Fig. 4: The E35K change partially restores K240E IN functionality in the context of charge-swapped E35K/K240E IN mutant in vitro. | Nature Communications

Fig. 4: The E35K change partially restores K240E IN functionality in the context of charge-swapped E35K/K240E IN mutant in vitro.

From: Oligomeric HIV-1 integrase structures reveal functional plasticity for intasome assembly and RNA binding

Fig. 4

a Salt bridge interactions formed between E35 in NTD1/NTD3 and K240 in CTD1/CTD3 within the outer two regions of the IN tetramer (the inset shows details of the NTD3-CTD3 interaction). Blue arrows point to the locations of the two other sets of E35 and K240 residues that do not interact with one another and are surface-exposed. b SEC analysis demonstrating the restoration of K240E tetramerization by the E35K change within the context of the E35K/K240E mutant (T: tetramer, D: dimer, M: monomer). c Concerted integration assay. Left panel: schematic representation of the assay using vDNA ends, supercoiled (s.c) plasmid DNA, and purified IN. Both half-site and concerted integration products are generated as outputs. Middle panel: integration products were detected on 1.5% agarose gel using a fluorescence scanner. The bands corresponding to half-site and concerted products are indicated. Right panel: bar graph showing the quantification of concerted integration within the middle panel. RAL, WT IN reactions in the presence of raltegravir IN inhibitor. The integration activity of WT IN was set to 100%, and the activity of mutant IN is presented as a percentage of the WT. The results are represented as mean ± SDs of independent experiments, n = 3, each performed with triplicate samples. d Left panel: Schematic of an AlphaScreen-based RNA bridging assay26. Each circle corresponds to an IN multimer. “A” and “D” indicate anti-digoxin Acceptor and streptavidin-coated Donor beads bound to either digoxin (DIG) or biotin, respectively. Right panel: quantification of purified IN protein interactions with HIV-1 TAR RNA. Average values are from three independent experiments, with error bars indicating SDs. Statistical significance was determined by the two tailed t-test in both (c) and (d) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The exact p values are reported on the figures. Source data are provided as a Source Data file.

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