A Small Molecule Inhibits and Misdirects Assembly of Hepatitis B Virus Capsids

Abstract

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. Hepatitis B virus (HBV) causes chronic and acute infections.Worldwide, more than 350 million people suffer from chronic infection, with an annual mortality rate of approximately 1 million people (21). HBV is an enveloped DNA virus with an icosahedral core, or capsid. In vivo, HBV capsids assemble around an RNA-reverse transcriptase complex (2). Assembly of the capsid is required for reverse transcription of the RNA pregenome to the mature DNA form (reviewed in references 7 and 21). In HBV, the dominant form of capsid (25) is composed of 120 copies of the capsid protein dimer (6,10,29). Though assembly is robust in vitro (3,23,29,35), even modest mutations of the capsid protein can have dramatic effects on the viability of progeny virus (12,18,19,30,31). This suggests that capsid assembly could be an effective therapeutic target for chronic HBV. Though interfering with assembly is a likely strategy for antiviral intervention, to the authors' knowledge this approach has not been used with any virus.Capsid formation using the assembly domain of the HBV capsid protein has been examined (29, 34). The truncated capsid protein consists of the first 149 amino acids and lacks the C-terminal, 34-residue RNA-binding domain. The assembly domain forms a stable dimer (29, 32), hereafter referred to as Cp149 2 . Cp149 2 assembles rapidly and efficiently in response to increased levels of ionic st...

Observed Hysteresis of Virus Capsid Disassembly Is Implicit in Kinetic Models of Assembly

“…Capsid Preparation-The dimeric HBV capsid protein assembly domain, Cp149 2 , was expressed in Escherichia coli and purified as described (18). Capsids for dissociation experiments were assembled by adding NaCl to Cp149 2 to final concentrations of 25 M Cp149 2 , 25 mM HEPES, pH 7.5, 2 mM dithiothreitol, 0.5 M NaCl (assembly buffer) then incubating at 4°C for 24 h. Capsids were isolated by SEC using a Superose-6 column equilibrated with assembly buffer.…”

Master equation approach to the assembly of viral capsids

“…It has since been extended to more involved scenarios [6,26,27], including a study of the energy landscape underlying assembly [7], that is similar to approaches in protein folding [2] or the energy landscape description of association reactions [21,22,23]. These results have been used to investigate the possibility of inhibiting assembly via an anti-viral drug in the case of Herpes Virus [28]. Related approaches include molecular dynamics studies of viral capsid assembly [13,14], and a molecular dynamics-like formalism that is implemented in connection with a "local rules" mechanism that regulates capsid assembly [1,18].…”

A method for in vitro assembly of hepatitis C virus core protein and for screening of inhibitors

“…Bis-ANS, a fluorescent probe that binds hydrophobic residues on protein, has been shown to inhibit in vitro HBV capsid assembly [27]. We showed that this compound also inhibits C1-82 assembly in vitro in a dose-dependent manner (Fig.…”

“…Bis-ANS was shown to trigger detrimental secondary effects in cells and is therefore not suitable for therapy in its present form [27]. However, the inhibitory effects of this compound confirm that targeting hydrophobic clusters of the core is relevant to the development of assembly inhibitors for HCV NLPs.…”

“…This peptide was derived from the hydrophobic glycine-rich cluster (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) located between the two basic clusters. This peptide does not interact with nucleic acids (Fig.…”