BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.     

Genetically Altering the Thermodynamics and Kinetics of Hepatitis B Virus Capsid Assembly Has Profound Effects on Virus Replication in Cell Culture

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.     

Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1

Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy.     

A theoretical model successfully identifies features of hepatitis B virus capsid assembly.

The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Interaction between nucleophosmin and HBV core protein increases HBV capsid assembly

Host factors are involved in Hepatitis B virus (HBV) genome replication and capsid formation during the viral life cycle. A host factor, nucleophosmin (B23), was found to bind to HBV core protein dimers, but its functional role has not been studied. This interaction promoted HBV capsid assembly and decreased the degree of capsid dissociation when subjected to denaturant treatments in vitro. In addition, inhibition of B23 reduced intracellular capsid formation resulting in a decrease of HBV production in HepG2.2.15 cells. These results provide important evidence that B23 acts on core capsid assembly via its interaction with HBV core dimers.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein

Assembly of virus particles in infected cells is likely to be a tightly regulated process. Previously, we found that in vitro assembly of hepatitis B virus (HBV) capsid protein is highly dependent on protein and NaCl concentration. Here we show that micromolar concentrations of Zn2+ are sufficient to initiate assembly of capsid protein, whereas other mono- and divalent cations elicited assembly only at millimolar concentrations, similar to those required for NaCl-induced assembly. Altered intrinsic protein fluorescence and highly cooperative binding of at least four Zn2+ ions (KD approximately 7 microM) indicated that binding induced a conformational change in capsid protein. At 37 degrees C, Zn2+ enhanced the initial rate of assembly and produced normal capsids, but it did not alter the extent of assembly at equilibrium. Assembly mediated by high zinc concentrations (> or =300 microM) yielded few capsids but produced a population of oligomers recognized by capsid-specific antibodies, suggesting a kinetically trapped assembly reaction. Comparison of kinetic simulations to in vitro assembly reactions leads us to suggest that kinetic trapping was due to the enhancement of the nucleation rate relative to the elongation rate. Zinc-induced HBV assembly has hallmarks of an allosterically regulated process: ligand binding at one site influences binding at other sites (cooperativity) indicating that binding is associated with conformational change, and binding of ligand alters the biological activity of assembly. We conclude that zinc binding enhances the kinetics of assembly by promoting formation of an intermediate that is readily consumed in the reaction. Free zinc ions may not be the true in vivo activator of assembly, but they provide a model for regulation of assembly.     

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser(106). PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser(106) mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.     

乙肝病毒核心蛋白钉突部位基因工程改造对其功能的影响

通过在乙肝病毒核心蛋白钉突部位插入标签蛋白EGFP及小片段多肽,研究各种改造对HBc功能的影响。采用RLIC方法,构建野生型HBc、HBc钉突部位带不同接头的EGFP融合重组体、缩短的EGFP融合重组体,并构建与HBc功能互补的质粒HBV1.1c－,将不同重组体与HBV1.1c－共转染HEK293细胞,通过观察荧光及Southern blotting检测病毒复制中间体,判断相应基因工程改造对重组蛋白中不同结构域功能的影响。RLIC方法可有效地用来进行片段缺失,且缺失片段大小及位置无明显限制。带柔性或刚性接头的重组HBc-EGFP均可产生绿色荧光,但荧光在细胞内分布形态不同,两种重组HBc-EGFP均不能支持正常的HBV复制,各种截短的插入片段以及aa79-80单独缺失体亦不能支持HBV复制。结果表明RLIC方法是一种基因工程改造的有力工具,不同类型接头对重组蛋白的结构和功能有不同影响,aa79-80对维持HBc的主要功能之一——支持HBV复制有重要作用。     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

Observed hysteresis of virus capsid disassembly is implicit in kinetic models of assembly

For many protein multimers, association and dissociation reactions fail to reach the same end point; there is hysteresis preventing one and/or the other reaction from equilibrating. We have studied in vitro assembly of dimeric hepatitis B virus (HBV) capsid protein and dissociation of the resulting T = 4 icosahedral capsids. Empty HBV capsids composed of 120 capsid protein dimers were more resistant to dissociation by dilution or denaturants than anticipated from assembly experiments. Using intrinsic fluorescence, circular dichroism, and size exclusion chromatography, we showed that denaturants dissociate the HBV capsids without unfolding the capsid protein; unfolding of dimer only occurred at higher denaturant concentrations. The apparent energy of interaction between dimers measured in dissociation experiments was much stronger than when measured in assembly studies. Unlike assembly, capsid dissociation did not have the concentration dependence expected for a 120-subunit complex; consequently the apparent association energy systematically varied with reactant concentration. These data are evidence of hysteresis for HBV capsid dissociation. Simulations of capsid assembly and dissociation reactions recapitulate and provide an explanation for the observed behavior; these results are also applicable to oligomeric and multidomain proteins. In our calculations, we find that dissociation is impeded by temporally elevated concentrations of intermediates; this has the paradoxical effect of favoring re-assembly of those intermediates despite the global trend toward dissociation. Hysteresis masks all but the most dramatic decreases in contact energy. In contrast, assembly reactions rapidly approach equilibrium. These results provide the first rigorous explanation of how virus capsids can remain intact under extreme conditions but are still capable of "breathing." A biological implication of enhanced stability is that a triggering event may be required to initiate virus uncoating.     

Conformational equilibria and rates of localized motion within hepatitis B virus capsids

Functional analysis of hepatitis B virus (HBV) core particles has associated a number of biological roles with the C terminus of the capsid protein. One set of functions require the C terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a protein-protein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T = 4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megaDalton complex.     

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.     

Surveying Capsid Assembly Pathways through Simulation-Based Data Fitting

Virus capsid assembly has attracted considerable interest from the biophysical modeling community as a model system for complicated self-assembly processes. Simulation methods have proven valuable for characterizing the space of possible kinetics and mechanisms of capsid assembly, but they have so far been able to say little about the assembly kinetics or pathways of any specific virus. It is not possible to directly measure the detailed interaction rates needed to parameterize a model, and there is only a limited amount of experimental evidence available to constrain possible pathways, with almost all of it gathered from in vitro studies of purified coat proteins. In prior work, we developed methods to address this problem by using simulation-based data-fitting to learn rate parameters consistent with both structure-based rule sets and experimental light-scattering data on bulk assembly progress in vitro. We have since improved these methods and extended them to fit simulation parameters to one or more experimental light-scattering curves. Here, we apply the improved data-fitting approach to three capsid systems—human papillomavirus (HPV), hepatitis B virus (HBV), and cowpea chlorotic mottle virus (CCMV)—to assess both the range of pathway types the methods can learn and the diversity of assembly strategies in use between these viruses. The resulting fits suggest three different in vitro assembly mechanisms for the three systems, with HPV capsids fitting a model of assembly via a nonnucleation-limited pathway of accumulation of individual capsomers while HBV and CCMV capsids fit similar but subtly different models of nucleation-limited assembly through ensembles of pathways involving trimer-of-dimer intermediates. The results demonstrate the ability of such data fitting to learn very different pathway types and show some of the versatility of pathways that may exist across real viruses.     

Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.     

Antisense oligodeoxynucleotides targeted against molecular chaperonin Hsp60 block human hepatitis B virus replication

The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.     

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.