The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of Ϸ44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with Ϸ22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at Ϸ12-Å resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.cryo-EM ͉ subviral particle ͉ virus structure H epatitis B (HB) virus (HBV) is a major cause of both acute and chronic liver disease. It is estimated that there are Ϸ350 million carriers of the virus, and a high proportion of these will develop serious liver diseases, including hepatocellular carcinoma. The double-layered mature infectious particle, known as the Dane particle, is an icosahedral structure comprising an inner core formed from 180 or 240 copies of the HB core antigen (HBcAg) arranged with triangulation (T) numbers 3 or 4 and an outer proteolipid HB surface Ag (HBsAg) layer. The HBsAg envelope glycoprotein of HBV occurs in three forms, denoted by L (large), M (medium) and S (small), which form a nested set of products sharing a common C-terminal domain, the SHBsAg polypeptide. The SHBsAg itself has a molecular mass of Ϸ24 kDa and is predicted to be intimately associated with lipid, having four or five membrane-spanning helices, and is found in two forms, glycosylated and unglycosylated, in approximately equal amounts. SHBsAg glycosylation is a single biantennary glycan at Asn-146 and raises its molecular mass to Ϸ27 kDa (1, 2). About 80% of HBsAg is in the S form, which assembles with M and L forms into both the outer surface of the Dane particle and smaller subviral particles. These HBsAg subviral particles, known as 22-nm particles because of their diameter, are stabilized by disulfide bonds (3).HBsAg particles greatly outnumber mature virus particles and presumably act as decoys for the immune system. Although the 22-nm particles are predominately SHBsAg, tubular assemblies, also observed in the serum of chronic carriers, tend to be richer in LHBsAg. Because HBV subviral particles share important immunological determinants with mature virus, they can be used as effective immunogens. Indeed, expression of SHBsAg particles in yeast in the 1980s led to the developm...