Paula Bubulya (Committee Member), David Goldstein (Committee Member), Mill Miller (Advisor)
Master of Science (MS)
The HIV Rev protein has the ability to bind tubulin heterodimers and depolymerize microtubules (MTs) in vitro (Watts et al. 2000). These interactions may account for MT defects observed in HIV infected cells or cells that over-express Rev. Watts et al. hypothesized 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. In this study, eight mutant Rev proteins (T34A, A37D, R39A, R42A, E47A, R50A, E57A, and E47A/E57A) were fused to YFP and over-expressed in HeLa cells. The ability of these cells to grow in culture was measured. Previous results show over-expression of wild-type Rev slows growth in culture and alters cell cycle progression. If these defects are due to Rev-MT interactions, mutation of residues critical for these interactions should mitigate these defects. T34A and R50A have the most restorative effects in cell morphology and growth in culture suggesting the affected amino acids are important for Rev function. R50A is expected to be an important mutation, as this residue is necessary for Kin13 function. Consequently, if the Rev-Kin13 hypothesis were correct, an amino acid substitution in Rev would be necessary for Rev function. T34A is a surprising result suggesting that this shared amino acid might be important for Kin13 function as well and merits investigation. Curiously, over-expression of Rev mutants R39A, R42A, and the double mutant E47A/E57A also alter doubling times and increase the frequency of multinucleated cells. Taken together, Rev over-expression can lead to defects not directly attributable to Rev-MT interactions.
Department or Program
Department of Biological Sciences
Year Degree Awarded
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