Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.     

Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus

The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.     

Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion.

The hepatitis delta virus (HDV) is coated with large (L), middle (M), and small (S) envelope proteins encoded by coinfecting hepatitis B virus (HBV). To study the role of the HBV envelope proteins in the assembly and infectivity of HDV, we produced three types of recombinant particles in Huh7 cells by transfection with HBV DNA and HDV cDNA: (i) particles with an envelope containing the S HBV envelope protein only, (ii) particles with an envelope containing S and M proteins, and (iii) particles with an envelope containing S, M, and L proteins. Although the resulting S-, SM-, and SML-HDV particles contained both hepatitis delta antigen and HDV RNA, only particles coated with all three envelope proteins (SML) showed evidence of infectivity in an in vitro culture system susceptible to HDV infection. We concluded that the L HBV envelope protein, and more specifically the pre-S1 domain, is important for infectivity of HDV particles and that the M protein, which has been reported to bear a site for binding to polymerized albumin in the pre-S2 domain, is not sufficient for infectivity. Our data also show that the helper HBV is not required for initiation of HDV infection. The mechanism by which the L protein may affect HDV infectivity is discussed herein.     

The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

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 (S-HBsAg) 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. ​(Fig.1).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 virions and SVPs follow distinct pathways for budding: the late endosomal multivesicular bodies (MVBs) for HBV virions, and the MVB-independent secretory pathway for SVPs (26, 28, 46). The HBV envelope proteins can also package the hepatitis delta virus (HDV) ribonucleoprotein (RNP), in case of HBV/HDV coinfection (5, 45), leading to the formation of HDV virions. Whether HDV uses the SVP secretion pathway rather than an MVB-dependent route is uncertain.Open in a separate windowFIG. 1.Schematic representation of HBV envelope proteins. The topology of the L-, M-, and S-HBsAg proteins at the viral membrane is represented. The pre-S2 domain of L- and M-HBsAg, and the determinants of viral entry, pre-S1 and AGL, are indicated. The M-HBsAg protein, represented in gray, is dispensable for infectivity. The myristic acid (Myr) linked to the L-HBsAg N terminus is indicated (closed box). Subdomains 2-48 and 49-75 of the pre-S1 infectivity determinant are indicated. Open boxes represent transmembrane regions in the S domain.L-HBsAg, but not M-HBsAg, is crucial to infectivity of both HBV and HDV particles (13, 31, 41, 42). L-HBsAg contains a major infectivity determinant located between amino acid residues 2 and 75 of its N-terminal pre-S1 domain (4, 30), including a myristoyl anchor linked to glycine-2 (1, 8, 18), a putative receptor binding site (RBS) between positions 2 and 48, and a domain of unknown function between amino acids 49 and 75. To date, the most compelling evidence that pre-S1 mediates receptor binding comes from studies demonstrating that myristoylated synthetic peptides specific for the N-terminal 2-to-48 pre-S1 domain can bind to hepatocyte plasma membranes and block infection in vitro (3, 16, 17) and in vivo (37). Beside pre-S1, a second determinant was recently identified in the antigenic loop (AGL) borne by the three HBV envelope proteins (Fig. ​(Fig.1).1). The AGL participation in viral entry was first established in the HDV model (23) and more recently directly in the HBV model (39). Interestingly, serine substitutions for the AGL cysteine residues, which prove detrimental to the conserved immunodominant “a” determinant, could also block viral entry. Note that the “a” determinant consists in conformational epitopes, which elicit highly neutralizing antibodies (22). Infectivity and the “a” determinant were also lost when virions were treated with membrane-impermeable inhibitors of thiol/disulfide isomerization (2). These findings clearly established a correlation between the AGL cysteine disulfide bonds network, the conformation of the “a” determinant, and infectivity. Hence, the strict conservation of the “a” determinant among all HBV genotypes is related to the AGL function at viral entry. The AGL determinant may operate in association with, or independently of pre-S1, in binding to receptors at the early step of entry and/or in the mechanism of envelope disassembly postentry.In the present study, we investigated the pre-S1 determinant by performing transcomplementation experiments between mutants of 3 pre-S1 subelements: the myristoyl anchor, subdomain 2-48, and subdomain 49-75. We analyzed the activity of the AGL determinant in the S- or L-HBsAg background (S- and L-AGL, respectively), and we examined the effect of introducing increasing amounts of infectivity-deficient pre-S1, or AGL, in the virion''s envelope on infectivity.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange

Hepatitis delta virus (HDV) particles are coated with the envelope proteins (large, middle, and small) of the hepatitis B virus (HBV). The large protein bears an infectivity determinant in its pre-S1 domain, whereas a second determinant has been proposed to map to the cysteine-rich antigenic loop (AGL) within the S domain of all three envelope proteins (G. Abou Jaoudé and C. Sureau, J. Virol. 79:10460-10466, 2006). In this study, the AGL cysteines were substituted by serine or alanine, and the mutants were evaluated for their function at viral entry using HDV particles and susceptible HepaRG cells. Mutations of cysteines 121 to 149 were tolerant of the production of HDV virions. The mutations altered the structure and antigenicity of the conserved “a” determinant of the AGL, as measured by conformation-sensitive antibodies, and they created a block to infectivity. Substitution of Cys-90 or Cys-221, located outside of the AGL, had no impact on the “a” determinant or viral entry. Furthermore, infectivity was maintained when the AGL CxxC motif at position 121 to 124 was modified by single-amino-acid deletion or insertion, suggesting that cysteines 121 and 124 are not catalyzers of thiol/disulfide exchange. However, membrane-impermeable inhibitors of thiol/disulfide isomerazation demonstrated a dose-dependent inhibition of infection in an in vitro assay when applied to the virus prior to inoculation or during the virus-cell interaction period. Overall, the results demonstrate the essential role of the AGL cysteines at viral entry, and they establish a correlation between the cysteine disulfide network, the conformation of the “a” determinant, and infectivity.     

O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection

BackgroundHepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg.MethodsHBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined.ResultsMass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(β1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro.ConclusionsGlycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection.General significanceThe detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.     

Properties of subviral particles of hepatitis B virus

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.     

A Function Essential to Viral Entry Underlies the Hepatitis B Virus “a” Determinant

The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant “a” determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the “a” determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the “a” determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the “a” determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan “a” determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.Hepatitis B virus (HBV) causes acute and chronic infections in humans; such infections are often associated with severe liver diseases, including cirrhosis and hepatocellular carcinoma (10). To date, it is estimated that approximately 350 millions individuals worldwide suffer from chronic infection despite the availability of an effective vaccine for more than 25 years. Remarkably, the development of a vaccine soon after the HBV discovery was, at least in part, the consequence of a very peculiar feature that is unique to members of the Hepadnaviridae family: viral envelope proteins are produced in quantities far exceeding the amounts required for assembly of HBV virions (6) and, owing to their capacity for autoassembly, the vast majority are secreted as subviral particles. Besides the practical consequences in the original vaccine development, in nature, the phenomenon of HBV envelope protein overexpression has provided a helper function to the hepatitis delta virus (HDV) (29). The HBV envelope proteins assist in packaging the HDV ribonucleoprotein (RNP) in case of HBV-HDV coinfection, thereby ensuring spreading of the satellite HDV. As a result, the coats of HBV and HDV particles are similar, consisting of cell-derived lipids and the HBV envelope proteins—large, middle, and small—bearing the HBV surface antigen (HBsAg) and referred to as L-HBsAg, M-HBsAg, and S-HBsAg, respectively (4, 14).The HBsAg includes an immunodominant determinant common to all HBV strains, referred to as “a,” and several mutually exclusive subtype-specific determinants referred to as “d”/“y” and “w”/“r” (21). The “a” determinant is defined by a specific conformation of the antigenic loop (AGL) polypeptide present at the surface of subviral, HBV, or HDV particles. The AGL itself resides between the transmembrane domain II (residues 80 to 100) and the hydrophobic carboxyl terminus (residues 165 to 226) of the envelope proteins S domain (see Fig. ​Fig.1).1). It is the “a” determinant that elicits the most effective neutralizing antibody response upon vaccination or infection (32). Surprisingly, a function in the HBV life cycle had never been assigned to the “a” determinant, the first identified HBV marker, until the recent demonstration of its involvement in HDV entry (2, 15). More precisely, it was shown that the AGL cysteine residues were critical for both the structure of the “a” determinant and HDV infectivity (2).Open in a separate windowFIG. 1.Schematic representation of the HBV envelope protein AGL. (A) The topology of the L-, M-, and S-HBsAg proteins (L, M, and S, respectively) is represented. The determinants of viral entry, pre-S1 and AGL, are indicated in red. The M-HBsAg protein, represented in gray, is dispensable for infectivity. Open boxes represent transmembrane regions in the S domain. (B) Alignment of the AGL amino acids sequences (positions 101 to 172 in the S domain) of HBV (genotype D, ayw3 phenotype), WMHBV, and WHV. The GenBank sequence numbers of the isolates are as follows: {"type":"entrez-nucleotide","attrs":{"text":"J02203","term_id":"329640","term_text":"J02203"}}J02203 (HBV), {"type":"entrez-nucleotide","attrs":{"text":"AY226578","term_id":"29501369","term_text":"AY226578"}}AY226578 (WMHBV), and {"type":"entrez-nucleotide","attrs":{"text":"NC_004107","term_id":"22256030","term_text":"NC_004107"}}NC_004107 (WHV). HBV amino acid residues important for infectivity (the present study) are indicated in blue. A hyphen denotes amino acid identity with the HBV sequence.It is now well established that both HBV and HDV entry rely on the pre-S1 domain of L-HBsAg as the primary infectivity determinant that is likely to promote attachment to a specific receptor at the surface of human hepatocytes (11). The AGL determinant could thus fulfill complementary functions for attachment, uptake, or particle disassembly after entry (2, 15).In the present study, the AGL infectivity determinant was mapped and confirmed to be closely related to the “a” determinant. Moreover, its essential function at viral entry was demonstrated in the HBV model.     

Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles.

Hepatitis delta virus (HDV) has an envelope composed of large-, middle-, and small-form hepatitis B surface antigens (HBsAgs) provided by the helper hepatitis B virus (HBV). In order to examine the roles of individual HBsAgs in HDV assembly, we constructed plasmids containing each specific HBsAg gene and then cotransfected each plasmid with HDV cDNA into a permissive human hepatoma cell line (HuH-7) to examine the effects on HDV production. Results indicated that the plasmids containing only the HBsAg genes were able to complement HDV cDNA as efficiently as the plasmid containing the complete HBV genome in generating HDV-like particles. Moreover, the small-form HBsAg alone was sufficient for HDV packaging. The particles produced from the cotransfection experiments have density and protein composition characteristics similar to those of naturally occurring HDV. With the electron microscope, they were identified as 36- to 38-nm-diameter particles. It was concluded that only the HBsAgs were able to help in the assembly of HDV-like particles.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

Plant expression,lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B.     

Genetic organization and replication strategy of hepatitis delta virus

Hepatitis delta virus (HDV) is a subviral satellite of human hepatitis B virus (HBV). The discovery in 1977 and subsequent demonstration of HDV as an infectious agent was primarily due to the work of Rizzetto and co-workers. In nature, HDV infections occur only if HBV is present. This is because HDV is a subviral satellite of HBV; HBV provides the envelope, or surface antigens, needed for the assembly of HDV particles. Other than this dependence, HDV seems fundamentally different from HBV; it has a single-stranded RNA genome and replicates via RNA-directed RNA synthesis. Five years ago the first nucleotide sequence of the genome was obtained and as a consequence we have progressively gained a picture of the genetic organization of this unusual agent and of its replication strategy.     

Failure To Cleave Murine Leukemia Virus Envelope Protein Does Not Preclude Its Incorporation in Virions and Productive Virus-Receptor Interaction

It is thought that complete cleavage of retroviral envelope protein into mature surface protein (SU) and transmembrane protein (TM) is critical for its assembly into virions and the formation of infectious virus particles. Here we report the identification of highly infectious, cleavage-deficient envelope mutant proteins. Substitution of aspartate for lysine 104, arginines 124 and 126, or arginines 223 and 225 strongly suppressed cleavage of the envelope precursor and yet allowed efficient incorporation of precursor molecules as the predominant species in virions that were almost as infectious as the wild-type virus. These results indicate that cleavage of the envelope precursor into mature SU and TM is not necessary for assembly into virions. Moreover, they call into question how many mature envelope protein subunits are required to complete virus entry, suggesting that a very few molecules suffice. The failure of host cell proteases to cleave these mutant proteins, whose substitutions are distal to the actual site of cleavage, suggests that the envelope precursor is misfolded, sequestering the cleavage site. In agreement with this, all cleavage mutant proteins exhibited significant losses of receptor binding, suggesting that these residues play roles in proper envelope protein folding. We also identified a charged residue, arginine 102, whose substitution suppressed envelope cleavage and allowed precursor incorporation but resulted in virions that were virtually noninfectious and that exhibited the greatest reduction in receptor binding. Placement of these cleavage mutations into envelope proteins of targeted retroviral vectors for human gene therapy may prevent loss of the modified surface proteins from virions, improving their infectivity and storage hardiness.     

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.     

Assembly of hepatitis B virus envelope proteins onto a lentivirus pseudotype that infects primary human hepatocytes

This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry. Also, virus particles that were able to infect cultured primary human hepatocytes (PHH) were released. Under optimal conditions, 50% of PHH could be infected. In addition, the susceptibility of PHH and the resistance of other cell types to the pseudotype particles were similar to those observed for HBV and hepatitis delta virus (HDV), which shares the same L, M, and S. Furthermore, the infection of PHH by the pseudotype was sensitive to known inhibitors of HBV and HDV entry. These findings of specific and efficient infection of hepatocytes could be applicable to liver-specific gene therapy and may help clarify the attachment and entry mechanism used by HBV and HDV.