==== Front PLoS Pathog PLoS Pathog plos plospath PLoS Pathogens 1553-7366 1553-7374 Public Library of Science San Francisco, CA USA 32226116-68-5213/journal.ppat.1008459 PPATHOGENS-D-19-01800 Research Article Biology and Life Sciences Biochemistry Proteins Post-Translational Modification Phosphorylation Biology and Life Sciences Microbiology Virology Viral Replication Viral Packaging Biology and life sciences Microbiology Medical microbiology Microbial pathogens Viral pathogens Hepatitis viruses Hepatitis B virus Medicine and health sciences Pathology and laboratory medicine Pathogens Microbial pathogens Viral pathogens Hepatitis viruses Hepatitis B virus Biology and life sciences Organisms Viruses Viral pathogens Hepatitis viruses Hepatitis B virus Research and Analysis Methods Electrophoretic Techniques Gel Electrophoresis Electrophoretic Blotting Southern Blot Biology and Life Sciences Molecular Biology Molecular Biology Techniques Molecular Probe Techniques Electrophoretic Blotting Southern Blot Research and Analysis Methods Molecular Biology Techniques Molecular Probe Techniques Electrophoretic Blotting Southern Blot Biology and Life Sciences Microbiology Virology Viral Structure Virions Biology and Life Sciences Microbiology Virology Viral Replication Research and Analysis Methods Electrophoretic Techniques Gel Electrophoresis Biology and life sciences Genetics DNA Forms of DNA Circular DNA Biology and life sciences Biochemistry Nucleic acids DNA Forms of DNA Circular DNA Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation HBc NTD phosphorylation and nucleocapsid disassemblyhttp://orcid.org/0000-0003-3713-5438Luo Jun ConceptualizationData curationFormal analysisInvestigationMethodologyWriting – original draftWriting – review & editing1 Xi Ji Data curationMethodologyWriting – review & editing1 Gao Lu Funding acquisitionProject administrationWriting – review & editing2 http://orcid.org/0000-0002-3967-2133Hu Jianming ConceptualizationFormal analysisFunding acquisitionProject administrationSupervisionWriting – original draftWriting – review & editing1* 1 Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America 2 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China Siddiqui Aleem Editor University of California, San Diego, UNITED STATES The authors have declared that no competing interests exist. * E-mail: savagestephanie@example.net 30 3 2020 3 2020 16 3 e100845926 9 2019 5 3 2020 © 2020 Luo et al2020Luo et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs. Author summary Hepatitis B virus (HBV) persistently infects hundreds of millions of people worldwide, causing viral hepatitis, cirrhosis and liver cancer. The basis of HBV persistence is the viral covalently closed circular (CCC) DNA, a nuclear episome, that drives all viral gene expression to sustain viral replication. CCC DNA is derived from the relaxed circular (RC) DNA, which is formed inside a proteinaceous shell, the viral capsid, but has to be released from the capsid in order to be converted to CCC DNA by host cell factors. We report here that the phosphorylation state of the capsid protein, regulated by host cell enzymes including one that is packaged inside the viral capsid, plays a critical role in regulating the release of RC DNA and thus controlling CCC DNA formation. Intense ongoing efforts are being directed at developing novel antiviral strategies to eliminate the HBV CCC DNA for curing persistence HBV infection, including those targeting the capsid protein. Our results should inspire novel approaches to targeting the HBV capsid and CCC DNA. Furthermore, uncoating is an essential step in the infection process for virtually all viruses that remains ill-understood. Thus, our results have broad implications for understanding viral infection in general. http://dx.doi.org/10.13039/100000060National Institute of Allergy and Infectious DiseasesR01AI043453, R01Al127670http://orcid.org/0000-0002-3967-2133Hu Jianming http://dx.doi.org/10.13039/100007013F. Hoffmann-La Rochehttp://orcid.org/0000-0002-3967-2133Hu Jianming This work was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (R01AI043453 and R01Al127670) to J.H. It was also supported by a Roche Post-doctoral Fellowship (RPF) to J.L. The NIH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Roche approved the submission of the manuscript. PLOS Publication Stagevor-update-to-uncorrected-proofPublication Update2020-04-09Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.Data Availability All relevant data are within the manuscript and its Supporting Information files. ==== Body Introduction Hepatitis B virus (HBV), the prototype member of Hepadnaviridae family, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide [1]. HBV has a small (ca. 3.2 kbp), partially double-stranded (DS), relaxed circular (RC) DNA genome [2] which is replicated via reverse transcription from an RNA intermediate, the pregenomic RNA (pgRNA) [3, 4]. The RC DNA genome is packaged into an icosahedral capsid composed of the HBV core protein (HBc), which in turn is enclosed in an envelope layer composed of host cell derived lipid bilayer studded with three viral envelope or surface proteins [5]. Upon infection of susceptible host cells, the HBV RC DNA genome is delivered to the nucleus where it is repaired to form the covalently closed circular (CCC) DNA [6, 7]. CCC DNA then serves as the viral transcriptional template to produce all viral RNAs, including pgRNA, required for replication. In addition to serving as the template for reverse transcription to replicate the viral DNA genome, pgRNA also serves as the mRNA for translating both HBc and the viral reverse transcriptase (RT) protein. Progeny virus assembly starts with the formation of a replication-competent nucleocapsid (NC) whereby the assembling HBc subunits (as homodimers) incorporate a single copy each of the RT protein and pgRNA, as a ribonucleoprotein (RNP) complex. Viral DNA synthesis ensues in the resulting pgRNA-containing NC to produce first the single-stranded (SS), minus strand DNA and then the RC DNA. NCs containing pgRNA or SS DNA are considered immature as they are not secreted outside the cells as virions; only RC DNA-containing (mature) NCs are competent for interaction with the viral envelope proteins and secreted extracellularly as complete virions [5]. Interestingly, the newly formed progeny RC DNA in intracellular mature NCs, like that from the incoming virion, can also be delivered to the host cell nucleus to form more CCC DNA [8–12]. This so-called intracellular CCC DNA amplification pathway effectively increases the HBV CCC DNA pool in the nucleus and is thought to be a critical feature of HBV replication for sustaining viral persistence [7, 13, 14]. HBc is a small (ca. 21 kDa) protein that plays multiple roles in HBV replication in addition to forming the shell of the NC [15]. It contains a globular N-terminal domain (NTD) and a C-terminal domain (CTD). The HBc NTD (amino acid 1–140), also known as the assembly domain, is essential for capsid formation [16, 17], but also plays a role in pgRNA packaging and reverse transcription [18]. The HBc CTD (amino acid 150–183) shows non-specific nucleic acid-binding activity and is essential for pgRNA packaging and reverse transcription [19–21]. In addition, the short (9-residues, 141–149) linker peptide between the NTD and CTD, traditionally thought to be simply a flexible spacer between the two domains, has been shown recently to play critical roles during multiple stages of viral replication [22]. With respect to the role of HBc in CCC DNA formation, current evidence suggests that HBc can control CCC DNA formation via at least two separate mechanisms. First, delivery of the RC DNA into the nucleus for conversion to CCC DNA is controlled by nuclear localization signals (NLSs) contained in the HBc CTD [23–25]. Also, mutations in the NTD such as L95A and K96A have been shown to increase CCC DNA formation, possibly also through their potential effects on nuclear import of RC DNA [26]. Second, HBc controls uncoating or disassembly of mature NCs to release RC DNA for CCC DNA formation [26, 27], with the generation of protein free (PF)-RC DNA, in which the RT protein covalently attached to the 5' end of the minus strand of RC DNA has been removed, being an indication of (at least partial) uncoating [10]. PF-RC DNA may in fact be a mixture of multiple species each with a slightly different fine structure, and it remains uncertain if any of these are true intermediates in CCC DNA formation from RC DNA [10, 11, 28]. On the other hand, one particular form of PF-RC DNA, the closed minus strand RC DNA or cM-RC DNA, in which the minus strand of RC DNA is covalently closed but the plus strand remains open, represent the most likely intermediate to date in RC DNA to CCC DNA conversion [29]. The HBc CTD is known to undergo dynamic phosphorylation and dephosphorylation, which regulate HBc functions in capsid assembly [30] as well as in pgRNA packaging and reverse transcription [31–44]. Three major sites, and four minor sites of CTD phosphorylation, have been identified [23, 32, 36, 45]. The host cell cyclin-dependent kinase 2 (CDK2) and Polo-like kinase 1 (PLK-1) have been shown to phosphorylate the serine-proline (SP) and non-SP sites, respectively [45, 46] although additional kinases such as protein kinase C and the serine-arginine protein kinase have also been reported [34, 47–50]. Furthermore, a cellular kinase(s) is well known to be packaged into HBV capsids (the so-called endogenous kinase), and recent evidence indicates that the cyclin-dependent kinase 2 (CDK2) represents the major endogenous kinase, at least for capsids isolated from cultured human hepatoma cells [45]. In contrast to extensive studies on the role of CTD phosphorylation in HBV replication, little is known about potential phosphorylation of HBc NTD and its role in viral replication. We therefore decided to target two highly conserved and putative sites of phosphorylation in the HBc NTD. Our results obtained with HBc mutants that either block phosphorylation or mimic constitutive phosphorylation at these two NTD sites are consistent with a role of transient NTD phosphorylation, following NC maturation, in destabilizing the mature NC and facilitating its disassembly. Furthermore, our results suggest that phosphorylation at these HBc NTD sites can enhance phosphorylation at the CTD and possibly elsewhere in HBc to facilitate uncoating, and the endogenous kinase may play a major role in mediating these phosphorylation events during capsid uncoating. Materials and methods Ethics statement Human primary hepatocytes from human liver chimeric mice (PXB cell) was purchased commercially from PhoenixBio (Hiroshima, Japan). Cell cultures and plasmids HepG2, HEK293T and Huh7 cells were maintained in Dulbecco modified Eagle F-12 (DMEM-F12) medium supplemented with 10% fetal bovine serum, 50 ug/ml of penicillin-streptomycin. The HepAD38 cells were maintained in DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS), 50 ug/ml of penicillin-streptomycin, 400 ug/ml G418, and 5 ug/ml of tetracycline (Tet). AD38 cells were induced to express HBV pgRNA upon removal of Tet from the culture medium [51]. The HepG2-NTCP cell line [29] was derived from the human hepatoma cell line HepG2 and stably expresses the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). HepG2-NTCP cells were maintained in DMEM-F12 supplemented with 10% FBS and 50 ug/ml of penicillin-streptomycin. pCI-HBc WT, pCI-HBc-3A, pCI-HBc-3E pCI-HBc-7A, pCI-HBc-7E have been previously described [30]. pCI-HBc-N2A and pCI-HBc-N2E were constructed by changing two NTD putative phosphorylation sites (S44 and S49) to A and E, respectively, via PCR-mediated mutagenesis. pCI-HBc-9A and pCI-HBc-9E were constructed by changing all nine phosphorylation sites (S44 and S49 from NTD and all 7 S/T from CTD) to A and E, respectively. The HBV replicon plasmid used here, pCIΔA-HBV-HBc-WT, was constructed by subcloning the entire HBV insert together with the linked human cytomegalovirus (HCMV) promoter sequences from pCMVHBV [52, 53] to the pCIΔA vector [26]. pCIΔA-HBV-N2A and pCIΔA-HBV-N2E were constructed by subcloning the N2A and N2E mutations into pCIΔA-HBV-HBc-WT and express the mutated HBV pgRNA encoding the same HBc NTD mutations as in pCI-HBc-N2A and pCI-HBc-N2E, respectively. Antibody The mouse monoclonal antibody (mAb) clone T2221 against the HBc NTD was purchased from Tokyo Future Style (Cat no. 2AHC24) [22]. The HBc CTD-specific rabbit mAb 25–7 and 6–1 were custom-made (by Abcam) using a non-phosphorylated CTD peptide (from residue 152 to the C terminus) as an immunogen and have been described before [30]. A701 is a mouse mAb prepared using a non-phosphorylated HBc CTD peptide from position 156 to 176 as the immunogen and has been described before [43]. The rabbit polyclonal antibody against HBc was purchased from Dako. Transient transfection HepG2 cells in 60-mm dishes were transfected with 4 μg (total) of plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche). HEK293T cells seeded in 60-mm dishes were transfected with 10 μg (total) of plasmid DNA using the CalPhos Mammalian Transfection Kit (Clontech). Cells and culture supernatant were harvested on day five post-transfection. Huh 7 cells in 100-mm dishes were transfected with 12 ug of (total) of plasmid DNA using the FuGENE 6 transfection reagent (Promega). HBV infection HBV infection in HepG2-NTCP was carried out as previously described [29], with slight modifications. Briefly, HepG2-NTCP cells were plated in collagen I-coated 35-mm tissue culture dishes. When the cells reached 50% confluence, they were pretreated with 2% DMSO for 7 days to arrest cell growth. The cells were then infected by using the HBV inoculum harvested from induced HepAD38 cells or transiently transfected Huh7 cells, at a multiplicity of infection (MOI) of ca. 200–400 genome equivalent (GE)/cell in the presence of 2% dimethyl sulfoxide (DMSO), 4% polyethylene glycol (PEG) 8000, in the presence or absence of the indicated compounds. Freshly plated primary human hepatocytes isolated from chimeric mice containing human hepatocytes (PXB cell; PhoenixBio) were also used for HBV infection. PXB cells were plated on type I collagen-coated plates by PhoenixBio and cultured in modified dHCGM (DMEM with 10% FBS, 100 U/mL Penicillin, 100 ug/mL Streptomycin, 20 mM HEPES, 44 mM NaHCO3, 15 ug/mL L-proline, 0.25 ug/mL Insulin, 50 nM dexamethasone, 5 ng/mL EGF, 0.1 mM Asc-2P, 2% DMSO) [54]. Upon delivery, the cultured medium was replaced with fresh modified dHCGM. The cells were infected the next day (day 0) by replacing the culture medium with infection medium (dHCGM with 4% PEG 8000 and 5 ul inoculum harvested from HepAD38 cells at an MOI of 400 GE/cell. The inoculum was incubated with the cells for 20–28 hrs. Subsequently, the culture medium was changed daily until day 3 when they were harvested for analysis of HBV CCC DNA. To inhibit the CDK2 activity in PXB cells during HBV infection, the CDKII inhibitor K03861 (Selleckchem, cat no. S8100) or CDK2 inhibitor III (Sigma, cat no. 238803) [45] was added along with the HBV inoculum. The cells were harvested three days post infection. Induction of rapid and synchronous formation of HBV CCC DNA in HepAD38 cells To test the role of CDK2 in intracellular CCC DNA amplification, we adopted the synchronized CCC DNA formation system using induced HepAD38 cells as reported recently [55]. Briefly, Tet was removed from the culture medium to induce transcription of HBV pgRNA. Two days after Tet removal, the reversible inhibitor of the HBV reverse transcriptase, phosphonoformic acid (PFA) [56, 57], was added to the culture medium at 2 mM concentration to allow synthesis of HBV SS DNA but not RC DNA. PFA treatment was maintained for four days to accumulate nucleocapsids containing SS DNA. PFA was then removed to allow synchronous synthesis of RC DNA and the resulting conversion of RC to CCC DNA, and Tet was added back simultaneously to block further pgRNA transcription. At the same time of PFA removal and Tet add back, the CDK2 inhibitor was added. Cells were harvested 24 hrs after PFA removal for analysis of HBV DNA. Isolation of viral DNA The HBV core and PF DNAs were isolated from transiently transfected cells as previously described [28, 29] with minor modifications. For isolation of core DNA, cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40, and 1X protease inhibitor [Roche]). After removal of the nuclear pellet by centrifugation, the supernatant (cytoplasmic lysate) was incubated with micrococcal nuclease (MNase) (Roche) (150 units/ml) and CaCl2 (5 mM) at 37°C for 90 min to degrade the nucleic acids outside NCs. The MNase was then inactivated by addition of 10 mM EDTA. Thereafter, proteinase K (0.6 mg/ml) and sodium dodecyl sulfate (SDS) (0.5%) were used to digest and disrupt NCs and viral DNA-protein complexes. HBV DNA released from NCs was then purified by phenol-chloroform extraction and ethanol precipitation. Hirt extraction was used for PF DNA isolation. Cells in a 60-mm dish were lysed in 1 ml SDS lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 150 mM NaCl, and 1% SDS). After incubation for 5 min at room temperature, the cell lysate was transferred to a 1.5-ml microcentrifuge tube, mixed with 0.25 ml of 2.5 M KCl, and incubated at 4°C overnight with gentle rotation. After being centrifugated at 14,000 x g for 20 min, the supernatant was extracted three times with phenol and once with chloroform. The DNA was precipitated with ethanol and washed with 70% ethanol three times, vacuum dried, resuspended in 200 ul TE (10 mM Tris-HCl–1 mM EDTA [pH 8.0]). Exonuclease treatment To remove the input plasmids from purified viral DNA, Dpn I digestion was used [10, 29]. The Dpn I-treated DNA was further digested with Exonuclease I (Exo I) and Exonuclease III (Exo III) (NEB) as described [29]. Briefly, 20 ul PF DNA (from total 200 ul per 6-cm dish of cells) was digested with 1 ul of Exo I (20 units) & 0.25 ul of Exo III (25 units) in the 1x Cutsmart buffer (NEB) at 37°C for 2–3 hrs [29]. Southern blot analysis of viral DNA The HBV core and PF DNAs were resolved on a 1.2% agarose gel and detected by Southern blot analysis as previously described [29, 53, 57], using a 32P-labeled HBV DNA probe or strand-specific RNA probes as indicated. Where indicated, viral DNA was also released from NCs in cytoplasmic lysate, without prior nuclease digestion or further purification, by SDS (0.6%)-proteinase K (0.5 ug/ul) digestion as described [26, 27]. The digested lysate was directly resolved by agarose gel electrophoresis and analyzed by Southern blot analysis. Capsid assembly and RNA packaging To determine the levels of assembled capsids and the amount of pgRNA packaged inside capsids, cytoplasmic lysates were resolved on a 1% native agarose gel (native agarose gel electrophoresis or NAGE). Upon transfer of the resolved capsids onto nitrocellulose membrane, the packaged pgRNA was detected using a 32P-labeled anti-sense RNA probe as described previously [53]. Subsequently, capsids were detected on the same membrane by using the rabbit polyclonal (Dako) or mouse monoclonal (T2221) anti-HBc antibody as described [22]. Virion secretion assay To analyze HBV virion secretion, cell culture supernatant was first precipitated with polyethylene glycol (PEG) and subjected to DNase I digestion to remove plasmid DNAs [57, 58]. The concentrated viral particles were resolved on a 1% native agarose gel and detected using a 32P-labeled HBV DNA probe following transfer to nitrocellulose membrane. The same membrane was subsequently probed with the indicated core- or surface-specific antibody to detect the core or surface proteins, as described [22, 58]. Endogenous kinase reaction (EKR) EKR was performed as described before [45, 57] with minor modifications. Briefly, 20 ul NP-40 lysate (600 ul total per 60 mm dish) from transfected HEK293T cells was digested with proteinase K for 1 hr (final concentration, 1 mg/ml). Then, the proteinase K was stopped by adding the proteinase K inhibitor (Calbiochem) to 1 mM and incubating the sample at room temperature for 10 min. The digested lysate was then mixed with the kinase reaction buffer (final concentration: 10 mM sodium phosphate, pH 7.0, 10 mM MgCl2, and 0.01% Brij-35), 1x EDTA-free protease inhibitor cocktail (Roche), 1 mM dithiothreitol (DTT), and 5 uCi [γ-32P]ATP (3,000 Ci/mmol, 10 mCi/ml; Perkin Elmer) and incubated at 37°C for one hr. The reaction mixture was loaded on a 1% agarose gel. After transfer to nitrocellulose membrane, the radiolabeled capsids were detected by phosphorimaging and the total capsid levels determined by western blot analysis using the anti-HBc antibody T2221. In addition, the EKR reactions were also resolved by SDS-PAGE. After transfer to polyvinylidene difluoride membrane, radiolabeled HBc proteins were detected by phosphorimaging and total HBc proteins detected by the anti-HBc antibody T2221. Phos-tag gel electrophoresis Phos-tag gel electrophoresis to separate different phosphor-isomer of HBc was conducted as described [50, 59, 60], with minor modifications. Briefly, cytoplasmic lysate containing HBc proteins was boiled in SDS sample buffer containing 1 mM MnCl2 and 10% β-ME for 10 min and then resolved on an SDS-polyacrylamide gel (10%, acrylamide:bis-acrylamide ratio 37.5:1) containing 100 uM Phos-tag acrylamide and 200 uM MnCl2. After electrophoresis, the gel was soaked in transfer buffer containing 10 mM EDTA with gentle shaking for 10 min each time, with one to three buffer changes, before western blot analysis. Quantification and statistical analysis All experiments were repeated at least three times (three biological repeats, e.g., three separate transfections). Radioactive signals from Southern blot analysis and EKR were detected by phosphor imaging (Typhoon 9500, GE Healthcare Life Sciences) and quantified using Quantity ONE (Bio-Rad). Protein signals from western blot analysis were detected and quantified using the Bio-Rad Chemi-Doc (ImageLab) system. Subtraction of background signals was done via either the global method when the background signal was fairly uniform and was subtracted from all sample lanes, or done separately for each individual lanes using appropriate background within that particular lane. All quantifications were performed within the linear range of the instruments in the absence of signal saturation. To control for possible variations in transfection efficiency and sample loading, viral parameters from each batch of transfected cells at each step of viral replication were normalized to those from the preceding step from the same batch of transfected cells, e.g., capsid to total HBc levels for capsid assembly efficiency, pgRNA packaging to capsid levels for RNA packaging efficiency, core DNA to RNA packaging levels for DNA synthesis efficiency, CCC DNA to core RC DNA levels for CCC DNA formation efficiency, etc. The PEG precipitation procedure to concentrate secreted virions (as well as naked capsids) from the cell culture supernatant was found to generate consistent results from multiple experimental repeats and consistent precipitation efficiency from multiple samples could also be verified by staining of total proteins precipitated following resolution by SDS-PAGE. Results Rationale Close inspection of the HBc NTD sequences revealed two conserved potential phosphorylation sites, S44 and S49 (Fig 1), both in S-P motifs, which are localized to the interior surface of capsids. Two major classes of so-called proline-directed kinases, which are the major cellular protein kinases known to phosphorylate S(T)-P motifs, are CDKs and MAPKs [61]. Also, these two putative NTD phosphorylation sites would be inaccessible to cellular kinases once the capsid is assembled but would be accessible to a kinase that is packaged inside the capsid. Given our recent discovery that the major endogenous kinase packaged in HBV capsids is CDK2 (or at least a closely related kinase) [45], we hypothesized that the NTD SP motifs can be phosphorylated by the packaged CDK2 during certain stage(s) of viral replication to regulate HBc functions. To begin to test such a possibility, we changed both S44 and S49 to Ala (N2A) to block their phosphorylation and to Glu (N2E) to mimic constitutive phosphorylation (and block dephosphorylation). Using transient transfection assay in human hepatoma cells that support HBV replication, we analyzed the effects of these mutations on different stages of HBV replication and on the phosphorylation state of HBc. 10.1371/journal.ppat.1008459.g001Fig 1 HBc dimer structure and location of the putative NTD phosphorylation sites S44 and S49. The structure of the two HBc NTD monomer (in brown and dark red, respectively) in an NTD dimer, based on the HBV capsid crystal structure [17], is shown. The two putative NTD phosphorylation sites S44 and S49, located on the interior surface of the capsid, are highlighted. Both N2A and N2E mutants showed WT levels of capsid assembly but N2E showed decreased levels of RNA packaging Using an overlength HBV genomic construct (replicon, see Materials and Methods) that is competent to produce pgRNA and initiate viral replication in human hepatoma cells, we tested the effects of the NTD mutations on HBc expression, capsid assembly, pgRNA packaging, reverse transcription, CCC DNA formation, and virion secretion. SDS-PAGE and western blot analysis of lysate of transfected cells showed HBc expression levels were not affected by either the N2A or N2E mutation (Fig 2A, top). Interestingly, the N2E mutant protein migrated on SDS-PAGE significantly slower than the WT HBc, either due to enhanced phosphorylation or conformational change (see below). 10.1371/journal.ppat.1008459.g002Fig 2 Effects of NTD phosphorylation mutants on capsid assembly and RNA packaging. The HBV replicon construct expressing the N2A or N2E mutant, or WT HBc was transfected into HepG2 cells. Transfected cells were lysed five days later. A. Levels of HBc proteins (top) were measured by western blot analysis using the T2221 anti-HBC NTD mAb following resolution by SDS-PAGE (top). Assembled capsids (bottom) and packaged RNA (middle) were detected by using a plus strand specific RNA probe and the Dako anti-HBc polyclonal antibody, respectively, following resolution by native agarose gel electrophoresis (NAGE) and transfer to nitrocellulose membrane. C, HBc protein; Ca, HBV capsid. RNA signals were detected by phosphorimaging scan and protein signals by chemiluminescence scan. B. Quantitative results from multiple experiments shown in A. Capsid assembly efficiency (top) was determined by normalizing the levels of capsids measured following NAGE to those of HBc proteins following SDS-PAGE, with the efficiency from WT HBc set to 1.0. RNA packaging efficiency (bottom) was determined by normalizing the levels of RNA packaging to those of capsids, with the efficiency from WT HBc set to 1.0. Capsid assembly and pgRNA packaging were then determined using the NAGE assay. We have previously shown under our experimental conditions, mostly pgRNA, but not (+) strand DNA, was detected in our RNA packaging assay [53, 58]. To further verify that our RNA packaging assay was detecting mostly the packaged HBV RNA, but not DNA, we included analysis of capsids harvested from cells that were treated with the HBV RT inhibitor (entecavir, ETV), which is known to block viral DNA synthesis but not pgRNA packaging. While HBV DNA-associated with capsids, detected by a riboprobe specific for the minus strand DNA, was decreased dramatically in the entecavir treated samples (S1 Fig, lanes 7–9 vs 10–12), the packaged RNA levels, detected by a riboprobe specific for the HBV RNA (and plus strand DNA), were not significantly changed (S1 Fig, lanes 1–3 vs. 4–6). Both in the presence and absence of entecavir, RNA packaging by N2E was decreased by 4–5 fold compared to the WT HBc whereas N2A showed little effect (Fig 2A, middle and bottom; Fig 2B; S1 Fig). On the other hand, capsids levels were not significantly affected by either mutant. These suggested that the N2E, but not N2A, capsids were partially defective in pgRNA packaging. N2A and N2E showed no specific defect in reverse transcription or secretion of complete virions To determine the nature and levels of the viral DNA synthesized from the packaged pgRNA in NCs, viral DNA was released from NCs (core DNA) by SDS/proteinase K treatment and detected by Southern blot analysis. Core DNA levels were obviously lower (by ca. 8-fold) in N2E NCs compared to the WT (Fig 3A, lanes 6 vs. 1–4), but this decrease could be largely accounted for by the decrease in pgRNA packaging shown in Fig 2 above. Upon normalization of core DNA levels to those of packaged RNA, neither N2E or N2A showed any significant reduction in core DNA (Fig 3D), thus indicating that these mutants showed little specific defect in reverse transcription. 10.1371/journal.ppat.1008459.g003Fig 3 Effects of NTD phosphorylation mutants on core DNA and CCC DNA levels. HepG2 cells were transfected as in Fig 2. A. Cytoplasmic lysate from the transfected cells was treated with SDS-proteinase K to release the HBV core DNA from NCs, which was then resolved on an agarose gel (1% agarose) (lanes 1–6). In addition, a portion of cytoplasmic lysate was digested first with MNase to remove input plasmid DNA (and any core DNA not protected by the capsid) before SDS-proteinase K treatment. The core DNA was then purified and resolved on an agarose gel (lanes 7–9). Core DNA was then detected by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); PI, plasmid DNA; RC, RC DNA; SS, single-stranded DNA. B. Viral particles released into the culture supernatant of the transfected HepG2 cells were concentrated by PEG precipitation and resolved on an agarose gel (1% agarose). Following transfer to nitrocellulose membrane, HBV DNA associated with virions (V) or naked capsids (Ca) was detected by Southern blot analysis using an HBV DNA probe. To facilitate a more clear visualization of the degree of N2E deficiency in DNA synthesis and virion secretion, as compared to the WT, serial dilutions (1/4th, 1/8th, 1/16th) of the WT samples were loaded (A and B, lanes 2–4). C. HBV PF DNA was extracted from the transfected HepG2 cells. The HBc F122V mutant (lane 4) defective in DNA synthesis was included as a negative control for PF DNA analysis. The extracted DNA was digested with Dpn I (to degrade the input plasmid DNA) (lanes 1–4), Dpn I plus the exonuclease I and III (I&III) (lanes 5–8), or Dpn I plus the exonuclease T5 (T5) (lanes 9–12) before resolution on an agarose gel (1.2% agarose) and detection by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); RC, RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. All Southern blot images shown in A, B, and C were from phosphorimaging scan. D. Quantitative results from multiple experiments. Left, levels of core DNA were normalized to those of RNA packaging measured in Fig 2; middle, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized DNA values from the WT HBc were set to 1.0. HBV DNA levels in virions released into the culture supernatant were then analyzed by the NAGE assay. The amount of virion DNA detected by Southern blot analysis in the WT or the NTD mutants was proportional to their respective intracellular RC DNA levels (Fig 3A and 3B), indicating no specific block in the secretion of complete virions by the mutants. Specifically, the N2E virion DNA levels were reduced by ca. 8-fold compared to the WT, similar to the reduction in N2E intracellular DNA levels. Similarly, the amount of HBV DNA in naked capsids, which are also released into hepatoma cell culture supernatant, was also proportional to the intracellular HBV DNA levels. N2E but not N2A increased PF-RC and CCC DNA levels during intracellular amplification The potential effects of the HBc NTD mutations on HBV CCC DNA formation was then analyzed. PF-DNAs, including PF-RC DNA and CCC DNA, extracted from transfected cells were analyzed by Southern blot analysis. Transfection input plasmid DNAs were digested by Dpn I. To further analyze the nature of PF DNA, Exo I/III or T5 nuclease digestion was carried out, which removes RC DNA [29]. T5 will also removes any RC DNA in which only one of the two strands is covalently closed due to its SS DNA endonuclease activity. As a negative control to monitor the digestion of non-viral DNA, another HBc mutant, F122V, which is defective in pgRNA packaging and viral DNA synthesis [62], was included in the analysis. HBV PF DNA levels were determined by Southern blot analysis and normalized to those of the core RC DNA, which is the ultimate precursor to both PF-RC DNA and CCC DNA. The results showed that levels of PF-RC DNA from N2E was increased by ca. 5-folds compared to the WT. Furthermore, CCC DNA levels from N2E were also increased by ca. 2.5-folds (Fig 3C and 3D). These results indicated that N2E produced PF-RC DNA and CCC DNA more efficiently. On the other hand, N2A tended to have less PF-RC and CCC DNA than WT although the difference did not reach statistical significance; the PF DNA levels from N2A appeared to be rather variable from experiment to experiment (Fig 3D) (see also Discussion below). We noticed a DNA species savagestephanie@example.net. 3.0 kb in the core DNA and PF DNA (Fig 3A and 3C; also see below). This could represent the minor form of the HBV genome, the double-stranded linear DNA as suggested earlier (10) but could also be an incomplete RC DNA species with a relatively short plus strand. The N2A mutant virus was impaired in CCC DNA formation during de novo infection To test if the NTD state of phosphorylation played a role in CCC DNA formation during viral infection, we prepared the WT and N2A mutant virus inoculum using transient transfection of Huh7 cells. Unfortunately, the N2E mutant secreted too little virus and it was thus difficult to make sufficient amounts of the N2E mutant virus for the infection experiments. We then infected both the HepG2-NTCP cells and PXB cells (primary human hepatocytes isolated from human liver-chimeric mice) [54] with WT or N2A mutant virus at the same MOI. As described in the Methods, HepG2-NTCP cells were pretreated with DMSO to arrest their growth so as to mimic the non-dividing hepatocytes in the human liver before infection. Analysis of HBV CCC DNA levels three days post-infection showed that the N2A mutant virus was clearly less efficient than the WT in CCC DNA formation during infection of the HepG2-NTCP cells (Fig 4A) and both batches of PXB cells (Fig 4B). 10.1371/journal.ppat.1008459.g004Fig 4 Effects of N2A on CCC DNA formation during infection. The WT or N2A mutant virus inoculum was prepared from transiently transfected Huh7 cells. The WT and N2A replicon constructs were transfected into Huh7 cells. The culture supernatant was harvested on day 5, 7 and 9 post-transfection, pooled and concentrated by PEG precipitation as described in the Methods. HepG2-NTCP and PXB cells were infected with the WT or N2A mutant virus. HBV PF DNA was extracted from the infected cells and measured by Southern blot analysis using a 32P-labeled HBV DNA probe. The Southern blot images shown were from phosphorimaging scan. A. A representative Southern blot autoradiogram of PF DNA from HepG2-NTCP cells extracted at the indicated days post-infection. Quantitative analysis of CCC DNA levels at day three post-infection from multiple infection experiments is presented in the graph to the right, with the CCC DNA level from WT HBV infected cells set to 1.0. M, DNA m.w. markers in kbp; B. Representative Southern blot autoradiograms of PF DNA from two batches of PXB cells extracted three days post-infection. Relative CCC DNA levels are indicated at the bottom of the autoradiograms with the CCC DNA level from WT HBV infected cells set to 1.0. Mature NCs of the N2E mutant was destabilized compared to WT The decrease in core DNA accompanied by the increase in PF-RC DNA and CCC DNA shown by N2E was reminiscent of other HBC NTD mutants that we reported before (such as L60A and I126A) [26], which cause hyper-destabilization (loss of integrity) of mature NCs (and thus more efficient uncoating than the WT) and more efficient release of mature RC DNA into the host cell for conversion to PF-RC DNA and CCC DNA. To test if N2E or N2A affected the integrity of NCs, we tested the ability of the mutant NCs to protect their DNA content from exogenous nuclease digestion as we described before [26, 27, 63]. Indeed, the N2E mutant failed to protect its RC DNA while able to protect the immature DNA species (Fig 3A, lane 8 vs. 6), indicative of a selective destabilization of mature NCs. In contrast, N2A protected both the RC and immature DNAs just like the WT HBc (Fig 3A, lanes 7 and 9 vs. 5 and 1). The N2E mutant increased the production of cM-RC DNA more than CCC DNA As we reported recently [29], a comparison of Exo I plus III vs. T5 nuclease digestion revealed the existence of the cM-RC DNA, as evidenced by the detection of a minus strand circular DNA (cM) upon digestion of the PF-RC DNA, which was preserved following Exo I plus III treatment but removed by Exo T5 (Fig 3C). Interestingly, the ratio of cM DNA to CCC DNA appeared to be affected by the NTD mutations. In particular, the N2E mutant produced more cM DNA relative to CCC DNA, as compared to the WT whereas N2A produced similar amounts of cM DNA to WT (Fig 3C, lanes 5–7). To verify that these circular SS DNA bands were indeed derived from minus strands but not plus strands (which could arise due to nicking of CCC DNA), we performed the Southern blot analysis of the PF DNA using the minus strand- or plus strand-specific RNA probe. The results showed that the circular SS DNA was only of minus (but not plus) strand polarity (Fig 5A), indicating it was indeed derived from the cM-RC DNA following removal of the open plus strands [29]. After normalization of the cM-RC DNA to core RC DNA (the precursor to cM-RC DNA), N2E showed ca. 10-fold increased levels of cM-RC DNA as compared to WT (Fig 5B). Also, after normalization of levels of CCC DNA (which is likely the product from cM-RC DNA) to those of cM-RC DNA, N2E showed ca. 2.5-fold decreased cM-RC DNA compared to the WT (Fig 5C). These results thus suggested that N2E preferentially stimulated the production of cM-RC DNA, an early step in CCC DNA formation, but didn't simulate as strongly as (or might even have inhibited) the later step of cM-RC DNA to CCC DNA conversion. Again, the N2A mutant didn't significantly affect the levels of CCC DNA or cM-RC DNA via intracellular amplification. These results suggested that the core protein may modulate different steps in CCC DNA formation prior to the covalent closing of the minus strand as well as subsequent steps following cM-RC DNA formation (see Discussion below). 10.1371/journal.ppat.1008459.g005Fig 5 NTD phosphorylation could affect cM-RC DNA formation. The same PF-DNA samples shown in Fig 3. were treated with the exonuclease I and III before detection by Southern blot analysis, using strand specific riboprobes to detect either the minus (-) or plus (+) strand DNA separately (A). M, DNA size marker in kilo-basepairs (kbp); CCC, CCC DNA; cM, closed minus strand DNA. The Southern blot images shown were from phosphorimaging scan. B and C. Quantitative results from multiple experiments. B. cM-RC DNA normalized to core RC DNA. C. CCC DNA normalized to cM-RC DNA. All normalized values from the WT HBc were set to 1.0. N2E capsids showed increased levels of phosphorylation in vitro by the endogenous kinase To test whether the HBc NTD S44/S49 sites could serve as phosphorylation sites by the endogenous kinase in HBV capsids, we isolated WT and mutant HBc capsids from human HEK293T cells and conducted the EKR in vitro. In addition to N2A and N2E, we also isolated several additional mutant capsids, including 3A/3E (with three major CTD sites of phosphorylation changed to A or E) [30], 7A/7E (with all seven CTD sites of phosphorylation changed to A or E) [30], and 9A/9E (with all seven CTD sites as well as the two putative NTD-S44/S49 sites changed to A or E), for the EKR analysis. It was notable that the EKR signal from N2E was stronger than WT (Fig 6), suggesting that phosphorylation of these two NTD sites might stimulate CTD phosphorylation. Indeed, all other E mutants tested (3E, 7E, 9E) showed higher EKR phosphorylation signals than the corresponding A mutants (Fig 6). These results suggested that HBc phosphorylation might be cooperative and coordinated (see also Discussion below). The 3E mutant showed EKR signals nearly as high as WT, suggesting that the four minor in vivo CTD sites of phosphorylation, as well as any other putative sites of phosphorylation outside CTD, were used in the EKR efficiently. That the 7A and 9A mutants showed almost no EKR signal and the 7E and 9E showed only very low EKR signals suggested that the endogenous kinase mostly phosphorylated CTD. Although the residual EKR signals from 7E and 9E on the agarose gel (Fig 6A) suggested that HBc could be phosphorylated by the endogenous kinase outside CTD during EKR, phosphorylation of the mutant HBc protein itself could not be confirmed by SDS-PAGE for these mutants (Fig 6B). Therefore, it remained possible that the EKR signals detected on the agarose gel (i.e., associated with the intact capsid) (Fig 6A) might be due to phosphorylation of some other capsid-associated protein(s), although no other significantly phosphorylated proteins were detected by SDS-PAGE in Fig 6B as we reported previously [45]. On the other hand, it was also possible that the 7E and 9E mutant HBc proteins were phosphorylated (i.e., outside CTD) during the EKR in vitro but only at very low levels, which were below the limit of detection by the SDS-PAGE analysis. 10.1371/journal.ppat.1008459.g006Fig 6 EKR using WT and mutant HBV capsids. The WT and mutant HBc expression constructs were transfected into HEK293T cells. In addition to the N2A and N2E mutants above, additional HBc mutants tested here included 3A/3E (with three major CTD sites of phosphorylation—all SP motifs—changed to A or E) [30], 7A/7E (with all seven CTD sites of phosphorylation changed to A or E), and 9A/9E (with all seven CTD sites as well as the two putative NTD-S44/S49 sites changed to A or E). Cytoplasmic lysate was prepared from the transfected cells using 1% NP-40 five days after transfection. The lysate was treated with 0.5 ug/ul proteinase K at 37°C for one hr before EKR in the presence of [γ-32P]ATP. The reaction products were resolved on the native agarose gel (A) or by SDS-PAGE (B). Total capsid (A) or total core protein (B) levels were measured by chemiluminescence western blot assay using the HBc antibody T2221 (top). Radiolabeled (phosphorylated) capsid or core protein levels resulting from the EKR were measured using phosphorimaging (bottom). Ca, capsids; C, HBc protein (subunit). C. Phosphorylation efficiency during EKR was determined by normalizing the levels of labeled core protein to total core protein from B, with that from the WT capsid set to 1.0. ND, not detectable. Phos-tag gel system revealed a high and variable degree of phosphorylation of the WT and mutant HBc proteins The EKR results above suggested that the N2E mutant might increase HBc phosphorylation in cells. To look further into this possibility, we adopted the Phos-tag gel system to analyze the HBc phosphorylation level for the WT and mutant HBc expressed in human cells in the absence of viral replication, which can affect HBc state of phosphorylation (see Introduction). Protein mobility on this type of gel is directly proportional to the degree of phosphorylation [50, 59, 60]. Compared to the non-phosphorylated HBc expressed in E coli, the predominant protein species of WT, N2A, and N2E HBc proteins from human cells all migrated much slower (Fig 7, lanes 1–4), consistent with a high degree of phosphorylation of these proteins in human cells, as shown recently for the WT HBc [50]. A few minor, less phosphorylated species, which migrated faster than the predominant species from human cells but still much slower than HBc from E. coli, were also detectable for all three proteins. Furthermore, consistent with enhanced phosphorylation of N2E than WT or N2A, the major N2E species migrated even slower than the WT HBc and N2A migrated similarly to the WT, as observed above with regular SDS-PAGE analysis (Fig 2A). 10.1371/journal.ppat.1008459.g007Fig 7 Phos-tag SDS-PAGE analysis of WT and mutant HBc proteins. HEK293T cells were transfected and cytoplasmic lysate from transfected cells were prepared as in Fig 5. The lysate was resolved on the Phos-tag gel, and HBc proteins were detected by chemiluminescence western blot assay using the mAb T2221 (HBc NTD) (A) or 6–1 (HBc CTD) (B). The HBc protein (non-phosphorylated) expressed and purified from E. coli was include as a control (lane 1). C-P, phosphorylated HBc; C-deP, dephosphorylated (non-phosphorylated) HBc. We also included the HBc 3A, 3E. 7A, 7E, 9A and 9E mutants isolated from human cells in the Phos-tag gel analysis. In contrast to the relatively homogenous (and high) degree of phosphorylation of WT, N2A and N2E, 3E, and esp. 3A exhibited a much lower but highly heterogeneous pattern of phosphorylation, as evidenced by the multiple intense bands migrating well below the predominant WT (or N2A or N2E) band but still migrating slower than HBc from E. coli (Fig 7, lanes 5, 6). The much slower mobility of 3E compared to 3A on the Phos-tag gel again indicated the much stronger phosphorylation 3E compared to 3A in human cells, consistent with the in vitro EKR results above (Fig 6). 7A and 7E, with all CTD sites of phosphorylation eliminated, migrated like the non-phosphorylated HBc from E. coli, indicating the NTD sites S44/S49 (or any other potential phosphorylation sites in NTD or the linker of HBc) were not phosphorylated to any significant degree in human cells in the absence of NC maturation (Fig 7, lanes 7, 8). Interestingly, the 9E mutant migrated clearly slower than 7A/7E or the E. coli HBc (Fig 7, lane 10), suggesting that phosphorylation at the NTD S44/S49 sites could stimulate HBc phosphorylation outside CTD, i.e., other potential sites within NTD and/or the linker, consistent with the EKR results above (Fig 6). The reason for the slightly slower mobility of 9A (Fig 7, lane 9) relative to 7A/7E remains unclear but could suggest that there was residual HBc phosphorylation in 9A but not in 7A or 7E. To exclude the possibility that the multiple HBc species detected on the Phos-tag gel were derived from degradation from the C-terminal end, which could conceivably occur due to the unstructured nature of the HBc CTD, we used two different anti-HBc antibodies to detect the HBc proteins resolved by the Phos-tag gel in Fig 7. In Fig 7A, the HBc proteins were detected using the NTD mAb, T2221, targeted to an epitope around HBc position 130–140 [22], which should detect all HBc proteins irrespective their state of phosphorylation. In Fig 7B, we used the mAb 6–1, which is directed at an epitope at the extreme C-terminal end of HBc (requiring the last HBc residue Cys183) [30]. The mAb 6–1 detected essentially all the HBc species detected by T2221, indicating that there was no significant C-terminal degradation and the multiple species detected following the Phos-tag gel resolution mostly, if not exclusively, represented the various HBc phosphor-isoforms (see Discussion below). Confirmation of enhanced CTD phosphorylation of N2E by phosphorylation state-specific antibodies The in vitro EKR results as well as the Phos-tag gel analysis indicated that N2E, mimicking HBc NTD phosphorylation, could stimulate CTD phosphorylation by the endogenous kinase and in host cells. Taking advantage of our recently developed CTD specific mAbs that selectively recognize phosphorylated vs. non-phosphorylated CTD [22, 30, 43], we determined directly the state of CTD phosphorylation of WT, N2A, and N2E HBc proteins. Thus, WT and mutants HBc proteins expressed in human cells were resolved by SDS-PAGE (total HBc proteins, assembled and any free subunits) or agarose gel electrophoresis (only assembled capsids) and detected using different CTD antibodies as well as the NTD mAb T2221, which was used to detect all HBc proteins (irrespective phosphorylation state) for normalization of phosphorylated or non-phosphorylated HBc proteins. The mAbs selective for phosphorylated CTD mAbs didn't reveal significant differences among the different HBc proteins, likely due to the fact that the vast majority of the WT HBc protein expressed in human cells are normally heavily phosphorylated at CTD (Fig 7 above) [30, 43, 50, 60] such that any increase in CTD phosphorylation in N2E would not be dramatic (e.g., from 90% to 95% of the total protein). In contrast, a decrease in the (normally minor) non-phosphorylated CTD (from 10% to 5% in the above example) could
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