Potent HIV fusion inhibitors against Enfuvirtide-resistant HIV-1 strains

Abstract

T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5- or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.

In the early 1990s, a number of synthetic peptides derived from the N- and C-heptad repeat (NHR and CHR) regions of the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 were discovered to have potent anti-HIV activity (1–6). Two of the CHR peptides, C34 and DP-178 (also known as T20), inhibit HIV infection at low nM levels. Biochemical and biophysical analyses suggest that these CHR peptides inhibit HIV-1 Env-mediated membrane fusion by interacting with the viral gp41 NHR region to form heterologous trimer of heterodimer and block gp41 six-helix bundle (6-HB) core formation, a critical step in virus–cell fusion (1, 7–9).

T20 (Enfuvirtide, Fuzeon), jointly developed by Trimeris and Roche, was licensed by the US FDA as the first member of a new class of anti-HIV drugs—HIV fusion inhibitors. Clinical data show that T20 is effective as a salvage therapy for HIV/AIDS patients who have failed to respond to current antiretroviral therapeutics, including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). However, T20 can easily induce drug resistance, resulting in increasing failure rates in T20-treated patients. Therefore, we sought to develop HIV fusion inhibitors that are effective against T20-resistant HIV.

Here we designed an HIV fusion inhibitory peptide, designated CP32M (Fig. 1), on the basis of findings from our previous studies on anti-HIV peptides containing a motif (⁶²¹QIWNNMT⁶²⁷) located at the upstream region of the gp41 CHR, immediately adjacent to the pocket-binding domain (10), which is critical for 6-HB formation and stability. Surprisingly, CP32M is exceptionally potent against T20-resistant HIV-1 strains, with great potential to be further developed as an anti-HIV drug for treatment of HIV/AIDS patients, in particular those unresponsive to the first generation HIV fusion inhibitor used in clinics.

Fig. 1.

Interactions of CP32M and other CHR peptides with NHR peptides. (A) Schematic view of the HIV-1_HXB2 gp41 molecule. FP, fusion peptide; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; TR, tryptophan-rich domain; TM, transmembrane domain; CP, cytoplasmic domain. (B) In the current fusion model, the CHR region of gp41 folds back to interact with the NHR region to form a hairpin structure. Three molecules of hairpins associate with each other to form a 6-HB. The dashed lines between the NHR and CHR regions indicate the interaction between the residues located at the e, g and the a, d positions in the NHR and CHR, respectively. The interaction of the pocket-binding domain in the CHR (amino acids 628–635, in blue) with the pocket-forming domain in the NHR (amino acids 565–581, in red) is critical for stabilization of the 6-HB. Both T20 and C34 peptides contain the sequences targeting the GIV motif (in purple) in the NHR, but C34 contains the pocket-binding sequence, while T20 does not. The peptide CP621–652 overlaps the pocket-binding domain but it has no sequence targeting the GIV motif. The residues preceding the pocket-binding domain are in green. (C) Based on the CP621–652 sequence, CP32M was engineered by replacing 11 of 32 residues (the mutated residues are in red) to improve its pharmacokinetic profiles and anti-HIV activity. The positively charged residues (e.g., K) or negatively charged residues (e.g., E) were introduced to form ion pairs (salt bridges) at i to i + 4 position of the helical conformation.

Results

Structure-Based Design of the Anti-HIV Peptide CP32M.

We have recently found that the ⁶²¹QIWNNMT⁶²⁷ motif located at the upstream region of the gp41 CHR, immediately adjacent to the pocket-binding domain, is critical for the NHR and CHR interhelical interactions, and that the peptide CP621–652 containing the ⁶²¹QIWNNMT⁶²⁷ motif possesses potent anti-HIV activity (10). We designed a peptide, designated CP32M, by using the peptide CP621–652 as a template, to improve the anti-HIV activity and drug-resistant profiles and the pharmacokinetics of the anti-HIV peptide with wild-type sequence. As shown in Fig. 1C, 11 of 32 residues (34.4%) in the peptide CP621–652 were mutated, while the residues that are important for the activity or stability of the peptide remained unchanged. The positively or negatively charged residues (e.g., K or E) were introduced into the CP32M to promote the formation of ion pairs (salt bridges) at the i to i + 4 position of the helical conformation (e.g., E⁶³⁶, K⁶⁴⁰, and K⁶⁴⁴). First residue Q⁶²¹ at the “a” position in the heptad repeat was replaced by a hydrophobic residue V to enhance its hydrophobic interaction with the NHR target. The residues I⁶²², N⁶²⁵, S⁶⁴⁰, and N⁶⁵¹, which are located at the b, c, e, and f positions in the α-helical wheel, were replaced by negatively or positively charged residues, E or K, respectively, to increase the hydrophilicity of the peptide. It was expected that introduction of these residues in the CP32M would improve its solubility and strengthen its ionic interactions with the NHR.

CP32M Is Highly Effective in Blocking HIV-1-Mediated Membrane Fusion and Inhibiting Infection by a Broad Spectrum of HIV-1 Isolates.

It was important to learn whether the engineered peptide CP32M maintained, or better yet improved its antiviral activity. First, we determined the inhibitory activity of CP32M on HIV-1 IIIB-mediated cell–cell fusion by a dye transfer assay. As shown in Fig. 2A, CP32M inhibited cell–cell fusion with an IC₅₀ of 4 nM, approximately sevenfold more potent than T20 (IC₅₀ = 28 nM). Then, we assessed inhibitory activity of CP32M on HIV-1 IIIB infection. Consistently, CP32M was found to be capable of inhibiting HIV-1 IIIB infection in MT-2 cells with an IC₅₀ of 5 nM (Fig. 2B), approximately fourfold more effective than T20 (IC₅₀ = 26 nM). Further, we evaluated the inhibitory activity of CP32M on infection of peripheral blood mononuclear cells (PBMCs) by a panel of primary group M HIV-1 isolates. As shown in Table 1, CP32M potently inhibited infection by primary viruses with distinct genotypes (subtypes A–G) and phenotypes (R5- and R5X4-tropic). Strikingly, CP32M effectively inhibited infection by HIV-1 group O virus (BCF02) with an IC₅₀ of 10 nM, whereas neither T20 nor C34 exhibited inhibitory activity even at a concentration as high as 2,000 nM (Fig. 2C). Therefore, CP32M is a potent HIV-1 fusion inhibitor against infection by both laboratory-adapted and primary HIV-1 strains with different genotypes and phenotypes.

Fig. 2.

Inhibition of CP32M on HIV-1-mediated cell–cell fusion (A), and on infection by HIV-1 IIIB (B) and BCF02 (C). T20 was tested as a control. Each sample was tested in triplicate and the data are presented as mean ± standard deviations. Numbers in parentheses indicate IC₅₀ (nM) values.

Table 1.

Inhibitory activity of CP32M against infection by primary HIV-1 isolates

Virus	Subtype	Coreceptor	nM ± SD
			IC50	IC90
RW92008	A	R5	51 ± 3	66 ± 3
94UG103	A	R5X4	30 ± 12	54 ± 14
92US657	B	R5	235 ± 42	423 ± 40
JR-FL	B	R5	23 ± 3	37 ± 4
93IN101	C	R5	37 ± 2	80 ± 5
93MW959	C	R5	32 ± 3	83 ± 10
92UG001	D	R5X4	40 ± 6	87 ± 8
92THA009	E	R5	94 ± 16	120 ± 30
93BR020	F	R5X4	23 ± 3	105 ± 7
RU570	G	R5	84 ± 12	132 ± 30

CP32M Is Exceptionally Potent Against T20-Resistant HIV-1 Strains.

Subsequently, we sought to determine whether CP32M is effective against HIV-1 strains resistant to T20. A panel of HIV-1_NL4-3 mutants including two T20-sensitive and five T20-resistant strains (11) were used in our experiments. We found that double substitutions in the gp41 NHR (V38A/N42D, V38A/N42T, V38E/N42S, and N42T/N43K) also conferred cross-resistance to the peptide C34 (Table 2). Strikingly, CP32M was extremely active against both T20- and C34-sensitive and -resistant viruses. CP32M inhibited infection by several HIV-1_NL4-3 mutants (N42S, V38A, and V38A/N42T) at picomolar (pM) levels. In particular, infection by HIV-1_NL4-3 bearing V38A/N42T double mutations was shown to be effectively inhibited by CP32M with an IC₅₀ of 2 pM, whereas it was highly resistant to both T20 and C34.

Table 2.

Potent inhibitory activity of CP32M against infection by C34 and T-20-resistant viruses

HIV-1NL4-3 (36G)	Phenotype*	IC50 ± SD, nM
		T20	C34	CP32M
Parental	S	35.820 ± 14.350	3.410 ± 0.300	4.250 ± 0.130
N42S	S	28.915 ± 0.881	0.611 ± 0.142	0.042 ± 0.004
V38A	R	>2,000.000	1.686 ± 0.620	0.220 ± 0.075
V38E/N42S	R	>2,000.000	79.660 ± 4.630	4.690 ± 0.630
N42T/N43K	R	>2,000.000	119.681 ± 26.160	1.016 ± 0.359
V38A/N42T	R	>2,000.000	50.049 ± 8.305	0.002 ± 0.000
V38A/N42D	R	>2,000.000	24.474 ± 9.246	76.251 ± 11.277

*S, sensitive; R, resistant.

CP32M Interacts with the gp41 NHR Region to Form Highly Stable α–Helical Complex and Efficiently Blocks the gp41 6-HB Core Formation.

We previously showed that the CHR peptide CP621–652 can interact with the NHR peptides to form typical 6-HBs (10). It was of interest to determine whether the engineered peptide CP32M would retain its ability to interact with the NHR peptides after substitution of over one-third of its residues. As shown in Fig. 3, CP32M could form typical α-helical complexes with the NHR peptides (N36 or T21), similar to the parent peptide CP621–652 as assessed by CD spectra. Impressively, thermal denaturation analysis demonstrated that CP32M also interacted with N36 or T21 with higher thermostability (Fig. 3 B and D). The complex of N36/CP32M had a Tm value of 79°C which was 15°C higher than its wild-type bundle N36/CP621–652 (Tm = 64°C). The T21/CP32M complex displayed a Tm value (94°C) 13°C higher than the T21/CP621–652 complex (Tm = 81°C). As a control, the bundle N36/C34, which has been considered to be a core structure of the fusion-active gp41, had a Tm value of 64°C (data not shown).

Fig. 3.

Biophysical characterization of CP32M by CD spectroscopy. (A) α-helical conformation of the complex formed by N36 and CP32M or CP621–652. (B) Thermostability of the complex formed by N36 and C peptides. The unfolding temperature of each complex was scanned at 222 nm by CD spectroscopy, and their Tm values were calculated. (C) α-helical conformation of the complex formed by T21 and CP32M or CP621–652. (D) Thermostability of the complex formed by T21 and C peptides. Final concentration of each peptide in PBS is 10 μM.

We then used an N-PAGE-based method to visualize the complex formed by CP32M and counterpart peptide T21. As shown in Fig. 4A, the NHR peptide T21 shows no band in the native gel because it carries net positive charges and could migrate up and off the gel, but the negatively charged peptide CP32M shows a specific band. When CP32M was mixed with T21, a specific band corresponding to the 6-HB appeared. Their specific binding was also confirmed by size-exclusion high-performance liquid chromatography (HPLC) (Fig. 4B). Sedimentation equilibrium ultracentrifugation demonstrated that the MW_app value of the CP32M and T21 complex was 26,600 Da (Fig. 4C). Compared to an expected molecular mass of 8,688 Da for CP32M/T21 heterodimer, we concluded that these two peptides associate to form a 6-HB structure consisting of three CP32M and T21 peptides, respectively.

Fig. 4.

Determination of the activity of CP32M to form 6-HB with T21 and to block 6-HB formation between N36 and C34. (A) N-PAGE for detection of 6-HB formation between T21 and CP32M. (B) Size-exclusion HPLC analysis for 6-HB formation between T21 and CP32M. (C) Molecular mass of the T21/CP32M complex determined by sedimentation equilibrium ultracentrifugation at concentrations of 25 μM in PBS buffer (pH 7.4) at a rotor speed of 33,000 rpm. The observed molecular mass is 26,600 Da (the calculated mass for a trimer is 26,064 Da). (D) Inhibition of C peptides on formation of 6-HB modeled by N36/C34 peptides.

The mechanism of NHR or CHR-derived anti-HIV peptides has been considered to inhibit the formation of the viral gp41 6-HB in a dominant-negative fashion (7, 12). To test whether the engineered CP32M could arrest the formation of 6-HBs, an ELISA-based method was developed, in which the 6-HB-specific mAb NC-1 was used as a capture antibody and the peptide C34 was biotinylated (see Materials and Methods). Consistently, NC-1 reacted specifically with the complex of N36 and C34 but not with the isolated peptides (data not shown). The results show that CP32M could efficiently inhibit the formation of 6-HB between the peptides N36 and C34-biotin in a dose-dependent manner, comparable to its parent peptide CP621–652 and C34 itself (Fig. 4D). However, T20 had no such effect at a concentration as high as 8,000 nM, consistent with our previous data (13). This result suggests that CP32M, unlike T20, is able to block 6-HB formation between the NHR and CHR in a dominant-negative fashion.

Discussion

In the present study, we designed an anti-HIV peptide, CP32M, on the basis of the structural and functional information of HIV-1 gp41 and a recently identified anti-HIV peptide (CP621–652) containing a motif (⁶²¹QIWNNMT⁶²⁷) that is critical for the 6-HB formation and stability (10). Our data have demonstrated that, like its parent peptide CP621–652 (10), the engineered CP32M maintains its potency in inhibiting HIV-1-mediated cell–cell fusion and infection by laboratory-adapted HIV-1 strains. CP32M is highly effective against a panel of primary HIV-1 strains with distinct genotypes (group M, subtypes A–G) and phenotypes (R5 and R5X4) (Table 1). Favorably, CP32M could potently inhibit infection by BCF02, one of the HIV-1 group O isolates, having a high genetic diversity compared to the major group of HIV-1. In comparison, T20 and C34 had no inhibitory activity against infection by HIV-1_BCF02 at a concentration as high as 2,000 nM. Therefore, the engineered peptide CP32M which has a broader anti-HIV spectrum than T20 may possess a property to overcome the genetic barrier of HIV-1 group O isolates (e.g., BCF02) and thus can be used for treatment of HIV-1 group O infection.

The most unique feature of the CP32M peptide is its exceptional potency against HIV-1 variants resistant to T20 and other CHR peptides including C34 and T1249, a second generation HIV fusion inhibitor. Although its parent peptide (CP621–652) is also effective against the drug-resistant viruses, CP32M exhibited much-improved antiviral activity against some T20-resistant mutants, with IC₅₀ in the picomolar range.

Why are CP32M and its parent peptide CP621–652 effective against HIV-1 variants resistant to T20, C34, and T1249? We believe that this is because they have different target sites in the gp41 NHR region. Early in vitro studies indicate that HIV-1 acquires T20 resistance by mutations in the “GIV” motif (positions 36–38 based on reference HIV-1_HXB2 gp41 numbering, underlined in Fig. 1B) of the gp41 NHR region (11). Subsequent clinical data show that HIV-1 isolates from patients failing therapy with T20 also contain mutations in the NHR region of gp41 (amino acids 36–45: GIVQQQNNLL) (14, 15). Although two changes within the amino acids 36–45 domain have been observed in some patients resistant to T20 therapy, in most cases a single mutation alone can mediate resistance (16, 17). These findings suggest that the GIV motif may be a critical binding site for T20. Indeed, Chang and colleagues have shown that the LLSGIV stretch in NHR is a crucial docking site for T20 (18, 19).

Our previous studies demonstrated that the CHR region of HIV-1 gp41 contains three functional domains (20): a pocket-binding domain (amino acids 628–635, blue in Fig. 1B) that can bind to the pocket-forming region in NHR (red in Fig. 1B), an HR-binding domain (amino acids 628–666) which is able to interact with the 4–3 heptad repeat (HR) sequences in the gp41 NHR, and a tryptophan-rich lipid-binding domain (amino acids 666–673, orange in Fig. 1B) that has a tendency to bind lipid membranes (20). The helical packing interactions between the NHR and CHR play an essential role in viral infectivity (21–23). In particular, the hydrophobic and ionic interactions of the deep pocket region in the C terminus of the NHR groove and pocket-binding residues from the CHR can determine the conformation and stability of the fusion-active gp41 6-HB structure (1, 19, 24). T20 contains the HR- and lipid-binding domains. It inhibits HIV fusion by binding to the HR sequences in NHR, including the GIV motif, through its N-terminal HR-binding domain and interacting with the lipid membrane on the target cell, via its C-terminal lipid-binding domain (25). Mutations of the conserved GIV motif may affect the binding of T20 to the HR sequence in NHR, resulting in significant reduction of T20-mediated inhibitory activity on HIV fusion and entry. C34 contains the pocket-binding domain and shares with T20 the HR-binding sequence and thus associates with NHR to form a 6-HB through its interaction with both pocket-forming and HR-sequences in NHR. The pocket-forming region and the LLSGIV stretch in NHR are critical docking sites for C34 (18, 19). Thus, mutation of the GIV motif in viral gp41 may also affect C34 binding to the HR sequence, leading to resistance to C34. However, binding of C34 to the hydrophobic pocket region may partially compensate the decreased binding of C34 to NHR. Therefore, the viruses with GIV mutation in gp41 may be less resistant to C34 than T20 (26). T1249, a second generation HIV fusion inhibitor, contains all three functional domains, including pocket-, HR- and lipid-binding sequences. However, it functions more like T20 (25). Therefore, T20-resistant strains with GIV mutations are also insensitive to T1249 (27).

Unlike T20 and C34, CP621–652 does not contain the GIV-binding sequence, but consists of the pocket-binding domain and the ⁶²¹QIWNNMT⁶²⁷ motif, which is located at the upstream region of the CHR and immediately adjacent to the pocket-binding domain and is highly important for the stabilization of the gp41 core structure (10). Therefore, the mutations of the GIV motif may have little or no effect at all on the interaction of CP621–652 with the viral gp41 NHR region and consequently on the effectivity of CP621–652 against T20-resistant HIV-1 strains.

Like its parent peptide CP621–652, CP32M contains no GIV-binding sequence (Fig. 1 B and C). It is expected to be efficient in inhibiting infection by T20-resistant HIV-1 variants with GIV mutations in the gp41 NHR region. After optimization of the CP621–652 sequence, the engineered CP32M demonstrated improved anti-HIV activity. Biophysical characterization showed that CP32M could form highly stable 6-HBs with the counterpart NHR peptide T21 that contains no GIV motif, and had a Tm value of 94°C, while the 6-HB formed by CP621–652 and T21 had a Tm of 81°C (Fig. 3D). This result suggests that CP32M may target the NHR with higher affinity than CP621–652. This may also explain why CP32M is much more potent than CP621–652 in inhibiting infection by HIV-1 strains resistant to T20, C34, and T1249. All these results suggest that CP32M, which has a shorter peptide sequence than T20 (36-mer) and T1249 (39-mer), has great potential to be further developed as a unique anti-HIV drug for treatment of HIV/AIDS patients who have failed to respond to the first and second generation HIV fusion inhibitors.

Materials and Methods

Peptide Synthesis.

A set of peptides derived from the NHR (N36 and T21) or CHR (CP621–652, C34, and T20) of HIV-1 gp41 and CP32M (Fig. 1) were synthesized by a standard solid-phase FMOC method using an Applied Biosystems model 433A peptide synthesizer. All peptides were acetylated at the N termini and amidated at the C termini. The peptides were purified to homogeneity (>95% purity) by HPLC and identified by laser desorption mass spectrometry (PerSeptive Biosystems, Framingham, MA). The concentration of peptides was determined by UV absorbance and a theoretically calculated molar-extinction coefficient ε (280 nm) of 5500 mol/L⁻¹·cm⁻¹ and 1490 mol/L⁻¹·cm⁻¹ based on the number of tryptophan (Trp) residues and tyrosine (Tyr) residues (all of the peptides tested contain Trp and/or Tyr), respectively.

Circular Dichroism (CD) Spectroscopy.

CD spectroscopy was performed as previously described (24). Briefly, an N peptide was incubated with a C peptide (10 μM) at 37°C for 30 min. The CD spectra of the isolated peptides and their mixtures were acquired on Jasco spectropolarimeter (Model J-715, Jasco Inc., Japan). The α-helical content was calculated from the CD signal by dividing the mean residue ellipticity at 222 nm by the value expected for 100% helix formation (i.e., 33,000° cm² dmol⁻¹) according to the previous studies (28, 29). Thermal denaturation was monitored at 222 nm by applying a thermal gradient of 2°C/min in the range of 4–98°C. The melting curve was smoothened, and the midpoint of the thermal unfolding transition (Tm) values was calculated using Jasco software utilities as described previously (30).

Native Polyacrylamide Gel Electrophoresis (N-PAGE) Assay.

N-PAGE was carried out to determine the 6-HB formation between the N and C peptides as described previously (31). Briefly, N peptide T21 was mixed with C peptide CP32M (40 μM) and was loaded onto a 10 × 1.0-cm precast 18% Tris-glycine gel (Invitrogen, Carlsbad, CA). Gel electrophoresis was carried out with 125 V constant voltage at room temperature for 2 h. The gel was then stained with Coomassie Blue and imaged with a FluorChem 8800 Imaging System (Alpha Innotech).

Binding Assays by Size-Exclusion Chromatography (32).

T21 was mixed with CP32M (final concentration = 0.20 mM) at a molar ratio of 1:1 in 50 mM sodium phosphate/150 mM NaCl (pH 7.2) and incubated at 37°C for 30 min. The mixture or peptide (30 μl) was applied to the TSK-G 3000SW_xl HPLC column equilibrated with 50 mM sodium phosphate/150 mM NaCl and eluted at 0.8 ml/min, and fractions were monitored at 214 nm.

Sedimentation Equilibrium Centrifugation.

Sedimentation equilibrium experiments were performed using an Optima XL-I analytical ultracentrifuge (Beckman) equipped with a standard two-channel cell in an An-60 Ti rotor (33). The designated peptide concentration was 25 μM in buffer consisting of 50 mM sodium phosphate/100 mM NaCl (pH 7.4), and the complex was composed of 12.5 μM N peptide (T21) and 12.5 μM C peptide (CP32M). The samples were run at 25,000 or 33,000 rpm at 20°C for 24 h. Absorbance monitoring was performed at 280 nm. The apparent molecular weight (MW_app) was obtained by fitting the data to self-association using the sedimentation analysis software supplied by Beckman. The partial specific volumes used for T21 and CP32M were 0.738 and 0.729 respectively, as calculated from the mass average of the partial specific volumes of the individual amino acids.

Inhibition of CP32M on 6-HB Formation.

Inhibitory activity of the peptides (CP32M, CP621–652, C34, and T20) on the 6-HB formation was measured by a modified ELISA-based method as previously described (10). Briefly, a 96-well polystyrene plate was coated with a 6-HB specific monoclonal antibody NC-1 IgG (4 μg/ml in 0.1 M Tris, pH 8.8). A test peptide at graded concentrations was mixed with C34-biotin (0.25 μM) and incubated with N36 (0.25 μM) at room temperature for 30 min. The mixture was then added to the NC-1-coated plate, followed by incubation at room temperature for 30 min and washing with a washing buffer (PBS containing 0.1% Tween 20) three times. Then, streptavidin-labeled horseradish peroxidase (Invitrogen) and the substrate 3,3′,5,5′-tetramethylbenzidine (Sigma) were added sequentially. Absorbance at 450 nm (A₄₅₀) was measured using an ELISA reader (Ultra 384, Tecan). The percentage of inhibition by the peptides and the IC₅₀ values were calculated as previously described (34).

Cell–Cell Fusion Assay.

A dye transfer assay was used for detection of HIV-1-mediated cell–cell fusion as previously described (35). Briefly, Calcein-AM-labeled H9/HIV-1_IIIB-infected cells were incubated with MT-2 cells (ratio = 1:5) at 37°C for 2 h in the presence or absence of the test peptide. The fused and unfused calcein-labeled HIV-1-infected cells were counted under an inverted fluorescence microscope (Zeiss) with an eyepiece micrometer disk. The percentage of inhibition of cell–cell fusion and the IC₅₀ values were calculated as described before (35).

Inhibition of HIV-1_IIIB and T20-Resistant Virus.

The inhibitory activity of CP32M, T20, or C34 on infection by various T20-resistant virus isolates and laboratory-adapted HIV-1 strain (HIV-1_IIIB) was determined as previously described (35). In brief, 1 × 10⁴ MT-2 cells were infected with HIV-1 isolates at 100 TCID₅₀ (50% tissue culture infective dose) in 200 μl culture medium in the presence or absence of the test peptide overnight. Then the culture supernatants were removed and fresh media were added. On the fourth day postinfection, 100 μl of culture supernatants were collected from each well, mixed with equal volumes of 5% Triton X-100, and assayed for p24 antigen by ELISA.

Inhibition of CP32M on Primary Viruses.

The inhibitory activity of CP32M against a panel of primary HIV-1 isolates was determined as previously described (35). Briefly, the PBMCs were isolated from the blood of healthy donors using a standard density gradient (Histopaque-1077, Sigma) centrifugation. After incubation at 37°C for 2 h, the nonadherent cells were collected and resuspended at 5 × 10⁵/ml in RPMI medium 1640 containing 10% FBS, 5 μg of phytohemagglutinin (PHA)/ml, and 100 U of interleukin-2/ml, followed by incubation at 37°C for 3 days. The PHA-stimulated cells were infected with the corresponding primary HIV-1 isolates at a multiplicity of infection (MOI) of 0.01 in the absence or presence of CP32M at graded concentrations. The supernatants were collected 7 days postinfection and tested for p24 antigen by ELISA.