In vitro activity and resistance profile of samatasvir, a novel NS5A replication inhibitor of hepatitis C virus.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50 shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50 ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 µM provided a selectivity index of >5 × 10(7). Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.

Carotid sinus syndrome, should we pace? A multicentre, randomised control trial (Safepace 2).

BACKGROUND: Cardioinhibitory carotid sinus hypersensitivity (CICSH) is highly prevalent among older people with falls. OBJECTIVE: To assess the efficacy of dual-chamber pacing in older patients with CICSH and unexplained falls. DESIGN: A multicentre, double blind, randomised controlled trial. SETTING: Selection from emergency room, geriatric medicine and orthopaedic departments. PATIENTS: Patients aged >50 years, with two unexplained falls and/or one syncopal event in the previous 12 months for which no other cause is evident apart from CICSH. INTERVENTIONS: Patients randomised to either a 700/400 kappa, rate responsive pacemaker or implantable loop recorder (Medtronic Reveal thera RDR, Medtronic, Minneapolis, Minnesota, USA). MAIN OUTCOME MEASURES: The primary outcome was the number falls after implantation. Secondary outcomes were time to fall event, presyncope, quality of life and cognitive function. RESULTS: 141 patients were recruited from 22 centres. Mean age was 78 years and mean follow-up 24 months. The overall relative risk of falling after device implantation compared with before was 0.23 (0.15 to 0.32). No significant reduction in falls was seen between paced and loop recorder groups (RR=0.79; 95% CI 0.41 to 1.50). Data were also consistent in both groups for syncope, quality of life and cognitive function. Conclusions These results question the use of pacing in CICSH. However, the study was underpowered and also patient characteristics differed from those in Safepace 1-participants were physically and cognitively frailer. Further work is necessary to assess cardiac pacing in this setting.

Optimised conditions for the production of hepatitis B virus from cell culture.

In chronically infected patients, hepatitis B virus (HBV) particles reach numbers as large as >10(9) genome equivalents (GE)/ml of serum. However, expression of infectious HBV particles in cell culture only yields 10(5)-10(6) GE/ml, which is insufficient for many studies. HBV transcription and possibly replication is dependent on hepatocyte-specific differentiation. Thus, we tested several cell culture parameters that have been reported to enhance the expression of hepatocyte-specific markers, such as growth on different extracellular matrices, different cell culture media, low concentrations of fetal calf serum (FCS) and the addition of dimethyl sulfoxide (DMSO) to the medium. Lower concentrations of FCS, growth on collagen and inclusion of DMSO in the medium only moderately enhanced HBV production in vitro when applied individually. However, combinations of these parameters optimised cell culture conditions and reproducibly increased the release of HBV particles about 100-fold to titres >10(8) GE/ml of culture medium.

Antiviral efficacy of lobucavir (BMS-180194), a cyclobutyl-guanosine nucleoside analogue, in the woodchuck (Marmota monax) model of chronic hepatitis B virus (HBV) infection.

Lobucavir (BMS-180194), a cyclobutyl-guanosine nucleoside analogue, effectively reduced WHV-viremia in chronically infected carrier woodchucks (Marmota monax) by daily per os treatment. WHV-viremia in the animals was measured by the serum content of hybridizable WHV-genomic DNA. Lobucavir, given at daily doses of 10 and 20 mg/kg body weight, reduced WHV-viremia by a 10- to 200-fold range during therapy. Lobucavir, given at 5 mg/kg, suppressed WHV-viremia by a 10- to 30-fold range, whereas a 0.5 mg/kg dose had no significant effect. WHV-viremia was also measured by hepadnaviral endogenous polymerase activity (EPA) in sera of animals treated for 6 weeks at 5 and 0.5 mg/kg. Changes in EPA in sera of lobucavir treated animals were comparable to changes in WHV DNA levels. Viremia in treated carriers recrudesced to pretreatment levels by 2 weeks of therapy cessation. These results indicated that the minimally effective antiviral daily per os dose of lobucavir in WHV-carrier woodchucks was approximately 5 mg/kg.

In vitro inhibition of hepadnavirus polymerases by the triphosphates of BMS-200475 and lobucavir.

The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) in 2.2.15 liver cells and in the woodchuck animal model system, respectively. This repression was presumed to occur via inhibition of the viral polymerase (Pol) by the triphosphate (TP) forms of BMS-200475 and lobucavir which are both produced in mammalian cells. To determine the exact mode of action, BMS-200475-TP and lobucavir-TP, along with several other guanosine analog-TPs and lamivudine-TP were tested against the HBV, WHV, and duck hepatitis B virus (DHBV) polymerases in vitro. Estimates of the 50% inhibitory concentrations revealed that BMS-200475-TP and lobucavir-TP inhibited HBV, WHV, and DHBV Pol comparably and were superior to the other nucleoside-TPs tested. More importantly, both analogs blocked the three distinct phases of hepadnaviral replication: priming, reverse transcription, and DNA-dependent DNA synthesis. These data suggest that the modest potency of lobucavir in 2.2.15 cells may be the result of poor phosphorylation in vivo. Kinetic studies revealed that BMS-200475-TP and lobucavir-TP competitively inhibit HBV Pol and WHV Pol with respect to the natural dGTP substrate and that both drugs appear to bind to Pol with very high affinities. Endogenous sequencing reactions conducted in replicative HBV nucleocapsids suggested that BMS-200475-TP and lobucavir-TP are nonobligate chain terminators that stall Pol at sites that are distinct yet characteristically two to three residues downstream from dG incorporation sites.

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection.

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.

Properties of tumour suppressor p53 in murine hepatocyte lines transformed by hepatitis B virus X protein.

Persistent infection by hepatitis B virus (HBV) correlates with the prevalence of hepatocellular carcinoma. It has recently been demonstrated that the complete viral genome very efficiently transforms the immortalized murine hepatocyte line FMH202 in vitro. Here it is shown that the viral transactivating protein X (HBx) is sufficient to transform FMH202 cells, albeit with lower efficiency. Clonal cell lines expressing HBx mRNA in moderate or high amounts grew in soft agar and formed tumours in nude mice. Growth efficiency in soft agar of HBx transformed cell lines was much lower than that of cell lines transformed with the complete genome, and latency of tumour induction in nude mice was significantly longer after inoculation of HBx than of HBV transformed FMH202 cell lines. A marker of complete transformation, p53, was found to be phosphorylated more strongly in HBx transfected cell lines than in controls, and a cellular kinase was found to be associated with p53 complexes from HBx transformed cell lines. p53 was of wild-type conformation and was located in the nucleus of transformed cells.

Generation of replication-competent hepatitis B virus nucleocapsids in insect cells.

The double-stranded DNA genome of human hepatitis B virus (HBV) and related hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase (Pol) harboring both priming and RNA- and DNA-dependent elongation activities. Although hepadnavirus replication occurs inside viral nucleocapsids, or cores, biochemical systems for analyzing this reaction are currently limited to unencapsidated Pols expressed in heterologous systems. Here, we describe cis and trans classes of replicative HBV cores, produced in the recombinant baculovirus system via coexpression of HBV core and Pol proteins from either a single RNA (i.e., in cis) or two distinct RNAs (in trans). Upon isolation from insect cells, cis and trans cores contained Pol-linked HBV minus-strand DNA with 5' ends mapping to the authentic elongation origin DR1 and also plus-strand DNA species. Only trans cores, however, were highly active for the de novo priming and reverse transcription of authentic HBV minus strands in in vitro endogenous polymerase assays. This reaction strictly required HBV Pol but not the epsilon stem-loop element, although the presence of one epsilon, or better, two epsilons, enhanced minus-strand synthesis up to 10-fold. Compared to unencapsidated Pol enzymes, encapsidated Pol appeared to be (i) highly processive, able to extend minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) more active for HBV plus-strand synthesis. These observations suggest possible contributions to the replication process from the HBV core protein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, and also fuel the development of new anti-HBV drug screens.

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.

Assembly and antigenicity of hepatitis B virus core particles.

Recent studies in Xenopus oocytes and other systems have led to an understanding of the HBV capsid, or core particle, assembly process. Nascent HBV core polypeptides rapidly dimerize. Accumulation of free dimers to a signature concentration (approximately 0.8 microM) then triggers a highly cooperative capsid assembly reaction. This dimer-to-capsid transition is accompanied by a switch from HBe to HBc antigenicity and appears to be nucleated by interaction between core protein and RNA: deletion of a protamine-like RNA binding domain at the C-terminus of the core protein markedly increases the concentration of dimers needed to drive capsid assembly. The simple assembly pathway seen for HBV capsids mirrors that of R17 bacteriophage.

Ribonucleoprotein complex formation by the human hepatitis B virus polymerase.

Human hepatitis B virus (HBV) polymerase (pol or RT), when expressed in Xenopus oocytes upon injection of synthetic minimal pol RNA (RT RNA), assembles into a higher molecular weight complex with the characteristics of a ribonuclear protein (RNP) complex. In vitro RNA competition binding data suggest that RT RNA is preferentially packaged into this complex even though it lacks the authentic viral encapsidation signal, epsilon, and viral capsid protein sequences. Consistent with this finding, the in vitro polymerase reaction performed in pol-expressing oocyte extracts generates primarily HBV-specific DNAs even when the pol template is challenged with a coinjected non-HBV competitor RNA. These results suggest that interaction between pol and its cognate RNA can be mediated by sequences other than the known packaging elements. We speculate that HBV RNP complexes containing at least polymerase and viral RNA may play a role in viral nucleocapsid assembly and may help to segregate HBV reverse transcription from the cellular milieu in vivo.

A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface.

Core particles of hepatitis B virus are assembled from dimers of a single 185-residue (subtype adw) viral capsid or core protein (p21.5) which possesses two distinct domains: residues 1 to 144 form a minimal capsid assembly domain, and the arginine-rich, carboxyl-terminal residues 150 to 185 form a protamine-like domain that mediates nucleic acid binding. Little is known about the topography of the p21.5 polypeptide within either the p21.5 capsids or dimers. Here, using site-specific proteases and monoclonal antibodies, we have defined the accessibility of p21.5 residues in dimers and capsids assembled from wild-type and mutant hepatitis B virus core proteins in Xenopus oocytes and in vitro. The data reveal the protamine region to be accessible to external reagents in p21.5 dimers but largely cryptic in wild-type capsids. Strikingly, in capsids the only protease target region was a 9-residue peptide covering p21.5 residues Glu-145 to Asp-153, which falls largely between the two core protein domains. By analogy with protease-sensitive interdomain regions in other proteins, we propose that this peptide constitutes a hinge between the assembly and nucleic acid binding domains of p21.5. We further found that deletion or replacement of the terminal Cys-185 residue greatly increased surface exposure of the protamine tails in capsids, suggesting that a known disulfide linkage involving this residue tethers the protamine region inside the core particles. We propose that disruption of this disulfide linkage allows the protamine region to appear transiently on the surface of the core particle.

Stability governs the apparent expression of "particulate" hepatitis B e antigen by mutant hepatitis B virus core particles.

The p21.5 capsid or core protein of hepatitis B virus carries two distinct classes of epitopes. Core (HBc) epitopes are found exclusively on the surface of the 28-nm viral icosahedral capsids or core particles, while HBe epitopes are normally expressed only by subparticulate forms of the core protein. Recent studies have suggested that a "particulate" form of HBe is expressed on the surface of capsid particles assembled from p17, a truncated core protein that lacks the carboxy-terminal protamine-like region of p21.5 and hence the ability to bind and encapsidate RNA. In this report we have used epitope-specific ELISAs in conjunction with capsids assembled from a series of carboxy-terminally truncated core proteins to address the mechanistic basis for particulate HBe. Specifically, we sought to test the idea that particulate HBe expression might be linked to the loss of RNA binding. However, our results strongly suggest that expression of HBe by mutant core particles is a result of their intrinsic instability which increases sharply when RNA binding is lost. We show that core particles assembled from mutant core proteins lacking Cys residues also express HBe, again because of capsid instability. We report mild conditions that can induce the dissociation of the mutant capsids and discuss our findings in terms of the factors that control capsid stability.

Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1.

The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.

A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes.

Assembly of hepatitis B virus capsid-like (core) particles occurs efficiently in a variety of heterologous systems via aggregation of approximately 180 molecules of a single 21.5-kDa core protein (p21.5), resulting in an icosahedral capsid structure with T = 3 symmetry. Recent studies on the assembly of hepatitis B virus core particles in Xenopus oocytes suggested that dimers of p21.5 represent the major building block from which capsids are generated. Here we determined the concentration dependence of this assembly process. By injecting serially diluted synthetic p21.5 mRNA into Xenopus oocytes, we expressed different levels of intracellular p21.5 and monitored the production of p21.5 dimers and capsids by radiolabeling and immunoprecipitation, by radioimmunoassay, or by quantitative enzyme-linked immunosorbent assay analysis. The data revealed that (i) p21.5 dimers and capsids are antigenically distinct, (ii) capsid assembly is a highly cooperative and concentration-dependent process, and (iii) p21.5 must accumulate to a signature concentration of approximately 0.7 to 0.8 microM before capsid assembly initiates. This assembly process is strikingly similar to the assembly of RNA bacteriophage R17 as defined by in vitro studies.

Increased growth of permanent mouse fibroblasts in soft agar after transfection with hepatitis B virus DNA.

Previously we have shown that a nontumorigenic mouse hepatocyte line harboring simian virus 40 large tumor antigen (SV 40 TAg) could be converted to a full-malignant phenotype by transfection with HBV DNA. Using a permanent SV 40 TAg-negative mouse fibroblast cell line (LTK-), we studied whether the in vitro-oncogenicity of HBV was dependent on simultaneous expression of SV 40 TAg or not. Three fibroblast lines stably transfected by full-length HBV DNA formed four times more colonies of large size in soft agar than nontransfected LTK- cells. All three clones expressed high levels of HBx protein, but variable levels of other HBV proteins. A second type of clone that was transfected by a partial HBV genome and that expressed HBV surface but no HBx proteins, did not acquire increased growth in soft agar. These data reveal that HBV DNA can enhance malignant growth independent of SV 40 TAg and suggest that HBx protein may act as an HBV oncogene at least in vitro.

In vitro tumorigenicity of hepatitis B virus DNA and HBx protein.

Persistent infection by hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma. This linkage may be caused by oncogenic HBV gene products. Our initial in vitro studies have revealed that a non-tumorigenic, fetal mouse hepatocyte line (FMH202-1), harboring simian virus 40 large tumor antigen (SV40TAg) as transgene, can be converted into a full-malignant phenotype by transfection with dimeric HBV-DNA Höhne, M., Schaefer, S., Seifer, M., Feitelson, M.A., Paul, D. and Gerlich, W.H. (1990) EMBO J 9, 1137-1145. The oncogenic effect was neither dependent on simultaneous expression of SV40TAg nor on the cell type, since HBV-transfected permanent mouse fibroblasts (LTK-) also displayed enhanced colony formation in soft agar. Transfection of FMH202-1 with the X region of HBV generated clones that also formed colonies in soft agar and were tumorigenic in nude mice. Growth in soft agar and induction of nude mice tumors both depended on high expression of HBx protein. Although HBx expression was stronger in X-transfected than HBV-transfected clones, the former did not grow well in soft agar, and the X-derived tumors developed more slowly. In the tumors, expression of HBx was almost shut off, but again high in the tumor-derived cell lines. Constitutive expression of c-fos was strongly enhanced in the X-transfected cell lines and tumors. Transfection of FMH202-1 with an isogenic HBx-deficient mutant fragment generated several clones, which expressed normal levels of HBx transcripts, but did not grow in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression pattern of the hepatitis B virus genome in transfected mouse fibroblasts.

Permanent mouse fibroblast LTK- cells were transfected with dimeric hepatitis B virus (HBV) DNA linked to the simian virus 40 (SV40) early promoter/enhancer. Many clones stably expressed high levels of polyadenylated RNAs encoding hepatitis B surface (HBs) proteins (2.1 kb), HBe protein (3.6 kb), and HBx protein (0.6 kb). Although a chimeric RNA (4.0 kb) probably starting from the SV40 promoter was also synthesized, transcription of viral RNAs was predominantly directed by HBV promoters and its terminator. In contrast to HBV-transfected liver cells, the fibroblasts expressed only pregenomic 3.6-kb transcripts starting 5' to, but not within, the precore sequence. Thus, no normal core protein could be synthesized, but the cells expressed and secreted HBe protein of heterogeneous size. Small and middle HBs proteins were strongly expressed, while large HBs protein was almost absent. HBx mRNA expression was more efficient in mouse fibroblasts than in human hepatoma cells and 18-kDa HBx protein was exclusively detected in purified nuclei. Expression of HBe, small and middle HBs, and HBx proteins apparently does not require hepatic factors. Underexpression of HBc mRNA and large HBs mRNA suggests that activity of their promoters depends on cell-type-specific transcription factors.

Replication of hepatitis B virus in transfected nonhepatic cells.

Permanent murine fibroblasts (LTK-) were transfected with a dimer of hepatitis B virus (HBV) DNA and a neomycin resistance gene which were both linked to the simian virus 40 (SV40) early promoter/enhancer. One of the stably transfected clones, LTK4/36, which secreted HBsAg, HBeAg, and HBV DNA was further analyzed. It contained eight to nine copies of integrated HBV DNA per haploid genome and low amounts of episomal HBV DNA. The secreted viral DNA was covalently linked to protein and was associated with particles which had the characteristic density of natural virions from serum of human viremic carriers. The particles contained an endogenous DNA polymerase, small and middle surface proteins, but in contrast to natural virions very little core protein and large surface protein. Instead of core protein, they contained incompletely processed HBe protein which is colinear to core protein. The fibroblast-derived virions were less stable than virions from human carriers or from transfected hepatoma cells. After several days of storage, their DNA was only partially protected against DNase. Obviously, nonhepatic cells can express HBV-like particles, even if liver-dependent gene products like large surface protein and core protein are missing.

Interaction between hepatitis B surface proteins and monomeric human serum albumin.

HBsAg is known to bind to human serum albumin polymerized by glutaraldehyde, human serum albumin has been found in preparations of HBsAg by several investigators. However, it is not yet known whether natural human serum albumin binds to hepatitis B virus under physiological conditions. We studied the binding between natural or recombinant HBsAg and monomeric human serum albumin by immunological, biochemical and biophysical methods. The binding capacity of 20-nm HBs spheres was variable but ranged up to six molecules HSA/sphere. A reversible binding site for human serum albumin was exclusively localized in the preS2 domain, whereas the S domain was inactive in vitro. Human serum albumin copurified with HBsAg of human origin during gel chromatography or sucrose-gradient centrifugation. This human serum albumin was monomeric in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preS2-bound part of the human serum albumin could be removed from HBsAg by high-salt, such as CsCl centrifugation, but another part could only be removed by treatment with a disulfide cleaving reagent. Most of this covalently bound human serum albumin was retained at the HBsAg particle after complete cleavage of medium-sized HBs protein with trypsin. This indicates a second way in which albumin binds irreversible to cysteine(s) of the small HBs protein (SHBs, P24 and GP27).