Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines

Journal of Virological Methods 234 (2016) 96–100

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Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines Yong Wu a,b,1 , Tian-Ying Zhang a,c,1 , Lin-lin Fang a,b , Zi-Xuan Chen d , Liu-Wei Song a,c , Jia-Li Cao a,c , Lin Yang a,b , Quan Yuan a,b,∗ , Ning-Shao Xia a,b a

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China c School of Life Science, Xiamen University, Xiamen 361102, China d Affiliated Quanzhou First Hospital by Fujian Medical University, Fujian, China b

a b s t r a c t Article history: Received 12 March 2016 Received in revised form 14 April 2016 Accepted 15 April 2016 Keywords: Hepatitis B virus Sleeping Beauty transposon Stable cell line

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A–D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. © 2016 Published by Elsevier B.V.

1. Introduction Chronic human hepatitis B virus (HBV) infection is a global public health problem(Schweitzer et al., 2015), which is a leading cause of severe liver disease such as hepatitis, cirrhosis, hepatocellular carcinoma (HCC) and other hepatic pathological changes. Over 240 million people worldwide are chronically infected with hepatitis B virus and about 600,000 patients die from HBV-relative liver diseases annually (Liaw, 2009; Schweitzer et al., 2015), (Ott et al., 2012). Current approved anti-HBV drugs, which are either interferon or nucleos(t)ide analogues, can only induce disease remission, but not eradicate the virus (European Associationfor the Study of

∗ Corresponding author at: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Xiamen University, Xiamen 361102, China. E-mail address: [email protected] (Q. Yuan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jviromet.2016.04.010 0166-0934/© 2016 Published by Elsevier B.V.

the Liver. Electronic address, 2016; Niederau, 2014). Therefore, the development of more effective new anti-HBV agents is urgently needed to improve the clinical management this disease. A robust in vitro system for evaluating the activity of new agents against HBV is an important tool for new drug development. Currently, transient transfection of human hepatoma cell lines (Huh7 and/or HepG2) with replication-competent HBV genome containing plasmids is the most commonly used system to analyze HBV replication and antiviral effects (Lentz and Loeb, 2010). However, HBV-replicating stable cell lines, which carry replicationcompetent HBV genome stably integrated into the genome of host cell, provide several advantages for applications requiring standardized conditions: essentially all cells are stably HBV productive, minimal experimental variation and improved reproducibility (Sun and Nassal, 2006). Two such cell lines, HepG2.2.15 and HepaAD38(Ladner et al., 1997; Sells et al., 1987) are widely used in antiviral research. The HBV genomes which were integrated in the two cell lines are both genotype D. There is a need to develop

Y. Wu et al. / Journal of Virological Methods 234 (2016) 96–100

cell lines in particular for HBV genotypes A–C for drug development and test antivriral activities. The Sleeping Beauty (SB) transposon is a recently developed non-viral vector that can mediate high-efficient integration of transgenes into the mammalian genome. The SB transposon can be viewed as natural gene delivery vehicles that integrate target gene into the host genome through a ‘cut-and-paste’ mechanism in the presence of transposase (Ivics and Izsvak, 2011). Since its reconstruction in 1997 from Tc1/mariner elements resident in the salmonid fish genome (Ivics et al., 1997), the SB system has been subjected to several modifications aimed at improving its efficacy (Baus et al., 2005; Geurts et al., 2003; Yant et al., 2004; Zayed et al., 2004). The development of the hyperactive transposase SB100X has an approximate 100-fold improved efficiency compared with the original SB10 transposase (Mates et al., 2009). These mobile DNA elements encode a transposase flanked by inverted terminal repeats (ITRs) that contain the transposase binding sites necessary for transposition. In this study, we investigated the feasibility to generate HBVreplicating stable cell lines by Sleeping Beauty transposon system. A new all-in-one vector was developed for robust generation of HBV stable cell lines. Using this system, we successfully constructed and characterized four cell lines carrying replication-competent HBV genome of genotype A–D. 2. Materials and methods 2.1. Plasmids The encoding sequence of SB100X and SV40 ployA were obtained by PCR reaction using the pCMV(CAT)T7-SB100 plasmid (addgene #34879). The PGK promoter and the PCR product of SB100X-SV40ployA were inserted into KpnI–SalI-digestedpT2HB vector (addgene #26557) to generate a SB100X-expression cassettes on a basic SB transposon vector (denoted pTS vector). Subsequently, an expression cassette (SV40p-mCherry-2A-PuroRBGHployA), which contained the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR), were inserted into EcoRI–BglIIdigested pTS vector to generate the pTSMP vector. The HBV genome construct used in this study was 1.3-copy length of the HBV genome, shared a common 5 terminal starting at nt970and 3 terminal ending at nt2043, which includes Enh I and II, the Xand pregenomic/core promoter regions, the origin of replication (DR I and II) and the entire polyadenylation site. A total of four HBV 1.3-copy genome, including genotype Ae (AY707087), genotype Ba (GU357842), Genotype Ce (GU357845) and genotype D1 (GU357846), were inserted into EcoRI–EcoRV-digested pTSMP vector. All ligations of DNA fragments into the vector in this study were performed by using the method of “Gibson Assembly” (Gibson et al., 2009). All plasmid DNAs for transfection were purified from bacteria using a Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany). 2.2. Cells Human hepatoma HepG2 cells (Originally from the China Centre for Type Culture Collection, Wuhan, China) were maintained in minimum essential medium (MEM) with 10% fetal bovine serum under 5% CO2 incubation at 37 ◦ C. HepG2 cells were seeded into100-mm-diameter culture dishes. Plasmid transfections were performed with X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany), according to the manufacturer’s instructions. Forty-eight hours after transfection, cells with activated red fluorescence (mCherry positive) were sorted out by BD FACS Aria III (BD Bioscience). The sorted mCherry-expressing cells

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were cultured in 10 cm diameter culture dish in the presence of puromycin selection (2 ␮g/ml, Invitrogen, Carlsbad, CA, USA). Fresh culture medium containing puromycin was replaced every 2 days. On the following day, when cell confluence was 70–80%, the cells were subjected to a second round of red fluorescence-activated FACS sorting and further puromycin selection. After about a 4round of red fluorescence-activated FACS sorting and puromycin resistant cell selection, over 90% cells were puromycin resistant and stably expressed mCherry. 2.3. Virological analyses Viral antigens (HBsAg and HBeAg) in culture medium were measured by chemiluminescence method using commercial assay kits (Wantai, Beijing, China). HBV DNA quantification assays were performed using a commercial real-time PCR kit (Kehua, Shanghai, China). Southern blot analyses for intracellular HBV DNA were performed using a DIG-labeled DNA fragment encompassing the HBx genome according to a previous study (Huang et al., 2012). Immunofluorescence analysis was performed according to standard procedures (Lempp et al., 2016). Briefly, pTSMP-HBV1.3 cells were seeded on 24-well chamber slides a day before detection. The cells were fixed in the dark with 4% paraformaldehyde at room temperature and permeabilized in 0.1% Triton X-100. The cells were then blocked with 2% BSA diluted in PBS at 37 ◦ C. Then the indirect immunofluorescence assay was performed using a fluorescence kit. Mouse anti-HBcAg (WD® Anti-HBc) purchased from Inovax Biotechnology (Beijing, China), while fluorescein isothiocyanate (FITC)-tagged donkey anti-mouse immunoglobulin G (IgG, H + L) purchased from Life technologies. After mounting in antifade reagent (SlowFade® Gold antifade reagent, Life technologies), cells were examined by an inverted fluorescence microscope (Olympus, Tokyo, Japan). The parental HepG2 cells served as negative control. 3. Results 3.1. Construction of the pTSMP vector and the pTSMP-HBV1.3 plasmids To construct an all-in-one Sleeping Beauty transposon vector, we generated the pTSMP which contained a SV40-mCherry-2A-PuroRBGHployA cassette and multiple cloning sites (MCS), situated in a pT2 SB transposon flanked left inverted repeat and right inverted repeat. In addition, a PGK promoter-driven SB100X hyperactive transposase cassette (PGK-SB100X-SV40ployA) was placed on the outside of the transposon in the same plasmid. The cis expression of transposase allowed effective mediation of gene transfer in cells. The cassette of SV40-mCherry-2A-PuroR-BGHployA, in which the PuroR was linked with mCherry by a “self-cleaving” P2A peptide was driven by a single SV40 promoter, which resulted in the expression of the two independent proteins from a single transcription event. This strategy allowed selection of transfected cells by antibiotic screening and/or red fluorescence-activated cell sorting. The constructs of 1.3-genome-length HBV DNA of genotype Ae, Ba, Ce and D1, which were able to produce replicative HBV, were cloned into the MCS to generate pTSMP-HBV1.3 plasmids with four different genotypes. The map of pTSMP-HBV1.3 plasmid and the genome organization of HBV1.3 used in this study are shown in Fig. 1 A and B, respectively. 3.2. Generation and characterization of HBV-replicating stable cell lines The pTSMP-HBV1.3 plasmids were transfected into HepG2 cells. Forty-eight hours after transfection, successful transfected cells with activated red fluorescence (mCherry positive) were selected

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A

nt2043

nt3215/1

EcRV SV40p

HBV1.3 Genome

mCherry 2A

pTsmP-HBV1.3

nt970

Puro BGHpA

11.5kb

EcRI IR/DR

IR/DR PGKp

F1 Origin

SB100X Amp R

B

ColE Origin

EnhI/Px EnhI/Px

pgRNA

pre

Core

L

M+S mRNA

preS1

S2

preS1

Polymerase

nt970

nt3215/1

Poly A

X

X pre

Core

nt2043

HBV1.3 Genome

Fig. 1. Schematic representation of the pTSMP vector (A) and HBV 1.3 genome construction (B).

Fig. 2. Generation of HBV cell lines of various genotype. (A) An illustration of mCherry-activated FACS sorting; (B) Immunofluorescence for HBcAg of HBV cell lines.

by FACS and were seeded into a new culture dish for expansion in the presence of puromycin. The percentage of mCherry-activated cells increased from 26.1% to 44.6% after initial puromycin selection (Fig. 2A). Further antibiotic selection and FACS sorting significantly increased the percentage of mCherry-activated among cell population. After four rounds of screening, >90% of cells were puromycin resistant and stably expressed mCherry. In our experience, the screening procedure could be completed within 3–4 weeks. According to the procedures described above, a total of 4 stable cell lines transfected with pTSMP-HBV1.3 plasmids of genotype Ae, Ba, Ce and D1 were generated.

These cells were further propagated for characterization. After 10 passages, we found nearly 100% of cells of the 4HBV cell lines persistently expressed mCherry and grew well in the presence of puromycin. Immunofluorescence analyses revealed almost 100% of these cells stained positive for HBcAg (Fig. 2B). As shown by southern blot hybridization (Fig. 3A), all 4 cell lines produced the expected and similar pattern of relaxed circular (RC) DNA and single-stranded (SS) DNA, however, the HBV1.3-B cells yielded significant higher levels of HBV DNA than the others. To further evaluate the levels of viral antigens and HBV DNA in culture media, these cells were maintained in 24-well plates. The culture medium were replenished and collected for measurement of viral antigens

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Fig. 3. The replication and viral protein expression profiles of four pTSMP-HBV derived cell lines. (A) Southern blotting analysis for HBV replicative intermediates in HBV cell lines. The dynamic change of HBsAg (B), HBeAg (C) and HBV DNA (D) of four HBV cell lines.

and HBV DNA every 2 days. All cells had produced detectable HBsAg, HBeAg and HBV DNA in their culture media (Fig. 3B–D). The longitudinal changes of the levels of these viral markers in supernatants for each cell line were minimal during 1-month monitoring, however, the average levels of these viral markers in supernatants varied in different cell lines. The HBV1.3-C cell line produced the highest extracellular HBsAg (Fig. 3B), with an average level of 71.5 ± 1.0 IU/mL, which was about 2-fold higher than that of HBV1.3-B (33.7 ± 0.7 IU/mL) and HBV1.3-D (45.2 ± 0.6 IU/mL), and was about 12-fold higher than that of HBV1.3-A (6.1 ± 0.1 IU/mL). With regard to the extracellular HBeAg (Fig. 3C), the cells of HBV1.3B and HBV1.3-D exhibited similar secretion of HBeAg, at a level which was about 3-fold higher than that of HBV1.3-A and HBV1.3C. Consistent with the intracellular HBV DNA levels derived from southern blot hybridization, the HBV1.3-B cells presented the highest HBV DNA level (about 6 × 106 IU/mL, averagely) in supernatants, which was about 2–3 fold higher than that of other cell lines.

4. Discussion With regard to produce HBV-replicating stable cell lines, previous reported strategies depended on random integration of foreign DNA containing replication-competent HBV genomes via cell transfection, which are less-efficient and time-consuming (Fu and Cheng, 2000; Ladner et al., 1997; Sells et al., 1987; Sun and Nassal, 2006; Zhang et al., 2014). The HepG2.2.15 cell was developed by transfection of a plasmid containing two head-to-tail dimers of the HBV genome and a neomycin resistance gene (NeoR) expression cassette, followed by selection of cell clone producing HBV from G418 resistant cell clones (Sells et al., 1987). The HepaAD38 cell line, which contains a 1.1 unit length HBV genome

under the control of a tetracycline regulatory promoter, was also generated by the same strategy. However, the process of random integration of foreign DNA is characterized by a low efficiency, usually with less than 1% of successfully transfected cells (Ladner et al., 1997). More convenient and efficient systems to construct HBVreplicating stable cells are certainly required both for virological study and new antiviral agent screening. Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression both in vitro and in vivo (Li et al., 2013). The Sleeping Beauty transposon system is the first successful nonviral transfer vector for integration of a gene cassette into host cell genome (Izsvak et al., 2009; Kowarz, 2015). The newly developed SB100X hyperactive transposase greatly improved stable gene transfer efficiencies that compare favorably to stable transduction efficiencies with integrating viral vectors, such as Lentiviral vectors (Matrai et al., 2010; Sakuma et al., 2012). Therefore, we developed a new system for robust generation of stable cell lines carrying replication-competent HBV genome based on SB transposon with the hyperactive SB100X transposase. In our new system, cis-expression of SB100X with transposon cassette in a single plasmid was utilized aiming to improve the transposition efficiency. It has been demonstrated that the ‘all-in-one’ cis vector performed better than the dual plasmid system for efficient gene delivery and persistent gene expression (Kabadi et al., 2014; Liang et al., 2015). To avoid potential inhibitory effect derived from overproduction of transposase, we chose to produce the transposase by the human phosphorglycerate kinase 1 (PGK) promoter, which has moderate promoter strength and had been demonstrated to present improved transgene expression in previous studies (Mizushima and Nagata, 1990). To improve

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the screening efficiency, we introduced two selectable markers, mCherry and PuroR, into this system. The dual-marker cassette enables rapid identification and enrichment of cells carrying insertions through red fluorescence-activated cell sorting and the use of PuroR conferring resistance to puromycin. Using this system, we successfully constructed four cell lines carrying HBV 1.3 genomes of genotype A–D. By combined use of red fluorescence-activated cell sorting and puromycin resistant cell selection, stably transfected cells could rapidly obtained with 4-week. More importantly, our results showed that all of the four cell lines could persistently produce viral proteins and HBV DNA at a relatively stable level. In summary, our data demonstrated the feasibility of the utilization of optimized Sleeping Beauty transposon for robust generation of HBV-replicating stable cell lines. The new system and the cell lines developed in this study should provide valuable tools to investigate in vitro virological phenotypes of various clinical HBV isolates, as well as to screening new antiviral agents. Author’s contributions Y. Wu, T.-Y. Zhang, Q. Yuan and N.-S. Xia designed this study; Y. Wu, L.-L. Fang, Z.-X. Chen, J.-L. Cao, L.-W. Song, L. Yang performed lab work; Y. Wu and L.-L. Fang participated in data analysis; Q. Yuan and T.-Y. Zhang wrote the main manuscript text and prepared figures; Q. Yuan and N.-S. Xia critically reviewed the manuscript. All the authors read and approved the final manuscript. Conflicts of interest The authors declare no competing interests. Funding National Science Fund (81371819) and the Excellent Youth Foundation of Fujian Scientific Committee (2015J06018) supported this work. References Baus, J., Liu, L., Heggestad, A.D., Sanz, S., Fletcher, B.S., 2005. Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol. Ther. 12, 1148–1156. European Association, 2016. European Associationfor the Study of the Liver. Electronic address, e.e.e. EASL Clinical Practice Guidelines: Vascular diseases of the liver, Journal of hepatology 64, 179–202. Fu, L., Cheng, Y.C., 2000. Characterization of novel human hepatoma cell lines with stable hepatitis B virus secretion for evaluating new compounds against lamivudine- and penciclovir-resistant virus. Antimicrob. Agents Chemother. 44, 3402–3407. Geurts, A.M., Yang, Y., Clark, K.J., Liu, G., Cui, Z., Dupuy, A.J., Bell, J.B., Largaespada, D.A., Hackett, P.B., 2003. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol. Ther. 8, 108–117. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison 3rd, C.A., Smith, H.O., 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345. Huang, C.H., Yuan, Q., Chen, P.J., Zhang, Y.L., Chen, C.R., Zheng, Q.B., Yeh, S.H., Yu, H., Xue, Y., Chen, Y.X., Liu, P.G., Ge, S.X., Zhang, J., Xia, N.S., 2012. Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors. J. Hepatol. 57, 720–729.

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