Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.     

Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.     

Cellular receptor traffic is essential for productive duck hepatitis B virus infection

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.     

Carboxypeptidase D Is an Avian Hepatitis B Virus Receptor

The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4 degrees C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

Carboxypeptidase D (gp180), a Golgi-Resident Protein, Functions in the Attachment and Entry of Avian Hepatitis B Viruses

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180–pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.     

Topology of the large envelope protein of duck hepatitis B virus suggests a mechanism for membrane translocation during particle morphogenesis.

We have investigated the membrane topology of the large envelope protein of duck hepatitis B virus (DHBV) by protease protection and Western blot analysis, using monoclonal antibodies specific for the pre-S and S regions of the DHBV envelope to characterize protease-resistant polypeptides. These studies showed that DHBV L protein exhibits a mixed membrane topology similar to that of human hepatitis B virus L, with approximately half of the L molecules displaying pre-S on the surface of virus particles and the remainder with pre-S sequestered inside the virus envelope. The C-terminal region of DHBV pre-S was susceptible to protease digestion on all DHBV particle L protein, indicating that this region was externally disposed. DHBV L protein pre-S was entirely cytosolic immediately after synthesis. Our data, therefore, suggested that an intermediate form of the DHBV L molecule exists in mature envelope particles in which L is partially translocated or exists in a translocation-ready conformation. Incubation of virus particles at low pH and 37 degrees C triggered conversion of this intermediate into a fully translocated form. We have proposed a model for pre-S translocation based on our results that invokes the presence of an aqueous pore in the virus envelope, most likely created by oligomerization of transmembrane domains in the S region. The model predicts that pre-S is transported through this pore and that a loop structure is formed because the N terminus remains anchored to the inner face of the membrane. This translocation process occurs during particle morphogenesis and may also be a prerequisite to virus uncoating during infection.     

Inhibition of Duck Hepatitis B Virus Infection by a Myristoylated Pre-S Peptide of the Large Viral Surface Protein

We have used the duck hepatitis B virus (DHBV) model to study the interference with infection by a myristoylated peptide representing an N-terminal pre-S subdomain of the large viral envelope protein. Although lacking the essential part of the carboxypeptidase D (formerly called gp180) receptor binding site, the peptide binds hepatocytes and subsequently blocks DHBV infection. Since its activity requires an amino acid sequence involved in host discrimination between DHBV and the related heron HBV (T. Ishikawa and D. Ganem, Proc. Natl. Acad. Sci. USA 92:6259-6263, 1995), we suggest that it is related to the postulated host-discriminating cofactor of infection.     

Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB).

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.     

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.     

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.     

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.     

A metastable form of the large envelope protein of duck hepatitis B virus: low-pH release results in a transition to a hydrophobic, potentially fusogenic conformation

We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.     

Monoclonal antibodies to a 55-kilodalton protein present in duck liver inhibit infection of primary duck hepatocytes with duck hepatitis B virus.

As an approach to identifying hepatocyte receptors for the avian hepadnavirus duck hepatitis B virus (DHBV), hybridomas were prepared from mice immunized with permissive duck hepatocytes. Monoclonal antibodies (MAbs) were screened for the ability to inhibit binding of DHBV particles to primary duck hepatocytes and to block infection. We identified two MAbs which partially blocked binding and caused marked inhibition of infection of primary duck hepatocytes with DHBV. Lack of cross-reactivity with DHBV envelope proteins suggested that inhibition of infection was due to specific interaction between the antibodies and a host cell surface molecule. Both MAbs immunoprecipitated a 55-kDa protein (p55) expressed in duck liver and several other duck tissues. p55 homologs were also identified in other birds and mammals. We predict from our data that only a small proportion of total cellular p55 molecules are expressed at the surfaces of hepatocytes and that p55 is involved in some early step in the infectious pathway.     

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles.

DNA from the pre-S region of the duck hepatitis B virus (DHBV) genome was inserted into an open reading frame vector designed to give high-level expression in Escherichia coli. The resulting fusion protein contained the first 8 amino acids of beta-galactosidase, 86 amino acids of the DHBV pre-S region, and 219 amino acids of chloramphenicol acetyltransferase at the C terminus (beta-gal:pre-S:CAT). Rabbit antiserum against purified beta-gal:pre-S:CAT was used to identify pre-S-containing polypeptides in DHBV particles by Western blotting. A dominant species of 36 kilodaltons (kDa) was identified. Antiserum against the major 17-kDa DHBsAg polypeptide also reacted with the 36-kDa protein. This suggests that the DHBV envelope gene polypeptides share the same carboxyl terminus, but differ in the sites from which translation is initiated. N-linked carbohydrate was not detected on either the 17- or 36-kDa envelope proteins. Anti-beta-gal:pre-S:CAT abolished infectivity of the virus in an in vitro assay. Thus, the pre-S region is exposed on the surfaces of infectious virions and may be directly involved in binding of virus to host-cell receptors.     

Maternally transferred antibodies from DNA-immunized avians protect offspring against hepadnavirus infection

The outcome and protective efficacy of maternal antibodies elicited by DNA immunization to the large (L) hepadnavirus envelope protein were studied using the duck hepatitis B virus (DHBV) model. Following genetic immunization of breeding ducks with a DHBV L protein gene-bearing plasmid, specific and highly neutralizing antibodies were transferred from the sera of immunized ducks, via the egg yolk, to the progeny of vaccinees. Interestingly, large amounts (60 to 100 mg/egg) of high-titer and L protein-specific yolk immunoglobulins (immunoglobulin Y) accumulated in the egg yolk. These results suggest that eggs from genetically immunized avians may represent a potent source of DNA-designed antibodies specific to viral antigen. Importantly, these antibodies are vertically transmitted and protect offspring against high-titer DHBV challenge.     

Antibodies induced by liposomal protein exhibit dual binding to protein and lipid epitopes

Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.