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PubMed1998

Point mutations in the integron integrase IntI1 that affect recombination and/or substrate recognition.

The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.

Amino Acid Sequence↗
PubMed2000

Interaction of the FimB integrase with the fimS invertible DNA element in Escherichia coli in vivo and in vitro.

The FimB protein is a site-specific recombinase that inverts the fimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fim switch.

Amino Acid Sequence↗
PubMed2002

IntI2 integron integrase in Tn7.

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.

3' Untranslated Regions↗
PubMed2003

Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea.

We found in the environmental strain Nitrosomonas europaea a chromosomal integron-like structure with an integrase gene, intI(Neu). We have tested the capacity of the IntINeu integrase to excise and integrate several resistance gene cassettes. The results allow us to consider IntINeu a new functional integron integrase.

Attachment Sites, Microbiological↗
PubMed1992

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.

Amino Acid Sequence↗
PubMed1993

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate.

Amino Acid Sequence↗
PubMed1994

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.

Amino Acid Sequence↗
PubMed1994

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.

Avian Myeloblastosis Virus↗
PubMed1995

Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene.

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity. However, during infection at the nonpermissive temperature of 39.5 degrees C, K136A/E138A is capable of only one round of integration. Mutant virions formed at 39.5 degrees C do not integrate but are indistinguishable from wild-type virions when scored for activity of reverse transcriptase and correct expression and processing of Gag and Pol proteins. We demonstrate that the defect responsible for the ts phenotype of K136A/E138A is localized to a step after proviral formation and integrase protein synthesis but prior to particle maturation. It is the temperature at which the K136A/E138A virion is synthesized, not the temperature at which infection occurs, which determines the ability of the virus to integrate.

Alanine↗
PubMed1995

Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity.

The integrase protein from human immunodeficiency virus type 1 (HIV-1) has generally been reported to require Mn2+ for efficient in vitro activity. We have reexamined the divalent metal ion requirements of HIV-1 integrase and find that the protein is capable of promoting efficient 3' processing and DNA strand transfer with either Mn2+ or Mg2+. The metal ion preference depended upon the reaction conditions. HIV-1 integrase displayed significantly less nonspecific nuclease activity in reaction mixtures containing Mg2+ than it did under the previously described reaction conditions with mixtures containing Mn2+.

Base Sequence↗
PubMed2001

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

3T3 Cells↗
PubMed2006

Integrase diversity and transcription of the maize retrotransposon Grande.

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.

Gene Dosage↗
PubMed2000

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

Amino Acid Sequence↗
PubMed2001

Inhibition of HIV-1 integrase by small molecules: the potential for a new class of AIDS chemotherapeutics.

HIV-1 integrase, one of three constitutive viral enzymes required for replication, is an attractive target for chemotherapeutic intervention in the treatment of AIDS. Unlike the retroviral reverse transcriptase and protease enzymes, successful drug candidates based on the inhibition of integrase have yet to emerge despite the multitude of laboratories working on the problem. In vitro inhibition of integrase in the laboratory has not always led to antiviral activity in cell culture. Indeed, most compounds found from broad screening efforts have fallen into this category. Only recently have compounds that inhibit enzymatic function as well as exhibiting an antiviral effect as a result been discovered. The reasons why certain structural classes of compounds fail to have antiviral activity are only beginning to be understood. In the last two years a new class of compounds has emerged that do produce potent inhibition of viral growth in cell culture. Further work in this area may yet produce clinically useful antiviral agents.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Viral and immunologic follow up of 4 to 9 years of AIDS treatments by quadruple combinations of virostatics including integrase inhibitors applied in short sequences differing by drug rotation.

AIM: To present the 4 to 9 years (median: 6 years) treatment follow up of 10 HIV1-AIDS patients, 9 at AIDS and 1 at A3 stages. METHODS: We have applied from 1992 to 1994, AZT combined with 2 integrase inhibitors, acriflavine and hydroxy-methyl-ellipticine. We could shift, in 1994, to combinations of 3 drugs including two more retrotranscriptase inhibitors (RTI), ddI and ddC, and, after 1995, to combinations of 4 drugs including also two other RTI, d4T and 3TC, and 3 protease inhibitors (PI), indinavir, ritonavir, and saquinavir. In 1998, as cobalamine was shown by an in vitro test, to act as integrase inhibitor, vitamin B12 was added in cycles of various lengths. Every three weeks, not only the investigations were repeated, but the virostatics were changed. RESULTS: No grade 2 virostatics toxicity has been registered. The viral loads (VL) decreased according to exponential curves. Their initial parts obeyed first order kinetics. The second parts were and still are asymptotic. The first parts could be rectilinear or sinuous. The sinuosities were associated to cofactors present before treatment (chimerism, UV irradiation, hepatitis C or B and C, brain toxoplasmosis). The asymptotic parts, whose VL were below PCR detectable levels, presented discrete, reversible HIV1 rebounds, associated to other cofactors (such as herpes zoster, herpes 6, CMV, flat condyloma, and influenza). Among immunologic parameters, the monocyte and CTL numbers increased and presented, during the rapidly decreasing part of VL curve, a significant inverse correlation with it. Neither CD4+ nor suppressor T-cell (STC) numbers presented such correlation. Near 100 % of CTL were CD28+. Later, vitamin B12 applications increased monocyte and CD28+ CTL numbers, and appeared to reinforce VL stabilization. CONCLUSION: The combinations of inhibitors affecting 3 retrovirus targets, retrotranscriptase, integrase, and protease have given to 10 out of 10 AIDS patients survivals varying today between 4 to 9 years, in excellent conditions. The UVA-pretreated patient is the only one presenting a not maximally reduced asymptotic VL, while his CD4+ and STC have been absent for 8 years. Other patient VL regressed exponentially to become asymptotic, below PCR detectable levels.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins.

To achieve a productive infection, the reverse transcribed cDNA of the human immunodeficiency virus type 1 (HIV-1) has to be inserted in the host cell genome. The main protein required to accomplish this reaction is the virally encoded integrase. In vitro, the recombinant integrase is capable of catalyzing the two subsequent reactions of the integration process, namely the 3' processing followed by the strand transfer, without other viral and/or cellular proteins. However, a number of studies indicate that the in vivo integration process also involves cellular proteins, assisting the virus to integrate in the cellular genome. These cellular proteins can play a role during different steps of the integration process, including nuclear import, integrase catalysis, integration site selection and DNA gap repair. In this review we summarize the candidate cellular proteins involved in the HIV-1 integration process identified so far and discuss their potential roles during HIV-1 replication.

Apoptosis↗
PubMedDate not supplied

[Integrase of lambda phage inhibits the activity of promotor Patt under in vivo and in vitro conditions].

In vitro and in vitro transcription of the region of lambda phage DNA containing promoter Patt has been studied. Active transcription was observed in the vicinity of the att site and at the promoter Patt in vivo during the first minutes after infection. This transcription considerably decreases at the 1th minute, and stops completely at the 15th minute after infection. Maximum synthesis of integrase also takes place at the 15th minute after infection. In vitro the partially purified int protein (integrase) completely inhibited transcription from the promoter Patt. Hence, integrase is most probably a repressor of Patt in vivo and in vitro.

Adenosine Triphosphate↗
PubMed1997

Do the integrases of LTR-retrotransposons and class II element transposases have a common ancestor?

The integrases of retrotransposons (class I) and retroviruses and the transposases of bacterial type elements (class II) were compared. The DDE signature that is crucial for the integration of these elements is present in most of them, except for the non-LTR retrotransposons and members of the hAT and P super-families. Alignment of this region was used to infer the relationships between class II elements, retrotransposons, and retroviruses. The mariner-Tc1 and the Pogo-Fot1 super-families were found to be closely related and probably monophyletic, as were LTR retrotransposons and retroviruses. The IS elements of bacteria were clustered in several families, some of them being closely related to the transposase of the mariner-Tc1 super-family or to the LTR retrotransposon and retrovirus integrases. These results plus that of Xiong and Eickbush (1990) were used to develop an evolutionary history suggesting a common ancestral origin(s) for the integrases and transposases containing the DDE signature. The position of the telomeric elements (Het-A and TART) was assessed by comparing their gag and reverse transcriptase domains (when present) to those of group II introns and non-LTR retrotransposons. This preliminary analysis suggests that telomeric elements may be derived from non-LTR retrotransposons.

Amino Acid Sequence↗