Peptide Ligands Selected with CD4-Induced Epitopes on Native Dualtropic HIV-1 Envelope Proteins Mimic Extracellular Coreceptor Domains and Bind to HIV-1 gp120 Independently of Coreceptor Usage

Abstract

During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.HIV-1 entry (reviewed in reference 2) is a multistep process initiated by binding of the outer envelope (Env) glycoprotein gp120 to the CD4 receptor. This results in conformational changes leading to exposure of CD4-induced (CD4i) epitopes for the subsequent obligatory interaction with the chemokine receptor CCR5 or CXCR4. Further conformational changes in gp120 activate the fusion peptide at the N terminus of the transmembrane glycoprotein gp41 and lead to the assembly of a six-helix bundle structure that triggers membrane fusion and internalization of the viral particles. This sequential HIV-1 entry process allows the virus to protect the functionally important and highly conserved Env entry epitopes by limiting their exposure to antibodies which may neutralize the infection (35). The complex HIV-1 entry process offers the opportunity for therapeutic intervention at multiple steps. Thus, the first HIV-1 entry inhibitor approved by the FDA (T20, enfuvirtide [Fuzeon]) is a peptide inhibiting the very last step of entry, i.e., membrane fusion, by interfering with the six-helix bundle formation (39). A second entry inhibitor (maraviroc [Selzentry]), targeting CCR5, was approved by the FDA in 2007 (46). This drug, like others whose development had to be discontinued due to liver toxicities, is a CCR5 antagonist, and its use is restricted to drug-experienced HIV-1...

A neutralization assay for chikungunya virus infections in a multiplex format

“…They enable studies without the need of using the native virus, which usually requires a higher safety level. Pseudotyping of retro-or lentiviral vectors is frequently used to study viral entry and to evaluate entry inhibitors (Siegert et al, 2005), or to harvest neutralizing antibodies at reduced safety levels (Schnierle et al, 1997;Seaman et al, 2010;Dervillez et al, 2010). It has been previously described that lentiviral vectors can be pseudotyped with CHIKV E1/E2 and transfer the CHIKV host range to these vectors (Salvador et al, 2009;Akahata et al, 2010;Kishishita et al, 2013).…”

Targeting Cellular Cofactors in HIV Therapy

“…The importance of the N-terminal domain, especially from the CCR5 receptor, for gp120 binding led to the derivation of synthetic peptides inhibiting HIV entry in the micromolar range [74][75][76]. We could select an entry inhibitory peptide (XD3) with remarkable homology to the N-terminus of CCR5 and CXCR4 by phage display using CD4i epitopes on native dualtropic Env as target [77]. For CXCR4, which is less dependent on its N-terminus for HIV entry, we further designed a peptide comprising ECL1 to 3 linked to short spacers, which showed selective CXCR4 binding and entry inhibition in the low micromolar range [78].…”

A Functionally Selective Synthetic Mimic of the HIV‐1 Co‐receptor CXCR4

“…In a cellular HIV‐1 infection assay22 it was found that CX4‐M1 (IC 50 =9.93 μ M ), but not CX4‐M5 (IC 50 >50 μ M ), which presents only ECL2 of CXCR4, is able to interfere with the infection of cells with HIV‐1 with X4‐tropic HIV‐1 (HxBc2) (Table 1). This inhibitory effect was even slightly enhanced for the R183A and R188A variants of CX4‐M1 (CX4‐M6, IC 50 =5.14 μ M , and CX4‐M8, IC 50 =4.95 μ M ), which is in accordance with the ELISA binding data.…”