During preliminary experiments to establish the proportion of virus-coded p24 protein to virus membrane-associated HLA-DR in gradient-enriched HIV-1 preparations, we became aware of a large variability between experiments. In order to determine whether HLA-DR-containing cellular material was contaminating the virus preparations, we carried out enrichment by gradient centrifugation of clarified supernatants from noninfected cells and tested this material for HLA-DR content. We found that, independently of the cell type used, gradient enrichment resulted in the isolation of large quantities of HLA-DR-containing material which banded at a density overlapping that of infectious HIV. Electron microscopy of gradient-enriched preparations from supernatants of virus-infected cells revealed an excess of vesicles with a size range of about 50-500 nm, as opposed to a minor population of virus particles of about 100 nm. Electron micrographs of infected cells showed polarized vesiculation of the cell membrane, and virus budding was frequently colocalized with nonviral membrane vesiculation. Analysis of the cellular molecules present in the fractions containing virus or exclusively cellular material demonstrated that virus and cellular vesicles share several cellular antigens, with the exception of CD43 and CD63, found mainly at the virus surface, and HLA-DQ, which was found only in the cellular vesicles.

The Kex2 protease of the yeast Saccharomyces cerevisiae is the prototype of a family of eukaryotic subtilisin homologs thought to process prohormones and other precursors in the secretory pathway. Deletion analysis of Kex2 protease shows that a sequence of 154‐159 residues carboxyl to the subtilisin domain is essential for the formation of active enzyme. Disruption of this region, termed the ‘P‐domain’, blocks the normally rapid intra‐molecular cleavage of the N‐terminal pro‐segment of pro‐Kex2 protease in the endoplasmic reticulum (ER). The C‐terminal boundary of the P‐domain coincides closely with the endpoint of similarity between Kex2 protease and its mammalian homologues. The conservation of and functional requirement for the P‐domain sharpens the distinction between a ‘Kex2 family’ of processing enzymes and degradative ‘subtilases’, and implies that the Kex2‐related enzymes have in common entirely novel structural features that are important in the maturation of precursor polypeptide substrates. Failure to cleave the N‐terminal pro‐domain, due either to truncation of the P‐domain or to mutation of the active site histidine or serine, results in stable, intracellular retention of pro‐enzyme, apparently in the ER. Thus pro‐Kex2 protease appears to contain an ER retention signal which is removed or destroyed by cleavage of the pro‐domain.

The majority of resting memory T cells infected during the course of a preceding cell activation produces virus persistently, without establishing a true HIV-1 latency. The described system could be used as a model for testing new drugs able to control residual HIV-1 replication in resting memory T cells.

The post-translational proteolytic conversion of somatostatin-14 precursors was studied to characterize the enzyme system responsible for the production of the tetradecapeptide either from its 15-kDa precursor protein or from its COOH-terminal fragment, somatostatin-28. A synthetic undecapeptide Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-AsnTyr(NH2), homologous to the amino acid sequence of the octacosapeptide at the putative Arg-Lys cleavage locus, was used as substrate, after 125I labeling on the COOH-terminal tyrosine residue. A 90-kDa proteolytic activity was detected in rat brain cortex extracts after molecular sieve fractionation followed by ion exchange chromatography. The protease released the peptide 1251-Ala-Gly-Ala-Lys-Asn-Tyr(NH2) from the synthetic undecapeptide substrate and converted somatostatin-28 into somatostatin-14 under similar conditions (pH 7.0). Under these experimental conditions, the product tetradecapeptide was not further degraded by the enzyme. In contrast, the purified 15-kDa hypothalamic precursor remained unaffected when exposed to the proteolytic enzyme under identical conditions. It is concluded that this Arg-Lys esteropeptidase from the brain cortex may be involved in the in vivo processing of the somatostatin-28 fragment of prosomatostatin into somatostatin-14, the former species being an obligatory intermediate in a two-step proteolytic mechanism leading to somatostatin-14.A number of neural, or hormonal, peptides have been shown to derive biosynthetically from larger molecular weight precursors by post-translational proteolytic cleavage. Elucidation of the amino acid sequences of many of these proforms has clearly underlined the importance of basic amino acid doublets as potential recognition sites for selective proteases. In addition, putative cleavage points corresponding either to a lone lysine or arginine residue, as well as double pairs of basic amino acids, were deduced from the established primary structures of some precursors (1-7). Although structural information on these potential cleavage points is available, little is known at this time about the enzymes involved in recognition of these sites.The predicted sequences of the prosomatostatins from pancreatic tissue (2) have indicated that the tetradecapeptide somatostatin-14 occupies the COOH-terminal end of the precursor and that an Arg-Lys doublet can be found at position -2, -1 from the NH2 terminus of the somatostatin-14 sequence. Furthermore, the finding in both hypothalamic and pancreatic tissues of octacosapeptides called somatostatin-28 (8-11) has suggested that the lone arginine residue observed in the precursor (at position -15 from the NH2-terminal alanine of somatostatin-14) may constitute the recognition signal for a specific protease responsible for the release of somatostatin-28. Therefore, prosomatostatin offers a model particularly well suited for the study of the proteolytic systems involved in the processing of proneuropeptides and exhibiting a specificity either for basic amino acid doublets and...

Selective and limited proteolysis is a key step in the post-translational modification of peptide hormone precursors. This process appears to involve a proteolytic machinery including highly specific endoproteases. Some of the enzyme systems possibly involved in the processing of pro-neuropeptides will be described and their mechanism of action discussed. Special emphasis will be on the following: i) the physico-chemical characteristics of proteolytic enzymes which are believed to be involved in the processing of some of these polypeptide hormone precursors; ii) the bio-specificity of these enzymes toward the substrates; iii) the importance of both secondary and tertiary structures of the cleavage domain in recognition by the selective proteases. These properties will be discussed in connection with the possible importance of the maturation enzymes in the in vivo regulation of hormone biosynthesis.