Fig. 2

Specific interaction of A1752 with HIV-1 NC and functional inhibition of NC-mediated chaperone activities. a The determination of binding affinity of A1752 to NC using SPR assay. b Tryptophan fluorescence quenching assay. Shown is intrinsic tryptophan quenching of NC by A1752. NC (5 μM) was used and excitation wavelength was 280 nm and emission was scanned from 310 to 450 nm as shown. c Effect of A1752 on NC-mediated HIV-1 Psi RNA dimerization (Top) and NC-Psi complex formation as determined by gel-shift assay (Bottom). Monomeric and dimeric Psi RNA and Psi RNA-NC complex were probed with a SYBR green staining method for RNA and a SYPRO Ruby staining for protein, respectively. Control bands for monomeric and heat-induced dimeric Psi RNA are also indicated in lane 1 and 2, respectively. NC was incubated with A1752 at increasing molar ratio (1:1, 1:10, 1:25, and 1:50), DMSO and AZT (1:50) as described in “Methods”. d Inhibition of NC-induced cTAR destabilization by A1752. NC (1 μM) were preincubated for 10 min with increasing A1752 concentrations or other inhibitors, DIBA and SAMT, as indicated and added to 0.1 μM of doubly-labeled Rh6G-5′-cTAR-3′-DABCYL DNA for 1 h. Fluorescence change was monitored at excitation and emission wavelength of 520 and 560 nm, respectively