Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry

Abstract

We tested the ability of 20 synthetic theta defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus theta defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human theta-defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K(d), 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human alpha defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that theta defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.

FIG. 1.

Protection of ME-180 cells from infection by HSV-1 and HSV-2 (MTT assay). Peptides (50 μg/ml) and viruses were coincubated for 2 h, before being added to target cell monolayers that also contained 50 μg of peptide/ml. After incubation at 37°C for 72 h, cytotoxicity was measured with a kit. The bars (black, HSV-1; gray, HSV-2) represent means ± standard errors of the means of two to five experiments with each peptide. Asterisks identify the peptides whose activities against HSV-1 and HSV-2 differed significantly (P < 0.001). Group A and group B peptides are further described in the text.

FIG. 2.

Protection against HSV-1 and HSV-2. The most active peptides against HSV-1 and HSV-2 were RTD 3, retrocyclin 1 (RC 100), and retrocyclin 2 (RC 100b). Each peptide (various concentrations) was incubated with HSV-1 or HSV-2 for 2 h and added to ME-180 cell monolayers with the same concentration of peptide. After a 24-h incubation, aliquots were harvested for plaque counting.

FIG. 3.

Comparative antiviral activity. Each peptide is represented by a closed circle, and the most active peptides are labeled with their identifier in Table 1. The x axis shows activity against HIV-1, and the y axis shows activity against HSV-2. The JR-CSF strain (R5) of HIV-1 is represented by closed circles, and the IIIB strain (X4) is represented by open triangles. The most active peptides, retrocyclin 2 (RC 100b), RTD 3, and retrocyclin 1 (RC 100) are labeled.

FIG. 4.

Structures of selected θ defensins. Four θ defensins are diagrammed. θ defensins are circular octadecapeptides with two antiparallel β sheets that are linked by β turns and bridged by an evenly spaced tridisulfide ladder. Their 18 residues represent two 9-residue peptides contributed by a truncated, α-defensin-like prepropeptide. Black arrows identify the sites where the individual nonapeptide elements would be spliced together in vivo. The white arrows show the direction of the peptide backbone and point from the N terminus toward the C terminus.

FIG. 5.

Effect of preincubation on activity. HSV-1 and HSV-2 were exposed to various concentrations (5, 10, 20, and 25 μg/ml) of retrocyclins 1 to 3. Some viruses were preincubated with peptide for 2 h before being added to the ME-180 target cells (“2h pre”). In other assays, the peptide and virus were added to the target cells without preincubation (“no pre”). Viral replication was assessed by plaque counting, and percent protection was equated with the percent reduction in PFU. Results are means of two separate experiments with very similar results.

FIG. 6.

Time signature of the antiviral effect. Cells were cooled to 4°C and inoculated with HSV-2 G in the presence of peptide-free control buffer, retrocyclin 2, or HNPs 1 to 3. Peptides were added at different times, as described in Materials and Methods. The bars, which show PFU formed in the presence of defensin as a percentage of PFU formed in the control wells, represent the means ± standard deviations of two independent experiments, each performed in duplicate.

FIG. 7.

VP16 transport and IC4 expression. (a) These assays examined the effects of retrocyclin 2 or HNPs 1 to 3 on VP16 transport to the nucleus and ICP4 expression after infection with HSV-2(G). Peptides were added during the 4°C binding period or at the temperature shift. After 4 h, nuclear extracts were prepared for VP16, and after 5 h, cell lysates were prepared for ICP4 expression. Lanes contained extracts or lysates from equivalent cell numbers, and VP16, ICP4, and β-actin (loading control) were detected by Western blotting. (b) To examine the effect on binding, cells were exposed to recombinant gB-2 (10⁻⁵ μg/cell) for 1 h at 37°C ± the indicated concentration of retrocyclin 2. Bound gB-2 was estimated by probing Western blots of cell lysates with anti-gB monoclonal antibody. Results are representative of three independent experiments.

FIG. 8.

Binding of θ defensins to gB2. Surface plasmon resonance binding curves (isotherms) of binding to immobilized gB2 are shown for retrocyclin 1 (RC 100a) (a), retrocyclin 2 (RC 100b) (b), RTD 3 (c), HNP 1 (d), HNP 2 (e), and HNP 3 (f). The concentration of peptide in each run is shown adjacent to its curve. The peptides were introduced into the flow cell at the 1-min mark, and 60 s later, at the 2-min mark, the sensor chips were perfused with peptide-free buffer to allow the off rate to be measured. Rate and affinity constants can be found in Tables 2 and 3.

FIG. 9.

Effect of deglycosylation on binding to gB2. Immobilized gB2 was selectively deglycosylated by incubation with sialidase A, endo-O-glycosidase, PNGase F, or a mixture of all three enzymes. Sialidase A removes nonreducing terminal and branched sialic acid residues. Endo-O-glycosidase removes O-linked glycans by cleaving Ser/Thr-linked unsubstituted Galβ(1-3)GalNAcα disaccharides. PNGase F removes N-linked glycans.

FIG. 10.

Lack of cytotoxicity. (a) Effects of three RTDs, 1 to 3, on human ME-180 cervical epithelial cells. (b) Effects of retrocyclins 1 to 3 on these cells. The sequences of these peptides can be found in Table 1.