12 research outputs found
GPG-NH<sub>2 </sub>acts via the metabolite αHGA to target HIV-1 Env to the ER-associated protein degradation pathway
<p>Abstract</p> <p>Background</p> <p>The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH<sub>2</sub>) 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-NH<sub>2 </sub>targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD) pathway during its maturation. However, the anti-viral effect of GPG-NH<sub>2 </sub>has been shown to be mediated by its metabolite α-hydroxy-glycineamide (αHGA), 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-NH<sub>2 </sub>was attributed to its metabolite αHGA.</p> <p>Results</p> <p>In the presence of fetal bovine serum, GPG-NH<sub>2</sub>, its intermediary metabolite glycine amide (G-NH<sub>2</sub>), and final metabolite αHGA all induced the degradation of gp160 through the ERAD pathway. However, when fetal bovine serum was replaced with human serum only αHGA showed an effect on gp160, and this activity was further shown to be completely independent of serum. This indicated that GPG-NH<sub>2 </sub>acts as a pro-drug, which was supported by the observation that it had to be added earlier to the cell cultures than αHGA to induce the degradation of gp160. Furthermore, the substantial reduction of Env incorporation into HIV-1 particles that occurs during GPG-NH<sub>2 </sub>treatment was also achieved by treating HIV-1 infected cells with αHGA.</p> <p>Conclusions</p> <p>The previously observed specificity of GPG-NH<sub>2 </sub>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-NH<sub>2 </sub>metabolite αHGA.</p
Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation â–¿
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-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2 was uncovered by examining Env expression and maturation in treated cells. GPG-NH2 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-NH2 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-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy
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
<div><p>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.</p></div
The influence of N- and C-terminal dimerization on the anti-viral effects of the MPER-derived peptides.
<p><b>A.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide QK26. <b>B.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide EK30. <b>C.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide EK37. <b>D.</b> Summary of the concentrations of peptides yielding a 50% and 80% reduction in tat-activated luciferase activity, as tested in B, C and D. The concentrations were estimated with GraphPad Prism. <b>E.</b> No cytotoxicity effect was observed for the MPER-derived dimeric peptides at 100 μM in TZM-bl cells. Monomeric and dimeric peptides (100 μM) were incubated with 10,000 Vero cells for 24 h. PrestoBlue cell viability reagent was subsequently added to the cells, and cytotoxicity effects were monitored as absorbance values (OD) at 570 nm and 600 nm (baseline).</p
Schematic representation of HIV-1 gp41 and partial sequence alignment of the gp41 from different groups.
<p>Partial HR2, the MPER and the TMD sequences of HIV-1 group M subtypes A, B, C and D; group O and the experimental strain of this study, HIV(NL4-3) were aligned[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134851#pone.0134851.ref015" target="_blank">15</a>]. Sequences of the anti-HIV-1 first and second generation fusion inhibitor, T20 and T1498, respectively, are shown together with the MPER-containing peptides tested in this study, EK37, EL30, QK26, QT19, LK21 and LK21-5W5A, and all are aligned with the MPER sequence. The MPER sequence is highlighted in bold with its conserved residues shaded. Peptide LK21-5W5A have all five tryptophan residues in MPER sequence substituted by Ala.</p
Schematic representation of the peptide N- and C-terminal dimerization strategies.
<p>N-terminal dimerization employed a linker molecule consisted of two serine residues branching from a lysine, to which two peptides were attached at their N-termini via a thiazolidine linkage. C-terminal dimerization employed a MBHA resin.</p
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 μM. 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
Expression and viral incorporation of viral structural proteins in the context of pseudotyped HIV-1.
<p><b>A.</b> p55/Gag-derived p24 levels in pseudovirus-producing HLtat cells. HIV(WT), HIV(W5A), HIV(W3A) and HIV(W2A) were produced by co-transfecting HLtat cells with pNLHIVxΔu∆ss and pNL1.5EU+, pNL1.5EU+W5A, pNL1.5EU+W3A, or pNL1.5EU+W2A, respectively. The p24 levels (ng) in cell lysates were quantified by the automated system Architect (Abbott). ***P < 0.001 as compared to WT by the unpaired Student’s t test. <b>B.</b> Steady-state intracellular levels of viral proteins in pseudovirus-producing HLtat cells. HLtat cells from A were harvested 48 h post-transfection and the lysates were resolved by SDS-PAGE and immunoblotted with antibodies against gp41, p24, Vif, Nef and β-actin. Vif and Nef expression served as the transfection control. Un-transfected HLtat cells served as negative control. <b>C.</b> Densitometric analysis of protein bands in blots from two independent experiments as described in in B was performed in ImageJ and presented as means ± SD, with gp160, p55/Gag, and p24 levels in HIV(WT) standardized to 100%. *P < 0.05; **P < 0.01 as compared to WT by the unpaired Student’s t test. <b>D.</b> p24 levels (ng) in the culture supernatants of pseudovirus-producing HLtat cells. p24 levels in the culture supernatant of HLtat cells in A was quantified by the automated system Architect (Abbott). ****P < 0.0001 as compared to WT by the unpaired Student’s t test. <b>E.</b> Env gp41, p55/Gag and p24 levels in precipitated HIV(WT), HIV(W5A), HIV(W3A) and HIV(W2A). Viral particles from the cell culture supernatant from A were precipitated, lysed, separated by SDS-PAGE and immunoblotted with antibodies against gp41 and p24. <b>F</b> Densitometric analysis of the blot in E was performed in ImageJ and presented as means, with gp41, p55/Gag and p24 levels in HIV(WT) standardized to 100%. <b>G.</b> Entry of the cell-free pseudotyped HIV-1 into TZM-bl cells. Cell culture supernatant from A containing HIV(WT), HIV(W5A), HIV(W3A) or HIV(W2A) was clarified through centrifugation and 0.45μm filtration, and applied to 10<sup>4</sup> TZM-bl cells. Seventy-two h post-infection, tat-activated luciferase activities in the TZM-bl cells were measured and plotted, with the luciferase activity in HIV(WT)-infected TZM-bl cells standardized to 100%.</p