کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1994232 1064753 2009 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Yeast two-hybrid detection of integrase–host factor interactions
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Yeast two-hybrid detection of integrase–host factor interactions
چکیده انگلیسی

Here we describe methods developed based on systematic yeast two-hybrid screenings that allowed us to identify several binding partners of HIV-1 integrase. We have developed an efficient strategy to perform large comprehensive screenings with different highly complex cDNA libraries derived both random- and oligo-dT primed reactions. A very efficient mating procedure was used for screening in yeast, allowing genetic saturation of positive clones. This importantly leads with confidence to the determination of the regions within the participating proteins responsible for the interactions. Several additional tools were used that allowed us to assess the specificity of the interactions detected, including rebound screens with cellular co-factors as baits performed against a library of random fragments of HIV-1 proviral DNA. For some of the identified cell factors, we have generated and characterized loss of affinity mutants of integrase, which, when combined with viral functional assays, validated the involvement of human lens epithelium-derived growth factor (LEDGF/p75) in the integration step of the HIV-1 replication cycle. All tolled, our studies identified LEDGF/p75, Transportin-SR2 (TNPO3), von Hippel–Lindau binding protein 1 (VBP1), and sucrose non-fermenting 5 (SNF5) as cellular binding partners of HIV-1 integrase.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 47, Issue 4, April 2009, Pages 291–297
نویسندگان
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