Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance

Das, Supratik; Cano, Jennifer; Kalpana, Ganjam V.

doi:10.1074/jbc.m808141200

Cited by 28 publications

(44 citation statements)

References 27 publications

(39 reference statements)

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“…SMARCB1 is known to inhibit HIV replication in cell lines, possibly through the stabilization of pre-intregration complexes in non-reactive conformations (Das et al, 2009; Maroun et al, 2006; Pyeon et al, 2015). XRCC6 (Ku70) and other components of the NHEJ pathway, however, have an unclear role in the HIV lifecycle, though some results suggest a role in post-integration expression (Baekelandt et al, 2000; Jeanson et al, 2002; Li et al, 2001; Manic et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

et al. 2016

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SUMMARY New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knock-out cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knock-out of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

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Section: Resultsmentioning

confidence: 99%

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

et al. 2016

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“…Here, as a first step toward understanding the mechanism of inhibition of HIV-1 late events by S6, we tested to determine whether cytoplasmic localization of S6 is necessary for inhibition. Recent studies from our laboratory have indicated that INI1 is a multimer and that some multimerization-defective mutants of S6 reversed the inhibitory effect (12). Because these mutants also disrupted the NES, it is unclear whether the reversal of inhibition is due to a defect in multimerization and/or nuclear export (12).…”

mentioning

confidence: 99%

“…Recent studies from our laboratory have indicated that INI1 is a multimer and that some multimerization-defective mutants of S6 reversed the inhibitory effect (12). Because these mutants also disrupted the NES, it is unclear whether the reversal of inhibition is due to a defect in multimerization and/or nuclear export (12). Furthermore, we tested to determine the stage/s of assembly that is affected by S6 using electron and fluorescence microscopy and pulse-chase analysis of Gag.…”

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confidence: 99%

“…INI1 binds tightly to IN in vitro and in vivo, is selectively incorporated into HIV-1 virions, and is found in the reverse transcription and preintegration complexes isolated from cells (17,19,26,39,46). Recombinant INI1 stimulates and inhibits in vitro integrase joining activity in a concentration-dependent manner (12,19). In addition, INI1 is a tumor suppressor gene that is biallelically deleted or mutated in aggressive pediatric cancers known as rhabdoid tumors (40).…”

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confidence: 99%

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Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Cano

Kalpana

2011

J Virol

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INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions.A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55 gag ) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding.

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“…INI1 not only binds integrase (IN), a viral enzyme that is absolutely required for the integration of the reverse transcribed viral double stranded DNA into the host chromatin, but also modulates the activity of IN [9]. Earlier studies have shown that INI1 stimulates as well as inhibits IN activity depending on the concentration of IN and the ratio of IN:INI1 [10]. Furthermore, these studies demonstrate that INI1 is a multimer and that multimerization of INI1 is essential for its ability to bind IN to form a high MW complex that is required for inhibition of IN activity [10].…”

Section: Introductionmentioning

confidence: 99%

Characterization of DNA Binding Property of the HIV-1 Host Factor and Tumor Suppressor Protein Integrase Interactor 1 (INI1/hSNF5)

et al. 2013

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Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105–183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K’d1 = 222 µM and K’d2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = –29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH’1 = 115.7 KJ/mole, TΔS’1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH’2 = –106.3 KJ/mole, TΔS’2 = –75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1.

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Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance

Cited by 28 publications

References 27 publications

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Characterization of DNA Binding Property of the HIV-1 Host Factor and Tumor Suppressor Protein Integrase Interactor 1 (INI1/hSNF5)

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