Peptides Containing Membrane-transiting Motifs Inhibit Virus Entry

Abstract

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 ...

Inhibition of Influenza Virus Infection by a Novel Antiviral Peptide That Targets Viral Attachment to Cells

“…With HSV-1, EB irreversibly inactivates virions, prevents entry at a postattachment site (6), and induces a resistance to infection in cells treated with peptide (H. Bultmann and C. Brandt, unpublished data). The EB peptide did not interfere with HSV-1 attachment to cells, in contrast to our data with influenza virus, and in fact enhanced binding at concentrations between 10 and 50 M due to aggregation (5,6). Further support for a specific interaction between EB and influenza comes from the observation that the EB peptide showed no antiviral activity against HIV, human astrovirus (data not shown), or adenovirus type VI (unpublished data).…”

“…The TAT series included TAT-C, derived from the protein transduction domain of the human immunodeficiency virus (HIV) Tat protein with an additional Cterminal cysteine and with a sequence-altered control peptide containing two norleucine substitutions at residues 50 and 51 ( n50,51 TAT-C) (Table 1) (5,6,18). The EB series included EB, composed of the FGF-4 signal sequence with a positively charged amino terminal sequence added to increase solubility, EB-D prepared with D-amino acids (to inhibit degradation by host proteases), and a sequence-scrambled control EBX (5,6,33 Immunofluorescence assay. MDCK cells seeded at a density of 5 ϫ 10 4 on glass coverslips were inoculated with medium alone, HK/483 (MOI of 0.5), or peptide-treated (0 to 30 M) virus for 1 h on ice and then washed with cold PBS to remove unbound virus.…”

“…Peptide synthesis and analysis were performed at the University of Wisconsin-Madison Biotechnology Center as previously described (5,6). The TAT series included TAT-C, derived from the protein transduction domain of the human immunodeficiency virus (HIV) Tat protein with an additional Cterminal cysteine and with a sequence-altered control peptide containing two norleucine substitutions at residues 50 and 51 ( n50,51 TAT-C) (Table 1) (5,6,18).…”

“…Peptide synthesis and analysis were performed at the University of Wisconsin-Madison Biotechnology Center as previously described (5,6). The TAT series included TAT-C, derived from the protein transduction domain of the human immunodeficiency virus (HIV) Tat protein with an additional Cterminal cysteine and with a sequence-altered control peptide containing two norleucine substitutions at residues 50 and 51 ( n50,51 TAT-C) (Table 1) (5,6,18). The EB series included EB, composed of the FGF-4 signal sequence with a positively charged amino terminal sequence added to increase solubility, EB-D prepared with D-amino acids (to inhibit degradation by host proteases), and a sequence-scrambled control EBX (5,6,33 Immunofluorescence assay.…”

A Herpes Simplex Virus Scaffold Peptide That Binds the Portal Vertex Inhibits Early Steps in Viral Replication

“…Direct addition of antennapedia and other membrane-penetrating peptides either alone or fused to unrelated short peptides can decrease viral entry when added to virions or to cells to which virions had been allowed to attach (58). This mechanism may be due to an induced aggregation of virions or perturbation of the virion envelope.…”