Figure 1.
Schematic diagram of DHBc domains and GST-DHBc CTD (DCC) fusion constructs.
A. DHBc contains an N-terminal assembly domain (NTD) from amino acids 1–185, a flexible linker region from amino acid 186 that extends into the CTD, which includes the phosphodomain from 230–262. B. 1–228, a CTD-deletion mutant. C. Each WT fusion protein contains a differing length of the CTD and was named for the amino acid position where the tail fragment begins (229 or 196). DCC229 contains the phosphodomain from residue 229 with all six known WT phosphorylation sites (S230, S232, T239, S245, S257, and S259, panel E). DCC196 contains the phosphodomain plus the additional upstream 33 amino acids of the linker region. DCC196-228 contains amino acid residues 196–228 (the linker region), deleting the entire phosphodomain with all six phosphorylation sites. D. The linker sequence with the basic cluster underlined. E. Alanine (A) or aspartic acid (D) substitutions were made in the full-length DHBc, as well as in the context of each WT fusion protein, with the resultant mutants the same length as the WT version. The full-length A and D mutants were named by the positions of the mutated phosphosites they contain: SSAAAA, SSDDDD, AAAAAA, and DDDDDD. The GST-DCC fusion proteins were named by their amino acid starting position and their phosphosite substitutions. The final four phosphorylation sites were substituted by A in 229-SSAAAA or D in 229-SSDDDD. 196-SSAAAA contained amino acids 196–262 with the last four phosphorylation sites mutated to A and maintained S230 and S232. Similarly, 196-SSDDDD maintained S230 and S232, while the last four phosphosites contained D substitutions. Finally, all six phosphorylation sites were substituted by A in 196-AAAAAA or D in 196-DDDDDD.
Figure 2.
Effect of DHBc phosphorylation site mutations on protein expression and DNA synthesis in HEK293T cells.
HEK293T cells were cotransfected with pCMV-DHBV/C-, which expresses a core-defective DHBV genome, and the indicated WT or mutant core expression plasmid. Five days post-transfection, cells were lysed and core DNA was isolated as previously described. DHBc was detected by western blotting with antibody against DHBc (bottom panel) and core DNA was detected by Southern blotting analysis with a DHBV DNA probe (top panel). RC, relaxed-circular DNA; SS, single-stranded DNAs; IC, internal control plasmid added during the DHBV DNA extraction.
Figure 3.
Metabolic labeling of GST-DCC constructs in HEK293T cells.
Cells were transfected with the indicated plasmids expressing either GST or GST-DCC fusion proteins, then were metabolically labeled with either [35S]cysteine/methionine or [32P]orthophosphate on the third day post-transfection. A. 35S- or B. 32P-labeled proteins were purified without RNase A digestion using GSH affinity resin and were visualized by silver staining (top panels) and autoradiography (bottom panels). Arrowheads indicate GST or GST-DCC fusion proteins. *, non-specific background bands that appeared in all transfections or untransfected cells.
Figure 4.
GST co-purification and MS analysis.
HEK293T cells were transfected with the indicated GST-DCC fusion constructs. GST fusion proteins (short arrows) were purified from RNase A-treated lysates using GSH affinity resin. Co-purifying proteins were resolved by SDS-PAGE and detected by CB staining. Protein bands of interest were excised, subjected to in-gel trypsinization, and identified by mass spectrometry (long arrows). *, non-specific background bands that appeared in all samples, including GST alone.
Figure 5.
Coimmunoprecipitation and pulldown experiments.
A. HEK293T cells were cotransfected with plasmids to express GST-DCC fusion constructs and 3X-Flag-tagged B23 protein. Coimmunoprecipitation experiments were conducted with an anti-Flag antibody. GST pulldown, using GSH affinity resin, was performed in parallel. B. HEK293T cells were transfected with GST-DCC fusion protein expression plasmids. GST pulldown, using GSH affinity resin, was performed. Samples were analyzed by western blotting with antibodies to detect B23 (top panel) and I2PP2A (middle panel). C. HEK293T cells were cotransfected with plasmids to express GST-DCC fusion proteins and I2PP2A-Flag-HA. Immunoprecipitation was conducted with a mixture of antibodies against Flag and HA. Samples were analyzed by western blotting with antibodies to detect I2PP2A (top panel) and GST (bottom panel). LC, light chain.
Figure 6.
GST pulldown with E. coli-derived DCC fusion proteins.
GST or GST-DCC fusion proteins were purified from E. coli. The purified proteins were added to RNase A-treated HEK293T cell lysates. HEK293T cellular proteins that were pulled down by the fusion proteins were visualized by silver staining (A, lanes 1–4) and western blotting (B, lanes 1–4). For comparison, the same GST-DCC fusion proteins were expressed and purified from HEK293T cells and the co-purifying cellular proteins were shown in A & B, lanes 5–7. *, non-specific background bands that appeared in all samples, including GST alone.
Figure 7.
Interaction between the in vitro-translated full-length DHBc proteins or GST-DCC fusion proteins and B23 and I2PP2A.
A. Full-length DHBc proteins or firefly luciferase were translated in RRL and interaction with B23 or I2PP2A purified from HEK293T cells was determined by coimmunoprecipitation. Lanes 1–7 show RRL-translated input proteins (autoradiogram). Lanes 8–14 show the B23 immunoprecipitate (autoradiogram, top panel, and CB stain, bottom panel) and lanes 15-21 show the I2PP2A immunoprecipitate (autoradiogram, top panel, and CB stain, bottom panel). B. GST-DCC fusion proteins were translated in RRL and interaction with B23 or I2PP2A purified from HEK293T cells was determined by coimmunoprecipitation. The WT DCC196 fusion protein was either dephosphorylated with CIAP or left untreated. Lanes 1–6 show RRL-translated input proteins. Lanes 7–12 show the B23 immunoprecipitate (autoradiogram, top panel, and CB stain, bottom panel) and lanes 13–18 show the I2PP2A immunoprecipitate (autoradiogram, top panel, and CB stain, bottom panel). I2, I2PP2A-Flag-HA.
Figure 8.
Interaction of the HBc CTD with B23 and I2PP2A.
A. Schematic diagram of HBc domains. Depicted are the N-terminal assembly domain (NTD) from amino acids 1–140 and the CTD that includes a flexible linker region from amino acid 141–149 and a phosphodomain from 150–183. B. GST-HBc CTD (HCC) fusion construct. The WT HCC141 fusion protein contains the CTD from residue 141 to the end of HBc. C. The sequence of the HBc CTD, with the three major phosphorylation sites (S155, S162, and S170) and alanine (A) or glutamic acid (E) substitutions indicated. The phosphosite mutant GST-HCC fusion proteins were named for the mutated phosphosites they contain: HCC141-AAA or HCC141-EEE. The four basic clusters are underlined. D. CB staining (bottom panel) and western blotting (top and middle panels) analysis of GST (lane 1) and GST-DCC fusion proteins (lanes 2–4) purified from HEK293T cells.
Figure 9.
Possible mechanisms for the differential binding of phosphorylated and unphosphorylated hepadnavirus core protein to B23 and I2PP2A.
A. The lack of phosphorylation in the DHBc phosphodomain permits an open conformation in the CTD and renders the upstream linker region containing the host protein binding site accessible for B23/I2PP2A interactions (I). The negative charges introduced by CTD phosphorylation decrease the overall positive charge of the CTD and thus weaken the electrostatic interactions with the acidic regions of B23 and I2PP2A (II). Alternatively, the negative charges introduced into the phosphodomain may enable it to fold over and interact with the basic upstream linker and thereby mask the host protein binding site located within the linker (III). B. Similarly, phosphorylation of the HBc CTD would result in a decreased overall positive charge of the CTD that weakens the electrostatic interactions with the acidic regions of B23 and I2PP2A (I and II). In this case, the positively charged host protein binding sites are interspersed with the phosphorylation sites.