Publication Date

2009

Document Type

Dissertation

Committee Members

Gerald Alter (Committee Member), David Cool (Committee Chair), Julian Gomez-cambronero (Committee Member), Mark Mamrack (Committee Member), Mill Miller (Advisor)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Rev is a 13 kDa regulatory HIV protein essential for viral replication. It trans-activates expression of late viral proteins by multimerizing onto target mRNA and promoting their export into the cytoplasm. During attempts to find the solution conditions needed to study Rev structure, Watts et al. (2000) discovered that Rev depolymerizes microtubules (MTs) in vitro through the formation of ringed tubulin intermediates called Rev-tubulin toroids (RTTs). Rev interactions with MTs are specific and are thought to mimic the mechanism of Kinesin-13 proteins, themselves potent MT depolymerases that regulate the assembly of the mitotic spindle. If Rev and Kinesin-13 proteins share a common mechanism, then Rev mediated MT depolymerization and RTT formation will require Rev multimerization and its arginine-rich motif (ARM).

If Rev multimerization is essential, then multimerization defective mutants should not depolymerize MTs and form RTTs. To test this hypothesis, a combination of sedimentation, gel filtration and visual assays were used to compare the activities of wild-type Rev with the multimerization defective mutant Rev M4 (M4). Both wild-type Rev and M4 are able to bind tubulin heterodimers and form high molecular complexes. However, these complexes are not RTTs. M4 also binds GMPCPP-stabilized MTs but unlike wild-type Rev, it neither depolymerizes MTs nor forms RTTs. These data show that Rev multimerization is important for MT depolymerization although it is unclear whether it is involved in targeting Rev to MT ends or provides the force required for depolymerization. Because M4 promotes MT bundling, this mutation is concluded to subtly affect Rev tertiary structure such that the relative orientation of Rev monomers within a multimer is altered in a manner that allows MT cross-linking.

These same assays were also used to test the hypothesis that the ARM is also important for Rev-tubulin interactions. In this instance, the activities of wild-type Rev were compared to the M6 mutant (M6), a well-characterized substitution-deletion mutant in the ARM predicted to perturb binding, depolymerization and RTT formation. The M6 mutation affects RTT formation when mixed with tubulin heterodimers, reducing both ring size and the amount of complexes that can be sedimented. In addition, M6 is unable to depolymerize GMPCPP-stabilized MTs through an apparent inability to bind MT. Results presented here suggest Rev possesses only a single MT binding motif present in the ARM. Some residues in this region are critical for binding MT ends where depolymerization occurs. These conclusions are also consistent with the hypothesis that Rev interacts with MTs by a mechanism shared with Kinesin-13 proteins, themselves potent cellular MT depolymerases.

Page Count

117

Department or Program

Biomedical Sciences

Year Degree Awarded

2009

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