We tested the ability of 20 synthetic defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human -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 ␣ 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 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.The worldwide AIDS epidemic has intensified interest in identifying naturally occurring antiviral molecules (5,29,34,57). Certain rabbit and human ␣ defensins were shown to protect cells from infection by herpes simplex virus type 1 (HSV-1) and HSV-2 almost 20 years ago (17,30), and more recent studies indicated that rabbit ␣ defensin NP-1 blocks HSV infection at a very early stage (46). Even adenoviruses, which are nonenveloped, are susceptible to ␣ defensins (3, 21), although the mechanism of this effect is unknown. In vitro, human ␣ defensins human neutrophil peptide 1 (HNP 1) to HNP 3 can protect cells from infection by human immunodeficiency virus type 1 (HIV-1), and release of these defensins from the ␣,  CD8-positive T cells of HIV-infected subjects may (60) or may not (11) correlate with their long-term clinical stability.Defensin peptides belonging to three subfamilies, designated ␣, , and defensins, have been identified in leukocytes and other cells of humans or nonhuman primates. ␣ defensins contain 29 to 35 residues and are produced as ϳ100-residue prepropeptides (16). Human neutrophils (polymorphonuclear leukocytes) contain four ␣ defensins, called HNPs 1, 2, 3, an...
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.
We compared the susceptibilities of Chlamydia trachomatis elementary bodies (EBs) to human defensin HNP-2 and porcine protegrin PG-1, cysteine-rich beta-sheet antimicrobial peptides produced by mammalian leukocytes. Although both peptides protected McCoy cell monolayers from infection by chlamydial EBs, protegrins were especially potent. Protegrin-mediated inactivation of chlamydiae occurred rapidly, was relatively independent of the presence of serum, and was effective against serovars L2, D, and H. Protegrin-treated EBs showed striking morphological changes, with obvious damage to their limiting membranes and loss of their cytoplasmic contents and nucleoid. Their effectiveness against chlamydial EBs and other sexually transmitted pathogens combined with their relative lack of cytotoxicity suggests that protegrins and related molecules could serve as prototypes for topical agents to prevent sexually transmitted chlamydial infection.
We tested 20 protegrins against Chlamydia trachomatis serovar L2 (L2/434/Bu). Five of the protegrins had native structures; the others included nonamidated, enantiomeric, and truncated variants and peptides with <2 disulfide bonds. Antichlamydial activity resided principally in residues 5 to 15 of native protegrin PG-1, and optimal activity required both intramolecular disulfide bonds. Cysteine-rich, endogenous antimicrobial peptides are widely distributed in the phagocytic cells and epithelial tissues of humans and other animals. For example, the neutrophils and small intestinal Paneth cells of humans contain ␣-defensins, a family of 3.5-kDa peptides with cysteine-stabilized -sheet structures and broad-spectrum antimicrobial efficacy (2, 9). The female human genital tract produces hBD-1 (1), a -defensin homologous to the antimicrobial peptides found in bovine (13) and avian (5, 6) leukocytes and various bovine epithelial cells (4, 12). Protegrins, the subject of this study, are unusually potent antimicrobial molecules that were originally isolated from porcine leukocytes (7). They are active against several important sexually transmitted pathogens, including Neisseria gonorrhoeae (11), Chlamydia trachomatis (15), and human immunodeficiency virus type 1 (14). Like defensins, protegrins possess a cystine-stabilized -sheet structure; however, they are much smaller than defensins and contain only two intramolecular disulfide bonds. We initiated this study to determine if structurally simpler, protegrin-like molecules would retain antichlamydial activity. Peptides. Synthetic PG-1 amide, PG-1 acid, enantio-PG-1, PG-2, PG-3, and PG-5 were prepared by SynPep (Dublin, Calif.), and purified as previously described (11, 15). The other protegrin variants used in this study were synthesized by Fmoc chemistry at the Macromolecular Structure Facility of
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