**4. Biochemical Properties of SVPs**

The proportion of the HBsAgL, -M, and -S proteins differ between Dane particles, filaments, and SVPs generated during a natural infection. It is estimated that the envelope of Dane particles contains HBsAgL, HBsAgM, and HBsAgS at a ratio of approximately 3:2:5, and filaments at a ratio of 1:1:4. SVPs contain less HBsAgM compared to filaments with trace amounts of HBsAgL [46,83,88]. HBsAgS SVPs have a molecular weight of 2–4 <sup>×</sup> 106, and are composed of protein (75% by weight), carbohydrates (in form of glycoproteins) and lipids (25% by weight) [89]. Approximately 100 HBsAgS proteins assemble with lipids into lipoprotein particles, fifty HBsAgS dimers were identified in SVPs purified from sera of transgenic mice [49,89,90]. Three different regions of the S-domain contribute to the oligomerization of the HBsAg proteins, the cytosolic loop, TM2, and the luminal loop (Figure 3) [57]. The SVPs are compact particles with a reported density of 1.21 g/mL in caesium chloride (CsCl) compared to a density of infectious virions between 1.24 and 1.26 g/mL [23,91,92]. The compact structure of the SVPs is due to the large number of intra- and inter-molecular disulfide bonds within and between the S-domains of the individual HBsAg subunits [62,65,90,93–95]. Kinetic studies demonstrated that disulfide-linked HBsAgS dimers are formed in the ER, then the immature particle precursors are transported to a post-ER, pre-Golgi compartment, which excludes the enzyme "protein disulfide isomerase" and allows the formation of HBsAgS oligomers [96]. Intracellular HBsAgS particles contain high-mannose oligosaccharide chains, and after secretion, SVPs contain complex oligosaccharide chains with terminal sialic acid *N*-acetylglucosamine residues representing glycosylation patterns conforming with the HBsAg movement from the ER through the Golgi cisternae [12,67,97].

Cryo-EM studies and biophysical analyses of the SVPs produced in cell culture or purified from sera of transgenic mice demonstrates a tight HBsAgS protein–lipid interaction. The lipid composition of HBsAgS SVPs purified from the plasma of several HBV carriers showed that phospholipids, in particular, phosphatidylcholine is a major lipid class; with palmitic, stearic, oleic, and linoleic acids being the major fatty acid components [89]. Consistently, SVPs produced in human hepatoma cell lines predominantly contain phospholipids, with phosphatidylcholine being the dominant component [98]. The tight protein–lipid interaction restricts lipid movement suggesting that the lipids are not aligned in a typical bilayer structure. HBsAg particles seem to contain the lipids in an unusual arrangement, with the lipids being closely intercalated with the proteins, located on the particle surface, and are hence likely arranged in a lipid monolayer [99,100]. HBsAg proteins contain a high content of alpha helices (45%–52%), which are lipid-associated, and provide an arrangement which allows the disposal of the loop regions in the particle interior or on its surface [100,101]. Assessing yeast (*Hansenula polymorpha*)-derived SVPs, the particles have an ordered and rigid lipid interface, possibly organized as a phospholipid monolayer, with a hydrophobic and fluid inner core. HBsAgS proteins penetrate into the lipid core, with parts of the protein protruding from the particle surface [89,102,103]. The lipids contribute to the antigenic activity of HBsAg particles [100], likely by stabilizing the proper helical structure of the HBsAg proteins and the conformation of their hydrophilic region, which contains the antigenic site. Removal of lipids decreases the helical content and reduces the antigenic activity of

the particles [100]. S-domain antigenic structures seem to be strongly impacted by the lipid–protein interface, which defines the formation of alpha-helical structures and accommodates the arrangement of proper disulfide bonding patterns and the correct folding of HBsAg.
