Electrostatic regulation of genome packaging in human hepatitis B virus

Abstract

Hepatitis B virus (HBV) is a contagious human pathogen causing liver diseases such as cirrhosis and hepatocellular carcinoma. An essential step during HBV replication is packaging of a pregenomic (pg) RNA within the capsid of core antigens (HBcAgs) that each contains a flexible C-terminal tail rich in arginine residues. Mutagenesis experiments suggest that pgRNA encapsidation hinges on its strong electrostatic interaction with oppositely charged C-terminal tails of the HBcAgs, and that the net charge of the capsid and C-terminal tails determines the genome size and nucleocapsid stability. Here, we elucidate the biophysical basis for electrostatic regulation of pgRNA packaging in HBV by using a coarse-grained molecular model that explicitly accounts for all nonspecific interactions among key components within the nucleocapsid. We find that for mutants with variant C-terminal length, an optimal genome size minimizes an appropriately defined thermodynamic free energy. The thermodynamic driving force of RNA packaging arises from a combination of electrostatic interactions and molecular excluded-volume effects. The theoretical predictions of the RNA length and nucleocapsid internal structure are in good agreement with available experiments for the wild-type HBV and mutants with truncated HBcAg C-termini.

Figure 1

Thermodynamic representation of pgRNA stability. (a) The RNA fragment outside the capsid is subject to nuclease digestion. (b) The stable nucleocapsid yields a minimum thermodynamic potential defined by Eq. 4. Shown here are thermodynamic potentials corresponding to the WT HBV and mutant 164. (c) Sequences of amino acid residues at the arginine-rich domains of WT HBcAg (183 amino acids) and the mutants with truncated C-termini (residues 164–179). These mutants are employed in both experiments and theoretical calculations. The electrostatic charge of each amino acid residue is coded with a different color: red, +1; teal, −1; and black, neutral. Three serine sites (S155, 162, and 170) are phosphorylated.

Figure 2

(a) Correlation between RNA length (in kilobases) and the net charge of the C-terminal tails in WT and mutated HBV capsids. The solid line is to guide the eye. (b) Individual contributions to the driving forces behind RNA packaging in WT capsids.

Figure 3

Local packing densities of RNA segments and C-terminal tails of HBV nucleocapsids. The local packing fraction for RNA/protamine tail segments is defined as η(r) = πρ(r)σ³/6, where ρ(r) represents the local number density and σ the segment diameter.

Figure 4

(a) Local packing fractions of amino acids in the C-terminal tail with different charges. (b) Distributions of the phosphorylated serine residues and the mean electrostatic potential inside the WT virus.