Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering

Abstract

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(–) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region (146RRGRKRKAEKQ156) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/–) and IBD(+/–) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.

Integrase, LEDGF/p75 and HIV replication

“…Knockout mice feed poorly in the perinatal period, leading to some mortality, but survivors display mild skeletal and neurobehavioral abnormalities and the derived MEFs grow normally [94]. In addition, stable knockdown is well tolerated in human cell lines, so far producing no discernible differences in growth rates, morphology or phenotypes other than lentivirus susceptibility [75,80,85,95,96,110].…”

“…The interaction between p75 and IN is direct [73] and it is confined to only one of the seven retroviral genera, lentivirinae [75,87,100]. A number of laboratories established the role of p75 in chromatin tethering, mapped relevant functional domains, and worked to answer the question of virological relevance [74][75][76][77][78][79][80][81][82][85][86][87][88][89][90][91]93,[95][96][97][100][101][102][103][104][105][108][109][110]. The first indication of the protein's significance was the clarification it provided about the vexing issue of the intracellular trafficking of retroviral integrase proteins.…”

“…In contrast, immunofluorescence studies with anti-IN antibodies or with small antigenic epitope-tagged versions of IN produce strict nuclear and cytoplasmic lentiviral IN distributions in the presence and absence of p75 respectively [75,80]; see also ref. [120] which first identified the nuclear location of epitopetagged HIV IN.…”

“…[120] which first identified the nuclear location of epitopetagged HIV IN. Further illustrating the point, fusions of IN to larger proteins, e.g., pyruvate kinase or beta-galactosidase, disrupt the p75-IN interaction entirely [75,80,123,125]. Therefore, fluorescent protein-IN fusions are no longer preferred for sub-cellular trafficking experiments.…”

“…4b and refs. [75,80]). Nuclear import must also be distinguished from nuclear localization, since whether IN protein is trapped by p75 after import or is co-transported with it through the nucleopore is not yet clear, although studies with nuclei from digitonin-permeabilized cells suggest nuclear import can occur independently of p75 [86].…”