Chromosomal integration enables human immunodeficiency virus (HIV) to establish a permanent reservoir that can be therapeutically suppressed but not eradicated. Participation of cellular proteins in this obligate replication step is poorly understood. We used intensified RNA interference and dominant-negative protein approaches to show that the cellular transcriptional coactivator lens epithelium-derived growth factor (LEDGF)/p75 (p75) is an essential HIV integration cofactor. The mechanism requires both linkages of a molecular tether that p75 forms between integrase and chromatin. Fractionally minute levels of endogenous p75 are sufficient to enable integration, showing that cellular factors that engage HIV after entry may elude identification in less intensive knockdowns. Perturbing the p75-integrase interaction may have therapeutic potential.

Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integraseto-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

We used recombinant chicken deoxyribonucleic acid clones containing embryonic and adult ,8-globin genes and "runoff" endogenous nuclear transcription to investigate the expression of embryonic and adult f-globin genes during hematopoiesis in the developing chicken embryo. Purified, cloned deoxyribonucleic acids were digested with various restriction enzymes, separated on agarose gels, blotted to nitrocellulose, and annealed with purified nuclear [3P]ribonucleic acid synthesized in vitro from embryonic or adult red cell nuclei. Transcription of the respective globin genes was assayed by hybridization of nuclear [3P]ribonucleic acid to specific restriction fragments containing adult or embryonic coding sequences. Our results indicate that little, if any, transcription from the adult or embryonic ,8-globin genes is detectable in the heterologous red cell nuclei, even under conditions in which ribonucleic acid processing probably does not occur.Differential expression of hemoglobin genes during avian development offers a model system for studying gene regulation at both the cellular and molecular levels. In the developing chicken embryo, hemoglobin first appears in first-generation erythroblasts at approximately 30 to 35 h of development. These cells then serve as the progenitors of six subsequent maturational generations of erythroblasts, each of which contains characteristic amounts of hemoglobin and displays unique morphological characteristics. These cells mature rather synchronously and, by day 6 of embryonic development, most have become erythrocytes. Between days 6 and 7, another lineage ofhematopoietic cells, the definitive embryonic lineage, enters the circulating erythrocyte population, arising from the endothelial lining of the blood vessels. The relationship between these two lineages and the possible generation of the definitive series from the immediate precursors of the primitive lineage are the focus of current investigation (2,16,17).The definitive series enters the circulating erythroid compartment between days 6 and 7 as fairly mature erythroblasts and by days 11 to 13 matures into inactive erythrocytes. These cells are the major erythroid cells in circulation until hatching, at which time a second definitive line appears. Although minor differences occur, by and large both definitive lines produce similar types of hemoglobin chains (1), and both differ from the primitive series in the synthesis of specific fi chains.In this and former studies, we have focused on the switch in f-chain synthesis between the primitive and first definitive series. Given the similarities in fl-chain synthesis between the two definitive lines, we have not distinguished between the two types of definitive cells, and, for convenience, refer to both as adult red cells since they both produce adult, chains. In contrast, given the distinctive embryonic, chains synthesized in the primitive series, we refer to these cells as embryonic red cells. In examining the control of embryonic and adult globin gene expressions, we previ...

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.

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type ( Shortly after infection of a cell by a retrovirus, reverse transcription of the RNA genome yields a linear cDNA copy, which along with the viral integrase (IN) and other proteins comprises the preintegration complex (PIC), the functional precursor to integration (5). Certain features of the IN structure are conserved among retroelements, and conserved amino acid residues that are critical for catalysis have been identified. Retroviral INs have three domains: an N-terminal domain, a central catalytic core domain, and a C-terminal domain (26). The N-terminal domain contains a zinc finger-like sequence that influences IN oligomerization (9). The C-terminal domain, which is the most divergent, binds DNA in a sequenceindependent manner (20, 58). IN has also been reported to play other roles in the lentiviral life cycle, in particular in nuclear import of the PIC (23).The genetic analysis of IN functions is not straightforward because the enzyme is generated by viral protease-mediated cleavage from the Gag/Pol precursor. Many IN mutations produce pleiotropic effects on Gag/Pol-derived functions, including particle formation and reverse transcription (17). Accordingly, two types of IN mutants are generally recognized. Nonspecific phenotypes (which have been termed class II) result from deletions, truncations, and numerous single amino acid changes (17,19,56). In contrast, nonpleiotropic (class I) mutations affect only the DNA cleaving and joining reaction, while leaving intact other measurable aspects of the virus life cycle, such as Gag/Pol precursor processing, particle formation, virion morphogenesis, reverse transcription, and PIC nuclear import (17,18,28,29). In human immunodeficiency virus type 1 (HIV-1), mutations of any of three residues that participate in the catalytic center (D64, D116, and E152) produce class I properties (15,18,29). These residues form a catalytic triad (DX 39-58 DX 35 E) that is broadly conserved in retroelement INs (5,17). Experimentally, class I IN mutants enable control designs that compare the fates of integration-defective, structurally normal particles differing in only one amino acid in a single enzyme that comprises a very small molar fraction of virion protein molecules (17).