Hermann Bultmann

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

ABSTRACT 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.

Modified FGF4 Signal Peptide Inhibits Entry of Herpes Simplex Virus Type 1

ABSTRACT 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 μM. Blocking preadsorbed virus requires higher EB concentrations. Cytotoxic effects (trypan blue exclusion) are first noted at 50 μM EB in serum-free medium and at ≥200 μM 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.

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

ABSTRACT 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.

Heat shock responses in polytene foot pad cells of Sarcophaga bullata.

Heat shock induces a single large puff (hs puff) near the tip of chromosome arm EL in polytene foot pad cells of fly pupae (Sarcophaga bullata). The inducible hs locus is constitutively active, invariably forming a small puff, which can be maximally activated in cells of the dorsal epidermis or in trichogen cells at any time during the lifetime of mature polytene chromosomes. Both in vivo and in cultured food pads, maximal puff induction occurs at 37° C. At the same temperature, normal development of puffing patterns continues undisrupted for several days. A few specific hs proteins are vigorously induced at 37° C, also without disrupting patterns of normal protein synthesis. Rates of normal protein synthesis in cultured food pads and rates of pupal development are enhanced up to about 39° C. During heat shock at 41°–44° C protein synthesis becomes completely dominated by the production of hs proteins. The severe or complete suppression of most of the proteins normally made is followed by developmental arrest. There is also a decline of transcription (chromosomal uridine incorporation) between 37° and 44° C, which appears to affect all chromosomal loci proportionally, including the hs locus. The hs puff is no longer maximally induced at 41°–44° C, but the expanded puff now persists indefinitely, whereas below 39° C, initial puff expansion is always followed by at least partial puff regression. The control of the duration of the puffing response appears to be entirely independent of protein synthesis, e.g., complete inhibition of protein synthesis by cycloheximide fails to prolong transient puffing responses. Canavanine also has no effect on puff regression. Heat shock above 45° C arrests all RNA and protein synthesis within 30 min. RNA synthesis is resumed immediately after shift-down to 25° C, not only at the hs locus, but at most or all previously active loci. Protein synthesis is also resumed immediately, but it is almost completely restricted to the production of the major hs protein (hsp-65, equivalent to hsp-70 of Drosophila melanogaster). Extreme heat shock also triggers maximal puffing responses at the hs locus, but actual puff expansion is delayed and only occurs hours after shift-down in the wake of a surge of hsp-65 synthesis. Following these delayed hs responses pupal thermotolerance starts increasing and protein synthesis returns to normal.

Induction of a heat shock puff by hypoxia in polytene foot pad chromosomes of Sarcophaga bullata

The single large heat-responsive puff (hs puff) in polytene foot pad cells of fly pupae (Sarcophaga bullata) is shown to be inducible by oxygen deprivation but not, as in other systems, by reoxygenation following an hypoxic treatment. The ambient oxygen concentration must drop below 2% for the hs puff to be maximally induced but the puff is fully inducible and transcriptionally active even in the complete absence of oxygen. Lack of oxygen is also compatible with continued transport of puff materials (formation and dissipation of puff droplets at the hs locus). Hypoxia-induced hs puffs persist indefinitely (>2 days) at maximal or intermediate size and only regress completely after oxygen is resupplied. The induction of the hs puff during hypoxia is highly specific and does not seem to involve activation of any other chromosomal loci, yet the reaction is not confined to the giant foot pad cells or to specific developmental stages. Azide poisoning of cultured foot pads simulates the in vivo effects of hypoxia. The induction of the hs puff by azide, heat, or other means is inhibited by sulfhydryl reagents (iodoacetamide, arsenite) and fluoride, but not by an inhibitor of substrate-linked phosphorylation (arsenate). Instead, arsenate, like other uncouplers (2,4-dinitrophenol) is an inducer of the hs locus. The hs puff can be fully induced by hypoxia at any temperature between 2° and 45° C. The rate of puff expansion is strictly temperature dependent and the temperature characteristics of this process are remarkably similar to those of a promoter RNA polymerase association. The major heat shock protein (hsp-65) in Sarcophaga foot pad cells is also slightly induced during hypoxia and its maximal synthesis during aerobic recovery is no longer completely dependent on concomitant RNA synthesis, indicating that some of the required transcripts are made earlier during hypoxia (presumably at the hs locus).

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

ABSTRACT 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 β-galactosidase, EB below 20 μM acted as an enzyme inhibitor, whereas above 40 to 100 μM EB, β-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 peptides 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.