The HIV-1 Rev protein enables the nucleocytoplasmic export of unspliced or partially spliced mRNAs that is required for the synthesis of structural proteins. By doing so, it regulates the switch to the late phase of the viral replication cycle (Cullen, 1992). This regulatory control over viral replication makes Rev an attractive target for anti-viral intervention. The development of anti-viral remedies is hindered because the three-dimensional structure of Rev has not yet been solved by X-ray crystallography and NMR. Rev, which polymerizes into regular hollow filaments at high concentrations, forms side-to-side and end-to-side interactions making it prone to aggregation and precipitation (Wingfield et al., 1991). Watts et al. (2000) in an attempt to solve the solubility of Rev discovered a novel interaction between Rev and tubulin. They observed that Rev filaments react with microtubules (MTs) to form Rev-tubulin toroidal (RTT) complexes showing that Rev is a microtubule depolymerizing agent that possibly mimics the mechanism used by Kinesin-13 proteins, themselves potent microtubule depolymerases.
The first goal of the experiments conducted here was to develop a sedimentation assay capable of measuring Rev stimulated microtubule depolymerization. Under the conditions employed here, Rev tubulin toroidal complexes (RTTs) were not formed due to limiting concentrations of magnesium ions so that the amount of tubulin released from microtubule polymers would not reform high molecular weight complexes that would sediment in our assays.
Initial experiments determined that bacterial expressed Rev was capable of depolymerizing GMPCPP stabilized microtubules. Depolymerization was not affected by the oligomeric state of Rev. Rev polymerized into filaments or maintained as monomers by the addition of high salt concentrations were equally able to depolymerize microtubules. Microtubule depolymerization appears to be partially dose dependent and occurs at concentrations as low as 300 nM. At low concentrations of Rev, more tubulin is released from the microtubule polymer than there is Rev present in the reaction. This suggests that Rev either has higher affinity for microtubule ends in the lattice or that Rev multimerization is important for depolymerization activity. Depolymerization occurs quickly which is consistent with the findings of Watts et al. (2000). In contrast to the findings of Watts et al. (2000) who demonstrated a complete disappearance of Taxol stabilized microtubules when treated with Rev, Rev was unable to completely depolymerize microtubule polymers stabilized by GMPCPP.