A mutant hepatitis B virus core protein mimics inhibitors of icosahedral capsid self-assembly

Abstract

Understanding self-assembly of icosahedral virus capsids is critical to developing assembly directed antiviral approaches and will also contribute to the development of self-assembling nanostructures. One approach to controlling assembly would be through the use of assembly inhibitors. Here we use Cp149, the assembly domain of the hepatitis B virus capsid protein, together with an assembly defective mutant, Cp149-Y132A, to examine the limits of the efficacy of assembly inhibitors. By itself, Cp149-Y132A will not form capsids. However, Cp-Y132A will coassemble with the wild-type protein on the basis of light scattering and size exclusion chromatography. The resulting capsids appear to be indistinguishable from normal capsids. However, coassembled capsids are more fragile, with disassembly observed by chromatography under mildly destabilizing conditions. The relative persistence of capsids assembled under conditions where association energy is weak compared to the fragility of those where association is strong suggests a mechanism of "thermodynamic editing" that allows replacement of defective proteins in a weakly associated complex. There is fine line between weak assembly, where assembly defective protein is edited from a growing capsid, and relatively strong assembly, where assembly defective subunits may dramatically compromise virus stability. Thus, attempts to control virus self-assembly (with small molecules or defective proteins) must take into account the competing process of thermodynamic editing.

Figure 1

HBV capsids are composed of dimers that are held together by relatively small hydrophobic contacts. Capsids are icosahedral (far left) with each icosahedral facet (central panel) composed of two classes of dimer, AB (red and blue, respectively) and CD (yellow and green), that form the icosahedral asymmetric unit. A pair of dimers, highlighted in the central panel, are shown as translucent surfaces over a ribbon diagram. Residue Y132 (magenta) contributes to approximately 10% of the buried surface at each dimer-dimer interface.

Figure 2

Cp149-Y132A co-assembles with Cp149. (A) Representative kinetics of assembly at 37°C observed by light scattering. Compared to control experiments of Cp149 without Cp149-Y123A (thin lines, offset for clarity) the presence of Cp149-Y132A leads to a great excess of light scatter. (B) Light scattering enhancement correlates with association energy, which is proportional to ionic strength, i.e. high ionic strength shows the greatest effect (24).

Figure 3

Co-assembly is saturable. Samples containing 1 μM (final concentration) Cp149 plus varying amounts of either Cp149 or Cp149-Y132A was induced to assemble at 21°C by adding NaCl to 1 M. Light scattering was read after 24 hr. Co-assembly with Cp149-Y132A extrapolates to about twice the scattering of 1μM Cp149. This enhanced light scattering indicates that Cp149 can only cooperate with a limited amount of mutant. Additional Cp149-Y132A does not poison co-assembly. For comparison to the co-assembly data, the scattering of corresponding concentrations of unassembled Cp149 is also shown (dashed line).

Figure 4

Comparing Cp149 assembly and Cp149-Y132A coassembly kinetics over 24 hours. At long times, the rate of capsid formation is equal to, and thus diagnostic for, the rate of nucleation. SEC of 11.7uM Cp149-Y132a coassembly with 5uM wildtype (open squares) results in more capsids than 5uM Cp149 alone (filled squares) but with a similar rate of assembly. The first point was taken at 8 minutes post mixing and every hour thereafter. Assembly was induced by addition of NaCl to 0.6mM in the presence of 1mM DTT and eluted in 50mM Hepes pH 7.5, 1.0M NaCl. Inset: Overlaid SEC traces of Cp149 assembly (solid line) and Cp149-Y132A/Cp149 coassembly (dashed line) at 23 hours.

Figure 5

Co-assembly of Cp149-Y132A with Cp149. (A) SEC under capsid stabilizing conditions shows that co-assembly slightly increases the yield of capsids (2.5 units corresponds to ~8 μM dimer in the capsid peak). There is a weak but significant (p < 0.02) increase in the yield of capsid with increased Y132A concentration for all but the 0.15M NaCl experiments. Assembly with 10 μM Cp149 and varying mutant was carried out at 21°C in 0.1M HEPES, pH 7.5 with the stated NaCl. After ~24 hr, reactions were separated on a 21 ml Superose-6 column equilibrated with HEPES and 1 M NaCl. (B) Negatively stained electron micrographs of 24 hr assembly reactions similar to those in panel A except these reactions have 20uM Cp149-Y132A.

Figure 6

Co-assembly of Cp149-Y132A with Cp149 results in fragile capsids. These experiments are similar to those shown in Figure 5A except reactions were incubated at 37°C resulting in ≥80% assembly at all ionic strengths. To examine particle stability, SEC was performed on Superose-6 column equilibrated in HEPES buffer with only 0.15 M NaCl, an ionic strength where capsids are nominally stable.

Figure 7

A model describing the roles that Cp149-Y132 plays in assembly. The mutant cannot nucleate assembly on its own. The relatively slow assembly that takes place in the presence of excess mutant suggests that it can play a slight role in retarding nucleation. When association energy between subunits is weak (low NaCl), incorporated subunits dissociate during assembly to yield capsids that are enriched in wildtype. When association is strong (high NaCl), mutant is retained in capsids with the long term effect that these capsids readily dissociate, as shown in Figure 6. Assembly inhibitors and defective subunits are expected to fill similar roles.