Fig 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.
Fig 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.
Fig 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.
Fig 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.
Fig 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.
Fig 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.
Fig 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.
Fig 8.
Analysis of effects of NTD mutations on CTD phosphorylation using CTD-phosphorylation state specific antibodies.
HepG2 cells were transfected and cytoplasmic lysate from transfected cells were prepared as described in Fig 2. The lysate was resolved on a native agarose gel (A) or by SDS PAGE (B). HBc proteins were detected by chemiluminescence western blot analysis using the indicated mAbs selective for dephosphorylated (de-P) HBc (mAb A701 and 25–7) or with no selectivity for the phosphorylation state of HBc (total HBc) (mAb T2221). Quantitative results from multiple experiments are shown to the right. Levels of dephosphorylated HBc were normalized to those of total HBc, with the normalized value from the WT HBc set to 1.0. C, HBc protein; Ca, Capsid.
Fig 9.
Effects of CDK2 inhibitors on CCC DNA formation during HBV infection.
The PXB cells were infected with HBV and treated with the CDK2 inhibitor K03861 at the indicated concentrations (A) or CDK2 inhibitor III (CDK2i III; 125 nM) (B) at the same time. HBV PF DNA was extracted from the cells three days after infection and measured by Southern blot analysis using a 32P-labeled HBV DNA probe. Shown are representative Southern blot autoradiograms (phosphorimaging scan) of PF DNA, with the relative levels of CCC DNA indicated at the bottom and CCC DNA level from the mock-treated cells set to 1.0.
Fig 10.
Effects of CDK2 inhibition on CCC DNA formation via intracellular amplification in HepAD38 cells.
HBV pgRNA transcription were induced in HepAD38 cells by Tet removal. To accumulate SS HBV DNA, PFA was added into the culture medium on day two after Tet removal and maintained for the next four days. PFA was then removed to allow the synthesis of RC DNA and formation of CCC DNA. At the same time of PFA removal, the CDK2 inhibitor (K03861) was added. Twenty four hours later, HBV core DNA and PF DNA were extracted from the cells and measured by Southern blot analysis using a 32P-labeled HBV DNA probe. A. Representative Southern blot autoradiograms (phosphorimaging scan) of HBV core DNA (lanes 1–4) and PF DNA (lanes 5–8). B. Quantitative analysis of Southern blot results from multiple independent experiments. Data are expressed as CCC DNA levels normalized to those of core RC DNA, with the normalized CCC DNA level from the mock-treated cells set to 1.0.
Fig 11.
Model of HBc phosphorylation-dephosphorylation-rephosphorylation cycle in regulating NC assembly, maturation, and disassembly.
Upon translation from pgRNA, HBc subunits are phosphorylated at CTD (pCTD) and assemble into immature NCs incorporating the RT protein and pgRNA in the cytoplasm (Cyto). During NC maturation, pgRNA is converted to RC DNA by the RT protein within maturing NCs while they undergo dephosphorylation to facilitate RC DNA synthesis and stabilization of mature NCs, although mature NCs are relatively unstable as compared to immature NCs (dashed vs. solid line of the hexagon). Rephosphorylation of HBc, at both NTD and CTD (pNTD/pCTD), in mature NCs further destabilizes them (broken hexagon) and facilitates their uncoating and the release of RC DNA into the nucleus (Nuc). Some HBc subunits may remain associated with RC DNA in the nucleus and modulate its conversion to cM-RC DNA (cM) and ultimately to CCC DNA (CCC). In the transfection experiments, plasmid DNAs harboring the HBV genome (HBV plasmid) serves as surrogate CCC DNA to initiate HBV gene expression and replication. See text for details.