Lentiviruses are associated with immunological impairment in

Lentiviruses are associated with immunological impairment in their respective hosts, and both human immunodeficiency and feline immunodeficiency viral infections increase the likelihood of secondary bacterial infections [23], [24]. Recently, Kubes et al. [25] demonstrated that feline neutrophils exhibited a marked (>90%) reduction of neutrophil chemotaxis toward fMLF following infection with FIV. Ueda et al. noted that the HIV-1 envelope GP-41 was able to downregulate the expression of FPR and several chemokine receptors at low nanomolar concentrations, and that the downregulation of FPR and the chemokine receptors was dependent upon expression of CD4 [26]. Peptides derived from the HR2 and proximal membrane regions of HIV-1, SIV, HRSV, human parainfluenza virus type 3, measles virus, and a coronavirus have all been shown to be able to block virus infection [27], [28], [29], [30]. An eight mer peptide derived from the proximal membrane region of FIV GP-36, WEDWVGWI, was a potent (low nanomolar) inhibitor of FIV infection [31]. The three W residues were all essential for activity whereas the other residues were unimportant for activity. We previously observed that FPR W190/N192 exhibited an enhanced affinity to bind some viral peptides but had a reduced affinity for the formyl peptide formyl-NleLeuPhe when compared with FPR R190/N192 or FPR R190/K192 [8]. Here we assessed whether the three W residues in WEDWVGWI essential to viral inhibition [31] are also are important in FPR binding. We also undertook a more extensive analysis of three FPR polymorphisms using a variety of formyl peptides of varying sequences in order to identify formyl peptides which might behave markedly different toward the different polymorphisms. We identified several formyl peptides that exhibit high selectivity for activation of R190/K192 and R190/N192 compared with W190/N192. We also identified possible secondary structure changes which might result from amino AS 101 changes by secondary structure prediction [32] and correlateld these structural changes with altered binding and activation of FPR.
Materials and methods
Results and discussion FIV peptides (N-acetylated and C-amidated) derived from the proximal membrane region of GP-36 have been assessed for antiviral activity [31]. Therefore N-acetylated and C-amidated, WEDWVGWI, AEDWVGVWI, WEDAVGWI, and WEDWVGAI were synthesized and evaluated for their ability to inhibit formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC binding to FPR. Fig. 1 shows the effect of these peptides on binding to W190/N192, R190/K192, and R190/N192. Only WEDWVGWI exhibited appreciable inhibition similar to what Giannecchini et al. [31] observed for inhibition of viral infection by FIV inhibition. The dose dependence of WEDWVGWI, formyl-NleWEDWVGWI, and formyl-NleNleWEDWVGWI inhibition of formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC binding to W190/N192, R190/K192, and R190/N192 was determined. The results are shown in Table 1. Thus the addition of a formyl-Nle had no affect on affinity and the addition of formyl-NleNle increased the affinity to FPR ∼2-fold. W190/N192 exhibited a ∼2 fold lower affinity for all three peptides. Since downregulation is a hallmark of receptor activation [33] and the trafficking of FPR expressed in CHO cells to endocytotic vesicles following treatment with fMLF has been thoroughly investigated [34], we chose this assay as a highly precise way to evaluate ligand activation of receptor. Fig. 2A shows a time course of loss of surface receptor for the three FPR variants following treatment with 3 μM formyl-Nle-Leu-Phe. All exhibited t1/2 of ∼5–6 min but W190/N192 downregulated to a lesser maximum extent. Fig. 2B shows recovery of surface receptor after treatment with formyl-Nle-Leu-Phe for 1 h. All three variants exhibited partial recovery of surface receptor after downregulation with slightly higher recovery observed with W190/N192. We then determined the peptides concentration dependent downregulation surface expression of the receptor to assess whether the peptides exhibited agonist, partial agonist, or antagonist activity. As we observed for other viral peptides [8], WEDWVGWI did not cause any downregulation at concentrations up to 25 μM, but 25 μM WEDWVGWI inhibited the downregulation with 300 nM formyl-Nle-Leu-Phe by ∼50% in all three FPRs (data not shown). However, formyl-NleWEDWVGWI behaved as a partial agonist (Fig. 2B) when compared with formyl-Nle-Leu-Phe (Fig. 3A). Only weak partial agonist activity was observed with W190/N192 (34±3%, P<0.0001 vs. either R190/K192 or R190/N192), and greater partial agonist activity was observed with R190/K192 (55±2%) and R190/N192 (58±4%). W190/N192 also exhibited a higher EC50 for downregulation than R190/K192 or R190/N192 but the difference was not significant. Formyl-NleNleWEDWVGWI behaved as a full agonist for all three FPRs (Fig. 4A), but the EC50 for W190/N192 (300±40 nM, P<0.0001 vs. either R190/K192 or R190/N192) was 5–7 fold higher than either R190/K192 (40±3 nM) or R190/N192 (60±8 nM) despite the observation that the Kis differed by only two fold. The observed EC50s were more than 10 fold below the respective Kis for R190/K192 and R190/N192 and 3 fold below the Ki of W190/R190, implying that formyl-NleNleWEDWVGWI was 3 times more effective in activating R190/K192 and R190/N192 than W190/N192. This large difference in EC50 vs. Ki is in marked contrast to what we observe with formyl-Nle-Leu-Phe (Fig. 3A) where the EC50s are similar to the Ki s we previously reported [8]; EC50=175±20 vs. Ki=160±25 for W190/N192; EC50=75±5 vs. Ki=62±8 for R190/K192; EC50=50±7 vs. Ki=67±16 for R190/N192. Thus, formyl-NleNleWEDWVGWI appears to behave as a “super agonist” with R190/K192 and R190/N192, so that occupation of only 5% of the receptors is able to bring about the internalization of 50% of them. We also tested formyl-NleNleWEDAVGWI (Fig. 4B) for its ability to cause downregulation of the receptor. Its behavior was essentially identical to that seen with formyl-NleNleWEDWVGWI. This is in contrast to WEDAVGWI which was>10 fold weaker than WEDWVGWI in binding to the three FPRs. We also tested formyl-NleNleWED. This peptide exhibited very little downregulation at concentrations up to 50 μM with all three FPRs. Thus the VGWI portion of the peptide, formyl-NleNleWEDWVGWI, appears to be essential for FPR activation.