Fig 1.
Structural and functional aspects of HBc.
(A) Domain organization. Numbers refer to aa positions. The immunodominant c/e1 epitope, in the 3D structure at the tips of the capsid spikes, and mutation F97L are indicated. Residues 141–149 (blue) link the assembly domain to the CTD which encompasses 16 basic R residues (red) and one acidic E residue (green). Phosphorylation of the S/T residues would reduce positive charge. (B) Non-sequence-specific RNA packaging by HBc in E. coli. HBc forms dimers that spontaneously assemble into CLPs. With HBc183, the basic CTDs mediate encapsidation of ~3–4 kb non-sequence-specific RNA. In HBV infection this would compete with specific encapsidation of viral pgRNA. (C) Basic functions of HBc in the HBV life-cycle. HBc is required to interact with different forms of viral genomic nucleic acid (NA) while avoiding interactions with irrelevant NAs. Progeny virus particles leaving a cell must be environmentally stable, yet upon infection of a new cell they must orderly release the viral NA for nuclear cccDNA formation. This implies temporal changes in NA binding capacity and capsid structure, including CTD disposition. (D) Options for capsid structure modulation. Regulated structure modulation is manifest by the selective, L protein dependent envelopment of dsDNA (or no NA) containing capsids (symbolized by the altered shape and color of the HBc dimer in the center) but not of capsids harboring RNA or ssDNA. Sensing the type of internal NA must involve the CTDs, and likely their phosphorylation status. The negatively charged phosphoryl groups could directly affect CTD—assembly domain interactions (i) and/or CTD disposition (ii), or act indirectly through altered NA binding (iii). Mutations like F97L evade this dependency, possibly by a priori promoting an envelopment-proficient structure.
Fig 2.
Coexpression with SRPK1 strongly reduces HBc CLP RNA content.
(A) Visualization of RNA content in HBc183_F97L CLPs by NAGE and RNA vs. protein staining. HBc183_F97L CLPs expressed in the absence (-) or presence (+) of NHisSRPK1ΔNS1 were enriched to sucrose gradient sedimentation. The indicated fractions were separated by NAGE in the presence of ethidium bromide (EB); subsequently proteins were stained by Coomassie Blue (CB). Note the high vs. low EB to CB signal ratios in the -SRPK1 vs. the +SRPK1 samples but their nearly identical mobility. Negative staining EM (EM) did not reveal differences between the two HBc183_F97L samples, nor between the respective wild-type HBc183 samples (S3B Fig). (B) Impact of NHisSRPK1ΔNS1 coexpression vs. CTD truncations on RNA content. CLPs from the indicated truncated HBc variants were subjected to NAGE in EB-free gels, then stained with Sybr Green 2 (SG2) for RNA; after documentation the gels were counterstained with Sypro Ruby (SR) for protein. Green and red signals were semiquantitatively evaluated by laser scanning at the indicated conditions (grey-scale panels). The ratios of SG2 to SR fluorescence in each sample are given as percent of the respective value for HBc183 CLPs expressed without kinase. (C) Similarly strong reduction in absolute CLP RNA content by NHisSRPK1ΔNS1 coexpression as by CTD deletion. RNA contents of the indicated HBc CLPs (in nt per HBc protein monomer (N/P)) were calculated from UV/VIS spectra [73]. Black bars show the mean N/P values ± SD (n≥3). For comparison, the relative values derived from (B) are shown as grey bars; the scale was set such that the 100% value (HBc183) matched the mean N/P value (~16) of the same sample. In either assay, the RNA content of HBc183 and HBc183_F97L CLPs coexpressed with SRPK1 was as low as that of HBc140 CLPs.
Fig 3.
Strong and selective retardation of SRPK1-coexpressed wild-type and F97L HBc183 in Phos-tag SDS-PAGE.
Samples from the indicated gradient-enriched CLPs expressed alone (-) or coexpressed with SRPK1 (+) were separated by normal SDS-PAGE (left panel) or by Mn2+ Phos-tag SDS-PAGE (right panel). The weak bands at the 45 kDa position marked by arrowheads (left panel) represent SRPK1 that had co-sedimented with the HBc CLPs.
Fig 4.
Accurate MS confirmation of seven SRPK1 phosphorylation sites in the HBc CTD.
(A) Genetic structure of the non-assembling CTD fusion constructs. The dual promoter vectors carried an ORF for non-His-tag kinase (SRPK1ΔNS1, or the catalytic domain of PKA) plus an ORF for a His-tagged GFP protein to which the CTD of HBc variant C183A was linked via a TEV protease recognition site; see text and S1 Protocol for details. (B) Calculated MH+ masses of the indicated CTD derivatives vs. observed m/z values. Note the excellent agreement for a seven-fold phosphorylated CTD species in the +SRPK1 sample. (C) SDS-PAGE of the purified CTD peptides from the indicated GFP fusion proteins. The sequence of the chemically synthesized N-acetylated sCTD peptide carrying the genuine C183 residue is shown in (B). (D) MALDI-TOF spectrum of the +SRPK1 CTD sample from (C). Note the predominance of the m/z peak corresponding to seven-fold phosphorylated CTD. Mass spectra for the other CTD peptides are shown in S6 Fig.
Fig 5.
Mapping the SRPK1 phosphorylation sites in the HBc CTD by mutation and MS.
(A) Position of S/T>A mutations, dominant m/z peak observed and calculated MH+ mass of best-fitting phospho-species. Note that S181A is the only single-site variant with a best-fit m/z for seven-fold phosphorylation; all other single-site mutants showed the best match to six phosphoryl groups. (B) Impact of number and position of phospho-sites on Phos-tag SDS-PAGE mobility. Following Phos-tag SDS-PAGE HBc proteins were analyzed by immunoblotting using the HBc assembly-domain specific mAb 1D8. All SRPK1-coexpressed proteins contained seven (HBc183, S181A) or six phosphoryl groups (all others) but variants S170A (*) and especially T160A and S162A (**) were less retarded, even though much more than three-fold PKAcd phosphorylated HB183. The impact of the mutations on recognition by the phospho-CTD specific mAb T2212 is shown in S12 Fig. (C) Impact on CLP RNA content. CLPs from the indicated HBc proteins were analyzed by NAGE and sequential SG2 vs. SR staining using an improved, background-reducing protocol (Materials and Methods). SG2: SR ratios are indicated in percent of that in non-phosphorylated HBc183 CLPs. Bars show the mean of ≥3 determinations; error bars represent standard deviation (SD). Original NAGE fluorograms plus data for additional variants are shown in S8 Fig. Note the exceptionally low SG2 to SR ratio for S181A and the higher ratio for S170A vs. all other single S/T>A variants (red arrows). The minor impact of PKA and PKC coexpression on RNA content (blue bars) correlated with the predominance of only three-fold phosphorylated species (S7 Fig) and their less pronounced retardation in Phos-tag SDS-PAGE (Fig 5B and Fig 9C).
Fig 6.
Little impact on SDS sensitivity of CLPs by high phosphorylation, low RNA content and F97L mutation.
About 5 μg HBc protein from the indicated CLP preparations were directly loaded (Ø), or after 30 min incubation in non-denaturing 6x DNA loading buffer (Ø*) or in SDS-containing DNA loading buffer (NEB Purple) at the indicated final SDS concentrations, then analyzed by NAGE. A 1 kb DNA ladder (M) plus 1 μg of untreated HBc183 CLPs served as markers. EB fluorescence signals (top) were recorded using a laser scanner (excitation 532 nm/O580 nm filter). Proteins were subsequently stained by CB. In all samples the intact CLP bands became fuzzier at 0.024% SDS compared to the untreated controls, and a distinct upward mobility shift occurred at 0.048% SDS. For the non-phosphorylated CLPs (-SRPK1) this was accompanied by visible release of RNA.
Fig 7.
Strong impact on CLP trypsin sensitivity by high level phosphorylation and low RNA content but not the F97L mutation.
(A) Full cleavage of accessible but not CLP-borne CTD regardless of phosphorylation. NHis-GFP-CTD fusion protein expressed with or without SRPK1, and HBc183 CLPs were incubated as indicated with 1% (w/v) trypsin; digestion was stopped by AEBSF and reactions were analyzed by SDS-PAGE and CB staining. Non-phosphorylated and phosphorylated GFP-CTD fusion protein were completely cleaved with comparable kinetics. In the CLPs less than half the HBc183 subunits were rapidly cleaved into a 15 kDa product; the 14 kDa lysozyme band (ly) in some samples originates from the cell lysis procedure. (B) Non-phosphorylated HBc183 CLPs. Trypsin incubation and inhibition were done as in (A). Reaction products were analyzed by SDS-PAGE and NAGE (lower panels) with EB and CB staining. Note the overall unaltered NAGE signals despite cleavage of about half the subunits; intact versus cleaved chains are labeled 1 and 4. (C) Non-phosphorylated HBc183_F97L CLPs. As for HBc183 CLPs, uncleaved chains (1) plus a 15 kDa band (4) were the major products (top panel); NAGE showed unaltered RNA content and CLP mobility (bottom). (D) SRPK1-phosphorylated HBc183 CLPs. Note the additional intermediate mobility bands 2 and 3 (top panel). Running the 16 h sample along a set of C terminally truncated HBc proteins (lower panel) allowed to approximately map the cleavage sites, as outlined in the scheme at the bottom. (E) SRPK1-phosphorylated HBc183_F97L CLPs. The cleavage pattern with bands 1, 2, 3, 4 and accumulation of band 3 (top panel) was as for SRPK1-coexpressed HBc183; also in NAGE (bottom panels) the much weaker RNA signals as well as the equally strong protein signals remained unaltered.
Fig 8.
CryoEM comparison between non-phosphorylated and seven-fold CTD-phosphorylated HBc183 CLPs.
(A) Surface representations from image reconstructions at 7.8 Å and 7.9 Å resolution. The A, B, C, and D monomers of one asymmetric unit in the T = 4 HBc183 CLP are colored blue, cyan, red and yellow. (B) Density representations of equatorial slices. Non-phosphorylated HBc183 CLPs display an unstructured shell of high density underneath the inner surface (top) which was absent from the SRPK1 coexpressed particles (bottom) with their much lower RNA content. (C) Impact of seven-fold phosphorylation and/or low RNA content on the structure of the assembly domain. The surface representations show close-ups of the two-fold symmetry axes at 7.8 Å and 7.9 Å resolution, sharpened with B-factors of -797 Å2 and -979 Å2, respectively. For clarity densities at radii <127 Å and small disconnected speckles were removed. Surface coloring reflects differences between the normalized unsharpened maps. Only differences with a confidence level of >99% (Student´s t-test) are shown. Colored areas highlight extra densities in non-phosphorylated (red) and phospho-HBc CLPs (blue). The length of the scale bar equals 2 nm. Numbers 2, 3 and 5 indicate the respectice symmetry axes. (D) Ordered internal protein density in seven-fold phosphorylated HBc183 CLPs. Shown are luminal close-ups of the five-fold symmetry axes; density accounted for by modelling the crystal structure of HBc149 CLPs (pdb: 1QGT) into the reconstructions is color-coded as in (A); not accounted for density is shown in white. Note the tube-like structures extending all the way to the feet of the spikes (one of the five highlighted as black continuous line) in the phospho-HBc CLPs. The extensions appear in direct contact with R112, part of the inner CLP lining (S11B Fig).
Fig 9.
The bulk of HBc183 in capsids from human hepatoma cells is highly phosphorylated.
(A) Enrichment of particulate HBc by Nycodenz gradient sedimentation. Cytoplasmic lysate from HBV producing HepG2.117 cells was sedimented through a Nycodenz gradient. Fractions were analyzed by SDS-PAGE and CB staining (top panel; asterisks mark a 21 kDa band possibly representing HBc183; the complete gel is shown in S12A Fig); by NAGE followed by immunoblotting with the anti-HBc assembly domain mAb 312 as PO conjugate (middle panel); and by hybridization with a 32P-labeled HBV probe. (B) Southern blot for capsid-borne HBV DNA in individual gradient fractions. M, DNA marker comprising HBV-specific fragments of the indicated sizes loaded in native ds form, or in heat-denatured ss form. (C) Phos-Tag SDS-PAGE immunoblot. Aliquots from the respective gradient fractions and recombinant HBc183 coexpressed with the indicated kinases, or not (ø), were separated by Phos-Tag SDS-PAGE and immuno-blotted with mAb 1D8. Short exposure showed one band with comparably strong retardation as SRPK1-phosphorylated HBc183. Longer exposure (left panel) revealed weak additional bands with mobilities similar to those of unmodified and PKC and PKA phosphorylated HBc183 (arrowheads). (D) Enveloped capsids contain phosphorylated HBc183. PEG-precipitated particles in supernatants from HepG2.117 cells, or from an HBc183 producing Huh7 line, H4-15 were analyzed by NAGE immunoblotting alongside E. coli HBc183 CLPs. The blot was sequentially probed with mAb T2212 (anti-phospho-CTD), mAb 312, and mAb 9H9 (anti-HBs). Note that T2212 detected only HBc from eukaryotic cells, including a low mobility species that comigrated with HBsAg and was absent from the H4-15 samples; it therefore represents enveloped capsids. Additional data employing mAb T2212 are presented in S12 Fig.
Fig 10.
Implications of the high HBc phosphorylation—Low CLP RNA content correlation for specific pgRNA encapsidation in HBV infection.
In E.coli nonphosphorylated HBc183 CTDs have maximal positive charge and thus maximal electrostatic RNA binding capacity, generating RNA-filled CLPs (Fig 1B). Seven-fold phosphorylation by SRPK1 neutralizes most positive charges, leading to virtually empty capsids. The similarly strong Phos-tag retardation of most HBc183 from human cells indicates similarly high phosphorylation, whether by SRPK1 or a combination of kinases. Strongly reduced RNA binding favors formation of empty capsids and then empty virions, perhaps the dominant pathway in vivo [52]. The backside of avoiding irrelevant RNA packaging is a loss in specific pgRNA interaction capability. As blocking just one of the seven SRPK1 phosphorylation sites restored substantial RNA packaging in bacteria we propose that the pgRNA/P protein complex, besides other host factors, also carries a protein phosphatase (PPase) activity. This PPase would dephosphorylate only nearby HBc CTDs, and thus locally unleash their RNA binding potential in proximity to pgRNA. This would go on with further HBc dimers until the shell is completed. Progressive dephosphorylation of the now internal CTDs by the PPase activity could maintain electrostatic homeostasis, especially during the near doubling of negative charges associated with dsDNA formation. Targeted nucleocapsid destabilization upon infection of a new cell could occur by CTD re-phosphorylation and completion of plus-strand DNA.