Interaction of HIV-1 gp41 Core with NPF Motif in Epsin

The human immunodeficiency virus, type 1 (HIV-1), gp41 core plays an important role in fusion between viral and target cell membranes. We previously identified an HIV-1 gp41 core-binding motif HXXNPF (where X is any amino acid residue). In this study, we found that Asn, Pro, and Phe were the key residues for gp41 core binding. There are two NPF motifs in Epsin-1-(470–499), a fragment of Epsin, which is an essential accessory factor of endocytosis that can dock to the plasma membrane by interacting with the lipid. Epsin-1-(470–499) bound significantly to the gp41 core formed by the polypeptide N36(L8)C34 and interacted with the recombinant soluble gp41 containing the core structure. A synthetic peptide containing the Epsin-1-(470–499) sequence could effectively block entry of HIV-1 virions into SupT1 T cells via the endocytosis pathway. These results suggest that interaction between Epsin and the gp41 core, which may be present in the target cell membrane, is probably essential for endocytosis of HIV-1, an alternative pathway of HIV-1 entry into the target cell.

The interaction of the viral envelope glycoprotein surface subunit gp120 with the primary receptor CD4 (1) and a chemokine coreceptor (CXCR4 or CCR5) (2) is the first step of human immunodeficiency virus, type 1 (HIV-1), 3 entry into the target cell. Then the fusion peptide at the gp41 N terminus inserts into the target cell membrane. Subsequently, the N-and C-terminal heptad repeat (NHR and CHR, respectively) regions associate to form a six-helix bundle (6-HB; also known as trimer-of-heterodimers or trimer-of-hairpins), which represents the gp41 core structure (3)(4)(5). Formation of the 6-HB is believed to bring the viral and target cell membranes into close proximity to facilitate their merging (3,6,7).
We have previously demonstrated that HIV-1 gp41 binds to some proteins with molecular masses of 45 and 62 kDa (P45 and P62, respectively) on the surface of T and B lymphocytes and monocytes via its N-or C-terminal domain (8,9). Others have reported that HIV-1 gp41 interacts with a 60-kDa heat-shock protein-like molecule (10) and human leukocyte elastase (11). Alfsen et al. (12) have shown that HIV-1 binds to the epithelial glycosphingolipid galactosylceramide, which is an alternative receptor for HIV-1, via a site involving the conserved ELDKWA epitope in gp41. It has been reported that lipid rafts, consisting of sphingolipids and cholesterol, are being utilized by HIV-1 to enter the target cells (13). Hovanessian et al. (14) have shown that gp41 binds to a major integral protein in the membrane of caveolae, caveolin-1, and forms a complex in the HIV-1-infected cells. We demonstrated that the HIV-1 gp41 core interacts with a hydrophobic motif ⌽XXXX⌽XX⌽ (where X is any amino acid and ⌽ is W, Y, or F) in the scaffolding domain of caveolin-1 (15). Wang and co-workers (16 -18) have reported that gp41 and the peptides derived from gp41, e.g. N36, T20, and T21, attract and activate human phagocytes by using G-protein-coupled formyl peptide receptors. We also identified a gp41 core-binding motif HXXNPF by screening the phage display peptide library (19) and found that the peptides containing the HXXNPF motif could inhibit HIV-1 envelope glycoprotein-mediated syncytium formation (20). These data suggest that HXXNPF motif-containing molecules may specifically bind to the HIV-1 gp41 core to block HIV-1 entry.
In this study, we sought to identify the cellular gp41 corebinding proteins. Mutational analysis demonstrated that the most conservative residues in the gp41-binding motif HXXNPF were Asn, Pro, and Phe. By searching the protein data base, we found that Epsin contains the NPF motifs in the C-terminal domain. Epsin plays an important role in the clathrin-mediated endocytosis (21). Epsin N-terminal homology (ENTH) domain binds to the cell membrane through the lipid PIP 2 and phosphatidylinositol 1,4,5-trisphosphate to induce positive membrane curvature and tubulation of plasma membrane. Its central region contains eight copies of DPW motifs that are necessary for binding of Epsin to the adaptor protein complex 2 (AP-2). A clathrin-binding site, which is adjacent to the DPW motifs in Epsin, can simultaneously interact with two clathrin terminal domains (22). The C-terminal region of Epsin consists of three NPF motifs responsible for association of Epsin with the Eps15 homology (EH) domain of Eps15, intersectin, and POB1 during the process of endocytosis (23).
We constructed a recombinant protein, Epsin-1-(470 -499), that spans the amino acid residues 470 -499 of Epsin and contains two NPF motifs. Epsin-1-(470 -499) bound significantly to the gp41 expressed in mammalian cells and interacted with the gp41 core. Endocytosis of HIV-1 particles into T cells could be blocked by the peptide containing the Epsin-1-(470 -499) sequence. These results suggest that via the unique NPF motifs, Epsin may interact with the gp41 core of HIV-1, perhaps in the target cell membrane. This interaction may involve the pathogenesis of HIV-1 infection as well as an alternative pathway, i.e. endocytosis.

EXPERIMENTAL PROCEDURES
Cell Lines and Transient Transfection-293T cells were grown in Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% fetal bovine serum. Transfection was performed using Vigofect reagent (Vigorous Biotechnology, Inc., Beijing, China) according to the manufacturer's protocol. Cells were plated in a 6-well tissue culture plate 24 h prior to transfection. Cells at 50 -70% confluency were transfected with 5 g of plasmid DNA. Cells were collected 48 h after transfection. SupT1 T cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin.
Construction of Mutants of L7.8-g3p*-We used a standard site-directed mutagenesis procedure to introduce mutations into the sequence of L7.8-g3p*, a fragment of the gene 3 protein (g3p) of the phage clone L7.8 containing the gp41 core-binding motif. Expression plasmids were constructed by inserting the DNA fragments encoding H19A, N22A, P23A, and F24A mutants of L7.8-g3p*, respectively, into an expression vector of the pET-28b series. After ligation and electroporation, recombinant clones were identified by PCR. The inserted DNA fragments were sequenced to exclude any undesirable mutations. The same procedures for expression and purification of the wild-type L7.8-g3p* (20) were used for the L7.8-g3p* mutants.
Surface Plasmon Resonance (SPR) Assay-The kinetics of the binding affinity of the mutants of L7.8-g3p* (H19A, N22A, P23A, and F24A) to N36(L8)C34 were determined by SPR using the Biacore 2000 system (GE Healthcare) at 25°C. N36(L8)C34 (10 g/ml) was immobilized onto the CM5 sensor chip according to the amine coupling protocol, and the unreacted sites were blocked with 1 M Tris-HCl (pH 8.5). The association reaction was initiated by injecting the peptide (0.5 M) at a flow rate of 5 l/min. The dissociation reaction was done by washing with PBS. Wild-type L7.8-g3p* was used as a control. At the end of each cycle, the sensor chip surface was regenerated with 0.1 M glycine-HCl (pH 2.5) for 30 s.
Pulldown Assay-The in vitro pulldown assay was carried out as described previously (23). The pellet of the 293T cells expressing the EGFP-tagged Epsin-1-(470 -499) was solubilized in 0.2 ml of ice-cold lysis buffer (1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na 3 VO 4 , 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, 5 mg/ml pepstatin) at 4°C for 30 min. The supernatants were collected after centrifugation at 12,000 rpm for 10 min at 4°C. GST-Eps15-EH2 domain conjugated glutathione-Sepharose beads were incubated with the supernatants on ice for 2 h. GST-conjugated glutathione-Sepharose beads were used as a control. The beads were washed five times with lysis buffer. The bound protein was detected by Western blot with rabbit polyclonal anti-EGFP antibody. Similar procedures were used for pulling down the complex of GST-N36(L8)C34 with EGFP-Epsin-1-(470 -499) expressed in the transfected 293T cells. EGFP was used as a control.
For pulling down the complex of GST-Epsin-1-(470 -499) with human IgG Fc-tagged recombinant soluble gp41 (rsgp41) expressed in the transfected 293T cells, 293T cells lysates were prepared by incubating 293T cells in 0.2 ml of lysis buffer at 4°C for 30 min. After centrifugation at 12,000 rpm for 30 min at 4°C, the supernatants containing human IgG Fc-tagged rsgp41 (rsgp41-Fc) were collected and incubated with GST and GST-Epsin-1-(470 -499), respectively. Protein G-Sepharose-beads (10 l) were added, followed by incubation on ice for 2 h with shaking. The beads were washed with TBS buffer (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl (pH 7.4)) five times. The bound gp41 was eluted by heating with SDS-PAGE sample buffer and detected by Western blot with the polyclonal anti-EGFP antibody.
Enzyme-linked Immunosorbent Assay (ELISA)-To test whether human IgG Fc-tagged rsgp41 (rsgp41-Fc) expressed in the cytoplasm of 293T cells could form core structures, 100 l of rsgp41-Fc-expressing 293T cell lysates at 4-fold serial dilutions in 0.1 M NaHCO 3 were coated in the wells of microplates. The lysates of wild-type 293T cells were used as a control. After blocking and washing, 50 l of the mAb NC-1 (10 g/ml) were added and incubated at room temperature for 2 h, followed by addition of peroxidase-conjugated anti-mouse IgG and substrate o-phenylenediamine dihydrochloride, sequentially. Absorbance at 450 nm (A 450 ) was measured.
Competition ELISA-Mouse anti-SJ-3136 antisera were obtained from BALB/c mice that were immunized with the peptide SJ-3136, which was conjugated with bovine serum albumin three times with Freund's adjuvant. To test whether SJ-3136 could compete with NC-1 in binding to the gp41 core, the wells of a microplate (Costar, High Binding) were coated with 100 l of rabbit IgG (2 g/ml) against the gp41 6-HB core formed by the peptides N36 and C34 (7). After blocking with blocking buffer, 100 l of N36(L8)C34 at 5 g/ml in PBS were added into each well and incubated at 37°C for 30 min. After extensive washes, 25 l of SJ-3136 at graded concentrations were added and incubated at 37°C for 60 min, followed by addition of 25 l of NC-1 (0.1 g/ml). After incubation at 37°C for 30 min, the amount of bound NC-1 was detected by adding biotin-goat anti-mouse IgG, streptavidin-labeled horseradish peroxidase, and 3,3Ј,5,5Ј-tetramethylbenzidine substrate solution, sequentially. Absorbance at 450 nm was recorded.
Preparation of HIV-1 Virions Labeled with GFP-293T cells were cotransfected with HIV-1 pNL4-3 proviral DNA (24 g) and pEGFP-Vpr (24 g) using Lipofectamine (60 l, Invitrogen) according to the manufacturer's protocol. After overnight culture at 37°C, the medium was replaced completely with fresh culture medium. Two days after transfection, supernatants of cells were collected and centrifuged at 4,000 rpm for 15 min. The supernatants were transferred to sterile ultracentrifuge tubes and then centrifuged in an SW28 rotor at 20,000 rpm at 4°C for 2 h to sediment the viral particles. The virions in the pellet were resuspended in RPMI 1640 complete medium (0.5 ml) and stored in aliquots at Ϫ80°C until use.
Flow Cytometric Analysis of HIV-1 Entry into Target Cells via Endocytosis and Fusion-HIV-1 entry into target cells through endocytosis and fusion was determined by flow cytometry as described previously (24). Briefly, human SupT1 T cells (2 ϫ 10 5 ) were suspended in fresh medium in the absence or presence of inhibitors, i.e. AMD3100 (40 g/ml), chlorpromazine (20 g/ml), and SJ-3136 (1 mg/ml) and combinations thereof, at 37°C for 30 min, followed by addition of GFP-labeled virions (200 ng of p24 Gag). After incubation at 37°C for 4 h, the cells were washed twice with PBS and then treated with a trypsin/ EDTA solution (1ϫ; Sigma) for 4 min at 37°C to remove surface-bound virions. Cells were fixed with 1% paraformaldehyde/PBS solution, and cellular GFP virion uptake was measured by flow cytometry (Canto, BD Biosciences).

Mutations of the Key Residues in the gp41
Core-binding Motif of the L7.8-g3p* Mutants Resulted in Loss of gp41 Core Binding Activity-To determine which key residues of the HXXNPF motif of L7.8-g3p* are responsible for binding to the gp41 core, His-19, Asn-22, Pro-23, and Phe-24, respectively, were substi-tuted with alanine by site-directed mutagenesis. Four mutants of L7.8-g3p* (named H19A, N22A, P23A, and F24A) were constructed. E. coli bearing the expression vectors was grown to log-phase, and fusion protein expression was induced by adding isopropyl ␤-D-thiogalactopyranoside. The His-tagged wildtype L7.8-g3p* and four L7.8-g3p* mutants purified by metal affinity chromatography from the cell lysates had a molecular mass of about 10 kDa (Fig. 1A). The respective binding affinities of the mutants to N36(L8)C34 were compared with that of the wild-type L7.8-g3p* by SPR. As shown in Fig. 1B, the mutant H19A showed significantly reduced binding affinity, and the mutants N22A, P23A, and F24A completely lost their ability to bind to N36(L8)C34. These results demonstrate that the residues Asn, Pro, and Phe in the HXXNPF motif were definitely essential for interaction with the gp41 core, suggesting that the protein containing the NPF motif may specifically bind to the HIV-1 gp41 core.
Interaction of Epsin-1-(470 -499) with Eps15-EH2-We learned from a search of the protein data base that the NPF motif is present in many proteins, e.g. Epsin (25, 26), Ap180   (27), Numb (28), and Hrb (29). No sequence homology was found in these proteins except for the presence of one or more copies of the NPF motif. The NPF motif was demonstrated to be indispensable in vivo for the binding of the identified proteins to the EH domains of Eps15 (30). Eps15 is important for an endocytic entry pathway utilized by HIV-1. Expression of a dominant-negative mutant of Eps15 leads to reduction of HIV entry in HeLa cells by about 95%, confirming that the endocytosis pathway is also necessary for causing infection (21). Epsin is the Eps15-interacting protein and promotes the assembly of clathrin. Therefore, we speculated that the gp41 core might be involved in the clathrin-dependent endocytosis through interaction with the NPF motif of Epsin. It was reported that the fragment of Epsin-(433-551), which contains three copies of NPF motifs, bound to the EH domain of Eps15 directly (23). In this study, Epsin-1-(470 -499) containing two copies of NPF motif (Table 1) was amplified by PCR. The EH2 domain of Eps15 was constructed as a GST fusion protein (GST-Eps15-EH2) ( Table 1) and purified with the glutathione-Sepharose 4B affinity column. As shown in Fig. 2A, the purified GST-Eps15-EH2 migrated through the SDS-PAGE revealing a band with an expected molecular size of 37 kDa.
Interaction of GST- Epsin-1-(470 -499) with Recombinant Soluble gp41 (rsgp41)-It has been reported that the purified rsgp41 has a tendency to form a core-like structure, i.e. its NHR and CHR regions may associate to form a 6-HB (31). The plasmid of pcDNA3.0-pIg-rsgp41 was transfected into the 293T cells to express human IgG Fc-tagged rsgp41 (rsgp41-Fc) fusion protein in cytoplasm. NC-1, a mAb that is specific for the gp41 core conformation (7), could react with the lysates of rsgp41-Fc-expressing 293T cells in ELISA, whereas the control wild-type 293T cells showed no binding activity (Fig. 4), suggesting that rsgp41-Fc contains the gp41 core structure. Therefore, it was of interest to investigate whether the rsgp41 containing the core structure could also bind to Epsin-1-(470 -499).
Inhibitory Activity of the Peptide SJ-3136 on Endocytosis of HIV-1 Particles-To further examine whether SJ-3136 could block the entry of HIV-1 virus via the endocytosis pathway, we constructed GFP-vpr-labeled X4-tropic HIV-1 virions and determined the inhibitory activity of SJ-3136 on endocytosis of HIV-1 virions. AMD3100, a fusion inhibitor targeting CXCR4, and chlorpromazine, a drug disrupting clathrin-mediated endocytosis, were used as controls. As shown in Fig. 7 and Table  2, after 4 h of incubation with a viral input of 200 ng of p24, AMD3100, chlorpromazine, and SJ-3136, when tested individually, effectively inhibited HIV-1 entry into SupT1 T cells (45.19, 56.27, and 36.44%, respectively). The inhibitory activity of the AMD3100/chlorpromazine combination on virus entry (97.08%) was significantly higher than that of AMD3100 or chlorpromazine alone. Combination of SJ-3136 and AMD3100 exhibited significantly higher inhibition (78.68%) of HIV-1 entry than SJ-3136 or AMD3100 alone. However, combination of SJ-3136 and chlorpromazine exhibited no significant increase in inhibition of virus entry (58.6%). These results suggest that SJ-3136, like chlorpromazine, inhibits HIV-1 virion entry via the endocytosis pathway.

DISCUSSION
HIV-1 uses fusion and endocytosis as the main and alternative pathways, respectively, to enter the target cells (24). In the case of fusion, the viral and target cell membranes are allegedly brought into close proximity by the gp41 core to facilitate merging of the membranes (3). Some virions may have lost their fusion ability, possibly resulting from the interaction between gp120 on virions and soluble CD4 molecules that are released from CD4 ϩ cells, triggering the gp41 core formation (31,32). Furthermore, the degraded fragments from HIV-1 gp41 may interact with the viral gp41 NHR and CHR regions to form heterologous 6-HBs, blocking further progress of membrane fusion. Although lacking fusion ability, these virions may still be capable of entering the target cells via endocytosis.
It has been shown that two principal types of endocytosis mediated by caveolae/raft and clathrin are used by different viruses (33,34). Manes et al. (16) have demonstrated that lateral assembly mediated by the membrane raft microdomain is required for HIV-1 entry. Our previous studies suggest that interaction of the HIV-1 gp41 core with the scaffolding domain of caveolin-1 may be involved in the endocytosis of HIV-1 (20). Eps15 plays an important role in clathrin-mediated endocytic entry pathway utilized by HIV-1 because a dominant-negative mutant of Eps15 results in about 95% reduction of HIV entry into HeLa cells (21). The EH domain of Eps15 interacts with the C-terminal region of Epsin during the process of endocytosis (23).
We previously identified an HIV-1 gp41 core-binding motif, HXXNPF, which also contains the NPF sequence (19). We thus hypothesized that the NPF sequence may be the key residues in the HIV-1 gp41 core-binding proteins. In this study, we confirmed that Asn, Pro, and Phe were indeed the key residues, because mutations of these residues in the gp41 core-binding motif of L7.8-g3p* resulted in loss of gp41 core-binding activity (Fig. 1).
Because the C-terminal region of Epsin contains three NPF motifs and Epsin is an essential accessory factor of clathrinmediated endocytosis, which controls the internalization of extracellular macromolecules and cell surface receptors (35), we envisaged that Epsin might interact via its NPF motif with the gp41 core to mediate the HIV-1 endocytosis. To prove this hypothesis, we constructed a fusion protein, GST-Epsin-1-(470 -499), containing two copies of NPF motifs, and tested its binding activity to Eps15. The results indicate that Epsin-1-(470 -499) can interact with Eps15-EH2 (Fig. 2B), confirming that Epsin-1-(470 -499) retains the binding ability of Epsin to Eps15. We further demonstrated that Epsin-1-(470 -499) could bind very well to the gp41 core formed by the polypeptide N36(L8)C34 (Fig. 3). It was also capable of capturing gp41 expressed in the cytoplasm (Fig. 5B), and which spontaneously formed 6-HBs (Fig. 4).
To determine the potential role of Epsin-1-(470 -499) in HIV-1 entry mediated by the gp41 core, a peptide containing the Epsin-1-(470 -499) sequence (SJ-3136) was synthesized and tested for its inhibitory activity on HIV-1 entry into the target cells. Schaeffer et al. (24) have employed flow cytometry to analyze the entry of GFP-Vpr-labeled HIV-1 particles into SupT1 cells and used AMD3100, a CXCR4-specific fusion inhibitor, and chlorpromazine, an endocytosis inhibitor, to determine the pathway of viral entry. Using the same approaches, we tested the potential inhibitory activity of SJ-3136 on HIV-1 entry into target cells via fusion or endocytosis. As shown in Fig. 7 and Table 2, the combination of AMD3100 and chlorpromazine resulted in significantly higher inhibitory activity on HIV-1 entry than that of AMD3100 and chlorpromazine tested separately. This confirms that these two compounds have different mechanisms of action and that their combination has synergistic effects on the inhibition of HIV-1 entry. Similarly, the combination of SJ-3136 with AMD3100 exhibited more potent inhibition of HIV-1 entry than that of SJ-3136 or AMD3100 alone. These results suggest that, like chlorpromazine, peptide SJ-3136 inhibits HIV-1 entry via the endocytosis pathway.
Based on these findings, we propose that the HIV-1 gp41 core may interact with the NPF motifs in Epsin to facilitate endocytosis of HIV-1 particles. This interaction is perhaps triggered with the assistance of a number of cellular proteins, such as clathrin, PIP 2 , AP-2, and dynamin. This hypothesis is in agreement with the observation that HIV-1 virions could traverse between cells by a clathrin-dependent endocytic process (36). In the cytosolic domain of gp41 there are two highly conserved tyrosine-based motifs ( 712 YSPL and 768 YHRL) and a conserved GYXXဧ motif, which can specifically bind to the clathrin-associated protein AP-2. This results in the internalization of the HIV-1 envelope glycoprotein (37,38) suggesting that the cytosolic domain of gp41 may also play an important role in the endocytosis of HIV-1. One may question where the HIV-1 gp41 extracellular core domain interacts with Epsin, which is normally localized inside cell. We speculate that the gp41 core may bind to Epsin in the target cell membrane. Previous studies indicated that the gp41 6-HB core could bind to the lipid membranes (39), and its NHR domain could further interact with the target cell membrane to facilitate fusion pore formation (40). Most recently, Benferhat et al. (41) reported that the caveolin-1 binding domain in the HIV-1 gp41, which partially overlaps with the CHR region, could penetrate the cell membrane to bind caveolin-1 inside the cell. Epsin can also bind to the plasma membrane through the interaction between the ENTH domain of Epsin and the lipid PIP 2 to initiate the membrane curvature and coat-pit formation and promote the assembly of clathrin. Mutations in the ENTH domain result in abolishment of Epsin binding to the lipid membrane (42). It was reported that Epsin could interact with the epithelial sodium channel expressed on the cell surface and mediate the clathrin-based endocytosis of the epithelial sodium channel (43).
This study presents the initial evidence for a direct interaction between the HIV-1 gp41 core and the NPF motif of Epsin, probably in the target cell membrane, which may facilitate the endocytosis of HIV-1 particles. The potential site(s) where gp41 core interacts with Epsin and the gp41 core-binding motif(s) in other proteins, such as AP180, Numb, and Hrb, also merit further investigation to provide information for understanding the endocytosis of HIV-1 virions and the pathogenesis of HIV-1 infection, as well as for rational design of novel HIV-1 entry inhibitors. a The concentrations of the compounds used are as follows: AMD3100 ϭ 40 g/ml; chlorpromazine (Chl) ϭ 20 g/ml; SJ-3136 ϭ 1 mg/ml. Positive control is cells ϩ virus; negative control is cells ϩ PBS. The percent inhibition of virus entry into target cells via fusion (e.g. AMD3100) or endocytosis (e.g. chlorpromazine) was determined as described under "Experimental Procedures."