Hepatitis B virus (HBV) causes 350 – 400 million chronic infections worldwide, and approximately 1 million deaths annually.1
Infected hepatocytes express two closely related viral antigens; the capsid or core-antigen (HBcAg), and a secreted protein known as e-antigen (HBeAg). Unlike HBcAg, HBeAg is not essential for infection, replication, or assembly,2; 3; 4
yet it is conserved in all members of the Hepadnaviridae
. It is thought to modulate both the innate 5; 6; 7
and adaptive 8; 9; 10
immune responses so as to favor persistent infection.11; 12; 13
HBcAg and HBeAg are both derived from the pre-C/C
gene but they are initiated from different in-frame start codons. The first residue of HBcAg is designated as (+1) and the wild-type protein terminates at residue 183. HBeAg on the other hand is initiated at an upstream start site and its first residue is by convention designated as (−29). This precursor is processed amino-terminally with the removal of a 19-residue leader peptide, and carboxy-terminally at position 154.14
Although the amino-terminus of serum-derived HBeAg has not yet been determined directly, it is assumed to be (−10) and HBeAg is therefore believed to have the form (−10)154, which is however susceptible to adventitious processing to the form (−10)149.14
HBcAg and HBeAg therefore share a common sequence, except for 10 and 29 (or 34) residues at their amino- and carboxy-termini, respectively, and both proteins are considered to be dimeric.15; 16
In the case of HBcAg the two polypeptide chains pair through the formation of a four-helix bundle and the resulting dimers polymerize into capsids17
whereas in HBeAg, despite a very similar primary structure, the dimers remain soluble. HBcAg and HBeAg also have several cysteine residues (reviewed in16
). Crucial among these are C61, which can oxidize to form an inter
-molecular disulfide bond across the dimer interface, and C(−7) in HBeAg, which can form an alternative intra
-molecular disulfide bond with C61.16
These cysteines play a central role in the structure and secretion of HBeAg.18; 19; 20
The structural and physiological differences between HBcAg and HBeAg are therefore attributable to the propeptide and its cysteine residue.
The goal of this study was to prepare soluble (dimeric) and particulate (capsid) forms of both HBcAg and HBeAg, thereby allowing direct comparison of the two proteins and insight as to the role of the propeptide. To this end, we expressed recombinant forms of HBcAg and HBeAg in Escherichia coli
. For HBcAg we employed a protein consisting of only the first 149 residues of the 183-residue wild-type protein, as these are known to suffice for capsid assembly.16
This protein forms capsids with two morphologies; one composed of 90 dimers (with T = 3 icosahedral symmetry, and a diameter of 32 nm) and one composed of 120 dimers (with T = 4 symmetry, and a diameter of 35 nm).21
These capsids are devoid of nucleic acid but otherwise appear to be indistinguishable – at least on their outer surface - from wild-type (Cp183) capsids, and are therefore well suited for structural studies.16
For HBeAg we employed a construct with the same 149-residue sequence as above but extended at its amino-terminus with the 10-residue propeptide, in other words, identical to the (−10)149 form of HBeAg ().
The wild-type sequence of the Cp(−10)149 variant used in this study
The cryo-EM structures of the T = 4 capsid22; 23; 24
have shown that dimers cluster around the 2- and 5-fold axes of symmetry and the crystal structure17
reveals that interactions between neighboring dimers are mediated by residues in helix 5 (residues 112–127), the proline rich loop (128–136), and the carboxy-terminal arm (137–142). Accordingly, we mutated residues in these regions, aiming to produce protein that forms capsids less readily, i.e. more soluble dimers.15; 25
With both proteins available as soluble dimers, the cysteine residues were then systematically mutated to investigate their contributions, and that of the propeptide, to protein stability. Stability was assessed in terms of thermal denaturation temperature and monitored by circular dichroism spectroscopy. As noted above, the native HBeAg is probably inherently dimeric; nevertheless, we determined conditions in which dimers can be induced to assemble into capsids and used these to localize the residual propeptide (residues −10 to −1) by cryo-electron microscopy.
The mutant proteins used in this study are named in accordance with previous nomenclature, slightly extended.15
Cp183 refers to the wild-type polypeptide chain of HBcAg. Cp149 refers to a polypeptide consisting of the first 149 residues of Cp183, and corresponds to the assembly domain of that protein. Cp(−10)149 refers to HBeAg. Dimer and capsid forms of both proteins are indicated by the subscripts “d” and “c”, respectively. The names of mutations are appended to that of the basic construct, for instance, Cp(−10)149d
.G123A. Disulfide bonds are specified by superscripts. For example, the above protein, with an intra-molecular disulfide bond between C(−7) in the propeptide and C61 in the assembly domain, is specified as Cp(−10)149d
. In the constructs used, the cysteines at positions 48 and 107, which are not involved in disulfide bonds,17
have been mutated to alanine, although this is not indicated explicitly in the name.