Peptide Fusion Inhibitors Research Articles

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin.

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand-binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand-binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol-conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41-Fc-chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV-1 strains, it was considerably more potent than the unconjugated C41-Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol-conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat-derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses.

Identification and characterization of a subpocket on the N-trimer of HIV-1 Gp41: implication for viral entry and drug target.

Crystallographic studies of HIV-1 gp41 demonstrate a stable six-helix bundle (6-HB) folded by trimeric N and C-terminal heptad repeats (NHR and CHR), and a deep hydrophobic pocket (pocket-1) on the NHR helices (N-trimer); however, previous crystal structures of 6-HB core were determined by peptide fragments missing the downstream sequence of pocket-1; thus, the structural features of this site could not be observed. We recently determined several 6-HB structures containing the pocket-1 and its downstream site. Here, we focused to investigate the structural features of N-trimer previously uncharacterized. Biophysical, biochemical and functional approaches were combined to characterize the downstream residues of pocket-1. A subpocket (designated pocket-2) was visualized on the C-terminal portion of N-trimer, which is formed by a cluster of seven residues, including Leu587, Lys588 and Glu584 on one NHR helix and Tyr586, Val583, Ala582 and Arg579 of another NHR helix. Mutagenesis studies demonstrated that the pocket-2 residues play essential roles for HIV-1 Env-mediated cell entry and critically determine the antiviral activity of NHR-derived peptide fusion inhibitor T21. Further, the pocket-2 mutations dramatically impaired the thermostability and conformation of 6-HB structure and reduced the binding affinity of CHR-derived inhibitor HP23 that specifically targets the deep pocket-1. These data have provided important information for the structure-function relationship of HIV-1 gp41 and for the development of antiviral entry inhibitors.

621. Fusion Between Neutrophils (PMN) and Target Cells Mediate Cytotoxicity During Measles Virus (MV) Oncolysis – A Novel Mechanism

We have previously shown that PMNs play a role in MV-mediated oncolysis (Grote et al) and that oncolytic vaccine strain of MV (as opposed to wild type) stimulates release of numerous cytokines with potential anti-tumor effects (Zhang et al). Recent data from our laboratory indicated that normal healthy PMNs were capable of specific killing of 35-40% of infected Jurkat target cells. On the basis that MV-induced cell-cell fusion amplifies IFN-alpha (α)/beta (β) production in infected cells, we sought to determine whether fusion between infected target cells and normal human PMNs specifically mediates cytotoxicity. First, we showed that PMN-mediated MV killing completely disappeared in the presence of FIP (fusion inhibitory peptide). Next, we examined the role of cell-cell fusion on PMN degranulation, reactive oxygen species (ROS) production and IFN production. We carried out all experiments with and without FIP. Jurkat cells were infected with MVNSe or mock-infected and incubated with PMNs isolated from healthy donors at 8:1 E:T ratio. At 24 hours, the cells were collected. PMN degranulation was determined by increase in cell surface expression of CD66b, CD63 and CD35 and ROS generation was determined by flow cytometry. IFN α and β production in the supernatant was quantified by ELISA. During co-culture of infected target cells and PMNs, IFN α (3000-5000pg/ml) and β (70-90pg/ml) were produced. No IFN α and β was produced by infected or uninfected Jurkats without PMNs present, confirming PMNs as the source. This was highly significantly abrogated in the presence of FIP (α – 200-300pg/ml; β – 0-3pg/ml). PMN degranulation and ROS generation followed the same pattern with abrogation in the presence of FIP, although the differences were not statistically significant. We confirmed that the α or β IFN was not directly responsible for cytotoxicity, since addition of the same quantity of exogenous IFN directly to Jurkat cells did not lead to cell death. Hence, we sought downstream cytotoxic effectors and evaluated TRAIL, which we know from our previous work, can be released from pre-formed granules in response to MV-infection of PMNs. The conditions that generated high levels of IFN also produced significant levels of soluble TRAIL (800-1500pg/ml), which was also abrogated in the presence of FIP (190-650pg/ml). Our data suggests infected target cells stimulate PMNs to display a cytotoxic effector phenotype, directly killing infected target cells. High levels of type I IFNs, are generated which are not in themselves responsible for the cytotoxicity. None of this occurs without target cell-neutrophil fusion as it is abrogated by FIP and does not occur in infected target cells alone or infected PMNs alone. This is a novel mechanism of stimulation of innate immunity by an oncolytic virus.

Real-Time Imaging Reveals that HIV-1 Vpr Dissociates from the Core and Accumulates in the Nucleus after Viral Fusion

Viral protein R (Vpr) is an HIV-1 accessory protein that associates with capsids during viral assembly and is important for infections in non-dividing cells. Vpr functions in host cells include induction of G2 cell-cycle arrest, and regulation of cellular proliferation and apoptosis. Vpr has two nuclear localization sequences that direct its transport to the nucleus. Fluorescently labeled Vpr (YFP-Vpr) is widely used to visualize HIV-1 cores in the cytoplasm during entry. Here we report on the dissociation of YFP-Vpr from HIV-1 cores shortly after viral fusion and its subsequent accumulation in the nuclei. Real-time live cell imaging showed that, under conditions of productive entry and infection, Vpr dissociated from cores post-fusion and accumulated in nuclei over time-scales that correlated with the kinetics of viral fusion (t1/2∼15 min). Nuclear accumulation of Vpr scaled with the number of cell-bound virions and could be blocked by lysosomotropic agents or a fusion-inhibitory peptide. These effects were observed in two cell lines and were independent of the fusion proteins incorporated into viral particles. Fluorescence recovery after photobleaching of YFP-Vpr within the nucleus revealed quick (t1/2∼3 min) recovery, indicating that Vpr dissociation from capsids is a rate-limiting step in Vpr post-fusion transport. Fluorescence correlation spectroscopy measurements on post-fusion nuclear YFP-Vpr, yielded fast and slow diffusive components (D∼10 μm2/s and 0.8 μm2/s, respectively) similar to those measured for YFP-Vpr over-expressed in cells. These diffusion coefficients reflect that nuclear Vpr exists in two forms - as a monomer, and in large complexes with host proteins or perhaps even chromatin structures. Current efforts are underway to explore the determinants of the stability of Vpr-capsid complexes. This work was supported by the NIH R01 GM054787 grant.

Enhanced HIV Fusion Inhibitors Efficacy Requires Membrane Affinity and Exposure of the Pocket Binding Domain of C34 Derivatives

Viral fusion inhibitors block the fusion between the membranes of an enveloped virus and a target cell, therefore preventing the entry of the viral content. It has been demonstrated that the combination of cholesterol-tagging and dimerization of the C34 peptide sequence resulted both in an increase of the antiviral potency and extension of the in vivo half-life of two new HIV fusion inhibitors: HIVP3 (C34-PEG4-cholesterol) and HIVP4 ([C34-PEG4]2-cholesterol). Given the importance of lipophilicity and selective affinity for different lipid domains, the aim was to evaluate the interaction of these HIV fusion inhibitor peptides with biomembranes model systems and human blood cells, in order to clarify where and how they are located. This study allowed the understanding of the mechanism of action of these peptides at the molecular level, and which strategies may be followed to increase their fusion inhibition efficacy. Membrane partition, dipole potential and surface pressure measurements indicated that HIVP3 and HIVP4 interact preferentially with cholesterol-rich liquid ordered membranes. HIVP3 and HIVP4 are able to interact with human erythrocytes and peripheral blood mononuclear cells to a similar degree as previously described for the simpler drug C34-cholesterol. However, the pocket binding domain(PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. The efficient blocking of HIV entry results from the synergic effect between the membranotropic behavior and the enhanced exposure of the PBD[1]. Maximizing antiviral activity requires finding the proper balance of membrane affinity and exposure of the peptide moiety, through variations in the lipid-binding domain, PEG spacer region, and number of peptide moieties in the construct.[1] Augusto MT et al.(2014)Improvement of the HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure.J Antimicrob Chemother,69:1286-1297

Synthesis of Cholesterol Conjugated Non-native Derived Heptad Repeat Sequence Peptides as Potent HIV-1 Fusion Inhibitors†

As addition of a cholesterol group to nature peptide fusion inhibitor can dramatically increase its antiviral potency,we designed and synthesized a series of cholesterol conjugated artificial peptide with low sequence homologous from HIV-1 gp41 as HIV-1 fusion inhibitors. The cholesterol moiety was introduced into C- or N-terminal of the peptide through a thioether in cysteine side chain and a β-alanine linker to study the effect of cholesterol modification to the activity of non-natural HR sequence and explore new method to overcome the problem of HIV drug resistance. Cell-cell fusion assays showed that the fusion inhibitory activity of the C-terminal cholesterol conjugated peptide was significantly increased,while the activity of N-terminal conjugated peptide was completely lost,consistent with the reported peptide conjugation using native peptide sequence,indicated that the designed non-natural sequences had a similar mechanism of action with natural sequences. This work provided a useful base for further drug optimization and discovery of highly active nonnatural peptide sequences.

Prevention of measles virus infection by intranasal delivery of fusion inhibitor peptides.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated and that helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immunocompromised hosts. Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion-inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small-animal models of MV infection, that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

Resistance to N-peptide fusion inhibitors correlates with thermodynamic stability of the gp41 six-helix bundle but not HIV entry kinetics.

BackgroundThe HIV-1 envelope glycoprotein (Env) undergoes conformational changes that mediate fusion between virus and host cell membranes. These changes involve transient exposure of two heptad-repeat domains (HR1 and HR2) in the gp41 subunit and their subsequent self-assembly into a six-helix bundle (6HB) that drives fusion. Env residues and features that influence conformational changes and the rate of virus entry, however, are poorly understood. Peptides corresponding to HR1 and HR2 (N and C peptides, respectively) interrupt formation of the 6HB by binding to the heptad repeats of a fusion-intermediate conformation of Env, making the peptides valuable probes for studying Env conformational changes.ResultsUsing a panel of Envs that are resistant to N-peptide fusion inhibitors, we investigated relationships between virus entry kinetics, 6HB stability, and resistance to peptide fusion inhibitors to elucidate how HR1 and HR2 mutations affect Env conformational changes and virus entry. We found that gp41 resistance mutations increased 6HB stability without increasing entry kinetics. Similarly, we show that increased 6HB thermodynamic stability does not correlate with increased entry kinetics. Thus, N-peptide fusion inhibitors do not necessarily select for Envs with faster entry kinetics, nor does faster entry kinetics predict decreased potency of peptide fusion inhibitors.ConclusionsThese findings provide new insights into the relationship between 6HB stability and viral entry kinetics and mechanisms of resistance to inhibitors targeting fusion-intermediate conformations of Env. These studies further highlight how residues in HR1 and HR2 can influence virus entry by altering stability of the 6HB and possibly other conformations of Env that affect rate-limiting steps in HIV entry.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0086-8) contains supplementary material, which is available to authorized users.

Circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection.

Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutinin-neuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo. Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

A cyclic GB virus C derived peptide with anti-HIV-1 activity targets the fusion peptide of HIV-1

The development of peptide fusion inhibitors based on short synthetic peptides represents a promising option in the fight against HIV-1 infection, especially in individuals infected with multiresistant HIV-1 strains. GBV-C has the beneficial effect of retarding the progression of AIDS in people who are co-infected with both the GBV-C and HIV viruses. In previous works, the E1(22–39) GBV-C sequence (E1P8lin) was found to be capable of inhibiting the interaction of HIV-1 FP with bilayers and its cyclic analogue (E1P8cyc) showed a higher anti-HIV-1 activity. In the present work, in an attempt to gain a better understanding of the interaction of E1P8 peptides with HIV-1 FP, we analyzed direct interactions between peptides at the molecular level. Our results support that E1P8cyc might be more potent at blocking HIV-1 entry than E1P8lin as a consequence of the structure induced in the complex formed with HIV-1 FP, which is able to modify the conformation adopted by this functional domain of the HIV-1 gp41 protein in target cell membranes.

Poster #T171 PIERRE RIVIÈRE VS. ANDERS BREIVIK: IS HISTORY REPEATING ITSELF? RATIONALITY, MADNESS, AND PSYCHOPATHOLOGY IN THE 19TH AND 21ST CENTURY

The objective of present work was to develop rapid, sensitive, selective, accurate and precise RP-HPLC method for analysis of Efavirenz from combination anti-HIV drug (Efavirenz-Enfuvirtide) incorporated into polymer-lipid hybrid nanoparticles (PLN). Chromatographic separation of Efavirenz was performed on Waters Spherisorb® 5 μm ODS (C18) column (4.6 x 250 mm) with acetonitrile and 10 mM phosphate buffer pH 6.8 (70:30, v/v) as mobile phase. The UV detection wavelength was 246 nm. The method was found to be linear between the concentration range of 500–20000 ng/ml with 160 ng/ml and 480 ng/ml as limit of detection and limit of quantitation respectively. Heteroscedasticity of calibration curve responses was minimized using weighted least square regression analysis. The method was found to be specific for analysis of Efavirenz in presence of Enfuvirtide (a fusion inhibitor peptide), formulation excipients and release media, accurate (average recovery rate: 99.9 ± 9.39%) and precise (%RSD < 2%). The validated RP-HPLC method could be effectively utilized to determine % entrapment efficiency (%EE), % drug loading (%DL), % cumulative drug release and drug content of Efavirenz from Efavirenz-Enfuvirtide co-loaded PLN.

Modular assembly of dimeric HIV fusion inhibitor peptides with enhanced antiviral potency

The HIV-1 envelope gp120/gp41 glycoprotein complex plays a critical role in virus-host cell membrane fusion and has been a focus for the development of HIV fusion inhibitors. In this Letter, we present the synthesis of dimers of HIV fusion inhibitor peptides C37H6 and CP32M, which target the trimeric gp41 in the pre-hairpin intermediate state to inhibit membrane fusion. Reactive peptide modules were synthesized using native chemical ligation and then assembled into dimers with varying linker lengths using Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ chemistry. Cell–cell fusion inhibition assays demonstrated that dimers with a (PEG)7 linker showed enhanced antiviral potency over the corresponding monomers. Moreover, the bio-orthogonal nature of the CuAAC ‘click’ reaction provides a practical way to assemble heterodimers of HIV fusion inhibitors. Heterodimers consisting of the T20-sensitive strain inhibitor C37H6 and the T20-resistant strain inhibitor CP32M were produced that may have broader spectrum activities against both T20-sensitive and T20-resistant strains.

A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

The peptides and peptidomimetics: HIV-1 fusion inhibitors

The fusion of HIV and target cell membranes is mediated by the transmembrane glycoprotein gp41. HIV-1 fusion inhibitors target gp41 to prevent the virus-cell membrane fusion, thus interrupt the initial steps of viral replication. The first peptide fusion inhibitor (T-20) was approved by the U.S. FDA in 2003. However, the application of T-20 was limited by drawbacks such as rapid proteolysis, highly clinical dosage, poor patient adhesive and ineffectiveness to drug resistant HIV-1 isolates. In recent years, considerable progresses have been made in the understanding of the mechanism of fusion inhibitors and in the design of novel fusion inhibitors, to overcome the drawbacks of T-20. Various peptides and peptidomimetics used as fusion inhibitors have been developed, and these novel fusion inhibitors target different part of HIV-1 gp41 and show higher activity and better pharmacokinetic properties. In this paper, the recent progresses in the design and development of novel peptides and peptidominetics used as HIV-1 fusion inhibitors are reviewed, providing a reference for related drug development and basic research.

Increase of anti-HIV activity of C-peptide fusion inhibitors using a bivalent drug design approach

We reported the design of fusion inhibitors with improved activity using a multivalent inhibitor design strategy. First, we chose C29 as the template sequence, which is a 29-mer peptide derived from HIV-1 gp41 CHR domain and has anti-HIV activity of IC50 118nM in a cell–cell fusion assay. We optimized the crosslink sites and linkers of the template peptide. We found that N-terminal crosslink caused activity improvement based on the multivalent co-operative effect. Especially, the IC50 of peptide (CAcaC29)2 was improved from 49.02 (monomeric form) to 5.71nM. Compared with long peptides, short peptides may be more suitable to analyze the co-operative effect. So we selected a shorter peptide C22 to synthesize the bivalent inhibitors. Due its weak helicity, no co-operative effect appeared. Therefore, we chose SC22EK, which were introduced salt bridges to consolidate the helicity based on the natural sequence C22. The cross-linked (CAcaSC22EK)2 was four times more potent than the monomer SC22EK in anti-HIV activity, with an IC50 value of 4.92nM close to the high active peptide fusion inhibitor C34. The strategy used in this study may be used to design new fusion inhibitors to interfere similar processes.

Effect of Amphipathic HIV Fusion Inhibitor Peptides on POPC and POPC/Cholesterol Membrane Properties: A Molecular Simulation Study

T-20 and T-1249 fusion inhibitor peptides were shown to interact with 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) (liquid disordered, ld) and POPC/cholesterol (1:1) (POPC/Chol) (liquid ordered, lo) bilayers, and they do so to different extents. Although they both possess a tryptophan-rich domain (TRD), T-20 lacks a pocket binding domain (PBD), which is present in T-1249. It has been postulated that the PBD domain enhances FI interaction with HIV gp41 protein and with model membranes. Interaction of these fusion inhibitor peptides with both the cell membrane and the viral envelope membrane is important for function, i.e., inhibition of the fusion process. We address this problem with a molecular dynamics approach focusing on lipid properties, trying to ascertain the consequences and the differences in the interaction of T-20 and T-1249 with ld and lo model membranes. T-20 and T-1249 interactions with model membranes are shown to have measurable and different effects on bilayer structural and dynamical parameters. T-1249’s adsorption to the membrane surface has generally a stronger influence in the measured parameters. The presence of both binding domains in T-1249 appears to be paramount to its stronger interaction, and is shown to have a definite importance in membrane properties upon peptide adsorption.

A Cholesterol Tag at the N Terminus of the Relatively Broad-Spectrum Fusion Inhibitory Peptide Targets an Earlier Stage of Fusion Glycoprotein Activation and Increases the Peptide's Antiviral Potency In Vivo

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG3) conjugated to the peptides' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG3-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide's insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.

HIV-1 Resistance Mechanism to an Electrostatically Constrained Peptide Fusion Inhibitor That Is Active against T-20-Resistant Strains

T-20EK is a novel fusion inhibitor designed to have enhanced α-helicity over T-20 (enfuvirtide) through engineered electrostatic interactions between glutamic acid (E) and lysine (K) substitutions. T-20EK efficiently suppresses wild-type and T-20-resistant variants. Here, we selected T-20EK-resistant variants. A combination of L33S and N43K substitutions in gp41 were required for high resistance to T-20EK. While these substitutions also caused resistance to T-20, they did not cause cross-resistance to other known fusion inhibitors.

An ancestral HIV-2/simian immunodeficiency virus peptide with potent HIV-1 and HIV-2 fusion inhibitor activity

To produce new fusion inhibitor peptides for HIV-1 and HIV-2 based on ancestral envelope sequences. HIV-2/simian immunodeficiency virus (SIV) ancestral transmembrane protein sequences were reconstructed and ancestral peptides were derived from the helical region 2 (HR2). The activity of one ancestral peptide (named P3) was examined against a panel of HIV-1 and HIV-2 primary isolates in TZM-bl cells and peripheral blood mononuclear cells and compared to T-20. Peptide secondary structure was analyzed by circular dichroism. Resistant viruses were selected and resistance mutations were identified by sequencing the env gene. P3 has 34 residues and overlaps the N-terminal pocket-binding region and heptad repeat core of HR2. In contrast to T-20, P3 forms a typical α-helical structure in solution, binds strongly to the transmembrane protein, and potently inhibits both HIV-2 (mean IC50, 63.8 nmol/l) and HIV-1 (11 nmol/l) infection, including T-20-resistant isolates. The N43K mutation in the HR1 region of HIV-1 leads to 120-fold resistance to P3 indicating that the HR1 region in transmembrane glycoprotein is the target of P3. No HIV-2-resistant mutations could be selected by P3 suggesting that the genetic barrier to resistance is higher in HIV-2 than in HIV-1. HIV-1-infected patients presented significantly lower P3-specific antibody reactivity compared to T-20. P3 is an HIV-2/SIV ancestral peptide with low antigenicity, high stability, and potent activity against both HIV-1, including variants resistant to T-20, and HIV-2. Similar evolutionary biology strategies should be explored to enhance the production of antiviral peptide drugs, microbicides, and vaccines.

Conjugation of Cholesterol to HIV-1 Fusion Inhibitor C34 Increases Peptide-Membrane Interactions Potentiating Its Action

Recently, the covalent binding of a cholesterol moiety to a classical HIV-1 fusion inhibitor peptide, C34, was shown to potentiate its antiviral activity. Our purpose was to evaluate the interaction of cholesterol-conjugated and native C34 with membrane model systems and human blood cells to understand the effects of this derivatization. Lipid vesicles and monolayers with defined compositions were used as model membranes. C34-cholesterol partitions more to fluid phase membranes that mimic biological membranes. Importantly, there is a preference of the conjugate for liquid ordered membranes, rich in cholesterol and/or sphingomyelin, as observed both from partition and surface pressure studies. In human erythrocytes and peripheral blood mononuclear cells (PBMC), C34-cholesterol significantly decreases the membrane dipole potential. In PBMC, the conjugate was 14- and 115-fold more membranotropic than T-1249 and enfuvirtide, respectively. C34 or cholesterol alone did not show significant membrane activity. The enhanced interaction of C34-cholesterol with biological membranes correlates with its higher antiviral potency. Higher partitions for lipid-raft like compositions direct the drug to the receptor-rich domains where membrane fusion is likely to occur. This intermediary membrane binding step may facilitate the drug delivery to gp41 in its pre-fusion state.