Domain Of Integrase Research Articles

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

BackgroundAmino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.Methodology/Principal FindingsTo determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.Conclusions/SignificanceThis data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.

Role of the 207–218 peptide region of Moloney murine leukemia virus integrase in enzyme catalysis

X-ray diffraction data on a few retroviral integrases show a flexible loop near the active site. By sequence alignment, the peptide region 207–218 of Mo-MLV IN appears to correspond to this flexible loop. In this study, residues H208, Y211, R212, Q214, S215 and S216 of Mo-MLV IN were mutated to determine their role on enzyme activity. We found that Y211A, R212A, R212K and Q214A decreased integration activity, while disintegration and 3′-processing were not significantly affected. By contrast H208A was completely inactive in all the assays. The core domain of Mo-MLV integrase was modeled and the flexibility of the region 207–216 was analyzed. Substitutions with low integration activity showed a lower flexibility than wild type integrase. We propose that the peptide region 207–216 is a flexible loop and that H208, Y211, R212 and Q214 of this loop are involved in the correct assembly of the DNA-integrase complex during integration.

Structural basis for HIV-1 DNA integration in the human genome

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the human genome is catalyzed by the viral integrase protein that requires the lens epithelium-derived growth factor (LEDGF), a cellular transcriptional coactivator. In the presence of LEDGF, integrase forms a stable complex in vitro and importantly becomes soluble by contrast with integrase alone which aggregates and precipitates. Using cryo-electron microscopy (EM) and single-particle reconstruction, we obtained three-dimensional structures of the wild type full length integrase-LEDGF complex with and without DNA [1]. The stoichiometry of the complex was found to be (integrase)4-(LEDGF)2 by mass spectrometry analysis and existing atomic structures were unambiguous positioned in the EM map. In vitro functional assays reveal that LEDGF increases integrase activity likely in maintaining a stable and functional integrase structure. DNA-Protein cross-linking experiments show specific interaction between viral DNA and the C-terminal domain of integrase. Upon DNA binding, IN undergoes large conformational changes. Cryo-EM structure underlines the path of viral and target DNA and a model for DNA integration in human DNA is proposed (see fig. ​fig.1,1, overleaf). Figure 1 Proposed mechanism for thei ntegration of viral cDNA into the host genome: The LEDGF envelope is represented in blue; the integrase tetramer is shown as atomic structures. The viral DNA is in orange and the target DNA in red. On target DNA binding, there ...

Boty-II, a novel LTR retrotransposon in Botrytis cinerea B05.10 revealed by genomic sequence

Botrytis cinerea is a necrotrophic pathogen causing pre- and post-harvest diseases in at least 235 plant species. It manifests extraordinary genotype and phenotype variation. One of the causes of this variation is transposable elements. Two transposable elements have been discovered in this fungus, the retrotransposon ( Boty ), and the transposon ( Flipper ). In this work, two complete ( Boty-II-76 and Boty-II-103 ) and two partial ( Boty-II-95 and Boty-II-141 ) long terminal repeat (LTR) retrotransposons were identified by an in silico genomic sequence analysis. Boty-II-76 and Boty-II-103 contain 6439 bp nucleotides with a pair of LTRs at both ends, and an internal deduced pol gene encoding a polyprotein with reverse transcriptase and DDE integrase domains. They are flanked by 5 bp direct repeats (ACCAT, CTTTC). In Boty-II-141 , two LTRs at both ends, and a partial internal pol gene encoding a protein with a DDE integrase domain were identified. In Boty-II-95 , a right LTR and a partial internal pol gene encoding a protein with no conserved domains were identified. Boty-II uses a self-priming mechanism to initiate synthesis of reverse transcripts. The sequence of the presumed primer binding site for first-strand reverse transcription is 5'-TTGTACCAT-3'. The polypurine-rich sequence for plus-strand DNA synthesis is 5'-GCCTTGAGCGGGGGGTAC-3'. Fourteen Boty-II LTRs that contain 125-158 bp nucleotides and share 69.1 ~ 100% identities with the short inverted terminal repeats of 5 bp (TGTCA...TGACA) were discovered. Analysis of structural features and phylogeny revealed that Boty-II is a novel LTR retrotransposon. It could potentially be used as a novel molecular marker for the investigation of genetic variation in B. cinerea.

Solution NMR structure of an integrase domain from protein SPA4288 from Salmonella enterica, Northeast Structural Genomics Consortium Target SlR105H

Solution NMR structure of an integrase domain from protein SPA4288 from Salmonella enterica, Northeast Structural Genomics Consortium Target SlR105H

CGIN1: A Retroviral Contribution to Mammalian Genomes

This study describes the origin and structural features of a mammalian gene, CGIN1 (Cousin of GIN1). CGIN1 proteins contain an NYN domain, retroviral RNase H and integrase domains, and a domain of unknown function (CGIN1 domain) that is also present in two other genes (N4BP1 and KIAA0323). We suggest that CGIN1 derives from the fusion of a KIAA0323-like gene with retroviral sequences, which occurred prior to the marsupial-eutherian split. Sequence and structural analyses indicate that the CGIN1 integrase domain is inactive but still retains the 3D folding observed in retroviral integrases. We hypothesize that CGIN1 may contribute to retroviral resistance in mammals by regulating the ubiquitination of viral proteins.

DNA binding and synapsis by the large C-terminal domain of C31 integrase

The integrase (Int) from phage ϕC31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL × attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein–protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD–CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL × attR. hCTDE449K was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.

Mutational Analysis and Homology-Based Modeling of the IntDOT Core-Binding Domain

Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

BackgroundHIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype.ResultsOur study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant.ConclusionOverall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of the IN-induced lethal phenotype in yeast.

The Solution Structure of the Simian Foamy Virus Protease Reveals a Monomeric Protein

In contrast to orthoretroviruses, foamy viruses (FVs) express their Pol polyprotein from a separate pol-specific transcript. Only the integrase domain is cleaved off, leading to a protease-reverse transcriptase (PR-RT) protein. We purified the separate PR domain (PRshort) of simian FV from macaques by expressing the recombinant gene in Escherichia coli. Sedimentation analyses and size exclusion chromatography indicate that PRshort is a stable monomer in solution. This allowed us to determine the structure of the PRshort monomer using 1426 experimental restraints derived from NMR spectroscopy. The superposition of 20 conformers resulted in a backbone atom rmsd of 0.55 Å for residues Gln8–Leu93. Although the overall folds are similar, the macaque simian FV PRshort reveals significant differences in the dimerization interface relative to other retroviral PRs, such as HIV-1 (human immunodeficiency virus type 1) PR, which appear to be rather stable dimers. Especially the flap region and the N- and C-termini of PRshort are highly flexible. Neglecting these regions, the backbone atom rmsd drops to 0.32 Å, highlighting the good definition of the central part of the protein. To exclude that the monomeric state of PRshort is due to cleaving off the RT, we purified the complete PR-RT and performed size exclusion chromatography. Our data show that PR-RT is also monomeric. We thus conclude adoption of a monomeric state of PR-RT to be a regulatory mechanism to inhibit PR activity before virus assembly in order to reduce packaging problems. Dimerization might therefore be triggered by additional viral or cellular factors.

A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

Bacteriophage ϕC31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, ϕC31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of ϕC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein–protein interactions.

Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase.

Bacteriophage lambda integrase catalyzes site-specific DNA recombination. A helical bundle domain in the enzyme, called the core-binding domain (Int(CB)), promotes the catalysis of an intermediate DNA-cleavage reaction that is critical for recombination and is not well folded in solution in the absence of DNA. To gain structural insights into the mechanism behind the accessory role of this domain in catalysis, an attempt was made to crystallize an Int(CB)-DNA complex, but crystals of free Int(CB) were fortuitously obtained. The three-dimensional structure of DNA-free Int(CB) was solved at 2.0 A resolution by molecular replacement using as the search model the previously available DNA-bound 2.8 A structure of the Int(CB) domain in a larger construct of lambda integrase. The crystal structure of DNA-free Int(CB) resembles the DNA-bound structure of Int(CB), but exhibits subtle differences in the DNA-binding face and lacks electron density for ten residues in the C-terminus that form a portion of a linker connecting Int(CB) to the C-terminal catalytic domain of the enzyme. Thus, this work reveals the domain in the absence of DNA and allows comparison with the DNA-bound form of this catalytically activating domain.

Persistent transcription of a nonintegrating mutant of simian immunodeficiency virus in rhesus macrophages

A nonintegrating mutant, SIVsmD116N, was derived from the infectious pathogenic SIVsmE543-3 clone by introducing an Asp (D) to Asn (N) mutation into the catalytic domain of integrase. Although SIVsmD116N generated all viral proteins following transfection, cell-free virus did not productively infect CEMx174 cells, macaque peripheral blood mononuclear cells (PBMCs) or monocyte-derived macrophages (MDM). Viral DNA and transcripts were observed transiently in SIVsmD116N-infected CEMx174 cells and macaque PBMC but persisted in MDM for as long as 20 days. Circular forms of viral DNA were detected but there was no evidence of integration detected by Alu PCR. We found that SIV D116N mutant remained transcriptionally active and expressed low levels of viral proteins persistently in MDM. These data are consistent with a role for macrophages as a persistent latent reservoir for AIDS viruses. The capacity of nonintegrating SIV to persistently generate viral products in macrophages suggests that nonintegrating lentiviral vectors could be engineered to efficiently and safely express proteins for vaccine purposes.

The complete nucleotide sequence of a New World simian foamy virus

We determined the complete nucleotide sequence of the New World simian foamy virus (FV) from spider monkey (SFVspm). Starting from a conserved region in the integrase (IN) domain of the pol gene we cloned fragments of the genome up to the 5′ end of the long terminal repeat (LTR) into plasmid vectors and elucidated their nucleotide sequence. The 3′ end of the genome was determined by direct nucleotide sequencing of PCR products. Each nucleotide of the genome was determined at least two times from both strands. All protein motifs described to be conserved among primate FVs were found in SFVspm. At both the nucleotide and protein levels SFVspm is the most divergent primate FV described to date, reflecting the long-term phylogenetic separation between Old World and New World primate host species ( Catarrhini and Platyrrhini, respectively). The molecular probes developed for SFVspm will allow the investigation of trans-species transmissions of this New World foamy virus to humans by serological assays.

Phosphorylation Regulates Integration of the Yeast Ty5 Retrotransposon into Heterochromatin

The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5's mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock's hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge.

A novel internally controlled real-time reverse transcription-PCR assay for HIV-1 RNA targeting the pol integrase genomic region

Given the worldwide increasing spread of HIV-1 genetic variants, it is mandatory that assays used for nucleic acid testing for HIV-1 detect all existing groups and subtypes of HIV-1. In this report the development and evaluation of a quantitative real-time HIV-1 RT-PCR assay that targets a conserved region within the pol integrase domain is described. As an internal control reaction, endogenous glyceraldehyde-3-phosphate-dehydrogenase transcripts were detected in a multiplex configuration. The detection limit (95% cut-off value) was determined by probit analysis and calculated as 281 IU/ml of HIV-1 RNA. Within-run and between-run coefficients of variation were below 15 and 27%, respectively, indicating high reproducibility. The described assay detected all tested HIV-1 isolates representing groups M, O and N. Within group M, quantitative test results correlated well with viral loads as determined by the automated Abbott RealTi me™ HIV-1 assay. Based on the testing of 1206 confirmed HIV-1 RNA negative blood donor samples, assay specificity was found to be 100%. The rate of inhibition was 0.37%. The described HIV-1 real-time RT-PCR was validated according to regulatory guidelines and is applicable to the screening of blood donors as well as the determination of HIV-1 viral load.

Characterization of AFLAV, a Tf1/Sushi retrotransposon from Aspergillus flavus

The plasmid, pAF28, a genomic clone from Aspergillus flavus NRRL 6541, has been used as a hybridization probe to fingerprint A. flavus strains isolated in corn and peanut fields. The insert of pAF28 contains a 4.5 kb region which encodes a truncated retrotransposon (AfRTL-1). In search for a full-length and intact copy of retrotransposon, we exploited a novel PCR cloning strategy by amplifying a 3.4 kb region from the genomic DNA of A. flavus NRRL 6541. The fragment was cloned into pCR 4-TOPO. Sequence analysis confirmed that this region encoded putative domains of partial reverse transcriptase, RNase H, and integrase of the predicted retrotransposon. The two flanking long terminal repeats (LTRs) and the sequence between them comprise a putative full-length LTR retrotransposon of 7799 bp in length. This intact retrotransposon sequence is named AFLAV (A. flavus Retrotransposon). The order of the predicted catalytic domains in the polyprotein (Pol) placed AFLAV in the Tf1/sushi subgroup of the Ty3/gypsy retrotransposon family. Primers derived from AFLAV sequence were used to screen this retrotransposon in other strains of A. flavus. More than fifty strains of A. flavus isolated from different geological origins were surveyed and the results show that many strains have extensive deletions in the regions encoding the capsid (Gag) and Pol.

Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences: HML expression in human tissues

The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1–10, also referred to as human endogenous retrovirus “HERV-K”. Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan ®-based assays for HML groups 1–7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML1–7 were used as standards. The RT and IN based QPCRs could detect 10 0–10 3 copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.

Integrase diversity and transcription of the maize retrotransposonGrande

Grande is an abundant gypsy-like retrotransposon present in the genera Zea and Tripsacum. Related retro transposon families can be found in sorghum, rice, and barley, but not in wheat or rye. We have amplified and sequenced several copies of part of the integrase domain derived from the Zea mays, Zea diploperennis, and Tripsacum dactyloides genomes. There are no significant differences in divergence or clustering between the integrase sequences of these species. The substitution rate for synonimous sites was found to be higher than those of non-synomymous sites; this indicates that Grande integrase has been under purifying selection for function. Grande is transcribed in leaves. The transcripts show sequence diversity similar to that of genomic sequences, but belong to restricted clades; this indicates that only some evolutionary branches of Grande have retained transcriptional competence.

Retroviral DNA integration: reaction pathway and critical intermediates

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.