Publication Date


Document Type


Committee Members

Paula Bubulya (Committee Member), Shulin Ju (Committee Member), Mill Miller (Advisor)

Degree Name

Master of Science (MS)


The HIV protein Rev regulates the expression of essential viral proteins during the course of infection by a mechanism that is well understood. It promotes nuclear export of viral transcripts, normally retained in the nucleus owing to the presence of introns, by interacting with host cell transport factors. However, over-expression of Rev in cells leads to defects in cell cycle progression, specifically slowing growth and impairing progression through mitosis (43). While it is possible that Rev may be altering the proteins in transport pathways, cell cycle defects may be attributed to Rev's interactions with other proteins.

In vitro experiments show that highly purified Rev has a high and specific affinity for a and ß tubulin present either as free heterodimers or polymerized into microtubules (MTs) (65). Moreover, Rev rapidly depolymerizes MTs in vitro producing intermediates that closely resemble the products of depolymerization reactions triggered by a variety of experimental conditions. Owing to structural similarities, Rev hypothetically depolymerizes MTs by a mechanism used by Kin-13 proteins that are potent MT depolymerizing enzymes.

To determine whether Rev is interacting with MTs in a Kin-13-like manner, point mutations were previously introduced into Rev substituting alanine for amino acids shared with Kin-13. Mutant proteins were tagged with YFP, over-expressed in HeLa cells and cell cycle progression was monitored by Chang and Miller (27). In contrast to expression of Rev, which lengthened doubling times and all stages of the cell cycle, each point mutant partially corrected the defect. These results are consistent with Rev acting in a manner similar to Kin-13. To determine whether Rev over-expression affects MT dynamics in cells, MT arrays were experimentally depolymerized and allowed to recover. If Rev inhibits MT nucleation or promotes depolymerization, then MT arrays in cells expressing Rev should require more time to recover.

Results show that MT arrays recover from depolymerization equally well in presence and absence of Rev. Because wild-type Rev accumulates largely in the nucleus and nucleoli, we used Rev mutants M4, M6, and Rev2.2 with mutations that impair Rev multimerization and nuclear import, and Rev attached to glucocorticoid hormone receptor respectively. These mutants typically maintain higher cytoplasmic expression levels than wild type Rev. However, exogenous expression of Rev mutants does not affect MT recovery after depolymerization. Furthermore, the ability of MTs to recovery after cold-treatment in Rev expressing metaphase cells was also studied when Rev localizes perichromosomally and is in a position to affect spindle behavior,. However, similar defects were observed in control cells suggesting there was no consequence attributable to Rev. These results suggest that the cell cycle defects observed in Rev-expressing cells are not mediated by Rev's ability to alter the polymerization state of MT. It therefore seems likely that cell cycle defects caused by Rev must be mediated by its interactions with other proteins, possibly B23 or Ran.

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Department or Program

Department of Biological Sciences

Year Degree Awarded


Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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