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PubMed2005

Iterative in vivo assembly of large and complex transgenes by combining the activities of phiC31 integrase and Cre recombinase.

We have used the phiC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than approximately 150 kb we have established a method that we call; 'Iterative Site Specific Integration' (ISSI). ISSI combines the activities of phiC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.

Animals↗
PubMed1998

Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Antiviral activity, pharmacokinetics, and dose response of the HIV-1 integrase inhibitor GS-9137 (JTK-303) in treatment-naive and treatment-experienced patients.

BACKGROUND: GS-9137 is a potent low-nanomolar strand transfer inhibitor of HIV-1 integrase. METHODS: The antiviral activity, tolerability, pharmacokinetics, and pharmacodynamics of GS-9137 were evaluated in a randomized, double-blind, placebo-controlled monotherapy study in 40 HIV-1- infected patients not receiving antiretroviral therapy with an HIV-1 RNA between 10,000 and 300,000 copies/mL and a CD4 count of 200 cells/microL or greater. GS-9137 or matching placebo was administered with food for 10 days at 5 dosage regimens (200, 400, or 800 mg BID, 800 mg QD, or 50 mg+100 mg ritonavir QD; 6 active, 2 placebo per dose level). The primary end point was the maximum reduction from baseline in log10 HIV-1 RNA. RESULTS: Forty patients were enrolled, with a mean baseline viral load of 4.75 log10 copies/mL and a CD4 count of 442 cells/microL. Each GS-9137 dosing regimen exhibited significant, exposure-dependent (mean reductions, -0.98 to -1.99 log10 copies/mL) antiviral activity compared with placebo (P<0.01). Twice-daily administrations of GS-9137 at doses of 400 or 800 mg or once-daily dosing of 50 mg with ritonavir demonstrated mean reductions from baseline in HIV-1 RNA of 1.91 log10 copies/mL or greater, with all patients exhibiting 1 log10 or greater and 50% having 2 log10 or greater reductions. No patient developed evidence of integrase resistance. GS-9137 showed an adverse event profile similar to placebo, and there were no study drug discontinuations. CONCLUSIONS: GS-9137 demonstrated substantial short-term antiviral activity and was well tolerated as monotherapy, thus warranting further study.

Acquired Immunodeficiency Syndrome↗
PubMed2002

X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds.

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the Zn K edge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess beta-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboom et al. (1997), Curr. Biol. 7, 739-746; (2000), J. Biomol. NMR, 18, 119-28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations with EXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 A (Debye-Waller-type factor 0.009 A(2)) and two imidazole ligands with the N atoms at 1.99 A (Debye-Waller-type factor 0.005 A(2)). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the Volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn-S and Zn-N distances of 2.3 A and 2.0 A, respectively).

Absorptiometry, Photon↗
PubMed2002

A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases.

The replication fork blocks are common in both prokaryotes and eukaryotes. In most cases, these blocks are associated with increased levels of mitotic recombination. One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae. It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S. cerevisiae, but the mechanistic aspects of these events are still poorly understood. Sequence profile searches revealed that Fob1p is related to retroviral integrases. Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p. Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.

Amino Acid Sequence↗
PubMed2004

Excision of the high-pathogenicity island of Yersinia pseudotuberculosis requires the combined actions of its cognate integrase and Hef, a new recombination directionality factor.

The Yersinia high-pathogenicity island (HPI) encodes the siderophore yersiniabactin-mediated iron uptake system. The HPI of Yersinia pseudotuberculosis I has previously been shown to be able to excise precisely from the bacterial chromosome by recombination between the attB-R and attB-L sites flanking the island. However, the nature of the Y. pseudotuberculosis HPI excision machinery remained unknown. We show here that, upon excision, the HPI forms an episomal circular molecule. The island thus has the ability to excise from the chromosome, circularize and reintegrate itself, either in the same location or in another asn tRNA copy. We also demonstrate that the HPI-encoded bacteriophage P4-like integrase (Int) plays a critical role in HPI excision and that, like phage integrases, it acts as a site-specific recombinase that catalyses both excision and integration reactions. However, Int alone cannot efficiently promote recombination between the attB-R and attB-L sites, and we demonstrate that a newly identified HPI-borne factor, designated Hef (for HPI excision factor) is also required for this activity. Hef belongs to a family of recombination directionality factors. Like the other members of this family, Hef probably plays an architectural rather than a catalytic role and promotes HPI excision from the chromosome by driving the function of Int towards an excisionase activity. The fact that the HPI, and probably several other pathogenicity islands, carry a machinery of integration/excision highly similar to those of bacteriophages argues for a phage-mediated acquisition and transfer of these elements.

Amino Acid Sequence↗
PubMed2005

Mutations in the amino-terminal domain of lambda-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.

Base Sequence↗
PubMed2005

Prevalence of Streptococcus pneumoniae isolates bearing macrolide resistance genes in association with integrase genes of conjugative transposons in Japan.

The relationship between susceptibility to macrolides and tetracycline, and the distribution of the resistance genes erm(B), mef(A) and tet(M) and the integrase gene of the conjugative transposon, Int-Tn, was examined in 43 isolates of Streptococcus pneumoniae from Gunma Prefecture, Japan. Thirty-five isolates were resistant to both macrolides and tetracycline and carried tet(M); erm(B) and mef(A) were detected in 19 and 16, respectively, of these isolates. Sixteen mef(A)-positive isolates were all identified as mef(E) variants. Int-Tn of Tn1545 was associated with 17 erm(B)- and 14 mef(A)-bearing isolates, and Int-Tn of Tn5252 was found in the remaining two mef(A)-carrying isolates. Pneumococcal strains with resistance to macrolides conferred by erm(B) or mef(A) in association with the integrase gene of conjugative transposon Tn1545 or Tn5252 appear to be prevalent in Japan.

Bacterial Proteins↗
PubMed2006

Cooperation between reverse transcriptase and integrase during reverse transcription and formation of the preintegrative complex of Ty1.

Reverse transcriptase (RT) and integrase (IN) play a central role in the replication and transposition of retroelements. Increasing evidence suggests that the interaction between these two enzymes is functional and plays an important role in replication. In the yeast Saccharomyces cerevisiae retrotransposon Ty1, the interaction of IN with RT is critical for the formation of an active conformation of RT. We show here that the RT associated with VLPs is active only if it is in close interaction with IN. To probe the IN-RT cis-trans relationship, we have used a complementation assay based on coexpressing two transposons. We show that IN acts in cis to activate RT and that a functional integrase provided in trans is not able to complement replication and transposition defects of IN deletion or IN active-site mutant elements. Our data support a model in which IN not only interacts closely with RT during reverse transcription but also remains associated with RT during the formation of the preintegrative complex.

Binding Sites↗
PubMed2005

Role of integrase in reverse transcription of the Saccharomyces cerevisiae retrotransposon Ty1.

Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

Amino Acid Sequence↗
PubMed1991

Conjugative transposition of Tn916 requires the excisive and integrative activities of the transposon-encoded integrase.

Transposon Tn916 is a 16.4-kb broad-host-range conjugative transposon originally detected in the chromosome of Enterococcus faecalis DS16. Transposition of Tn916 and related transposons involves excision of a free, nonreplicative, covalently closed circular intermediate that is substrate for integration. Excisive recombination requires two transposon-encoded proteins, Xis-Tn and Int-Tn, whereas the latter protein alone is sufficient for integration. Here we report that conjugative transposition of Tn916 requires the presence of a functional integrase in both donor and recipient strains. We have constructed a mutant, designated Tn916-int1, by replacing the gene directing synthesis of Int-Tn by an allele inactivated in vitro. In mating experiments, transfer of Tn916-int1 from Bacillus subtilis to E. faecalis was detected only when the transposon-encoded integrase was supplied by trans-complementation in both the donor and the recipient. These results suggest that conjugative transposition of Tn916 requires circularization of the element in the donor followed by transfer and integration of the nonreplicative intermediate in the recipient.

Bacillus subtilis↗
PubMed1997

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.

Bacteriophages↗
PubMed2001

Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR.

Forty-eight clinical Acinetobacter isolates with different epidemic behavior were investigated for the presence of integrons and plasmids and for antibiotic susceptibility. Integrons were demonstrated in 50% of the strains by an integrase gene PCR. Epidemic strains of Acinetobacter baumannii were found to contain significantly more integrons than nonepidemic strains. Also, the presence of integrons was significantly correlated with simultaneous resistance to several antibiotics. Plasmids were detected in 42% of the strains. However, there was no significant correlation between the numbers of plasmids and integrons in Acinetobacter species strains, no significant difference in the number of plasmids between epidemic and nonepidemic A. baumannii strains, and no significant correlation between the presence of plasmids and antibiotic resistance. Hence, it is likely that integrons play an important role in antibiotic resistance and thereby in the epidemic behavior of A. baumannii. Because the integrase gene PCR identified almost three-quarters of the epidemic A. baumannii isolates (17 of 23), this seems to be a rapid and simple technique for the routine screening and identification of clinical A. baumannii isolates with epidemic potential.

Acinetobacter↗
PubMed2006

Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication.

A recent report sought to demonstrate that acetylation of specific lysines within integrase (IN) by the histone acetyltransferase (HAT) p300 regulates human immunodeficiency virus type 1 (HIV-1) integration and is essential for viral replication (A. Cereseto, L. Manganaro, M. I. Gutierrez, M. Terreni, A. Fittipaldi, M. Lusic, A. Marcello, and M. Giacca, EMBO J. 24:3070-3081, 2005). We can corroborate the efficient and specific acetylation of the IN carboxyl-terminal domain (CTD) (amino acids 212 to 288) by p300 using purified recombinant components. Although arginine substitution mutagenesis of the isolated CTD confirms that the majority of p300 acetylation occurs at lysine residues 264, 266, and 273, the pattern of acetylation is not uniform and a hierarchy of reactivity can be established. Several combinatorial mutations of the CTD lysines modified by p300 in vitro were reconstructed into an otherwise infectious proviral plasmid clone and examined for viral growth and frequency of productive chromosomal integration. In contrast to the findings of Cereseto and coworkers, who used epitope-tagged viruses for their experiments, we find that an untagged mutant virus, IN K(264/266/273)R, is fully replication competent. This discrepancy may be explained by the use of an acidic epitope tag placed at the extreme carboxyl terminus of integrase, near the target site for acetylation. Although the tagged, wild-type virus is viable, the combination of this epitope tag with the RRR substitution mutation results in a replication-defective phenotype. Although IN belongs to the very small set of nonhistone proteins modified by HAT-mediated activity, an obligate role for acetylation at the reactive CTD lysines in HIV-1 IN cannot be confirmed.

Acetylation↗
PubMed1990

Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.

Amino Acid Sequence↗
PubMed1992

Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase.

Concerted integration of retroviral DNA termini, which produces a characteristic duplication of sequences at the integration site and formation of the proviral state, is a necessary step of the retroviral life cycle. We investigated the pairwise integration reaction catalyzed by purified avian retrovirus integrase by measuring the response to solution parameters and how the sequences of the viral termini, which comprise the avian imperfect inverted repeat, affect the reaction. When we optimized the reaction, an efficiency was achieved which approached that measured in systems using cytoplasmic extracts from virus-infected cells. The response of purified avian integrase to solution parameters was similar to that of the integration activity derived from cellular extracts. For strand transfer, the U3 viral terminal sequences were preferred to those of the U5 termini, a result we previously showed for the trimming reaction. That the sequence preference was the same for trimming and strand transfer may be further evidence that only one catalytic site is used for both reactions. A significant number of integration sites were sequenced. Interesting trends were found for the fidelity of the host duplications to the avian 6-bp duplication size, the clustering of the integration sites in the nonessential region of the lambda host DNA, and the sequence characteristics of the duplication sites.

Avian Myeloblastosis Virus↗
PubMed1993

Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate.

The human immunodeficiency virus type 1 integrase protein can be specifically cross-linked to viral long terminal repeat substrate oligonucleotides in vitro by using UV light. Site-directed mutagenesis and deletion analyses were used to define the domains involved in the interaction of integrase with the viral DNA substrate. Our results showed that mutation of conserved residues Pro-109 and Asp-116, which are found to be critical for the endonuclease and integration activities of IN protein, abolished the ability of the protein to cross-link to its DNA substrate. Furthermore, deletion analysis experiments showed that removal of 39 amino acids from the amino terminus and deletion of 15 amino acids from the carboxyl terminus abolished DNA cross-linking.

Amino Acid Sequence↗
PubMed1994

Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli.

Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein. We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography. The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration. FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies. In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack. In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction. We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.

Amino Acid Sequence↗