Virus Infection in Human Lymphocytes and Macrophages - NCBI

Vol. 68, No. 9

JOURNAL OF VIROLOGY, Sept. 1994, p. 5714-5720

0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Multibranched V3 Peptides Inhibit Human Immunodeficiency Virus Infection in Human Lymphocytes and Macrophages NOUARA YAHI,' JACQUES FANTINI,' KAMEL MABROUK,' CATHERINE TAMALET,2 PHILIPPE DE MICCO,2 JURPHAAS VAN RIETSCHOTEN,' HERVE ROCHAT,' AND JEAN-MARC SABATIERI*

CNRS URA 1455, Laboratoire de Biochimie, Ing6ni6rie de Proteines, Faculte de Medecine Secteur Nord, 13916 Marseille Cedex 20,' and Laboratoire de Virologie, CHRU de la Timone, 13005 Marseille,2 France Received 16 March 1994/Accepted 20 June 1994

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gpl20 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 ,uM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 ,uM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients. loop, elicited type-specific responses (29). The rationale for using V3 SPCs instead of monomeric V3 peptides as anti-HIV compounds is that they may have enhanced ligand avidity due to the high local density of binding sites for the potential V3 recognition sites. A similar strategy was recently developed by Nomizu et al. (31), who found that multimerization of a short peptide derived from the laminin Bi chain resulted in a large increase in antitumor properties.

The third variable region of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gpl20 (V3 loop) appears to play a key role in HIV-1 infection and pathogenesis (25). This domain was reported to be essential for virus infectivity and tropism (17, 25) and is the major immunodominant epitope for the generation of neutralizing antibodies (27). However, because of the variability of the V3 loop among HIV-1 isolates, neutralization by anti-V3 antibodies is generally isolate specific (32). The development of vaccines and immunotherapy for HIV is therefore difficult (4). The V3 loop of HIV-1 gpl20 does not seem to be involved in the binding of gpl20 to its main cellular receptor, CD4, a 55-kDa glycoprotein on the surface of a subclass of T lymphocytes and on macrophages (20). Nevertheless, several studies indicate that the V3 loop may be involved in the postbinding events necessary for viral entry into the cells (14, 25). Several attempts have been made to inhibit HIV infection by V3 loop-related peptides. The results have been various and contradictory. For instance, De Rossi et al. (10) found that some V3 peptides enhanced viral infectivity while Nehete et al. (30), using other V3 peptides, found inhibition of HIV-1 infection in lymphocytes. Recently, we reported that multimeric forms of the V3 consensus motif GPGRAF displayed a potent anti-HIV activity in vitro while the monomer GPGRAF was not active (12). Such synthetic polymeric constructions (SPCs), consisting of an uncharged poly-Lys core matrix (which accounts for about 15% of the total weight), have been used as immunogens (36) and, in the case of the HIV-1 V3 *

MATERIALS AND METHODS Peptide synthesis. Chemical synthesis of SPCs was performed by the solid-phase technique (23). The peptide chains were elongated stepwise on 4-(oxy-methyl)-phenylacetamidomethyl resin by using optimized t-butyloxycarbonyl-benzyl chemistry as previously described (33). Amino acid analysis of the purified SPCs agreed with the deduced amino acid ratios. [GPGRAF]8-SPC (see below for nomenclature) was further characterized by electrospray mass spectrometry (experimental Mr, 5,671.1; deduced Mr, 5,671.6). The consensus sequence of the HIV-1 V3 loop was obtained from LaRosa et al. (19). The nomenclature used for SPCs gives the peptide sequence in brackets, followed by the index of polymerization and the term SPC. For instance, [GPGRAF]8-SPC is an SPC containing eight GPGRAF motifs, i.e., (GPGRAF)8-(K)4-(K)2-K-PA. [GPG(D-Arg)AF]8-SPC is a derivative of [GPGRAF]8-SPC containing a D-Arg residue in the fourth position of the motif. In [GPGRAF]8-SPCD, all residues in the motif were D-amino acids. Two irrelevant SPCs were used as negative controls: [PPPYVEPTlTQC]4-SPC was derived from region 124 to 135 of human T-cell leukemia virus type II Gag, and [GKCMNRK]8-SPC was from basic region 27 to 33 of

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kaliotoxin. Stock solutions of peptides (10-' M) were filter sterilized on 0.22-,um-thick low-protein-binding membranes (Costar) and stored aliquoted at -20GC. Cell culture. Human peripheral blood mononuclear cells (PBMCs) were obtained from a healthy donor, activated with phytohemagglutinin (PHA; Sigma), and cultivated in RPMI 1640 containing 10% fetal calf serum with interleukin-2 (Biotest) (complete medium) as described previously (38). Human mononuclear cells were isolated from leukaphoresis units enriched for monocytes by Ficoll-Hypaque density separation. Macrophages were purified by adherence to plastic in RPMI 1640 supplemented with 10% fetal calf serum and 5% human AB serum. The adherent cells were cultured for 5 days in the presence of granulocyte macrophage-colony-stimulating factor (1 ng/ml) and were positive for the human macrophage marker (clone 25F9, which recognizes an 86-kDa protein expressed by cultivated blood monocytes [Boehringer Mannheim]). Human T-lymphoblastoid cell lines CEM and CEMx174 were cultured in RPMI 1640 supplemented with 10% fetal calf serum. Viruses. Laboratory strains HIV-1(LAI) (2), HIV-1(NDK) (34), and HIV-2(ROD) (8) were produced in CEM cells. Macrophage-tropic HIV-1(89.6) (9) was produced in CEMx174 cells as previously described (35). Cultured supernatants from infected cells were harvested at the peak of viral production, filtered on 0.22-p.m-thick filters, and stored at -80°C. Identities of the viruses were verified by PCR and/or radioimmunoprecipitation assay (39). Fresh primary isolates were obtained by cocultivation of PBMCs from HIV-1- or HIV-2-infected patients with PHA-activated, fresh, normal PBMCs. In vitro zidovudine sensitivity was determined by a cell-free isolate sensitivity assay (5). Assay of HIV-i-induced syncytium formation. 8E5 cells (5 x 104) chronically infected with a reverse transcriptase (RT)deficient HIV-1(IIIB) isolate (13) were cocultivated with 15 x 104 PHA-stimulated PBMCs in the presence of various peptides in 96-well microplates as previously described (16). Syncytium formation was determined after 24 h of incubation in the continued presence of the peptides. HIV-i and HIV-2 infection assays. Samples of 6 x 106 PBMCs or 4 x 106 CEM cells were exposed to HIV-1 or HIV-2 (multiplicity of infection, 0.001 50% tissue cultureinfective doses [TCID50] per cell) for 1 h at 37°C in the presence of different SPC concentrations. After thorough washing, cells were cultured in their respective media with the corresponding SPC. The state of infection was assessed by HIV-1 p249ag measurements (Dupont kit; cutoff, 10 pg/ml) in cell-free supernatants. The peptides did not interfere with p249ag measurements with this kit. When indicated (especially for HIV-2 isolates), infection was evaluated by detection of RT activity after pelleting the virus by ultracentrifugation as described previously (38). Under these conditions, the SPCs tested did not interfere with the RT assay. Human macrophages (5 x 105 cells) were treated for 45 min with different SPC concentrations and subsequently exposed to macrophage-tropic isolate HIV-1(89.6) at a multiplicity of infection (1 TCID50 per cell) which induced the formation of numerous large syncytia in macrophage cultures. After thorough washing, the cells were refed with SPC-containing medium and analyzed for HIV-1 p249ag production and syncytium formation. HIV-1 infectivity assay. [GPGRAF]8-SPC was evaluated for an inhibitory effect on HIV-1 infectivity by measuring residual viral infectivity after incubation of HIV-1(NDK) with the multibranched peptide (10-4 and 10-5 M) for 1 h at 37°C. At the end of the incubation, the virus was ultracentrifuged at

V3 PEPTIDES INHIBIT HIV INFECT'ION

5715

TABLE 1. Inhibition of HIV-1-induced cell fusion by V3 SPCsa Syncytium formation or toxicity at peptide concn of:

Peptide

0.5 ,uM

5 pM

50 uM

[GPGRAF]8-SPC [IGPGRAF]8-SPC

+++ + +++

+++ -

[GPGRA]8-SPC

+++

[GPGR]8-SPC

[GPGRAF]8-SPCD

+++ + ++ ++++

+++ +++ +++

[Ac-GPGRAF]8-SPC

+++

+++

[RKSIHIGPGRAFYT]4-SPC [RKSIHKGPGRAFYT]4-SPC [RKSIHTGPGRAFYT]4-SPC RAFVTIGK [RAFVTIGK]8-SPC [PPPYVEPTITQC]4-SPC

+ ++ + ++ + ++ +++ +++ +++

-

GPGRAF

[GPG(D-Arg)AF]8-SPC

+ +++ +++

+++ +++ +++

T +++ -

T T T T T

[GKCMNRK]8-SPC +++ +++ +++ a HIV-1-expressing cells chronically infected with an RT-deficient HIV1(IIIB) isolate were cocultivated with PHA-stimulated PBMCs in the presence of the peptides indicated, as described in Materials and Methods. The number of syncytia was determined after 24 h of incubation in the continued presence of the peptides. Symbols: + + +, number of syncytia present in the well was similar to that in control untreated wells (35 to 40 syncytia per well); -, total absence of syncytia in the well; ±, presence of 1 to 3 syncytia in the well. SPCs by themselves did not induce syncytium formation in PBMCs or CEMx174 or 8E5 cells. Toxicity (T) was evaluated by the MTT assay after 24 h of incubation.

100,000 rpm (Beckman TL100) for 7 min and rinsed twice in RPMI 1640. The virus was then titrated by dilution in CEM cells. Infection was monitored by measurement of p249ag production, and the number of TCID50s was determined. Cytotoxicity studies. Viability of lymphocytes (CEM or PBMCs) in the presence of SPCs was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells in 24-well microplates were incubated for an appropriate period in the absence or presence of SPCs at 37°C in duplicate. MTT (Sigma) was dissolved in phosphatebuffered saline at 5 mg/ml (stock solution). A 10-,ul volume of MTT stock solution was added to 100 ,ul of cell suspension and incubated at 37°C for 3 h in 96-well microplates. At the end of the incubation, the cells were rinsed and resuspended in ethanol to dissolve the dark blue formazan salts, and the optical density was measured at a wavelength of 570 nm. RESULTS Blockade of HIV-induced cell fusion by V3 SPCs. Syncytium formation between HIV-1-infected cells and uninfected CD4+ cells involves an interaction between the CD4 receptor and the HIV-1 surface envelope glycoprotein (7). This process is blocked by soluble CD4 and by anti-CD4 and anti-V3 antibodies (25). To document the possible effect of V3 SPCs on the formation of syncytia, we used a cell-to-cell assay based on the fusion between CEM-derived cell clone 8E5 chronically infected with HIV-1(IIIB) and PHA-stimulated human PBMCs (Table 1 and Fig. 1). 8E5 cells, which produce noninfectious HIV-1 particles because of a point mutation in the RT gene, express HIV-1 envelope glycoproteins and produce syncytia when mixed with CD4+ lymphocytes (16). Syncytium formation in this test was blocked by anti-CD4 antibodies OKT4A (anti-CDR2) and 13B8-2 (anti-CDR3), consistent with previous reports (3, 37). Under these experimental conditions, [GPGRAF]8-SPC (see Materials and Methods for SPC nomenclature) at a concentration as low as 0.5 ,uM induced a

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