Human serum protein enhances HIV-1 replication and up-regulates the transcription factor AP-1.

In vitro studies on HIV (HIV-1) replication and neutralization are usually performed in human cell cultures supplemented with FBS instead of human serum (HS). Here we show that in contrast to FBS, addition of increasing amounts of human serum from noninfected donors to the cell culture directly correlates with an increase in HIV-1 replication in vitro. This effect is independent of cell line, virus strain, or batch of pooled human serum used. We found that human serum affects viral transcription in a dose-dependent manner by activating the activator protein-1 (AP-1) member proteins c-FOS, JunD, and JunB in TZM-bl cells. Analysis of the human serum component responsible for this effect indicates that it is a protein having a molecular mass between 250 and 300 kDa. This serum protein, HIV-1 enhancing serum protein (HESP), might promote viral transcription in vivo and consequently play a role in disease progression.

GPG-NH2 acts via the metabolite alphaHGA to target HIV-1 Env to the ER-associated protein degradation pathway.

BACKGROUND: The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH2) was previously shown to abolish the ability of HIV-1 particles to fuse with the target cells, by reducing the content of the viral envelope glycoprotein (Env) in progeny HIV-1 particles. The loss of Env was found to result from GPG-NH2 targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD) pathway during its maturation. However, the anti-viral effect of GPG-NH2 has been shown to be mediated by its metabolite alpha-hydroxy-glycineamide (alphaHGA), which is produced in the presence of fetal bovine serum, but not human serum. In accordance, we wanted to investigate whether the targeting of gp160 to the ERAD pathway by GPG-NH2 was attributed to its metabolite alphaHGA. RESULTS: In the presence of fetal bovine serum, GPG-NH2, its intermediary metabolite glycine amide (G-NH2), and final metabolite alphaHGA all induced the degradation of gp160 through the ERAD pathway. However, when fetal bovine serum was replaced with human serum only alphaHGA showed an effect on gp160, and this activity was further shown to be completely independent of serum. This indicated that GPG-NH2 acts as a pro-drug, which was supported by the observation that it had to be added earlier to the cell cultures than alphaHGA to induce the degradation of gp160. Furthermore, the substantial reduction of Env incorporation into HIV-1 particles that occurs during GPG-NH2 treatment was also achieved by treating HIV-1 infected cells with alphaHGA. CONCLUSIONS: The previously observed specificity of GPG-NH2 towards gp160 in HIV-1 infected cells, resulting in the production of Env (gp120/gp41) deficient fusion incompetent HIV-1 particles, was most probably due to the action of the GPG-NH2 metabolite alphaHGA.

Small molecule targets Env for endoplasmic reticulum-associated protein degradation and inhibits human immunodeficiency virus type 1 propagation.

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH(2)), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH(2) was uncovered by examining Env expression and maturation in treated cells. GPG-NH(2) treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH(2) negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH(2), which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.

Alzheimer's Disease: Erythrocyte 2,3-diphosphoglycerate Content and Circulating Erythropoietin.

BACKGROUND: Alzheimer's Disease (AD) features the accumulation of ß-amyloid in erythrocytes. The subsequent red cell damage may well affect their oxygen-carrying capabilities. 2,3- diphosphoglycerate (2,3-DPG) binds to the hemoglobin thereby promoting oxygen release. It is theorized that 2,3-DPG is reduced in AD and that the resulting hypoxia triggers erythropoietin (EPO) release. METHODS & OBJECTIVE: To explore this theory, we analyzed red cell 2,3-DPG content and EPO in AD, mild cognitive impairment, and the control group, subjective cognitive impairment. RESULTS: We studied (i) 2,3-DPG in red cells, and (ii) circulating EPO in AD, and both markers were unaffected by dementia. Disturbances of these oxygen-regulatory pathways do not appear to participate in brain hypoxia in AD.

Erythrocyte Amyloid Beta Peptide Isoform Distributions in Alzheimer and Mild Cognitive Impairment.

INTRODUCTION: We recently showed that Amyloid Beta (Aß)40 accumulates in erythrocytes and possibly causes cell damage as evidenced by an increased number of assumed injured low-density (kg/L) erythrocytes. Furthermore, we have suggested a separation technique to isolate and concentrate such damaged red blood cells for subsequent analysis. OBJECTIVES: We isolated high- and low-density erythrocytes and investigated the accumulation patterns of the Aß peptides (Aß40, Aß42, and Aß43) in Alzheimer (AD), mild cognitive impairment (MCI), and Subjective Cognitive Impairment (SCI). METHODS: Whole blood was fractionated through a density gradient, resulting in two concentrated highand presumed injured low-density erythrocyte fractions. After cell lysis, intracellular Aß40, Aß42, and Aß43 were quantified by ELISA. RESULTS: In both high- and low-density erythrocytes, Aß40 displayed the lowest concentration in MCI, while it was equal and higher in AD and SCI. Aß40 was detected at a 10-fold higher level than Aß42, and in injured low-density erythrocytes, the lowest quantity of Aß42 was found in AD and MCI. Aß40 exhibited a 100-fold greater amount than Aß43, and lighter erythrocytes of MCI subjects displayed less intracellular Aß43 than SCI. CONCLUSION: Red blood cell accumulation patterns of Aß40, Aß42, and Aß43 differ significantly between AD, MCI, and SCI. The data must be verified through larger clinical trials. It is, however, tenable that Aß peptide distributions in erythrocyte subpopulations have the potential to be used for diagnostic purposes.

Membrane-Active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-Containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins.

The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics.

Structure-activity relationship of tumor-selective 5-substituted 2-amino-3-carboxymethylthiophene derivatives.

Methyl-2-amino-5-[2-(4-methoxyphenethyl)]thiophene-3-carboxylate (8 c) is the prototype of a well-defined class of tumor-selective agents. Compound 8 c preferentially inhibited the proliferation of a number of tumor cell lines including many human T-lymphoma/leukemia cells, but also several prostate, renal, central nervous system and liver tumor cell types. Instead, a broad variety of other tumor cell lines including B-lymphomas and HeLa cells were not affected. The tumor selectivity (TS; selectivity index or preferential suppression of CEM lymphoma (IC50 =0.90 µM) versus HeLa tumor cell carcinoma (IC50 =39 µM)) amounted up to ~43 for 8 c. At higher concentrations, the compound proved cytotoxic rather than cytostatic. The antiproliferative potency and selectivity of 8 c could be preserved by replacing the ethyl linker between the 2-amino-3-carboxymethylthiophene and the substituted aryl by a thioalkyl but not by an oxyalkyl nor an aminoalkyl. Among >50 novel 8 c derivatives, the 5-(4-ethyl- and 4-isopropylarylmethylthio)thiophene analogues, methyl-2-amino-5-((4-ethylphenylthio)methyl)thiophene-3-carboxylate (13 m) and methyl-2-amino-5-((4-isopropylphenylthio)methyl)thiophene-3-carboxylate (13 n), were more potent (IC50 : 0.3-0.4 µM) and selective (TS: 100-144) anti-T-lymphoma/leukemia agents than the prototype compound.

Glycine-amide is an active metabolite of the antiretroviral tripeptide glycyl-prolyl-glycine-amide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH(2)) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH(2)), proline (P-OH), and glycine-amide (G-NH(2)) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH(2) has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH(2) exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH(2) concentration that inhibited virus replication by 50% (IC(50)) was equimolar to that of GPG-NH(2) and ranged from 3 to 41 microM. Transmission electron microscopy revealed that the effect of G-NH(2) on HIV-1 morphology was equivalent to that of GPG-NH(2) and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH(2) for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH(2) might act as a prodrug and that G-NH(2) is an active antiretroviral metabolite.