Prototype Foamy Virus Integrase Research Articles

Structural basis of Mos1 transposase inhibition by the anti-retroviral drug Raltegravir.

DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg2+ or Mn2+, without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg2+ or Mn2+ ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

A Homology Model of HIV-1 Integrase and Analysis of Mutations Designed to Test the Model

Although there are structures of the different domains of human immunodeficiency virus type 1 (HIV-1) integrase (IN), there is no structure of the entire protein. The recently determined crystal structures of the prototype foamy virus (PFV) IN tetramer, in complexes with viral DNA, led to the generation of models of full-length HIV-1 IN. These models were generated, in part, by superimposing the structures of the domains of HIV-1 IN onto the structure of full-length PFV IN. We developed a model for HIV-1 IN—based solely on its sequence alignment with PFV IN—that differs in several ways from the previous models. Specifically, in our model, the junction between the catalytic core domain and C-terminal domain adopts a helix–loop–helix motif that is similar to the corresponding segment of PFV IN and differs from the crystal structures of these two HIV-1 IN domains. The alignment of residues in the C-terminal domain also differs from the previous models. Our model can be used to explain the phenotype of previously published HIV-1 IN mutants. We made additional mutants, and the behavior of these new mutants provides additional support for the model.

Substrate recognition and motion mode analyses of PFV integrase in complex with viral DNA via coarse-grained models.

HIV-1 integrase (IN) is an important target in the development of drugs against the AIDS virus. Drug design based on the structure of IN was markedly hampered due to the lack of three-dimensional structure information of HIV-1 IN-viral DNA complex. The prototype foamy virus (PFV) IN has a highly functional and structural homology with HIV-1 IN. Recently, the X-ray crystal complex structure of PFV IN with its cognate viral DNA has been obtained. In this study, both Gaussian network model (GNM) and anisotropy network model (ANM) have been applied to comparatively investigate the motion modes of PFV DNA-free and DNA-bound IN. The results show that the motion mode of PFV IN has only a slight change after binding with DNA. The motion of this enzyme is in favor of association with DNA, and the binding ability is determined by its intrinsic structural topology. Molecular docking experiments were performed to gain the binding modes of a series of diketo acid (DKA) inhibitors with PFV IN obtained from ANM, from which the dependability of PFV IN-DNA used in the drug screen for strand transfer (ST) inhibitors was confirmed. It is also found that the functional groups of keto-enol, bis-diketo, tetrazole and azido play a key role in aiding the recognition of viral DNA, and thus finally increase the inhibition capability for the corresponding DKA inhibitor. Our study provides some theoretical information and helps to design anti-AIDS drug based on the structure of IN.

Aromatic interaction profile to understand the molecular basis of raltegravir resistance

Integrase (IN) is the enzyme of human immunodeficiency virus (HIV) which inserts the viral DNA (vDNA) into the host genome for successful viral replication leading to the infection. However, the chemical basis of HIV IN catalysis is speculative due to lack of complete co-crystal structure. Using the recently published prototype foamy virus IN crystal structure, we developed a model structure of HIV IN showing interaction of vDNA, the metal (Mg2+) cofactor, and raltegravir (RLT) in the active site. Molecular docking and dynamics simulations studies showed that RLT uses it core central ring with diketo motif for Mg2+ chelation and bridge interaction with DDE motif. The triple arene interactions mediated by RLT with neighboring molecular motifs (Y143, cytosine, and adenine) is maintained during long simulation in wild type (WT). The fluorobenzyl and oxadiazole moieties of RLT forms aromatic stacking with cytosine base (head stacking) aromatic side chain of Y143 (tail stacking), respectively, while central ring further establishes aromatic stacking with distorted adenine base of vDNA (central stacking). The novel triple stacking systems were further explored to understand the molecular basis of drug resistance by molecular simulation. The in silico mutation (N155H, Q148H, and Q148H + G140S) and simulation studies elucidated the structural mechanism of resistance to RLT. The simulation studies provided the molecular basis for interdependency observed for the primary and secondary (Q148H and G140S) mutations and also explained the mechanism of viral fitness regain. Our study reveals that triple stacking and its consequence in terms of VdW energetic profile acts as a critical point to understand the drug-resistance. Here, we demonstrate that the root mean square deviation of centroid system (aromatic stacking) can be used as a major determinant of RLT binding toward the fold resistance. This is first kind of report, which discloses a strategy to explore the molecular level of drug resistance profile using aromatic interactions.

Solution Conformations of Prototype Foamy Virus Integrase and Its Stable Synaptic Complex with U5 Viral DNA

Using small-angle X-ray and neutron scattering (SAXS/SANS), in combination with analytical centrifugation and light scattering, we have determined the solution properties of PFV IN alone and its synaptic complex with processed U5 viral DNA and related these properties to models derived from available crystal structures. PFV IN is a monomer in solution, and SAXS analysis indicates an ensemble of conformations that differ from that observed in the crystallographic DNA-bound state. Scattering data indicate that the PFV intasome adopts a shape in solution that is consistent with the tetrameric assembly inferred from crystallographic symmetry, and these properties are largely preserved in the presence of divalent ions and clinical strand transfer inhibitors. Using contrast variation methods, we have reconstructed the solution structure of the PFV intasome complex and have located the distal domains of IN that were unresolved by crystallography. These results provide important insights into the architecture of the retroviral intasome.

Study on the interactions between diketo-acid inhibitors and prototype foamy virus integrase-DNA complex via molecular docking and comparative molecular dynamics simulation methods

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an important drug target for anti-acquired immune deficiency disease (AIDS) treatment and diketo-acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN. Due to lack of three-dimensional structures including detail interactions between HIV-1 IN and its substrate viral DNA, the drug design and screening platform remains incompleteness and deficient. In addition, the action mechanism of DKA inhibitors with HIV-1 IN is not well understood. In view of the high homology between the structure of prototype foamy virus (PFV) IN and that of HIV-1 IN, we used PFV IN as a surrogate model for HIV-1 IN to investigate the inhibitory mechanism of raltegravir (RLV) and the binding modes with a series of DKA inhibitors. Firstly, molecular dynamics simulations of PFV IN, IN-RLV, IN-DNA, and IN-DNA-RLV systems were performed for 10 ns each. The interactions and inhibitory mechanism of RLV to PFV IN were explored through overall dynamics behaviors, catalytic loop conformation distribution, and hydrogen bond network analysis. The results show that the coordinated interactions of RLV with IN and viral DNA slightly reduce the flexibility of catalytic loop region of IN, and remarkably restrict the mobility of the CA end of viral DNA, which may lead to the partial loss of the inhibitory activity of IN. Then, we docked a series of DKA inhibitors into PFV IN-DNA receptor and obtained the IN-DNA-inhibitor complexes. The docking results between PFV IN-DNA and DKA inhibitors agree well with the corresponding complex of HIV-1 IN, which proves the dependability of PFV IN-DNA used for the anti-AIDS drug screening. Our study may help to make clear some theoretical questions and to design anti-AIDS drug based on the structure of IN.

3′-Processing and strand transfer catalysed by retroviral integrasein crystallo

Retroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di- or trinucleotides from viral DNA ends to expose 3'-hydroxyls attached to the invariant CA dinucleotides (3'-processing reaction). Second, it inserts the processed 3'-viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3'-processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3'-processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3'-processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules.

Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

BackgroundWe applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.Methodology/ResultsPhotoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.ConclusionAnalysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

Molecular Dynamics Approaches Estimate the Binding Energy of HIV-1 Integrase Inhibitors and Correlate with In Vitro Activity

The design of novel integrase (IN) inhibitors has been aided by recent crystal structures revealing the binding mode of these compounds with a full-length prototype foamy virus (PFV) IN and synthetic viral DNA ends. Earlier docking studies relied on incomplete structures and did not include the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV-1 complex and developed a molecular dynamics (MD)-based approach that correlates with the in vitro activities of novel compounds. Four well-characterized compounds (raltegravir, elvitegravir, MK-0536, and dolutegravir) were used as a training set, and the data for their in vitro activity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild-type (WT) IN data. Three additional compounds were docked into the IN-DNA complex model and subjected to MD simulations. All three gave interaction potentials within 1 standard deviation of values estimated from the training set, and the most active compound was identified. Additional MD analysis of the raltegravir- and dolutegravir-bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV-1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug-resistant mutants, making it possible to streamline synthesis and testing.

Resistance to raltegravir highlights integrase mutations at codon 148 in conferring cross-resistance to a second-generation HIV-1 integrase inhibitor

Raltegravir is the first integrase strand-transfer inhibitor (INSTI) approved for use in highly active antiretroviral therapy (HAART) for the management of HIV infection. Resistance to antiretrovirals can compromise the efficacy of HAART regimens. Therefore it is important to understand the emergence of resistance to RAL and cross-resistance to other INSTIs including potential second-generation INSTIs such as MK-2048. We have now studied the question of whether in vitro resistance selection (IVRS) with RAL initiated with viruses derived from clinical isolates would result in selection of resistance mutations consistent with those arising during treatment regimens with HAART containing RAL. Some correlation was observed between the primary mutations selected in vitro and during therapy, initiated with viruses with identical IN sequences. Additionally, phenotypic cross-resistance conferred by specific mutations to RAL and MK-2048 was quantified. N155H, a RAL-associated primary resistance mutation, was selected after IVRS with MK-2048, suggesting similar mechanisms of resistance to RAL and MK-2048. This was confirmed by phenotypic analysis of 766 clonal viruses harboring IN sequences isolated at the point of virological failure from 106 patients on HAART (including RAL), where mutation Q148H/K/R together with additional secondary mutations conferred reduced susceptibility to both RAL and MK-2048. A homology model of full length HIV-1 integrase complexed with viral DNA and RAL or MK-2048, based on an X-ray structure of the prototype foamy virus integrase–DNA complex, was used to explain resistance to RAL and cross-resistance to MK-2048. These findings will be important for the further discovery and profiling of next-generation INSTIs.

Patterns of resistance development with integrase inhibitors in HIV

Raltegravir, the only integrase (IN) inhibitor approved for use in HIV therapy, has recently been licensed. Raltegravir inhibits HIV-1 replication by blocking the IN strand transfer reaction. More than 30 mutations have been associated with resistance to raltegravir and other IN strand transfer inhibitors (INSTIs). The majority of the mutations are located in the vicinity of the IN active site within the catalytic core domain which is also the binding pocket for INSTIs. High-level resistance to INSTIs primarily involves three independent mutations at residues Q148, N155, and Y143. The mutations significantly affect replication capacity of the virus and are often accompanied by other mutations that either improve replication fitness and/or increase resistance to the inhibitors. The pattern of development of INSTI resistance mutations has been extensively studied in vitro and in vivo. This has been augmented by cell-based phenotypic studies and investigation of the mechanisms of resistance using biochemical assays. The recent elucidation of the structure of the prototype foamy virus IN, which is closely related to HIV-1, in complex with INSTIs has greatly enhanced our understanding of the evolution and mechanisms of IN drug resistance.

Minimal size of prototype foamy virus integrase for nuclear localization

We have reported previously that the prototype foamy virus (PFV) integrase (IN) has a strong nuclear localization signal (NLS) in its C-terminal domain, in particular in a region of aa 306-334 including highly karyophilic arginines or lysines at positions 308, 313, 318, 324, and 329. In this study, we used various mutants of the C-terminal domain to further analyze its karyophilic determinants. Plasmids expressing these mutants fused to maltose binding protein (MBP) and enhanced green fluorescent protein (EGFP) were transfected to COS-1 cells and subcellular localization of these fluorescent fusion proteins was determined by fluorescent microscopy. The results revealed that a maximum karyophilicity was exhibited by a region longer than the previously described one of 29 aa (aa 306-334), in particular by a 64 aa region (aa 289-352) with Arg341 and Lys349 as critical determinants.

Non-Enzymatic Functions of Retroviral Integrase: The Next Target for Novel Anti-HIV Drug Development

Integrase (IN) is a retroviral enzyme that catalyzes the insertion of viral DNA (vDNA) into host chromosomal DNA, which is necessary for efficient viral replication. The crystal structure of prototype foamy virus IN bound to cognate vDNA ends, a complex referred to as the intasome, has recently been resolved. Structure analysis of the intasome revealed a tetramer structure of IN that was required for its catalytic function, and also showed the inhibitory mechanism of the IN inhibitor. Genetic analysis of IN has revealed additional non-enzymatic roles during viral replication cycles at several steps other than integration. However, the higher order structure of IN that is required for its non-enzymatic functions remains to be delineated. This is the next major challenge in the field of IN structural biology hoping to be a platform for the development of novel IN inhibitors to treat human immunodeficiency virus type 1 infectious disease.

Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with first- and second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTI-bound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/A mutations in drug-resistant viral strains.

Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3' processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the alpha4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl(2) concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg(2+). In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. (15)N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.

Characterization of Biochemical Properties of Feline Foamy Virus Integrase

In order to study biochemical properties, the integrase (IN) protein of feline foamy virus (FFV) was over-expressed from Escherichia coli, purified by two-step chromatography; Talon column and heparin column, and characterized in biochemical aspects. For the three enzymatic reactions of the 3' -processing, strand transfer, and disintegration activities, Mn2+ ion was essentially required as a cofactor. Interestingly, Co2+ and Zn2+ ions were found to act as effective cofactors, while other transition elements such as Ni2+, Cu2+, La3+, Y3+, Cd2+, Li1+, Ba2+, Sr2+, V3+, and so on were not. Regarding to the substrate specificity, FFV IN has low substrate specificities as it cleaved in a significant level prototype foamy virus (PFV) U5 LTR substrate as well as FFV U5 LTR substrate, while PFV IN did not. Finally, the 3' -processing activity was observed in the high concentrations of several solvents such as CHAPS, Glycerol, Tween 20 and Triton X-100, which are generally used for dissolution of chemicals in inhibitor-screening. Therefore, as it is the first report showing biochemical properties, FFV IN is proposed to have low specificities on the use of cofactor and substrate for enzymatic reaction when it is compared with other retroviral INs.

Functional and structural characterization of the integrase from the prototype foamy virus

Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.

Characterization of nuclear localization signals of the prototype foamy virus integrase

To analyse the potential karyophilic activity of prototype foamy viruses (PFVs), we expressed the PFV integrase (IN) and its mutants as fusion proteins with enhanced green fluorescence protein. The subcellular localization of the fusion proteins was investigated by fluorescence microscopy. The PFV IN was found to be karyophilic and targeted the fusion protein to the nucleus. Mutational analyses demonstrated that the PFV IN contains a potent but non-transferable nuclear localization signal (NLS) in its C-terminal domain and contains five arginine and lysine residues between amino acids 308 and 329 that are critical for its NLS function.