The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription

Abstract

Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.

Importance: The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.

FIG 1

HBV Cp structure and substitution design at the interdimer interface. (A) HBV Cp149 dimer structure with HAP1 (cyan spheres) bound (Protein Data Bank accession code 2G34). (B) Residue V124 (yellow spheres) contacts HAP1 (cyan spheres) at the Cp interdimer interface viewed from the interior of the capsid. (C) Hydrophobic residues used as replacements at residue V124 and the structure of HAP12. Me, methyl. Panels A and B are adapted from Tan et al. (45).

FIG 2

Thermodynamic study of Cp149-V124X. (A) The equilibrium concentrations of capsid and dimer were determined by size exclusion chromatography. Two representative chromatographs show the elution of 10 μM Cp149-WT and Cp149-V124F assembled at 100 mM NaCl. mAU, milli-absorbance units. (B) Based on the capsid-dimer equilibrium, we extracted the average pairwise association energy between dimers. There is a linear correlation between the association energy and the hydrophobic surface area at residue 124. The Cp149-WT, -V124L, -V124F, and -V124W association energies were measured at 100 mM NaCl, while the Cp149-V124A association energy was measured at 700 mM NaCl and extrapolated to that at 100 mM NaCl.

FIG 3

Electron micrographs of Cp149-V124X assembly products. (Top) Under conditions favoring capsid formation, Cp149-V124X assembled into capsid-like particles. Notably, Cp149-V124L mainly yielded irregular noncapsid polymers. Cp149-V124W was assembled at 50 mM NaCl, Cp149-V124F and -V124L were assembled at 100 mM NaCl, Cp149-WT was assembled at 500 mM NaCl, and Cp149-V124A was assembled at 1 M NaCl. (Middle) At 500 mM NaCl, Cp mutants with strong association energies and fast kinetics (V124W, V124F, and V124L) formed complexes with trapped assembly defects. Cp149-V124W had some partial or irregular capsids. Cp149-V124F and -V124L mainly yielded noncapsid polymers. (Bottom) Except for Cp149-V124W, 20 μM HAP12 (with assembly induced at 50 mM NaCl) resulted in the formation of large aberrant Cp149-V124X structures. The scale bar applies to all micrographs.

FIG 4

Assembly kinetics of Cp149-V124X at 500 mM NaCl observed by stopped-flow light scattering. (A) Assembly kinetics of Cp149-V124W and -V124F at 1.5 to 3.5 μM protein concentrations. (B) Assembly kinetics of Cp149-WT, -V124L, and -V124A at 3.5 to 40 μM protein concentrations.

FIG 5

Cp149-V124X dimer elution positions on a Superose 6 column. Though all mutants had nearly the same mass, Cp149-Y132A eluted the fastest, indicating that it has the largest Stokes radius. Cp149-V124W eluted the slowest, indicating that it has the most compact conformation. Cp149-V124F eluted between the WT and the V124W mutant. The elution position correlates with assembly kinetics and is consistent with allosteric changes in the Cp149 average conformation and, thus, the allosteric regulation of HBV assembly.

FIG 6

HAP12 titration on Cp149-V124X assembly. Cp149-V124X (10 μM) was induced to assemble at 50 mM NaCl and different HAP12 concentrations. The dimer fraction at equilibrium was used to indicate the assembly extent. The sensitivity to HAP12 correlated with the size of the V124X residue, with Cp149-V124W being nearly insensitive to the small molecule. Data for WT and V124W are from Tan et al. (45) and are included for comparison.

FIG 7

V124X substitutions affect capsid formation, pgRNA packaging, and reverse transcription in Huh7 cells. (A) A Western blot shows that Cp183-V124X mutants accumulated to similar levels in the cytoplasm. Relative sample volumes are indicated below the image. (B) Velocity sedimentation profiles of Cp183-V124X assembly obtained by Western blotting show that capsid formation correlates with in vitro assembly. Cp183-WT capsids sedimented at fractions 7 to 9, with dimers sedimenting at fractions 1 to 3. Cp183-V124W and -V124A had capsid sedimentation profiles similar to the WT profile, though Cp183-V124W had less free dimer and Cp183-V124A had more free dimer. Cp183-V124W capsid sedimentation was shifted to a slightly lower density. Cp183-V124F and -V124L had a large proportion of capsid assembly products sedimenting at intermediate fractions. Lane U, a sample of unfractionated cell lysate. (C) pgRNA was detected in capsid fractions 7 to 9 by Northern blotting. Cp183-V124W and -V124A capsids packaged much less pgRNA than the WT. Cp183-V124W is more defective in pgRNA packaging than Cp183-V124A. The amount of pgRNA packaged by Cp183-V124F and -V124L was too low to be detected. (D) A Southern blot shows that Cp183-V124X differentially affected ssDNA and rcDNA production.