Characterization of HIV Rev and Tubulin Interactions

Abstract

<p>The HIV Rev protein has the ability to bind tubulin heterodimers and depolymerize microtubules (MTs) in vitro producing bilayered rings called Rev-tubulin toroids (RTTs) (Watts et al. 2000. J. Cell Biol. 150: 349-360). These interactions may account for MT defects observed in HIV infected cells or cells that over- express Rev. Watts et al. hypothesize Rev interacts with MTs by a mechanism shared with Kinesin-13 (Kin13) proteins owing to the presence of a shared amino acid sequence. Kin13 proteins are potent MT depolymerizing agents affecting MT behavior during mitosis. To test this hypothesis, point mutations were introduced into Rev substituting amino acids shared with Kin13. My lab aims to compare the abilities of each mutant to the wild-type protein to bind tubulin, depolymerize microtubules and alter spindle function. My role in this project is to purify recombinant and wild-type and mutant Rev proteins expressed in bacteria using a two- column FPLC method. Briefly, bacterial cell lysates will be clarified by low speed centrifugation and applied to a MonoQ ion exchange column. Rev eluting from the column will be applied to a heparin sepharose column where Rev is predicted to elute at salt concentrations greater than 1.5 M. The eluted protein will be denatured with 6 M urea and refolded by sequential dialysis in solutions such that Rev monomers will polymerize into 13 nm thick filaments. At this point, each protein will be analyzed using functional assays that measure Rev-tubulin interactions.</p>