Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro

Abstract

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.

Keywords: LLPS biomolecular condensates; human immunodeficiency virus; integrase; integration mechanism regulation; intrinsically disordered region; macromolecular crowding; viral DNA.

Figure 1

Both IN and LEDGF/p75 proteins are predicted with several intrinsically disordered regions.A, prediction of the disorder probability of LEDGF/p75 (left) and IN (right) using PrDOS software. Positions above the red line were considered as disordered residues, with a standard False Positive (FP) rate set at 5% by the software. B, structure prediction (LEDGF/p75 (left) and IN (right)) using Alphafold2. Color code of the structure according to the confidence (pLDDT: predicted local-distance difference test) are estimated by Alphafold2.

Figure 2

Both LEDFG/p75 and IN-LEDGF/p75 complexes are able to form LLPS in vitro. Fluorescent LLPS were imaged with a X100 objective (scale bars = 5 μm), using Dye-490 labeled LEDGF/p75 from E. coli (A), IN-LEDGF/p75 from E. coli (B) and from mammalian cells (C). Top panels correspond to brightfield images (BF) whereas bottom panels correspond to the fluorescence of the Dye 490. Images were recorded either without LLPS enhancer (column 1) or in presence of 10% PEG-4000 (column 2), or with both 10% PEG-4000 and 10% 1,6-Hexanediol (LLPS inhibitor) (column 3). Yellow triangles point the structures consider as aggregates (not spherical, bigger shape, and not as refringent in BF images). Quantification of LLPS number per field (D) and size in nm (E). All the analyses were performed using the “Particles Analyzer” module from the ImageJ software. Standard deviations (STD) were calculated from five different images for each condition. LLPS of LEDGF/p75 are depicted in yellow, IN-LEDGF/p75 from E. coli in blue, and IN-LEDGF/p75 from mammalian cells in green. Dash bars correspond to the size estimated after exclusion of the biggest particles >1 μm considered as aggregates.

Figure 3

Characterization of IN-LEDGF/p75 complex LLPS.A, size evolution of LLPS size plotted versus time in min. Size estimation was performed using the “Particles Analyzer” module from the ImageJ software. Standard deviations (STD) were calculated from five different images for each condition. B, fluorescent LLPS were imaged from longer kinetics (indicated time after mixing with LLPS reagent) with an X100 objective (scale bars = 5 μm), using Dye-490 labeled IN-LEDGF/p75 complex from mammalian cells. C, quantification of LLPS number per field (left panel) and size in nm (right panel) depending on the concentration of IN-LEDGF/p75 complex. All the analyses were performed using the “Particles Analyzer” module from the ImageJ software. Standard deviations (STD) were calculated from five different images for each condition. D, fluorescent LLPS were imaged using other LLPS reagents with an X100 objective, using Dye-490 labeled IN-LEDGF/p75 complex from mammalian cells. Scale bar = 5 μm.

Figure 4

Intasomes constituted with IN-LEDGF/p75 and DNA form LLPS in vitro.A, fluorescent LLPS were imaged with a X100 objective, in presence of viral DNA labeled in 5′ with the 6FAM fluorophore. Column 1 is without PEG-4000; column 2 is a control without protein, column 3 is the condition with 10% PEG-4000 and the protein. Top panels correspond to the brightfield images (BF) whereas bottom panels correspond to the fluorescence of the 6-FAM Dye (scale bars = 10 μm). B, fluorescent LLPS were monitored in the presence of the 2 DNA molecules forming the complete intasomes: one viral DNA (6-FAM labeled) and one target DNA (Cy5 labeled). Column 1: brightfield image, column 2: 6-FAM signal (viral DNA), column 3: Cy5 signal (target DNA) and column 4: overlay (scale bars = 5 μm). Yellow triangles point to the structures consider as aggregates (not spherical, bigger shape, and not as refringent in BF images). C, LLPS were imaged with an X100 objective, with 10% PEG-4000, using Dye-490 labeled CPSF5/6 complex alone (top panel) and with IN-LEDGF/p75 complex in presence of target DNA labeled in 5′ with the Cy5 fluorophore (bottom panel). Column 1 corresponds to the CPSF5/6 fluorescent signal (Dye-490), Column 2 to the IN-LEDGF/p75 complex fluorescent signal (Cy5 target DNA) and Column 3 is the overlay.

Figure 5

3′ processing activity is drastically reduced in an LLPS environment.A, the IN-LEDGF/p75 complex is able to form LLPS in the 3′ processing reaction buffer (scale bars = 5 μm). B, fluorescence anisotropy was recorded at 25 °C in the presence (square) or absence (circle) of 10% PEG-4000. C, quantification of the slope observed in B, normalized at 100% without PEG-4000. Controls without protein and with an uncleavable random DNA are also shown. D, quantification of 3′ processing activity regarding the LLPS reagent used. Anisotropy slope were calculated and normalized at 100% in the condition without PEG-4000. Black bars correspond to the IN-LEDGF/p75 complex alone and white bars corresponds to the complex in the presence of 1,6 Hexanediol. E, gel separation of unprocessed DNA (40 nucleotides DNA, top signal) and processed DNA (38 nucleotides, bottom signal). The left part of the gel was performed in the absence of PEG-4000 whereas right part was performed using 10% PEG-4000. Kinetics were monitored from 0 to 6 h at 37 °C. F, the ratio of product signal to substrate signal was calculated and then plotted versus time. Squares correspond to data in the presence of PEG-4000 whereas circles describe the data in the absence of PEG-4000. Solid lines correspond to the fits of the data with equations described in the table under the graph. Half-life are in min.

Figure 6

Strand transfer activity of IN-LEDGF/p75 is strongly enhanced in LLPS environment.A, the IN-LEDGF/p75 complex is able to form LLPS in the strand transfer reaction buffer (scale bars = 5 μm). B, luminescence signals were measured performing the strand transfer assay either without or with indicated LLPS reagent. Black bars correspond to the IN-LEDGF/p75 complex alone and white bars corresponds to the complex in presence of 1,6 Hexanediol. C, integration assay using 601 nucleosomes was performed in the presence of either PEG, Ficoll or Dextran as indicated in the Experimental procedures section and products formed after 10 min reaction and deproteinization were loaded onto 8% polyacrylamide native gel. Lane one corresponds to the reaction without IN-LEDGF/p75. D, integration assay performed as in C and run for 5, 10, and 15 min before monitoring of the integration products after deproteinization and 8% polyacrylamide gel running. E, increasing concentrations of 1.6 HD were added to integration reactions run for 10 min before analysis F, similar experiments were performed using biotinylated nucleosomes trapped to streptavidin beads, and integration products formed after 10 min of reactions in the different conditions were monitored by quantifying the remaining radioactivity bound to the captured beads Data are reported as mean of at least three independent experiments ± SD. Blue = no reagent; Orange = + 10% PEG-4000; Grey = + 10% Ficoll-400; and Yellow = + 10% Dextran-5000. Dashed lines correspond to the presence of 10% of 1,6 Hexanediol.

Figure 7

Dynamic model of pre-integration events emerging from our data. After the entrance into the nucleus, reverse transcription ended into the capsid (1). Nascent viral 3′ processed DNA together with IN (purple circles) is released in the nucleus in a condensates environment allowed by the presence of CPSF5/6 (green circles) in the close proximity to the p24 capsid protein (2). The IN-protein recruits then its cofactor LEDGF/p75 (blue circles) required for the chromatin targeting, to form the functional intasome that will perform the final integration reaction (3). At this step, the intasome may leave the CPSF5/6 condensates to form its own LLPS environment. Figure adapted from https://scienceofhiv.org/wp/life-cycle/(license: https://creativecommons.org/licenses/by-nc-sa/4.0/).

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