INTRODUCTION
Globally, more than 250 million patients are chronically infected with hepatitis B virus (HBV) (
44), but a functional cure of chronic hepatitis B (CHB) is rarely achieved even after years of treatment with nucleos(t)ide analogues (NAs) such as entecavir (ETV) and tenofovir disoproxil fumarate (TDF) (
1). Pegylated interferon alpha (IFN-α) enhances antiviral immune response, but the cure rate remains low, and side effects are often difficult to tolerate (
2,
3). The major obstacles to curing CHB include the persistence of the episomal covalently closed circular DNA (cccDNA) and an immune system that is tolerized to HBV, likely due to the excess amount of circulating hepatitis B surface antigen (HBsAg) levels (
4–6).
The HBV envelope proteins preS1, preS2, and HBsAg are synthesized in the endoplasmic reticulum and are secreted as both viral and subviral particles (
7,
8). HBV virions are double-shelled particles with an outer lipoprotein bilayer containing the envelope proteins and an inner nucleocapsid that encloses the HBV DNA and viral polymerase. The subviral particles devoid of nucleocapsids and HBV DNA (
9,
10) are up to 100,000-fold in excess relative to the virions in the blood of infected patients (
11). Such high levels of subviral particles are believed to play a key role in immune tolerance and maintenance of persistent HBV infection (
5,
6). In patients with chronic hepatitis B, HBV-specific T cells are depleted or functionally impaired (
12–15), and circulating and intrahepatic antiviral B cells are defective in the production of antibodies against HBsAg, with an expansion of atypical memory B cells (
16,
17). HBsAg has also been linked to the inhibition of innate immunity and functionality of other immune cell types (
18). Therefore, antiviral strategies that aim to target the HBV RNA transcripts could suppress HBsAg production and may break the immune tolerance state to potentially increase the functional cure rate.
Regulation of HBV RNA metabolism involves the posttranscriptional regulatory element (PRE), which is a stretch of ribonucleotides spanning positions 1151 to 1582 on the viral transcripts that is essential to HBV subgenomic RNA (sRNA) nuclear export and regulation of pregenomic RNA (pgRNA) splicing (
19–22). The PRE contains three subelements, PREα, PREβ1, and PREβ2. Each subelement is sufficient to support sRNA nuclear export and HBsAg production, but all three together exhibit much greater activity (
23,
24). RNA secondary structure prediction and phylogenetic covariations analysis suggest that two stem-loop structures (stem-loop alpha [SLα] and SLβ1) localized either in PREα or PREβ1 subelements may exist
in vivo and serve as protein binding sites (
23,
25). These two stem-loop structures are highly conserved not only in HBV variants but also throughout the different mammalian hepadnaviruses, and mutations in the stem regions reduced HBsAg production (
23). The PRE is complexed with several RNA binding proteins, including T-cell intracellular antigen 1, La protein, polypyrimidine tract binding protein, ZC3H18, and ZCCHC14 (
26–32). These PRE binding proteins may serve to regulate the export and stability of HBV RNAs. In particular, the CAGGC pentaloop sequence/structure of SLα within the PREα subelement has been predicted to bind sterile alpha motif domain-containing proteins (
24). Recently, ZCCHC14 (a sterile alpha motif-containing protein), together with PAPD5 and PAPD7 [the noncanonical poly(A) RNA polymerase-associated domain-containing proteins 5 and 7], were identified as the cellular binding proteins that interacted with the HBV SLα sequence (
33).
The small-molecule compound, RG7834, targets PAPD5/7 and destabilizes HBV RNAs (
34–37). Using a genome-wide CRISPR screen, it was subsequently observed that
ZCCHC14 and
PAPD5 were associated with the antiviral activity of RG7834 (
32). Interestingly, individual knockdown of
PAPD5 or
PAPD7 had minimal effect against HBsAg production, while knockdown of
ZCCHC14 or double knockdown of
PAPD5/
7 had a profound anti-HBsAg activity similar to that observed when cells were treated with RG7843 (
32,
37). It was further demonstrated that double knockout of
PAPD5/7 reduced guanosine incorporation frequency within HBV RNA poly(A) tails, leading to a proposed model in which HBV RNA recruits the PAPD5/7-ZCCHC14 complex via the CNGGN pentaloop of PRE SLα to enable the extension of mixed tailing on HBV poly(A) tails, which subsequently protects the viral RNAs from cellular poly(A) ribonucleases (
33).
To gain further insights into how small-molecule inhibitors destabilize HBV RNAs, mechanistic studies were performed using AB-452, an analogue of RG7834, to evaluate its effect in HBV-replicating cells and in cells transfected with constructs containing mutations within the PRE sequence. To better understand how PAPD5 and PAPD7 coordinate in the protection of HBV RNAs, both HBV RNA transcripts and their poly(A) tails were analyzed in cells with PAPD5 and/or PAPD7 knockout. Our results reveal that HBV utilizes two layers of protection mechanism provided by PAPD5 and PAPD7 to protect their poly(A) tail integrity and RNA stability.
DISCUSSION
Current therapies for chronic hepatitis B patients rarely achieve functional cure, which is characterized as sustained loss of HBsAg with or without HBsAg antibody seroconversion (
41). The discovery of RG7834 has raised significant interest, as this class of small-molecule inhibitors has the potential to reduce both HBV RNA and viral proteins, which are distinct from direct-acting antivirals targeting the HBV polymerase and capsid proteins (
34,
36,
37). AB-452 is an analog of RG7834 with a similarly broad antiviral effect against multiple HBV replication intermediates. It has been appreciated that integrated HBV DNA is a major source of HBsAg expression in HBeAg-negative patients (
42). Our data indicate that AB-452 can reduce HBsAg produced from cccDNA in HBV-infected cells as well as from integrated HBV DNA in patient-derived hepatocellular carcinoma cells (
Table 1). Furthermore, oral administration of AB-452 substantially reduced HBV DNA, HBsAg, HBeAg, and intrahepatic HBV RNA from AAV-HBV-infected mice (
Fig. 2). Our studies here provide insights into the mode of action for AB-452 and further characterize the RNA stabilization mechanisms utilized by the virus. Our results demonstrate that the
cis-acting SLα viral sequence and the transacting host factors PAPD5 and PAPD7 coordinate to protect viral RNA. Interference of such viral-host interactions through small-molecule compounds treatment or genetic mutations led to destabilization of viral transcripts and reduction of HBsAg.
The requirement of PAPD5/7 and ZCCHC14 to form a complex with HBV RNA through the PRE element for stabilizing HBV RNA has been described (
32,
33). Since the ZCCHC14/PAPD5/7 complex is recruited onto the SLα sequence, it is conceivable that mutating the SLα sequence may disrupt the binding of the ZCCHC14/PAPD5/7 complex and consequently affect HBV RNA stability. Here, our studies provided the genetic evidence that an intact SLα sequence is indeed critical for maintaining HBV poly(A) tail integrity and stability, as inverting or deleting this sequence both destabilize HBV RNA. Notably, the phenotype of the SLα deletion and inversion mutants resembled the antiviral effect of AB-452: cells treated with AB-452 display the phenotypes of HBV poly(A) tail shortening, reduced guanosine incorporation, and HBV RNA degradation.
Initial studies suggest that PAPD5 and PAPD7 may provide redundant if not identical role(s) in protecting HBV RNA stability (
32,
33,
37,
43). However, our results from the
P5_KO and
P7_KO cell lines would argue that PAPD5 and PAPD7 may serve two lines of protection in maintaining the stability of HBV RNA.
P5_KO, but not
P7_KO, impaired poly(A) tail integrity. Moreover, the phenotypic measurements we monitored so far indicate that the
P7_KO cells were similar to WT cells, further supporting that PAPD5 expression alone could support viral RNA integrity and stabilization (
Fig. 7). These data suggest that PAPD7 did not actively contribute to HBV RNA protection in the presence of PAPD5 but, instead, served as a second line of protection by moderately extending the HBV poly(A) tail when PAPD5 was depleted (
Fig. 7I to
K). Results from the enzymatic assays show that PAPD5 was more robust than PAPD7 in the extension of poly(A) tails (data not shown), supporting our argument that PAPD5 would be the major host factor in protecting HBV RNA. Immune precipitation experiments conducted by two independent research groups indicated that both PAPD5 and PAPD7 were bound to HBV mRNA, with PAPD7 at a lower level compared to PAPD5 (
33,
43). Further studies would be required to clarify the role of PAPD7 in HBV RNA metabolism in WT cells.
Another noteworthy observation from this study is that the two HBV RNA destabilizers, AB-452 and RG7834, displayed different inhibitory efficiencies against PAPD5 and PAPD7. Both compounds were 5- to 7-fold less efficient against the enzymatic activities of PAPD7 than PAPD5, which was, in turn, consistent with the results from cell-based studies in which AB-452 and RG7834 displayed a 6- to 10-fold reduction in activities against HBsAg production in the P5_KO cells (in which PAPD7 is present) compared to those from the WT and P7_KO cells (in which PAPD5 is present). These data suggest that it may not be critical to completely inhibit PAPD7 to achieve HBV RNA destabilization. These data, together with the genetic studies, support the hypothesis that PAPD5 could be more essential than PAPD7 in stabilizing HBV RNA. Our results further suggest that developing PAPD5-selective inhibitors of HBV replication could be pharmacologically feasible.
Here, we propose a working model of the interplay between HBV transcripts and the cellular ZCCHC14/PAPD5/7 RNA metabolism machineries (
Fig. 8). Maintenance of HBV RNA stability is a dynamic process regulated by canonical and noncanonical poly(A) polymerases and deadenylases. PAPD5 could form a complex with ZCCHC14, which directs the noncanonical polymerase onto the viral transcripts through the SLα within the HBV PRE sequence. Assembly of the ZCCHC14/PAPD5 onto SLα within the HBV PRE sequence facilitates the addition of G while extending the poly(A) tail. This guanylation process may stall the cellular poly(A) exonuclease and terminate further deadenylation, thus protecting the RNA from degradation. When PAPD5 is depleted, ZCCHC14/PAPD7 complex may bind to HBV RNA and protects its degradation; however, PAPD7 is less effective for poly(A) extension and guanylation incorporation. When HBV is challenged by HBV RNA destabilizers such as AB-452 or PRE mutations, viral RNA integrity and stability are disrupted due to either the inhibition of PAPD5/7 enzymatic activities or disarraying of the ZCCHC14/PAPD5/7 complex from interacting with the SLα sequence, respectively.
MATERIALS AND METHODS
Cell lines and culture.
HepG2.2.15, HepAD38, and PLC/PRF/5 cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Corning, NY, USA), supplemented with 10% fetal bovine serum (Gemini, CA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. Huh-7 cells (Creative Bioarray, NY, USA) were cultured in RPMI 1640 medium (Basel, Switzerland) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. HepG2-hNTCP-C4 cells were cultured in DMEM medium (Gibco, MA, USA) containing 10% fetal bovine serum and 10 mM HEPES (Gibco).
Antiviral studies.
HepG2.2.15 (30,000 cells/well) were plated in 96-well collagen-treated plates and incubated at 37°C. Compounds were half-log serially diluted and added in duplicate to 96-well microtiter plates. The plates were incubated for a total duration of 6 days, after which the culture medium was removed for the HBsAg and HBeAg CLIA assays (AutoBio Diagnostics Co., China) performed according to the manufacturer’s instructions. Secreted HBV DNA was extracted (Realtime Ready cell lysis kit; Roche, Mannheim, Germany) and quantified in a qPCR assay (LightCycler 480 SYBR green I master mix; Roche) with the 5′-GGCTTTCGGAAAATTCCTATG-3′ (sense) and 5′-AGCCCTACGAACCACTGAAC-3′ (antisense) primers using the PCR conditions of denaturing at 95°C for 5 min followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 30 s. Antiviral selectivity studies against a panel of different DNA and RNA viruses were performed at ImQuest BioSicences (Frederick, MD, USA). Briefly, test compounds were evaluated using 6 doses, including the highest concentration of 30 μM and five serial half-logarithmic dilutions in triplicate for the antiviral assays.
Infection of HepG2-hNTCP-C4 cells and PHHs.
HepG2-hNTCP-C4 cells were seeded into collagen-coated 10-cm dishes at a density of 8.6 × 106 cells per dish and cultured in 10 ml complete DMEM medium containing 2% dimethyl sulfoxide (DMSO). One day later, the cells were infected with HBV at 100 to 250 genome equivalent (GE)/cell in DMEM containing 4% polyethylene glycol 8000 (PEG 8000). The inoculums were removed 24 h later, and the infected cells were washed 4 times with phosphate-buffered saline (PBS) and seeded in 96-well plates at a density of 5 × 104/well following trypsinization. Serial dilutions of compounds were added to the plates and refreshed at days 4, 8, and 11 postinfection. The supernatants were harvested for HBsAg and HBeAg analysis (enzyme-linked immunosorbent assays [ELISAs]; International Immuno-Diagnostics, CA, USA). HBV DNA was extracted from cell lysates per the manufacturer’s instructions (Qiagen DNeasy 96 blood and tissue kit; Qiagen, Hilden, Germany). HBV DNA was detected by qPCR using primers and probe as follows: 5′-GTC CTC AAY TTG TCC TGG-3′ (sense), TGA GGC ATA GCA GCA GGA-3′ (antisense), and Probe/56-FAM/CTG GAT GTG TCT GCG GCG TTT TAT CAT/36-TAMSp/. For the infection of PHHs, cells were placed in collagen-coated 96-well plates (65,000 cells/well) overnight and then infected with HBV at 250 GE per cell in media containing 4% PEG 8000. The inoculums were removed 24 h later, and compounds were added or replenished on days 4, 7, 9, 11, and 14 postinfection. On day 16, medium was removed, and HBV antigens and DNA and RNA quantification were monitored as described above.
Detection of intracellular HBV RNA by Northern blotting analysis.
Total cellular RNA was extracted from HepG2.2.15 or HepAD38 cells treated with or without antiviral compounds using TRIzol reagent according to the manufacturer's directions (Thermo Fisher Scientific, Waltham, MA). Northern blotting was performed as described previously (
1). Briefly, total RNA was separated in a 1.5% agarose gel and transferred onto an Amersham Hybond-XL membrane (product no. GERPN303S; GE Healthcare, IL, USA). Membranes were probed with an [α-
32P]UTP (PerkinElmer, CT, USA)-labeled minus strand-specific full-length HBV riboprobe (3.2 kb) transcribed from plasmid pSP65_HBV(+) DNA. Membranes were exposed to a phosphoimager screen, and the hybridization signals were quantified using Image Studio software (Li-Cor Biosciences, NE, USA).
Detection of intracellular HBV DNA by Southern blotting.
Total intracellular viral core-associated DNA was extracted as described previously (
1) and analyzed by Southern blotting hybridization with an [α-
32P]UTP-labeled full-length HBV riboprobe (3.2 kb) that specifically hybridized to minus strand of viral DNA. Membranes were exposed to a phosphoimager screen, and the hybridization signals were quantified using Image Studio software.
Detection of intracellular HBcAg, PAPD5, and β-actin by Western blotting.
HepG2.2.15 or HepAD38 cells cultured in a 12-well plate were lysed with 200 μl Laemmli sample buffer (Bio-Rad, PA, USA) supplemented with 2.5% 2-mercaptoethanol (Sigma-Aldrich, MO, USA). Cell lysates were subjected to denaturing gel electrophoresis with 12% Criterion TGX stain-free precast gels and Tris-glycine-SDS running buffer (Bio-Rad). Proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS)-0.1% Tween for 1 h and incubated with the primary antibody overnight at 4°C. After washing with TBS containing 0.1% Tween 20 (TBST), the membrane was incubated with the secondary antibody. Membranes were again washed 3 times with TBST and soaked with 200 μl Clarity Western ECL substrate (Bio-Rad) and imaged with the iBright imaging systems (Thermo Fisher Scientific). The primary antibodies used in the present study include anti-HBc antibody (catalog no. B0586; Dako, United Kingdom), anti-PAPD5 antibody (catalog no. HPA042968; Atlas Antibodies, Bromma, Sweden), and anti-β-actin antibody (catalog no. ab8227; Abcam, Cambridge, United Kingdom).
Particle gel for viral nucleocapsid and encapsidated HBV DNA analysis.
For intracellular viral nucleocapsid analysis, HepG2.2.15 cells were lysed in buffer containing 10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40. Cell debris was removed by centrifugation, and the viral particles were fractionated through nondenaturing 1% agarose gels electrophoresis and transferred to a nitrocellulose filter by blotting with TNE buffer (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 1 mM EDTA). To detect HBV core antigens, membranes were probed with polyclonal antibody against HBV core protein (catalog no. B0586; Dako, United Kingdom). Bound antibodies were revealed by horseradish peroxidase (HRP)-labeled secondary antibodies (Thermo Fisher Scientific) and visualized with the iBright imaging systems according to the protocol of the manufacturer. For the detection of encapsidated HBV DNA, the DNA-containing particles on the membrane were denatured with a solution containing 0.5 M NaOH and 1.5 M NaCl, and this step was followed by neutralization with a solution containing 1 M Tris-HCl (pH 7.6) and 1.5 M NaCl. HBV DNA was detected by hybridization with an [α-32P]UTP-labeled full-length HBV riboprobe (3.2 kb) that specifically hybridized to the minus strand of viral DNA.
Detection of encapsidated pgRNA.
To detect intracellular encapsidated pgRNA, HepG2.2.15 or HepAD38 cells were lysed with 300 μl of lysis buffer (10 mM Tris-HCl [pH 7.6], 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40) per well. Cell debris and nuclei were removed by centrifugation, and the supernatants were digested with 20 U/ml of micrococcal nuclease (MNase) at 37°C for 30 min. The core particles were precipitated with 35% PEG 8000 dissolved in 1.5 M NaCl on ice for 60 min, isolated by centrifugation, and dissolved in TNE buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 1 mM EDTA). Encapsidated pgRNA in core particles was extracted with TRIzol reagent, and pgRNA was quantified in a qPCR assay (SuperScript III Platinum SYBR Green one-step qPCR kit w/ROX; Invitrogen) with the 5′-GGT CCC CTA GAA GAA GAA CTC CCT-3′ (sense) and 5′-CAT TGA GAT TCC CGA GAT TGA GAT-3′ (antisense) primers using the PCR conditions of 50°C for 30-min hold (cDNA synthesis), denaturing at 95°C for 5 min, followed by 40 cycles of amplification at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s.
In vivo antiviral activity in a mouse model of HBV.
HBV mouse experiments were conducted at Arbutus Biopharma (Burnaby, Canada) in accordance with Canadian Council on Animal Care (CCAC) Guidelines on Good Animal Practices and using protocols approved through the CCAC-certified Institutional Animal Care and Use program. Male C57BL/6J mice, 6 weeks old, were each inoculated with 1E11 genomes of adeno-associated virus (AAV) vector AAV-HBV1.2 containing a 1.2× overlength sequence of HBV genome (genotype D; GenBank accession no.
V01460) (
2). Mice were administered the AAV2/8-type vector via intravenous tail vein injection. Twenty-eight days after AAV infection, animals were randomized into groups (
n = 5) based on serum HBsAg concentration. Animals were administered vehicle only or AB-452 at 0.1, 0.3, or 1 mg/kg by oral gavage twice daily for 7 days and terminated 12 h after the last dose. Serum and liver HBsAg concentrations were determined using the Bio-Rad enzyme immunoassay (EIA) GS HBsAg 3.0 kit according to the manufacturer's instructions. Serum HBV DNA concentrations were measured from total extracted DNA using primer/probe sequences described previously (
3). Total HBV RNA concentrations in the liver were quantified using a branched DNA assay (QuantiGene 2.0; Thermo Fisher Scientific) with probes targeting the shared 3′ region of HBV transcripts, whereas 3.5 kb HBV RNA concentrations were quantified with probes targeting the unique 5′ region of the pgRNA; in both cases, signal was normalized against mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Purification of recombinant PAPD5 and PAPD7 proteins.
Molecular cloning, expression, and purification of the recombinant human PAPD5 and PAPD7 proteins were performed at Xtal BioStructures (MA, USA). PAPD5 (amino acids 186 to 518; GenBank accession no.
XM_011523275) and PAPD7 (amino acids 226 to 558; GenBank accession no.
NM_006999.6) genes comprising of nucleotidyltransferase and PAP-associated domains were codon optimized, synthesized, and inserted in the expression vector pET-24a(+) for
E. coli expression with 10×His-Flag-tobacco etch virus (TEV) tags at the N terminus. The pET-24a(+) constructs were transformed into
E. coli-competent cells BL21(DE3). Cells were grown in TB-kanamycin (Kan) medium (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 72 mM K
2HPO
4, 17 mM KH
2PO
4 plus 50 μg kanamycin/ml plus 100 mM sodium phosphate, pH 7.0, and 2 mM MgSO
4) at 37°C until optical density at 600 nm (OD
600) of ∼0.7 was reached. Expression of protein was induced for 16 h with 0.2 mM IPTG (isopropyl-β-
d-thiogalactopyranoside) at 18°C. Cells were lysed in lysis buffer (25 mM HEPES, pH 7.6, 300 mM KCl, and 5% glycerol) containing lysozyme (1 mg/ml) and EDTA-free protease inhibitor tablets (cOmplete protease inhibitor; Roche). Cell suspension was sonicated 8 × 20 s at 27 to 30 W (power level, 3.5 to 4.0) with 40-s breaks between each pulse. Lysate was cleared by centrifugation in a 50-ml conical tube at 20,000 ×
g for 30 min (Fiberlite F13-14 × 50cy rotor in a Sorvall RC6 centrifuge). Supernatant was incubated with Ni-charged MagBeads (GenScript; catalog no. L00295) equilibrated with the binding buffer [25 mM HEPES, pH 7.5, 300 mM KCl, 5% glycerol, and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)]. Bound proteins were washed with 50 ml binding buffer and eluted in elution buffer containing a gradient of imidazole ranging from 5 to 500 mM. Peak fractions were pooled and stored in 50% glycerol at −80°C.
PAPD5 and PAPD7 ATP depletion assay.
Reactions were carried out in duplicate in 96-well low-profile, skirted, white plates (catalog no. AB0800WL; Thermo Fisher) where each well contained 10 μl of the reaction mixture consists of purified recombinant PAPD5 or PAPD7 proteins (12.5 nM) in 10 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2, 250 nM RNA substrate (CALM1) 5′-GCC UUU CAU CUC UAA CUG CGA AAA AAA AAA-3′, 750 nM ATP, 0.1 mM EDTA, 1 mM TCEP, and 0.002% NP-40. The reaction mixtures were incubated at room temperature for 3 h, and ATP depletion was monitored by using Kinase-Glo luminescent kinase kit following the manufacturer’s instructions (catalog no. V6712; Promega, WI, USA).
Poly(A) tail length analysis of HBV transcripts.
The poly(A) tail of HBV transcripts was measured with the poly(A) tail-length assay kit (catalog no. 76455; Thermo Fisher) per the manufacturer’s instructions. Total RNA was added with poly(G/I) tail at the 3′ end and reverse transcribed by the primer specific to the poly(G/I) tail. HBV mRNA poly(A) tail was amplified by HBV-specific primer (5′-
CAC CAG CAC CAT GCA ACT TT-3′, nucleotides [nt] 1806 to 1825 on HBV genome) and the universal primer that anneals to the G/I tail. HBV gene-specific PCR (GSP) was conducted using primers targeting nt 1633 to 1702 of HBV RNA with 5′-
CCG AAT GTT GCC CAA GGT CT-3′ (sense) and 5′-
CTC AAG GTC GGT CGT TGA CA-3′ (antisense). ACTB mRNA poly(A) tail was amplified by (sense) 5′-
TTG CCA TCC TAA AAG CCA CC-3′ and the universal primer. ACTB GSP primers include 5′-
CCC AGC ACA ATG AAG ATC AAG-3′ (sense) and 5′-
GAC TCG TCA TAC TCC TGC TTG-3′(antisense). Amplified products were used as loading control. The obtained amplicon products were resolved on a 2% agarose gel. HBV poly(A) PCR products were sequenced on PacBio Sequel sequencing platform. Circular consensus sequencing (CCS) reads were generated from raw subreads using PacBio SMRTLINK software (v5.1). Accuracy cutoff default was 0.9. PCR primer sequences (allowing two mismatches) were used to identify segments potentially containing poly(A) tails in CCS reads. A CCS read could contain multiple such segments because of PCR conditions. These segments were processed separately in downstream analysis. Poly(A) tails were identified in each segment. Note: it was unsuccessful to sequence the sample “DMSO-treated WT (parent) cells” initially (
Fig. 6J and
K). In the second sequencing, two samples, including “DMSO-treated WT (parent) cells” and “DMSO-treated P7_KO (T2-22) cells,” were sequenced, and the sample “DMSO-treated P7_KO (T2-22) cells” served as a bridge to normalize two batches of sequencing data.
HBV PRE cis-elements analysis.
Constructs containing either HBsAg or the
Gaussia luciferase (Gluc) reporter genes were synthesized (GenScript). The H133 encodes the full HBsAg transcript sequence (spanning nt 2 to 1991; GenBank accession no.
U95551) under the regulation of tetracycline-controlled cytomegalovirus (CMV) promoter. The H133_dSLα and H133_dLa are derived from H133 with either the SLα sequence (nt 1294 to 1322) or the La protein binding site (nt 1271 to 1294) deleted, respectively. In the luciferase-based constructs, the HBsAg encoding region was replaced with Gluc (the Gluc constructs). Variants were introduced into the Gluc constructs in which the HBx coding sequence was deleted with either wild-type SLα (Gluc_dHBx) or an inverted SLα sequence (Gluc_rcSLα). Huh-7 cells were transfected with the HBsAg or luciferase reporter-derived plasmids per the manufacturer’s instructions (Lipofectamine 3000; Invitrogen, MA, USA). Cells were treated with the indicated compounds for 5 days. Culture supernatants were used for HBsAg or luciferase measurement (Pierce Gaussia Luciferase Glow assay kit; Thermo Fisher Scientific, Waltham, MA, USA). Cells were collected for HBV RNA transcript and cellular rRNA analysis by Northern blotting.
CRISPR-Cas9 knockout generation.
PAPD5 (GeneID 64282), PAPD7 (GeneID 11044), and ZCCHC14 (GeneID 23174) were knocked out with the CRISPR-Cas9 gene editing in the HepG2-hNTCP-C4 cells at GenScript Biotech Corporation (NJ, USA). Briefly, genomic RNAs (gRNAs) were designed and expressed in the plasmid pSpCas9 BB-2A-GFP PX458. The following three gRNAs were evaluated for each gene, and the gRNA with the highest cleavage efficiency was selected to knock out the target gene: PAPD5 gRNA (5′-GAC ATC GAC CTA GTG GTG TTT GG-3′), PAPD7 gRNA (5′-ATA TTT GGC AGC TTT AGT ACA GG-3′), and ZCCHC14 gRNA (5′-GCG TGA GAC CCG CAC CCC CG-3′). HepG2-hNTCP-C4 cells were transfected with validated gRNA-Cas9 plasmids. Transfected cells were sorted by fluorescence-activated cell sorter (FACS) through enhanced green fluorescent protein (EGFP). The obtained cell pool was expanded for single-cell cloning. Genomic DNA from the single-cell clones was extracted for PCR amplification with primers flanking the target site followed by Sanger sequencing.
ACKNOWLEDGMENTS
We thank Ingrid Graves, Agnes Jarosz, Chris Pasetka, and Alice Li for the analysis of in vivo data. We thank Jorge Quintero for scaling up the production of cmpdA. We thank Tianlun Zhou for providing HBsAg-encoded adenoviruses.
All the authors listed in the manuscript are either current or previous employees of Arbutus Biopharma. A.C.H.L., F.G., A.S.K., A.M., L.D.B., and R.R. were Arbutus employees at the time of data generation.
F.L. and M.G. conceived and designed the research. F.L., A.C.H.L., F.G., A.S.K., H.M.M.S., A.M., L.D.B., X.W., S.C., S.G.K., and A.G.C. performed the research. All authors analyzed the data. F.L., A.C.H.L., and M.G. wrote the paper; A.G.C., D.G., B.D.D., and R.R. designed plasmids; and A.L. and M.J.S. revised the paper.
This study was sponsored by Arbutus Biopharma.