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.

Capsids of spherical viruses share a common architecture: an icosahedral arrangement of identical proteins. We suggest that there may be a limited number of common assembly mechanisms for such viruses. Previous assembly mechanisms were proposed on the basis of virion structure but were not rigorously tested. Here we apply a rigorous analysis of assembly to cowpea chlorotic mottle virus (CCMV), a typical, small, positive-strand RNA virus. The atomic resolution structure of CCMV revealed an interleaving of subunits around the quasi-sixfold vertices, which suggested that capsid assembly was initiated by a hexamer of dimers (Speir et al., 1995, Structure 3, 63-78). However, we find that the capsid protein readily forms pentamers of dimers in solution, based on polymerization kinetics observed by light scattering. Capsid assembly is nucleated by a pentamer, determined from analysis of the extent of assembly by size-exclusion chromatography. Subsequent assembly likely proceeds by the cooperative addition of dimers, leading to the T = 3 icosahedral capsid. At high protein concentrations, the concentration-dependent nucleation reaction causes an overabundance of five-dimer nuclei that can be identified by classical light scattering. In turn these associate to form incomplete capsids and pseudo-T = 2 capsids, assembled by oligomerization of 12 pentamers of dimers. The experimentally derived assembly mechanisms of T = 3 and pseudo-T = 2 CCMV capsids are directly relevant to interpreting the structure and assembly of other T = 3 viruses such as Norwalk virus and pseudo-T = 2 viruses such as the vp3 core of blue tongue virus.

Supplementary data are available at Bioinformatics online.

In multistep reactions, stability of intermediates is critical to the rate of product formation and a significant factor in generating kinetic traps. The capsid protein of cowpea chlorotic mottle virus (CCMV) can be induced to assemble into spherical particles of 30, 60, and 90 dimers. Based on examining assembly kinetics and reaction end points, we find that formation of uniform, ordered structures is not always a result of reactions that reach equilibrium. Equilibration or, alternatively, kinetic trapping can be identified by a straightforward analysis. Altering the assembly path of "spherical" particles is a means of controlling the distribution of products, which has broad applicability to self-assembly reactions.

We take advantage of the self-assembly properties of the coat protein of a spherical virus to form uniform tubular nanostructures. Critical to redirecting assembly are the weak interprotein association energy inherent to virus assembly and the relatively rigid nature of the double-stranded DNA scaffold at their core.

alpha-Synuclein is a major constituent of glial cytoplasmic inclusions (GCIs), which are pathognomic for multiple system atrophy (MSA). We have previously demonstrated that in normal human brain, alpha-synuclein mRNA has a restricted pattern of neuronal expression and no apparent glial expression. The current study used double-label in situ hybridization to determine if alpha-synuclein mRNA is expressed by oligodendroglia of MSA cases. Analysis of MSA brain tissue revealed depletion of regional signal for this transcript in many brain areas due to extensive neurodegeneration. Cellular analysis of oligodendroglia in crus cerebri, a GCI-rich region ventral to substantia nigra, revealed an absence of alpha-synuclein mRNA signal in control and MSA cases. However, an abundance of this transcript was detected in melanin-containing neurons of substantia nigra. Therefore, oligodendroglia do not express alpha-synuclein mRNA in control and MSA cases suggesting that involvement of alpha-synuclein in GCI pathology of MSA is due to its ectopic presence in oligodendroglia.

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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...