Title

Three-dimensional structure determination of a subunit of the HIV-1 packaging signal

Date of Award

1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Philip N. Borer

Keywords

Immune deficiency, SL3, Nucleocapsid, HIV-1, Packaging signal

Subject Categories

Biophysics

Abstract

The Human Immunodeficiency Viruses (HIV), responsible for the Acquired Immunodeficiency Syndrome (AIDS), are members of the retroviral family. The RNA genomic material is packaged in new infective virions during the last stage of the retroviral life cycle, a process common to all retroviruses. Only full-length retroviral RNA genome is packaged efficiently. A relatively small region of the HIV-1 genome has been found to be critical for efficient packaging of the virus. This region, known as the "packaging signal", $\Psi$, folds in a secondary structure composed of three to four stem-loop hairpins, named SL1 to SL4. No X-ray or NMR three-dimensional structures have been reported for retroviral packaging signals or for their subunits. Structural studies of the "packaging signal" may provide clues about the HIV packaging mechanism and suggest tools to defeat AIDS.

This thesis is based upon the three-dimensional structure determination of a 20-nucleotide stem-loop RNA SL3 subunit of the HIV-1 packaging signal, using Nuclear Magnetic Resonance techniques. NMR is currently the only technique that allows the determination of high resolution three-dimensional structures of biomolecules in solution under nearly physiological conditions.

SL3 RNA was synthesized by transcription of a synthetic DNA template by the enzyme T7 RNA polymerase. UV melting curves, native gel electrophoresis and 1D H$\sb2$O imino proton spectra were analyzed to confirm the secondary structure of the SL3 molecule. Two-dimensional COSY-type, NOESY, heteronuclear $\sp1$H-$\sp{31}$P-COSY NMR experiments were acquired, processed and analyzed to assign resonances and determine distance and dihedral angle constraints. The NMR constraints were used to refine the three-dimensional structure of SL3 by running restrained Molecular Dynamics calculations.

The final structure of SL3 has revealed an A-form stem and a quite flexible GGAG tetraloop with the second (G10) and fourth (G12) bases extruded from the normal stacking arrangement. The H-bonding loci of G10, A11, and G12 are unoccupied in the free RNA structure and exposed to the solvent available to be easily recognized by possible binding agents. In collaboration with Dr. Summers (UMBC) we have shown that SL3 forms a stable 1:1 complex with the nucleocapsid NCp7, and determined the three-dimensional structure of the SL3-NCp7 complex.

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