The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

Tying up the package: The HIV‐1 RNA packaging signal ψ is a complex RNA structure located within the 5′‐end of unspliced HIV‐1 RNAs (see picture). Upon binding of the viral polyprotein Gag to the ψ RNA, a process mediated by the nucleocapsid protein NCp7, two RNA genomes are packaged into the assembling virions. By using phage display technology peptides were selected that bind specifically to different RNA structures derived from the ψ RNA.

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CCR5 is a chemokine receptor that mediates entry of human immunodeficiency virus-1 (HIV-1). Two monoclonal antibodies (mAbs) that block HIV-1 entry, 3A9 and 5C7, were used to select peptide mimotopes of sequences on CCR5 from phage displayed peptide libraries. The selected mimotofpes comprised motifs at the N-terminus and on the first and third extracellular loops (ECL1 and ECL3) of CCR5. Amino acids in these motifs were exchanged for alanines by site-directed mutagenesis (sdm) in the cDNA for human CCR5. Ensuing effects on antibody binding to CCR5, cellular entry of HIV-1 and chemokine-induced signalling were analysed by transfection of mutant cDNAs into HEK293.CD4 cells. For both mAbs, fluorescence-activated cell sorting analysis was used to define overlapping conformational epitopes on CCR5 at the N-terminus, on ECL1 and ECL3. Mutation of the N-terminal motif 10 YD 11 prevented HIV-1 entry into transfected cells as judged by single round infection assays with R5 and R5X4 HIV-1 isolates, as did mutation of the motif 96 FG 97 in ECL1, whereas mutation of the motif 274 RLD 276 in ECL3 had only a minor effect. None of the motifs in CCR5 relevant to HIV-1 entry disrupted chemokine-induced signalling. Thus, peptide mimotopes of conformational contact sites of CCR5 with the paratope of mAbs 3A9 and 5C7 represent sites on CCR5 that are essential for HIV-1 entry. Structural knowledge of these mimotopes could help elucidate the nature of the interaction between CCR5 and HIV-1, and thus the derivation of specific inhibitors of entry of HIV-1 into susceptible cells without interference with chemokine signalling. The chemokine receptor CCR5 is a G-protein-coupled receptor (GPCR) that is the major cellular coreceptor for primary human immunodeficiency virus-1 (HIV-1) isolates, and hence has been identified as an attractive therapeutic target in HIV-1 infection. 1 CCR5-dependent viral strains (R5 virus) are predominantly found in newly infected patients and in asymptomatic individuals. The several anti-viral molecules that target CCR5 include chemokines, modified chemokines, monoclonal antibodies (mAbs) and small molecule inhibitors. 2,3 Some agents including small molecule inhibitors have already entered clinical trials: these are the first to target host receptors rather than viral structures in therapy of HIV-1 infection. Individuals that lack functional CCR5 on their cell surface because of a homozygous deletion of 32 bp in their ccr5 genes (CCR5D32) are resistant to HIV-1-R5 viruses, which is encouraging for drug development based on antagonism of CCR5. 1,2 mAbs to CCR5 have been generated that interfere with HIV-1 infection, ligand-induced signalling or both; 2,4-8 two such mAbs, 3A9 and 5C7, proved capable of inhibiting the binding of gp120 of HIV-1 without interference with chemokine binding or signalling. 9 Accordingly, knowledge of these epitopes should help to define regions of CCR5 specifically required for coreceptor function. Previous attempts to identify such CCR5 domains were based on receptor chimeras in wh...

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