Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41aa) and CHR (34aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-Helix protein exists as a trimer and is highly alpha-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis.     

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.     

Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120

Fuzeon (also known as T-20 or enfuvirtide), one of the C-peptides derived from the HIV-1 envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (CHR) region, is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. It has been widely believed that T-20 shares the same mechanism of action with C34, another C-peptide. The C34 is known to compete with the CHR of gp41 to form a stable 6-helix bundle (6-HB) with the gp41 N-terminal heptad repeat (NHR) and prevent the formation of the fusogenic gp41 core between viral gp41 NHR and CHR, thereby inhibiting fusion between viral and target cell membranes. Here we present data to demonstrate that, contrary to this belief, T-20 cannot form stable 6-HB with N-peptides derived from the NHR region, nor can it inhibit the 6-HB formation of the fusogenic core. Instead, it may interact with N-peptides to form unstable or insoluble complexes. Our data suggest that T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. We also demonstrate that the T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. These new findings imply that T-20 inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors.     

Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides

HIV-1 fusion with its target cells is mediated by the glycoprotein 41 (gp41) transmembrane subunit of the viral envelope glycoprotein (ENV). The current models propose that gp41 undergoes several conformational changes between the apposing viral and cell membranes to facilitate fusion. In this review we focus on the progress that has been made in revealing the dynamic role of the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions within gp41 to the fusion process. The involvement of these regions in the formation of the gp41 pre-hairpin and hairpin conformations during an ongoing fusion event was mainly discovered by their derived inhibitory peptides. For example, the core structure within the hairpin conformation in a dynamic fusion event is suggested to be larger than its high resolution structure and its minimal boundaries were determined in situ. Also, inhibitory peptides helped reveal the dual contribution of the NHR to the fusion process. Finally, we will also discuss several developments in peptide design that has led to a deeper understanding of the mechanism of viral membrane fusion.     

Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621-652 complexed by T21. We find that the (621)QIWNNMT(627) motif does not maintain the α-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621-652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.     

Syncytin-A mediates the formation of syncytiotrophoblast involved in mouse placental development.

Syncytin-A, a new mouse endogenous retroviral envelope protein expressed in placenta, can mediate cell fusion in vitro. But its physiological function was still unknown. We proposed a role for syncytin-A in syncytiotrophoblast (SynT) formation derived from the differentiation of trophoblast stem (TS) cells during placental development. To evaluate this hypothesis, we analyzed the involvement of syncytin-A in the differentiation of mouse TS cells. After withdrawing fibroblast growth factor 4 (FGF4), TS cells can fuse to form SynT cells. We found syncytin-A mRNA and protein expression are colinear with fusion index increase during TS cell differentiation. Expression of syncytin-A is localized in SynT cells through in situ immunofluorescent staining. By using specific antibody and antisense oligonucleotides, we demonstrated that inhibition of syncytin-A lead to obvious decrease of SynT cell formation. These results present evidence in support of the direct role for syncytin-A in mouse TS cell fusion and differentiation involved in placental development.     

The Conserved Residue Arg46 in the N-Terminal Heptad Repeat Domain of HIV-1 gp41 Is Critical for Viral Fusion and Entry

During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR) of gp41 interacts with the C-terminal heptad repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54-70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35-53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB's stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.     

Addition of artificial salt bridge by Ile646Lys mutation in gp41 coiled-coil domain regulates 6-helical bundle formation

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. The C34 which has 34 amino acid residues is known as the core structure in CHR. A highly anti-HIV peptide inhibitor derived from C34 was designed. An artificial salt bridge was added in the 6-helical bundle by substitution of lysine for Ile646. With a cholesterol modification at C-terminal, the inhibitor containing I646K mutation represented higher anti-viral activity than C34–cholesterol combination without mutation.     

Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC₅₀ increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors.     

On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.     

Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects

Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N- and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable α-helical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, T(m) , of 86.8 ± 2.0°C and van't Hoff enthalpy, ΔH(vH) , of -28.2 ± 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed α-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3-6.1) had dramatic effects (ΔT(m) = 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion.     

Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.     

分子内二硫键对HERV囊膜蛋白融合核心结构的影响

合胞素(Syncytin)是一类由人俘获的逆转录病毒囊膜蛋白,与胎盘的形态发生中细胞滋养层到合胞滋养层的分化过程十分相关。Syncytin与人免疫缺陷病毒I型(HIV-1)囊膜蛋白(Env)在结构上具有相似的特点,二者可能具有相似的膜融合机制。本文通过PCR对融合核心部位七肽重复区HR1和HR2之间linker中自然存在的一对保守的分子内二硫键进行定点突变,表达纯化该突变蛋白,并进行了相应的结构及稳定性探讨,通过与未突变蛋白的性质比较确证该分子内二硫键在蛋白结构的正确形成及稳定性上起着一定的作用。     

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett. 2015, 25 2853–59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC₅₀ = 37.81 µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.     

Inhibition of Ebola virus entry by a C-peptide targeted to endosomes

Ebola virus (EboV) and Marburg virus (MarV) (filoviruses) are the causative agents of severe hemorrhagic fever. Infection begins with uptake of particles into cellular endosomes, where the viral envelope glycoprotein (GP) catalyzes fusion between the viral and host cell membranes. This fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike that ultimately result in formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Infection by other viruses employing similar viral entry mechanisms (such as HIV-1 and severe acute respiratory syndrome coronavirus) can be inhibited with synthetic peptides corresponding to the native CHR sequence ("C-peptides"). However, previously reported EboV C-peptides have shown weak or insignificant antiviral activity. To determine whether the activity of a C-peptide could be improved by increasing its intracellular concentration, we prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, which is known to accumulate in endosomes. We found that this peptide specifically inhibited viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. We determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Mechanistic studies suggested that the peptide acts by blocking a membrane fusion intermediate.     

Discovery of HIV fusion inhibitors targeting gp41 using a comprehensive α-helix mimetic library

The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic pocket recognizing an intramolecular CHR α-helix provided a detailed depiction of structural features required for binding and led to the discovery of small molecule inhibitors (K(i) 0.6-1.3 μM) that not only match or exceed the potency of those disclosed over the past decade, but that also exhibit effective activity in a cell-cell fusion assay (IC(50) 5-8 μM).     

Permanent inhibition of viral entry by covalent entrapment of HIV gp41 on the virus surface

HIV entry occurs by concerted conformational changes in the envelope protein complex on the surface of the virus. This complex is made up of a trimer of heterodimers of two subunits: surface subunit, gp120, and transmembrane subunit, gp41. Conformational changes in the envelope complex allow gp41 to mediate membrane fusion leading to exposure of two gp41 regions: N-heptad repeat (NHR) and C-heptad repeat (CHR). Peptides from the NHR or the CHR have been found to inhibit HIV entry. Herein we show that we can covalently inhibit HIV viral entry by permanently trapping the gp41 intermediate on the virus surface using a covalently reactive group on inhibitory peptides. This is evidence showing that vulnerable conformational intermediates exist transiently during HIV viral entry, and the details presented herein will facilitate development of envelope as a target for therapeutics and potential chemopreventive agents that could disable the virus before contact with the host cell.     

Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here 'Fusoselect', a universal procedure allowing the identification and engineering of molecular determinants for cell-cell fusion-activity by directed evolution. The system couples cell-cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a gamma-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell-cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect.     

Molecular Dynamics Studies of the Inhibitor C34 Binding to the Wild-Type and Mutant HIV-1 gp41: Inhibitory and Drug Resistant Mechanism

Mutations on NHR (N-terminal heptad repeat) associated with resistance to fusion inhibitor were observed. In addition, mutations on CHR (C-terminal heptad repeat) accompanied NHR mutations of gp41 are noted in many cases, like N43D/S138A double mutation. In this work, we explored the drug resistant mechanism of N43D mutation and the role of S138A second mutation in drug resistance. The binding modes of the wild type gp41 and the two mutants, N43D and N43D/S138A, with the HIV-1 fusion inhibitor C34, a 34-residue peptide mimicking CHR of gp41, were carried out by using molecular dynamics simulations. Based on the MD simulations, N43D mutation affects not only the stability of C34 binding, but also the binding energy of the inhibitor C34. Because N43D mutation may also affect the stable conformation of 6-HB, we introduced S138A second mutation into CHR of gp41 and determined the impact of this mutation. Through the comparative analysis of MD results of the N43D mutant and the N43D/S138A mutant, we found that CHR with S138A mutation shown more favorable affinity to NHR. Compelling differences in structures have been observed for these two mutants, particularly in the binding modes and in the hydrophobic interactions of the CHR (C34) located near the hydrophobic groove of the NHR. Because the conformational stability of 6-HB is important to HIV-1 infection, we suggested a hypothetical mechanism for the drug resistance: N43D single mutation not only impact the binding of inhibitor, but also affect the affinity between NHR and CHR of gp41, thus may reduce the rate of membrane fusion; compensatory mutation S138A would induce greater hydrophobic interactions between NHR and CHR, and render the CHR more compatible to NHR than inhibitors.     

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.The entry process of the human immunodeficiency virus type 1 (HIV-1) has become a major target for new antiviral drugs. Viral entry is initiated by binding of the HIV-1 envelope glycoprotein subunit gp120 to the CD4 receptor and a chemokine coreceptor, generally CCR5 or CXCR4. Upon coreceptor binding, the viral transmembrane subunit gp41 undergoes conformational changes that eventually lead to the formation of the six-helix bundle (6HB) and membrane fusion. The 6HB is composed of a central trimeric coiled-coil structure formed by the N-terminal heptad repeat (NHR) domains of three gp41 molecules and the corresponding C-terminal heptad repeats (CHRs) that pack into the longitudinal grooves on the surface of the NHR coiled-coil in an antiparallel orientation (23). C-peptide fusion inhibitors (CFI) derived from the CHR of gp41 compete with the viral CHR for binding to the NHR trimer, thus blocking 6HB formation and viral entry (18).T-20 (enfuvirtide) is the first clinically approved CFI with high antiviral activity and a low-toxicity profile. However, as with many anti-HIV-1 drugs, resistance can emerge rapidly (13). The majority of the resistance mutations are found in the NHR of gp41 among the amino acids 544 to 553 (32, 35) (numbering refers to gp160 of the HIV-1 HXB2 strain throughout the article). Most of these mutations cause resistance by reducing the affinity of the NHR target region to inhibitory C peptides (13). Additionally, viral entry kinetics were found to correlate with the baseline susceptibility of different HIV strains to CFI. Determinants for viral entry kinetics are found in gp41 as well as in gp120 (1, 14, 35). Here, the influence of coreceptor affinity on virus entry kinetics and CFI susceptibility has been studied extensively (28, 30, 31). Recently, a statistical approach was used that highlighted positions in gp120 that underwent mutations in patients under enfuvirtide treatment (38). However, to our knowledge, selected CFI resistance mutations outside of gp41 have never been confirmed experimentally.Previously, we developed a retroviral vector expressing a membrane-anchored antiviral C peptide (maC46) that efficiently inhibits a broad range of different HIV-1 isolates. Enfuvirtide-resistant HIV-1 strains with mutations in the GIV motif of NHR were fully susceptible to maC46 (10). In the present study, we selected an HIV-1 variant with reduced sensitivity to maC46 by passaging an enfuvirtide-resistant BaL strain of HIV-1 on cells expressing increasing concentrations of maC46. Mutations in gp120 and gp41 were found to contribute to maC46 resistance.