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In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75
- Source :
- Journal of Molecular Biology, Journal of Molecular Biology; Vol 410
- Publication Year :
- 2011
-
Abstract
- Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture. ispartof: JOURNAL OF MOLECULAR BIOLOGY vol:410 issue:5 pages:811-830 ispartof: location:Netherlands status: published
- Subjects :
- musculoskeletal diseases
Nuclear Localization Signals
Mutant
Oligonucleotides
Enzyme-Linked Immunosorbent Assay
HIV Integrase
Plasma protein binding
Biology
Models, Biological
Structure-Activity Relationship
03 medical and health sciences
chemistry.chemical_compound
Structural Biology
Humans
NLS
skin and connective tissue diseases
Molecular Biology
030304 developmental biology
0303 health sciences
030302 biochemistry & molecular biology
DNA
AT Rich Sequence
Molecular biology
biological factors
Long terminal repeat
Protein Structure, Tertiary
3. Good health
Integrase
Chromatin
Kinetics
Solubility
nervous system
chemistry
HIV-1
biology.protein
Intercellular Signaling Peptides and Proteins
Biological Assay
Mutant Proteins
sense organs
Nuclear localization sequence
Protein Binding
Subjects
Details
- Language :
- English
- ISSN :
- 00222836
- Volume :
- 410
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular Biology
- Accession number :
- edsair.doi.dedup.....40d10d9947ed81d5385150fb4c2568dd
- Full Text :
- https://doi.org/10.1016/j.jmb.2011.03.073