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PubMed2005

The Salmonella genomic island 1 is an integrative mobilizable element.

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).

Amino Acid Sequence↗
PubMed2001

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Amino Acid Sequence↗
PubMed2006

Detection and molecular characterization of foamy viruses in Central African chimpanzees of the Pan troglodytes troglodytes and Pan troglodytes vellerosus subspecies.

BACKGROUND: Foamy viruses are exogenous retroviruses that are highly endemic in non-human primates (NHPs). Recent studies, mainly performed in North America, indicated frequent simian foamy virus (SFV) infection in persons occupationally exposed to NHPs. This zoonotic infection was demonstrated mainly after bites by chimpanzees [Pan troglodytes (P. t.)] of the West African P. t. verus subspecies in primatology centers or zoos in the USA. METHODS: We studied 32 chimpanzees from the Central African subspecies P. t. troglodytes and P. t. vellerosus, originating from Cameroon (29 cases) or Gabon (3 cases). We screened first plasma or sera of the animals with a Western blot detecting the SFVs Gag doublet proteins. Then, we performed two nested polymerase chain reactions (PCRs) amplifying a fragment of the integrase and LTR regions and, finally, we made phylogenetical analyses on the sequences obtained from the integrase PCR products. RESULTS: By serological and/or molecular assays, we detected foamy viruses (FVs) infection in 14 chimpanzees. Sequence comparison and phylogenetic analyses of a 425 bp fragment of the integrase gene obtained for 10 of the 14 positive apes, demonstrated a wide diversity of new FVs strains that belong phylogenetically either to the P. t. troglodytes or P. t. vellerosus foamy viral clade. CONCLUSIONS: This study shows that chimpanzees living in these areas of Central Africa are infected by several specific foamy viruses. This raises, in such regions, the potential risk of a human retroviral infection of zoonotic origin linked to chimpanzees contacts, as already exemplified for STLV-1 and SIV infections.

Amino Acid Sequence↗
PubMed1990

Interaction of integration host factor from Escherichia coli with the integration region of the Haemophilus influenzae bacteriophage HP1.

The specific DNA-binding protein integration host factor (IHF) of Escherichia coli stimulates the site-specific recombination reaction between the attP site of bacteriophage HP1 and the attB site of its host, Haemophilus influenzae, in vitro and also appears to regulate the expression of HP1 integrase. IHF interacts specifically with DNA segments containing the att sites and the integrase regulatory region, as judged by IHF-dependent retardation of relevant DNA fragments during gel electrophoresis. The locations of the protein-binding sites were identified by DNase I protection experiments. Three sites in the HP1 attP region bound IHF, two binding sites were present in the vicinity of the attB region, and one region containing three partially overlapping sites was present in the HP1 integrase regulatory segment. The binding sites defined in these experiments all contained sequences which matched the consensus IHF binding sequences first identified in the lambda attP region. An activity which stimulated the HP1 site-specific integration reaction was found in extracts of H. influenzae, suggesting that an IHF-like protein is present in this organism.

Bacterial Proteins↗
PubMed1997

Integration specificities of two lambdoid phages (21 and e14) that insert at the same attB site.

It was shown previously that phage 21 and the defective element e14 integrate at the same site within the icd gene of Escherichia coli K-12 but that 21 integrase and excisionase excise e14 in vivo very infrequently compared to excision of 21. We show here that the reverse is also true: e14 excises itself much better than it excises an adjacent 21 prophage. In vitro integrase assays with various attP substrates delimit the minimal attP site as somewhere between 366 and 418 bp, where the outer limits would include the outermost repeated dodecamers suggested as arm recognition sites by S. J. Schneider (Ph.D. dissertation, Stanford University, Stanford, Calif., 1992). We speculate that the reason 21 attP is larger than lambda attP (240 bp) is because it must include a 209-bp sequence homologous to the 3' end of the icd transcript in order to allow icd expression in lysogens. Alteration of portions of 21 attP to their e14 counterparts shows that 21 requires both the arm site and core site sequences of 21 but that replacements by e14 sequences function in some positions. Consistent with Schneider's in vivo results, and like all other known integrases from lambdoid phages, 21 requires integration host factor for activity.

Bacterial Proteins↗
PubMed1999

Protein-DNA complexes in mycobacteriophage L5 integrative recombination.

The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

Base Sequence↗
PubMed1991

Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex.

Cytoplasmic extracts prepared from cells infected with metabolically radiolabeled virions of human immunodeficiency virus type 1 contain viral DNA in association with labeled viral proteins. Viral DNA can be purified from these extracts by gel filtration chromatography and sucrose gradient sedimentation as a part of a nucleoprotein complex containing integrase as the only viral protein detectable by immunoprecipitation and gel electrophoretic analysis. The purified complex contains no detectable gag gene products, including p17, p24, p7, or p6, and contains no additional pol gene products, including the p10 protease, p66 and p51 polymerase, or the p15 RNase H. Nearly all of the purified nucleoprotein complexes are capable of integrating into heterologous DNA targets in vitro. These observations demonstrate that integrase is a component of the human immunodeficiency virus type 1 preintegration complex and suggest that integrase may be the only viral protein necessary for the integration of retroviral DNA.

Attachment Sites, Microbiological↗
PubMed1993

Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex.

The preintegration complex of human immunodeficiency virus type 1 (HIV-1) is a large nucleoprotein complex containing viral nucleic acids in association with products of the viral gag and pol genes. One of these proteins, integrase, is absolutely required for the integration and formation of the provirus. Although HIV-1-specific 2-LTR circles from nuclei of HIV-1-infected cells were found to be associated within a high-molecular-weight nucleoprotein complex, antibodies to HIV-1 integrase failed to precipitate this form of viral DNA. This result indicates that circular forms of HIV-1 DNA are not associated with integrase. These viral DNA forms seem to exist in a context of a nucleoprotein complex that is different from a preintegration complex of HIV-1.

Cell Line↗
PubMed1995

Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1.

Certain human immunodeficiency virus type 1 (HIV-1) isolates are able to productively infect nondividing cells of the monocyte/macrophage lineage. We have used a molecular genetic approach to construct two different HIV-1 integrase mutants that were studied in the context of an infectious, macrophage-tropic HIV-1 molecular clone. One mutant, HIV-1 delta D(35)E, containing a 37-residue deletion within the central, catalytic domain of integrase, was noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages. The HIV-1 delta D(35)E mutant, however, exhibited defects in the assembly and/or release of progeny virions in transient transfection assays, as well as defects in entry and/or viral DNA synthesis during the early stages of monocyte-derived macrophage infection. The second mutant, HIV-1D116N/8, containing a single Asp-to-Asn substitution at the invariant Asp-116 residue of integrase, was also noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages but, in contrast to HIV-1 delta D(35)E, was indistinguishable from wild-type virus in reverse transcriptase production. PCR analysis indicated that HIV-1D116N/8 entered monocyte-derived macrophages efficiently and reverse transcribed its RNA but was unable to complete its replication cycle because of a presumed block to integration. These data are consistent with the hypothesis that integration is an obligate step in productive HIV-1 infection of activated peripheral blood mononuclear cells and primary human macrophage cultures.

Amino Acid Sequence↗
PubMed2003

Human immunodeficiency virus type 1 (HIV-1) Vpr enhances expression from unintegrated HIV-1 DNA.

Retroviral DNA synthesized prior to integration, termed unintegrated viral DNA, is classically believed to be transcriptionally inert and to serve only as a precursor to the transcriptionally active integrated proviral DNA form. However, it has recently been found to be expressed under some circumstances during human immunodeficiency virus type 1 (HIV-1) replication and may play a significant role in HIV-1 pathogenesis. HIV-1 Vpr is a virion-associated accessory protein that is critical for HIV-1 replication in nondividing cells and induces cell cycle arrest and apoptosis. We find that Vpr, either expressed de novo or released from virions following viral entry, is essential for unintegrated viral DNA expression. HIV-1 mutants defective for integration in either the integrase catalytic domain or the cis-acting att sites can express unintegrated viral DNA at levels similar to that of wild-type HIV-1, but only in the presence of Vpr. In the absence of Vpr, the expression of unintegrated viral DNA decreases 10- to 20-fold. Vpr does not affect the efficiency of integration from integrase-defective HIV-1. Vpr-mediated enhancement of expression from integrase-defective HIV-1 requires that the viral DNA be generated in cells through infection and is mediated via a template that declines over time. Vpr activation of expression does not require exclusive nuclear localization of Vpr nor does it correlate with Vpr-mediated cell cycle arrest. These results attribute a new function to HIV-1 Vpr and implicate Vpr as a critical component in expression from unintegrated HIV-1 DNA.

Cell Line↗
PubMed2006

[Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana].

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.

Cloning, Molecular↗
PubMed1994

Triplex-mediated inhibition of HIV DNA integration in vitro.

Integration of human immunodeficiency virus (HIV) DNA into the genome of host cells is an obligatory step in the replicative cycle of the virus. The overall process is carried out in vitro by a single viral protein, the integrase, which binds to short sequences located at the ends of viral DNA long terminal repeats (LTRs). These end sequences are highly conserved in all HIV genomes and are therefore attractive targets for selective DNA binding compounds. The integrase-binding site located in U3 LTR contains a purine motif, 5'-GGAAGGG-3' which can be selectively targeted by oligonucleotide-intercalator conjugates. Under neutral pH and physiological temperature, these conjugates readily form a stable complex with the viral DNA which involves a short DNA triplex. Triple-helix formation prevents the catalytic functions of the integrase in vitro which results in a sequence-specific inhibition of the U3 integration process.

Base Sequence↗
PubMed1998

Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.

We have examined the expression and function of a gene, vlf-1, of Autographa californica nuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis revealed that vlf-1 is expressed during the late phase of infection, primarily from 15 to 24 h postinfection. VLF-1 localized in the cell nucleus and was also present in the nucleocapsids of virus particles. Mapping of vlf-1 mRNA by primer extension showed that transcription initiates at a TAAG motif 71 bp upstream of the vlf-1 open reading frame. Disruption of this TAAG motif abolished the ability of vlf-1 to stimulate transcription from the very late polyhedrin gene (polh) promoter in transient expression assays, suggesting that vlf-1 expression is controlled by the TAAG motif. Using a highly efficient system to construct recombinant viruses with modifications in vlf-1, we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutants of vlf-1 failed, suggesting that vlf-1 is an essential gene for virus replication. Computer-assisted sequence homology searches place vlf-1 in the lambda phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found in vlf-1 could be changed in the viral genome, implying that the putative integrase activity of VLF-1 is associated with the essential function of vlf-1. However, mutation of a crucial active-site tyrosine did not affect the ability of vlf-1 to transactivate the polh promoter in transient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity.

Animals↗
PubMed2002

Integration site selection by lentiviruses: biology and possible control.

Retroviruses integrate into naked DNA in a generally sequence nonspecific fashion, but closer study reveals a variety of forces that influence target site selection. Primary sequence of the target plays a small but detectable role. Proteins bound to target DNA can inhibit integration by blocking access of integration complexes or stimulate integration by distorting DNA. An important example of the latter is DNA distortion in nucleosomal DNA. In vivo integration has not yet been convincingly shown to be biased in favor of any identifiable sequence features, though this could still change in future studies. Many applications of retroviral vectors could be facilitated by targeting integration in vivo to predetermined sites. Towards this end, several groups have studied the properties of fusions of integrase proteins to sequence-specific DNA-binding domains. To date such studies establish that targeting can work well in reactions in vitro, but a variety of obstacles complicate applications in vivo. However, naturally occurring retrotransposons do carry out highly targeted integration using retrovirus-like integrase proteins, fueling long-term hopes for targeting with retroviral integrases as well.

DNA, Viral↗
PubMed2005

Targeted modification of mammalian genomes.

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination. Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or PhiC31 integrase) are usually 30-50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.

Animals↗
PubMed2004

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.

Amino Acid Sequence↗
PubMed2006

Transient gene expression by nonintegrating lentiviral vectors.

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

Antigens, CD34↗
PubMedDate not supplied

[Structure and function of integrons].

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Integrons have an integrase gene (intI), an attachment site (attI), into which individual resistance genes are inserted and a promotor sequence (Pant), allowing expression of resistance genes (cassette-associated genes), which do not have promotors. Integrase recognizes 59-be, a specific sequence in certain resistance genes, which is captured by recombination at the attI attachment site. The fragment intI - attI is highly conserved in all integrons and is called 59 -CS. Integrons have been classified according to the sequence of their integrase and the ones most frequently detected in isolated clinical strains belong to Class I. Class I integrons contain the 59 -CS region followed by gene cassettes in a variable region and finally, a conserved region known as 39 -CS containing two genes, the quaternary ammonium resistance gene (qacEDI) and the sulphonamide resistance gene (sul1); both genes are fixed in this structure. Accordingly, the structure of a Class 1 integron would be IntI - attI [R11 R21.] - qacED1 - sul1. Integrons are probably not mobile, but they are often found in transposons within conjunctive plasmids, which assures their mobility, as can be seen by their wide diffusion among bacteria.

Drug Resistance, Microbial↗