STRUCTURAL AND FUNCTIONAL DETERMINANTS IN ADENOVIRUS TYPE-2 PENTON BASE RECOMBINANT PROTEIN

Discrete domains involved in structural and functional properties of a denovirus type 2 (Ad2) penton base were investigated with site-directe d mutagenesis of the recombinant protein expressed in baculovirus-infe cted cells, Seventeen substitution mutants were generated and phenotyp ed for various functions in insect and human cells as follows, (i) Pen tamerization of the penton base protein was found to be dependent on t hree amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556, (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, t o a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549, (iv) Penton base mutants were also assayed fo r HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad mediated gene delivery. The results obtained suggested that the pr eviously identified integrin-binding motifs RGD(340) and LDV287 were f unctionally and/or topologically related to other discrete regions whi ch include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which we re involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.