Stability and assembly in vitro of bacteriophage PP7 virus-like particles

Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles in vitro were established.The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble in vitro in a reaction that requires RNA.Viruses and VLPs are currently under investigation for a variety of uses that include confinement of chemical reactions, as templates for materials synthesis, as molecular electronics components, as platforms for polyvalent display of antigens and other ligands, and for targeted drug delivery. For some relevant examples see references [1-13]. The single-strand RNA bacteriophages offer certain advantages for such applications. VLPs can be produced in large quantities by self-assembly of a single coat protein polypeptide expressed from a plasmid, thus allowing extensive genetic manipulation of the capsid without the constraints imposed by the necessity to maintain virus viability [14]