*3.3. Topology of HBsAgL*

The large HBsAg protein adopts two distinguished transmembrane topologies facilitating an internal or external location of the preS1/S2 domain (Figure 1A) [17,79–81]. The internal orientation allows preS1/S2 to interact with the viral capsid, a critical step in viral morphogenesis. The preS2 region of the HBsAgL protein serves as a possible spacer to facilitate conformational changes of the preS1/S2 domain [17,81,82]. During the maturation process, the preS1/S2 domain is translocated to adopt an external orientation, which is essential for virus attachment to the host cell through a specific interaction with heparan sulfate proteoglycan [34,35] and binding to the entry receptor [36,37]. In addition, the preS1 domain is myristoylated at the N-terminus, which is required for efficient HBV

entry into hepatocytes (Figure 1B) [83,84]. The potential *N*- and *O*-glycosylation sites in the HBsAgL preS1/S2 domain are not utilized due to the cytosolic orientation of the preS1/preS2 domain after translation. HBsAgL molecules have molecular weights of 39 kD (p39) or 42kD (gp42) depending on the glycosylation status at position N146 in the S-domain (Figure 1B). Expression of HBsAgL in mammalian cells in the absence of HBsAgS and HBsAgM does lead to particle formation, but not secretion, and it is retained in post-ER and pre-Golgi compartments [85]. HBsAgL causes a dose-dependent inhibition of particle release if co-expressed with HBsAgS [86,87].

Taken together, the S-domain is shared by the HBV envelope proteins and defines the backbone of the particle due to the presence of the topogenic transmembrane regions, glycans at position N146, cysteine residues to form inter- and intra-molecular disulfide bonds, and the external loop region. The correct folding of the external loop defines the HBsAg-specific antigenic determinants.
