Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain

During the life cycle of hepatitis B virus (HBV), the large envelope protei n (L) plays a pivotal role. Indeed, this polypeptide is essential for viral assembly and probably for the infection process. By performing mutagenesis experiments, we have previously excluded a putative involvement of the pre -S2 domain of the L protein in viral infectivity. In the present study, we have evaluated the role of the pre-S1 region in HBV infection. For this pur pose, 21 mutants of the L protein were created. The entire pre-S1 domain wa s covered by contiguous deletions of 5 amino acids. First, after transfecti on into HepG2 cells, the efficient expression of both glycosylated and ungl ycosylated L mutant proteins was verified. The secretion rate of envelope p roteins was modified positively or negatively by deletions, indicating that the pre-S1 domain contains several regulating sequences able to influence the surface protein secretion. The ability of mutant proteins to support th e production of virions was then studied. Only the four C-terminal deletion s, covering the 17 amino acids suspected to interact with the cytoplasmic n ucleocapsids, inhibited virion release. Finally, the presence of the modifi ed pre-S1 domain at the external side of all secreted virions was confirmed , and their infectivity was assayed on normal human hepatocytes in primary culture. Only a short sequence including amino acids 78 to 87 tolerates int ernal deletions without affecting viral infectivity. These results confirm the involvement of the L protein in the infection step and demonstrate that the sequence between amino acids 3 and 77 is involved in this process.