Hydrophobic mutations in buried polar residues enhance HIV-1 gp41 N-terminal heptad repeat–C-terminal heptad repeat interactions and C-peptides’ anti-HIV activity
Zheng, Baohuaa,b; Wang, Kunb; Lu, Luc; Yu, Feid; Cheng, Maoshenga; Jiang, Shiboc,d,∗; Liu, Kelianga,b,∗; Cai, Lifengb,∗
aMOE Key Laboratory of Structure-Based Drug Design & Discovery, Shenyang Pharmaceutical University, Shenyang
bDepartment of Medicinal Chemistry, Beijing Institute of Pharmacology & Toxicology, Beijing
cKey Laboratory of Medical Molecular Virology of MOE/MOH, Shanghai Medical College, Fudan University, Shanghai, China
dLindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.
∗Lifeng Cai, Keliang Liu and Shibo Jiang contributed equally to this work.
Correspondence to Lifeng Cai, PhD, Beijing Institute of Pharmacology & Toxicology, 27 Taiping Road, Haidian District, Beijing 100850, China. Tel: +86 10 66930640; fax: +86 10 68211656; e-mail: email@example.com.
Received 11 November, 2013
Revised 23 January, 2014
Accepted 5 February, 2014
To investigate the effect of mutations in a highly conserved buried polar area on the function of HIV-1 gp41.
During HIV-1 entry, a six helical bundle (6-HB) formation between the C-terminal and N-terminal heptad repeat (CHR and NHR) of gp41 provides energy for virus cell membrane fusion. In 6-HB, residues at a and d (a–d) positions of CHR directly interact with NHR and are buried. They are considered critical residues for 6-HB stability and for anti-HIV-1 activity of CHR-derived peptides (C-peptides). Most of a–d residues in CHR are hydrophobic, as buried hydrophobic residues facilitate protein stability. However, HIV-1 gp41 CHR contains a highly conserved polar area with four successive buried a–d polar residues: S649/Q652/N656/E659. We mutated these buried polar residues to hydrophobic residues, either Leu or Ile, and studied its effect on the gp41 NHR–CHR interactions and anti-HIV activities of the C-peptides.
We measured the C-peptide mutants’ ability to form 6-HB with NHR, thermal stability of the 6-HBs and C-peptides’ inhibitory activity against both T20-sensitive and resistant HIV-1 strains.
All the mutated C-peptides retained their ability to form stable 6-HB with NHR and strongly inhibited HIV-1 replication. Strikingly, S649L and E659I mutations endow C-peptide with a significantly enhanced activity against T20-resistant HIV-1 strains.
The highly conserved buried a–d polar residues in HIV-1 gp41 CHR can be mutated as a means of developing new fusion inhibitors against drug-resistant HIV-1 strains. The concept can also be utilized to design fusion inhibitors against other viruses with similar mechanisms.
HIV-1 enters and infects host cells through an envelope glycoprotein (Env)-mediated virus cell membrane fusion [1,2]. HIV-1 Env is a complex of noncovalently associated surface unit gp120 and transmembrane subunit gp41. The gp41 ectodomain contains a fusion peptide, an N-terminal heptad repeat (NHR), a C-terminal heptad repeat (CHR) and a membrane-proximal external region (MPER) (Fig. 1a). During HIV-1-cell membrane fusion, a coiled-coil six-helical bundle (6-HB) formation between the gp41 NHR and CHR brings the viral and cellular membranes in close apposition, thus promoting virus cell membrane fusion . The gp41 CHR-derived peptides (C-peptides) are highly potent HIV fusion inhibitors [4,5]. They interact with viral gp41 NHR to form heterologous nonfunctional 6-HB, thereby preventing formation of the homologous functional 6-HB [2,6]. T20 (gp41638-673, Fuzeon, enfuvirtide), a 36-mer C-peptide , was approved for clinical use in 2003 by the U.S. Food and Drugs Administration (FDA) [7,8]. However, its application is limited as a consequence of the high cost of peptide synthesis, rapid proteolysis and high potential to induce drug-resistant viral strains. These drawbacks call for a new generation of fusion inhibitors with improved antiviral and pharmacokinetic profiles [9–11].
Peptide engineering has been applied to the design of next-generation peptide fusion inhibitors based on wild-type HIV-1 gp41 CHR sequences . HIV-1 gp41 6-HB crystal structures have shown that three NHRs form an inner core using residues at a, d (a–d) positions in their helices for self-association and e–g residues to interact with a–d residues in CHR [3,12,13]. In a typical 6-HB, a–d–e residues of the outer CHR helices face the inner NHR helices and are largely buried, while the b–c–f–g residues are largely solvent exposed . In fusion inhibitor design, C-peptides are usually regarded as ligands that interact with the NHR target . The solvent-exposed b–c–f–g residues are considered not critical to CHR–NHR interaction, as they make no direct contact with NHR, and they are also less conserved among HIV-1 strains . Extensive mutations have been made to solvent-exposed residues to increase the activity and pharmacokinetic profiles of C-peptide fusion inhibitors [10,11,15]. Otaka et al. systematically replaced b–c–f–g residues in C34 (gp41628–661) with E-E-K-K residues, respectively, to form double-salt bridges to stabilize α-helical structure, which resulted in the identification of highly potent fusion inhibitors with high solubility that were therefore more suitable for clinical use [16–18]. Dwyer et al. used both salt bridge and alanine substitutions to systemically replace the b-c-f-g residues to enhance helical structure of a C-peptide, C38 (gp41626–663). The resultant peptides formed more stable 6-HBs with the NHR, and some peptides showed high potency against drug-resistant HIV-1 strains . He et al. have carried out detailed structural analysis of CP621–652 peptide and its analogous [19–22]. These studies and other designed peptide fusion inhibitors, such as Sifuvirtide  and T1249 [23,24], which demonstrated that all the solvent-exposed residues could be subjected to mutation to get fusion inhibitors with increased potency and improved pharmacokinetic profiles.
Compared with solvent-exposed residues, the buried face of the HIV-1 gp41 CHR composed of a–d–e residues remains poorly explored in peptide engineering for HIV fusion inhibitor design. These residues are regarded as binding sites of C-peptides that specifically interact with the respective residues in NHR, and they are also highly conserved. In the context of drug design, binding sites and highly conserved groups between natural ligands and their targets are considered critical, thus eliminating the need for change. Formation of HIV-1 gp41 6-HB between NHR and CHR is essentially the result of a coiled-coil interaction wherein hydrophobic residues prefer occupying the a–d positions of the outer helices . The a–d residues in the N-terminal half of C34 (gp41628–661) are hydrophobic or aromatic residues. However, the presence of four successive polar a–d residues in the C-terminal half of C34 (S649/Q652/N656/E659) form a polar area, which spans 13 residues (gp41647–659) (Table 1) [12,13,26]. The CHR polar area is highly conserved among HIV-1, HIV-2 and Simian immunodeficiency virus (SIV) (Fig. 1), and it is considered to match a polar area in NHR required for the formation of polar interaction networks, including salt bridge and hydrogen bond [12,28–32]. In the context of protein folding, hydrophobic interactions of buried hydrophobic residues contribute more energy than electrostatic and polar interaction to stabilize a protein structure, and replacing buried polar residues with hydrophobic ones usually results in a more stable protein . Therefore, we made the presumption that replacing the buried polar residues with hydrophobic residues in the HIV-1 gp41 CHR would enhance its coiled-coil interaction with NHR, thus promoting its use for novel C-peptide fusion inhibitor design.
In this study, we generated hydrophobic mutations in the four successive buried a–d polar residues in C36 (gp41628–663) and investigated their effect on the biological activities of C-peptides and NHR-CHR 6-HB stability. We measured the activity of C36-mutants against HIV-1 Env-mediated cell–cell membrane fusion, as well as their ability to interact with NHR-peptide to form stable 6-HB. We also assessed the potency of these peptides against T20-sensitive and T20-resistant HIV-1 strains. This is the first systematic study reporting on the buried face of HIV-1 gp41 CHR. By complementing previously reported mutations in solvent-exposed residues [10,11,15], we can provide a full view of peptide engineering for C-peptide fusion inhibitors, thus shedding new light on the mechanisms of HIV-1 gp41 NHR–CHR interactions and identifying new directions for novel fusion inhibitor design.
Materials and methods
Peptides were synthesized on a CEM Liberty peptide synthesizer (CEM, Matthews, North Carolina, USA) with a standard solid phase Fmoc-protocol using Rink amide resin (0.44 mmol/g; Nankai Hecheng, Tianjin, China). The carboxyl-termini were amidated and the amino-termini were acetylated. Peptides were cleaved from the resin and deprotected with Reagent K (TFA/m-cresol/water/ethanedithiol at a ratio of 85 : 10 : 2.5 : 2.5). The crude products were precipitated with cold diethyl ether, lyophilized and purified by preparative reverse-phase, high-performance liquid chromatography (PrepLC 4000, Waters) to more than 95% purity. The molecular weight of the peptides was confirmed by MALDI-ToF-MS (Autoflex III; Bruker Daltonics Inc., Billerica, Massachusetts, USA). Peptide concentrations were determined by measuring the ultraviolet (UV) absorbance in 6 mol/l guanidinium hydrochloride at 280 nm, using extinction coefficients 12 660 mol/l/cm for C-peptides and 5690 mol/l/cm for N38.
Cell–cell fusion assay
Cell–cell fusion assays were performed as described earlier [34–36]. HL2/3 cells, which express the cleaved HIV-1 molecular clone HXB2/3gpt  as the effector cells and TZM-bl cells, which express high levels of CD4 and CCR5, along with endogenously expressed CXCR4 as the target cells , were obtained from the NIH AIDS Research and Reference Reagent Program. TZM-bl cells in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum were plated in a 96-well plate (2.5 × 104 cell/well, Corning Costar) and cultured overnight at 37°C. All culture supernatants were removed and fresh culture medium was supplied. Inhibitors were then added, followed by addition of HL2/3 cells (7.5 × 104 cell/well). After coculture for 6 h at 37°C, cell–cell fusion was determined by measuring luciferase activity using the Luciferase Assay System (Promega, Madison, Wisconsin, USA) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, California, USA).
HIV-1 infection assay
The inhibitory activities of C36-mutants on infection by T20-sensitive and T20-resistant HIV-1 strains were determined as previously described . T20-sensitive strain, HIV-1 pNL4-3 with N36G mutation in gp41 (#9489) and its mutants that are resistant to T20 were obtained from the NIH AIDS Research and Reference Reagent Program. Briefly, 1 × 104 MT-2 cells were infected with an HIV-1 strain at 100 TCID50 (50% tissue culture infective dose) overnight in 200 μl of RPMI 1640 medium containing 10% foetal bovine serum in the presence or absence of a test peptide. The culture supernatants were then removed and fresh media were added without inhibitors. 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 as previously described .
Circular dichroism spectroscopy
Circular dichroism spectra were acquired on a MOS-450 system (BioLogic, Claix, France) using 4.0 nm bandwidth, 0.1 nm resolution, 0.1 cm pathlength, 4.0 s response time and 50 nm/min scanning speed. N-peptide was incubated with respective C-peptides at 37oC for 30 min in PBS, pH 7.2. The mixture was then cooled to room temperature for measurement. Spectra were corrected by subtraction of solvent blank. The α-helical content was calculated from the circular dichroism signal by dividing the mean residue ellipticity at 222 nm by the value expected for 100% helix formation (33 000 degree.cm2.d/mol), according to previous studies [40,41]. For circular dichroism thermal denaturation analysis, the temperature was controlled by a BioLogic TCU250 system and circular dichroism spectra were monitored at 222 nm from 15°C to 90°C at 2°C/min scan speed.
Tris-glycine gels (20%) and a BayGene Mini Cell were used for native PAGE (N-PAGE) analysis. N-peptide in water was mixed with C-peptides in PBS and incubated at 37°C for 30 min (final concentration of N and C-peptide 50 μmol/l). The samples were mixed 1 : 1 with 2xTris-glycine native sample buffer (BioRad, Hercules, California, USA) and then loaded onto the gels. Gel electrophoresis was carried out at 120 V constant voltage at room temperature for 2.5 h, and the gel was subsequently stained with Coomassie blue R250.
Selection of N and C-peptides and introduction of mutations in the C-peptides
C36 (HIV-1 gp41628–663) was selected as a template. It contains two more residues than C34 at the C-terminus, thereby providing one additional a residue and having a larger contact interface with NHR (Fig. 1). Consequently, N38 (gp41544–581) was selected to interact with C36 based on the N36-C34 6-HB crystal structure and the antiparallel interaction between the gp41 NHR and CHR . Three solvent-exposed hydrophobic residues (M629, L641, L661, at b, g, f position, respectively) were mutated to polar residues to increase peptide solubility without significantly affecting the NHR–CHR interaction based on previously employed strategies applied to second and third-generation peptide fusion inhibitors [10,11]. The resulted C36M0 showed properties similar to those of C36 with respect to N38 interaction and inhibitory potency in the cell–cell fusion assay (Table 1). It was therefore used as the template for mutation analysis.
In C36M0, a total of 11 a–d residues in direct NHR contact are buried in the 6-HB. Six a–d residues at the N-terminal half are hydrophobic or aromatic residues and interact hydrophobically with the NHR, as typically found in coiled-coil interactions [1,25,42]. Four successive a–d residues at the C-terminal half of C36M0 (S649/Q652/N656/E659) are polar residues. These buried polar residues, despite being highly conserved across HIV-1, HIV-2 and SIV , do not follow the law of protein folding associated with coiled-coil interaction . To better understand the mechanism of gp41 NHR-CHR interaction for fusion inhibitor design, we substituted these polar a–d residues with hydrophobic residues and studied their effect on the biological activity of C36 and on 6-HB formation. Single mutations (S649L, Q652I, N656L and E659I) were introduced to generate peptides C36M1–C36M4, respectively (Table 1).
Replacement of buried polar residues by hydrophobic residues retained the high activity of C-peptides against HIV-1 Env-mediated cell–cell fusion
We first tested the inhibitory effect of the respective C36-mutations on HIV-1 Env-mediated membrane fusion using a cell–cell fusion assay . Native C-peptides are highly potent HIV-1 fusion inhibitors [2,6]. C36 achieved 50% inhibition at a concentration (IC50) of 3.92 nmol/l, and C36M0 exhibited similar inhibitory activity (IC50 = 5.86 nmol/l) (Table 1). All the tested C36-mutants with mutation in the buried polar region showed similar low nmol/l levels of inhibitory potency in the cell–cell fusion assay (Fig. 2), suggesting that mutations could be introduced into critical C-peptide binding sites as a means of designing novel fusion inhibitors or improving the pharmacokinetics of peptide drugs.
C36 mutants retained the ability to form stable 6-HB with N38
C-peptide fusion inhibitors interact with HIV-1 gp41 NHR to form a hetero 6-HB, thus preventing fusogenic 6-HB formation [3,43–45]. 6-HB formation between N and C-peptides is characterized by an increased α-helical content concomitant with the formation of a high molecular weight complex [39,45]. We first examined the effect of the mutations on 6-HB formation between C36-mutants and N38 by circular dichroism spectroscopy. C36 and N38 formed random and partial helical structures in solution, respectively, when separated, as characterized by a smaller circular dichroism signal at 222 nm (Fig. 3b). When they were mixed, the circular dichroism signals at 222 nm were significantly increased, and a double minima at 208/222 nm, typical for dispersed α-helical folding, was observed. The circular dichroism signal of the C36-N38 mixture (C36/N38) was significantly larger than that of the mathematical sum of those of isolated peptides (C36 and N38), indicating the induction of a large α-helical structure upon interaction, which is typical for 6-HB formation [1,3,40]. C36M0–C36M4 all interacted with N38 to form complexes with highly α-helical content (Fig. 3c), indicating that they retained their ability to interact with the gp41 NHR to form 6-HBs.
We next used N-PAGE to further confirm 6-HB formation between respective C36 mutants and N38. Only stable and tightly associated complexes can form new bands in N-PAGE, which would confirm the circular dichroism results . Generally, C-peptides migrate rapidly through N-PAGE; however, migration slows down after interacting with N-peptide to form stable 6-HBs, and a new band with a lower migration rate is observed. N-peptides usually do not show up in N-PAGE as a result of their net positive charge, which causes them to migrate into the solvent . Respective C36 mutants were mixed with N38 and subjected to N-PAGE analysis, using N36/C34 as a control [3,40]. As observed for C34/N36, all C36 mutants formed stable 6-HBs with N38 as evidenced by new bands migrating at a lower rate (higher molecular weight) concomitant with the fading or disappearance of the C-peptide bands (Fig. 3a). N-PAGE analysis results also indicated a stronger interaction between either C36M1 or C36M4 and N38 than interactions between C36M2 or C36M3 and N38 based on the densities of the residue C-peptide bands observed in the gel.
Hydrophobic mutation in buried C-terminal heptad repeat polar residues can increase thermal stability of 6-HBs formed between C36 mutants and N38
The thermal stability of the 6-HBs was determined by thermal denaturation analysis. The melting temperature (Tm) of 6-HB represents an index of thermal stability associated with protein folding correlated with binding affinity between N and C-peptides and activities of C-peptides [10,11]. The thermal denaturation analysis of the 6-HBs was performed by monitoring circular dichroism signals at 222 nm as a function of temperature. As shown in Fig. 3e, all 6-HBs exhibited a cooperative, thermal-induced unfolding transition, which is typical for well folded proteins [31,40]. Hydrophobic substitutions at S649 (C36M1) and E659 (C36M4) resulted in more stable 6-HBs with a 5°C increase in the Tm, whereas substitutions at Q652 (C36M2) and N656 (C36M3) less affected thermal stability of respective 6-HBs (Table 1).
S649L and E659I C36 mutants showed significantly enhanced inhibitory potency against T20-resistant HIV-1 strains
Next, we tested C36-mutants’ inhibitory activities against T20-sensitive and T20-resistant HIV-1 strains. Consistent with cell–cell fusion assay results, all the C36 mutants showed similar IC50 values against infection by a T20-sensitive HIV-1 pNL4-3 strain (Table 2) . C36M0 displayed a significantly lower activity against T20-resistant HIV-1 strains, resulting in up to 19-fold potent decrease. C36M1 and C36M4 showed significantly improved inhibitory profiles against T20-resistant HIV-1 strains, and they are highly active against most of T20-resistant strains. However, C36M2 and C36M3 are out of our expectation, as they showed poorer inhibitory activity than C36M0 against some T20-resistant strains. This is consistent with thermal denaturation results and possibly due to the mismatching of the residues in the NHR interface, from polar interaction, conformation or steric effect. Further investigation of these two mutant peptides is warranted.
The inhibitory profiles of the C36 mutants against T20-resistant strains paralleled their 6-HB thermal stability, suggesting that the enhanced interaction between C-peptide fusion inhibitor and NHR could, by the introduction of hydrophobic residues at the buried polar area of CHR, restore the impaired interactions between C-peptide fusion inhibitors and gp41 NHR target of drug-resistant HIV-1 strains.
Despite similar activity in the cell–cell fusion assay, C36M0 showed poorer activity against T20-resistant HIV-1 strains than C36 (Table 2). This suggests that mutations at ‘noncritical’ sites of CHR, which have seen widespread use for fusion inhibitor design [10,11], do affect the activity of C-peptide fusion inhibitors. Therefore, we reversed the mutations in solvent-exposed residues from C36M1 to C36M4 and designed C36M1A to C36M4A (Table 1). Interestingly, the reversed C36 mutants C36M1A and C36M4A showed significantly higher antiviral activity against four of the five T20-resistant HIV-1 strains than C36, although C36M2A and C36M3A showed better activity than C36 against only one or two T20-resistant HIV-1 strains. A similar trend was observed in circular dichroism and thermal denaturation experiments (Fig. 3d, f). On the basis of these results, we may conclude that substitution of some conserved buried gp41 polar residues, such as 649L or 659I, with hydrophobic residues, can increase the potency of C-peptide fusion inhibitor against T20-resistant HIV-1 strains.
Since the discovery of T20, efforts have been made to develop a new generation of HIV-1 fusion inhibitors to overcome the drawbacks of T20 . Most highly potent HIV fusion inhibitors are C-peptides that possess a–d–e residues that face and directly interact with gp41 NHR. As these residues are considered critical for NHR–CHR interaction, they have remained unchanged during the process of designing new fusion inhibitors. In contrast, b–c–f–g residues exposed to the solvent have been extensively mutated during the design of next-generation peptide fusion inhibitors [10,11,15,18].
Current work focused on the buried face of the HIV-1 gp41 CHR. We selected a highly conserved CHR polar area that contains four successive polar a–d residues, which are buried in 6-HB structure. We introduced hydrophobic residues to replace them. Our results showed that mutations changing these polar residues to hydrophobic ones had no adverse effects on the anti-HIV activity of C36. Instead, S649L and E659I mutations enhanced CHR–NHR interactions, and the respective peptides showed significantly increased activity against T20-resistant HIV-1 strains. These mutations in conserved binding sites between drug target and its native ligands are counterintuitive in the context of drug design. However, the results agree with the principle of protein folding for a coiled-coil interaction, wherein hydrophobic interactions from buried hydrophobic residues provide larger free energy to stabilize protein structure than polar interactions .
Two faces exist and define HIV-1 gp41 NHR–CHR interaction. First, a specific interaction between gp41 NHR and CHR involves matched residues at the binding interface. Second, the interaction is a typical coiled-coil interaction resulting in the formation of a 6-HB between a trimerized NHR inner core and three CHR domains binding in an antiparallel manner [3,25]. The significance of the specific interactions has been highlighted in C-peptide fusion inhibitor design and optimization [1,10,11,46,47]. Despite various viruses that use 6-HB structure folding as a means of completing the virus cell membrane fusion process, studies have shown that only peptides derived from envelope glycoproteins of identical viruses interact in a proper manner with their counterparts to inhibit infection with the same virus. Artificially designed peptide fusion inhibitors based solely on coiled-coil interaction have usually shown two to three orders lower potency than native C-peptides and their derivatives , but their activities can be greatly increased by introducing target-specific interaction and hydrophobic interactions .
The current work highlighted the coiled-coil nature of the HIV-1 gp41 NHR–CHR interaction. Considering the combined studies by Otaka et al. and Dwyer et al. [10,11], this work suggested that the HIV-1 gp41 CHR could sustain extensive mutations in both solvent-exposed residues and residues comprising the buried binding interface, as long as the mutated peptides retain the ability to form a coiled-coil structure. This observation also reflected the hyperplasticity of the HIV genome and the various emerging drug-resistant HIV-1 isolates.
Compared with mutations made to solvent-exposed residues, mutations in residues at the gp41 NHR–CHR interface had a bigger effect on the affinity between C and N-peptides, as well as the secondary structure of C-peptides. Dwyer et al.  showed that replacement of polar a–d residues with hydrophobic residues significantly increased the thermal stability of 6-HB formed between C and N-peptides, although this enhanced interaction did not always transfer into higher anti-HIV potencies. Introduction of the hydrophobic residues at the a–d positions promotes the secondary structure formation of peptides, and some C-peptides form stable self-associated trimers with high α-helical content, thus showing longer in-vivo half-time . Peptide engineering on buried face of gp41 CHR will result in the design of C-peptide fusion inhibitors with higher potency and improved pharmacokinetic and pharmacodynamic profiles against drug-resistant HIV isolates.
We demonstrated that the highly conserved a–d polar residues in the HIV-1 gp41 CHR could be mutated as a means of generating new fusion inhibitors against drug-resistant HIV-1 strains. Replacement of buried polar residues with hydrophobic residues enhanced HIV-1 gp41 NHR–CHR interactions and increased the activity of C-peptides against T20-resistant HIV-1 strains. Our work highlights the nature of coiled-coil interaction between HIV-1 gp41 NHR and CHR. This physicochemical-based evidence can inspire more comprehensive investigations to understand the detailed mechanisms of viral cell membrane fusion for novel fusion inhibitor design and development.
The authors thank NIH AIDS Research and Reference Reagent Program for providing cell line and HIV-1 isolates for cell–cell fusion assay and HIV infection assay. We also thank Xiaolei Bao at Consulting Center of Biomedical Statistics, Academy of Military Medical Science, for help and suggestion on statistical analysis. This work was supported by Natural Science Foundation of China Grants (Nos. 81273434, 81373266, 81361120378 and 81261120382) and Key Tech. of National S & T Major Project of Original New Drug Research Grant (2012ZX09301003).
L.C. conceived the concept; L.C., K.L., S.J. supervised the project; L.C., B.Z. designed the experiment; B.Z., K.W., L.L., F.Y. performed experiment; L.C., B.Z., M.C., S.J., K.L. analysed data and wrote the article.
Conflicts of interest
The authors have no commercial or other association that might pose a conflict of interest.
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envelop glycoprotein; fusion inhibitor; gp41; HIV-1; mutation; peptide
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