Template-based coiled-coil antigens elicit neutralizing antibodies to the SARS-coronavirus

The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.     

Molecular determinants of antiviral potency of paramyxovirus entry inhibitors

Hendra virus (HeV) and Nipah virus (NiV) constitute the Henipavirus genus of paramyxoviruses, both fatal in humans and with the potential for subversion as agents of bioterrorism. Binding of the HeV/NiV attachment protein (G) to its receptor triggers a series of conformational changes in the fusion protein (F), ultimately leading to formation of a postfusion six-helix bundle (6HB) structure and fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions, the first (the N-terminal heptad repeat [HRN]) adjacent to the fusion peptide and the second (the C-terminal heptad repeat [HRC]) immediately preceding the transmembrane domain. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing activated F molecules from forming the 6HB structure that is required for fusion. We previously reported that a human parainfluenza virus 3 (HPIV3) F peptide effectively inhibits infection mediated by the HeV glycoproteins in pseudotyped-HeV entry assays more effectively than the comparable HeV-derived peptide, and we now show that this peptide inhibits live-HeV and -NiV infection. HPIV3 F peptides were also effective in inhibiting HeV pseudotype virus entry in a new assay that mimics multicycle replication. This anti-HeV/NiV efficacy can be correlated with the greater potential of the HPIV3 C peptide to interact with the HeV F N peptide coiled-coil trimer, as evaluated by thermal unfolding experiments. Furthermore, replacement of a buried glutamic acid (glutamic acid 459) in the C peptide with valine enhances antiviral potency and stabilizes the 6HB conformation. Our results strongly suggest that conserved interhelical packing interactions in the F protein fusion core are important determinants of C peptide inhibitory activity and offer a strategy for the development of more-potent analogs of F peptide inhibitors.     

Inhibition of hendra virus fusion

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.     

Nonhelical leash and alpha-helical structures determine the potency of a peptide antagonist of human T-cell leukemia virus entry

Viral fusion proteins mediate the entry of enveloped viral particles into cells by inducing fusion of the viral and target cell membranes. Activated fusion proteins undergo a cascade of conformational transitions and ultimately resolve into a compact trimer of hairpins or six-helix bundle structure, which pulls the interacting membranes together to promote lipid mixing. Significantly, synthetic peptides based on a C-terminal region of the trimer of hairpins are potent inhibitors of membrane fusion and viral entry, and such peptides are typically extensively alpha-helical. In contrast, an atypical peptide inhibitor of human T-cell leukemia virus (HTLV) includes alpha-helical and nonhelical leash segments. We demonstrate that both the C helix and C-terminal leash are critical to the inhibitory activities of these peptides. Amino acid side chains in the leash and C helix extend into deep hydrophobic pockets at the membrane-proximal end of the HTLV type 1 (HTLV-1) coiled coil, and these contacts are necessary for potent antagonism of membrane fusion. In addition, a single amino acid substitution within the inhibitory peptide improves peptide interaction with the core coiled coil and yields a peptide with enhanced potency. We suggest that the deep pockets on the coiled coil are ideal targets for small-molecule inhibitors of HTLV-1 entry into cells. Moreover, the extended nature of the HTLV-1-inhibitory peptide suggests that such peptides may be intrinsically amenable to modifications designed to improve inhibitory activity. Finally, we propose that leash-like mimetic peptides may be of value as entry inhibitors for other clinically important viral infections.     

Conserved salt bridge between the N- and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys⁵⁷⁴ in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp⁶³² substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, nonconserved substitutions of Asp⁶³² also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp⁶³² significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.     

Interactions between HIV-1 gp41 core and detergents and their implications for membrane fusion

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.     

Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.     

Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope

While it has been established that peptides modeling the C-helical region of human immunodeficiency virus type 1 gp41 are potent in vivo inhibitors of virus replication, their mechanism of action has yet to be determined. It has been proposed, but never directly demonstrated, that these peptides block virus entry by interacting with gp41 to disrupt the formation or function of a six-helix bundle structure. Using a six-helix bundle-specific monoclonal antibody with isolate-restricted Env reactivity, we provide the first direct evidence that, in receptor-activated viral Env, C-peptide entry inhibitors bind to the gp41 N-helical coiled-coil to form a peptide/protein hybrid structure and, in doing so, disrupt native six-helix bundle formation.     

The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.     

Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic Cavity in Membrane Fusion

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an alpha-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.     

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.     

Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy

Structural changes for a series of antimicrobial peptides in various solvents were investigated by a combined approach of FTIR and CD spectroscopy. The well-characterized and potent antimicrobial peptides indolicidin and tritrpticin were studied along with several analogs of tritrpticin, including Tritrp1 (amidated analog of tritrpticin), Tritrp2 (analog of Tritrp1 with Arg-->Lys substitutions), Tritrp3 (analog of Tritrp1 with Pro-->Ala substitutions) and Tritrp4 (analog of Tritrp1 with Trp-->Tyr substitutions). All peptides were studied in aqueous buffer, ethanol and in the presence of dodecylphosphocholine (DPC) micelles. It was shown that tritrpticin and its analogs preferentially adopt turn structures in all solvents studied. The turn structures formed by the tritrpticin analogs bound to DPC micelles are more compact and more conformationally restricted compared to indolicidin. While several peptides showed a slight propensity for an alpha-helical conformation in ethanol, this trend was only strong for Tritrp3, which also adopted a largely alpha-helical structure with DPC micelles. Tritrp3 also demonstrated along with Tritrp1 the highest ability to interact with DPC micelles, while Tritrp2 and Tritrp4 showed the weakest interaction.     

Structural characterization of the SARS-coronavirus spike S fusion protein core

The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.     

Helical interactions in the HIV-1 gp41 core reveal structural basis for the inhibitory activity of gp41 peptides

The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors.     

Synthesis of cyclic α-MSH peptides

Summary Cyclic lactam analogs of α-melanocyte stimulating hormone (α-MSH) have been shown to be potent agonists in the frog skin bioassay [Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized cyclic α-MSH(1–13) and α-MSH(4–10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization procedure: side chains of Asp⁵ and Lys¹⁰ from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an α-MSH analog cyclized via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions at position 7 dramatically influenced the selectivity for the rMC4 receptor.     

Buried polar interactions and conformational stability in the simian immunodeficiency virus (SIV) gp41 core

For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule.     

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.     

Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct alpha-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.     

Binding of HIV-1 gp41-Directed Neutralizing and Non-Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.     

The pH-sensitive action of cholesterol-conjugated peptide inhibitors of influenza virus

Influenza viruses are major human pathogens, responsible for respiratory diseases affecting millions of people worldwide, with high morbidity and significant mortality. Infections by influenza can be controlled by vaccines and antiviral drugs. However, this virus is constantly under mutations, limiting the effectiveness of these clinical antiviral strategies. It is therefore urgent to develop new ones. Influenza hemagglutinin (HA) is involved in receptor binding and promotes the pH-dependent fusion of viral and cell endocytic membranes. HA-targeted peptides may emerge as a novel antiviral option to block this viral entry step. In this study, we evaluated three HA-derived (lipo)peptides using fluorescence spectroscopy. Peptide membrane interaction assays were performed at neutral and acidic pH to better resemble the natural conditions in which influenza fusion occurs. We found that peptide affinity towards membranes decreases upon the acidification of the environment. Therefore, the released peptides would be able to bind their complementary domain and interfere with the six-helix bundle formation necessary for viral fusion, and thus for the infection of the target cell. Our results provide new insight into molecular interactions between HA-derived peptides and cell membranes, which may contribute to the development of new influenza virus inhibitors.