Inhibition of murine leukemia virus envelope protein (env) processing by intracellular expression of the env N-terminal heptad repeat region

A conserved structural motif in the envelope proteins of several viruses consists of an N-terminal, alpha-helical, trimerization domain and a C-terminal region that refolds during fusion to bind the N-helix trimer. Interaction between the N and C regions is believed to pull viral and target membranes together in a crucial step during membrane fusion. For several viruses with type I fusion proteins, C regions pack as alpha-helices in the grooves between N-helix monomers, and exogenously added N- and C-region peptides block fusion by inhibiting the formation of the six-helix bundle. For other viruses, including influenza virus and murine leukemia virus (MLV), there is no evidence for comparably extended C-region alpha-helices, although a short, non-alpha-helical interaction structure has been reported for influenza virus. We tested candidate N-helix and C-region peptides from MLV for their ability to inhibit cell fusion but found no inhibitory activity. In contrast, intracellular expression of the MLV N-helix inhibited fusion by efficiently blocking proteolytic processing and intracellular transport of the envelope protein. The results highlight another mechanism by which the N-helix peptides can inhibit fusion.     

Trimeric, coiled-coil extension on peptide fusion inhibitor of HIV-1 influences selection of resistance pathways

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.     

Design of an engineered N-terminal HIV-1 gp41 trimer with enhanced stability and potency

HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six-helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30-fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1-resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 A crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six-helix bundle. These results provide an initial model of the pre-fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides.     

Binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of F-glycoprotein of HVJ (Sendai virus): possible implication in the fusion reaction

Specificity of the binding of sterols and related compounds with purified F-protein (fusion protein) of the HVJ (Sendai virus) was studied by binding competition with [3H]cholesterol. Requirement for cholesterol or the A/B ring trans structure and nonrequirement for the 3-hydroxyl group were found in this binding. Binding of 125I-labeled Z-Phe-Tyr, an inhibitory peptide of viral membrane-cell membrane fusion, was studied by using purified proteins and virions. F-Protein and virions showed a specific binding with the peptide, whereas the result was negative with hemagglutinin and neuraminidase protein. Thermolysin-truncated F-protein (an F-protein derivative deprived of a 2.5-kDa fragment from the N-terminal of the F1 subunit and without fusogenic activity) exhibited a considerably diminished binding ability both to cholesterol and to inhibitory peptides. Therefore, the N-terminal hydrophobic sequence that was previously assigned as fusogenic seems to be the binding site of these molecules. In support of this, the binding of cholesterol with F-protein was inhibited by Z-Phe-Tyr and other fusion inhibitory peptides, whereas it was not affected with non-fusion-inhibitory Z-Gly-Phe. These results are discussed in relation to the notion that the binding of the N-terminal portion of the F1 subunit of F-protein with cholesterol in the target cell membranes facilitates the fusion reaction.     

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.     

Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions

Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.     

Design and characterization of viral polypeptide inhibitors targeting Newcastle disease virus fusion

Paramyxovirus infections can be detected worldwide with some emerging zoonotic viruses and currently there are no specific therapeutic treatments or vaccines available for many of these diseases. Recent studies have demonstrated that peptides derived from the two heptad repeat regions (HR1 and HR2) of paramyxovirus fusion proteins could be used as inhibitors of virus fusion. The mechanism underlying this activity is in accordance with that of class I virus fusion proteins, of which human immunodeficiency virus (HIV) and influenza virus fusion proteins are members. For class I virus fusion proteins, the HR1 fragment binds to HR2 to form a six-helix bundle with three HR1 fragments forming the central coiled bundle surrounded by three coiled HR2 fragments in the post fusion conformational state (fusion core). It is hypothesized that the introduced exogenous HR1 or HR2 can compete against their endogenous counterparts, which results in fusion inhibition. Using Newcastle disease virus (NDV) as a model, we designed several protein inhibitors, denoted HR212 as well asHR121 and 5-Helix, which could bind the HR1 or HR2 region of fusion protein, respectively. All the proteins were expressed and purified using a GST-fusion expression system in Escherichia coli. The HR212 or GST-HR212 protein, which binds the HR1 peptide in vitro, displayed inhibitory activity against NDV-mediated cell fusion, while the HR121 and 5-Helix proteins, which bind the HR2 peptide in vitro, inhibited virus fusion from the avirulent NDV strain when added before the cleavage of the fusion protein. These results showed that the designed HR212, HR121 or 5-Helix protein could serve as specific antiviral agents. These data provide additional insight into the difference between the virulent and avirulent strains of NDV.     

Peptides derived from the heptad repeat region near the C-terminal of Sendai virus F protein bind the hemagglutinin-neuraminidase ectodomain

Previously, we showed that Sendai virus fusion protein (F) acts as an inhibitor of neuraminidase activity of hemagglutinin-neuraminidase (HN) protein. Here we report that synthetic peptides derived from the heptad repeat region proximal to the transmembrane domain (HR2) of Sendai virus F inhibit fusion and enhance the enzymatic activity of the HN. This occurs on the virus-bound HN and on its soluble globular head. The enhancing effect on virus-bound HN is reversible and depends on the presence of F. The data indicate that, by binding to the HN ectodomain, the HR2 peptides abolish the F inhibition of HN and disrupt the communication between the F and HN essential to promote virus-cell fusion.     

Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein

Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC(50) values of 0.14 and 1.19microM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.     

A New Water-Soluble Analogue of the Fusion Peptide of Influenza Virus Hemagglutinin: Synthesis and Properties

A water-soluble analogue F32 of the fusion peptide from influenza virus hemagglutinin was synthesized. It consisted of 32 aa residues and retained the ability to interact with lipid membranes; its N-terminal sequence 1–24 coincided with that of the fusion protein from hemagglutinin (strain A/PR/8/34), whereas residues 25–32 (GGGKKKKK) provided its solubility in water. The peptide induced the conductivity fluctuations in planar bilayer lipid membranes characteristic of active fusion peptides. Conditions were found using CD spectroscopy under which the structure of F32 inside detergent micelles, where it can be studied by high-resolution ¹H NMR spectroscopy, is close to the structure of the peptide during its interaction with phospholipid liposomes.     

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.     

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.     

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

The stability of the intact envelope glycoproteins is a major determinant of sensitivity of HIV/SIV to peptidic fusion inhibitors

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC(50)<10nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC(50)>100nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

New water-soluble analog of the fusion peptide of influenza virus hemagglutinin: synthesis and properties

A water-soluble analogue F32 of the fusion peptide from the influenza virus hemagglutinin was synthesized. It consisted of 32 aa residues and retained the ability to interact with lipid membranes; its N-terminal sequence 1-24 coincided with that of the fusion protein from hemagglutinin (strain A/PR/8/34), whereas residues 25-32 (GGGKKKKK) provided its solubility in water. The peptide induced the conductivity fluctuations in planar bilayer lipid membranes characteristic of active fusion peptides. Conditions were found using CD spectroscopy under which the structure of F32 inside detergent micelles, where it can be studied by high-resolution 1H NMR spectroscopy, is close to the structure of the peptide during its interaction with phospholipid liposomes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Rationally designed anti-HIV peptides containing multifunctional domains as molecule probes for studying the mechanisms of action of the first and second generation HIV fusion inhibitors

We have previously shown that the first generation human immunodeficiency virus (HIV) fusion inhibitor T20 (Fuzeon) contains a critical lipid-binding domain (LBD), whereas C34, another anti-HIV peptide derived from the gp41 C-terminal heptad repeat, consists of an important pocket-binding domain (PBD), and both share a common 4-3 heptad repeat (HR) sequence (Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). T1249, the second generation HIV fusion inhibitor, has both LBD and PBD but a different HR sequence, suggesting that these three anti-HIV peptides may have distinct mechanisms of action. Here we rationally designed a set of peptides that contain multiple copies of a predicted HR sequence (5HR) or the HR sequence plus either LBD (4HR-LBD) or PBD (PBD-4HR) or both (PBD-3HR-LBD), and we compared their anti-HIV-1 activity and biophysical properties. We found that the peptide 5HR exhibited low-to-moderate inhibitory activity on HIV-1-mediated cell-cell fusion, whereas addition of LBD and/or PBD to the HR sequence resulted in a significant increase of the anti-HIV-1 activity. The peptides containing PBD, including PBD-4HR and PBD-3HR-LBD, could form a stable six-helix bundle with the N-peptide N46 and effectively blocked the gp41 core formation, whereas the peptides containing LBD, e.g. 4HR-LBD and PBD-3HR-LBD, could interact with the lipid vehicles. These results suggest that the HR sequence in these anti-HIV peptides acts as a structure domain and is responsible for its interaction with the HR sequence in N-terminal heptad repeat, whereas PBD and LBD are critical for interactions with their corresponding targets. T20, C34, and T1249 may function like 4HR-LBD, PBD-4HR, and PBD-3HR-LBD, respectively, to interact with different target sites for inhibiting HIV fusion and entry. Therefore, this study provides important information for understanding the mechanisms of action of the peptic HIV-1 fusion inhibitors and for rational design of novel antiviral peptides against HIV and other viruses with class I fusion proteins.     

Identification of a critical motif for the human immunodeficiency virus type 1 (HIV-1) gp41 core structure: implications for designing novel anti-HIV fusion inhibitors

Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif ((621)QIWNNMT(627)) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD ((628)WMEWEREI(635)). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the (621)QIWNNMT(627) motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T(m)] value of 82 degrees C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T(m) of 64 degrees C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.