Fig 1.
Summary of the effects of HBc linker mutants on different steps of viral replication.
A. Alignment of HBc linker sequence (`141–149), including the last residue of NTD (140), with the consensus PP2A-B56 binding motif. B. Mutations in the B56 binding motif and predicted effects on B56 binding, and observed effects of the mutations on HBc CTD dephosphorylation at two distinct mAb epitopes—A701 (T160/S162) and 25–7 (S178)—both normalized to total HBc levels, capsid assembly (Ca assem), pgRNA packaging (pgRNA/NC), SS DNA synthesis (SS/pgRNA), RC DNA synthesis (RC/SS), mature NC stability, CCC DNA synthesis (CCC/RC), and DNA virion secretion (virion DNA/RC), with the level for each parameter from the WT HBc set to 1.0. N/A, not applicable due to the lack of detection of RC DNA; -/+, very weak signal detected; -, below the limit of detection; unstab, unstable NC; stab, stable NC as WT; *, CCC DNA decreased during infection. +, CCC DNA generated but no detectable core RC DNA, so CCC DNA/RC DNA normalization was impossible. The red font in the mutant LC indicates the amino acid change from the WT sequence (S141T and VV148/149II).
Fig 2.
Effects of HBc linker mutations on CTD state of phosphorylation.
A. Schematic of HBc domain structure and mAb epitope locations. The NTD and CTD are depicted as pink and blue boxes, with the linker peptide in between as a thin line. Epitopes recognized by the HBc-specific mAbs: 1D8 for NTD (ca. 70–80), independent of CTD state of phosphorylation; A701 for unphosphorylated S162/T160; and 25–7 for unphosphorylated S178. B. HepG2 cells were transfected with indicated HBc expression constructs and were harvested seven days post-transfection. Cytoplasmic lysates from the transfected cells were resolved by SDS-PAGE (top) or NAGE (bottom) and detected by western blot analysis, using the NTD-specific mAb 1D8 (for total HBc proteins) and two non-phosphorylated CTD-specific mAbs, A701 and 25–7. C. The CTD dephosphorylation signals from total HBc (quantified following SDS-PAGE; black bars) or capsids (quantified following NAGE; gray bars) were normalized to total HBc or capsid signal (A701/1D8, top; 25-7/1D8, bottom). The normalized A701 or 25–7 signals of the linker mutants are shown relative to that of the WT HBc, which was set to 1.00. *, P<0.05; **, P<0.01;***, P<0.001.
Fig 3.
Phos-Tag SDS-PAGE and immunoblot analysis of HBc linker mutants.
HepG2 cells were transfected with HBc mutants expression constructs and harvested seven days post-transfection. Cytoplasmic lysates of the transfected cells were resolved by Phos-tag SDS-PAGE and detected by western blot analysis, using the HBc NTD mAb 1D8 for total HBc (A) or 25–7 for dephosphorylated S178 (B). 1 & 2, hyperphosphorylated HBc species; 3, hypophosphorylated HBc species; 4, non-phosphorylation HBc, *, non-specific signal. The top and bottom panels in A are long and short exposure of the same membrane.
Fig 4.
Effect of linker mutations on capsid assembly and pgRNA packaging.
HepG2 were co-transfected with the indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and harvested seven days post-transfection. A. Cytoplasmic lysates from the transfected cells were resolved by NAGE and transferred to nitrocellulose membrane. The anti-sense HBV RNA probe and the mAb 1D8 were used to detect packaged pgRNA and assembled capsids respectively (top panel). Total HBc levels were measured, following SDS-PAGE and transfer to PVDF membrane, using mAb 1D8 against HBc NTD (bottom panel). Ca, capsid; C, HBc monomer. B. Quantitative results from multiple experiments shown in A. Capsid assembly efficiency was determined by normalizing the levels of capsids to those of total HBc protein, and RNA packaging efficiency by normalizing the levels of RNA packaging to those of capsids, with the efficiency from WT set to 1.0. *, P<0.05; **, P<0.01;***, P<0.001.
Fig 5.
Effect of linker mutations on core DNA.
HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and were harvested seven days post-transfection. HBV NC-associated DNA (core DNA) was extracted from cytoplasmic lysate without (A) or with TURBO DNase digestion (B), and detected by Southern blot analysis. Input plasmid DNA (but not viral replicative DNA) was removed with Dpn I before Southern blot analysis in A. The viral DNA signals (RC and immature DS DNA) digested by the nuclease were marked by the dotted boxes (B). RC, RC DNA; SS, SS DNA. C. Quantitative results from multiple experiments shown in A. SS DNA synthesis efficiency was determined by normalizing the levels of SS DNA to the pgRNA signals in Fig 4. RC DNA synthesis efficiency was determined by normalizing the levels of RC DNA to the SS DNA signals. The efficiencies from WT was set to 1.0. *, P<0.05; **, P<0.01;***, P<0.001.
Fig 6.
Effect of linker mutations on virion secretion.
HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and the culture supernatant was collected seven days post-transfection. A. Concentrated culture supernatant was resolved by agarose gel electrophoresis and transferred to nitrocellulose membrane. HBV DNA in virions and naked capsids were detected by using the HBV DNA probe. B. DNA virion secretion efficiency was determined by normalizing levels of virion DNA to cytoplasmic RC DNA levels (as shown in Fig 5). The efficiency from WT was set to 1.0. V, virion; Ca, capsid. **, P<0.01;***, P<0.001.
Fig 7.
Effects of linker mutations on CCC DNA formation.
HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids (A), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA (B), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P<0.05; **, P<0.01;***, P<0.001.
Fig 8.
Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein.
The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L- derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P<0.01.
Fig 9.
Effect of linker mutations on infection.
HBV inocula were prepared from HepG2 cells co-transfected with the HBc-expression construct plus the HBc-defective replicon as shown in Fig 6, and used to infect HepG2-NTCP cells. PF DNA was extracted three days post-transfection and analyzed by Southern blot analysis, with (lane 1–6) or without (lane 7–12) pretreatment with exonuclease I and III (Exo I&III). Quantitative results are shown in the graphs to the right. The small m. w. DNA smear present in certain linker mutants (lane 3–6) is indicated by the bracket. PF-RC, PF-RC DNA; CCC, CCC DNA; ND, not detected. **, P<0.01;***, P<0.001.
Fig 10.
Effects of PP2A inhibitors on HBc dephosphorylation in vitro.
The WT and 3E mutant HBc proteins were translated in RRL. The translated HBc, without any further treatment (untreated) or treated with NEBuffer 3 plus CIAP overnight at 37°C (CIP), with NEBuffer 3 plus a mixture of non-specific phosphatase inhibitors over night at 37°C (PPI), NEBuffer 3 alone overnight at 37°C (mock 1), with NEBuffer 3 plus 0.01% DMSO overnight at 37°C (CIAP) (mock 2), with NEBuffer 3 plus increasing concentrations of Fostriecin (10 nM, 20 nM, 40 nM, 80 nM) or Okadaic acid (1 nM, 10 nM, 100 nM), before further analysis. A. The 35S-labeled HBc proteins (WT and 3E) were resolved by SDS-PAGE and detected by autoradiography or western blot analysis using the HBc CTD dephosphorylation specific mAb 25–7. B. The HBc 3E reactions were resolved by NAGE. The 35S-labeled HBc proteins were detected by autoradiography (total HBc) or western blot analysis using the HBc specific polyclonal antibody (Dako). C. The HBc 3E reactions were resolved by Phos-tag SDS-PAGE, with the no-template translation reaction as a negative control, and 35S-labeled HBc proteins were detected by autoradiography. a & b: hyperphosphorylated 3E; c-e: hypophosphorylated 3E; f: non-phosphorylated 3E.
Fig 11.
Effect of PP2A inhibition on HBc dephosphorylation and viral DNA replication in cell culture.
A. Scheme for rapid and synchronized NC maturation and CCC DNA formation in cell culture. Following removal of tetracyclin (tet) from HepAD38 cells for two days to induce pgRNA expression packaging, PFA was added to arrest HBV DNA synthesis at the single-stranded (SS) DNA stage. Following 4 days of PFA treatment to accumulate SS DNA NCs, PFA was removed to allow rapid and synchronized synthesis of RC DNA from SS DNA and RC DNA conversion to CCC DNA during the ensuing one day before the cells were harvested. At the same time of PFA removal, tetracyclin was added back to turn off pgRNA synthesis and thus suppress further production of immature NCs. Fostriecin (20 μM, 40 μM, 60 μM) was added at the same time when PFA was removed. The cells were then harvested for analysis of HBc dephosphorylation (B) and viral DNA synthesis (C). B. HBc was resolved by SDS-PAGE and detected by western blot analysis, using the HBc CTD dephosphorylation (S178) specific mAb (25–7) or NTD mAb (1D8, for total HBc). The dephosphorylated HBc signal was normalized to total HBc signal. The normalized dephosphorylation signal of HBc treated with Fostriecin is shown in the graph relative to that of mock treatment, which was set to 1.00. C. HBV NC-associated DNA (core DNA, lane 1–6) and PF DNA (lane 7–12) were detected by Southern blot analysis. Lane 1 and 7 (induced): DNA from control cells cultured in the absence of tetracyclin or PFA for 7 days; lane 2 and 8 (induced-PFA arrest): DNA from cells harvested right at the end of PFA treatment before PFA release. RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA. D. Quantitative results of CCC DNA normalized to core RC DNA. The normalized CCC DNA signals from the cells treated with Fostriecin is shown relative to that of the mock treatment, which was set to 1.00. *, P<0.05; **, P<0.01.
Fig 12.
Effect of PP2A inhibition on HBV infection.
HepG2-NTCP cells were infected with HBV for one day. Following removal of the inoculum, the cells were mock treated or treated with the PP2A inhibitor Fostriecin (20 μM, 40 μM, 80 μM). HBV PF DNA was extracted three days post-infection and detection by Southern blot analysis. PF-RC, PF-RC DNA; CCC, CCC DNA. In the graph, PF-RC DNA and CCC DNA signals from Fostriecin-treated cells were compared to mock treatment, which was set to 1.00. *, P<0.05; **, P<0.01.
Fig 13.
Model of interactions between HBc linker and host protein phosphatase (kinase) in regulating multiple steps of viral replication.
The HBc domains (NTD, linker, CTD) are shown in the middle as horizontal boxes, with the linker sequence highlighted. Also highlighted are the last residue of NTD (L140, part of the PP2A-B56 consensus binding motif) and the three CTD sites where phosphorylation state was monitored in this study. The linker sequence is proposed to regulate dynamic CTD phosphorylation and dephosphorylation to control different stages of viral replication, among other mechanisms. The red and green arrows on the top denote the recruitment of cellular phosphatase (PP) and kinase (KI), respectively, by the indicated linker/NTD sites to modulate the indicated CTD phosphorylation sites. Shown at the bottom is a simplified scheme of HBV replication cycle, with the different stages affected by the different CTD phosphorylation sites and PP2A/CDK2 highlighted. The stimulatory and inhibitory effects of certain linker mutations on CCC DNA formation during intracellular amplification and infection, respectively, are denoted by the green and red arrows. See text for details.