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

Abstract

During the life cycle of hepatitis B virus (HBV), the large envelope protein (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 purpose, 21 mutants of the L protein were created. The entire pre-S1 domain was covered by contiguous deletions of 5 amino acids. First, after transfection into HepG2 cells, the efficient expression of both glycosylated and unglycosylated L mutant proteins was verified. The secretion rate of envelope proteins 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 the production of virions was then studied. Only the four C-terminal deletions, covering the 17 amino acids suspected to interact with the cytoplasmic nucleocapsids, inhibited virion release. Finally, the presence of the modified 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 internal 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.

FIG. 1

(A) Expression vector of the L protein. The thick line indicates HBV sequence; the thin line indicates plasmid pSV-SPORT 1 sequences with its simian virus 40 early promoter-origin region (PO SV40); boxes indicate ORFs for viral X, P, C, and S proteins. The envelope ORF is divided into pre-S1, pre-S2, and S domains. The approximate locations of the posttranscriptional regulatory element (PRE) (20) and the polyadenylation site (pA) in the HBV sequence are shown. (B) Amino acid deletions in the pre-S1 region of the S gene cloned in different L protein expression plasmids. Deletions (L x/y, where x is the WT position of the N-terminal amino acid flanking the deletion and y is the WT position of the C-terminal amino acid flanking the deletion) are indicated above.

FIG. 2

Analysis of intra- and extracellular surface viral proteins. HepG2 cells were transfected with 20 μg of different L expression vectors: NC, plasmid without an HBV insert (negative control); WT, expression plasmid driving the synthesis of the WT L protein; L x/y, expression plasmids driving the synthesis of different mutant L proteins. (A and B) Proteins were extracted from cells (A) or precipitated from the culture medium (B) and studied by Western blotting. Samples were analyzed by electrophoresis through a 12.5% polyacrylamide–sodium dodecyl sulfate gel. The primary monoclonal antibody was directed against the pre-S2 region. gp 42, p 39, ggp 36, and gp 33 indicated the migration positions of the glycosylated and unglycosylated L proteins and the diglycosylated and glycosylated M proteins, respectively. (C) HBsAg, secreted by transfected HepG2 cells, was measured by a conventional radioimmunoassay in culture supernatants collected 6 days posttransfection. The data represent the percentage of secreted HBsAg compared to the average of the two WT controls.

FIG. 3

Southern blot analysis of HBV DNA from intracellular core particles and from extracellular complete viral particles. HepG2 cells were transfected with the L-defective genome complemented with different L expression vectors. NC, plasmid without an HBV insert (negative control); WT, expression plasmid driving the synthesis of the WT L protein; L x/y, expression plasmids driving the synthesis of different mutant L proteins. (A) An anti-HBc antibody was used to immunoprecipitate cytoplasmic core particles from transfected cells. (B) Complete viral particles were immunoprecipitated with a polyclonal anti-HBs antibody from HepG2 cell supernatants collected between days 3 and 6 posttransfection. DNA was extracted from the immunoprecipitates and analyzed on a 1.5% agarose gel. Molecular size markers are indicated in kilobases to the left; the positions of relaxed-circular DNA (RC) and single-stranded DNA (SS) are shown to the right.

FIG. 4

Infectivity of complete viral particles containing mutant pre-S1 proteins. Hepatocytes were incubated with concentrated supernatants obtained from HepG2 cells transfected with L-defective genome complemented with different L expression vectors: NC, plasmid without an HBV insert (negative control); WT, expression plasmid driving the synthesis of the WT L protein; L x/y, expression plasmids driving the synthesis of different mutant L proteins. (A) HBsAg, secreted by human hepatocytes following in vitro infection assays, was measured by a conventional radioimmunoassay in hepatocyte primary culture supernatants collected 10 days postinfection. The data represent the percentage of secreted HBsAg compared to the average of the two WT controls. (B) Southern blot analysis of HBV cccDNA in human hepatocytes following in vitro infection assays. Supercoiled viral DNA was selectively extracted from hepatocytes collected 10 days postinfection and analyzed on a 1.5% agarose gel. Molecular size markers are indicated in kilobases to the left; the position of cccDNA is shown to the right (CCC).