Specific interaction of conformational polypeptides derived from HIV gp120 with human T lymphocyte CD4 receptor

Immunology Letters 63 (1998) 27 – 32

Specific interaction of conformational polypeptides derived from HIV gp120 with human T lymphocyte CD4 receptor Mingfang Liu a,*, Jin Zeng b, Frank A. Robey a a

Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA b Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received 3 March 1998; received in revised form 9 April 1998; accepted 14 April 1998

Abstract Specifically cross-linked peptides (peptomers) have been prepared from the repeating sequences of the C4 domains of glycoproteins 120 present in different isolates of human immunodeficiency virus (HIV). In order to investigate if the HIV C4 peptomers could function as gp120 protein, we have used a novel protein-binding assay to examine if and which components of the peptomers could interact with CD4 receptor in vitro. Here, we demonstrate that all the polymeric components of the HIV-1 C4 peptomer could bind to recombinant soluble CD4 protein. A similar result was also obtained with HIV-2 C4 peptomer except that the binding occured only in those of constituents having molecular weights higher than that of trimer. Remarkably, the CD4-binding was demonstrated to be specific to the HIV C4 peptomers as it did not occur with control peptomers such as Poly V3 MN and Poly NINA whose peptide sequences bore no homology to those of the HIV C4 peptomers. Furthermore, consistent with previous findings, no interaction of HIV-1 C4 monomeric peptide (419 – 436) with CD4 was detected under the same conditions. Since it is known that the HIV C4 peptomers have much higher contents of a-helical conformation than those of their monomeric peptides, we conclude that the secondary structure is a pivotal determinant for the successful CD4-binding by the peptomers. Our finding reveals a more defined molecular nature of the gp120-CD4 interaction and may be important for designing HIV vaccines and therapeutics which target the first step in the virus infection. © 1998 Elsevier Science B.V. All rights reserved. Keywords: gp120-CD4 interaction; Synthetic C4 peptide; Peptomer; Peptomer-CD4 binding

1. Introduction Peptomers are polymers of specifically cross-linked synthetic peptides [1 – 5]. They are made of a mixture of polypeptides with increased chain lengths. Unlike most of monomeric peptides which have very little, if any, secondary structure, certain peptomers have conformations which mimic those found for peptides in native proteins. Consequently, they display the physiological * Corresponding author. Present address: CIBP, Institut Pasteur de Lille, 1 Rue Calmette, 59019 Lille, France. Tel.: +33 3 20877890; fax: +33 3 20152092. 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00051-0

activities of their parent proteins and could, in principle, be used in diagnostics, therapeutics and vaccine development. HIV-1 C4 peptomer (419–436) is one of the examples, which is derived from the amphipathic peptide sequence (the 419–436 residues) in the fourth conserved (C4) domain of human immunodeficiency virus (HIV) type 1 glycoprotein 120 (gp120) [6]. The secondary structure content of the peptomer has been calculated from circular dichroism (CD) spectrum to be more than 25-fold higher than that of its monomeric peptide. Furthermore, the peptomer induces the production of rabbit antibodies which recognizes recombinant and

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native gp120 whereas the monomeric C4 peptide (419– 436) does not [6]. Since the conformation of HIV-1 C4 peptomer (419–436) is comparable to that of the C4 domain of gp120 as suggested by the immunogenecity study, one could expect that the peptomer performs at least some of biological functions of the envelope glycoprotein. One of the major functions of gp120 is to interact with CD4 receptor in T lymphocytes [7], the first step which initiates a cascade of molecular events leading to HIV infection [8–10]. Several regions of gp120, most notably the C4 domain, are implicated in the binding process [11–14]. A tryptophan in this domain is critical as loss of CD4-binding occurs when the amino acid is mutated [15]. Consequently, the virus carrying a mutation at this position is non-infectious [15]. Although the glycoprotein has been shown to interact with the membrane receptor with strong avidity (Kd =10 − 9 M) [16], synthetic C4 peptides do not exhibit the binding capacity [6]. In order to mimic the binding activity of the envelope glycoprotein, we have prepared several peptomers from the C4 sequences that surround the aromatic amino acid. Using dot-blot assay, we have also obtained some preliminary evidences which indicates that the peptomers could interact with CD4 [6]. Nevertheless, we wanted to prove the peptomer-CD4 interaction and to address several unanswered important questions concerning the binding process using a more sensitive and specific method. In this work, we employed a novel protein-binding assay to demonstrate that the HIV C4 peptomers could indeed bind to recombinant soluble CD4 molecule in vitro. More importantly, we find that only certain polymeric forms of the HIV C4 peptomers could interact with the receptor, demonstrating that a-helical conformation is essential to the successful CD4 binding. Significantly, the CD4-binding was shown to be specific as several control peptomers from dissimilar sequences to that of the C4 domain could not form complexes with the receptor protein.

2. Materials and methods

the N-terminal. A peptomer was formed by polymerization of the haloacetylated peptide in 10 mM Tris–HCl, pH 8.0 buffer containing 1 mM EDTA at 25°C for several hours.

2.2. Labeling of CD4 protein with biotin Recombinant soluble CD4 protein (rsCD4; a gift from Genentech., South San Francisco, CA) at a concentration of 1 mg/ml in Tris buffered saline (TBS) was dialyzed against 0.1 M NaHCO3, pH 8.5. 1 ml of the dialyzed rsCD4 was then supplemented with 12.3 ml of biotin succinimide ester (Pierce Chemicals) dissolved in N,N%-dimethylformamide at a concentration of 10 mg/ ml. The reaction mixture was rotated for 1 h at 4°C, dialyzed into TBS and stored at 4°C until use.

2.3. Modified western blot analysis for in 6itro binding of peptomers to CD4 molecule Peptomers (25 mg) were first dissolved in non-reducing sample buffer and were separated on a 4–20% linear gradient SDS-PAGE (Novex, Encino, CA). The peptomers were then transferred onto a PVDF membrane (Immobilon P. Millipore, Bedford, MA), and the filter was washed five times with TBS to remove SDS. The membrane was incubated in TBS overnight, allowing the peptomers to renature. After rinsing with TBS for three times, the filter was blocked from non-specific binding with 5% of non-fat milk (Bio Rad) in TBS supplemented with 0.1% Tween-20 at room temperature for 2 h overnight. The blot was then briefly rinsed and incubated with 2.5 mg/ml of the biotin-labeled rsCD4 protein in the TBS/Tween-20 solution at 4°C for 2 h, following by several times of wash with the same buffer/detergent at room temperature. Upon incubation with strepavidin-horseradish peroxidase conjugate (TAGO, Burlinnggame, CA) diluted 1:150000 in TBS/ Tween-20 solution for another 2 h at 4°C, the blot was washed five times with the TBS/detergent buffer and then developed with enhanced chemiluminescent (ECL) reagent (Amersham) according to the manufacturer’s instructions.

2.1. Peptide and peptomer syntheses 3. Results and discussion The peptides were synthesized by Merrifield’s solid phase method on an automated peptide synthesizer model 430 purchased from Applied Biosystems (Forster City, CA). All chemicals used in the synthesis were from the same company. Peptomers were synthesized according to the methods described in detail previously [1 – 6]. In brief, a peptide was specially designed to contain a cysteine residue which was in the amide form at the C-terminal and to have a bromoacetyl- or a choloroacetyl-group at

3.1. HIV-1 C4 peptomer (419 – 436) binds to CD4 protein in 6itro In a series of experiments aiming at studying the biological activities of HIV C4 peptomers, we have taken the first step to investigate if they could retain the primary function of gp120 in CD4-binding. Since a peptomer is a mixture of polymers, we also wanted to find out which component(s) of the HIV C4 peptomers

M. Liu et al. / Immunology Letters 63 (1998) 27–32 Table 1 Peptide sequences of peptomers used in the CD4-binding study C4 Peptomer (419–436)a C4 Peptomer (412 – 429)a Poly CD4-D.A. Poly V3 MN Poly NINA

KIKQIINMWQEVGKAMYA (the residues 419 – 436 of HIV-1 gp120) HIEQIINTWHKVGKNVYL (the residues 412 – 429 of HIV-2 gp120) SLKLENKEAK-NH2 (a part of CD4 sequence) KRKRIHIGPGRAFY (a part of gp120 v3 loop sequence) MTEYKLVVVGAGGVGKSALTIQLIQ (a part of ras sequence)

a

Bold letters indiate amino acids residues that are homologous between HIV-1 (MN isolate) and HIV-2 (ISYR) glycoprotein 120.

is (are) functionally effective. Firstly, C4 peptomer (419 –436) (Table 1) derived from the sequence of the MN isolate of HIV-1 was used in the study as the peptomer has been thoroughly characterized physically and immunologically [6]. We used a modified Western blot analysis to examine if HIV-1 C4 peptomer (419 – 436) could interact with CD4 in vitro. A fixed amount of the peptomer was first separated by a linear gradient SDS-PAGE. As shown in

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Fig. 1 (lane 5), the peptomer displays as a ladder of Coomassie Blue-stained bands and is a mixture of polymers with different molecular weights. The dimer of approximately 6 kDa is the smallest component of the peptomer. After blotting onto a PVDF membrane, the peptomer was reacted with biotin-labeled recombinant soluble CD4 protein (rsCD4). Complex formation was visualized on the blot as a band detectable by ECL reagent. Fig. 1 (lane 4) illustrates that the reagent could reveal a ladder of bands after successive incubation with biotin-labeled rsCD4 protein and strepavidinhorseradish peroxidase conjugate. The pattern of the bands is similar to that stained with Coomassie Blue after gel electrophoresis of the peptomer (lane 5), indicating that all the polymeric components of HIV-1 peptomer (419–436) could bind to the soluble receptor protein in vitro. By contrast, ECL reagent could not detect any signal when biotin-labeled rsCD4 (lane 1) or strepavidin-horseradish peroxidase conjugate (lane 2) or both of them (lane 3) were omitted in the Western blot analyses, demonstrating the specificity of the method.

Fig. 1. Modified western blot analysis for in vitro binding of HIV-1 C4 peptomer (419 – 436) to CD4 molecule. Twenty-five micrograms of HIV-1 C4 peptomer (419 – 436) was fractionated on a 4–20% linear gradient SDS-PAGE before blotting onto a PVDF membrane. After renaturation of the peptomer in PBS, the blot was probed with 2.5 mg/ml of the biotin-labeled rsCD4 protein. The filter was incubated with 1:150000 diluted strepavidin-horseradish peroxidase conjugate (TAGO, Burlinggame, CA) in TBS for another 2 h at 4°C and then developed with an enhanced Chemilluminescent (ECL) reagent (Amersham). Lanes: 1, HIV-1 C4 peptomer (419 – 436) detected with strepavidin horseradish peroxidase conjugate alone; 2, HIV-1 C4 peptomer (419–436) alone; 3, HIV-1 C4 peptomer (419 – 436) detected with biotin-labeled recombinant soluble CD4 alone; 4, HIV-1 C4 peptomer (419–436) detected with biotin-labeled recombinant soluble CD4 protein plus strepavidin horseradish-peroxidase conjugate; and 5, gel electrophoresis, Coomassie-Blue staining, the band with the lowest molecular mass was the dimer of HIV-1 C4 peptomer (419–436). Note that a slight difference between the band images of Western blot and electrophoresis was due to the small change in the size of the membrane after incubation. HRP-Strepavidin, strepavidin horseradish-peroxidase conjugate; Biotin-rsCD4, biotin-labeled recombinant soluble CD4. HRP-Strepavidin, strepavidin horseradish-peroxidase conjugate.

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Fig. 2. Modified western blot analysis for in vitro binding of HIV-2 C4 peptomer (412 – 429) to CD4 molecule. Twenty-five micrograms of HIV-2 C4 peptomer (412 – 429) was separated on a 4–20% linear gradient SDS-PAGE. The binding of the peptomer to CD4 protein was detected using Western blot analysis as described in Fig. 1. Lanes: 1, HIV-2 C4 peptomer (412 – 429) detected with strepavidin horseradish-peroxidase conjugate alone; 2, HIV-2 C4 peptomer (412–429) alone; 3, HIV-2 C4 peptomer (412 – 429) detected with biotin-labeled recombinant soluble CD4 alone; 4, HIV-2 C4 peptomer (412–429) detected with biotin-labeled recombinant soluble CD4 protein plus horseradish-peroxidase conjugate; and 5, gel electrophoresis, Coomassie-Blue staining, the band with the lowest molecular mass was the dimer of HIV-2 C4 peptomer (412–429). HRP-Strepavidin, strepavidin horseradish-peroxidase conjugate; Biotin-rsCD4, biotin-labeled recombinant soluble CD4.

3.2. HIV-2 C4 peptomer (412 – 429) also binds to CD4 protein in 6itro We then extended the binding study to another peptomer derived from the residues 412 – 429 of the ISYR isolate of HIV-2 gp120 protein (Table 1). Fig. 2 (lane 5) shows the result of gel electrophoresis of HIV-2 C4 peptomer (412–429). As expected, the pattern is comparable to that of HIV-1 C4 peptomer (419 – 436) (Fig. 1, lane 5). Upon incubation with rsCD4 protein (lane 4), the HIV-2 C4 peptomer also formed complex with the receptor molecule. However, unlike the HIV-1 C4 peptomer, only the components of HIV-2 C4 peptomer with molecular weights higher than that of trimer could associate with the receptor protein in vitro (lane 4). Likewise, the CD4-binding signal of the HIV-2 peptomer could only be observed when both biotin-labeled rsCD4 and strepavidin-horseradish peroxidase conjugate were present (lanes 1 – 3).

peptomers could interact with the receptor molecule in vitro. Fig. 3 (lanes 1–3) shows that like HIV-1 C4 peptomer (419–436), peptomers poly CD4-DA and poly V3 MN (Table 1) derived from sequence segments of CD4 molecule and V3 loop of gp120, respectively, could be fractionated into a mixture of different polymers. This figure also displays that, in contrast to HIV-1 C4 peptomer (419–436), peptomer poly V3 MN failed to associate with rsCD4 (lane 6). The same result was also obtained for another control peptomer poly NINA prepared from a peptide sequence of ras oncogene protein (data not shown). A relatively weak binding signal is seen after incubation of peptomer poly CD4-DA with rsCD4 (lane 5), indicating that CD4 molecules are capable of self-association. This result is expected as CD4 is known to dimerize with affinity lower than 4× 105 M − 1 [17]. Hence, we conclude that the interaction of the HIV C4 peptomers with the receptor protein in vitro is specific.

3.3. Specific interaction of HIV C4 peptomers with CD4 protein in 6itro

3.4. HIV-1 C4 monopeptide (419 – 436) does not bind to CD4 receptor molecule in 6itro

To determine the specificity of binding of the HIV C4 peptomers to CD4 protein, we tested if various control

Since HIV-1 C4 peptomer (419–436) contains a much higher helical content than that of its monomeric

M. Liu et al. / Immunology Letters 63 (1998) 27–32

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peptide [6], it is of significant interest to evaluate if the secondary structure is important for in vitro CD4-binding. Hence, we carried out the binding assay for the HIV C4 monomeric peptide. An equal amount of the HIV-1 C4 peptomer and its monopeptide was fractionated on SDS-PAGE. The result shown in Fig. 4 (lanes 1 – 2) displays a characteristic ladder of bands for the peptomer and a relatively diffused and dense band for the monopeptide. Remarkably, in contrast to the peptomer (lane 3), the C4 monopeptide could not produce an ECL reagent-detectable signal after incubation with CD4 molecule (lane 4), indicating its inability of binding to the receptor under the in vitro conditions. This result is consistent with previous findings by other methods [6,18]. It demonstrates that the high content of a-helical conformation is essential for the strong CD4binding by the peptomers. We believe that the interaction of the monomeric peptide with CD4 is very inefficient. Firstly, very weak CD4-binding by the peptomer poly CD4-DA could be observed (Fig. 3, lane 5). Secondly, the HIV-1 C4 dimer

Fig. 4. Modified western blot analysis for comparison of the relative binding affinities of HIV-1 C4 peptomer (419 – 436) and its monopeptide to CD4 protein. Twenty-five micrograms of HIV-1 C4 peptomer (419 – 436) and its monopeptide were used in a western blot analysis as described in Fig. 1. Lanes: 1 and 2, gel electrophoresis, CoomassieBlue staining; 3 and 4, Western blot analysis, ECL detection. Peptomer (419 – 436) (lanes 1 and 3), HIV-1 C4 peptomer (419–436); Monopeptide (419 – 436) (lanes 2 and 4), HIV-1 C4 monopeptide (419 – 436). The band with the lowest molecular mass was the dimer of HIV-1 C4 peptomer (419 – 436) (lane 1).

Fig. 3. Specificity of the binding of HIV-1 peptomer C4 (419–436) to CD4 receptor. Twenty-five micrograms of various peptomers were fractionated on a 4 – 20% linear gradient SDS-PAGE. Biotin-labeled CD4 was used in a Western blot analysis to monitor the binding as described in Fig. 1. Lanes: 1–3, gel electrophoresis, Coomassie-Blue staining; 4 – 6, Western blot analysis, ECL detection. Peptomer (419 – 436) (lanes 1 and 4), HIV-1 C4 peptomer (419–436); Poly CD4-DA (lanes 2 and 5) and Poly V3 MN (lanes 3 and 6), peptomers derived from sequence segments of CD4 molecule and gp120 V3 loop, respectively.

which occupies only a small fraction in the polymeric components of the peptomer shows a strong CD4-binding signal (Fig. 4, lane 3) whereas the monomeric peptide used at the same amount as the peptomer exhibits no interaction with the receptor (Fig. 4, lane 4). When the peptide sequence of the HIV-1 C4 peptomer (419–436) is arranged into the helical wheel model [6], it becomes clear that there are two faces to the resulting conformation, a hydrophobic face and a hydrophilic face [6]. This theoretical treatment suggests that the peptide could fold into a-helical conformation within an appropriate environment, such as that present in an intact protein or in a polymerized state as mentioned before. Due to this potential, a single step of dimerization of the peptide results in a markedly enhanced helical content (44%). With further polymerization, the percentage of the secondary structure is again increased to a final average of 53% for the peptomer.

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Because of such a high degree of conformational constraincy, all the polymeric constituents of the HIV-1 C4 peptomer bind to CD4 effectively (Fig. 1, lane 4). Like the HIV-1 C4 peptomer, HIV-2 C4 peptomer (412 –429) could associate with the receptor protein in vitro (Fig. 2, lane 4). This is expected from the helical wheel model [6] because the sequence of HIV-2 C4 peptide (Table 1) also has two sides like those in the HIV-1 peptide. Therefore, it possesses a potential for helical formation. However, unlike the HIV-1 C4 peptomer, only the components of the HIV-2 C4 peptomer with molecular weights higher than that of trimer display the CD4-binding. This could be explained by the fact that the HIV-2 C4 peptomer has about 30% of average helical content which is considerably lower than that of the HIV-1 C4 peptomer (53%) [6]. The HIV-2 C4 peptide contains about 40% of different amino acid residues from those of the HIV-1 peptide, some of them may contribute a relatively stronger helical destabilizing effect than their counterparts in the HIV-1 peptide. Since the negative effect on helical formation from an unfavorable amino acid residue has to be offseted with a corresponding level of polymerization as predicted by the physical chemistry of polymer [19], it is logical that the dimer and the trimer of the HIV-2 C4 peptomer could not interact effectively with CD4 (Fig. 2, lane 4). Moreover, in view of the findings with the HIV C4 peptomers, one could predict that HIV-2 gp120 protein contains a lower percentage of helical content and consequently should bind to the receptor with a weaker affinity than the HIV-1 protein. Indeed, Moore has reported that the HIV-2 gp120 has 25-fold lower affinity for soluble CD4 than the HIV-1 protein [20]. Taken together, we conclude that the CD4-binding affinities of gp120 proteins and their peptomers are positively correlated with their helical contents. Significantly, the binding of the HIV C4 peptomers to CD4 protein in vitro is specific as the control peptomers such as poly V3 MN and poly NINA (Fig. 3 and data not shown) could not form complexes with the receptor under the same conditions. This result also confirms that, in addition to a high helical content, an appropriate peptide sequence is also required for a productive association with CD4 molecule. Malvoisin and Wild [21] have shown that HIV gp120 interacts with both soluble and the native CD4 proteins, albeit giving different forms of complex. One could expect that the HIV C4 peptomers would bind to the native protein as well although we used only sCD4 in this study. Because the HIV C4 peptomers could recapitulate the binding event, they seem to be better analytical tools for dissecting the functions of different regions of gp120 protein than their monomeric peptides and may .

be useful for the current vaccine and therapeutics designs which target the first step in the HIV infection process.

Acknowledgements The timely support to F.A. Robey from the Director’s Discretionary Fund of the Office of AIDS Research, NIH made continuation of this work possible. We are pleased to acknowledge Genetech in South San Francisco, California for the generous gifts of recombinant gp120 and CD4.

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