Multimerization and DNA binding properties of INI1/hSNF5 and its functional significance

Abstract

INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183-294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183-294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication.

FIGURE 1.

Active INI1 is a multimer. A, Coomassie Blue staining of protein preparations from different stages of His-INI1 purification, viz. Ni-NTA, hydroxylapatite, and Mono-Q Sepharose. B, in vitro integration assay with increasing amounts of IN (0.25, 0.5, and 1 pmol) and INI1 (0.5 and 1 pmol) as indicated. C, in vitro integration assay at physiological salt concentration as indicated. For stimulation, 0.25 pmol of IN and 0.5 pmol of INI1 were used. For inhibition, 0.5 pmol of IN and 2 pmol of INI1 were used. D, in vitro integration assay with increasing concentrations of IN (0.25 and 0.5 pmol) and LEDGF (0.25, 0.5, 1, and 2 pmol) as indicated and with 5.3 μm integrase inhibitor S-1360. Lane 14 is a control for the addition of inhibitor, where vehicle (DMSO) has been added. E, 15–35% glycerol gradient centrifugation of <5 nm Mono-Q His-INI1. Fractions were analyzed by Western blot using anti-His antibody as probe. BSA, aldolase, and catalase were run in parallel gradients. F, 15–35% glycerol gradient centrifugation of >100 nm Mono-Q His-INI1. Analysis was carried out as described in E. Aldolase was used as a molecular weight marker. G, GST pulldown assay using GST, GST-tagged INI1 immobilized on glutathione-Sepharose beads, and purified His-INI1. Bound proteins were analyzed by Western blot using anti-His antibody as probe. H, coimmunoprecipitation (IP) of FLAG-INI1 using HA-INI1 as bait. 293T cells were transfected with either HA-INI1 or FLAG-INI1 or both, and cell lysates were immunoprecipitated using anti-HA agarose. Immunoprecipitated proteins were analyzed by Western blot (WB) using anti-FLAG and anti-HA antibodies as probes. STP, strand transfer product.

FIGURE 2.

A region containing the Rpt1 and Rpt2 motifs comprises the minimal multimerization domain of INI1. A, schematics of wild type (WT) INI1 and various INI1 deletion mutants fused to GAL4AD. Rpt1, repeat 1 motif; Rpt2, repeat 2 motif; CC, coiled coil domain. B, quantitative liquid yeast two-hybrid assay (β-galactosidase/ONPG assay) of LexADBD-fused wild type INI1 and different deletion fragments and wild type INI1 fused to GAL4AD. C, Coomassie Blue staining and Western blot analysis using the indicated antibodies of Mono-Q Sepharose-purified INI1-(141–304). D, Coomassie Blue staining of INI1-(141–304) purified in the presence and absence of detergent (IGEPAL) showing the faster migrating band to be an artifact of detergent solubilization. E, Superdex 200 HR 10/30 gel filtration of Mono-Q Sepharose-purified INI1-(141–304). Fractions collected were analyzed by Western blot using anti-His antibody as probe. Chymotrypsinogen A (25 kDa) and ovalbumin (43 kDa) were used as molecular mass standards.

FIGURE 3.

Isolation and characterization of INI1-(183–294) mutants defective for multimerization. Amino acid residues involved in multimerization of INI1 are overlapping but distinct from those involved in IN binding. A, identity and position of multimerization-defective mutants of INI1-(183–294). B, quantitative yeast two-hybrid assay (β-galactosidase/ONPG assay) of GAL4AD-fused wild type and mutant INI1-(183–294) with full-length INI1 fused to LexADBD. C, quantitative yeast two-hybrid assay (β-galactosidase/ONPG assay) of GAL4AD-fused wild type and mutant INI1-(183–294) with LexADBD-fused wild type IN. D, Western blot analysis of wild type and mutant INI1-(183–294) containing yeast extracts using anti-GAL4AD antibody as probe. E, quantitative yeast two-hybrid assay (β-galactosidase/ONPG assay) of GAL4AD-fused wild type and mutant INI1-(183–294) and LexADBD-fused wild type INI1. F, quantitative yeast two-hybrid assay (β-galactosidase/ONPG assay) of GAL4AD-fused wild type and mutant INI1-(183–294) and LexADBD-fused wild type IN. G, Western blot analysis of wild type and mutant INI1-(183–294) containing yeast extracts using anti-GAL4AD antibody as probe.

FIGURE 4.

Requirement of multimerization and nuclear export properties for the inhibition of particle production by INI1-S6-(183–294). A, location of DD5 and DD6 mutations with respect to NES. B, confocal microscopy of GFP-fused S6 and DD5 and DD6 mutants. C, subcellular localization of S6 and DD5 and DD6 mutants. Values are expressed as percentages. D, intracellular, virion-associated, and ratio of virion-associated to intracellular p24 in the presence of wild type INI1 or S6, DD5, and DD6 mutants. E, expression of INI1, S6, DD5 and DD6 proteins in 293T cells. F, coimmunoprecipitation (IP) of YFP-IN with HA-tagged S6, DD5, and DD6. Inputs of YFP-IN and HA-tagged proteins are shown. WB, Western blot; PI, propidium iodide.

FIGURE 5.

Co-localization of IN with INI1 and mutants. A, confocal microscopy of cells expressing full-length INI1 harboring DD5 and DD6 mutations. B, confocal microscopy to determine the co-localization of yellow fluorescent protein (YFP)-IN with cyan fluorescent protein (CFP)-INI1 and its mutants. C, graphic representation of percentage of cells exhibiting co-localization of IN and INI1 harboring either DD5 or DD6 mutants. PI, propidium iodide.

FIGURE 6.

Effect of multimerization-defective mutants of full-length INI1 on in vitro integration. A, 15–35% glycerol gradient centrifugation of wild type INI1 and DD mutants. Fractions were analyzed by Western blot using anti-His antibody as probe. B, GST pulldown experiment with GST or GST-fused INI1 and HAP eluate of wild type and mutant His-INI1 as shown. Bound proteins were analyzed by Western blot using anti-His antibody as probe. The same blots were stripped and probed with anti-GST antibodies. C, GST pulldown of wild type INI1 and DD mutants and E3 with glutathione-Sepharose 4B beads coupled to GST or GST-IN at different salt concentrations. Bound proteins were analyzed by Western blot using anti-His antibody as probe. The same blots were stripped and probed with anti-IN (integrase) antibodies. D, in vitro integration (stimulation) assay with wild type (WT), DD mutants, and E3 INI1 HAP eluate at 100 mm salt. STP, strand transfer product. E, in vitro integration (inhibition) assay with wild type, DD mutants and E3 INI1 HAP eluate at 100 mm salt.

FIGURE 7.

Correlation of multimerization and DNA binding properties of INI1 to modulate in vitro integration. A, 15–35% glycerol gradient centrifugation of integrase and integrase incubated with wild type INI1 or I264T,I268T. Fractions were analyzed by Western blot using anti-INI1 and anti-integrase antibodies as probes. B, gel retardation assay with HAP eluate (8 and 16 pmol) of wild type INI1 (WT) and DD mutants (including E3) and pCDNA plasmid as explained under “Materials and Methods.” C, EMSA of HAP eluate of INI1 (5 pmol) with 23-mer viral LTR double-stranded DNA radiolabeled with γ-³²P ATP in the presence of 10× cold oligos. 250× scrambled LTR DNA was used for the competition experiment. His-INI1 was immunodepleted with anti-His antibody. D, EMSA of HAP INI1 (5 pmol) with radiolabeled viral LTR DNA in the presence of increasing amounts of distamycin A and DAPI as indicated. E, EMSA of HAP wild type and mutant INI1 (5 pmol) with radiolabeled viral LTR DNA.