codon s201 and TTA to TAA switch at codon s216) created a stop codon in the S proteins, without affecting the overlapping polymerase gene. By MCC method HBsAg was not produced by 2 isolates (nos. 6 and 11): one (no. 6) was replicative defective, the other (no. 11) was replication competent, but DS of the input DNA showed the presence of the s216 stop codon, possibly selected by the cloning step. The SS DNA and HBsAg levels across the different isolates (excluding no. 11 for the reasons explained above) showed a positive correlation in the MCC method (Spearman correlation: R = 0.696; p = 0.017). On the contrary, a negative correlation was observed in the Günther method, where higher HBsAg levels were found in isolates with lower levels of replication (Spearman correlation: R = −0.437; p = 0.179). To avoid possible interference due to the variability in the ratio between viral replication and antigen expression in the different genotypes, a sub-analysis was run in genotype D isolates only. In genotype D isolates the correlation between HBsAg levels and viral replication was even higher in the MCC system (Spearman correlation: R = 0.928; p = 0.008), but not in GM method (Spearman correlation: R = −0.395; p = 0.439).
120
post transfection time (h)
Fig. 4. Kinetics of single strand HBV-DNA production by the 3 different approaches. The intensity of SS DNA band was measured by densitometric analysis and then a reference HBV marker was used to convert the intensity value in copies (cp).
The present study investigated the reasons for the low replication efficiency of the Günther’s method and found that a major limiting step is the low proportion (<20%) of SapI digested DNA in the DNA used for transfection. The DS showed that the majority of the input DNA is not replication competent, since the continuity of the Basic Core Promoter (BCP) and Pre-Core (PC) regions is disrupted by the presence of the primers tails, that prevent DNA circularization. In the attempt to optimize the digestion conditions, SapI was replaced by BspQI enzyme, but replication was improved only in 2 of the 8 replication competent isolates. DS analysis showed that the 2 genomes with high replication efficiency
D. Cavallone et al. / Journal of Virological Methods 189 (2013) 110–117
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Post Transfection time (h) 24 48 96 0.0 15.3 65.5 153.5 0.0E+00 0.0E+00 0.0E+00 0.0E+00
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Fig. 5. Correlation between HBsAg production (•) and intrahepatic HBV replicative intermediates (o) in the 4 transfection conditions: (A) NRC, (B) GM, (C) MLSE, and (D) MCC. A positive correlation between HBV RI and HBsAg production could be observed in panels B, C and D but not in the replication defective system (panel A). All the values showed in the graphics are reported in panel E.
(Fig. 6A, patients number 3 and 8) were those more efficiently digested by BspQI enzyme. On the contrary, heterologous primer sequences were detected as prevalent population in all the other genomes with lower or absent replication. The evidence that the input DNA in the Günther’s method results from a mixture of digested and undigested full length HBV-DNA identifies a major confounding factor for the in vitro study of viral replication, whose efficiency appears to be influenced not only by viral features but also by technical artifacts. Accordingly, sequencing the full length of all genomes excluded that mutations affecting viral replication could have been eventually introduced by PCR amplification. Probably the use of High Fidelity Taq DNA polymerase could explain our results: accordingly, Günther reported that the frequency of the mutations decreased significantly when Taq DNA polymerase without proofreading activity was substituted by an enzyme with proofreading activity (High Fidelity) (Günther et al., 1998). In the attempt to improve further in vitro HBV replication the method was modified by transfecting, after cloning, pure replicative competent DNA either as Monomer Linear Sticky Ends DNA
(MLSE method), which still requires intracellular circularization, or Monomer Circular Closed DNA (MCC method), which is competent for replication immediately after transfection (Cavallone et al., 2010). Accordingly, by the latter approach (MCC method) HBV replicative intermediates were detected earlier than with Günther’s or MLSE methods (24 vs. 48 h) (Fig. 3B). In addition, the transfection with MLSE DNA or MCC DNA was two and six times more efficient in terms of viral replication than the original Günther’s method (Fig. 4). Similar results were recently reported by Qin et al. (2011) who used a similar approach. Using the MCC method very high level of replicative intermediates were produced by 7 of 12 HBV isolates (Fig. 6B), whereas 3 isolates showed only linear double-strand HBV-DNA (Fig. 6B, nos. 4, 9 and 10). Nevertheless, in the setting of MCC method the detection of linear double strand HBV-DNA (3.2 Kb) has to be considered a true expression of viral replication. In fact, at variance with Günther’s method where traces of the input 3.2 Kb DSL-DNA can contaminate the results, in the case of the MCC approach the circularized input DNA migrates with a molecular weight of 2 Kb.
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Fig. 6. Analysis of the replication fitness of 12 isolates from HBeAg positive patients by the in house Gunther’s method (A) and MCC DNA method (B). HuH7 cells transfected with 3 g of HBV-DNA full length isolated from patients (referred as number 1–12) by GM (A) or of MCC method (B): lanes correspond to DNA extracted from viral core particles derived from cell lysates. The figure shows a representative result of experiments performed in triplicate. In the mock control lane (mc) the cells were not transfected. Lane M, DNA size markers (Kb) Filters were autoradiographed at −80 ◦ C for 10 h.
Accordingly, when an isolate is unable to replicate in vitro with the MCC method no background band was observed (Fig. 6B, patients 1 and 6). Furthermore, in all 3 isolates where only DSL-DNA was present at Southern blotting, HBsAg was detected in the medium (Fig. 6B, nos. 4, 9 and 10), proving the presence of viral replication because in this setting HBsAg production results exclusively from viral replication. At variance with Gunther’s method, where linear undigested full length HBV-DNA, that is not replication competent but represents a significant proportion of input DNA, may produce HBsAg independently from HBV replication (Fig. 3C). Indeed, the mixture of different (digested and undigested) DNA forms could be responsible for the asymmetry between viral replication and HBsAg production that leads in the GM method to the inverse correlation between HBsAg levels and replicative intermediates, when different isolates are compared. By MCC method the HBsAg secretion showed a significant correlation with HBV replication particularly when the analysis was restricted to genotype D isolates, to avoid possible interference due to the variability in the ratio between viral replication and antigen expression that was described in the different genotypes (Sugiyama et al., 2006). Overall these findings suggest that the MCC method consistently reproduces the in vivo conditions, representing a reliable approach for the in vitro study of HBV biology. Particularly, the MCC method appears adequate for a precise analysis of the correlation between viral replication and HBsAg production, because only circularized HBV-DNA is transfected. The in vivo complexity of the interplay between viral replication and HBsAg production was underlined in the recent years by several clinical studies, suggesting the possible interference of both viral (genotype) and host (immune competence) factors (Brunetto, 2010; Brunetto et al., 2010). The possibility to study specifically the biological features of each HBV isolate, without loosing the chance of the population approach by creating a clone pool could warrant a better understanding of the virologic
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