A selection system to study protein-RNA interactions: functional display of HIV-1 Tat protein on filamentous...

The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle and belongs to the RNA binding proteins of the arginine-rich motif (ARM) family. Very little is known about their mechanism of RNA recognition. To study the principles of RNA-protein recognition we constructed a system to display HIV-1 Tat on the surface of the filamentous bacteriophage M13. HIV-1 Tat (1-72) and a mutant Tat lacking five cysteine residues were cloned into the pAK phagemid system, which allows fusion of the tat gene to a supershort version of the gene for minor M13 coat protein. Expression of the resulting fusion proteins was shown via western blot analysis. Phages displaying functional Tat could be selected from phages without Tat or with a non-functional Tat variant via binding to biotinylated TAR using streptavidin coated paramagnetic beads. By randomizing certain amino acid positions of Tat and screening of the resulting phage libraries for affinity and specificity, we are now able to study the role and importance of amino acids of HIV-1 Tat for affinity and specificity to TAR RNA.