Full-length Integrase Research Articles

Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance.

HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1-280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination (R(2)) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 10(4) cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Structural dynamics of full-length retroviral integrase: a molecular dynamics analysis

HIV integrase catalyzes the integration between host and viral DNA and is considered as an interesting target for treating HIV. Knowledge of the complete structure of integrase is inevitable to describe the communicative inter-domain interactions affecting the HIV integration and disintegration process and hence the study on full-length integrase turns out to be an essential task. In this investigation, a structure of full-length integrase is designed to analyze the global dynamics of integrase dimer and monomers (with and without the C-terminal, 270–288 amino acids) for a period of 20 ns. The molecular dynamics analysis and the subsequent DynDom analysis reveal (i) a stable dynamics of dimeric CCD and NTD domains and (ii) CCD-α11-mediated rotational-cum-translational CTD motion as the functional dynamics of IN dimer. This observation supports that (i) aggregation enhances the integrase activity and (ii) flexible CTD for its cis and trans coordination with CCD. The role of C-loop over the dynamics of integrase is also explored, which unveils that the spatial arrangement of integrase domains is changed during dynamics in the absence of C-loop. In essence, here we report a C-loop-dependent structural dynamics of integrase and the active dynamics of integrase in dimer. Further studies on C-loop sensing mechanism and the multimerization of integrase would provide insight into HIV integration and disintegration processes. Supplementary material. Movies generated from molecular dynamics trajectory showing the CTD dynamics of IN structures (monomers with & without C-loop and dimer) are linked online to this article. The remaining supplementary data can be downloaded from the author’s server at the URL http://ramutha.bicpu.edu.in.

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

HIV-1 integrase strand-transfer inhibitors: Design, synthesis and molecular modeling investigation

This study is focused on a new series of benzylindole derivatives with various substituents at the benzene-fused ring, suggested by our 3D pharmacophore model developed for HIV-1 integrase inhibitors (INIs). All synthesized compounds proved to be active in the nanomolar range (6–35 nM) on the strand-transfer step (ST). In particular, derivative 4-[1-(4-fluorobenzyl)-5,7-dimethoxy-1 H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid ( 8e), presenting the highest best-fit value on pharmacophore model, showed a potency comparable to that of clinical INSTIs GS 9137 ( 1) and MK-0518 ( 2). The binding mode of our molecules has been investigated using the recently published crystal structure of the complex of full-length integrase from the prototype foamy virus in complex with its cognate DNA (PFV-IN/DNA). The results highlighted the ability of derivative 8e to assume the same binding mode of MK-0518 and GS 9137.

Characterization of HIV Type 1 Genetic Diversity Among South African Participants Enrolled in the AIDS Vaccine Integrated Project (AVIP) Study

The genetic diversity of HIV-1 strains circulating among HIV-1-infected South Africans was investigated in a cohort of 420 individuals enrolled as part of the AIDS Vaccine Integrated Project (AVIP) study. Representative samples (10%) were randomly selected from treatment-naive participants. Viral RNA was extracted for reverse transcriptase-initiated amplification and population-based sequencing of partial pol (encompassing protease and reverse transcriptase) and full-length integrase. Overall, HIV-1 sequences confirmed that 97.1% and 96.9% were HIV-1 subtype C in pol and integrase, respectively. Two participants were infected with unique A1/C and C/A1 recombinants in pol/integrase. Further pol sequence analysis identified mutation patterns associated with high level resistance to NNRTIs in two participants, whereas no primary mutations conferring resistance to integrase inhibitors were detected. The predominance of HIV-1 subtype C in South African populations is therefore confirmed in the AVIP cohort finalized for testing preventive or therapeutic vaccines against HIV-1 infection.

Insights into the Functions of a Molecular Swiss Army Knife: The Intasome Structure Reveals Inhibitor Action

Evaluation of: Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P: Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464(7286), 232–236 (2010). During HIV replication, integrase is responsible for the integration of the cDNA copy of the viral RNA genome into the host chromatin. In the recent history of anti-HIV therapy integrase has become an established drug target for HAART, but development of second-generation integrase inhibitors has been hampered by the lack of structural information. Hare et al. have now described the full-length integrase structure of a distant cousin of HIV-1, the prototype foamy virus. This insight into the mechanism of catalysis will boost the rational design of second-generation strand-transfer inhibitors.

Retroviral intasome assembly and inhibition of DNA strand transfer

Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell chromosome. The structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking. Furthermore, although clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains speculative. Herein we report a crystal structure of full-length integrase from the prototype foamy virus in complex with its cognate DNA. The structure reveals the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive protein-DNA and protein-protein interactions. Binding of strand transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex. Our findings define the structural basis of retroviral DNA integration and will allow modeling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

Integrase and integration: biochemical activities of HIV-1 integrase

Integration of retroviral DNA is an obligatory step of retrovirus replication because proviral DNA is the template for productive infection. Integrase, a retroviral enzyme, catalyses integration. The process of integration can be divided into two sequential reactions. The first one, named 3'-processing, corresponds to a specific endonucleolytic reaction which prepares the viral DNA extremities to be competent for the subsequent covalent insertion, named strand transfer, into the host cell genome by a trans-esterification reaction. Recently, a novel specific activity of the full length integrase was reported, in vitro, by our group for two retroviral integrases (HIV-1 and PFV-1). This activity of internal cleavage occurs at a specific palindromic sequence mimicking the LTR-LTR junction described into the 2-LTR circles which are peculiar viral DNA forms found during viral infection. Moreover, recent studies demonstrated the existence of a weak palindromic consensus found at the integration sites. Taken together, these data underline the propensity of retroviral integrases for binding symmetrical sequences and give perspectives for targeting specific sequences used for gene therapy.

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.

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Molecular dynamics studies of the full-length integrase–DNA complex

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140–149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic activity of HIV-1 integrase.

Mutations in the amino‐terminal domain of λ‐integrase have differential effects on integrative and excisive recombination

Lambda integrase (Int) forms higher-order protein-DNA complexes necessary for site-specific recombination. The carboxy-terminal domain of Int (75-356) is responsible for catalysis at specific core-type binding sites whereas the amino-terminal domain (1-70) is responsible for cooperative arm-type DNA binding. Alanine scanning mutagenesis of residues 64-70, within full-length integrase, has revealed differential effects on cooperative arm binding interactions that are required for integrative and excisive recombination. Interestingly, while these residues are required for cooperative arm-type binding on both P'1,2 and P'2,3 substrates, cooperative binding at the arm-type sites P'2,3 was more severely compromised than binding at arm-type sites P'1,2 for L64A. Concomitantly, L64A had a much stronger effect on integrative than on excisive recombination. The arm-binding properties of Int appear to be intrinsic to the amino-terminal domain because the phenotype of L64A was the same in an amino-terminal fragment (Int 1-75) as it was in the full-length protein.

Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design

We report structural models of the full-length integrase enzyme (IN) of the human immunodeficiency virus type 1 (HIV-1) and its complex with viral and human DNA. These were developed by means of molecular modeling techniques using all available experimental evidence, including X-ray crystallographic and NMR structures of portions of the full-length protein. Special emphasis was placed on obtaining a model of the enzyme's active site with the viral DNA apposed to it, based on the hypothesis that such a model would allow structure-based design of inhibitors that retain activity in vivo. This was because bound DNA might be present in vivo after 3'-processing but before strand transfer. These structural models were used to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors (many of them preferentially inhibiting strand transfer) for which no experimentally derived complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevent the exposure of the 3'-processed end of the viral DNA to human DNA.

Analysis of the full-length integrase–DNA complex by a modified approach for DNA docking

A model of the full-length HIV-1 integrase dimer was constructed assembling the experimentally determined structures of the single domains. Subsequently, the three-domain protein–viral DNA complex was generated for the first time through an automated docking algorithm, obtained modifying the ESCHER program, a well-known method for protein–protein docking. A detailed study of the contacts established with DNA by the enzyme revealed that the predicted model reproduced the results of mutagenesis and cross-linking experiments, confirming the validity of our docking approach in predicting the base specificity in the DNA–protein interaction.

Modeling HIV-1 integrase complexes based on their hydrodynamic properties.

We present a model structure of a candidate tetramer for HIV-1 integrase. The model was built in three steps using data from fluorescence anisotropy, structures of the individual integrase domains, cross-linking data, and other biochemical data. First, the structure of the full-length integrase monomer was modeled using the individual domain structures and the hydrodynamic properties of the full-length protein that were recently measured by fluorescence depolarization. We calculated the rotational correlation times for different arrangements of three integrase domains, revealing that only structures with close proximity among the domains satisfied the experimental data. The orientations of the domains were constrained by iterative tests against the data on cross-linking and footprinting in integrase-DNA complexes. Second, the structure of an integrase dimer was obtained by joining the model monomers in accordance with the available dimeric crystal structures of the catalytic core. The hydrodynamic properties of the dimer were in agreement with the experimental values. Third, the active sites of the two model dimers were placed in agreement with the spacing between the sites of integration on target DNA as well as the integrase-DNA cross-linking data, resulting in twofold symmetry of a tetrameric complex. The model is consistent with the experimental data indicating that the F185K substitution, which is found in the model at a tetramerization interface, selectively disrupts correct complex formation in vitro and HIV replication in vivo. Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors.

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.