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.Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are widespread human pathogens. Between 70 and 90% of adults in the United States are infected with HSV-1, and 22% of individuals over the age of 12 are seropositive for HSV-2 (7, 61). Both viruses infect mucous membranes causing ulcerative lesions that are usually self-limited in immunocompetent individuals. However, serious infections can occur, including lethal neonatal HSV, encephalitis, and blinding keratitis (34,61). A number of antivirals are approved for HSV treatment, and the use of these drugs has had a significant impact on the disease. These antivirals reduce the frequency of recurrent viral shedding and disease and reduce transmission of genital herpes between discordant partners (12,42,46). Although these treatment regimens significantly reduce disease, they are not completely effective and cannot clear latent infections. One significant problem in dealing with HSV infections is the ability of the virus to persist in the host in the form of latent infection (45). Preventing the establishment of a persistent infection, which could be accomplished either by blocking the initial infection or preventing the establishment of latency, would be ideal for dealing with this virus.One strategy for preventing infection is to block the attachment of virus. Attachment of HSV can be inhibited by heparin, which competes directly with the binding of gC and gB to cell surface heparan sulfate (HS) on cells (23,24,(26)(27)(28). Other polymeric anions and polyoxotungstate compounds have been shown to inhibit attachm...

Inhibition of Herpes Simplex Virus Type 1 Infection by Cationic β-Peptides

“…The cationic ␤-peptides are also likely to bind to heparan sulfate, raising the question of why the cationic ␤-peptides do not block attachment. In our studies with TAT peptides, we found that attachment was not inhibited to a significant extent, suggesting that TAT peptides and HSV-1 recognize different structural features in heparan sulfate (9). Thus, it is possible that the cationic ␤-peptides act similarly to TAT and therefore do not inhibit attachment of the virus to cells.…”

“…In addition to inhibiting entry, the added cysteine gave the peptide the capacity to inactivate virions in solution and induce a state of resistance to infection in cells pretreated with the peptides. The peptide composed entirely of D-amino acids (TAT-CD) was equally as effective as the L-amino acid version, indicating that chirality is not critical (9). The TAT-C peptide has previously been shown to have low toxicity both in vitro and in vivo (1).…”

“…If so, then it might be expected that binding to virion sialic acid would inactivate the virus. However, previous work in one of our laboratories showed that the TAT peptide itself (GRKKRRQRRR) inhibited entry but was not virucidal and did not induce cellular resistance to infection (9). Virucidal and cell resistance activities of the TAT peptide required the addition of a C-terminal cysteine (TAT-C).…”

“…One of these cell-penetrating peptides, the cationic peptide GRKKRRQRRR derived from the HIV tat protein, was shown to inhibit HSV-1 infection at the entry step in cell culture with a 50% effective concentration (EC 50 ) of 1 M (7). Recently, we showed that the antiviral activity of TAT peptide could be improved by adding a single cysteine residue to the C terminus of the TAT peptide (TAT-C) (9). In addition to inhibiting entry, the added cysteine gave the peptide the capacity to inactivate virions in solution and induce a state of resistance to infection in cells pretreated with the peptides.…”

Synthesis of 1,3,4-oxadiazole derivatives from α-amino acid and acyl hydrazides under thermal heating or microwave irradiation conditions

“…27,28 On the other hand, amino acids have emerged as important building blocks for the synthesis of a range of different compounds. [29][30][31] Moreover, the interest on the biological and medicinal properties of chalcogen amino acids has also been increasingly appreciated, mainly due to their antioxidant, 32 antitumor, 33 antimicrobial, 34 and antiviral 35 properties.…”