Characterization of the infectivity determinants of the envelope proteins of Hepatitis B Virus

The human Hepatitis B virus (HBV) is a small hepatotropic DNA virus, which consists of a nucleocapsid and an envelope with three membrane-embedded surface proteins (large (L), middle (M), and small (S)). While the S protein is required for budding and is the major component of the envelope, the L protein is crucial for infectivity. Several infectivity determinants have previously been described: (i) The N-terminal myristic acid moiety together with aa 2-48 are indispensable for virus infectivity and specifically bind a yet unknown receptor. Consistent with this, a peptide HBVpreS/2-48myr, composed of a myristoyl group and the N-terminal 47 residues of the L protein, inhibits HBV entry at picomolar concentrations. (ii) Amino acids 49-78 and the first transmembrane domain of the L protein are also required for virus infectivity. However, their function is not clear. (iii) Recently, it has been shown that the S-domain, which is common to all three surface proteins, also contains an infectivity determinant in its antigenic loop. Currently, it is not fully understood how the virion orchestrates these infectivity determinants during entry process and how exactly the preS-derived peptide HBVpreS/2-48myr interferes with virus entry. The major obstacle, restricting such investigations for a long time, was the lack of easily accessible in vitro infection systems. A recently established human hepatoma cell line (HepaRG), susceptible for HBV infection upon differentiation in vitro, resolved this issue and allowed us to analyze the role of the HBV envelope proteins in virus entry. In the present work, several approaches were undertaken: (i) systematic optimization of HepaRG cells for HBV infection, (ii) establishment of a reverse genetics approach that allows production of virions with mutated envelope proteins, (iii) an extensive mutation analysis within the L and S protein to determine viral assembly and infectivity, (iv) infection-competition study using preS-derived peptides, and (v) design of membrane-anchored inhibitory peptides by replacing the myristoyl group with a type II transmembrane protein. With an optimized infection assay, over 30% of differentiated HepaRG cells could be infected. The optimized infection assay facilitated further infectivity analyses of HBV virions generated by complementation, in which the L and S proteins were separately expressed. The following was observed in the present work: (i) Besides acting as a simple myristoylation signal, the N-terminus of the L-protein (aa 2-8) bears a more complex function for virus entry, since the substitution with heterologous myristoylation motifs abolished virus infectivity. This conclusion is strengthened by a complementary approach showing that the inhibitory potential of the peptide was also severely reduced when amino acids 2-8 were deleted or substituted. (ii) Expression of a membrane-anchored peptide prevents HepaRG cells from HBV infection, probably due to an interference with virus receptor on the plasma membrane. (iii) Amino acids 49-78 of L protein do not tolerate insertions and point mutations at their conserved region, and the peptide comprising aa 49-78 did neither interfere with HBV infection nor inhibit the antiviral activity of HBVpreS/2-48myr. (iv) While the M protein is dispensable for both HBV assembly and infectivity, the preS2 domain as an internal domain of the L protein is needed for virion release in a length-dependent but sequence-independent manner. Notably, HBV with an L protein carrying a mostly scrambled preS2 domain fully supported virion formation and virus infectivity. (v) Cysteine mutations in cytosolic loop-I of L protein drastically reduced virus infectivity, indicating that a properly formed cytosolic loop-I is also required for HBV entry. (vi) The essentiality of the antigenic loops during virus entry is mainly contributed by those in the context of S protein, and is correlated with the binding activity of the virion to heparin. In summary, this data indicates that the myristoyl chain of the L protein provides an anchor into the hepatocyte membrane thereby allowing the subsequent interaction with a hepatocyte-specific receptor. The N-terminal amino acids 2-8 of the HBV L protein serve as an adapter between the myristoyl moiety and the receptor-binding domain that orients the essential receptor binding site into the right position. The preS2 domain is dispensable for the HBV infectivity in vitro. However, it acts as a linker within the L-protein during virus assembly. The S protein may participate in primary attachment of virion to hepatocytes via the antigenic loop, which is critical for virus infectivity. Last but not the least, the membrane-anchored inhibitory peptide presents a promising approach to identify the virus receptor and may be used in a gene therapy approach against HBV infection.