θ Defensins Protect Cells from Infection by Herpes Simplex Virus by Inhibiting Viral Adhesion and Entry

Abstract

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...

Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

“…However, PG-1 manifests appreciable cytotoxicity, and the peptide is hemolytic for human erythrocytes. In marked contrast, -defensins such as RC-2 are nonhemolytic and noncytotoxic, even when tested at high concentrations (52).…”

“…The ability of -defensins to bind carbohydrates (50) also makes them the smallest known lectins. Their broad antiviral spectrum encompasses human immunodeficiency virus type 1, influenza A, and herpes simplex viruses (31,51,52), and its mechanisms have been the focus of many of our recent studies.…”

HIV-1 Adapts to a Retrocyclin with Cationic Amino Acid Substitutions That Reduce Fusion Efficiency of gp41

“…Bacteria can acquire resistance to cationic antimicrobial peptides via genetic or regulatory responses that decrease the negative charge of their membrane phospholipids, teichoic acids, or regions of their LPS (13)(14)(15)(16). Enveloped viruses, such as HIV-1, HSV-1, and HSV-2, are believed to be susceptible to RCs, because their net positive charge and lectinlike behavior permit binding to anionic or carbohydrate-containing viral or host cell membrane domains, including sites implicated in membrane fusion and viral uptake (7,9,17,18). This binding is thought to antagonize HIV-1 entry into target cells, but the precise interactions and mechanism of viral inhibition by RCs are poorly understood.…”

θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

“…Retrocyclins and other -defensins exert broad spectrum antiviral properties in vitro and can protect cells from infection by HIV-1 (6 -9), herpes simplex (10), and influenza A viruses (11).…”