Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.     

Modulation of HIV-1 integrase activity by single-stranded oligonucleotides and their conjugates with eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Molecular dynamics studies of the wild-type and double mutant HIV-1 integrase complexed with the 5CITEP inhibitor: mechanism for inhibition and drug resistance

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138-149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations.     

Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design

A fragment-based screen against human immunodeficiency virus type 1 (HIV) integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF) binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.     

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.     

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.     

Insight into the inhibitory mechanism and binding mode between D77 and HIV-1 integrase by molecular modeling methods

Integrase is an essential enzyme in the life cycle of Human immunoficiency virus type 1 (HIV-1) and also an important target for designing integrase inhibitors. In this paper, the binding modes between the wild type integrase core domain (ICD) and the W131A mutant ICD with the benzoic acid derivative--D77 were investigated using the molecular docking combined with molecular dynamics (MD) simulations. The result of MD simulations showed that the W131A substitution affected the flexibility of the region 150-167 in both the monomer A and B of the mutant type ICD. In principle, D77 interacted with the residues around the Lens Epithelium-Derived Growth Factor (LEDGF/p75) binding site which is nearby the HIV-1 integrase dimer interface. However, the specific binding modes for D77-wild type integrase and D77-mutant integrase systems are various. According to the binding mode of D77 with the wild type ICD, D77 can effectively intervene with the binding of LEDGF/p75 to integrase due to a steric hindrance effect around the LEDGF/p75 binding site. In addition, we found that D77 might also affect its inhibitory action by reducing the flexibility of the region 150-167 of integrase. Through energy decomposition calculated with the Molecular Mechanics Generalized Born Surface Area approach to estimate the binding affinity, it seems likely that W131 and E170 are indispensable for the ligand binding, as characterized by the largest binding affinity. All the above results are consistent with the experimental data, providing us with some helpful information not only for the understanding of the mechanism of this kind of inhibitor but also for the rational drug design.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Structural analysis of a mutant of the HIV-1 integrase zinc finger domain that forms a single conformation

HIV-1 integrase consists of three functional domains, an N-terminal zinc finger domain, a catalytic core domain and a C-terminal DNA binding domain. NMR analysis of an isolated N-terminal domain (IN(1-55)) has shown that IN(1-55) exists in two conformational states [E and D forms; Cai et al. (1997) Nat. Struct. Biol. 4, 567-577]. The two forms differ in the coordination of the zinc ion by two histidine residues. In the present study, structural analysis of a mutant of IN(1-55), Y15A, by NMR spectroscopy indicated that the mutant protein folds correctly but takes only the E form. Since the Y15A mutation abrogates the HIV-1 infectivity, Y15 might have some important role in the full-length integrase activity during the virus infection cycle. Our results suggest a possible role of Y15 in structural transition between the E and D forms of HIV-1 integrase to allow the optimal tetramerization.     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

Crystal structures of catalytic core domain of BIV integrase: implications for the interaction between integrase and target DNA

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45? and 2.2?, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 ?, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.     

Interaction of HIV-1 integrase with DNA repair protein hRad18

We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65-226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process.     

Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance

Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of approximately 2:1 (molar ratio). In addition, we show that INI1/hSNF5 is not incorporated into related primate lentiviral and murine retroviral particles despite the abundance of the protein in producer cells. We have found that the specificity in incorporation of INI1/hSNF5 into HIV-1 virions is directly correlated with its ability to exclusively interact with HIV-1 integrase but not with other retroviral integrases. This specificity is also reflected in our finding that the transdominant mutant S6, harboring the minimal integrase interaction domain of INI1/hSNF5, blocks HIV-1 particle production but not that of the other retroviruses in 293T cells. Taken together, these results suggest that INI1/hNSF5 is a host factor restricted for HIV-1 and that S6 acts as a highly specific and potent inhibitor of HIV-1 replication.     

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.