Date of Award

12-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Philip Borer

Keywords

HIV-1, Tat, Tat Protein Characterization, Tat Protein Expression

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

Efficient transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the viral protein Tat with the trans-activation response (TAR) stem-loop of the long-terminal repeat (LTR) portion of nascent viral RNA. The production of viable transcripts is enhanced dramatically by the interaction of HIV-1 Tat with the host protein human Cyclin T1. Interaction with hCycT1 remodels Tat protein contributing a single cysteine residue that is critical to the formation of the second of two zinc fingers (Zn2). Here we suggest that it is the presence of this critical cysteine residue and not the presence of arginine residues from human Cyclin T1 that imparts high affinity and specificity to the interaction with HIV-1 TAR RNA. Crucial structural features of this interaction remain unresolved by NMR or existing crystal structures. Specifically, the structure of the Tat activation domain (AD), and Tat interaction with hCycT1 while bound to HIV-1 TAR RNA remain elusive. Much of the difficulty in obtaining structural data is a result of the notoriously difficult expression of native HIV-1 Tat caused in large part by the high cysteine count, and poor solubility of the Tat protein. This work presents a protocol for the expression and purification of a high affinity recombinant chimeric protein which includes the full 101 amino acid Tat protein fused to an essential minimal portion of CycT1m) necessary for TAR binding in sufficient purity and concentration for structural study by nuclear magnetic resonance (NMR).The elucidation of this critical region has the potential for profound impact in the structural based drug design of HIV-1 therapeutics.

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Open Access

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