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.

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…S1). This model is based on the crystal structure of the prototype foamy virus intasome (62) and our FRET analysis of the HIV-1 SSC and its interaction with LEDGF/p75 (31). The assembly of the SSC requires a tetramer of IN with two subunits tightly interacting with viral DNA and other two subunits providing a supporting Fig.…”

“…Preparation of SSC-SSCs were prepared as previously described using 400 nM IN with ϳ 20% forming the SSC (31). SSCs were then mixed with purified 4 M TNPO3 and allowed to bind for 1 h at 4°C.…”

“…SSCs were then mixed with purified 4 M TNPO3 and allowed to bind for 1 h at 4°C. Mixtures were then passed through PCR Kleen Spin Columns (Bio-Rad) as described for LEDGF/p75 binding to SSCs (31). The resulting flow-through material was then analyzed by SDS-PAGE and Western blot using anti-hexahistidine (anti-HIS) and anti-IN antibodies (HIV-1 IN mono-clonal antibody (8G4) from NIH AIDS Research and Reference Reagent Program).…”

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

“…MS-based protein footprinting studies of full length IN, the results of which are summarized in Fig. 7A in the context of the IN structural model (54), allowed us to identify protein-protein interactions in the CCD and CTD that extend beyond the inhibitor binding sites and contribute to higher-order IN multimerization. A recent study (41), which investigated the multimerization of IN domains in the presence of ALLINI GSK1264 using sedimentation velocity and turbidity assays, has also revealed the significance of the CTD in addition to the CCD for the formation of insoluble protein aggregates.…”

“…In the NMR structure of the isolated CTD, which is seen as a dimer with Lys-264 and Lys-266 being fully surface-exposed and positioned on the opposite side of the CTD-CTD interface (28 -30). In the HIV-1 intasome models, which have been generated based on the analogous prototype foamy virus intasome structure, the CTD domains do not engage in protein-protein interactions and instead interact with viral DNA (10,54,55). In particular, Lys-264 and Lys-266 directly contact viral DNA (Fig.…”