Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN ...interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.
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.
Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and ...mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH₂ groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.
HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that ...involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69⁻75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.
Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking ...myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the “dimer interface” is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.
Human T-cell leukemia virus type-1 (HTLV-1) is estimated to infect 15-25 million people worldwide, with several areas including southern Japan and the Caribbean basin being endemic. The virus is the ...etiological agent of debilitating and fatal diseases, for which there is currently no long-term cure. In the majority of cases of leukemia caused by HTLV-1, only a single viral gene, hbz, and its cognate protein, HBZ, are expressed and their importance is increasingly being recognized in the development of HTLV-1-associated disease. We hypothesized that HBZ, like other HTLV-1 proteins, has properties and functions regulated by post-translational modifications (PTMs) that affect specific signaling pathways important for disease development. To date, PTM of HBZ has not been described. We used an affinity-tagged protein and mass spectrometry method to identify seven modifications of HBZ for the first time. We examined how these PTMs affected the ability of HBZ to modulate several pathways, as measured using luciferase reporter assays. Herein, we report that none of the identified PTMs affected HBZ stability or its regulation of tested pathways.
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 helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient 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.
XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting ...and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98-219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA-DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA-DNA junction interactions.
Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1, like all retroviruses, stably integrates its vDNA copy into host chromatin, a ...process allowing for permanent infection. This essential step for HIV-1 replication is catalyzed by viral integrase (IN) and aided by cellular protein LEDGF/p75. In addition, IN is also crucial for proper virion maturation as it interacts with the viral RNA genome to ensure encapsulation of ribonucleoprotein complexes within the protective capsid core. These key functions make IN an attractive target for the development of inhibitors with various mechanisms of action. We conducted a high-throughput screen (HTS) of ∼370,000 compounds using a homogeneous time-resolved fluorescence-based assay capable of capturing diverse inhibitors targeting multifunctional IN. Our approach revealed chemical scaffolds containing diketo acid moieties similar to IN strand transfer inhibitors (INSTIs) as well as novel compounds distinct from all current IN inhibitors including INSTIs and allosteric integrase inhibitors (ALLINIs). Specifically, our HTS resulted in the discovery of compound 12, with a novel IN inhibitor scaffold amenable for chemical modification. Its more potent derivative 14e similarly inhibited catalytic activities of WT and mutant INs containing archetypical INSTI- and ALLINI-derived resistant substitutions. Further SAR-based optimization resulted in compound 22 with an antiviral EC50 of ∼58 μM and a selectivity index of >8500. Thus, our studies identified a novel small-molecule scaffold for inhibiting HIV-1 IN, which provides a promising platform for future development of potent antiviral agents to complement current HIV-1 therapies.
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-yloxy-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 small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.
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