**3. Role of HBsAg in HBV, Filament, and SVP Formation**

An essential step in the formation of virions, filaments, or SVPs is the cotranslational insertion of the HBsAgS protein into the membrane of the endoplasmic reticulum (ER) with a short luminal exposed N-terminal sequence, two transmembrane regions separated by a cytosolic loop, and a luminal domain, followed by a hydrophobic C-terminal region. The luminal domain corresponds to the external loop region of the S-domain, which contains multiple epitopes, including the immunodominant "a"-determinant region that is common to all HBV genotypes, and the allelic antigenic determinants "d/y" and "w/r" [17,31]. A conformational heparan sulfate binding site also overlaps with the "a"-determinant region and is essential to infectivity [32].

HBV virion formation depends on the presence of the viral capsid containing rcDNA and the HBsAg proteins for envelopment. The preS1 region of HBsAgL is essential for the assembly of infectious HBV particles by interacting with the capsid [17,33], possibly contributes to the binding to a proteoglycan attachment site [34,35], and is required for binding to the hepatocyte entry receptor, sodium taurochlorate cotransporting polypeptide (NTCP) [36,37]. Chaperones of the heat shock protein

(hsp) 70 family facilitate the interaction of the HBsAgL preS1 domain with the capsid by retaining the preS1/preS2 sequence in the cytosolic (internal) orientation at the ER [38–40]. Virion secretion depends on host factors of the endosomal sorting complex required for transport (ESCRT) and sorting into late endosomal multivesicular bodies (MVBs), and finally, release of its intraluminal content at the hepatocyte surface [41–43]. The mature HBV virions are spherical, enveloped particles with a diameter of 42 nm with an inner capsid of 22 nm in diameter [11–13]. Due to the elevated presence of HBsAgL in virions and filaments compared to the SVPs, filaments seem to follow the secretion pathway taken by the virions (Figure 2) [44]. The filaments have a width of approximately 20 nm and are variable in length [12,23,45].

**Figure 2.** Illustration of HBsAg protein synthesis and assembly pathways during a natural infection (**A**), HBsAgS expression and assembly in mammalian cells in the absence of other viral gene products (**B**), or expressed in yeast (**C**). During a natural infection, virions and filaments are formed by budding from multivesicular bodies. The spherical subviral particles (SVPs) are produced and secreted through the endoplasmic reticulum (ER)-Golgi complex. The HBsAg subunits of the virions, filaments, and SVPs form intra- and intermolecular disulfide bonds, and are partially glycosylated (not shown) (**A**). Expression of the HBsAgS gene in mammalian cell lines leads to the formation of SVPs exclusively composed of HBsAgS permitting the formation of disulfide bonds and partial glycosylation (not shown) (**B**). HBsAgS protein expressed in yeast accumulates in the ER, causes an extended ER and forms multilayered lamellar structures. HBsAgS protein complexes are harvested from the cell lysate, and SVPs are assembled during down-stream processing (**C**). cccDNA: covalently closed circular DNA.

In contrast to the formation of infectious particles, the formation of SVP does not depend on the preS1 domain. HBsAgS proteins have the ability to assemble into secretion-competent SVPs and are composed solely of envelope proteins, lipids, and glycans. Contrary to virion secretion, SVP assembly follows the constitutive secretory pathway of the host cell for release [43,44]. The non-infectious SVPs are 17 to 25 nm in diameter. SVPs isolated from HBV carriers show spike-like features protruding from the surface similar to the surface projections observed on filaments and infectious virions (Dane particles) [24,45–48]. An alternative structure for recombinant SVPs expressed in transgenic mice has been proposed to possess an octahedral symmetry [49]. Mammalian cell lines expressing HBsAgS in the

absence of any other viral component secrete SVPs, which are morphologically indistinguishable from serum-derived SVPs (Figure 2) [50–54]. Thus, particle morphogenesis substantially differs between SVPs and virions, but they have identical antigenic structures due to the S-domain, which is encoded by all HBV envelope proteins (Figure 1A,B).
