Isolation and characterization of a small antiretroviral molecule affecting HIV-1 capsid morphology
© Abdurahman et al; licensee BioMed Central Ltd. 2009
Received: 22 December 2008
Accepted: 08 April 2009
Published: 08 April 2009
Formation of an HIV-1 particle with a conical core structure is a prerequisite for the subsequent infectivity of the virus particle. We have previously described that glycineamide (G-NH2) when added to the culture medium of infected cells induces non-infectious HIV-1 particles with aberrant core structures.
Here we demonstrate that it is not G-NH2 itself but a metabolite thereof that displays antiviral activity. We show that conversion of G-NH2 to its antiviral metabolite is catalyzed by an enzyme present in bovine and porcine but surprisingly not in human serum. Structure determination by NMR suggested that the active G-NH2 metabolite was α-hydroxy-glycineamide (α-HGA). Chemically synthesized α-HGA inhibited HIV-1 replication to the same degree as G-NH2, unlike a number of other synthesized analogues of G-NH2 which had no effect on HIV-1 replication. Comparisons by capillary electrophoresis and HPLC of the metabolite with the chemically synthesized α-HGA further confirmed that the antiviral G-NH2-metabolite indeed was α-HGA.
α-HGA has an unusually simple structure and a novel mechanism of antiviral action. Thus, α-HGA could be a lead for new antiviral substances belonging to a new class of anti-HIV drugs, i.e. capsid assembly inhibitors.
During or concomitant with the HIV-1 release from infected cells, the Gag precursor protein (p55) is cleaved sequentially into matrix (MA/p17), capsid (CA/p24), nucleocapsid (NC/p7) and p6. Thus, proteolytic cleavage of p55 within the budded particle triggers a morphological change of the core which transforms it from a spherical  to a conical core structure consisting of approximately 1,500 p24 molecules [2–4]. The conical core formation not only results as a consequence of the proteolytic cleavage of p55 but also from substantial conformational changes and rearrangements of the p24  which is connected to one another through N-terminal hexamer and C-terminal dimer formations [5–8]. Acquisition of virion infectivity, reverse transcription, and subsequent dissociation of the capsid core are all critically dependent on just the right semi-stability of the capsid cone structure, which in turn is made up of multiple semi-stable non-covalent p24-p24 interactions . Thus, proper structural rearrangement of p24 after Gag cleavage is a crucial step and is a highly conserved feature in most retroviruses . This makes p24 of interest as a target for developing new antiviral drugs.
There are twenty-five approved drugs that belong to six different antiretroviral classes for the treatment of HIV-patients . The majority of these drugs control HIV-1 infection by targeting the two viral enzymes reverse transcriptase and protease . A 36 amino acid peptide binding to the transmembrane glycoprotein gp41 inhibiting the fusion of the viral envelope with the plasma membrane is also used [13, 14]. Two other classes of antiretroviral drugs, a CCR5 co-receptor antagonist entry inhibitor  and an integrase inhibitor [16, 17], have also recently been approved. Other drugs being developed include zinc finger inhibitors affecting the RNA binding of the nucleocapsid protein (NC, p7) [18, 19], and capsid maturation inhibitors [20–22].
We have previously shown that the tripeptide glycyl-prolyl-glycineamide (GPG-NH2) cleaved to G-NH2 by dipeptidyl peptidase CD26, present in both human and fetal calf serum, affects proper HIV-1 capsid assembly and infectivity [23–26]. Here we show that G-NH2 by itself does not affect HIV-1 replication, but displays antiviral effect only when converted to a metabolite by a yet uncharacterized enzyme present in porcine or bovine serum but not in human serum. The metabolite was identified as the small molecule α-hydroxy-glycineamide (α-HGA) having a molecular mass of only 90 Daltons, a molecule which we recently showed could inhibit HIV-1 replication .
The effect of serum on the antiviral activity of glycineamide (G-NH2)
To further test if G-NH2 was converted to the active antiviral substance by an enzyme present in porcine or calf serum, HIV-1-infected H9 cells were cultured in medium containing normal PS or boiled PS (BPS). The cells were then treated with 100 μM G-NH2, with the DS at a 1/10 dilution or were left untreated. A typical experiment is depicted in Fig. 1C. Infected cells without any test compound and cultured in medium containing PS or BPS served as controls (Fig. 1C, Untreated). In contrast to what was observed in cells cultured with BPS and treated with DS, G-NH2 showed no antiviral activity in cells cultured with medium containing BPS (Fig. 1C). DS, however, repeatedly inhibited HIV replication regardless of the infected cells being cultured in the presence of PS or BPS (Fig. 1C, DS).
HPLC analysis of the unknown metabolite of G-NH2
Identification of metabolite-X (Met-X) by NMR
Based on the NMR analysis (1H NMR, coupled 1H-13C NMR, and 2D 1H-15N HSQC NMR) one of the possible structures of the unknown compound Met-X was determined as α-hydroxy-glycineamide (α-HGA).
Comparison of Met-X with α-HGA
Both α-HGA and Met-X treatment at concentrations corresponding to 10 μM resulted in similarly significant changes in virion core morphology (data not shown). Pleomorphic virus particles with distorted, irregular packing of aberrant core structures, partly devoid of dense core material, were seen. Virions having double core structures and occasionally viral cores bulging off from viral envelope were also observed.
Anti-HIV activities of α-HGA and other related test compounds
In this study, we were able to identify, isolate and characterize a novel antiretroviral glycineamide (G-NH2)-derived metabolite (Met-X) obtained after incubation of G-NH2 in porcine (PS) or fetal calf (FCS) serum. Dialysis of G-NH2 against FCS at 4°C or boiled PS gave no Met-X, indicating that the enzyme responsible for converting G-NH2 to Met-X is temperature-dependent and heat-sensitive. Furthermore, unlike in FCS or in PS, G-NH2 could not be converted to Met-X when incubated in human serum at 37°C, suggesting that humans lack either the active enzyme or a necessary co-factor. Interestingly, humans seem to share this inability to convert G-NH2 with mice, rats and birds. However, other species such as non-human primates can readily convert G-NH2 to Met-X.
Here we characterized Met-X by NMR and this unknown compound was identified as α-hydroxy-glycineamide (α-HGA). In addition, with NMR, HPLC and capillary electrophoresis analysis of Met-X and the synthesized α-hydroxy-glycineamide the same chemical structure was determined. Therefore, it is very likely that these two compounds are identical chemical entities. The antiviral activity of the Met-X purified by cation exchange chromatography and identified as α-HGA by NMR was also confirmed both in H9 cells infected with the HIV-1 SF-2 virus and chronically infected ACH-2 cells. Consistent with previous reports on GPG-NH2 and G-NH2, the addition of Met-X or α-HGA to the culture medium of infected cells resulted in HIV-1 particles with aberrant core morphology.
The reduction in infectivity was not due to cytotoxicity, since neither Met-X nor α-HGA at concentrations up to 1,000 μM has any effect on the cell viability of PBMC or a number of other cell lines . Furthermore, α-HGA had no mitogenic activity against human PBMCs at concentrations of up to 2,000 μM.
Two other compounds that inhibit or interfere with the HIV-1 capsid (p24/CA) maturation or assembly have previously been reported [20, 21, 28]. PA-457 [20, 22], is a compound that binds to the proteolytic cleavage site of the p24 precursor (p25/CA-SP1) and thereby affects its maturation to p24. α-HGA does not affect the proteolytic processing of p25 . The other compound reported by Tang et al. describes the binding of N-(3-chloro-4-methylphenyl)-N'-2-(5-[dimethylamino-methyl]-2-furyl)-methylsulfanyl-ethyl urea (CAP-1) to the N-terminal domain of p24 . CAP-1 affects HIV-1 capsid cone formation but did not prevent virus release . However, α-HGA, which is comparatively a small molecule, specifically affected HIV-1 CA assembly and cone formation, possibly by binding to the hinge region between the N- and C-terminal domains of p24 . A 12-mer alpha-helical peptide (CAI) was also shown to interfere with p24 dimerization, but not with HIV-1 replication in cell culture due to the lack of cell penetration [28, 29]. However, more recently a structure-based rational design was used to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP) displaying antiviral activity .
In this study, we have reported that G-NH2 by itself has no anti-viral activity but is converted to a small (molecular mass 90) anti-retroviral compound when incubated in some animal sera. The new compound was identified as α-HGA, which has an unusually simple structure and a novel mechanism of antiviral action. Thus, α-HGA could be a lead for new antiviral substances belonging to a new class of anti-HIV drugs, i.e. capsid assembly/maturation inhibitors.
Cells, media and reagents
Peripheral blood mononuclear cells (PBMC), H9 and ACH-2 cells were cultured in complete RPMI-1640, and HeLa-tat cells was cultured in complete DMEM medium supplemented with 10% serum and antibiotics. Porcine and human sera (PS and HS) (Biomeda), fetal calf serum (FCS; Invitrogen) oxamic acid and oxamide (Sigma) were used. Glycineamide (G-NH2) and α-hydroxy glycineamide (α-HGA; manufactured to order by Pharmatory Oy, Oulu, Finland) were kindly provided by Tripep AB, Stockholm, Sweden. 13C2/15N-labeled Fmoc-glycine (Isotech) was transformed to 13C2/15N-labeled G-NH2 by Fmoc peptide synthesis. 13C2/15N-labeled G-NH2 was dialyzed against PS or FCS to produce 13C2/15N-labeled metabolite of G-NH2 which will be referred to as Met-X (Fig. 2A, B and 2C).
Inhibition of viral infectivity
HIV-1 stock of SF-2 from H9 cells was prepared as described previously , and 50% tissue culture infectious dose (TCID50) was determined. H9 cells were infected with SF-2 at 100 TCID50 by incubating for 2 hours at 37°C. The cells were then pelleted, washed and resuspended in complete RPMI medium containing HS or PS, and the test compound was added. Cells were cultured for 11 days, and the growth medium was changed seven days post-infection. The HIV-1 p24 antigen contents were assayed at day 7 and 11 post infection essentially as described elsewhere  (see below). For RT-assay, the manufacturer's procedure was followed (Cavidi Tech AB, Uppsala, Sweden).
HPLC analysis and purification of Met-X
13C2/15N-labeled or unlabeled G-NH2 was enzymatically transformed to Met-X by dialysis against FCS or PS at 37°C. Dialysis was performed with 10 ml of serum in a dialysis tubing (5 kD MWCO) that was prewashed by dialyzing 5× against PBS under constant stirring. After 24 hours, the dialysis solution (DS) containing Met-X was analyzed by injecting it onto a 250 × 10 mm, 5 μm cationic ion-exchange column, Theoquest Hypersil SCX, (Thermo), with 90% 0.1 M KH2PO4pH 4.5/10% acetonitrile as mobile phase at isocratic flow. The absorbance was measured at 206 nm. Lyophilized 13C2/15N-labeled Met-X was also analyzed as above except that a mobile phase of 90% 2.5 mM formic acid pH 3/10% acetonitrile was used. All HPLC chromatograms were compared using retention time as an indicator. Once the structure of Met-X was indicated by NMR (see below) to be α-HGA, the HPLC properties of Met-X and chemically synthesized α-HGA were analyzed under the same conditions.
Compound characterization by NMR spectroscopy
The HPLC peak fraction containing 13C2/15N-labeled Met-X was isolated, lyophilized, and analyzed with NMR. Due to the low natural abundance of 13C- and 15N-nuclei, a commercially available labeled glycine with two 99% 13C- and one 99% 15N-isotopes (Fig. 4A) was used as starting material. The labeled glycine was transformed into G-NH2 (Fig. 4B) which was dialyzed against PS or FCS to obtain labeled Met-X. The 13C/15N-labeled Met-X was purified by HPLC and concentrated by lyophilization before being analyzed with NMR. The samples were analyzed on a Bruker DPX 300 MHz, a Jeol Eclipse+500 MHz and Bruker DMX 600 MHz spectrometers.
Comparison of Met-X with α-HGA by capillary electrophoresis
Capillary electrophoresis experiments were carried out at 20°C with a BioFocus 3000 system (Bio-Rad) which was equipped with a fast scanning UV-Vis detector. Fused silica tubing (50 and 365 μm inner and outer diameter, respectively) was purchased from MicroQuartz and cut to a length of 23 cm (with 18.5 cm effective length). Sodium phosphate buffer (0.05 M) at pH 7.4 was used as a background electrolyte. The polarity was set from positive to negative (with the detection point closer to the cathode). The capillary was flushed with the buffer for 1 minute before each run. The Met-X solution obtained from the dialysis procedure was diluted two fold in the buffer solution and filtered through a syringe disc filter (Ultra free-MC 5 000 NMWL, Millipore) prior to injection by pressure (3 psi·s). α-HGA was dissolved in the buffer at 10 mM concentration and injected by pressure (3 psi·s). The applied voltage was 10 kV in all experiments resulting in 50 μA current.
p24-ELISA of infected cell culture supernatants was performed essentially as described elsewhere . Briefly, rabbit anti-p24 coated micro-well plates (MWP) were blocked with 3% BSA in PBS at 37°C for 30 minutes. Supernatants from infected cells were added to the plates, followed by incubated at 37°C for 1 hour. The MWPs were washed three times, and biotinylated anti-p24 antibody (1:1 500) was added. One hour after incubation, the MWPs were washed and incubated with HRP-conjugated streptavidine (1:2 000) for 30 minutes. Finally, the MWPs were washed and detected by adding the substrate o-Phenylenediamine Dihydrochloride (Sigma). Recombinant p24 at fixed concentrations was used as a standard. The plates were read in a Labsystems multiscan MS spectrometer. For RT-ELISA, the manufacturer's procedure was followed (Cavidi Tech).
Anti-HIV activities of α-HGA and other related test compounds
The antiviral activity of α-HGA and some other structurally related compounds was tested in infected H9 cells in the presence of FCS at drug concentrations of 100 μM. H9 cells were infected as described above and cultured in medium containing oxamic acid, oxamide, α-methoxy glycineamide and Boc-α-methoxy glycineamide.
Conversion of G-NH2 to α-HGA by different sera
The sera from different animal species were diluted 10-fold in 50 mM potassium phosphate buffer pH 8.0. To 100 μl (10% serum) were added 0.1 μCi [14C]G-NH2 (radiospecificity: 56 mCi/mmol), and the samples were incubated for 1, 6 or 24 hours at 37°C. At these time points, 200 μl cold methanol was added, and the samples were left on ice for another 15 minutes. After centrifugation at 15,000 rpm, the supernatants were subjected to HPLC analysis using a SCX-partisphere column (Whatman). The following gradient was used to separate G-NH2 and Met-X (α-HGA): 5 mM buffer B (5 mM NH4H2PO4 pH 3.5) (10 minutes); linear gradient to 83% buffer C (0.3 M NH4H2PO4 pH 3.5) (6 minutes); equilibration 83% buffer C (2 minutes); linear gradient to 100% buffer B (6 minutes); equilibration 100% buffer B (6 minutes). The retention times of G-NH2 and α-HGA were 12 and 2 minutes, respectively.
Transmission electron microscopy (TEM)
H9 cells were infected with HIV-1 SF-2 at 100 TCID50 by incubating for 2 hours at 37°C. After seven days of incubation, medium containing Met-X or α-HGA was added. Cells were cultured for an additional four days, and progeny virus was analyzed by transmission electron microscopy (TEM). The HIV-1-infected H9 cells were fixed freshly upon embedding in epon, essentially as described before . Sections were made approximately 60 nm thick to allow accommodation of the volume of the core structure parallel to the section plane. Duplicate samples were used and minimal beam dose technique was employed throughout. Evaluation of morphology was done with series of electron micrographs to depict different categories of virus morphology. Similar results were also obtained with chronically infected ACH-2 cells induced to replicate HIV-1.
We thank Pia Österwall and Sung Oun Stenberg for help with the dialysis and antiviral assay. We thank the original donors of the following reagents that were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: the CD4+ cell lines H9  and ACH-2 , and the CD4- cell line HeLa-tat-III . This work was supported by grants from the K.U. Leuven (GOA-05/19), Swedish Research Council (grant no. K2000-06X-09501-10B), Swedish International development Cooperation Agency, SIDA (grant no. HIV-2006-050) and Tripep AB.
- Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM, Johnson MC: The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol. 2004, 11: 672-675.View ArticlePubMedGoogle Scholar
- Lanman J, Lam TT, Barnes S, Sakalian M, Emmett MR, Marshall AG, Prevelige PE: Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. J Mol Biol. 2003, 325: 759-772.View ArticlePubMedGoogle Scholar
- Li S, Hill CP, Sundquist WI, Finch JT: Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature. 2000, 407: 409-413.View ArticlePubMedGoogle Scholar
- Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW, Sowder RC, Barsov E, Hood BL, Fisher RJ, et al: Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol. 2006, 80: 9039-9052.PubMed CentralView ArticlePubMedGoogle Scholar
- Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI, Hill CP: Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell. 1996, 87: 1285-1294.View ArticlePubMedGoogle Scholar
- Momany C, Kovari LC, Prongay AJ, Keller W, Gitti RK, Lee BM, Gorbalenya AE, Tong L, McClure J, Ehrlich LS, et al: Crystal structure of dimeric HIV-1 capsid protein. Nat Struct Biol. 1996, 3: 763-770.View ArticlePubMedGoogle Scholar
- Berthet-Colominas C, Monaco S, Novelli A, Sibai G, Mallet F, Cusack S: Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab. Embo J. 1999, 18: 1124-1136.PubMed CentralView ArticlePubMedGoogle Scholar
- Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, Wang H, McCutcheon JP, Sundquist WI, Hill CP: Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science. 1997, 278: 849-853.View ArticlePubMedGoogle Scholar
- Forshey BM, von Schwedler U, Sundquist WI, Aiken C: Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol. 2002, 76: 5667-5677.PubMed CentralView ArticlePubMedGoogle Scholar
- Mortuza GB, Haire LF, Stevens A, Smerdon SJ, Stoye JP, Taylor IA: High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature. 2004, 431: 481-485.View ArticlePubMedGoogle Scholar
- De Clercq E: The design of drugs for HIV and HCV. Nat Rev Drug Discov. 2007, 6: 1001-1018.View ArticlePubMedGoogle Scholar
- De Clercq E: Emerging anti-HIV drugs. Expert Opin Emerg Drugs. 2005, 10: 241-273.View ArticlePubMedGoogle Scholar
- Root MJ, Steger HK: HIV-1 gp41 as a target for viral entry inhibition. Curr Pharm Des. 2004, 10: 1805-1825.View ArticlePubMedGoogle Scholar
- Yu H, Tudor D, Alfsen A, Labrosse B, Clavel F, Bomsel M: Peptide P5 (residues 628–683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection. Retrovirology. 2008, 5: 93-PubMed CentralView ArticlePubMedGoogle Scholar
- Groeschen HM: Novel HIV treatment approved. Am J Health Syst Pharm. 2007, 64: 1886-View ArticlePubMedGoogle Scholar
- Traynor K: Integrase inhibitor gains FDA approval. 2007, 64: 2310-Google Scholar
- Anker M, Corales RB: Raltegravir (MK-0518): a novel integrase inhibitor for the treatment of HIV infection. 2008, 17: 97-103.Google Scholar
- Darlix JL, Garrido JL, Morellet N, Mely Y, de Rocquigny H: Properties, functions, and drug targeting of the multifunctional nucleocapsid protein of the human immunodeficiency virus. Adv Pharmacol. 2007, 55: 299-346.View ArticlePubMedGoogle Scholar
- Musah RA: The HIV-1 nucleocapsid zinc finger protein as a target of antiretroviral therapy. Curr Top Med Chem. 2004, 4: 1605-1622.View ArticlePubMedGoogle Scholar
- Li F, Goila-Gaur R, Salzwedel K, Kilgore NR, Reddick M, Matallana C, Castillo A, Zoumplis D, Martin DE, Orenstein JM, et al: PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc Natl Acad Sci USA. 2003, 100: 13555-13560.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang C, Loeliger E, Kinde I, Kyere S, Mayo K, Barklis E, Sun Y, Huang M, Summers MF: Antiviral inhibition of the HIV-1 capsid protein. J Mol Biol. 2003, 327: 1013-1020.View ArticlePubMedGoogle Scholar
- Smith PF, Ogundele A, Forrest A, Wilton J, Salzwedel K, Doto J, Allaway GP, Martin DE: Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-o-(3',3'-dimethylsuccinyl)betulinic acid (bevirimat) against human immunodeficiency virus infection. Antimicrob Agents Chemother. 2007, 51: 3574-3581.PubMed CentralView ArticlePubMedGoogle Scholar
- Su J, Andersson E, Horal P, Naghavi MH, Palm A, Wu YP, Eriksson K, Jansson M, Wigzell H, Svennerholm B, Vahlne A: The nontoxic tripeptide glycyl-prolyl-glycine amide inhibits the replication of human immunodeficiency virus type 1. J Hum Virol. 2001, 4: 1-7.PubMedGoogle 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.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.View ArticlePubMedGoogle Scholar
- Andersson E, Horal P, Jejcic A, Hoglund S, Balzarini J, Vahlne A, Svennerholm B: Glycine-amide is an active metabolite of the antiretroviral tripeptide glycyl-prolyl-glycine-amide. Antimicrob Agents Chemother. 2005, 49: 40-44.PubMed CentralView ArticlePubMedGoogle Scholar
- Abdurahman S, Vegvari A, Youssefi M, Levi M, Hoglund S, Andersson E, Horal P, Svennerholm B, Balzarini J, Vahlne A: Activity of the small modified amino acid alpha-hydroxy glycineamide on in vitro and in vivo human immunodeficiency virus type 1 capsid assembly and infectivity. Antimicrob Agents Chemother. 2008, 52: 3737-3744.PubMed CentralView ArticlePubMedGoogle Scholar
- Sticht J, Humbert M, Findlow S, Bodem J, Muller B, Dietrich U, Werner J, Krausslich HG: A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol. 2005, 12: 671-677.View ArticlePubMedGoogle Scholar
- Ternois F, Sticht J, Duquerroy S, Krausslich HG, Rey FA: The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor. Nat Struct Mol Biol. 2005, 12: 678-682.View ArticlePubMedGoogle Scholar
- Zhang H, Zhao Q, Bhattacharya S, Waheed AA, Tong X, Hong A, Heck S, Curreli F, Goger M, Cowburn D, et al: A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J Mol Biol. 2008, 378: 565-580.PubMed CentralView ArticlePubMedGoogle Scholar
- Vahlne A, Horal P, Eriksson K, Jeansson S, Rymo L, Hedstrom KG, Czerkinsky C, Holmgren J, Svennerholm B: Immunizations of monkeys with synthetic peptides disclose conserved areas on gp120 of human immunodeficiency virus type 1 associated with cross-neutralizing antibodies and T-cell recognition. Proc Natl Acad Sci USA. 1991, 88: 10744-10748.PubMed CentralView ArticlePubMedGoogle 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.PubMed CentralView ArticlePubMedGoogle Scholar
- Popovic M, Sarngadharan MG, Read E, Gallo RC: Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984, 224: 497-500.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
- Rosen CA, Haseltine WA, Lenz J, Ruprecht R, Cloyd MW: Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences. J Virol. 1985, 55: 862-866.PubMed CentralPubMedGoogle Scholar
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