Influence of three-dimensional structure on the immunogenicity of a peptide expressed on the surface of a plant virus.

The influence of peptide structure on immunogenicity has been investigated by constructing a series of cowpea mosaic virus (CPMV) chimaeras expressing the 14 amino acid NIm-1A epitope from human rhinovirus 14 (HRV-14) at different positions on the capsid surface. Biochemical and crystallographic analysis of a CPMV/HRV chimaera expressing the NIm-1A epitope inserted into the betaC'-betaC" loop of the S protein revealed that, although the inserted peptide was free at its C-terminus, it adopted a conformation distinct from that previously found when a similarly cleaved peptide was expressed in the betaB-betaC loop of the S protein. Adjustment of the site of insertion within the betaB-betaC loop resulted in the isolation of a chimaera in which cleavage at the C-terminus of the epitope was much reduced. Crystallographic analysis confirmed that in this case the epitope was presented as a closed loop. Polyclonal antisera raised against the CPMV/ HRV chimaera presenting the NIm-1A epitope as a closed loop had a significantly enhanced ability to bind to intact HRV-14 particles compared with antisera raised against chimaeras presenting the same sequence as peptides with free C-termini. These results demonstrate that the mode of presentation of an epitope on a heterologous carrier can dramatically affect its immunological properties.

Energetics of quasiequivalence: computational analysis of protein-protein interactions in icosahedral viruses.

Quaternary structure polymorphism found in quasiequivalent virus capsids provides a static framework for studying the dynamics of protein interactions. The same protein subunits are found in different structural environments within these particles, and in some cases, the molecular switching required for the polymorphic quaternary interactions is obvious from high-resolution crystallographic studies. Employing atomic resolution structures, molecular mechanics, and continuum electrostatic methods, we have computed association energies for unique subunit interfaces of three icosahedral viruses, black beetle virus, southern bean virus, and human rhinovirus 14. To quantify the chemical determinants of quasiequivalence, the energetic contributions of individual residues forming quasiequivalent interfaces were calculated and compared. The potential significance of the differences in stabilities at quasiequivalent interfaces was then explored with the combinatorial assembly approach. The analysis shows that the unique association energies computed for each virus serve as a sensitive basis set that may determine distinct intermediates and pathways of virus capsid assembly. The pathways for the quasiequivalent viruses displayed isoenergetic oligomers at specific points, suggesting that these may determine the quaternary structure polymorphism required for the assembly of a quasiequivalent particle.

The acid deoxyribonuclease of neutrophils: a possible participant in apoptosis-associated genome destruction.

Human neutrophils are terminally differentiated cells that spontaneously undergo apoptosis in tissue culture. Apoptosis in these cells can be delayed by culture in the presence of granulocyte colony-stimulating factor or other inflammatory mediators. Neutrophils were found to contain an acid endonuclease that appeared to be responsible for the internucleosomal DNA cleavage that accompanies apoptosis. As measured by a plasmid nicking assay, this endonuclease had a molecular weight (M(r)) of 35,000, a pH optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8. It was weakly inhibited by divalent cations (Ca2+, Mg2+, and Zn2+) and more strongly inhibited by aurintricarboxylic acid and N-bromosuccinimide. DNA from neutrophils treated with nigericin in buffers of defined pH displayed nucleosomal ladders whose prominence varied with pH in a manner that paralleled the pH dependence of the plasmid cleavage assays, consistent with internucleosomal DNA cleavage by the acid endonuclease. We have previously shown that neutrophils undergo acidification to a pH value as low as 6.0 during apoptosis; we suggest that this endonuclease may be responsible for the DNA cleavage seen in apoptotic neutrophils.

Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase.

Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program.