- Open Access
GPG-NH2acts via the metabolite αHGA to target HIV-1 Env to the ER-associated protein degradation pathway
© Jejcic et al; licensee BioMed Central Ltd. 2010
- Received: 13 December 2009
- Accepted: 15 March 2010
- Published: 15 March 2010
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 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-NH2 was attributed to its metabolite αHGA.
In the presence of fetal bovine serum, GPG-NH2, its intermediary metabolite glycine amide (G-NH2), 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-NH2 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-NH2 treatment was also achieved by treating HIV-1 infected cells with αHGA.
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 αHGA.
- Post Transfection
- Total Particle Number
- Reference Reagent Program
- ERAD Pathway
- Capsid Formation
The HIV-1 envelope glycoprotein (Env) is co-translationally translocated into the endoplasmic reticulum (ER) as the precursor protein gp160. It is a is a type 1 membrane protein that in the ER obtains ~30 N-linked glycans and forms 10 disulphide bonds during a slow and extensive folding process . The mature gp160 trimerizes prior to its export to the Golgi, where it is being processed into the trans-membrane unit, gp41, and the highly glycosylated surface unit, gp120, which remain non-covalently associated to each other [2, 3]. These trimeric gp120/gp41 complexes are then transported to the cell surface for incorporation into the assembling particles.
During the course of studying its anti-viral mechanism it was discovered that GPG-NH2 is metabolized via glycine amide (G-NH2) into α-hydroxy-glycine amide (αHGA) in cell culture media containing fetal bovine serum (FBS) (Fig. 2A) [11, 12]. Both metabolites have been found to retain the ability to inhibit HIV-1 propagation in the presence of FBS and in serum from several other species . However, in HS only αHGA still possesses its anti-viral activity against HIV-1, which indicates that the unidentified enzyme responsible for the transition of G-NH2 into αHGA is not present in HS . This strongly suggests that the anti-viral activity previously ascribed to GPG-NH2 is actually an attribute of its final metabolite αHGA. In this study we therefore further examined if the potent ability of GPG-NH2 to target gp160 for ERAD is also dependent on it metabolizing into αHGA.
GPG-NH2, G-NH2and αHGA treatment all decrease the molecular mass, steady-state levels and processing of gp160
αHGA does not require FBS to affect gp160
αHGA targets gp160 for degradation more rapidly than GPG-NH2
We have previously shown that GPG-NH2 does not generally effect cellular glycoproteins, but acts rather selectively on gp160 . Here, we examined the glycoprotein expression profile in the HeLa-tat III cells upon treatment with αHGA added to the cultures at seeding and collected 24 h and 48 h later. The total protein content increased two fold and three fold, respectively, during incubation time (data not shown). As for GPG-NH2, αHGA showed no general effect on glycoproteins at 24 h or 48 h as only a single unidentified high-molecular-mass-protein (~150 kDa) slightly increased its mobility at 50 μM and 100 μM αHGA (Fig. 4E; only 24 h blot is shown).
αHGA decreases the content of Env in HIV-1 particles
In this study we examined whether either of the two GPG-NH2-metabolites retained the ability to target gp160 for destruction in the same manner as GPG-NH2. Here we show that when replacing FBS with HS or in complete absence of serum the effect of GPG-NH2 on gp160 was completely abolished, which strongly indicates that GPG-NH2 is not the molecule responsible for targeting gp160 for ERAD. αHGA, on the other hand was active against gp160 both in the presence of HS and under serum free conditions. The intermediate metabolite G-NH2 was not able to target gp160 for destruction in HS but showed some activity in absence of serum. This means that either some of the enzymatic activity converting G-NH2 to αHGA remained after washing of the cells and HS prevented its conversion to αHGA or G-NH2 was able to affect gp160 by itself but was inhibited by HS. GPG-NH2 had to be added much earlier than αHGA to the cell cultures in order to be effective against gp160. The comparably slow onset of GPG-NH2 also supports that GPG-NH2 needs conversion to αHGA to target gp160 for ERAD. In addition, viral particles produced in the presence of αHGA showed a dramatic loss in their gp120/gp41 content with respect to the capsid protein p24. Therefore, the effect on gp160 resulting in reduced gp120/gp41 content in progeny viral particles rendering them fusion incompetent that was previously ascribed to GPG-NH2 is most likely due to its metabolite αHGA. Although, deletion of the 19 N-terminal amino acids (aa) of the 30 aa long gp160 signal sequence has been shown to render gp160 resistant to αHGA treatment, the exact site of αHGA interaction remains to be identified .
We have previously shown that αHGA also causes a diversity of abnormal capsid formations in progeny viral particles . These two effects may be completely independent of each other as αHGA is believed to bind to the hinge region of p24 thereby preventing it from forming proper capsids . However, the gp41 deficiency in the particles could also contribute to the distorted capsid formation. The exceptionally long cytosolic tail of gp41, which stretches 150 aa into the particles, interacts with p55Gag and cellular proteins and may therefore play a role in the formation of proper internal viral structures [13–16]. Although important, it is difficult to evaluate which of the two effects is mostly responsible for the overall antiviral effect and whether they are related or are two separate phenomena. In an effort to solve this, we are now trying to induce the αHGA resistant gp160 signal sequence mutations into infectious clones of HIV-1 to see if the resulting clones are infectious and if so whether αHGA retains its anti-viral activity to such mutated virus.
In this study, we have reported that it is not GPG-NH2 but its small metabolite (90 Da) αHGA that targets gp160 for destruction via the ERAD pathway, which results in production of gp120/gp41 deficient HIV-1 progeny particles.
Reagents and Antibodies
GPG-NH2 and G-NH2 were purchased from Bachem Feinchemikalien and αHGA from Chemilia AB. The monoclonal antibody to gp41 (Chessie 8)  was obtained through the NIH AIDS Research and Reference Reagent Program, and the antibody to p24 (EF7) has previously been described .
Cell Lines and Plasmids
The cell lines HeLa-tat III and ACH-2 [19, 20] and the infectious HIV-1 expressing plasmid pNL4-3  were obtained through NIH AIDS Research and Reference Reagent Program. The expression plasmids for gp160 from the HIV-1 strain NL43 (pNL1.5EU)  and for Rev (pBRev) were kindly provided by Dr. S. Schwartz (Uppsala University, Sweden). PCRR3.1/CAT expresses chloroamphenichol acetyltransferase and was purchased from Invitrogen.
Transfection and drug treatments
HeLa-tat III cells (~3 × 105 cells/dish) were treated with the indicated concentrations of GPG-NH2, G-NH2 and αHGA prior to or post transfection with the gp160, and the transfection efficiency control CAT expressing plasmids using FuGENE 6 (Roche). The cells were rinsed twice in PBS and lysed 20-24 h post transfection in RIPA buffer containing 50 mM Tris-HCl pH 7.4, 1% Triton-X-100, 1% deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1% SDS and supplemented with Complete protease inhibitor cocktail (Roche).
PNGase F digestion
Cell lysates in RIPA buffer were supplemented with 1% β-mercaptoethenol and denaturated for 10 min at 95°C. Addition of 1% NP-40 and 16 U PNGase F (New England Biolabs) was followed by incubation at 37°C for 1 h.
Western Blot and ELISA
Cells and precipitated virus were lysed in RIPA buffer, standardized to CAT or p24 levels respectively, denatured and resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted. The membranes were exposed to film for the appropriate time and band intensities were quantified using GeneTools analysis software (SynGene). For probing against cellular glycoproteins peroxidase conjugated Concanavalin A (Sigma) was used according to manufacturer's protocol. In brief, the membranes were incubated in PBS containing 2% Tween, rinsed in PBS and probed over night in solution containing 2 μg/ml Concanavalin A, 0,05%Tween, 1 mM of CaCl2, MnCl2 and MgCl2. For detection of total protein the membranes were stained with 0.1% Naphthol Blue Black (Sigma) dissolved in 25% isopropanol and 10% acetic acid. P24 levels in cell culture supernatants were quantified using p24-ELISA  and CAT concentrations in cell lysates were quantified using the CAT ELISA kit (Roche).
Virus expression, precipitation of HIV-1 particles and immune EM
ACH-2 cells (8 × 105 cells/ml) were cultured with 100 nM 12-phorbol-13-myristate acetate (PMA) and with or without αHGA. Three days later the cell culture supernatants were collected, cleared by centrifugation at 300 × g for 10 min, passed through 0.45 μm filters and the particles were precipitated at 4°C for 48 h in 1:6 (v/v) with 40% poly ethylene glycol 6000 containing 0.667 M NaCl. The precipitated particles were allowed to sediment at 16,000 × g for 20 minutes at 4°C and the virus pellets were then dissolved in RIPA buffer. Sample preparation of hydrated ACH-2 cells for immunocytochemical analysis was performed as previously described using 10 nm colloidal gold labeling of anti-gp41 monoclonal antibody [17, 24]. Areas surrounding the infected cells were used for calculating the number of Au-labeled particles.
We thank Dr Robert Daniels for critical reading of the manuscript. We also thank the original donors and the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID for the cell lines HeLa-tat III from Dr William Haseltine and Dr. Ernest Terwilliger and ACH-2 from Dr Thomas Folks. We are grateful for the anti-gp41 antibody (Chessie 8) from Dr. George Lewis and the plasmid pNL4-3 from Dr Malcolm Martin. This work was supported by grants from the Swedish Medical Foundation (grant no. K2000-06X-09501-10B), Swedish International development Cooperation Agency, SIDA (grant no. HIV-2006-050) and by Tripep AB.
- Land A, Zonneveld D, Braakman I: Folding of HIV-1 envelope glycoprotein involves extensive isomerization of disulfide bonds and conformation-dependent leader peptide cleavage. Faseb J. 2003, 17: 1058-1067. 10.1096/fj.02-0811com.View ArticlePubMedGoogle Scholar
- Lu M, Blacklow SC, Kim PS: A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol. 1995, 2: 1075-1082. 10.1038/nsb1295-1075.View ArticlePubMedGoogle Scholar
- McCune JM, Rabin LB, Feinberg MB, Lieberman M, Kosek JC, Reyes GR, Weissman IL: Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988, 53: 55-67. 10.1016/0092-8674(88)90487-4.View ArticlePubMedGoogle Scholar
- Gomez C, Hope TJ: The ins and outs of HIV replication. Cell Microbiol. 2005, 7: 621-626. 10.1111/j.1462-5822.2005.00516.x.View ArticlePubMedGoogle Scholar
- Kilgore NR, Salzwedel K, Reddick M, Allaway GP, Wild CT: Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope. J Virol. 2003, 77: 7669-7672. 10.1128/JVI.77.13.7669-7672.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Ray N: Maraviroc in the treatment of HIV infection. Drug Des Devel Ther. 2009, 2: 151-161.PubMed CentralPubMedGoogle Scholar
- Dwek RA, Butters TD, Platt FM, Zitzmann N: Targeting glycosylation as a therapeutic approach. Nat Rev Drug Discov. 2002, 1: 65-75. 10.1038/nrd708.View ArticlePubMedGoogle Scholar
- Jacob GS: Glycosylation inhibitors in biology and medicine. Curr Opin Struct Biol. 1995, 5: 605-611. 10.1016/0959-440X(95)80051-4.View ArticlePubMedGoogle Scholar
- Tierney M, Pottage J, Kessler H, Fischl M, Richman D, Merigan T, Powderly W, Smith S, Karim A, Sherman J, et al: The tolerability and pharmacokinetics of N-butyl-deoxynojirimycin in patients with advanced HIV disease (ACTG 100). The AIDS Clinical Trials Group (ACTG) of the National Institute of Allergy and Infectious Diseases. J Acquir Immune Defic Syndr Hum Retrovirol. 1995, 10: 549-553. 10.1097/00042560-199512050-00008.View ArticlePubMedGoogle Scholar
- Jejcic A, Daniels R, Goobar-Larsson L, Hebert DN, Vahlne A: Small molecule targets Env for ER-associated protein degradation and inhibits HIV-1 propagation. J Virol. 2009, 83 (19): 10075-84. 10.1128/JVI.01700-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Abdurahman S, Vegvari A, Levi M, Hoglund S, Hogberg M, Tong W, Romero I, Balzarini J, Vahlne A: Isolation and characterization of a small antiretroviral molecule affecting HIV-1 capsid morphology. Retrovirology. 2009, 6: 34-10.1186/1742-4690-6-34.PubMed CentralView ArticlePubMedGoogle Scholar
- Balzarini J, Andersson E, Schols D, Proost P, Van Damme J, Svennerholm B, Horal P, Vahlne A: Obligatory involvement of CD26/dipeptidyl peptidase IV in the activation of the antiretroviral tripeptide glycylprolylglycinamide (GPG-NH(2)). Int J Biochem Cell Biol. 2004, 36: 1848-1859. 10.1016/j.biocel.2004.02.017.View ArticlePubMedGoogle Scholar
- Blot G, Janvier K, Le Panse S, Benarous R, Berlioz-Torrent C: Targeting of the human immunodeficiency virus type 1 envelope to the trans-Golgi network through binding to TIP47 is required for env incorporation into virions and infectivity. J Virol. 2003, 77: 6931-6945. 10.1128/JVI.77.12.6931-6945.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Dorfman T, Mammano F, Haseltine WA, Gottlinger HG: Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol. 1994, 68: 1689-1696.PubMed CentralPubMedGoogle Scholar
- Kim JT, Kim EM, Lee KH, Choi JE, Jhun BH, Kim JW: Leucine zipper domain of HIV-1 gp41 interacted specifically with alpha-catenin. Biochem Biophys Res Commun. 2002, 291: 1239-1244. 10.1006/bbrc.2002.6583.View ArticlePubMedGoogle Scholar
- Murakami T, Freed EO: Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail. J Virol. 2000, 74: 3548-3554. 10.1128/JVI.74.8.3548-3554.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Abacioglu YH, Fouts TR, Laman JD, Claassen E, Pincus SH, Moore JP, Roby CA, Kamin-Lewis R, Lewis GK: Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies. AIDS Res Hum Retroviruses. 1994, 10: 371-381. 10.1089/aid.1994.10.371.View ArticlePubMedGoogle Scholar
- Devito C, Levi M, Broliden K, Hinkula J: Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents. J Immunol Methods. 2000, 238: 69-80. 10.1016/S0022-1759(00)00141-1.View ArticlePubMedGoogle Scholar
- Clouse KA, Powell D, Washington I, Poli G, Strebel K, Farrar W, Barstad P, Kovacs J, Fauci AS, Folks TM: Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol. 1989, 142: 431-438.PubMedGoogle Scholar
- Terwilliger E, Proulx J, Sodroski J, Haseltine WA: Cell lines that express stably env gene products from three strains of HIV-1. J Acquir Immune Defic Syndr. 1988, 1: 317-323.PubMedGoogle Scholar
- Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986, 59: 284-291.PubMed CentralPubMedGoogle Scholar
- Schwartz S, Felber BK, Fenyo EM, Pavlakis GN: Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J Virol. 1990, 64: 5448-5456.PubMed CentralPubMedGoogle Scholar
- Horal P, Hall WW, Svennerholm B, Lycke J, Jeansson S, Rymo L, Kaplan MH, Vahlne A: Identification of type-specific linear epitopes in the glycoproteins gp46 and gp21 of human T-cell leukemia viruses type I and type II using synthetic peptides. Proc Natl Acad Sci USA. 1991, 88: 5754-5758. 10.1073/pnas.88.13.5754.PubMed CentralView ArticlePubMedGoogle Scholar
- Hoglund S, Su J, Reneby SS, Vegvari A, Hjerten S, Sintorn IM, Foster H, Wu YP, Nystrom I, Vahlne A: Tripeptide interference with human immunodeficiency virus type 1 morphogenesis. Antimicrob Agents Chemother. 2002, 46: 3597-3605. 10.1128/AAC.46.11.3597-3605.2002.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.