The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1-4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5-7). For example, the γ-retroviruses favor integration near transcription start sites, whereas lentiviruses favor integration within transcription units. These observations have suggested that different cellularbinding partners of retroviral integrases are likely to be responsible for integration target-site selection. However, to date, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes (8-14). Cellular cofactors of other retroviral genera are currently unknown.The molecular mechanisms of γ-retroviral murine leukemia virus (MLV) integration are of particular significance because MLV-based vectors are used for human gene therapy. In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. 15-18). The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.In this report, we have identified the bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as the cellularbinding partners of MLV IN and demonstrate their significance for stimulating and targeting MLV integration at transcription start sites. (Table 1, Table S1, and Fig. S1). Of these, Brd4 and Brd3 were the top hits in NIH 3T3 and Sup-T1 cells, respectively. Differential pull-down levels of these proteins (Table 1) could be attributable to the varying expression le...

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra-and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.Integration of the reverse-transcribed RNA genome into a host chromosome is an obligatory step for HIV-1 3 replication (reviewed in Ref. 1). This process is catalyzed by the retroviral enzyme integrase (IN) in two reaction steps. In the first step, which is called 3Ј-processing and takes place shortly after the cDNA is made, IN hydrolyzes a GT dinucleotide from each end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. The sites of attack on the two target DNA strands are separated by 5 bp, which leads to dissociation of the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. HIV-1 IN consists of three distinct structural and functional domains. The N-terminal domain (NTD) (residues 1-50) contains conserved pairs of histidine and cysteine residues that bind zinc (2, 3), which contributes to IN multimerization and its catalytic function (4, 5). The catalytic core domain (CCD) (residues 51-212) contains three acidic residues, Asp-64, Asp-116, and Glu-152, which play a key role in coordinating active site divalent metal ions (6, 7). The C-terminal domain (CTD) (residues 213-288) also contributes to functional IN multimerization (8, 9). Results of structural biology studies revealed each individual domain as a dimer (3,6,7,10,11) and more recent two-domain crystal structures comprised of the CCD and CTD (12) or NTD and CCD (13)...

Background:The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs). Results: The A128T substitution does not significantly alter ALLINI IC 50 values for IN-LEDGF/p75 binding but confers marked resistance to ALLINI-induced aberrant integrase multimerization. Conclusion: Allosteric perturbation of HIV-1 integrase multimerization underlies ALLINI antiviral activity. Significance: Our findings underscore the mechanism of ALLINI action and will facilitate development of second-generation compounds.

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helixturn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPAdeficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.

HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the smallmolecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.

Background: TNPO3 is a key cellular factor involved in early steps of HIV-1 replication. Results: TNPO3 is highly structured, interacts with the HIV-1 intasome by engaging the C-terminal domain of integrase, and does not directly bind capsid tubes. Conclusion: TNPO3 interacts with HIV-1 intasomes and not capsid cores. Significance: Our findings aid future genetic analysis to elucidate the role of TNPO3 in HIV-1 replication.

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