Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor

Abstract

Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle-Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a K(i) of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC(50) of approximately 25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPgammaS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked beta arrestin translocation and receptor downregulation induced by formyl-Nle-Leu-Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections.

Fig. 1

Transmembrane model of FPR. Residues in black are residues that have been shown in previous crosslinking and mutagenesis studies to be important in ligand binding , , . Residues in gray are sites of known polymorphisms .**R or W, *N or K.

Fig. 2

Determination of affinity of ligand binding. Binding of formyl-Nle–Leu–Phe–Nle–Tyr–Lys–FITC to FPR variants R190/N192, R190/K192, or W190/N192 was determined as a function of ligand concentration.

Fig. 3

Determination of affinity of Alexa Fluor labeled ETYIKWPWWVWL. Binding of Alexa Fluor labeled ETYIKWPWWVWL to W190/N192 FPR, R190/K192 FPR, and R190/N192 FPR and to CHO-S cells not expressing FPR are shown as a function of concentration. K_ds for W190/N192, R190/K192, and R190/N192 were 99 ± 10 nM, 115 ± 10 nM, and 121 ± 18 nM, respectively.

Fig. 4

ETYIKWPWWVWL labeled with Alexa Fluor 488 is internalized in CHO cells expressing FPR. CHO cells expressing FPR W190/N192, FPR R190/N192 or FPR R190/ K192 were incubated with indicated concentrations of Alexa Fluor labeled peptide for 37 °C for 1 h and the amount of internalized ligand was determined. The very low internalization of CHO cells not expressing FPR has been subtracted.

Fig. 5

Stimulation of GTPγS binding by formyl-Nle–Leu–Phe, formyl-MYVKWPWYVWL, MYVKWPWYVWL or ETYIKWPWWVWL in cells expressing W190/N192, R190/K192, or R190/N192 FPR.

Fig. 6

Inhibition of stimulation of GTPγS binding induced by formyl-Nle–Leu–Phe by ETYIKWPWWVWL in FPR variants R190/N192, R190/K192, or W190/N192. Inhibition of FPR activation by viral peptides. Inhibition of stimulation of GTPγS binding induced by formyl-Nle–Leu–Phe by ETYIKWPWWVWL. ETYIKWP, or WWVWL in W190/N192 FPR.

Fig. 7

Inhibition of FPR activation by viral peptides. Inhibition of stimulation of GTPγS binding induced by formyl-Nle–Leu–Phe by ETYIKWPWWVWL. ETYIKWP, or WWVWL in W190/N192 FPR.

Fig. 8

Arrestin translocation in the presence of various FPR ligands. Relative arrestin translocation was determined in non-expressing CHO cells, R190/N192 FPR expressing cells, R190/K192 FPR expressing cells, or W190/N192 FPR expressing cells in response to 10 μM formyl-Nle–Leu–Phe, 10 μM ETYIKWPWWVWL, or 10 μM formyl-Nle–Leu–Phe + 10 μM ETYIKWPWWVWL. 10 μM ETYIKWPWWVWL inhibited translocation in response to 10 μM formyl-Nle–Leu–Phe by 18 ± 4% in R190/N192 FPR expressing cells, 20 ± 9% in R190/K192 FPR expressing cells, and 77 ± 10% in W190/N192 expressing cells (P < 0.006 for W190/N192 FPR vs. either R190/N192 or R190/K192).

Fig. 9

Down regulation of FPR surface expression in response to various ligands. Surface expression of FPR in W190/N192 FPR expressing cells, R190/K192 FPR expressing cells, or R190/N192 FPR expressing cells was determined after exposure for 1 h at 37° to either 10 μM formyl-Nle–Leu–Phe, 10 μM ETYIKWPWWVWL, or 10 μM formyl-Nle–Leu–Phe + 10 μM ETYIKWPWWVWL. ETYIKWPWWVWL inhibited receptor downregulation in response to formyl-Nle–Leu–Phe by 16 ± 2% in R190/N192 FPR expressing cells, 20 ± 2% in R190/K192 FPR expressing cells, and 64 ± 6% in W190/N192 FPR expressing cells. (P < 0.001 W190/N192 vs. either R190/N192 or R190/K192).