AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 7, Number 10, 1991 Mary Ann Liebert, Inc., Publishers

N-Terminal Residues 105-117 of HIV-1 gpl20 Are Not Involved in CD4 Binding W. EL

AHMAR,1

P.

POUMBOURIOS,1

DA.

McPHEE,2 and B.E. KEMP1

ABSTRACT

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gpl20 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gpl20-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp 120, is likely to be due to indirect structural changes in gpl20 rather than representing an integral part of the CD4 binding domain.

INTRODUCTION

BINDING (HIV-1) envelope glycoprotein gpl20

OF THE HUMAN IMMUNODEFICIENCY vims

type 1

CD4 is the first ' step in the infection of susceptible cells. '2 Several groups have mapped the respective domains of gpl20 and CD4 that mediate this binding event. Mutational analyses of the CD4 molecule have indicated that domain 1 of CD4 is primarily involved in gpl20 binding, since the gpl20-CD4 interaction is adversely affected by the introduction of single or double amino acid substitutions to the sequence encompassed by residues 38to

67.3"9

The CD4 binding site has been mapped to the carboxyl terminal domain of gpl20.1013 Two monoclonal anti-gpl20 antibodies which block the gpl20-CD4 interaction have epitopes in the sequences Gln-422-Lys-432 and Phe-423-Trp427, respectively."12 Furthermore, introduction of deletion mutations or substitutions into this sequence resulted in a marked reduction in CD4 binding, consistent with this carboxyl terminal region containing the CD4 binding domain." Amino acid insertions at positions 362 and 42710 and amino acid substitutions at Thr-257, Asp-368, and Glu-37013 also affected CD4 binding, indicating that this carboxyl terminal domain may also be involved in CD4-binding. On the other hand, extensive deletion of amino terminal residues from gpl20,14 deletion of

residues 38-46 or 82-95 of gpl60,15 as well as deletion of residues 108-116 of gpl2016 all resulted in loss of CD4-binding

ability. Whether these deletions directly modify the CD4 binding site or whether they result in conformational changes in gpl20 that lead to the loss of CD4 binding remains to be

determined. We have previously found that a synthetic peptide gp 120( 105117), corresponding to the conserved T-cell epitope,'7 has antiviral activity.18 Since deletion of a corresponding region (108-116) from gpl20 was reported to destroy CD4 binding, we were prompted to investigate whether this antiviral peptide acts as an inhibitor of gpl20-CD4 association, but found that this was not the case. Furthermore, an antibody to the peptide gp 120(99-119) which also recognized intact gpl20, did not inhibit the binding of gp 120 to CD4, indicating that this region is not directly involved in CD4 binding.

MATERIALS AND METHODS

Synthetic peptides Synthetic peptides were synthesized using the Merrifield an Applied Biosystems Model 430 synthesizer as procedure with described. '9 The synthetic peptides gp 120(99-119), gp 120( 103117) and gp 120(105-117) sequence sperm whale

were

derived from the HIV-l(BRU) The

"DMVEQMHEDIISLWDQSLKPC119.20

myoglobin peptide 105EFIFEAIIHVLHSRHPC121

(SWM) was used as a control.

'St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. 2AIDS Cellular Biology Laboratory, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Fairfield, Victoria 3078, Australia.

855

EL AHMAR ET AL.

856 Recombinant proteins Purified recombinant

gpl20 (rgpl20)

of the HIV-1 (SF2)

strain, residues (31-509) was a kind gift of Dr. N. L. Haigwood

(Chiron Corp, Emeryville, USA). It should be noted that in the HIV-1 (SF2) strain, Gin replaces His at position 105. Soluble recombinant CD4 (rCD4) lacking the cytoplasmic and transmembrane domains was a kind gift of Dr. Kenneth Powell (Wellcome Research Laboratories, Beckenham, UK).

temperature). Plates were washed, individual wells excised, and radioactivity determined by solid-phase scintillation spectroscopy. In inhibition assays, rgpI20 was preincubated with serial dilutions of sheep antiserum for 2 h at room temperature prior to its addition to radioiodinated rCD4. Conversely, radioiodinated rCD4 was preincubated with serial dilutions of synthetic pepanti-CD4 monoclonal antibodies for 6 h at temperature prior to its addition to rgpl20. tides

or

Antibody production

RESULTS

Synthetic peptide gp 120(99-119) was coupled to soybean trypsin inhibitor using A'-succinimidyl-3(2-pyridithic) propionate (SPDP). Sheep were immunized intramuscularly on each hind flank with 1 mg of peptide conjugate in complete Freund's adjuvant followed by 5 booster injections at 28-day intervals (1 mg conjugate in incomplete Freund's adjuvant). Immunoglobulin G was isolated from the sheep anti-gp 120(99-119) serum (S15) by affinity chromatography on protein G-sepharose (Pharmacia, Uppsala, Sweden) as recommended by the supplier. Polyclonal human anti-gp 120 IgG was purified from pooled HIV-1+ human plasma using an affinity column comprising rgpl20 coupled to CNBr-activated Sepharose-CL4B (Pharmacia). Anti-CD4 monoclonal antibodies OKT4 and OKT4A were obtained from Ortho Diagnostic Systems Inc., Raritan, NJ.

Enzyme-linked immunosorbent assay (ELISA) Synthetic peptides (50 p.1 of 10 u-g/ml in phosphate-buffered

saline, PBS) or rgpl20 (50 p.1 of 5 p.g/ml in PBS) were coated onto flat-bottomed microtiter ELISA plates (Nunc Immunopiate Maxisorb) for 2 h. The wells were then blocked with bovine serum albumin (BSA) solution (100 p.1 of 10 mg/ml in PBS) for 1 h at room temperature. Serial log2 dilutions of S15 were added to the antigen-coated plates and left at room temperature for 6 h. Plates were developed with horseradish peroxidase-conjugated donkey anti-sheep IgG (Silenus Labs, Melbourne, Australia) and 2,2'-azino-èw(3-ethylbenzthiazoline-sulfonic acid).

Radioiodination of proteins

The specificity of S15 was determined in EIA (Fig. 1). A 1:800 dilution of the antiserum resulted in 50% of maximal binding to peptides gp 120( 103-117) and gp 120(105-117) while a 1/2400 dilution was required for 50% of maximal binding to the immunogen peptide gp 120(99-119). With rgp 120, a 1:3200 dilution of S15 gave 50% of maximal binding. The presence of Gin at position 105 within rgp 120 of the HIV-1(SF2) strain did not abrogate antibody binding. This indicates that His-105 is not an essential amino acid within the epitope of S15. The capture radioimmunoassay was employed to determine if S15 inhibits gpl20-CD4 binding. To determine the appropriate amount of gpl20 to use in the plate capture radioimmunoassay, varying concentrations of rgp 120 were incubated with a constant amount of soluble 125I-rCD4 and then captured on the antibodycoated microtiter plate. Maximum [125I]rCD4 capture was achieved with 625 ng/ml of rgp 120 (data not shown). A subsaturating amount of rgp 120 (312 ng/ml) was used in subse-

quent competition experiments.

Varying amounts of S15 were preincubated with constant rgpl20 before the addition of [125I]rCD4. It is evident (Fig. 2A) that S15, at a starting dilution of 1:5, did not inhibit rgp 120 binding to [125I]rCD4. All S15 epitopes on rgpl20 were saturated with antibody molecules since complete inhibition of binding of [125I]S15 IgG to rgp 120 was achieved with an S15 dilution of 1:40 (Fig. 2B). To verify that the binding of [125I]rCD4 to rgp 120 was specific, the ability of the anti-CD4 monoclonal antibody

Recombinant CD4 and protein G-Sepharose-purified S15 IgG radioiodinated using the lactoperoxidase iodination procedure.21 Radioiodinated proteins were purified by gel filtration on PD-10 columns (Pharmacia).

1.0

were

gpl20-CD4 binding

room

assay

gpl20-CD4 binding was determined using a microtiter plate capture radioimmunoassay. Round well poly vinyl chloride microdilution plates (Titertek) were coated with sheep-antihuman immunoglobulins ( 1/500 dilution in PBS, Silenus) for 90 min at

temperature. Wells were then blocked with BSA as described above. Affinity-purified human anti-gp 120 IgG (1/30 dilution in 5 mg/ml of BSA in PBS containing 0.05% Tween-20) was added to wells and left for 2 h at room temperature. In separate wells, [125I]rCD4 (75,000 cpm) was allowed to react with unlabelled rgpl20 (50 p.1 in 5 mg/ml BSA in PBS containing 0.05% Tween-20) for 2 h at room temperature. Radioiodinated rCD4-rgp 120 complexes were then captured on the antibody-coated microtiter plate (2.5 h incubation at room

r

0.9 -

0.8

-

0.7

-

S

0.6

g

0.5

-

-

room

0.0

I—I_i_1_I_I_i— o

m

i

o o

£VI

-

O o

TO ~

ANTISERUM

o

o OJ

o o

o o

C

5

CO

S

DILUTION^

CM

FIG. 1. Binding of S15 to synthetic peptides gp 120(99-119), , gpl20(103-117), , gpl20(105-117), a spermwhale myoglobin peptide, x, and rgp 120, O, in EIA. ,

gpl20 RESIDUES

AND CD4 BINDING

857

0-1-,-,-,-,-,-,-,-,-1-,-,-,-,-,-,-,-r— 1/5 1/10 1/20 1/40 1/80 1/160 1/320 1/640

1/5 1/10 1/20 1/40 1/80 1/160 1/320 1/640

ANTISERUM DILUTION

FIG. 2. (A) Examination of the effect of S15 on the gpl20CD4 interaction. Serial dilutions of S15, , or the preimmune serum, , were preincubated with rgpl20 (312 ng/ml) for 2 h at room temperature. [125I]rCD4 (75,000 cpm) was added to each sample and left to incubate for a further 2 h. Recombinant

gpl20-[125I]rCD4 complexes

were

captured

on

OKT4A to block this interaction was determined. It has been shown previously that OKT4A binds to CD4 residues 58-60 and 64-66 in domain 1 and has the ability to block the binding of CD4 to gpl20.1013 OKT4A inhibited the gpl20-CD4 interaction in a dose-dependent manner (Fig. 3A), with 20 ng/ml of

CONCENTRATION

(ng/ml)

INHIBITOR PEPTIDE CONCENTRATION

(iiM)

FIG. 3. (A) Examination of the effect of anti-CD4 monoclonal antibodies on the CD4-gp 120 interaction. Serial dilutions of the anti-CD4 monoclonal antibodies OKT4, O and OKT4A, , were preincubated with [125I]rCD4 (75,000 cpm) for 2 h at ,

temperature, after which rgpl20 (312 ng/ml) was added and left to incubate for a further 2 h. The complexes were then captured on the antibody coated plate by incubation for 2.5 h at room temperature. (B) Examination of the effect of the antiviral peptide gp 120(105-117) and synthetic peptide homologues on the gpl20-CD4 interaction. Serial twofold dilutions of the synthetic peptides gpl20(105-l 17), A, gpl20( 103-117), A, gp 120(99-119), O, and the control sperm whale myoglobin peptide, o, were preincubated with [I25I]rCD4 for 6 h after which rgp 120 (312 ng/ml) was added and left to incubate for a further 2 h. The complexes were then captured on the antibody coated plates by incubation for 2.5 h at room temperature. room

(Fig. 3B).

antibody-

coated plates by incubation for 2.5 h at room temperature. (B) Verification that rgp 120 is saturated with S15. Serial dilutions of S15, , or the preimmune serum, , were preincubated with rgp 120 (312 ng/ml) for 2 h at room temperature. Protein G-sepharose purified S15 IgG, labeled with 125I (100,000 cpm), was added to each sample and left to incubate for a further 2 h. Immune complexes were then captured on antibody-coated plates by incubation for 2.5 h at room temperature.

INHIBITOR ANTIBODY

OKT4A resulting in 50% inhibition. In contrast, another antiCD4 monoclonal antibody, OKT4, which binds to domain 4 of CD4 did not inhibit gpl20 binding. The capture radioimmunoassay was used to monitor the effect of the antiviral peptide gp 120(105-117) on the gpl20-CD4 interaction. Radioiodinated rCD4 was treated with varying amounts of the antiviral peptide gp 120(105-117) and the elongated homologues, gpl20(99-l 19) and gpl20(103-l 15) for 6 h at room temperature prior to the addition of rgp 120. No inhibition of binding of 125I-rCD4 to gpl20 was caused by the synthetic peptides in the concentration range 9-1200 p,M

DISCUSSION The ability of the HIV-1 envelope glycoprotein gpl20 to associate with CD4 and hence facilitate the attachment of the virus to a susceptible cell, is a functional property that is conserved among the different isolates of HIV-1 and HIV-2 (reviewed in Ref. 22). It was reported that deletion of the amino terminal gpl20 residues, 108-116, abrogated rgpl20 binding to CD4 suggesting that this region may be important for CD4binding.16 We have shown in this study that gpl20 residues 99-119, which encompass the deleted amino acids, are not directly involved in CD4 binding since an antibody raised against a synthetic peptide corresponding to these residues did not inhibit the gpl20-CD4 interaction. It appears, therefore, that the loss in CD4 binding ability resulting from the deletion of '6 gp 120 residues 108-116 is due to indirect structural changes in the gpl20 molecule. Our results are consistent with the report that nonconservative amino acid substitutions of residues 102, 103, 106, 113, and 117 have no affect on CD4 binding ability.13 This region may be oriented away from the CD4 binding domain of gpl20, since occupation of this site by an IgG molecule causes no steric hindrance to CD4 binding. The study of Syu et al.,16 however, showed that polyclonal antibodies raised to a relatively large recombinant gpl20 amino terminal fragment (residues 42-129) could block CD4 binding. This suggests that there are residues in the amino terminal domain of gpl20 which are proximal to or in the same orientation as the CD4 binding

domain. Our results also indicate that a synthetic peptide, gpl20(105117) which was shown to have antiviral activity,18 did not inhibit CD4 binding even at peptide concentrations well in excess of those required for the inhibition of HIV-1 infection of CEM cells.18 The results obtained suggest that the synthetic peptide gp 120(105-117) does not exert its antiviral activity by preventing the CD4-gpl20 interaction. Previous studies have indicated that residues 362-389 and 413-456 in the carboxyl terminal domain of gpl20 are involved in

CD4-binding.10"18

These two sequences are brought into proximity by two intrachain disulfide bonds, Cys-385— Cys-418 and Cys-378—Cys-435.23 Indeed, the disulfide bonding patterns are clustered in two groups with 6 in the amino terminal domain and 3 in the carboxyl terminal domain of gpl20. The region close to gpl20 residues 99-119 is not connected by disulfide bonds to these carboxyl-terminal CD4 binding domains23 and may thus be distal to them and is therefore unlikely to participate directly in CD4-binding.

close

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EL AHMAR ET AL.

ACKNOWLEDGMENTS We extend our thanks to Dr. Hans-Gerhard Schneider for advice on iodination procedures, Simon A. Cumming for antiserum production, Ken Mitchellhill for peptide synthesis, and Frosa Katsis for amino acid analysis. This work was supported by the National Health and Medical Research Council, the National Centre for AIDS Virology Research through the Commonwealth AIDS Research Grants Program and the American

Foundation for AIDS Research.

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9056. 4. Ashkenazi A, Presta LG, Marsters SA, Camerato TR, Rosenthal KA, Fendly BM, and Capon DJ: Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis. Proc Nati Acad Sei (USA) 1990;87:7150-7154. 5. Peterson A and Seed B: Genetic analysis of monoclonal antibody and HIV binding sites on the human lymphocyte antigen CD4. Cell

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Steinbrich R, Ramachandran H, Husain Y, and Reinherz EL: Substitution of murine for human CD4

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QJ, Clapham PR; Weiss RA, McDougal JS, Pietropaolo C, Axel R, Truneh A, Maddon PJ, and Sweet RW: Identification of the residues in human CD4 critical for the binding of HIV. Cell 1989;57:469-481. 8.

Brodsky MH, Warton M, Myers RM, and Littman DR: Analysis of the site in CD4 that binds to the HIV envelope glycoprotein. J

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Address reprint requests to: Pantelis Poumbourios St. Vincent's Institute of Medical Research 41 Victoria Parade Fitzroy, Victoria 3065, Australia