Maturation-induced conformational changes of HIV-1 capsid protein and identification of two high affinity sites for cyclophilins in the C-terminal domain.

Viral incorporation of cyclophilin A (CyPA) during the assembly of human immunodeficiency virus type-1 (HIV-1) is crucial for efficient viral replication. CyPA binds to the previously identified Gly-Pro90 site of the capsid protein p24, but its role remained unclear. Here we report two new interaction sites between cyclophilins and p24. Both are located in the C-terminal domain of p24 around Gly-Pro157 and Gly-Pro224. Peptides corresponding to these regions showed higher affinities (Kd approximately 0.3 microM) for both CyPA and cyclophilin B than the best peptide derived from the Gly-Pro90 site ( approximately 8 microM) and thus revealed new sequence motifs flanking Gly-Pro that are important for tight interaction of peptide ligands with cyclophilins. Between CyPA and an immature (unprocessed) form of p24, a Kd of approximately 8 microM was measured, which corresponded with the Kd of the best of the Gly-Pro90 peptides, indicating an association via this site. Processing of immature p24 by the viral protease, yielding mature p24, elicited a conformational change in its C-terminal domain that was signaled by the covalently attached fluorescence label acrylodan. Consequently, CyPA and cyclophilin B bound with much higher affinities ( approximately 0.6 and 0.25 microM) to the new, i.e. maturation-generated sites. Since this domain is essential for p24 oligomerization and capsid cone formation, CyPA bound to the new sites might impair the regularity of the capsid cone and thus facilitate in vivo core disassembly after host infection.

The V3 loop of human immunodeficiency virus type-1 envelope protein is a high-affinity ligand for immunophilins present in human blood.

Human immunodeficiency virus type-1 (HIV-1) infection requires binding of the envelope protein gp120 to host CD4 receptors and the action of the chemokine receptors CXCR4 or CCR5, which define cell tropism. The proline-containing V3 loop of gp120 determines the selection of the chemokine receptor and participates in conformational changes on binding of gp120 to CD4. In this study, we show that macrophage-tropic and T-cell-tropic V3 loop peptides bind specifically to the active site of the immunophilins FK506-binding protein (FKBP12), and cyclophilins A and B. Macrophage-tropic and T-cell-tropic V3 loop peptides inhibited the peptidyl-prolyl cis-trans isomerase (PPIase) activities of the immunophilins. Kd values in the range 0.036-4.1 microM were determined with V3 loop peptides labeled with an environmentally sensitive fluorophore. The observed binding properties of the V3 loop peptides reveal structural motifs of linear water-soluble peptidic substrates for tight interaction with immunophilins. FKBP12, and cyclophilins A and B were found to be present in normal human blood in the ranges 0.8-1.7, 1.4-2.3 and 2.4-3.1 nM, respectively, as demonstrated by PPIase activity measurements and western blot analysis. Cyclophilins A and B levels in serum of HIV-1-infected individuals were increased 3.6-fold and 1.6-fold. Due to the interaction of immunophilins with V3 loop peptides and with the envelope protein gp120, a role of immunophilins in HIV pathogenesis as conformases or docking mediators seems possible, since immunophilin receptors on cell membranes and immunophilin-related virulence factors of pathogens have been identified.

Apoptosis-induced concomitant release of cytosolic proteins and factors which prevent cell death.

In the course of the apoptotic cell death, cells fragment into apoptotic bodies, the elimination of which by phagocytosis is thought to avoid the release of cytosolic constituents whose occurrence is indicative for necrotic cell death. Confluent cultures of chicken embryo fibroblasts, however, show a different behaviour. After serum deprivation, they transiently released with the same time course mitogenic activity, lactate dehydrogenase and cytosolic peptidyl prolyl cis-trans isomerases into the serum-free culture medium. The release correlated in time with a decrease of the cell number which started approximately 3 h after serum removal and ceased within approximately 10 h at about half of the initial cell density. Morphological features like cell shrinkage, membrane blebbing and cell fragmentation as well as internucleosomal DNA fragmentation indicated apoptotic cell death whereas necrotic cell death could be excluded. Conditioned medium (M(r) > or = 30 kDa) from serum-deprived cultures of chicken embryo fibroblasts completely prevented chicken embryo fibroblasts to undergo apoptosis as did phorbol 12-myristate, 13-acetate and, to -60%, L-cysteine. Cycloheximide had no effect on serum deprivation-induced apoptosis. From the present results it can be concluded that chicken embryo fibroblasts and possibly other cells undergoing apoptosis release cytosolic components and endogenous survival factor(s) which prevent apoptosis.