HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop

<div><p>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 <em>flaviviridae</em> 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.</p> </div

E2-derived peptides do not alter CD4 or coreceptor surface expression.

<p>CD4, CXCR4 and CCR5 expression on PBMC (A) and TZM-bl cells (B) after preincubation with E2 peptides were determined by flow cytometry analysis. Positive controls like PMA (phorbol 12-myristate 13-acetate) lead to down-regulation of CD4, SDF-1α (CXCR4-ligand) and RANTES (CCR5-ligand) to down-regulation of the respective coreceptors, while E2 peptides P4-7 and P6-2 including the negative control P28 had no relevant effect on HIV-1 receptor presentation. Percentages illustrate the ratio-to-cell-negative (RTCN) values that were calculated from the percentage of positive cells and the fluorescence intensity of peptide-incubated cells compared to mock-incubated cells. Columns show average values ±SD of two independent experiments each performed in triplicate. *: p<0.05; **: <i>p</i><0.01 (mock- vs. inhibitor-incubated cells).</p

The N-termini of GBV-C E2 and HIV-1 gp120 share the same binding region within the gp41.

<p>Competitive binding of E2 and gp120 peptides (N35, N35s) with recombinant E2_340-Fc protein to immobilized recombinant gp41_MN. Simultaneously to E2₃₄₀-Fc incubation peptides of E2 or gp120 were added with increasing amounts. The graphs show average values of three independent experiments each performed in duplicate. *: <i>p</i><0.05; **: <i>p</i><0.01 (N35 or N35s vs. P28; P4-7 or P6-2 vs. P28 [data not shown]).</p

Model of the N-terminal region (residues 1–195) of the E2-protein.

<p>The globular Ig-fold domain (residues 76–195), for which a structural model could be generated, is shown in backbone presentation and colored according to the secondary structure type. The N-terminus (residues 1–75), which is not predicted to adopt a globular three-dimensional structure, is schematically depicted as circles indicating the identity of the respective amino acids. Cysteines, which may form disulfide bonds, are shown as diamonds and their sequence position is indicated. Residues belonging to the P4-7 and P6-2 peptides investigated in the present study are highlighted in red and blue, respectively. Overlapping residues present in both peptides are shown in magenta.</p

E2-derived peptides bind to the disulfide loop of gp41.

<p>(A) Illustration of gp41 variants (strains IIIB, MN) and peptides (N36, C34) used in ELISA as well as the binding epitopes of different monoclonal gp41 antibodies. Gp41 consists of the N-terminal fusion peptide (FP), the N- and C-heptad repeats (NHR, CHR) that are connected by the disulfide loop region (C-C loop), the membrane proximal external region (MPER), the α-helical transmembrane-spanning domain (TM), and the cytoplasmic tail (CP). (B) Inhibitory activity on 6-Helix-Bundle (6-HB) formation of peptides P4-7, P6-2, and P28, as well as C34 as control was measured by ELISA using the NC-1 monoclonal antibody that detects the 6-HB formation between C34-biotin and N36 peptides. Each sample was tested in triplicate. This experiment was repeated twice and similar results were obtained. The statistical significance (<i>p</i><0.01) was achieved for all measure points (C34 vs. P28). (C) Competitive binding of E2 peptides and different gp41 targeting mAbs to immobilized recombinant gp41_MN. Simultaneously to mAb incubation E2 peptides were added with increasing amounts. The graphs show average values of three independent experiments each performed in duplicate. *: <i>p</i><0.05 (P4-7 or P6-2 vs. P28) (D) Binding of recombinant E2₃₄₀-Fc protein and Fc protein as control to cyclic (Loop36ox) and linear (Loop36s) peptides presenting the HIV-1 gp41 disulfide loop (residues 588–623). The graphs show average values of three independent experiments each performed in duplicate. The statistical significance (<i>p</i><0.05) was achieved with more than 0.003 µM concentrations of each protein (Loop36ox+E2₃₄₀-Fc vs. Loop36ox+Fc).</p

E2-derived peptides inhibit the HIV-1 fusion process.

<p>The HIV-inhibitory activity of known HIV-1 entry inhibitors or respective E2 peptides was determined using a virus-cell fusion assay under standard (respective inhibitors were added simultaneously to HIV NL4-3_BlaM-Vpr inoculum: pre-CD4 binding) or under TAS (temperature-arrested state) conditions (respective inhibitors were added at low temperature of 23°C for 1 h after spin-inoculation [4°C] and removal of unbound HIV-1 particles: post-CD4 binding). Subsequently, HIV-1 fusion was enabled via a temperature shift to 37°C. Antibodies b12 (anti [a]-CD4-binding site [bs]) and B4 (a-CD4) are early inhibitors interfering with CD4 engagement, whereas 2F5 (MPER antibody) and AMD-3100 (CXCR4 antagonist) act after CD4 binding. The E2 peptide P28 served as a negative control. The extent of virus-cell fusion was determined from the ratio of blue (460 nm) and green (510 nm) emission upon exciting the cells at 405 nm using a fluorescence plate reader. Percentages were calculated in relation to mock-treated cells. Columns show average values ±SD of five independent experiments each performed in triplicate. **: p<0.01.</p

Recombinant E2₃₄₀-Fc protein and E2-derived peptides bind HIV-1 gp41.

<p>(A) 293T cells expressing NL4-3 envelope (X4<i>env</i>) or empty plasmid as control were incubated with sCD4 and FITC-conjugated E2 peptides, respectively. Peptide binding was analyzed by flow cytometry. Percentages illustrate the increase of cell binding to X4<i>env</i>-transfected cells. The increase is normalized to the background binding of E2 peptides to mock-transfected cells by calculating the RTCN value. Columns show average values ±SD of three independent experiments each performed in duplicate. *p<0.05 (vector- vs. X4<i>env</i>-transfected cells) (B) Binding of recombinant E2₃₄₀-Fc protein and Fc protein as control to recombinant HIV-1 glycoproteins (gp120_IIIB, gp120_MN, gp41_IIIB, or gp41_MN). (C) E2 peptides with increasing amounts were added to recombinant E2₃₄₀-Fc protein (at constant concentration) and then transferred to immobilized recombinant gp41_MN. All ELISA graphs show average values of three independent experiments each performed in duplicate. *: <i>p</i><0.05; **: <i>p</i><0.01 (E2₃₄₀-Fc, Fc, P4-7, or P6-2 vs. P28).</p

Local similarities between the N-termini of gp120 and E2.

<p>(A) Similarities of the two local sequences in the N-termini of HIV-1 gp120 and GBV-C E2. The detected E2 stretches (residues 33–46 and 54–70) are shown in the context of the entire E2 N-terminus and are color coded in green and orange, respectively. Illustrated are the sequences of the peptides that proved to be potent in HIV-1 entry inhibition <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054452#pone.0054452-Koedel1" target="_blank">[33]</a>. These peptides almost exclusively cover the E2 sequence stretch that exhibits similarities to the gp120 N-terminus. Molecules are shown in space-filled presentation and the functionally important regions are colored. (B) Structure of monomeric gp120. The N-terminal region of gp120 that exhibits local similarity with the active E2-derived peptides is shown in blue. Residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054452#pone.0054452-Caffrey1" target="_blank">[36]</a>) are additionally shown in green. (C) Structure of trimeric gp41. The disulfide-bonded loops that are recognized by the T32 antibody (residues 596–612) are shown in red. Residues 592–596 additionally present in the F240 epitope are shown in orange. Residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054452#pone.0054452-Caffrey1" target="_blank">[36]</a>) are additionally shown in green. The coordinates for structure presentations were taken from PDB entries 3JWD and 2EZO for gp120 and gp41, respectively.</p

E2-derived peptides do not influence HIV-1 gp120 binding to CD4 and CCR5.

<p>(A) 293T cells expressing CD4 were preincubated with E2-derived peptides and specific controls that bind to gp120 (sCD4; VRC01, directed against CD4 binding site [bs]) or CCR5 (Tak-779). Subsequently, cells were incubated with soluble Fc-gp120 (HIV-1_JRCSF) and binding to CD4 was analyzed by flow cytometry using a FITC-conjugated anti-human IgG pAb. Binding to cells in the absence of inhibitor was set as 100%. Columns show average values ±SD of three independent experiments each performed in duplicate. *: p<0.05 In (B) and (C) 293T cells expressing CD4, CCR5, or CXCR4 were preincubated with E2-derived peptides, E2₃₄₀-Fc and Fc protein as well as specific controls that bind to gp120 (sCD4, VRC01, 447-52D, F425 B4e8), gp41 (5F3), or CCR5 (Tak-779, Maraviroc). Soluble HIV-1 gp120 was added to the CD4-, CCR5-, or CXCR4-expressing cells and binding was investigated by separating the cell lysates on SDS-PAGE and visualized by performing Western Blot analyses. No influence of E2 peptides or intact E2 protein on the binding efficiency between gp120 and CD4, CCR5, or CXCR4 respectively, was observed after normalization of band intensity with the gel loading control Hsp90.</p