Production of HIV-1 by resting memory T lymphocytes.

BACKGROUND: The persistence of HIV-1 within resting memory CD4 T cells constitutes a major obstacle in the control of HIV-1 infection. OBJECTIVE: To examine the expression of HIV-1 in resting memory CD4 T cells, using an in-vitro model. DESIGN AND METHODS: Phytohaemagglutinin-activated peripheral blood mononuclear cells were challenged with T cell-tropic and macrophage-tropic HIV-1 clones, and with a replication-incompetent and non-cytotoxic HIV-1-derived vector (HDV) pseudotyped by the vesicular stomatitis virus glycoprotein G. To obtain resting memory CD4 T cells containing HIV-1 provirus, residual CD25(+), CD69(+) and HLA-DR(+) cells were immunodepleted after a 3 week cultivation period. RESULTS: In spite of the resting phenotype, the majority of provirus-harbouring T cells expressed HIV-1 genomes and produced infectious virus into cell-free supernatant. The expression of HDV dropped by only 30% during the return of activated HDV-challenged cells into the quiescent phase. Although resting memory T cells generated in vitro expressed HIV-1 and HDV genome when infected during the course of the preceding T cell activation, they were resistant to HIV-1 and HDV challenge de novo. The infected culture of resting memory T cells showed a higher resistance to the cytotoxic effects of HIV-1 in comparison with the same cultures after reactivation by phytohaemagglutinin. CONCLUSION: 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.

Caprine arthritis encephalitis virus: evidence for a B/D-type assembly pathway in a C-type lentivirus replication.

Lentiviruses, among which is caprine arthritis encephalitis virus (CAEV), are known to concomitantly assemble and bud at the plasma membrane of infected cells, in a C-type defined pathway. Electron microscopy analysis of CAEV-infected cells demonstrated viral particles budding at the plasma membrane and into intracellular membrane-surrounded vesicles. Furthermore, nonenveloped immature virus-like particles, resembling intracytoplasmic type-A particles (ICAPs), accumulated within the cytoplasm of those cells. Fractionation on sucrose density gradients of cytoplasmic lysates from CAEV-infected cells revealed that enveloped immature or mature viral particles had a density of 1.16--1.17 g/ml, whereas ICAPs sedimented at a density of 1.2--1.27 g/ml. Endogenous reverse transcriptase activity was only associated with the 1.16--1.17 g/ml density particles despite the presence of viral RNA in both populations. The intracellular enveloped particles were found to be infectious. The CAEV Gag precursor by itself was shown to direct assembly, budding, and release of immature virus-like particles when expressed in goat primary synovial membrane cells using the same pathways of assembly and budding as observed in CAEV-infected cells. These data suggest that CAEV assembly, driven by the Gag precursor, could unusually proceed via two simultaneous pathways characteristic of type-C and type-B/D retroviruses.

Tryptophan 95, an amino acid residue of the Caprine arthritis encephalitis virus vif protein which is essential for virus replication.

The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region.

Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations.

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.

A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease.

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.

Relationship between endo- and exopeptidases in a processing enzyme system: activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase.

The somatostatin-28 convertase activity involved in vitro in the processing of somatostatin-28 into the neuropeptides somatostatin-28-(1-12) and somatostatin-14 is composed of an endoprotease and a basic aminopeptidase. We report herein on the purification to apparent homogeneity of these two constituents and on their functional interrelationship. In particular we observed that after various physicochemical treatments, the 90-kDa endoprotease activity was recovered both at this molecular mass and as a 45-kDa entity. Moreover, the production of [Arg-2,Lys-1]somatostatin-14 from somatostatin-28 by the action of the endoprotease was activated in a cooperative manner by the aminopeptidase B-like enzyme. A 10-fold activation occurred when the exopeptidase was inhibited by 6.5 mM diisopropyl fluorophosphate and allowed the determination of a half-maximal activation constant (K1/2) of approximately equal to 13 nM. These observations strongly suggest that both enzymes act in a concerted manner in vitro and that they may form a complex in vivo.

Role of peptide substrate structure in the selective processing of peptide prohormones at basic amino acid pairs by endoproteases.

Three putative processing enzymes, each with defined action in a prohormone system, a 'pro-ocytocin-neurophysin convertase' from bovine neurohypophysis secretory granules, a 'Leu-enkephalin Arg6 generating enzyme' from human CSF and the endoprotease from the 'S-28 convertase' complex of rat brain cortex, were tested for their ability to hydrolyze peptides deriving from pro-ocytocin, pro-enkephalin B and pro-somatostatin, respectively at pairs of basic amino acids. The observations suggest that structural parameters specified by the peptide region around the dibasic moieties govern recognition by the enzyme and define which peptide bond is hydrolyzed.

Somatostatin-28 and pro-ocytocin/neurophysin convertases: basic pair selective endoproteases involved in pro-hormone processing in the rat brain cortex and bovine corpus luteum.

Two neuropeptide precursor processing enzyme systems were characterized in the rat brain cortex and bovine neurohypophysis and corpus luteum. The first one combines the action of a 90 kDa endoprotease which cleaves somatostatin-28 before the Arg-Lys doublet and that of an aminopeptidase B-like enzyme. The second system associates the action of a 58 kDa endoprotease cleaving pro-ocytocin/neurophysin (1-20) after the Lys-Arg dibasic moiety and a carboxypeptidase B-like activity. Both systems appear to be located in membrane-limited secretory vesicles of the producing organs, and to exhibit the properties of metallo-enzymes sensitive to divalent cation chelators. In contrast, they do not show the characteristics of serine-proteases and of trypsin-like enzymes. Studies with substrate analogs selectively modified at the basic doublet indicated that the integrity of both basic amino acids is essential but that conformational parameters, probably governed by the amino acid sequences flanking the basic doublet, play an important role. These data will be discussed in relation to a hypothesis on the predicted preferred secondary structure of these restriction loci.

Proteolytic enzymes in the post-translational processing of polypeptide hormone precursors.

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.

Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex.

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.

[Proteolytic events in the maturation of pro-neuropeptides. The somatostatin model]. / Evénements protéolytiques de la maturation des proneuropeptides. Le modèle de la somatostatine.

The post-translational processing (maturation) of the precursors was studied on the model of the prosomatostatin. We have shown the presence of a single and common precursor to both somatostatin -28 and -14 in mouse hypothalamus, in contrast with the situation in the Teleostean fish, Lophius piscatorius. The search for a maturation activity was carried out using a synthetic undecapeptide substrate including in its sequence the cleavage site for somatostatin-14 release. Using this peptide, we characterized in rat brain cortex extracts a specific enzyme activity of 90 kD. This "maturase", colocalized in the neurosecretory granules with the somatostatin products, generates both the N-terminal peptide S-28, and the tetradecapeptide hormone (S-14) from the somatostatin-28, acting as a "S-28 convertase" producing free Arg and Lys residues present at the pair of basic amino acids signal. We propose a model where three peptide bonds are cleaved by this enzymatic activity. In the teleostean fish: Lophius piscatorius, two precursors coding for two different somatostatin were predicted by the determination of cDNA sequence. In this system, we observed the presence of a unique form of the tetradecapeptide hormone. We show that the final maturation product of the second precursor is a new 28 amino acid hormone called Somatostatin-28 II. Moreover, the product of this second gene after the action of the Somatostatin-28 convertase from rat brain cortex is the (Tyr7, Gly 10)S-14 derivative predicted by the clone.(ABSTRACT TRUNCATED AT 250 WORDS)

The somatostatin-28 convertase of rat brain cortex is associated with secretory granule membranes.

An Arg-Lys esteropeptidase that converts somatostatin-28 in vitro into somatostatin-14 was previously characterized in extracts of rat cerebral cortex. Both the octacosapeptide somatostatin-28 and a synthetic undecapeptide containing the sequence around the Arg-Lys site, i.e. Peptide I: Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-125 I-Tyr (NH2), were used as substrates. We demonstrate that the converting activity is associated with neurosecretory granule fractions prepared from both cortical and hypothalamic tissue. This activity co-sediments with ghosts obtained from intact vesicles by osmotic shock. After solubilization either by mild ionic strength or sonication of vesicle membranes, the converting activity appears to possess properties indistinguishable from the convertase prepared directly from unfractionated tissue. It cleaves Peptide I to Ala-Gly-Ala-Lys-Asn-125I-Tyr (NH2) (Peptide II) and generates both the NH2- and COOH-terminal fragments of somatostatin-28, i.e. somatostatin-28 (1-12) and somatostatin-14, when the octacosapeptide is used as substrate. The selectivity appears to be strict and to depend upon the sequence around the Arg-Lys pair, as inferred from competition studies conducted with structural analogs possessing either an Arg-Lys or Arg-Arg doublet. It is concluded that this convertase could represent the enzyme system involved in the in vivo production of both the dodeca and tetradeca peptides from their common somatostatin-28 precursor.

The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28.

The products generated after addition of the ARG-LYS esteropeptidase activity purified from rat brain to synthetic somatostatin-28 were analyzed using radioimmunoassay, HPLC and amino acid analysis. In addition to somatostatin-14, both free arginine and free Lysine were identified together with somatostatin-28. The dipeptide ARG-LYS was not present, which indicates that three peptide bonds were hydrolyzed in order to achieve excision of the doublet. Since it is likely that the octacosapeptide is a precursor for both somatostatin-14 and somatostatin-28, these observations add further support to the hypothesis that the convertase is also involved in the in vivo processing of endogenous somatostatin-28.

Proteolytic events in the post-translational processing of somatostatin precursors from rat brain cortex and anglerfish pancreatic islets.

An Arg-Lys esteropeptidase which converts somatostatin-28 (S-28) into somatostatin-14 (S-14) was detected in rat brain cortical extracts using a synthetic undecapeptide substrate mimicking the octacosapeptide sequence at the restriction site. This enzyme system was unable to release either the octacosapeptide or S-14 from the 15,000 mol wt (15K) rat hypothalamic precursor. This argues in favor of sequential degradation of the precursor into S-14 via S-28 as an obligatory intermediate. Another in vivo processing system was analyzed in the anglerfish pancreatic Brockmann organs. Here, cloning of two cDNA corresponding to two mRNA species predicts two distinct somatostatins precursors, called prosomatostatins I and II (Hobart et al., Nature 288:137, 1980). While a single S-14 can be detected in extracts made from this pancreatic tissue, indistinguishable from the mammalian species, two S-28 species could be separated by HPLC. Immunochemical and biochemical evidence indicates that the second species should correspond to anglerfish S-28 (AF S-28), the product of prosomatostatin-II processing in vivo. Amino acid analysis, together with the determined complete amino acid sequence of this peptide, demonstrates that this is indeed the case and that AF S-28 contains in its C-terminal half the [Tyr7, Gly10] derivative of S-14. These observations give an example of a AF S-28 being a terminal active product of prosomatostatin processing. They suggest that this octacosapeptide, which is potent on the inhibition of growth hormone release by anterior pituitary cells, may play such a role in the gastrointestinal tract of the anglerfish. These results, while not excluding alternative routes, give support to a sequential processing of the 15 K precursor----S-28----S-14.

Characterization of a somatostatin-28 containing the (Tyr-7, Gly-10) derivative of somatostatin-14: a terminal active product of prosomatostatin II processing in anglerfish pancreatic islets.

Anglerfish (Lophius piscatorius) Brockmann organs contain a form of somatostatin-14, identical to the hypothalamic tetradecapeptide, and two distinct forms of somatostatin-28, which can be separated by reversed-phase high-pressure liquid chromatography (HPLC). Analysis of the NH2-terminal amino acid sequence and comparison of the ability to incorporate 125I indicate that one of these forms corresponds to an octacosapeptide including in its sequence the (Tyr-7, Gly-10) derivative of somatostatin-14 (somatostatin II). Exposure of this somatostatin-28 species to an endopeptidase activity from the rat brain cortex generates a peptide immunologically related to somatostatin and undistinguishable from synthetic (Tyr-7, Gly-10) somatostatin-14 II by HPLC. This somatostatin-28 II exhibits a potent inhibitory effect on growth hormone release by rat anterior pituitary cells, comparable to the other somatostatin-28 form. Since (Tyr-7, Gly-10) somatostatin-14 II cannot be detected in anglerfish pancreatic islets, these results indicate that somatostatin-28 II represents the terminal active product of prosomatostatin II processing, whose structure was predicted from the cDNA nucleotide sequence corresponding to the second mRNA cloned from anglerfish Brockmann organs [Hobart, P., Crawford, R., Shen, L. P., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141].

Enzymes processing somatostatin precursors: an Arg-Lys esteropeptidase from the rat brain cortex converting somatostatin-28 into somatostatin-14.

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-Asn-Tyr(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 125I-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.