Title

Affinity of the HIV-1 nucleocapsid protein for SL3 RNA: Effects due to salt and variation due to measurement technique

Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Philip N. Borer

Keywords

HIV-1, Nucleocapsid protein, Stem-loop RNA

Subject Categories

Chemistry

Abstract

The mature nucleocapsid protein of HIV-1 (NCp7) and the NC-domains in gag-precursors are attractive targets for anti-AIDS drug discovery. NCp7 binds tightly to a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA). The structure and stoichiometry of the NCp7-SL3 complex depend on the balance between sequence-specific binding of NCp7 to the G-rich loop bases of SL3 and non-sequence-specific interactions that are mainly electrostatic. This balance is primarily affected by the nature and concentration of the added salt, the concentration of wild-type NCp7 and SL3, and effects due to non-ideality of solutions. All these factors affect the measured or apparent dissociation constant (K d ). NC-domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The affinity of NCp7 for SL3 was studied using fluorescence (monitoring the fluorescence of Trp-37, the only tryptophan in the NCp7 sequence, which is quenched in a sequence-specific complex with SL3), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and electrophoretic mobility shift assay (EMSA). High salt concentrations screen the ionic interactions between NCp7 and SL3 (net charges of +9 and -19, respectively, at neutral pH). It was found that 0.018 M MgCl 2 and 0.200 M added NaCl produce the same affinity for a 1:1 sequence-specific complex between NCp7 and SL3. Acetate ions stabilize the complex by more than a factor of 2 over the chloride ion and sulfate ions stabilize the complex by another factor of 2-3 over acetate. No change in the affinity of the complex occurs when substituting K + for Na + . The order of addition of NCp7 and SL3 in titrations monitored by tryptophan fluorescence and ITC, and hence the concentration of NCp7 and SL3, does not affect the balance between sequence-specific and non-sequence-specific binding in the physiological range of salt concentration. The presence of only one species of a 1:1 complex between NCp7 and SL3 at 0.200 M NaCl was also confirmed by EMSA.

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