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.

1 repeats may also promote binding of the I 1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125 I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I 1-5 binding sites, independent of the III 1 and III 12-14 repeats, may be involved in fibrillogenesis.Fibronectin is required for normal growth and development (1) and plays an important role in regulating cell attachment and movement, wound healing, and tumorigenesis (for review, see Refs. 2 and 3). It is a 500-kDa disulfide-bonded dimer consisting of similar subunits and is found as a soluble glycoprotein in blood and other body fluids and as an insoluble fibrous matrix component in tissues. Each subunit of fibronectin consists of three different types of repeating sequences, called types I, II, and III, which are arranged into discrete structural and functional modules.Assembly of dimeric fibronectin into the extracellular matrix involves multiple consecutive binding interactions with integrin receptors, with itself, and with matrix components such as type I collagen (for review, see Refs. 4 -6). Although the ␣ 5 ␤ 1 integrin appears to be the primary fibronectin receptor involved in matrix assembly (7-12), at least two other integrins, ␣ IIb ␤ 3 and ␣ v ␤ 3 , can also support fibronectin fibrillogenesis (13,14). High affinity binding interactions between these integrins and the RGD site in the 10th type III repeat (III 10 ) of fibronectin are thought to promote fibrillogenesis by exposing appropriate self-assembly sites in fibronectin. Such sites may become exposed through local integrin-induced conformational changes in III 10 repeats (15) or through integrin-mediated stretching (reversible unfolding) of one or a whole array of type III repeats in fibronectin in response to cell movements (13, 16).Self-assembly of fibronectin dimers into fibrils is currently thought to involve primarily interactions between the first five type I repeats (I 1-5 ) and the first type III (III 1 ) repeat (17, 18). The I 1-5 repeats are critical for matrix assembly, i.e. peptides including these repeats block assembly of fibronectin into fibrils, and fibronectin dimers lacking these repeats will not be incorporated into fibrils (19 -22). The III 1 repeats are also important for fibril formation, and either anti-III 1 monoclonal antibodies or peptides derived from III 1 repeats can block assembly of fibronectin into matrix (23, 24). The mechanism, however, by which I 1-5 -III 1 interactions affect matrix assembly remains controversial. For example, Hocking et al. (25) have shown that III 1 repeats will interact not only with I 1-5 repeats but also with heat-denatured III 10 repeats. They have proposed that the latter interaction activates the III 1 repeat thereby allowing it to function as a receptor for the amino termini of a second fibronectin dimer. In contrast, Sechler et al. (26) have shown that fibronectin dimers lacking III 1-7 repeats are readily polymerized into fib...

Entry of herpes simplex virus type 1 (HSV-1) into host cells occurs through fusion of the viral envelope with the plasma membrane and involves complex and poorly understood interactions between several viral and cellular proteins. One strategy for dissecting the function of this fusion machine is through the use of specific inhibitors. We identified a peptide with antiviral activity that blocks HSV-1 infection at the entry stage and during cell-to-cell spreading. This peptide (called EB for "entry blocker") consists of the FGF4 signal sequence with an RRKK tetramer at the amino terminus to improve solubility. The activity of EB depends exclusively but not canonically on the signal sequence. Inhibition of virus entry (hrR3) and plaque formation (KOS) strongly depend on virus concentrations and serum addition, with 50% inhibitory concentrations typically ranging from 1 to 10 M. Blocking preadsorbed virus requires higher EB concentrations. Cytotoxic effects (trypan blue exclusion) are first noted at 50 M EB in serum-free medium and at >200 M in the presence of serum. EB does not affect gC-dependent mechanisms of virus attachment and does not block virus attachment at 4°C. Instead, EB directly interacts with virions and inactivates them irreversibly without, however, disrupting their physical integrity as judged by electron microscopy. At subvirucidal concentrations, EB changes the adhesive properties of virions, causing aggregation at high virus concentrations. This peptide may be a useful tool for studying viral entry mechanisms.Herpes simplex virus type 1 (HSV-1) is a significant human pathogen causing mucocutaneous lesions primarily in the oral mucosa but also in other sites as well. More serious manifestations of HSV-1 infection include encephalitis and blinding keratitis. Indeed, HSV-1 is the leading cause of sporadic viral encephalitis and the leading cause of blindness due to infection in the United States (65). HSV-1 also has the capacity to latently infect the host, with important consequences for transmission, treatment, and difficulties in eradication of the latent infection. Obtaining more precise information about many aspects of the viral life cycle is critical for designing prevention and intervention strategies. One critical aspect of the life cycle is the ability of HSV to enter host cells. Previous studies have shown that entry occurs at the cell surface due to fusion of the viral envelope with the plasma membrane of the cell (54). The combined processes of attachment and entry involve a number of components and require a number of steps. This complexity makes the study of these processes exceedingly difficult.HSV encodes at least 12 glycoproteins, 10 of which are structural components of the viral envelope (54). Of these, five are clearly involved in attachment and entry. The proteins involved in attachment and entry are gC, gB, gD, and a complex of the gH and gL proteins (4,10,13,21,24,25,31,34,38,47,62). Of these five, four (gB, gD, gH, and gL) are essential in that their loss results in noninf...

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.Protein-protein interactions are critical in many biological processes including numerous steps in viral infection. Peptides that interrupt protein-protein interactions thus have considerable potential as tools to elucidate the basic mechanisms underlying these processes and as pharmaceutical agents. As an example, the carboxyl terminal 9 amino acids of the small subunit of herpes simplex virus (HSV) 1 ribonucleotide reductase (RR2) can disrupt the RR1:RR2 complex and inhibit enzymatic activity (1, 2). A peptidomimetic derived from the nonapeptide was subsequently shown to have antiviral activity and inhibit replication in vivo (3). A series of synthetic peptides have also been used to identify potential heparin sulfate binding sites in the HSV gC protein (4), and a similar strategy was used to map the interaction site between the HSV UL42 protein and DNA polymerase (5). These findings support the potential use of peptides for numerous purposes; however, since the majority of peptides do not enter cells, their potential as antiviral agents is severely limited.Recently, several exceptional peptides have been identified that can cross plasma membranes and deliver various covalently linked moieties into cells (6 -10). Initially, we were interested in determining whether the membrane-transiting proteins (MTPs) could transport a known protein-protein disrupting peptide into cells, resulting in inhibition of viral replication. We chose to use the carboxyl terminal nonapeptide from the HSV RR2 (1, 2) coupled to one or more MTPs to test the strategy. While testing these MTPs for their ability to cotransport potential antiviral peptides into cells we previously found that a modified version of one, denoted EB, inhibited HSV in ...