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. 1998 Jul;72(7):5573-8.
doi: 10.1128/JVI.72.7.5573-5578.1998.

Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity

J Le Seyec  1 , P Chouteau, I Cannie, C Guguen-Guillouzo, P Gripon
Affiliations

Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity

J Le Seyec et al. J Virol. 1998 Jul.

Abstract

Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.

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Figures

FIG. 1
FIG. 1
(A and B) Expression vectors of the L protein. The heavy line indicates the 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) and the polyadenylation site (pA) in the HBV sequence are shown. (C) Amino acid deletions in the pre-S2 region of the S gene cloned in different L protein expression plasmids. Deletions (L x/y; x is the wild-type position of the N-terminal amino acid flanking the deletion; y is the wild-type position of the C-terminal amino acid flanking the deletion) are indicated below as thin lines.
FIG. 2
FIG. 2
Western blot analysis of the L protein expressed in transfected cells. HepG2 cells were transfected with 20 μg of different L expression vectors: control plasmid without HBV insert (lane 1), L WT expression plasmid driving the synthesis of the WT L protein (lane 2), and L x/y expression plasmids driving the synthesis of different mutant L proteins (lanes 3 to 8). Proteins were extracted from cells 7 days after transfection and analyzed by electrophoresis through a 10% polyacrylamide–SDS gel. The primary polyclonal antibody was directed against the pre-S1 region. gp 42 and p 39 indicated the migration positions of the glycosylated and unglycosylated L proteins, respectively.
FIG. 3
FIG. 3
Southern blot analysis of HBV DNA from intracellular core particles. An anti-HBc antibody was used to immunoprecipitate core particles from cells transfected with the L-defective genome complemented with different L expression vectors: control plasmid without HBV insert (lane 1), L WT expression plasmid driving the synthesis of the WT L protein (lane 2), and L x/y expression plasmids driving the synthesis of different mutant L proteins (lanes 3 to 8). DNA was extracted and analyzed on a 1.5% agarose gel. Molecular size markers are indicated in kilobases; the positions of relaxed-circular DNA (RC) and single-stranded DNA (SS) are shown.
FIG. 4
FIG. 4
Southern blot analysis of HBV DNA in particles secreted by transfected HepG2 cells. Cells were transfected with the L-defective genome complemented with different L expression vectors: control plasmid without HBV insert (lanes 1 and 9), L WT expression plasmid driving the synthesis of the WT L protein (lanes 2 and 10), and L x/y expression plasmids driving the synthesis of different mutant L proteins (lanes 3 to 8 and 11 to 16). Complete viral particles were immunoprecipitated from HepG2 supernatants, collected between days 3 and 6 posttransfection, with a polyclonal anti-HBs antibody (left panel) or with a monoclonal anti-pre-S1 antibody (right panel). Then DNA was extracted from the immunoprecipitates and analyzed on a 1.5% agarose gel. Molecular size markers are indicated in kilobases; the positions of relaxed-circular DNA (RC) and single-stranded DNA (SS) are shown.
FIG. 5
FIG. 5
HBsAg secretion by human hepatocytes following in vitro infection assays. Hepatocytes were incubated with PBS (T−) or with concentrated supernatants obtained from either 2.2.15 cell cultures (T+) or HepG2 cells transfected with the L-defective genome complemented with different L expression vectors: control plasmid without HBV insert, L WT expression plasmid driving the synthesis of the WT L protein, or L x/y expression plasmids driving the synthesis of different mutant L proteins. HBsAg was measured by a conventional radioimmunoassay in primary hepatocyte culture supernatants collected 10 days postinfection.
FIG. 6
FIG. 6
Southern blot analysis of HBV cccDNA in human hepatocytes following in vitro infection assays. Hepatocytes were incubated with PBS (T−) or with concentrated supernatants obtained from either 2.2.15 cell cultures (T+) or HepG2 cells transfected with the L-defective genome complemented with different L expression vectors: control plasmid without HBV insert, L WT expression plasmid driving the synthesis of the WT L protein, or L x/y expression plasmids driving the synthesis of different mutant L proteins. 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).
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