The virucidal EB peptide protects host cells from herpes simplex virus type 1 infection in the presence of serum albumin and aggregates proteins in a detergent-like manner.

The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634-2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 µM concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 µM, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (≥0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for ß-galactosidase, EB below 20 µM acted as an enzyme inhibitor, whereas above 40 to 100 µM EB, ß-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins.

Addition of a C-terminal cysteine improves the anti-herpes simplex virus activity of a peptide containing the human immunodeficiency virus type 1 TAT protein transduction domain.

Previous studies have shown that peptides containing the protein transduction domain (PTD) of the human immunodeficiency virus tat protein (GRKKRRQRRR) were effective inhibitors of herpes simplex virus type 1 (HSV-1) entry (H. Bultmann and C. R. Brandt, J. Biol. Chem. 277:36018-36023, 2002). We now show that the addition of a single cysteine residue to the C terminus of the TAT PTD (TAT-C peptide) improves the antiviral activity against HSV-1 and HSV-2. The principle effect of adding the cysteine was to enable the peptide to inactivate virions and to induce a state of resistance to infection in cells pretreated with peptide. The TAT-C peptide acted extracellularly, immediately blocked entry of adsorbed virus, prevented VP16 translocation to the nucleus, and blocked syncytium formation and cell-cell spread. Thus, TAT-C peptides are fusion inhibitors. The induction of the resistance of cells to infection was rapid, recovered with a half-life of 5 to 6 h, and could be reinduced by peptide treatment. TAT-C bound to heparan sulfate but was a poor competitor for viral attachment. The antiviral activity depended on the net positive charge of the peptide but not on chirality, and a free sulfhydryl group was not essential for antiviral activity because TAT-C dimers were at least as effective as monomers. The unique combination of antiviral activities and low toxicity combine to make TAT-C a strong candidate for further development as a drug to block HSV infection.

Inhibition of influenza virus infection by a novel antiviral peptide that targets viral attachment to cells.

Influenza A viruses continue to cause widespread morbidity and mortality. There is an added concern that the highly pathogenic H5N1 influenza A viruses, currently found throughout many parts of the world, represent a serious public health threat and may result in a pandemic. Intervention strategies to halt an influenza epidemic or pandemic are a high priority, with an emphasis on vaccines and antiviral drugs. In these studies, we demonstrate that a 20-amino-acid peptide (EB, for entry blocker) derived from the signal sequence of fibroblast growth factor 4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo, even when administered postinfection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor, preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic.

Peptides containing membrane-transiting motifs inhibit virus entry.

Several exceptional peptides have been identified that can cross plasma membranes and deliver various covalently linked moieties into cells. We report the surprising observation that each of four structurally distinct transiting peptides tested displayed antiviral activity and inhibited herpes simplex virus entry into cells. All four peptides inhibited infection at concentrations in the low micromolar range. Some of the peptides selectively and reversibly blocked entry without inactivating virions in a persistent manner. For other peptides, the effects on virus entry were not readily distinguishable from virus inactivation. High concentrations of nearly all peptides lead to irreversible inactivation of virions. By various criteria, the peptides differed in their ability to inactivate virions and in the temperature dependence of inactivation. Testing of peptides with modifications known to disrupt transport revealed that, in some instances, transport activity did not correlate with antiviral activity. These results identify inhibition of viral entry as another common property of membrane-transiting peptides in addition to their ability to cross membranes and transport materials into cells. These or related peptides may be useful as agents to prevent infection and to study the process of viral entry.