An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.     

Human immunodeficiency virus (HIV) gp41 escape mutants: cross-resistance to peptide inhibitors of HIV fusion and altered receptor activation of gp120

Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.     

Sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition

Viral glycoproteins catalyze the fusion between viral and cellular membranes. The fusion protein (F(1)) of Sendai virus has two fusion peptides. One is located at its N-terminus and the other, highly homologous to the HIV-1 and RSV fusion peptides, in the interior of the F(1) protein. A synthetic peptide corresponding to the internal fusogenic domain, namely, SV-201, was found to inhibit virus-cell fusion without interfering with the binding of the virus to the target cells, thus highlighting the importance of this region in Sendai virus-induced membrane fusion. However, its detailed mechanism of inhibition remains unknown. Here, we synthesized a shorter version of SV-201, namely, SV-208, an elongated one, SV-197, and two mutants of SV-201, and compared them functionally and structurally with SV-201. In contrast to SV-201, SV-208 and the two mutants do not inhibit virus-cell fusion. The differences in the oligomerization state of these peptides in aqueous solution and within the membrane, and in their ability to bind to Sendai virions, enabled us to postulate a possible mechanism of viral entry inhibition: SV-201 binds to its target in Sendai virions before the F(1) internal fusion peptide binds to the membrane, therefore blocking the F(1) conformational change required for fusion. In addition, we further characterized the fusogenic activity of the internal fusion peptide, compared to the N-terminal one, and determined its structure in the membrane-bound state by means of fluorescence, CD, and ATR-FTIR spectroscopy. Remarkably, we found that SV-201 and its elongated form, SV-197, are highly potent in inducing fusion of the highly stable large unilamellar vesicles composed of egg phosphatidylcholine, a property found only in an extended version of the HIV-1 fusion peptide. The inhibitory activity of SV-201 and its fusogenic ability are discussed in terms of the "umbrella" model of Sendai virus-induced membrane fusion.     

Suitable fusion of N-terminal heptad repeats to achieve covalently stabilized potent N-peptide inhibitors of HIV-1 infection

N-terminal heptad repeat (NHR)-derived peptide (N-peptide) fusion inhibitors, which are derived from human immunodeficiency virus (HIV) envelope glycoprotein 41 (gp41), are limited by aggregation and unstable trimer conformation. However, they could function as potent inhibitors of viral infection by forming a coiled-coil structure covalently stabilized by interchain disulfide bonds. We previously synthesized N-peptides with potent anti-HIV-1 activity and high stability by coiled-coil fusion and covalent stabilization. Here, we attempted to study the effects of NHRs of chimeric N-peptides by fusing de novo coiled-coil isopeptide bridge-tethered T21 peptides of different NHR lengths. Peptides (T21N23)₃ and (T21N36)₃ was a more potent HIV-1 fusion inhibitor than (T21N17)₃. The site of isopeptide bond formation was precisely controlled and had little influence on N-peptide properties. The N-peptide (T21N36)₃, which had a similar conformation as the NHR trimer and interacted well with the C34 peptide, may be useful for screening other C-peptides and small-molecule fusion inhibitors, and for studying the interactions between the NHR trimer and C-terminal heptad repeats.     

Direct evidence that the N-terminal heptad repeat of Sendai virus fusion protein participates in membrane fusion.

Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.     

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.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

The pre-transmembrane region of the human immunodeficiency virus type-1 glycoprotein: a novel fusogenic sequence

We have investigated membrane interactions and perturbations induced by NH(2)-DKWASLWNWFNITNWLWYIK-COOH (HIV(c)), representing the membrane interface-partitioning region that precedes the transmembrane anchor of the human immunodeficiency virus type-1 gp41 fusion protein. The HIV(c) peptide bound with high affinity to electrically neutral vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1), and induced vesicle leakage and lipid mixing. Infrared spectra suggest that these effects were promoted by membrane-associated peptides adopting an alpha-helical conformation. A sequence representing a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, was equally unable to induce vesicle fusion, and adopted a non-helical conformation in the membrane. We conclude that membrane perturbation and adoption of the alpha-helical conformation by this gp41 region might be functionally meaningful.     

Design and properties of N(CCG)-gp41, a chimeric gp41 molecule with nanomolar HIV fusion inhibitory activity.

The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.     

Synthetic peptides derived from an N‐terminal domain of the E2 protein of GB virus C in the study of GBV‐C/HIV‐1 co‐infection

Synthetic peptides derived from GB virus C (GBV‐C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV‐C that have the capacity to interfere with the HIV‐1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV‐1 infectivity in a dose‐dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N‐terminal sequence of the GBV‐C E2 protein. Immunoassays and cell–cell fusion assays were performed to evaluate their diagnostic value to detect anti‐GBV‐C antibodies in HIV‐1 patients, as well as their putative anti‐HIV‐1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7–26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV‐1‐mediated cell–cell fusion nor improved sensitivity in the detection of GBV‐C antibodies in HIV‐1 co‐infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7–26) region for the development of new peptide‐based immunosensor devices for the detection of anti‐GBV‐C antibodies in HIV‐1 co‐infected patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.     

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.     

HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.     

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.     

Structural characterization of mumps virus fusion protein core

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.     

Coiled-coil domains in glycoproteins B and H are involved in human cytomegalovirus membrane fusion

Human cytomegalovirus (CMV) utilizes a complex route of entry into cells that involves multiple interactions between viral envelope proteins and cellular receptors. Three conserved viral glycoproteins, gB, gH, and gL, are required for CMV-mediated membrane fusion, but little is known of how these proteins cooperate during entry (E. R. Kinzler and T. Compton, submitted for publication). The goal of this study was to begin defining the molecular mechanisms that underlie membrane fusion mediated by herpesviruses. We identified heptad repeat sequences predicted to form alpha-helical coiled coils in two glycoproteins required for fusion, gB and gH. Peptides derived from gB and gH containing the heptad repeat sequences inhibited virus entry when introduced coincident with virus inoculation onto cells or when mixed with virus prior to inoculation. Neither peptide affected binding of CMV to fibroblasts, suggesting that the peptides inhibit membrane fusion. Both gB and gH coiled-coil peptides blocked entry of several laboratory-adapted and clinical strains of human CMV, but neither peptide affected entry of murine CMV or herpes simplex virus type 1 (HSV-1). Although murine CMV and HSV-1 gB and gH have heptad repeat regions, the ability of human CMV gB and gH peptides to inhibit virus entry correlates with the specific residues that comprise the heptad repeat region. The ability of gB and gH coiled-coil peptides to inhibit virus entry independently of cell contact suggests that the coiled-coil regions of gB and gH function differently from those of class I, single-component fusion proteins. Taken together, these data support a critical role for alpha-helical coiled coils in gB and gH in the entry pathway of CMV.     

Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region

Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, approximately 90 degrees C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of approximately 50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.     

The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.     

Temperature-dependent intermediates in HIV-1 envelope glycoprotein-mediated fusion revealed by inhibitors that target N- and C-terminal helical regions of HIV-1 gp41

Peptides derived from the N- (N-HR) and C- (C-HR) terminal heptad repeat regions adjacent to the fusion peptide and transmembrane domains, respectively, of human immunodeficiency virus (HIV)-1 gp41 inhibit HIV-1 viral envelope glycoproteins (Env)-mediated cell fusion specifically. The mechanism of HIV-1 Env-mediated cell fusion and its inhibition by agents that target the N- and C-HR regions was investigated. Priming experiments with Env-expressing cells indicate that the N-HR region but not the C-HR region is exposed by treatment with sCD4 at 31 degrees C, whereas both the N- and C-HR regions are exposed at 37 degrees C.