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Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. Liver diseases related to the human hepatitis B virus (HBV) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. Some 15 million of these people are also infected with the hepatitis D virus (HDV), which is a satellite virus of HBV, and this places them at an even higher risk of liver diseases, including cancer. The viruses are known to enter liver cells by binding to receptors on their surface before being engulfed. Both HBV and HDV have outer coats that consist of three kinds of envelope proteins, and a region called the pre-S1 domain in one of them is known to have a central role in the interaction between the viruses and the receptors and, therefore, in infecting the cells. However, the identity of the HBV receptor has remained a mystery. Now Yan et al. have identified this receptor to be sodium taurocholate cotransporting polypeptide. This protein, known as NTCP for short, is normally involved in the circulation of bile acids in the body. In addition to humans, only two species are known to be susceptible to infection by human HBV and HDV—chimpanzees and a small mammal known as the treeshrew. Yan et al. started by isolating primary liver cells from treeshrews, and then used a combination of advanced purification and mass spectrometry analysis to show that the NTCP on the surface of the cells interacts with the pre-S1 domain in HBV. The authors then performed a series of gene knockdown experiments on liver cells of both human and treeshrew origin: when the gene that codes for NTCP was silenced, HBV infection was greatly reduced. Moreover, they were able to transfect HepG2 cells—which are widely used in research into liver disease, but are not susceptible to HBV and HDV infection—with NTCP from humans and treeshrews to make them susceptible. Similarly, although monkeys are not susceptible to HBV, replacing just five amino acids in monkey NTCP with their human counterparts was enough to make the monkey NTCP a functional receptor for the viruses. In the past, basic research into HBV and the development of antiviral therapeutics have both been hindered by the lack of suitable in vitro infection systems and animal models. Now, the work of Yan et al. means that it will be possible to use NTCP-complemented HepG2 cells for challenges as diverse as fundamental studies of basic viral entry/replication mechanisms and large-scale drug screening. It is also possible that HBV and HDV infection might interfere with some of the important physiological functions carried out by NTCP, so the latest work could also be of interest to medical scientists working on other diseases related to these infections.

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV. Here the authors look at why macaque is non-susceptible to hepatitis B virus by focusing on the protein structure of the host receptor homolog. They show that macaque-derived host receptor homolog has multiple structural defects against virus binding.

Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.

Even though an approved vaccine for hepatitis B virus (HBV) is available and widely used, over 257 million individuals worldwide are living with chronic hepatitis B (CHB) who require monitoring of treatment response, viral activity, and disease progression to reduce their risk of HBV-related liver disease. There is currently a lack of predictive markers to guide clinical management and to allow treatment cessation with reduced risk of viral reactivation. Novel HBV biomarkers are in development in an effort to improve the management of people living with CHB, to predict disease outcomes of CHB, and further understand the natural history of HBV. This review focuses on novel HBV biomarkers and their use in the clinical setting, including the description of and methodology for quantification of serum HBV RNA, hepatitis B core-related antigen (HBcrAg), quantitative hepatitis B surface antigen (qHBsAg), including ultrasensitive HBsAg detection, quantitative anti-hepatitis B core antigen (qAHBc), and detection of HBV nucleic acid-related antigen (HBV-NRAg). The utility of these biomarkers in treatment-naïve and treated CHB patients in several clinical situations is further discussed. Novel HBV biomarkers have been observed to provide critical clinical information and show promise for improving patient management and our understanding of the natural history of HBV.

Hepatitis B virus (HBV) remains a global health challenge. Capsid assembly modulators (CAMs) represent a promising class of antiviral agents that disrupt HBV core antigen (HBcAg) function. Understanding the structural and dynamic impact of CAMs on HBcAg is crucial for the development of next-generation antiviral therapies. This study employed molecular dynamics (MD) simulations to evaluate the conformational behavior of capsid monomers in unbound and ligand-bound states. Different classes of CAMs, Heteroaryldihydropyrimidine (HAP), Sulfamoylbenzamide (SBA), and Ciclopirox, were analyzed to assess their impact on HBcAg stability, flexibility, and interaction energy. RMSD analysis revealed that HAP binding stabilized HBcAg, reducing backbone fluctuations, whereas SBA and PPA increased HBcAg flexibility. RMSF calculations demonstrated that CAM interactions influenced loop and terminal region dynamics. PCA suggested ligand-specific alterations in HBcAg’s essential motions, with Sulfamoylbenzamide inducing the highest variance. Salt bridge analysis indicated that Ciclopirox formed the strongest electrostatic interactions, stabilizing its binding. DSSP secondary structure analysis showed that CAMs disrupted α-helical content, with Sulfamoylbenzamide and Ciclopirox exhibiting the most pronounced structural rearrangements. This study provides novel insights into CAM-induced conformational changes in HBcAg. While HAP stabilizes the core protein, SBA and Ciclopirox increase flexibility, potentially leading to misassembled or destabilized capsids. These findings contribute to the rational design of CAM-based antiviral therapies and highlight key structural determinants for future drug optimization.

Most proteins fulfil their function as part of large protein complexes. Surprisingly, little is known about the pathways and regulation of protein assembly. Several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virion and thus resemble ideal model systems for studying protein complex formation. Even for these systems, the mechanism for self-assembly is still poorly understood, although it is generally thought that smaller oligomeric structures form key intermediates. This assembly nucleus and larger viral assembly intermediates are typically low abundant and difficult to monitor. Here, we characterised small oligomers of Hepatitis B virus (HBV) and norovirus under equilibrium conditions using native ion mobility mass spectrometry. This data in conjunction with computational modelling enabled us to elucidate structural features of these oligomers. Instead of more globular shapes, the intermediates exhibit sheet-like structures suggesting that they are assembly competent. We propose pathways for the formation of both capsids. Although most proteins fulfil their role as part of large protein complexes, little is known about the pathways of complex assembly. Here, ion mobility–mass spectrometry is used to monitor and structurally characterize the assembly intermediates of viral protein shells, called capsids, of two major human pathogens, norovirus and hepatitis B virus.

Since the discovery of the Australia antigen, now known as the hepatitis B surface antigen (HBsAg), significant research has been conducted to elucidate its physical, chemical, structural, and functional properties. Subviral particles (SVPs) containing HBsAg are highly immunogenic, non-infectious entities that have not only revolutionized vaccine development but also provided critical insights into HBV immune evasion and viral assembly. Recent advances in cryo-electron microscopy (cryo-EM) have uncovered the heterogeneity and dynamic nature of spherical HBV SVPs, emphasizing the essential role of lipid–protein interactions in maintaining particle stability. In this review, recent progress in understanding the molecular architecture of HBV SVPs is consolidated, focusing on their symmetry, lipid organization, and disassembly–reassembly dynamics. High-resolution structural models reveal unique lipid arrangements that stabilize hydrophobic residues, preserve antigenicity, and contribute to SVP functionality. These findings highlight the significance of hydrophobic interactions and lipid–protein dynamics in HBV SVP assembly and stability, offering valuable perspectives for optimizing SVP-based vaccine platforms and therapeutic strategies.

Electron cryo-tomography produces 3D pictures of unique objects such as viruses, organelles or cells. This is a protocol for macromolecular structure determination from tomographic data using subtomogram averaging in the RELION software. Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ . Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein a protocol for subtomogram averaging from cryo-ET data using the RELION software ( http://www2.mrc-lmb.cam.ac.uk/relion ). RELION was originally developed for cryo-EM single-particle analysis, and the subtomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate 3D models for the contrast transfer function (CTF) that describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified hepatitis B capsid particles and Saccharomyces cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimization guidelines, high-resolution maps can be obtained in which secondary structure elements are resolved subtomogram.

Agents with novel mechanisms are clinically needed for the functional cure of chronic hepatitis B virus (HBV) infection. This study aimed to identify compounds that inhibit an interaction between large hepatitis B surface (LHBs) and hepatitis B core (HBc) proteins, which are considered to be important for HBV envelope formation. Using a high-throughput screening method to evaluate the protein-protein interaction in HepG2 cells by the NanoBRET system, 3,200 compounds from our library were tested. Then, the inhibitory effects of the hit compounds on HBV particles with the envelope were evaluated in HBV-expressing HepG2.2.15.7 cells. 40 hit compounds were found to inhibit the interaction between LHBs and HBc, of which 10 compounds inhibited HBV particles. The top 5 compounds with the least variation were considered as candidates. These compounds showed little effects on HBV proteins and RNAs, whereas a hit compound 16, which showed the lowest IC50 (0.9 µM), decreased precore mRNA significantly. When the culture supernatant from cells with hit compound 16 was subjected to sucrose density gradient centrifugation, HBV particles with the envelope were found to be reduced. In conclusion, we identified compounds that might inhibit the envelope formation of HBV through disturbing the interaction between LHBs and HBc.