The N-terminal pre-S domain of the large hepatitis B virus (HBV) envelope protein plays a pivotal role at the initial step of the viral entry pathway. In the present study, the entire pre-S domain was mapped for infectivity determinants, following a reverse-genetics approach and using in vitro infection assays with hepatitis delta virus (HDV) or HBV particles. The results demonstrate that lesions created within the N-terminal 75 amino acids of the pre-S region abrogate infectivity, whereas mutations between amino acids 76 and 113, overlapping the matrix domain, had no effect. In contrast to the results of a recent study (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. 103:6730-6734, 2006), the deletion of a cell membrane translocation motif (TLM) located between amino acids 148 and 161 at the C terminus of pre-S2 did not interfere with the infectivity of the resulting HDV or HBV mutants. Furthermore, a series of large deletions overlapping the pre-S2 domain were compatible with infectivity, although the efficiency of infection was reduced when the deletions extended to the pre-S1 domain. Overall, the results demonstrate that the activity of the pre-S domain at viral entry solely depends on the integrity of its first 75 amino acids and thus excludes any function of the matrix domain or TLM.Hepatitis B virus (HBV) is characterized by a narrow host range that reflects the specificity of interaction between envelope proteins and human hepatocyte receptors at viral entry (41). The HBV envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (S-HBsAg) are transmembrane glycoproteins that differ from each other in the sizes of their N-terminal ectodomains (22). L-HBsAg contains an N-terminal pre-S1 domain, a central pre-S2 region, and a C-terminal S domain. M-HBsAg is composed of the pre-S2 and S domains (Fig. 1), and S-HBsAg consists of the S domain only. Envelope protein synthesis occurs at the endoplasmic reticulum membrane, and particles, mostly empty subviral particles (SVPs), assemble from aggregates at a pre-Golgi membrane. Envelopment of the HBV nucleocapsid requires the presence of LHBsAg (but not that of M-HBsAg) in addition to S-HBsAg (6). L-HBsAg displays a dual-membrane topology: N-terminal pre-S (pre-S1 plus pre-S2) is either external (Le-HBsAg), at the surface of the particles, or internal (Li-HBsAg), facing the inner side of the particle (9,35,38). Le-HBsAg is thought to assume a receptor-binding function at viral entry, and LiHBsAg is thought to serve as a matrix protein for HBV nucleocapsid envelopment (10, 29). In addition to their propensity to form SVPs, the HBV envelope proteins can package the hepatitis delta virus (HDV) ribonucleoprotein (RNP) in the case of HBV-HDV coinfection (4, 47). This interaction leads to the formation of HDV particles, which are infectious when L-HBsAg is included in the envelope (44). L-HBsAg contains a major infectivity determinant in its pre-S1 domain, including a myristoyl anchor link...

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The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation ( The hepatitis B virus (HBV) is characterized by a most peculiar budding mechanism, which is nucleocapsid independent and driven by its viral envelope proteins at a cellular internal membrane (17,35,36). The three envelope proteins, encoded by a unique open reading frame on the HBV genome, bear the hepatitis B virus surface antigen (HBsAg) and are referred to as large, middle, and small (L-HBsAg, M-HBsAg, and S-HBsAg, respectively) because they differ in the sizes of their respective amino-terminal ends (16). Interestingly, the driving force of the viral particle budding process is provided by the sole S-HBsAg protein (32), which is produced in abundance in infected cells. All three envelope proteins are synthesized at the endoplasmic reticulum (ER) membrane, where they aggregate through protein-protein interactions leading primarily to the secretion of empty S-HBsAg-coated subviral particles (SVPs) (16). It is only when L-HBsAg is present in the envelope protein aggregates at the ER membrane that the HBV nucleocapsid can be recruited in the budding complex and released as a mature virion (4). Owing to the overwhelming activity of S-HBsAg for self-assembly, in comparison to that of L-HBsAg, HBV virion formation occurs only on rare occasions.In addition to the formation of SVPs and mature HBV virions, the HBV envelope proteins can also assist in the assembly of hepatitis delta virus (HDV) particles (2, 49). HDV is an occasional satellite of HBV. Its genome consists of a circular single-stranded RNA molecule with only one open reading frame from which a protein is known to be translated (50). The latter is synthesized in two isoforms, the small and large hepatitis delta antigens (S-HDAg and L-HDAg, respectively). HDV RNA replicates without any assistance from the helper HBV, and it assembles with mul...

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides-estimated at three to four per virion-is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S-and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.Hepatitis B virus (HBV) remains a major public health concern worldwide, affecting more than 350 millions of chronically infected individuals. Since the discovery of HBV, substantial information has been gathered on the viral replication cycle, but our understanding of the viral entry mechanism remains limited, and the identity of the receptor(s) for HBV is still unknown (15). HBV displays a very narrow host range, which is likely determined at viral entry by a highly specific interaction between the HBV envelope proteins and receptors at the surface of human hepatocytes. The envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (SHBsAg) are membrane-spanning glycoproteins that differ from each other by the size of their N-terminal ectodomain (21). L-HBsAg contains a N-terminal pre-S1, central pre-S2, and C-terminal S domains. M-HBsAg is shorter than L-HBsAg in lacking pre-S1, whereas S-HBsAg consists of the S domain only (Fig. 1). Envelope protein synthesis occurs at the endoplasmic reticulum (ER) membrane. Empty subviral particles (SVPs) assemble from aggregates at a pre-Golgi membrane and exit the cell through the secretory pathway (36). Assembly of mature HBV virions requires, in addition to S-HBsAg, the presence of L-HBsAg as a matrix protein for nucleocapsid envelopment (6). Recent findings indicate that HBV viri...

Worldwide there are approximately 240million individuals chronically infected with the hepatitis B virus (HBV), including 15-20million coinfected with the hepatitis delta virus (HDV). Treatments available today are not fully efficient and often associated to important side effects and development of drug resistance. Targeting the HBV/HDV entry step using preS1-specific lipopeptides appears as a promising strategy to block viral entry for both HBV and HDV (Gripon et al., 2005; Petersen et al., 2008). Recently, the human Sodium Taurocholate Cotransporting Polypeptide (hNTCP) has been identified as a functional, preS1-specific receptor for HBV and HDV. This groundbreaking discovery has opened a very promising avenue for the treatment of chronic HBV and HDV infections. Here we investigated the ability of FDA approved therapeutics with documented inhibitory effect on hNTCP cellular function to impair viral entry using a HDV in vitro infection model based on a hNTCP-expressing Huh7 cell line. We demonstrate the potential of three FDA approved molecules, irbesartan, ezetimibe, and ritonavir, to alter HDV infection in vitro.