FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

Abstract

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN-viral DNA and IN-LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN-viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN-LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.

Figure 1.

Scaled-up preparations of the SSC. (A) Experimental design. (B) Optimization of IN concentrations for the SSC assembly. Purified SSCs were subjected to SDS–PAGE and the IN band was visualized by western blot. Lane 1, IN load; lane 2, protein markers: MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA); lanes 3–9, SSCs assembled with increasing IN concentrations. At the optimal protein concentrations (200–400 nM), ∼20% of total IN was assembled in the SSC. (C) Comparison of HIV-1 IN interactions with specific and non-specific DNA (nsDNA). Lane 1, protein markers: MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA); lane 2, IN load; lane 3, no DNA was included in the reaction mixture; lanes 4 and 5, SSC assembly with nsDNA; lanes 6 and 7, SSC assembly with viral DNA. (D) Agarose gel electrophoresis: Lane 1, viral DNA alone; lane 2, DNA markers: GeneMate Quanti-Marker 1 kb (BioExpress, Kaysville, UT, USA); lane 3, the initial (1×) scale for SSC preparations as reported previously (17); lane 4, 10-fold scale-up; lane 5, 100-fold scale-up.

Figure 2.

Effects of the order of viral DNA and LEDGF/p75 additions to HIV-1 IN on the SSC assembly. (A) SDS–PAGE analysis of SSCs. Lane 1: 1/10 of IN load, lane 2: protein markers: MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA), lane 3: no DNA was included in the reaction mixture, lane 4: the SSC assembly with IN and viral DNA, lane 5: LEDGF/p75 was pre-incubated with IN and then viral DNA was added to the reaction. IN was visualized by western blot using the respective antibody as described in ‘Materials and Methods’ section. (B) Non-denaturing agarose gel electrophoresis. Lane 1, DNA markers: GeneMate Quanti-Marker 1 kb (BioExpress, Kaysville, UT, USA); lane 2, no IN was included in the reaction mixture; lane 3, the SSC assembly with IN and viral DNA; lane 4, LEDGF/p75 was pre-incubated with IN and then viral DNA was added to the reaction. Free DNA and the SSC were visualized by ethidium bromide staining. (C) Experimental design to probe LEDGF/p75 interactions with the SSC. (D) SDS–PAGE analysis of LEDGF/p75 interactions with the SSC. Lane 1, protein markers: MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA); lanes 2–6, the following samples were incubated in the buffer containing 750 mM NaCl and then subjected to size exclusion chromatography as shown in (C): IN alone (lane 2), the purified SSC (lane 3), LEDGF/p75 plus the SSC (lane 4), LEDGF/p75 alone (lane 5), LEDGF/p75 plus viral DNA (lane 6). IN and LEDGF/p75 were visualized by western blot using respective antibodies as described in ‘Materials and Methods’ section.

Figure 3.

Scheme illustrating design of protein–protein FRET experiments. Two IN proteins are prepared in parallel: one labeled with the D probe and another with the A probe. The protein concentration range in the reaction mixture is 200–400 nM, where free IN is predominantly a dimer. Two sites (A1/A2 and D1/D2) are labeled in each dimer. Two IN preparations are mixed in ice-cold buffer to minimize the subunit exchange between free dimeric IN proteins. Subsequent addition of viral DNA or LEDGF/p75 promotes IN tetramerization. Three different populations of IN–viral DNA or IN–LEDGF/p75 complexes are formed. Of these, only the complex containing D1–D2 and A1–A2 pairs yields FRET. For the cartoon, a molecular model of full-length HIV-1 IN in complex with LEDGF/IBD was employed.

Figure 4.

Site-selective labeling of HIV-1 IN with a fluorophore. (A) In parallel experiments, C56/65S and C56/65/280S mutants were subjected to treatment with Alexa 488 maleimide. The reactions were quenched with DTT and subjected to SDS–PAGE. Images of the same gel following coomassie staining (upper panel) and UV-light exposure (lower panel) are shown. No fluorescence signal was detected for the C56/65/280S protein (lane 2), while the C56/65S mutant (lane 1) was effectively labeled with the dye. (B) Assembly of SSCs with the labeled mutant IN. Lane 1, wild-type IN load; lane 2, load of the IN (C56/65S) mutant; lane 3, protein markers; lane 4, no DNA control; lane 5, the SSC assembly with wild-type IN and viral DNA; lane 6, the IN (C56/65S) mutant without DNA; lane 7, the SSC assembly with the IN (C56/65S) mutant. (C) Concerted integration assays of wild-type and mutant (C56/65S) IN proteins: Lane 1: DNA markers, lanes 2 and 3: increasing concentration of the IN (C56/65S) mutant, lanes 4 and 5: wild-type IN activities.

Figure 5.

Ss-FRET plots for IN–viral DNA and IN–LEDGF/p75 complexes. (A) IN alone, (B) IN–LEDGF/p75 complex, (C) IN–viral DNA complex, (D) overlay of the spectra from experiments shown in panels A–C. For control experiments, Alexa 488-labeled IN(C56/65S) was mixed with the unlabeled protein (blue circles in A) and then incubated with LEDGF/p75 (blue circles in B) or viral DNA (blue circles in C). The fluorescence intensities for the D–A pairs are depicted with diamonds and color coded as follows: IN alone, orange; IN–LEDGF/p75 complex, cyan; IN–viral DNA complex, magenta. Fluorescence quenching at 520 nm and concomitant increase of emission intensities at 610 nm demonstrate FRET. The spectra was normalized by defining the maximum intensity of the donor fluorophore in each donor alone experiment as 100%.

Figure 6.

Tr-fluorescence decay plots for IN–viral DNA and IN–LEDGF/p75 complexes. (A) IN alone; (B) IN–LEDGF/p75 complex, (C) IN–LEDGF/p75 complex was preformed and then exposed to viral DNA; (D) IN–viral DNA complex; (E) IN–viral DNA complex was preformed and then LEDGF/p75 was added to the nucleoprotein complex. Blue plots show fluorescence decays for donor only control, where Alexa 488-labeled IN(C56/65S) was mixed with the unlabeled protein. Magenta plots show data for the D–A pairs.

Figure 7.

Molecular modeling of IN, viral DNA and LEDGF/IBD interactions. (A) The assembly of the fully functional nucleoprotein complex. First, IN interacts with viral DNA to form the SSC. Then, LEDGF/IBD tightly binds the SSC by bridging between the two dimers. Four individual subunits of IN are colored orange, cyan, magenta and green. The cyan and magenta protomers directly interact with viral DNA, while green and orange subunits play supporting roles. LEDGF/IBD is depicted in gray. (B) IN interactions with LEDGF/IBD. Colors for IN subunits and LEDGF/IBD are the same as in A. Locations of C280 in each subunit are shown by red spheres.