Fejerlectin, a Lectin-like Peptide from the Skin of Fejervarya limnocharis, Inhibits HIV-1 Entry by Targeting Gp41

Abstract

Human immunodeficiency virus type 1 (HIV-1) is mainly transmitted by sexual intercourse, and effective microbicides preventing HIV-1 transmission are still required. Amphibian skin is a rich source of defense peptides with antiviral activity. Here, we characterized a lectin-like peptide, fejerlectin (RLCYMVLPCP), isolated from the skin of the frog Fejervarya limnocharis. Fejerlectin showed significant hemagglutination and d-(+)-galacturonic acid-binding activities. Furthermore, fejerlectin suppressed the early entry of HIV-1 into target cells by binding to the N-terminal heptad repeat of HIV-1 gp41 and preventing 6-HB formation and Env-mediated membrane fusion. Fejerlectin is the smallest lectin-like peptide identified to date and represents a new and promising platform for anti-HIV-1 drug development.

Figure 1

Identification and characterization of fejerlectin. (A) cDNA and the deduced amino acid sequence of fejerlectin. The signal peptide is shaded in gray and is followed by an acidic spacer domain with KR residues at the end (in red bold). The stop codon is indicated with an asterisk (*), and the sequence of mature fejerlectin is boxed. (B) Purity of synthesized fejerlectin detected by HPLC. (C) Molecular weight of synthesized fejerlectin confirmed by mass spectrometry.

Figure 2

Binding reaction of fejerlectin with d-(+)-galacturonic acid. (A) Effects of d-(+)-galacturonic acid on the HA activity of fejerlectin. The second row shows the HA activity of fejerlectin at final concentrations between 20 and 0.3125 μM. The third row shows the HA activity of different concentrations of d-(+)-galacturonic acid (from left to right: 8, 4, 2, 1, 0.5, 0.25, and 0.125 mM). The fourth row shows the HA activity of 20 μM fejerlectin incubated with d-(+)-galacturonic acid at concentrations corresponding to the third row. (B) Flow cytometry of the binding reaction between fejerlectin and bacteria. Staphylococcus aureus and Escherichia coli were incubated with fluorescein isothiocyanate (FITC)-fejerlectin (3.75, 7.5, 15, and 30 μM) at 37 °C for 15 min before flow cytometry analysis. (C) Bacterial agglutination induced by fejerlectin. S. aureus and E. coli diluted to 2 × 10⁸ cells/mL in Tris-buffered saline (TBS) were incubated with bovine serum albumin (BSA) (a, d), 5 μM fejerlectin (b, e), or 5 μM fejerlectin premixed with an equal volume of 4 mM d-(+)-galacturonic acid (c, f) for 1 h at room temperature and then stained with Gram dye. (D) Isothermal titration calorimetry (ITC) analysis of binding reaction of fejerlectin with d-(+)-galacturonic at 25 °C. The top panels displayed thermo changes of each injection at different time points, while the bottom panel presented the change of enthalpy as a function of ligand/target molar ratio. (E) Surface plasmon resonance imaging (SPRi) analysis of d-(+)-galacturonic acid binding to fejerlectin immobilized on a gold chip. Data were fit using a single-site binding model using the MicroCal Origin software package.

Figure 3

Anti-HIV-1 activity of fejerlectin and its cytotoxicity to host cells. The inhibitory activities of fejerlectin against HIV-1 infectious clones including HIV-1_SF162 (A), HIV-1_NL4-3 (B), and HIV-1_{81A and NL4-3} (C). Maraviroc, AMD3100, and AZT were used as positive controls, respectively. (D) In vitro cytotoxicity of fejerlectin on TZM-bl cells. Experimental data are expressed as mean ± standard deviation (SD) (n = 3).

Figure 4

Time-of-addition assay with fejerlectin: (A) R5-monotropic HIV-1_SF162, (B) X4-monotropic HIV-1_NL4-3, and (C) X4R5 dual-tropic HIV-1_{81A and NL4-3}. Experimental data are expressed as mean ± SD (n = 3).

Figure 5

Effect of fejerlectin on the HIV-1 infection cycle. Inhibitory activities of fejerlectin against HIV-1_JR-FL (A), HIV-1_HXB2 (B), and VSV-G (C) pseudotyped viruses. Maraviroc, AMD3100, and T-20 were used as positive controls. (D) Effects of fejerlectin on the formation of syncytia between CHO-WT and MT-2 cells. ADS-J1 was chosen as a positive control. Experimental data are expressed as mean ± SD (n = 3).

Figure 6

Effect of fejerlectin on the formation of 6-HB. The inhibitory effect of fejerlectin on the formation of 6-HB was analyzed by enzyme-linked immunosorbent assay (ELISA) (A) and CD spectra (B). ADS-J1 was chosen as a positive control. Experimental data are expressed as mean ± SD (n = 3). (C) Binding affinity between fejerlectin and N36 peptide derived from HIV-1_JR-FL gp41 analyzed by SPRi.