Immunology and Infection
Published: November 7th, 2018
We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1–2 copies) integration using inverse nested PCR.
Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.
HBV is a double-stranded DNA virus that can cause life-long chronic infection, leading to liver cirrhosis and hepatocellular carcinoma (HCC)1,2,3. While there are multiple molecular mechanisms driving HBV persistence4 (e.g., high stability of the epigenetic viral transcriptional template, evasion of immune surveillance, and low turnover of hepatocytes in the liver) and its associated risk of HCC initiation5,6 (e.g., chronic inflammation and activation of cellular stress pathways), HBV DNA integration into the host cell genome (a reported mechanism for both of these phenomena) has been poorly studied. A major reason is the lack of suitable in vitro infection systems for HBV that allow reliable detection of integration events. Here, we describe a recently-developed protocol for both in vitro generation and detection of HBV DNA integrations that can be used to elucidate the underlying molecular mechanisms and consequences thereof.
HBV replication and the formation of HBV DNA integration has been previously reviewed in detail7. Briefly, HBV enters hepatocytes using the Sodium Taurocholate Co-transporting Peptide (NTCP) as the main cellular receptor for infection8,9. The HBV nucleocapsids containing the HBV-relaxed circular DNA (rcDNA) genome enters the nucleus, where the rcDNA is converted into episomal covalently closed circular DNA (cccDNA). The nuclear cccDNA acts as the transcriptional template for viral mRNAs and pre-genomic RNA (pgRNA)10. HBV polymerase and pgRNA are then packaged into newly-formed nucleocapsids (composed of HBV core protein dimers). HBV pgRNA is reverse-transcribed within the nucleocapsid, resulting in either an rcDNA genome or a double-stranded linear DNA (dslDNA) genome11,12. These mature nucleocapsids containing HBV DNA genomes are finally enveloped and exported as virions.
Infection of hepatocytes by enveloped particles containing dslDNA molecules can result in viral integration into the host cell genome13, leading to replication-incompetent forms of HBV DNA7,14,15. HBV DNA integration occurs at the site of chromosomal double-stranded DNA breaks15. Accumulating evidence suggests that each integration event occurs in an essentially random position within the host cell genome16,17. In addition, HBV DNA integration occurs somewhat rarely, at a rate of 1 per 104 cells13,18,19,20. Important questions regarding HBV DNA integration remain unanswered, particularly regarding the exact molecular pathways involved, the dependence on viral and host factors, the viral antigens expressed from integrated forms, and their possible contribution to viral persistence7. We have set up an in vitro model to shed light on some of these questions.
The rarity of HBV DNA integration events (both with respect to integration rate per cell and to the copy number of each unique integration) in in vitro HBV infection models make them challenging to detect. Cell mitosis is limited in our in vitro system, as dividing cells do not support efficient infection. Thus, unlike in patient liver tissues where significant clonal expansion of hepatocytes occurs18,19,20, very few (1 to 2) copies of each integration are present in a given pool of cells infected in vitro. We have also found that integration mainly occurs during the initial infection of hepatocytes (and not continuously in chronically-infected hepatocytes)13. Accordingly, HBV DNA integration cannot be increased by simply culturing cells for a longer period.
In general, methods previously used to detect integrated HBV DNA, including Southern blot hybridization21,22,23,24,25, Alu-PCR26,27, cassette-mediated ligation PCR28, and whole genome sequencing29,30,31,32,33, do not have the sensitivity to detect single-copies of integrations. We and other researchers have used inverse nested PCR (invPCR) to detect hepadnaviral DNA integration in duck, woodchuck, and human infected livers13,14,18,19,34,35,36. Others have introduced procedural modifications to the invPCR technique that may alter the generation of false-positive signals and restrict the ability for quantification37. If the assay is carried out as described in this protocol, invPCR represents a specific and sensitive assay that identifies and quantifies (in absolute copy numbers) multiple HBV DNA integrations. The HBV-cell DNA junction is sequenced at a single-base pair resolution, allowing bioinformatical studies of viral and host DNA sequences at the sites of integration13.
We have previously described38 results from invPCR on DNA of HBV-infected tissue extracted from a large range of sources, including snap-frozen liver tissues, paraffin-embedded liver sections, and extremely small tissue samples isolated by laser-microdissection19. This protocol outlines an updated version of an invPCR assay using DNA extracted from cell culture-derived tissues after in vitro infection, in which low copy numbers of integrations (1–2 copies per integration) are generated. The HBV DNA integrations formed in vitro resemble those found in patient tissues with respect to their distribution over the cellular genome and junction within the viral sequence that is integrated13,16, suggesting a comparable pathway to within an infected liver.
1. Cell Infection and DNA Extraction
2. Inversion of DNA
3. Nested PCR
4. PCR Product Isolation and Gel Extraction
5. Sequence Analysis
A schematic representation of the invPCR technique is shown in Figure 1. An example agarose gel electrophoresis of a successful invPCR is shown in Figure 2 before and after the PCR product isolation. Figure 3 shows the BLAST analysis output from step 5.1 in the cases of (i) amplification of virus-cell DNA junction and (ii) amplification of an HBV DNA replicative intermediate.
Expected results from positive controls: Huh7-NTCP cells infected with heparin-purified HBV stocks as described above (Figure 1) can serve as a positive control. In this instance, single PCR products should be achieved by the second or third 1:3 dilution of each sample (corresponding to ~104 cell equivalents per dilution, Figure 2). At these dilutions, approximately 50% of products will represent true virus-cell DNA junctions, while the other half represents amplification of HBV DNA intermediates (Figure 3).
In previous studies13, we have found few instances of repeated virus-cell junctions, suggesting no cellular genomic sites of preferential HBV DNA integration. We also find that >80% of virus-cell junctions occur between nucleotides 1732 and 1832 (according to the nucleotide numbering of HBV genotype D, GenBank Accession #U95551.1), where the latter represents the right-hand terminus of the HBV dslDNA form. Sequences of true virus-cell junctions found through our method in in vitro experiments are publicly available on GenBank (Accession numbers MH057851 to MH058006).
Expected results from negative controls: Either uninfected Huh7-NTCP cells or inoculated Huh7-NTCP cells pre-treated with Myrcludex B can act as negative controls. InvPCR analysis of DNA extracted from these cells should produce no PCR products. Running these negative-control samples on the same PCR plate as positive samples allows for testing of cross-contamination during the nested-PCR process. The most stringent test for cross-contamination is the running of a negative-control sample in wells A-D and the positive sample in wells E-H on a 96-well plate. In this case, the most concentrated dilution of the positive sample is run in a row directly adjacent to the least concentrated dilution of the negative sample. If amplification products are observed in negative-control samples, institute the measures suggested in the discussion to minimize cross-contamination events.
Figure 1: Schematic figure of the invPCR process to detect HBV DNA integrations. After HBV infection, only a minority of Huh7-NTCP cells contain HBV dslDNA (red) integrated into the host cell genome (red), depicted in the top section. Total DNA is then extracted from the cells (step 1), inverted (step 2), and amplified by nested PCR (step 3). Shown are the relative positions of the restriction enzyme sites (NcoI and BsiHKAI) in the excised virus-cell DNA junction. The figure is adapted from a previous publication13. Please click here to view a larger version of this figure.
Figure 2: Agarose gel electrophoresis and subsequent gel extraction of a successful invPCR. "M" represents DNA marker ladder. Each row of 12 represents technical replicates at a single dilution on the PCR plate. Two inverted DNA samples can be run per PCR plate/agarose gel. The PCR products for each dilution should titrate out in a ~1:3 ratio. Extraction of the products from the two least concentrated dilutions where single bands can be easily isolated is generally sufficient, as HBV DNA integration rate is determined by end-point titration. Please click here to view a larger version of this figure.
Figure 3: BLAST analysis of invPCR product sequences. As long tracts of sequence are generated from Sanger sequencing, the source of DNA sequences is generally unambiguous. Strong alignment to HBV and cellular genomes is observed in different areas of the FASTA sequence file generated from Sanger sequencing in the top panel, suggesting a true virus-cell DNA junction. In the bottom panel, the sequence aligns only to fragments of the HBV genome, suggesting a product generated by the amplification of a rearranged HBV DNA genome. Please click here to view a larger version of this figure.
Before performing the protocol, it is important to note that this invPCR assay is a highly sensitive technique, capable of amplifying single copies of DNA template. Therefore, limiting contamination from PCR products is of utmost importance. General strategies to limit PCR product contamination include the following. (i) Establish physically separate areas for different steps of the method. Each area should have separate lab coats, and gloves should be changed when moving between these areas. We have listed these areas below in order from most to least likely to be contaminated: PCR product extraction and sequencing reaction set-up area (post-PCR); PCR template addition and flamed-pin transfer area (we have used PCR hoods with a decontaminating UV lamp with good results); DNA extraction and inversion area (pre-PCR); and a "DNA template-free" area used solely for preparing stock and PCR solutions. (ii) Be aware of air flow in the lab as a potential driver of cross-contamination. In particular, cross-contamination of PCR reactions during the flamed pin transfer step is most likely to occur and will lead to inaccurate quantification of integration frequency. PCR hoods can be used to limit these cross-currents. Negative control wells (e.g., Myrcludex B-treated samples or no template controls) in the PCR can be also used to test for cross-contamination. (iii) Limit potential PCR contaminants on pipettes and work surfaces by regularly wiping them down with a DNA degradation solution.
The protocol specified here is arranged to detect integration of a known infectious HBV clone generated from the HepAD38 cell line42. If the inoculum used is of a different HBV DNA sequence (e.g., from patient serum), then the HBV genome should be first sequenced to confirm compatibility of PCR primers and inversion design. In previously published studies19, we have used 3 sets of primers that bind conserved HBV DNA sequences flanking the expected site of HBV DNA integration junctions. Other primer sequences and protocols have been described to determine the genomic sequence of HBV44,45,46,47,48 and may work successfully.
Furthermore, different HBV-susceptible cells (e.g., primary human hepatocytes, differentiated HepaRG, HepG2-NTCP cells) can be used; however, we have found that Huh7-NTCP cells provide the greatest signal-to-noise ratio, when considering the number of virus-cell DNA junctions that are amplified compared to the virus intermediate DNA that is amplified13. In particular, terminally differentiated cells such as primary human hepatocytes or differentiated HepaRG do not efficiently undergo mitosis, resulting in a high level of amplifiable HBV DNA replicative intermediates remaining within the cells. We have found that ~90% of amplified sequences represent HBV DNA rearrangements (and not integration events) in terminally-differentiated cells, compared to 70–50% in hepatoma cell lines13. These products are generally HBV DNA genomes containing large deletions in the surface and core open reading frames, or they can represent HBV quasi-species with an additional NcoI site prior to the SphI site. The amplification of these HBV species (including a schematic diagram) have been described in detail previously13.
There are several drawbacks to this method. Due to the laborious and multi-day nature of this protocol, invPCR is not suited to high-throughput analyses of a large number of samples. Furthermore, as it relies on limiting dilution titration, our method is not highly precise in quantification; although, it should easily measure log-level changes in integration frequency.
Moreover, our inversion protocol is only suited to detect integrations occurring between the DR2 and DR1 region of the HBV genome, as the majority of HBV integrations occur within this region49. NGS analysis of HBV patient tissues has shown that a large minority (up to ~50%) may also occur outside of this region49. New invPCR designs are theoretically able to detect these other integration sites, though they have not (to our knowledge) been carried out yet. Relatedly, due to the necessary restriction enzyme sites required downstream from the virus-cell junction sequence for the inversion reaction, invPCR does not detect all integrations occurring within the DR2 and DR1 regions of the HBV genome (i.e., we have estimated that ~10% of all integrations are detectable using in silico simulations13). However, when applied to a focused batch of samples with small numbers of different treatments, invPCR is one of the only practical methods for detecting integrated HBV DNA at a single base-pair resolution.
We therefore envision applications of this method serving a key role in finding viral (via mutations of the HBV inoculum), cellular (through knockout or overexpression of specific cellular genes, or through application of various drugs), and environmental (e.g., exposure to oxidative stress) factors that induce HBV DNA integration. With this method and newly developed HBV infection systems, we enable unprecedented control of these factors so that they can be well-isolated and better characterized. We also expect that this will lead to a key understanding of the cellular consequences of HBV DNA integration, including to what extent viral antigens (e.g., HBx or HBsAg50) are expressed from the integrated form, what controls this expression, and whether or not HBV integration significantly changes the cellular phenotype towards a more pro-oncogenic state. The results of these future studies will have a profound impact on the therapeutic strategies used to treat chronic hepatitis B and on the basic understanding of the virus itself.
S.U. is a co-applicant and co-inventor of patents protecting Myrcludex B as an HBV/HDV entry inhibitor. T.T. declares no conflicts of interest.
This work received funding from the German Centre for Infection Research (DZIF), TTU Hepatitis Projects 5.807 and 5.704, the Deutsche Forschungsgemeinschaft (DFG) TRR179 (TP 15), and the Australian Centre for HIV and Hepatitis Virology Research.
We are indebted to Professor William Mason for his part in developing the original invPCR method and demonstrating it to us. We would like to thank Drs. Yi Ni and Florian A. Lempp for reagents (cell lines and HBV inoculum). We acknowledge Anja Rippert, Franziska Schlund, Sarah Engelhardt, and Dr. Katrin Schöneweis for technical assistance. We are grateful to Miriam Kleinig for proofreading, and to Professors Nicholas Shackel and Ralf Bartenschlager for continuous support.
|Thermo Fisher Scientific
|Fetal bovine serum
|Heat-inactivate before use
|Penicillin, 10000U/ml; Streptomycin 10mg/ml, 100×
|Trypsin, 0.5 mg/ml; EDTA, 0.22mg/ml, 1×
|PEG, MW 8000
|Stock at 40% w/v in 1xPBS, autoclave before use
|DMSO for spectroscopy
|Dissolve in DMSO
|Dissolve in DMSO
|NucleoSpin Tissue kit
|NanoDrop 2000/2000c Spectrolphotometer
|Thermo Fisher Scientific
|T4 DNA ligase
|Sodium dodecyl sulfate
|Dextran (35 -45 kDa)
|Amplitaq Gold Taq kit
|Thermo Fisher Scientific
|Use for first nest PCR
|Silicon sealing Mats for 96-Well PCR Plates
|DNAZap PCR DNA Degradation Solution
|Thermo Fisher Scientific
|96-well PCR plates
|Thermo Fisher Scientific
|GoTaq Flexi PCR kit
|Use for second nest PCR, use green buffer for easy loading of agarose gel
|Biozym LE Agarose
|QIAEX II Gel extraction kit
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