Hepatitis b virus capsid

References 1. The global burden of viral hepatitis from to findings from the Global Burden of Disease Study Lancet ; —8. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between and Lancet ; — Sci Rep ; 7 Zlotnick A, Stray SJ. How does your virus grow?

Understanding and interfering with virus assembly. Trends Biotechnol ; 21 — Low-level secretion of human hepatitis B virus virions caused by two independent, naturally occurring mutations P5T and L60V in the capsid protein.

J Virol ; 74 — Structure, assembly, and antigenicity of hepatitis B virus capsid proteins. Adv Virus Res ; 64 — Chain BM, Myers R. Variability and conservation in hepatitis B virus core protein. BMC Microbiol ; 5 Infect Disord Drug Targets ; 7 —6. Antiviral activity, safety, and pharmacokinetics of capsid assembly modulator NVR in patients with chronic HBV infection. Gastroenterology ; — Therapeutic strategies for hepatitis B virus infection: towards a cure.

Nat Rev Drug Discov ; 18 — Discovery of hepatitis B virus capsid assembly inhibitors leading to a heteroaryldihydropyrimidine based clinical candidate GLS4. Bioorg Med Chem ; 25 — Effects of ketoconazole and rifampicin on the pharmacokinetics of GLS4, a novel anti-hepatitis B virus compound, in dogs.

Acta Pharmacol Sin ; 34 —6. Outcomes of long-term treatment of chronic HBV infection with entecavir or other agents from a randomized trial in 24 countries. Clin Gastroenterol Hepatol ; 18 —67, e New antivirals for the treatment of chronic hepatitis B. Expert Opin Investig Drugs ; 26 — Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.

Science ; —6. TLR7 agonist increases responses of hepatitis B virus-specific T cells and natural killer cells in patients with chronic hepatitis B treated with nucleos t ide analogues. Th1 and Th2 immune response in chronic hepatitis B patients during a long-term treatment with adefovir dipivoxil. Mediators Inflamm ; Final results of a phase 1b day study of ABI-H, a novel core inhibitor, in non-cirrhotic viremic subjects with chronic hepatitis B.

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J Infect Dis ; — HBV RNA virion-like particles produced under nucleos t ide analogues treatment are mainly replication-deficient. J Hepatol ; 68 —9. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein. Gane EJ. It was then transferred to an oil bath and heated just below the boiling point for 1 hour.

Hot ethanol 2 mL was added and the mixture was stirred until homogeneous, and was then cooled to room temperature. The resulting solid was collected by suction filtration and washed with a minimum quantity of cold ethanol, then with boiling water approx. To a solution of oxazolone 2a 0. Solid calcium carbonate 0. The suspension was filtered to remove calcium salts, and the resulting solution was evaporated to dryness. Prepared by the analogous procedure starting with 2-fluorobenzaldehyde.

Observation of kinetics by light scattering was performed as previously described Briefly, scattering was observed with a Photon Technology International fluorometer set for nm for both excitation and emission: nm was chosen as the shortest wavelength for which no reaction component absorbs light. Scattering was initially observed for a sample containing approximately twice the final protein concentration in buffer containing no phenylpropenamide or NaCl.

After ca. After a second observation period of ca. Light scattering is reported in arbitrary units. All experiments were repeated three times with the exception of the 0.

After conducting LS experiments, samples were incubated for 24 hours to allow the reactions to approach equilibrium. Previous studies have shown that there is no significant increase in product concentration of Cp assembly reactions with longer incubation times; similar experiments have shown that assembly reactions in the presence of the phenylpropenamides reach steady state in approximately half that time.

The column was mounted on a Shimadzu-HPLC system equipped with a temperature-controlled autoinjector to facilitate the long time courses and many samples. Equilibrated samples from light scattering experiments were quantified to determine the concentrations of reaction products after 24 hours; Cp alone shows no further increase in product concentration beyond 24 hours, and assembly reactions in the presence of the phenylpropenamides reached a constant concentration of capsid in less than half that incubation period Long term kinetic experiments were performed by sampling a single reaction mix at every hour and separating reactants and products as described.

SEC chromatographs at nm were quantified after manual baseline correction using the supplied LCSolutions software for the quantification of reactants and products Shimadzu. The concentrations of assembled capsid and free dimer subunit were used to determine the apparent dissociation constant and the pairwise contact energies between subunits as described previously Briefly, capsid assembly was considered an equilibrium reaction of dimers assembling into a single capsid, from which the equilibrium constant K capsid can be expressed as.

Given the capsid geometry, wherein tetravalent subunits form pairwise contacts, a statistical term that describes this degeneracy can be used to derive the bimolecular K contact as follows:. From this value, the pairwise contact energies for assembly under a given set of conditions can be determined by.

An additional useful value, K D, , can be determined from K capsid. K D,app is the apparent dissociation constant, where concentrations of dimer and capsid are equal. K D,app is thus determined by the following:. Capsid assembly can be modeled as a variation of classical polymer theory As assembly depends on the interactions of many subunits, below a pseudocritical concentration of subunit, capsids will not form.

However, the concentration of free subunit varies with total subunit concentration and is thus an inconvenient value for assembly reaction comparisons. We had previously used the apparent dissociation constant, K D,app Equation 4 as an index of assembly stability. Unfortunately, K D,app occurs at the point where free subunit concentration just begins to plateau and capsids just begin to appear, making the molar quantities difficult to determine.

For this reason we now define a new index, K 10 , that is approximately equal to the experimentally observable pseudo-critical concentration of free subunit and is readily calculable. Formally, K 10 is the concentration of free subunit when the equilibrated concentration of assembled capsid is at high excess compared to K D,app , arbitrarily defined here as ten times K D,app. The influence of effectors on capsid assembly can be quantified from the change in capsid stability.

The average change of subunit contact energy in the presence of the phenylpropenamides can be converted to a weighted average of the contact energies of free and compound-bound Cp dimers, or. Kinetic parameters were derived from light scattering traces. Early time points were visually inspected to determine the region of greatest quantifiable slope. A linear regression of these points provided a numerical value for this maximum slope that was used in subsequent calculations of kinetic index Equation 3 , a dimensionless number relating the observed reaction rate to effector concentration.

This was calculated as:. Samples from light scattering experiments were adsorbed to glow-discharged carbon over paralodian copper grids EM Sciences. National Center for Biotechnology Information , U. ACS Chem Biol. Author manuscript; available in PMC Dec Sarah P. Katen , 1 Srinivas Reddy Chirapu , 2 M. Author information Copyright and License information Disclaimer. Copyright notice. See other articles in PMC that cite the published article.

Abstract Understanding the biological self-assembly process of virus capsids is key to understanding the viral life cycle, as well as serving as a platform for the design of assembly-based antiviral drugs. Open in a separate window.

Figure 1. Figure 2. Figure 3. Figure 4. Quantifying thermodynamics and kinetics of assembly From the SEC data, we calculated the effect of the phenylpropenamides on capsid stability, which is reported in terms of an analytical evaluation of the pseudo-critical concentration of assembly, K Figure 5. Phenylpropenamides primarily alter reaction kinetics The phenylpropenamide assembly effectors increase both the rate and extent of assembly, and yet produce normal capsids.

Figure 6. Phenylpropenamides as probes of the HBV assembly pathway Under aggressive assembly conditions, the phenylpropenamides offer a new view of HBV assembly. Figure 7. Propenamide synthesis Compounds AT and B were synthesized following a previously reported procedure 28 as shown in Figure 1. Representative procedures and characterization data are as follows: Z 2-methoxybenzylidene 4-nitrophenyl oxazol-5 4H -one 2a 4-Nitrohippuric acid 1 , 0.

E -N- 1-bromo 2-methoxyphenyl oxo piperidinyl propenyl nitrobenzamide AT To a solution of oxazolone 2a 0. E -N- 1-bromo 2-fluorophenyl oxo piperidinyl propenyl nitrobenzamide B Prepared by the analogous procedure starting with 2-fluorobenzaldehyde. Light Scattering Observation of kinetics by light scattering was performed as previously described Calculation of Kinetic and Thermodynamic Parameters SEC chromatographs at nm were quantified after manual baseline correction using the supplied LCSolutions software for the quantification of reactants and products Shimadzu.

Electron Microscopy Samples from light scattering experiments were adsorbed to glow-discharged carbon over paralodian copper grids EM Sciences. References 1. Katen SaZA. The Thermodynamics of Virus Capsid Assembly. Methods Enzymol. Ganem D, Schneider RJ. Hepadnaviridae: The Viruses and Their Replication. Ganem D, Prince AM. N Engl J Med. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev. The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport.

J Virol. Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication. Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol. Physical principles in the construction of regular viruses.

Zlotnick A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes. J Mol Biol. Ceres P, Zlotnick A. Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids. Zhang T, Schwartz R. Biophys J. Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein.

Hagan MF, Chandler D. Dynamic Pathways for Viral Capsid Assembly. Rossmann MG. Constraints on the Assembly of Spherical Virus Particles. In hepatitis B virus HBV , the capsid consists of identical protein monomers that dimerize and then arrange themselves into pentamers or hexamers on the capsid surface. By applying atomistic molecular dynamics simulation to an entire solvated HBV capsid subjected to a uniform mechanical stress protocol, we monitor the capsid-disassembly process and analyze the process down to the level of individual amino acids in 20 independent simulation replicas.

The strain of an isotropic external force, combined with structural fluctuations, causes structurally heterogeneous cracks to appear in the HBV capsid. Analysis of the monomer-monomer interfaces reveals that, in contrast to the expectation from purely mechanical considerations, the cracks mainly occur within hexameric sites, whereas pentameric sites remain largely intact.