Chemically Induced Morphogenesis of P22 Virus-like Particles by the Surfactant Sodium Dodecyl Sulfate.

In the infectious P22 bacteriophage, the packaging of DNA into the initially formed procapsid triggers a remarkable morphological transformation where the capsid expands from 58 to 62 nm. Along with the increase in size, this maturation also provides greater stability to the capsid and initiates the release of the scaffolding protein (SP). (2,4) In the P22 virus-like particle (VLP), this transformation can be mimicked in vitro by heating the procapsid particles to 65 °C or by treatment with sodium dodecyl sulfate (SDS). (5,6) Heating the P22 particles at 65 °C for 20 min is well established to trigger the transformation of P22 to the expanded (EX) P22 VLP but does not always result in a fully expanded population. Incubation with SDS resulted in a >80% expanded population for all P22 variants used in this work. This study elucidates the importance of the stoichiometric ratio between P22 subunits and SDS, the charge of the headgroup, and length of the carbon chain for the transformation. We propose a mechanism by which the expansion takes place, where both the negatively charged sulfate group and hydrophobic tail interact with the coat protein (CP) monomers within the capsid shell in a process that is facilitated by an internal osmotic pressure generated by an encapsulated macromolecular cargo.

Bacteriophage P22 ejects all of its internal proteins before its genome.

Double-stranded DNA bacteriophages are highly pressurized, providing a force driving ejection of a significant fraction of the genome from its capsid. In P22-like Podoviridae, internal proteins ("E proteins") are packaged into the capsid along with the genome, and without them the virus is not infectious. However, little is known about how and when these proteins come out of the virus. We employed an in vitro osmotic suppression system with high-molecular-weight polyethylene glycol to study P22 E protein release. While slow ejection of the DNA can be triggered by lipopolysaccharide (LPS), the rate is significantly enhanced by the membrane protein OmpA from Salmonella. In contrast, E proteins are not ejected unless both OmpA and LPS are present and their ejection when OmpA is present is largely complete before any genome is ejected, suggesting that E proteins play a key role in the early stage of transferring P22 DNA into the host.

Determination of antioxidant capacity using the biological system bacteriophage P22/bacterium Salmonella typhimurium.

Bacteriophage/bacterium systems have been employed in the past in assays for virucidal activity. A novel application of one such system is proposed here for the in vivo determination of antioxidant capacity. It was shown that an antioxidant such as gallic acid can effectively protect against oxidative damage brought about by H2O2-but only within a narrow range of concentrations (i.e., from 250 to 500 mg L-1); ascorbic acid, on the other hand, did not exhibit any protective effect against H2O2. Finally, neither ascorbic nor gallic acid demonstrated a virucidal effect. The P22/Salmonella typhimurium model system thus proved to be useful in the assessment of antioxidant capacity in vivo, at least using those two alternative model antioxidants.

Electrostatic interactions govern both nucleation and elongation during phage P22 procapsid assembly.

Icosahedral capsid assembly is an example of a reaction controlled solely by the interactions of the proteins involved. Bacteriophage P22 procapsids can be assembled in vitro by mixing coat and scaffolding proteins in a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here, we investigated the effect of the buffer composition on the interactions necessary for capsid assembly. Different concentrations of various salts, chosen to follow the electroselectivity series for anions, were added to the assembly reaction. The concentration and type of salt was found to be crucial for proper nucleation of procapsids. Nucleation in low salt concentrations readily occurred but led to bowl-like partial procapsids, as visualized by negative stain electron microscopy. The edge of the partial capsids remained assembly-competent since coat protein addition triggered procapsid completion. The addition of salt to the partial capsids also caused procapsid completion. In addition, each salt affected both assembly rates and the extent of procapsid formation. We hypothesize that low salt conditions increase the coat protein:scaffolding protein affinity, causing excessive nuclei to form, which decreases coat protein levels leading to incomplete assembly.

Intracellular trapping of a cytoplasmic folding intermediate of the phage P22 tailspike using iodoacetamide.

Critical steps in polypeptide chain folding within the bacterial cytoplasm have been difficult to identify. Salmonella cells infected with temperature-sensitive folding mutants of the P22 tailspike protein at restrictive temperature accumulated a metastable folding intermediate with a half-life of 6 min at 39 degrees C. The native trimeric tailspike contains 24 buried cysteines (8/chain) but neither disulfide bonds nor active site cysteines. Eighteen of the 24 cysteines are involved in strong hydrogen bonds (Thomas, G. J., Jr., Becka, R., Sargent, D., Yu, M.-H., and King, J. (1990) Biochemistry 29, 4181-4187). Cyanide and iodoacetamide prevented the folding and association of the restrictive temperature folding intermediate to the native state after shift to permissive temperature. The cytoplasmic folding intermediate was covalently modified by iodoacetamide within infected cells. Chains which had reacted with iodoacetamide were unable to proceed through the folding pathway. Iodoacetamide also reacted with a folding intermediate during the refolding of purified tailspike chains in vitro, inhibiting further folding. No reaction occurred with native tailspike in vivo or in vitro. The target residues in the intermediates were in the carboxyl terminus of the chain and may be a unique set of cysteine residues that are activated during protein folding, but not in the native state.

Inhibition of viral capsid assembly by 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid).

The precursor shells of dsDNA bacteriophages are assembled by the polymerization of competent states of coat and scaffolding subunits. The fluorescent dye 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid) (bisANS) binds to both the coat and scaffolding proteins from the Salmonella typhimurium bacteriophage P22. It displays little affinity for the polymerized forms of the proteins. The subunits with bound bisANS are incapable of assembling into procapsids. The binding constants of bisANS for both coat and scaffolding protein monomers have been measured and are 7 and 6 microM, respectively. Binding of bisANS to coat protein has little effect on the conformation as determined by circular dichroism and susceptibility to proteolysis. Binding of bisANS to scaffolding protein induces a change in the secondary structure consistent with a loss of alpha-helix, and an altered susceptibility to proteolysis. We suggest that the bisANS is probably binding at sites responsible for intersubunit interactions and thereby inhibiting capsid assembly.