An Essential Role for LEDGF/p75 in HIV Integration

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Science  20 Oct 2006:
Vol. 314, Issue 5798, pp. 461-464
DOI: 10.1126/science.1132319


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.

Integration enables human immunodeficiency virus type 1 (HIV-1) to establish a permanent genetic reservoir that can initiate new virion production, evade immune surveillance, and replicate through mitosis. Integrated proviruses that persist in long-lived T cells ensure rapid HIV recrudescence if antiviral drugs are withdrawn. Integration is catalyzed by the viral integrase (IN). When expressed as a free protein in cells rather than within its normal context as an intravirion cleavage product of the HIV Gag-Pol precursor, IN becomes tethered to chromatin by cellular lens epithelium–derived growth factor/p75 (p75) (13), which is a transcriptional coactivator (4). Accordingly, both proteins display tight colocalization with chromatin throughout the cell cycle; short hairpin RNA (shRNA)–mediated knockdown of p75 untethers IN, redistributing it from an entirely nuclear to an entirely cytoplasmic location (3). Molecular tethering results from specific linkages formed by p75's discrete functional modules: the N-terminal Pro-Trp-Trp-Pro (PWWP) and A/T-hook elements bind to chromatin (5), and a C-terminal integrase-binding domain (IBD) binds to IN (6, 7). p75 also protects the HIV-1 IN protein from rapid degradation in the 26S proteasome (8). In the bona fide viral context, drastic knockdown of p75 changed the genomic pattern of HIV-1 integration by reducing the viral bias for active genes, which suggests that p75 influences integration targeting (9). However, changes in overall levels of HIV integration and replication have been either absent or modest, and single-cycle infection analyses in cell lines have consistently detected no effect, which has led to questions about the overall importance of p75 in the viral life cycle (3, 7, 912).

Previously, we observed that a nuclear localization signal–mutant p75 protein became constitutively chromatin-trapped in stable cell lines (7). In the present work, we hypothesized the existence in previous severely RNA interference (RNAi)–depleted HIV-susceptible cells of a very small yet virologically potent chromatin-associated p75 residuum. We reasoned that a fractionally minute residual pool with a spatially favorable location (colocalized with chromatin) could explain the inability to demonstrate substantial, reproducible impairments in integration or viral replication in cells lacking detectable p75. Such a reservoir would be inadequate to affect observable properties of ectopically expressed IN but might be sufficient to engage the vastly less abundant incoming viral preintegration complex.

To test this hypothesis, we performed subcellular fractionation and interrogated chromatin, using a deoxyribonuclease (DNase) I– and salt-based extraction protocol (13). These methods detected a chromatin-associated p75 residuum in virally permissive knockdown cell lines. We then developed a new strategy to eradicate the residual pool, using RNAi with intensified lentiviral vector-based RNAi (ilvRNAi) [panel i in fig. S1A and (13)]. Human CD4+ T cells with no immunoblot-detectable p75 in whole-cell lysates [Fig. 1A, TL1 cells; see (13) for nomenclature] but with a scant detectable residue of p75 in the DNase I– and salt-extractable chromatin fraction (Fig. 1B, S2 fraction) were marginally impaired for single-cycle HIV infection (Fig. 1C). In contrast, in S2 fraction–negative (S2FN) cells in which ilvRNAi resulted in undetectable p75 in the S2 fraction as determined by direct Western blotting (Fig. 1B, TL2 cells) or immunoprecipitation (fig. S2A), HIV luciferase reporter virus (HIVluc) infection was reduced to 3.5% of control SupT1 cells transduced equivalently with lentiviral vectors encoding a control shRNA (Fig. 1C). The requirement for radical depletion of p75 to block infection was observed repeatedly in independently derived cell lines with both mCherry- and enhanced green fluorescent protein (eGFP)–marking lentiviral vectors (n = 7 experiments; mean fold inhibition, 28 ± 5%). In 46 experiments conducted with two independently derived S2FN T cell lines, the mean inhibition compared to control lines was 31-fold (fig. S3). The required RNAi intensification was also evident at the mRNA level: Only ilvRNAi-derived cells in which mRNA was reduced to less than 3% of baseline levels were S2FN and resistant to infection (fig. S2B). Equivalent transduction with the p75-specific shRNA and control shRNA ilvRNAi vectors was documented by both Southern blotting for vector DNA and marker protein fluorescence (fig. S4, A and B). The control vector, which differed from the p75-specific vector only within the 21 base pairs of the shRNA hairpin, had no effect on susceptibility to HIV infection in any of seven different ilvRNAi-derived cell lines.

Fig. 1.

Eradication of detectable p75 from chromatin and susceptibility to retroviral infection. Polyclonal SupT1 cell lines were derived by ilvRNAi (13). See fig. S1A (panel i) for construct structures and (13) for the cell line nomenclature system. Lentiviral vectors expressed either a control (TC cells) or a p75-specific shRNA (TL cells). TL1 and TL2 are two independently derived polyclonal TL cell lines. Seven TC lines (TC1 to TC7), independently derived in parallel for pairwise comparison with any given TL line, showed HIV-1 susceptibility equivalent to parental SupT1 cells. TC2 was used in this experiment. (A) Detection of p75 in whole-cell lysates by immunoblotting with p75 monoclonal antibody (mAb). (B) Detection of p75 in subcellular fractions by immunoblotting with p75 mAb. T, total cell lysate; S1, Triton-X100–extractable, non–chromatin-bound fraction; S2, Triton-X100–resistant chromatin-bound fraction released by further DNase I– and salt-based extraction. p75 was detected in abundance in S2 in the absence of p75-specific RNAi (TC cells); only radical S2 depletion blocked viral infection. See figure 3 of (5) for the fractionation scheme and method validation. (C) Analysis of single-round HIV reporter virus infection. Cells were infected with HIVluc, and luciferase enzymatic activity was determined 5 days later. (D) p75 eradication from the S2 fraction blocks HIV-1 and FIV infection but not MLV infection. S2FN cell lines were infected in parallel with three different retroviral vectors (HIV-1, FIV, and MLV), and luciferase activity was determined 5 days later. (E) p75 reexpression rescues HIV infectivity in S2FN T cell lines but p75IBD– does not. p75 [wild-type (WT) or IBD-deleted (ΔIBD)] was reexpressed in S2FN cells by gamma-retroviral vector expression of shRNA-resistant alleles (fig. S1B). Relevant immunoblotting is shown in fig. S5. (F) Differential effect of p75 versus HRP-2 reexpression. A single cell clone of TLH1, TLH1.1, was back-complemented with p75, HRP-2, or both and reexpression was verified by immunoblotting. Five additional clones (TLH1.2 to TLH1.6) showed the same results. Error bars in (C) to (F) represent SD of duplicate determinations.

Feline immunodeficiency virus (FIV), a nonprimate lentivirus with a p75-interacting IN (3, 14), was similarly blocked by p75-specific ilvRNAi (Fig. 1D). In contrast, a gamma retrovirus [murine leukemia virus (MLV)] was completely unimpaired in S2FN cell lines (Fig. 1D), a result consistent with the lack of interaction between MLV IN and p75 (3, 14). This lentiviral specificity enabled us to use MLV vectors to reexpress p75 (fig. S5), which fully rescued susceptibility to HIV-1 (Fig. 1E) and FIV (fig. S6A) but had no effect on gamma-retroviral infection (fig. S6B). Moreover, no rescue was seen when p75 had a specific deletion of the IBD, establishing that this component of the molecular tether is needed (Fig. 1E and fig. S5, panel iii). The results were corroborated by analyses of individual cell clones of ilvRNAi-derived cell lines, where 25- to 65-fold lentiviral-specific inhibition compared to control lines was observed (fig. S7, A and B).

Additional specificity was revealed when we targeted the p75-related protein hepatoma-derived growth factor–related protein 2 (HRP-2), which shares the p75 IBD and binds to HIV-1 IN (6, 7). HRP-2 can also rescue nuclear targeting of IN in p75-deficient cells (7). However, in contrast to p75, HRP-2 is fully extractable in Triton-X100 and does not constitutively tether IN to chromatin (7) or detectably influence integration-site patterns (9). HRP-2–specific ilvRNAi had no effect on HIV infection, whether introduced alone (fig. S7C, TH lines) or additively to p75-specific ilvRNAi (TLH1 cells and six clones, TLH1.1 to TLH1.6)(Fig. 1F and fig. S7C). p75 rescued double-knockdown cells but HRP-2 did not (Fig. 1F). FIV and MLV were also completely unaffected by HRP-2 ilvRNAi; all viral infection impairments were again both lentiviral-specific and attributable only to p75 (fig. S7D).

We also examined productive replication of HIV-1 (Fig. 2). Viral replication was blocked in S2FN cells at both low (0.001 to 0.01) and high (0.1) multiplicity of infection (MOI) when either polyclonal or clonal lines were challenged with replication-competent HIV-1 NL4-3, an infectious molecular clone. Again, p75 fully rescued viral replication (Fig. 2 and fig. S8), whereas HRP-2 did not (Fig. 2B).

Fig. 2.

HIV-1 replication in cell lines and clones with and without rescue of p75- and/or HRP-2RNAi. Each line or clone was compared to its respective back-complement. Equivalent CD4 and CXCR4 expression were verified by fluorescence-activated cell sorting analyses (fig. S4C). Cells were infected with HIV-1 clone NL4-3 at three different MOIs (0.1, 0.01, and 0.001). Experiments at the two higher MOIs are shown. (A) MOI = 0.1, polyclonal TL2 cell line. (B) Double-knockdown single cell clone TLH1.1 was back-complemented with p75, HRP-2, or both. Cells shown here were infected at an MOI value of 0.1. Lower MOI infection values (0.01 and 0.001) produced proportionate delays; for example, at an MOI value of 0.01, p24 values reached 20 ng/ml in TC2 and TLH1.1 cells on days 7 and 57, respectively. This viral replication delay was rescued by p75 reexpression (p24 values reached 9.2 ng/ml on day 9).

We next determined whether any mechanisms could be assigned to the virus producer cell [similar to effects seen with the negatively acting factor APOBEC3G (15)] or to transcriptional effects. However, the origin of challenge HIV in S2FN versus control T cells had no influence on infectivity. All effects were solely attributable to target cell p75 status for both single-cycle HIV-1 vectors and replication-competent HIV-1. We also depleted p75 with ilvRNAi after HIVluc infection, which produced no change in viralexpression. Conversely, reexpression of p75 in S2FN cells after HIVluc infection yielded no change in luciferase or p24 levels. To further test for transcriptional or promoter-specific effects, we infected TC and TL cells and clones with the internally promoted reporter virus HIVc-luc (fig. S1C). HIVc-luc yielded the same phenotype as HIVluc, confirming that p75-specific ilvRNAi does not interfere with Tat protein transactivation (fig. S9). In addition, MLV was uninhibited in S2FN cells, whether reporter expression was driven by the native retroviral U3 or an internal cytomegalovirus promoter.

To further investigate the mechanism of action of p75 in the viral life cycle, we tested the functional capabilities of a chromatin-binding mutant, p75P–A– (fig. S1B). p75P–A– lacks PWWP and A/T-hook domains, which compose the N-terminal chromatin-binding link of the molecular tether (5). Like wild-type p75, p75P–A– is nuclear and binds IN at the IBD, but it is not chromatin-bound and therefore it does not tether IN to chromatin (5). An IN stability rescue assay showed that p75P–A– does retain the function of protecting IN from proteasomal degradation (fig. S10). However, the HIV infection–rescuing function is lost (Fig. 3A), showing that such rescue requires not only the IBD interaction but also the IN-to-chromatin tethering capacity. Consistent with this evidence that the mechanism involves interaction with IN at chromatin, the defect for HIV infection in S2FN cells was determined to be at the integration step by real-time quantitative polymerase chain reaction (PCR) for stage-specific viral and viral-host junction DNAs (Fig. 3, B to D). Full-length reverse transcription products were equivalent at multiple time points in control and S2FN cells (Fig. 3B). In contrast, 2–long terminal repeat (LTR) circles were increased in S2FN cells (Fig. 3C); this finding is consistent with a block to integration with consequent routing of nuclear cDNA to circularization. In direct support of this idea, Alu-PCR products were reduced by more than 10 times in S2FN T cells (Fig. 3D). Southern blotting of genomic DNA with reporter virus–specific probes for an internal viral fragment (fig. S11A) and viral-host DNA junctions (fig. S11B) corroborated the integration block.

Fig. 3.

Mechanism of p75 action. (A) p75P–A– did not rescue HIV-1 infection. Luciferase activity was measured 5 days after HIVluc infection. (B to D) Analyses of viral DNA forms in cells infected with HIVluc. (B) Late cDNA. (C) 2-LTR circles. (D) Alu-PCR results 14 days after infection. (E) Dominant-negative inhibition by the p75 IBD. Each line was derived from SupT1 cells with a single lentiviral vector that expressed either eGFP or eGFP that was fused in frame to the IBD, with or without an shRNA cassette (fig. S1A, panel ii). p75 IBD fragments were wild type or contained a single Asp→Asn mutation at p75 residue 366. The five single lentiviral vectors that were used to derive the lines contained the following expression elements: lane 1, control shRNA with GFP; lane 2, GFP-IBD (D366N); lane 3, GFP-IBD (WT); lane 4, p75 shRNA with GFP; lane 5, p75 shRNA with GFP-IBD (WT). Cells were infected with HIVluc and analyzed 5 days later for luciferase activity. U, units. Error bars in (D) and (E) represent SD of duplicate determinations.

Reciprocal passages of outgrowth viruses between parental and knockdown cell lines did not yield evidence for viral adaptation to the lack of p75 in S2FN cells, indicating that the eventual viral outgrowth resulted from slow accrual of wild-type virus encountering stringent restriction, rather than mutational adaptation. These results suggest that the p75-IN interaction may be a target for small-molecule or dominant-negative therapeutic strategies. To investigate the dominant-negative concept and to further confirm the role of p75, we expressed an eGFP-IBD fusion protein in which amino acids 340 to 417 of p75 were fused in frame to the C terminus of eGFP (fig. S1A, panel ii). eGFP-IBD, which interacts with IN in cells (7), also protected IN from proteasomal degradation and had a seven-fold inhibitory effect on single-round HIV-1 infection in p75–wild-type cells (Fig. 3E). Introduction of a single amino acid mutation, Asp366→Asn366 (D366N), which abrogates IN-IBD interaction (16), had no effect. Combining p75-specific ilvRNAi and fusion protein expression produced a 555-fold reduction in HIV-1 susceptibility (Fig. 3E).

Our studies show that p75 acts through a tethering mechanism as a potent cofactor for HIV-1 integration. More generally, we conclude that the cellular factors that engage the incoming HIV substructure can have virological efficacy in low concentrations and may be missed by RNAi screens of lower intensity. The apparent lack of redundancy, with p75 but not HRP-2 being required for integration, may indicate a potential therapeutic opportunity. Small molecules that could disrupt the interface, and perhaps dominant-negative approaches, are of interest for further study. Given the skewed HIV integration pattern we observed previously in adherent cells that knocked down for p75 yet were still S2 fraction–positive, in which the bias for integrating into active genes was reduced (9), it will also be interesting to determine the genomic pattern of the low-level integration that occurs in S2FN T cells.

Supporting Online Material

Materials and Methods

Figs. S1 to S11


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