Solid-state [13C–15N] NMR resonance assignment of hepatitis B virus core protein

Abstract

Each year, nearly 900,000 deaths are due to serious liver diseases caused by chronic hepatitis B virus infection. The viral particle is composed of an outer envelope and an inner icosahedral nucleocapsid formed by multiple dimers of a ~ 20 kDa self-assembling core protein (Cp). Here we report the solid-state C andN resonance assignments of the assembly domain, Cp149, of the core protein in its capsid form. A secondary chemical shift analysis of the 140 visible residues suggests an overall alpha-helical three-dimensional fold matching that derived for Cp149 from the X-ray crystallography of the capsid, and from solution-state NMR of the Cp149 dimer. Interestingly, however, at three distinct regions the chemical shifts in solution differ significantly between core proteins in the capsid state versus in the dimer state, strongly suggesting the respective residues to be involved in capsid assembly.

Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae

“…The basic fold of HBV capsid polypeptide chains, their pairing as dimers, and the arrangement of dimers in T = 3 and T = 4 lattices ( Fig 2) have been well established by cryo-EM [3,8], X-ray crystallography [4,5], and NMR [6,9,10]. There have also been indications that the chain conformations, while similar, are not identical [7][8][9]53].…”

“…The virion is composed of a structurally distinctive capsid (called core-antigen) containing the genome and polymerase, and surrounded by a lipid envelope with embedded surface-antigen protein. The capsid structure has been extensively studied by electron microscopy, X-ray crystallography, and NMR spectroscopy [3][4][5][6][7][8][9][10]. Capsids are composed of a 183-residue polypeptide, the first 140 residues of which suffice for capsid assembly [11].…”

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

“…We here investigated E. coli self-assembled Cp149 HBV capsids forming homogeneous T = 4 icosahedra with a typical diameter of 30 AE 3 nm ( Figure 1a). The 13 C and 15 N resonances of the Cp149 protein were assigned using 3D spectroscopy, [23] and the annotated 2D 13 C-13 C Dipolar Assisted Rotational Resonance [24] (DARR) spectrum of the capsids is displayed in Figure 1b. Sequential assignments reveal that a subset of residues experiences resonance splitting, with up to 2.5 ppm of chemical-shift differences amongst themselves in the 13 C and/or 15 N dimensions.…”

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

“…The HBV capsid has recently been subject to solid-state NMR studies on capsids isolated from E. coli cells where they auto-assemble ( Figure 3 f). The sequential 1 H/ 13 C/ 15 N assignments have been established as a basis for further studies [ 35 , 106 ]. The NMR spectra yielded high-resolution NMR fingerprints ( Figure 3 g), with linewidths similar to those obtained from the HIV capsids, confirming that this type of symmetrical object is well in the scope of solid-state NMR.…”

“…The NMR spectra yielded high-resolution NMR fingerprints ( Figure 3 g), with linewidths similar to those obtained from the HIV capsids, confirming that this type of symmetrical object is well in the scope of solid-state NMR. A comparison between the chemical shifts of the core protein in its dimeric and capsid states revealed the residues which structurally adapt upon shell assembly [ 106 ]. The slight non-symmetry (“quasiequivalence”) between the four subunits was reflected in the NMR spectra [ 55 ], in which multiple peaks are observed for ~20% of residues located at the dimer–dimer interfaces at the five- and six-fold symmetry axes ( Figure 3 g,h), as shown at the example of Ala137.…”