Identifying the Amino Acids Important for HIV Rev-Tubulin Interactions

The Rev protein is Human Immunodeficiency virus\u27s switch from events occurring early in infection to later events. Early in infection, the 13 KDa Rev protein begins to accumulate in the host cell nucleus. Once enough Rev is produced, Rev stimulates a switch in viral gene expression by multimerizing onto nuclear viral RNAs and stimulating their export into the cytoplasm. Multimerization occurs on an RNA structure called the Rev Response Element (RRE). Several Rev monomers bind the RRE and once that threshold is met the Rev-RRE complex is exported out of the nucleus. Once out of the nucleus the Rev-RRE complex dissociates and Rev imports back into the nucleus for another cycle of export. Rev\u27s unique function makes it a theoretically ideal target for inhibiting viral replication. Consequently, understanding the three-dimensional structure of Rev will promote drug design. Obtaining structural information is difficult because Rev aggregates. While trying to find solutions conditions for crystallography, Watts et al. (2000) discovered Rev depolymerizes microtubules in vitro forming bilayered rings called Rev-Tubulin Toroids (RTTs). RTTs also form when Rev is mixed with tubulin heterodimers. Similar rings form when MCAK and other members of the Kinesin 13 family of microtubule-associated proteins (Kin-13) are mixed with tubulin. The similar primary and secondary structure of Rev (amino acids 34-57) and MCAK (amino acids 506-530) has prompted Watts et al. to hypothesize that the two proteins interact with tubulin and microtubules by a shared mechanism. Studies have shown mutating amino acids within this shared region has a detrimental affect on Kin-13 ability to depolymerize microtubules and form spindles. Therefore, Rev may serve as a model to further the understanding how Kin-13 proteins function. To test Rev\u27s ability to be used as a model for Kin-13 interaction with tubulin point mutations were introduced into the shared region, (A37D, R42A, E47A, and E57A). Then purified proteins were mixed with tubulin heterodimers to see if RTTs form. The A37D, E47A, and E57A mutations do not have any meaningful affect on Rev structure. All were able to form hollow filaments at high concentrations comparable to filaments formed with wtRev. When mixed with tubulin, A37D, E47A, and E57A form RTTs with similar ring diameter and thickness as wild-type rings. These results suggest that the mutated amino acids are unimportant for Rev-tubulin interactions. These data are somewhat consistent with data published for MCAK. The A -\u3e D substitution in MCAK also has no affect on MCAK activity. The glutamic acid corresponding to E47 has not been tested in MCAK and this warrants testing. The glutamic acid corresponding to E57 in MCAK behaves differently. This residue is essential for MCAK activity whereas it appears to have no effect on Rev-tubulin interactions. These data suggest that Rev and MCAK may work by different mechanisms. Mutation of RevR42 had significant effects on RTT formation. R42A does not interact with tubulin heterodimers and RTTs do not form. Moreover, the mutation affects the thickness of Rev filaments suggesting that this amino acid is important for Rev-Rev and Rev-tubulin interactions. Mutating the corresponding amino acid in MCAK will be an interesting test of the hypothesis that Rev and MCAK act by a shared mechanism