A pocket-factor-triggered conformational switch in the hepatitis B virus capsid

Abstract

Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid-envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the "synergistic double interaction" hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

Keywords: Triton; hepatitis B virus; hydrophobic pocket; solid-state NMR.

Fig. 1.

Structure and maturation of HBV virions. (A) Mature HBV is made by a nucleocapsid containing rcDNA, surrounded by an envelope in which the three L, M, and S surface proteins are inserted. (B) The three surface proteins share the S domain, and M and L have additional N-terminal preS1 and preS2 extensions. (C) The core protein shows an assembly domain and a CTD, which can be phosphorylated. The assembly domain forms a dimer, 120 copies of which assemble into the T = 4 capsid. Genotype G (Cp-G) shows a 12 amino-acid extension at the N terminus of core. (D) The core protein assembles, via its CTD, around the pgRNA/polymerase complex to form the immature core. The RNA is reverse transcribed into ssDNA and then into partially double-stranded rcDNA. The CTD is subsequently, at least partially, exposed (71). The mature capsid becomes enveloped, and the virion is secreted. The maturation signal and single strand blocking hypothesis aim at providing possible rationales for envelopment regulation. Mutations can cause altered genome maturity (16, 55). (E) Empty capsid envelopment can occur without the need for L. The resulting particles are then secreted as empty virions.

Fig. 2.

All Cp show two conformations except for genotype G and cell-free synthesized capsids. (A) Overlay of 2D ¹³C DARR spectra of Cp183 E. coli (blue) and Cp183-pgRNA (pink). Examples of CSDs are framed and labeled with the corresponding residue number. (B) Zoom on selected resonances from A. (C) Negative-stain EM of the different Cps: Cp183, P7-Cp183, Cp183-F97L, Cp183-EEE, Cp149, and Cp-GenotypeG (Cp-G) form T = 4 capsids in both E. coli and upon reassembly; Cp140 assembles mainly in T = 3 capsids. (D) I59 Cδ1-Cβ peaks of 2D DARR spectra for E. coli isolated versus in vitro reassembled capsids. For Cp149, Cp183, Cp183-F97L, Cp183-EEE, and P7-Cp183, E. coli–isolated capsids conform mainly to form A, and reassembled capsids mainly to form B. Exceptions are E. coli–isolated Cp140 capsids, which comprised a mixture of A and B forms and Cp-G capsids that conformed exclusively to B, regardless of the preparation procedure. Color-coding of the NMR peaks is according to the capsid in C. (E) 3D proton-nitrogen-alpha carbon correlation (hCANH) spectrum extract showing W62 of cell-free synthesized CF-Cp183 (green) versus Cp149 E. coli (red) and reassembled (gray) capsids. CF-Cp183 are in form B. (F) Percentage of conformation B in the different capsid forms. In addition, two Cp149 E. coli capsid preparations left for 1 mo at different temperatures are shown as well as Cp149 and Cp-G E. coli capsids after a gel filtration step. The percentage of B was calculated from six representative and isolated peaks in the DARR spectrum (see Materials and Methods). Error bars represent SDs.

Fig. 3.

Conformational differences between forms A and B. (A) Histogram showing CSDs between A and B forms ($CSD=\hspace{0.17em}\left|{\delta }_C\left[\text{A}\right]\hspace{0.17em}-\hspace{0.17em}{\delta }_C\left[\text{B}\right]\right|$). A single bar is plotted for each assigned carbon atom (see for example V27, where the five transparently superimposed bars correspond to the CSDs of the five assigned carbon resonances). The protein sequence up to residue 140 is given below the graph, and CSDs >0.6 ppm are shown in red, <0.3 ppm in gray, and in between in orange. (B) Residues with CSDs >0.6 ppm are shown as spheres on the X-ray structure of a Cp149 dimer [chains C and D of Protein Data Bank (PDB) accession code 1QGT (29)] and localized at the base of the spike. Color code is as follows: dark gray and red for chain C; light gray and light red for chain D. (C) Proximities in the 3D structure (≤4 Å) between residues with CSDs >0.6 ppm are shown by black lines. (D) Residues from the hydrophobic pocket according to ref. . (E) Residues showing large CSDs between A and B forms (in red in A) in surface mode on the X-ray structure (color coded according to residue type as follows: white, hydrophobic; red, acidic; blue, basic; green, polar; and yellow, cysteine).

Fig. 4.

Mutational obstruction of the hydrophobic pocket blocks access to form A, while adding Triton X-100 to wild-type capsids restores form A. In silico mutated W5 (A) and W60 (B) as predicted by PyMol (Schrödinger, LLC) highlighted on the Cp149 structure [PDB: 1QGT (29)], picturing how the P5W and L60W replacements can obstruct the hydrophobic pocket. Residues involved in the conformational A/B switch are colored in red. (C) I59 Cδ1-Cβ correlation peak extracted from a 2D DARR recorded on Cp183 capsids isolated from E. coli in form A (in blue), reassembled with pgRNA in form B (in pink), and compared to mutants P5W (in green), L60W (in yellow), P5G (in orange), and L60G (in gray). P5W and L60W capsids isolated from E. coli adopted >95% form B, while P5G and L60G showed a broad intermediate peak. Addition of Triton X-100 at a 8:1 molar ratio to capsids reassembled in form B (Right) fully restored the form A (in cyan).

Fig. 5.

CTD phosphorylation, RNA content, and capsid geometry have little impact on the capsid structure. (A–E) ¹³C CSDs mapped on the amino-acid sequence in a compact form with the same color coding as in Fig. 2A (for full plots, see SI Appendix, Fig. S12 A–E) and (F–J) CSDs plotted on the 3D structures. CSDs are shown for F97L mutation on Cp183 (A and F); empty capsids with and without CTD (Cp149 versus P7-Cp183) (B and G); absence/presence of RNA (Cp149 versus Cp183) (C and H); CTD phosphorylation and nucleic acid content (Cp183 versus P7-Cp183) (D and I); and T = 3 versus T = 4 capsid geometry (Cp140 versus Cp149) (E and J); on the 3D structure [PDB 1QGT (52)], residues with medium and large CSDs are shown as orange and red spheres, respectively. E113 which lost more than 50% of intensity in presence of the CTD is shown in blue and the mutated residue F97 in green.

Fig. 6.

Cp mutants with impaired access to form A support capsid-internal HBV replication but not capsid envelopment. HepG2 cells were transfected with a wild-type HBV expression vector (pCHwt) or cotransfected with an analogous Cp-deficient HBV vector (pCH_core⁻) plus a separate HBc expression vector encoding wild-type Cp (wt) or the indicated mutants. Particles in the culture supernatants were preseparated in Nycodenz gradients according to their buoyant densities (virions ∼1.18 g/mL; naked capsids ∼1.22 g/mL) and then via native agarose gel electrophoresis by electrophoretic mobility. Signals represent HBV-specific DNA in virions and in naked capsids, as revealed by hybridization with a radioactively labeled HBV probe. The presence of envelope proteins plus Cp (virions) versus only Cp (naked capsids) at the respective gel positions was shown by immunoblotting, as detailed in SI Appendix, Figs. S13 and S14. Note the lack of DNA signals at the virion position for all mutant Cps, perhaps except very faint signals for the L60 mutants (arrows), despite wild-type Cp-like DNA signals at the naked capsid position; hence, the four Cp mutants support capsid-internal replication but are deficient for envelopment. The lighter areas between virion and capsid positions arise from nonviral bulk protein in the polyethylene glycol (PEG) precipitated particle preparations.

Fig. 7.

Possible role of the two different capsid forms in the context of viral particle formation. (A) The presence of the CTD, its phosphorylation, and the presence of nucleic acids or the T = 3/T = 4 symmetry do not induce significant conformational change. (B) In scenario 1, capsid maturation changes the hydrophobic pocket so that is becomes proficient for interaction, likely with the preS matrix domain, represented as a green square. (C) In scenario 2, a pocket factor, causing a conformation similar to form A, binds the capsid. The bound pocket factor enables preS binding at a different site in the capsid. The pocket factor is then either released upon preS binding (as shown) or remains in the enveloped particle. (D) Cartoon of the “synergistic double interaction” hypothesis, where two different mechanisms combine to produce the observed phenotype (e.g., mainly empty and mature capsids are enveloped). (E) In the F97L mutant, the pocket is structurally modified, thereby favoring the interaction with preS and resulting in premature envelopment. (F) Genotype G capsids as well as Cp183-P5 and L60 mutant capsids have no or only poorly accessible hydrophobic pockets, resulting in reduced or impaired envelopment. (G) Binding of a ligand to the hydrophobic pocket results in an antiviral effect through inhibition of preS binding, therefore preventing envelopment.