Background:  RNAi knock-down of the transcriptional co-activator LEDGF/p75 disrupts the tight chromatin association of lentiviral integrase proteins and causes them to relocate from the nucleus to the cytoplasm of cells. To determine the basis for the cellular trafficking of LEDGF/p75 and integrase, we have mapped human LEDGF/p75 for nuclear localization signals and identified a domain that is necessary and sufficient for integrase interaction.

Methods:  We characterize the subcellular distribution of LEDGF/p75 mutants in cell lines we made stably deficient in endogenous LEDGF/p75 by immunofluorescence. All constructs were evaluated by immunoblotting for correct protein sizes.

Results:  A single classical nuclear localization signal in the N-terminal region (¹⁴⁶RRGRKRKAEKQ¹⁵⁶) was identified by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the nuclear localization signals are critical for its activity. Deletion of C-terminal residues 340 to 417 preserved nuclear and chromatin localization of LEDGF/p75, but abolished HIV-1 integrase interaction and tethering of integrase to chromatin. Transfer of this integrase-binding domain was sufficient to confer HIV-1 integrase interaction to GFP. Strikingly, however, stable expression studies with nuclear localization signals (+/–) and integrase-binding domain (+/–) mutants revealed that the nuclear localization signals, while responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and integrase. When stably introduced into cells, both wild type LEDGF/p75 and nuclear localization signal-mutant LEDGF/p75 are entirely chromatin-associated in cells in all phases of the cell cycle, and each protein tethers integrase to chromatin in an integrase-binding domain-dependent manner.

Conclusions:  LEDGF/p75 is a multi-domain adaptor protein that interacts with the nuclear import apparatus, lentiviral integrase proteins, and chromatin by means of a nuclear localization signal, an integrase-binding domain, and additional chromatin-interacting domains.

Keywords: HIV-1 Integrase; LEDGF/p75; Chromatin