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.     

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.     

Identification of HIV-1 integrase inhibitors via three-dimensional database searching using ASV and HIV-1 integrases as targets

Integration of viral DNA into the host cell genome is a critical step in the life cycle of HIV. This essential reaction is catalyzed by integrase (IN) through two steps, 3'-processing and DNA strand transfer. Integrase is an attractive target for drug design because there is no known cellular analogue and integration is essential for successful replication of HIV. A computational three-dimensional (3-D) database search was used to identify novel HIV-1 integrase inhibitors. Starting from the previously identified Y3 (4-acetylamino-5-hydroxynaphthalene-2,7-disulfonic acid) binding site on the avian sarcoma virus integrase (ASV IN), a preliminary search of all compounds in the nonproprietary, open part of the National Cancer Institute 3-D database yielded a collection of 3100 compounds. A more rigorous scoring method was used to rescreen the 3100 compounds against both ASV IN and HIV-1 IN. Twenty-two of those compounds were selected for inhibition assays against HIV-1 IN. Thirteen of the 22 showed inhibitory activity against HIV-1 IN at concentrations less than 200 microM and three of them showed antiviral activities in HIV-1 infected CEM cells with effective concentrations (EC50) ranging from 0.8 to 200 microM. Analysis of the computer-generated binding modes of the active compounds to HIV-1 IN showed that simultaneous interaction with the Y3 site and the catalytic site is possible. In addition, interactions between the active compounds and the flexible loop involved in the binding of DNA by IN are indicated to occur. The structural details and the unique binding motif between the HIV-1 IN and its inhibitors identified in the present work may contribute to the future development of IN inhibitors.     

Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.

In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.     

Constructing HIV-1 integrase tetramer and exploring influences of metal ions on forming integrase-DNA complex

HIV-1 integrase (IN) is essential for the replication of HIV-1 in human cells. At present, the complete structure of complex IN-DNA has not been resolved. In this paper, a HIV-1 IN tetramer model was built with homology modeling and molecular dynamics simulation approach, in which two Mg2+ ions were reasonably located in each catalytic core domain. Moreover, it was found that the AB and CD chains of HIV-1 IN tetramer were different in the structures and metal ions of HIV-1 IN tetramer might have great influences on DNA locating on IN. These findings may provide a more complete structural basis for guiding drug discovery and revealing integration mechanism.     

Functional nucleotides of U5 LTR determining substrate specificity of prototype foamy virus integrase

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.     

Binding mode prediction of biologically active compounds from plant Salvia Miltiorrhiza as integrase inhibitor

Integrase (IN), an essential enzyme for HIV-1 replication, has been targeted in antiretroviral drug therapy. The emergence of HIV-1 variants clinically resistant to antiretroviral agents has lead to the development of alternative IN inhibitors. In the present work, binding modes of a high potent IN inhibitor, M₅22 and M₅32, within the catalytic binding site of wild type (WT) IN were determined using molecular docking calculation. Both M₅22 and M₅32 displayed similar modes of binding within the IN putative binding pocket and exhibited favorable interactions with the catalytic Mg²⁺ ions, the nearby amino acids and viral DNA through metal-ligand chelation, hydrogen bonding and π-π stacking interactions. Furthermore, the modes of action of these two compounds against the mutated Y212R, N224H and S217H PFV IN were also predicted. Although the replacement of amino acid could somehow disturb inhibitor binding mode, almost key interactions which detected in the WT complexes were fairly conserved. Detailed information could highlight the application of M₅22 and M₅32 as candidate IN inhibitors for drug development against drug resistant strains.     

Design and synthesis of novel pyrimidone analogues as HIV-1 integrase inhibitors

A series of novel pyrimidone analogues have been designed and synthesized as HIV-1 integrase (IN) inhibitors. This study demonstrated that introducing a substituent in the N1-position of the pyrimidone scaffold does not significantly influence IN inhibitory activity. Molecular docking studies showed these compounds could occupy the IN active site and form pi–pi interactions with viral DNA nucleotides DC16 and DA17 to displace reactive viral DNA 3′OH and block intasome activity.     

Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.     

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.     

Understanding the effect of drug-resistant mutations of HIV-1 intasome on raltegravir action through molecular modeling study

Raltegravir is the first FDA-approved drug targeting the strand transfer step of HIV-1 integration. However, the rapid emergence of viral strains that are highly resistant to raltegravir has become a critical problem. Unfortunately, the detailed molecular mechanism of how HIV-1 integrase (IN) mutations actually confer drug resistance is not well understood. In the present study, starting from our previously constructed complex of HIV-1 IN and viral DNA, we employed molecular dynamics (MD) simulation and molecular mechanics generalized Born surface area (MM-GBSA) calculation, to uncover the molecular mechanism behind the resistant mechanism of HIV-1 IN to raltegravir. The values of the calculated binding free energy follow consistently the experimentally observed ranking of resistance levels. A detailed analysis of the results of MD simulation suggests that the Tyr143 located in the 140s loop (e.g., residues from Gly140 to Gly149) is a key anchoring residue that leads to stable raltegravir binding. The decrease in the interaction at this residue is one of the key reasons responsible for the resistance of HIV-1 IN to raltegravir. Additionally, the calculation results also proved that the 3' adenosine flip in different conformations in the wild-type and mutant HIV-1 IN-viral DNA complexes play an important role in raltegravir binding. Our results could provide a structural and energetic understanding of the raltegravir-resistant mechanism at the atomic level and provide some new clues on how to design new drugs that may circumvent the known resistance mutations.     

A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid

Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg²⁺ cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg²⁺ cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.     

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.     

Peptides that bind the HIV-1 integrase and modulate its enzymatic activity--kinetic studies and mode of action

Several peptides that specifically bind the HIV-1 integrase (IN) and either inhibit or stimulate its enzymatic activity were developed in our laboratories. Kinetic studies using 3'-end processing and strand-transfer assays were performed to study the mode of action of these peptides. The effects of the various peptides on the interaction between IN and its substrate DNA were also studied by fluorescence anisotropy. On the basis of our results, we divided these IN-interacting peptides into three groups: (a) IN-inhibitory peptides, whose binding to IN decrease its affinity for the substrate DNA - these peptides increased the K(m) of the IN-DNA interaction, and were thus inhibitory; (b) peptides that slightly increased the K(m) of the IN-DNA interaction, but in addition modified the V(max) and K(cat) values of the IN, and thus stimulated or inhibited IN activity, respectively; and (c) peptides that bound IN but had no effect on its enzymatic activity. We elucidated the approximate binding sites of the peptides in the structure of IN, providing structural insights into their mechanism of action. The IN-stimulating peptide bound IN in several specific sites that did not bind any of the inhibitory peptides. This may account for its unique activity.     

HIV-1 Integrase and Virus and Cell DNAs: Complex Formation and Perturbation by Inhibitors of Integration

HIV-1 integrase (IN) catalyzes integration of viral DNA into cell DNA through 3′-processing of viral DNA and strand transfer reactions. To learn on binding of IN to DNAs and IN inhibition we applied spectroscopy (circular dichroism, fluorescence) in a simplified model consisting in a peptide analogue (K156) of α4 helix involved in recognition of viral and cell DNA; an oligonucleotide corresponding to the U5′ LTR DNA end; and an inhibitor (TB11) of the diketo acid (DKA) family. Results extrapolated to IN show that: the enzyme binds viral DNA with high affinity and specificity, but cell DNA with low affinity and specificity; the affinity of TB11 for IN is high enough to impair the binding of IN to cell DNA, but not to viral DNA. This explains why TB11 is an inhibitor of strand transfer but not of 3′-processing. These results can help in the search of new IN inhibitors.     

Subunit-specific protein footprinting reveals significant structural rearrangements and a role for N-terminal Lys-14 of HIV-1 Integrase during viral DNA binding

To identify functional contacts between HIV-1 integrase (IN) and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys- and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The foot-printing studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes.     

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages

Background

HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain.

Methods

Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD).

Results

Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC₅₀s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N -Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those displayed by fungal molecules from Fusarium sp., shown in the 1990s to inhibit HIV-1 integration.

Conclusion

The 3D model presented here supports the idea that INSTIs dock at the putative acceptor DNA-binding site in a IN/viral DNA complex. This mechanism of enzyme inhibition, likely to be exploited by some natural products, might disclose future strategies for inhibition of nucleic acid-manipulating enzymes.