Oriented display of HIV-1 Env trimers by a novel coupling strategy enhances B cell activation and phagocytosis.

Introduction: Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display. Methods: To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner. Results: Microspheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation. Discussion: In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.

Bind&Bite: covalently stabilized heterodimeric coiled-coil peptides for the site-selective, cysteine-free chemical modification of proteins.

Ensuring site-selectivity in covalent chemical modification of proteins is one of the major challenges in chemical biology and related biomedical disciplines. Most current strategies either utilize the selectivity of proteases, or are based on reactions involving the thiol groups of cysteine residues. We have modified a pair of heterodimeric coiled-coil peptides to enable the selective covalent stabilization of the dimer without using enzymes or cysteine moieties. Fusion of one peptide to the protein of interest, in combination with linking the desired chemical modification to the complementary peptide, facilitates stable, regio-selective attachment of the chemical moiety to the protein, through the formation of the covalently stabilized coiled-coil. This ligation method, which is based on the formation of isoeptide and squaramide bonds, respectively, between the coiled-coil peptides, was successfully used to selectively modify the HIV-1 envelope glycoprotein. Covalent stabilization of the coiled-coil also facilitated truncation of the peptides by one heptad sequence. Furthermore, selective addressing of individual positions of the peptides enabled the generation of mutually selective coiled-coils. The established method, termed Bind&Bite, can be expected to be beneficial for a range of biotechnological and biomedical applications, in which chemical moieties need to be stably attached to proteins in a site-selective fashion.

Smaller, Stronger, More Stable: Peptide Variants of a SARS-CoV-2 Neutralizing Miniprotein.

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro.

BACKGROUND: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. METHODS: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. RESULTS: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive ß-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. CONCLUSIONS: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection.

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.

A PROSS-designed extensively mutated estrogen receptor α variant displays enhanced thermal stability while retaining native allosteric regulation and structure.

Protein stability limitations often hamper the exploration of proteins as drug targets. Here, we show that the application of PROSS server algorithms to the ligand-binding domain of human estrogen receptor alpha (hERα) enabled the development of variant ERPRS* that comprises 24 amino acid substitutions and exhibits multiple improved characteristics. The protein displays enhanced production rates in E. coli, crystallizes readily and its thermal stability is increased significantly by 23 °C. hERα is a nuclear receptor (NR) family member. In NRs, protein function is allosterically regulated by its interplay with small molecule effectors and the interaction with coregulatory proteins. The in-depth characterization of ERPRS* shows that these cooperative effects are fully preserved despite that 10% of all residues were substituted. Crystal structures reveal several salient features, i.e. the introduction of a tyrosine corner in a helix-loop-helix segment and the formation of a novel surface salt bridge network possibly explaining the enhanced thermal stability. ERPRS* shows that prior successes in computational approaches for stabilizing proteins can be extended to proteins with complex allosteric regulatory behaviors as present in NRs. Since NRs including hERα are implicated in multiple diseases, our ERPRS* variant shows significant promise for facilitating the development of novel hERα modulators.

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV-1 Inhibition by a Peptide Derived from Human Pegivirus-1 Envelope Protein E2.

Co-infection with the human pegivirus 1 (HPgV-1) often has a beneficial effect on disease progression in HIV-1-infected individuals. Several HPgV-1 proteins and peptides, including a 20-mer peptide (P6-2) derived from the N-terminal region of the HPgV-1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C-terminal negative charge as key factors for the HIV-1 inhibitory activity of P6-2. Analysis of mutations in P6-2-resistant HIV-1 indicated a binding site for the peptide in the HIV-1 envelope glycoprotein gp120. In fact, P6-2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV-1 inhibitory activity of P6-2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6-2 with the gp120 V3 loop.

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, ß- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

High-resolution crystal structures of two prototypical ß- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of ß- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these ß- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

Untargeted Proteomics-Based Profiling for the Identification of Novel Processing-Induced Protein Modifications in Milk.

Nonenzymatic post-translational protein modifications (nePTMs) affect the nutritional, physiological, and technological properties of proteins in food and in vivo. In contrast to the usual targeted analyses, the present study determined nePTMs in processed milk in a truly untargeted proteomic approach. Thus, it was possible to determine to which extent known nePTM structures explain protein modifications in processed milk and to detect and identify novel products. The method combined ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with bioinformatic data analysis by the software XCMS. The nePTMs detected by untargeted profiling of a ß-lactoglobulin-lactose model were incorporated in a sensitive scheduled multiple reaction monitoring method to analyze these modifications in milk samples and to monitor their reaction kinetics during thermal treatment. Additionally, we identified the structures of unknown modifications. Lactosylation, carboxymethylation, formylation of lysine and N-terminus, glycation of arginine, oxidation of methionine, tryptophan, and cysteine, oxidative deamination of N-terminus, and deamidation of asparagine and glutamine were the most important reactions of ß-lactoglobulin during milk processing. The isomerization of aspartic acid was observed for the first time in milk products, and N-terminal 4-imidazolidinone was identified as a novel nePTM.

Inhibition of HIV-1 infection by human pegivirus type 1-derived peptides is affected by human pegivirus type 1 genotype and HIV-1 coreceptor tropism.

OBJECTIVE(S): Up to 40% of HIV-1 infected individuals are coinfected with human pegivirus type 1 (HPgV-1). The majority of studies, but not all, have reported a beneficial effect of HPgV-1 coinfection on HIV-1 disease progression. So far, the impact of different HPgV-1 genotypes on different HIV-1 subtypes remains unclear. METHODS: Peptides derived from HPgV-1 envelope protein E2, and representing different viral genotypes, were synthesized using Fmoc/t-Bu-based solid phase peptide synthesis. The inhibitory effect of these peptides on the infection of reporter cell lines was tested using an HIV-1 subtype panel representing clades A (n = 2), AG (n = 2), B (n = 6), C (n = 2), D (n = 2), F (n = 2), G (n = 1), G/H (n = 1), and group O (n = 2). RESULTS: HIV-1 infection was blocked more efficiently by peptides derived from HPgV-1 GT2 than GT1 (P = 0.05). The HIV-1 subtype did not affect the degree of inhibition by a peptide derived from HPgV-1 GT2. All CXCR4-/dual-tropic isolates (n = 12), but only half (four out of eight) CCR5-tropic viruses were inhibited by this peptide (P = 0.014). CONCLUSION: Our data indicate that the inhibitory effect of peptides derived from HPgV-1 E2 protein is dependent on the genotype, suggesting that coinfection with HPgV-1 GT1 is less likely to confer a beneficial effect on HIV-1 disease progression than GT2. The preferential suppression of more pathogenic CXCR4-tropic HIV-1 by peptides derived from HPgV-1 GT2 may explain the favorable effect in patients harboring these HIV-1 isolates. Consequently, HPgV-1 genotype and HIV-1 coreceptor tropism are likely determinants for the beneficial effect of HPgV-1 co-infection in HIV-1-infected individuals.

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains.

BACKGROUND: Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming. RESULTS: This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells. CONCLUSIONS: The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

A Monosaccharide Residue Is Sufficient to Maintain Mouse and Human IgG Subclass Activity and Directs IgG Effector Functions to Cellular Fc Receptors.

Immunoglobulin G (IgG) glycosylation modulates antibody activity and represents a major source of heterogeneity within antibody preparations. Depending on their glycosylation pattern, individual IgG glycovariants present in recombinant antibody preparations may trigger effects ranging from enhanced pro-inflammatory activity to increased anti-inflammatory activity. In contrast, reduction of IgG glycosylation beyond the central mannose core is generally believed to result in impaired IgG activity. However, this study reveals that a mono- or disaccharide structure consisting of one N-acetylglucosamine with or without a branching fucose residue is sufficient to retain the activity of the most active human and mouse IgG subclasses in vivo and further directs antibody activity to cellular Fcγ receptors. Notably, the activity of minimally glycosylated antibodies is not predicted by in vitro assays based on a monomeric antibody-Fcγ-receptor interaction analysis, whereas in vitro assay systems using immune complexes are more suitable to predict IgG activity in vivo.

Turning Peptide Ligands into Small-Molecule Inhibitors of Protein-Protein Interactions.

Proline makeover: Truncation and extensive chemical modification of a peptide ligand yielded a biologically active, cell-permeable, peptidomimetic, small-molecule inhibitor of a protein-protein interaction. A key step in this transformation was the replacement of a tetraproline motif by two conformationally constrained diproline units that retain the molecule's PPII helix.

Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction.

Ligand selectivity of a synthetic CXCR4 mimetic peptide.

The chemokine receptor CXCR4 belongs to the family of seven-transmembrane G-protein coupled receptors (GPCRs). It is activated by its natural ligand SDF-1α. In addition, CXCR4, along with CCR5, serve as coreceptors during HIV-1 entry into its target cell. Recently, we introduced a CXCR4 mimetic peptide, termed CX4-M1, which presents the three extracellular loops (ECLs) of the receptor. CX4-M1 was shown to selectively bind to gp120 of X4-tropic, that is, CXCR4 using, HIV-1, as well as to peptides that present the V3-loops of these gp120 proteins. Furthermore, CX4-M1 selectively inhibits infection of cells with X4-tropic HIV-1. We have now adapted the sequence of the ECLs presented by CX4-M1 to the recently published crystal structure of CXCR4. The binding behavior, as well as the effect on HIV-1 infection, of the resulting peptide (CX4-Mc) was very similar to CX4-M1, validating retrospectively the original design of CX4-M1. A peptide presenting the ECLs of CCR5 (CR5-M), on the other hand, did neither bind to gp120 from X4-tropic HIV-1, nor did it inhibit infection of cells with X4-tropic HIV-1. Furthermore, we could show that CX4-M1, as well as CX4-Mc, but not CR5-M, are selectively recognized by anti-CXCR4 antibodies, bind to SDF-1α, and also inhibit SDF-1α signaling, extending the scope of selective functional CXCR4 mimicry through CX4-M1.

HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.