ADDITION OF A C-TERMINAL CYSTEINE IMPROVES THE ANTI-HERPES SIMPLEX VIRUS ACTIVITY OF THE TAT PROTEIN TRANSDUCTION DOMAIN PEPTIDE

Department of Ophthalmology and Visual Sciences, Program in Cell and Molecular Biology, and Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706

^* To whom correspondence should be addressed. Email: crbrandt{at}facstaff.wisc.edu.

	Abstract

Previous studies have shown that peptides containing the protein transduction domain (PTD) of the HIV tat protein (GRKKRRQRRR) inhibited HSV-1 entry (J. Biol. Chem. 277:36018-36023). 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 effects of adding the cysteine were to add the capability to inactivate virions (EC₅₀ 100 µM) and to induce a state of cellular resistance to infection (EC₅₀ 5 µM). TAT-C acted extracellularly, blocking entry of adsorbed virus immediately without eluting virions. TAT-C also prevented VP16 translocation to the nucleus and syncytium formation indicating that TAT-C is a fusion inhibitor. The induction of cellular resistance was rapid, recovered with a t of 5-6 hours, and could be rapidly re-induced by peptide treatment. TAT-C bound to cell surface heparan sulfate (HS) but was a poor competitor for adsorption suggesting that TAT-C and virions recognize different structural features of HS. The replacement of positively charged lysine or arginine residues with norleucine reduced the antiviral activity indicating net charge was critical. Peptides composed entirely of d-amino acids were equally effective as l-amino acid versions, thus chirality does not play a role. The free sulfhydryl group was not essential for antiviral activity but TAT-C dimers were more effective on a molar basis. The unique combination of antiviral activities and the low toxicity make TAT-C a strong candidate for further development as a drug to block HSV infection.