Laboratoires de Biologie Cellulaire et Virologie, EA 2639 Analyse Structurale des Antigènes, Faculté de Médecine, Institut Fédératif de Recherche Transposons et Virus, Université François Rabelais, 2 bis Boulevard Tonnellé, F-37032 Tours Cedex, France1
Laboratoire de Virologie Moléculaire, EA 2637 Processus Infectieux et Tumoraux, Faculté des Sciences Pharmaceutiques Philippe Maupas, Institut Fédératif de Recherche Transposons et Virus, Université François Rabelais, Tours, France2
Author for correspondence: Philippe Roingeard. Fax +33 2 47 36 60 90. e-mail roingeard{at}med.univ-tours.fr
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Abstract |
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Previous studies have shown that immature nucleocapsids containing pregenomic RNA are incompetent for envelopment and that minus-strand DNA synthesis must be completed for core particle envelopment to occur (Wei et al., 1996 ; Gerelsaikhan et al., 1996
). This suggests that DNA synthesis within the capsid is coupled to the appearance of a signal on the exterior of the nucleocapsid that is essential for nucleocapsid envelopment. The molecular nature of this envelopment signal is unknown, but it is thought to involve the specific binding of DNA-containing core particles, but not of RNA-containing core particles, to HBV envelope proteins (Hui et al., 1999
; Nassal, 1999
). We have recently investigated the coreenvelope interactions involved in the maturation of HBV, using in vitro assays to determine the capacity of HBV core particles to bind a panel of synthetic peptides that map to HBV envelope proteins (Poisson et al., 1997
). A short domain of the large (L) envelope protein (aa 96116 in the ayw subtype) and a short domain in the cytosolic loop of the small (S) envelope protein (aa 5680) were found to bind HBV core particles efficiently. The L envelope domain found to interact with HBV core particles was subsequently shown to be essential for virion morphogenesis in two independent studies with envelope mutants, in which this domain was substituted by random sequence (Bruss, 1997
) or deleted (Le Seyec et al., 1998
). A recent study with the S domain confirmed that deletions and/or amino acid substitutions within this domain hinder subviral particle morphogenesis and therefore provide no information about the role of this domain in virion packaging (Jenna & Sureau, 1998
).
Our previous assays of the binding of HBV core particles to HBV envelope peptides were performed with purified, liver-derived core particles containing HBV DNA (Poisson et al., 1997 ). In this study, we used similar assays to compare the binding of these DNA-containing core particles with that of empty HBV core particles, obtained by using the baculovirus expression system. These particles, called HBV
core particles, were obtained by expression of the sequence encoding the first 144 amino acids of the HBV core gene (excluding the arginine-rich C-terminal stretch of 39 amino acids responsible for pregenome binding) and have been described elsewhere (Touzé et al., 1999
). HBV envelope peptides (Fig. 1
) and binding assays were described previously (Poisson et al., 1997
; Hourioux et al., 1998
). We used two 25-residue peptides, S1 and S2, that together covered the cytosolic loop of the HBV S protein. To cover the cytosolic domain of the HBV L protein, we used five peptides of 28, 28, 29, 23 and 55 residues named L1, L2, L3, L4 and L5, respectively (L1L4 corresponded to the pre-S1 domain and L5 corresponded to the pre-S2 domain). Two additional peptides overlapping peptide L4 were used: peptide L4a (the last 10 residues of peptide L3 plus the first eight residues of peptide L4) and peptide L4b (the last 13 residues of peptide L4 plus the first eight residues of peptide L5).
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We found that HBV core particles bound the L and S HBV envelope peptide domains with the same affinity as liver-derived, HBV DNA-containing capsids (Table 1
). We cannot rule out the hypothesis that, in the absence of their C-terminal part, our truncated capsids could adopt a structure that resembles that of DNA-containing capsids, thus explaining their binding to envelope peptides. This is unlikely, however, because others have demonstrated the ability of full-length, E. coli-derived HBV capsids to bind to HBV L envelope protein expressed in rabbit reticulocyte lysates (Tan et al., 1999
). Therefore, our results suggest that the selection of replication-competent nucleocapsids for envelopment is not related to a particular capacity of DNA-containing nucleocapsids to bind HBV envelope proteins.
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Acknowledgments |
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References |
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Received 29 November 1999;
accepted 13 January 2000.
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