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PubMed2003

Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.

Amino Acid Sequence↗
PubMed2000

Microvirus of chlamydia psittaci strain guinea pig inclusion conjunctivitis: isolation and molecular characterization.

The authors report the isolation and molecular characterization of a bacteriophage, φCPG1, which infects CHLAMYDIA: psittaci strain Guinea pig Inclusion Conjunctivitis. Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the φX174 family of bacteriophages. The single-stranded circular DNA genome of φCPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described CHLAMYDIA: bacteriophage (Chp1) that infects avian C. psittaci. Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees. Lesser sequence similarities were seen between two ORF products and the major capsid protein of the φX174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids. Phage φCPG1 is the second member of the genus CHLAMYDIAMICROVIRUS:, the first to infect a member of a CHLAMYDIA: species infecting mammals. Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae. This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of φCPG1. This finding suggests that C. pneumoniae has been infected by a phage related to φCPG1 and that infection resulted in integration of some of the phage genome into the C. pneumoniae genome.

Amino Acid Sequence↗
PubMed2005

Integration and excision by the large serine recombinase phiRv1 integrase.

The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.

Attachment Sites, Microbiological↗
PubMed1997

Characterization of the mycobacteriophage L5 attachment site, attP.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

Bacteriophages↗
PubMed2006

Viral load, E2 gene disruption status, and lineage of human papillomavirus type 16 infection in cervical neoplasia.

The clinical utility of human papillomavirus (HPV) load and integration status remains unclear. We applied refined methods to delineate the viral load, integration status, and lineage of 104 women with HPV-16 monotype infection, including 19 with normal cervices, 9 with histologically proven cervical intraepithelial neoplasia (CIN) 1, 24 with CIN 2, 27 with CIN 3, and 25 with squamous cell carcinoma (SCC). Higher crude viral load, as determined by real-time polymerase chain reaction (PCR) targeting the E7 gene, was observed for SCC but became insignificant after normalization for cell content. Integration was located and quantified by real-time PCRs targeting, respectively, the carboxyl, amino, and hinge domains of the E2 gene. Pure episomal, integrated, and mixed forms were observed in all disease groups. Most E2 gene disruptions involved the amino-terminal, but sparing the hinge region that has been frequently used as a surrogate marker of integration. Large-fragment disruption involving all 3 E2 regions was observed only in the CIN 3 and SCC groups. Altogether, 33.3% of the CIN 3 group and 28.0% of the SCC group harbored pure episomal genomes. The Asian lineage was associated with a higher risk for CIN 3/SCC than the European lineage, and 6 of the 7 large-fragment E2 disruptions were from Asian lineage. The link between viral lineage, integration pattern, and oncogenesis deserves further study.

Adolescent↗
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↗
PubMed1994

Integration site of human papillomavirus type-18 DNA in chromosome band 8q22.1 of C4-I cervical carcinoma: DNase I hypersensitivity and methylation of cellular flanking sequences.

The C4-I cell line derived from a non-keratinizing squamous cell carcinoma of the uterine cervix contains integrated human papillomavirus-18 DNA. Fluorescence in situ hybridization of C4-I cells demonstrated a single viral integration site at 8q22.1 on a derivative chromosome originating from an 8q;12q translocation. 8q22 is a site of chromosome fragility and is also recombinogenic in several human malignancies. DNase I hypersensitivity of the integration site was studied with a cellular flanking probe. A hypersensitive site was detected within 3 kb from viral DNA. The integration sites are undermethylated in C4-I and HeLa cells and fully methylated in tumor cell lines of other origin, such as lymphoid cells.

Carcinoma, Squamous Cell↗
PubMed2002

Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370.

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.

Attachment Sites, Microbiological↗
PubMed2006

Molecular evolution of the HERV-E family in primates.

More than 50 copies of HERV-E family elements have been estimated to exist in the human genome. Here we examined the recent evolutionary history of the HERV-E family by a PCR approach using genomic DNA from hominoid primates and a human monochromosomal panel. From the HERV-E family, 25 and 68 env fragments, were identified and analyzed from hominoid primates and human chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, X, and Y, respectively. They showed 76.7-99.6% sequence similarity to that of HERV-E (accession no. M10976). Phylogenetic analysis of HERV-E env family distinctively divided into two groups (groups I and II) that each contained three subgroups. Divergence times of the two groups were estimated as 10.7 MYr for group I and 41.3 MYr for group II using an average evolutionary rate of 0.3% per MYr. These data are consistent with that of PCR analysis, which showed a band of the HERV-E family in the genomes of the hominoids, Old World monkeys, and New World monkeys. Therefore, the HERV-E family may have integrated into the primate genome after prosimians and New World monkeys diverged. Then they proliferated extensively in the genome of humans and great apes during primate evolution.

Animals↗
PubMed2004

BAF: roles in chromatin, nuclear structure and retrovirus integration.

Barrier-to-autointegration factor (BAF) is an essential protein that is highly conserved in metazoan evolution. BAF binds directly to double-stranded DNA, nuclear LEM-domain proteins, lamin A and transcription activators. BAF is also a host cell component of retroviral pre-integration complexes. BAF binds matrix, a retroviral protein, and facilitates efficient retroviral DNA integration in vitro through unknown mechanisms. New findings suggest that BAF has structural roles in nuclear assembly and chromatin organization, represses gene expression and might interlink chromatin structure, nuclear architecture and gene regulation in metazoans.

Amino Acid Sequence↗
PubMed2002

The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome.

The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E. coli chromosome.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of HIV-1 integration by mono- & bi-functionalized triple helix forming oligonucleotides.

HIV-1 DNA integration is carried out by integrase, a viral protein which binds to specific sequences located on both extremities of the HIV-1 DNA LTR. Inhibition of integration was observed with submicromolar concentrations of mono- or bifunctionalized 11-mer oligonucleotide-intercalators, which were designed to form an alternate strand triple helix with the U5 LTR end containing two adjacent purine tracts on opposite strands 5'-GGAAAATCTCT-3'/3'-CCTTTTAGAGA-5'.

Base Sequence↗
PubMed2007

Human herpesvirus 6 DNA levels in cerebrospinal fluid due to primary infection differ from those due to chromosomal viral integration and have implications for diagnosis of encephalitis.

The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log(10) copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log(10) copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (>/=3.5 log(10) copies/ml) or whole blood (>/=6.0 log(10) copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log(10) copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log(10) copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood.

Adult↗
PubMed2004

Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK(CS)) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK(CS)-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PK(CS) gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Animals↗
PubMed2004

Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes.

Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the "physiologically" elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by "natural" infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3' deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.

Cells, Cultured↗
PubMed2001

Rapid detection of the ERV-K(C4) retroviral insertion reveals further structural polymorphism of the complement C4 genes in old world primates.

The fourth component of complement (C4) is coded for by two tandem-duplicated genes located in the class III region of the MHC of humans as well as a number of primates. A C4 gene size polymorphism giving rise to two gene variants of 16 and 22.3 kb length can be attributed to a complete endogenous retroviral insertion of 6.3 kb termed ERV-K(C4) in intron 9 of the long C4 genes. We developed a simple PCR-based screening assay to detect the presence of this insertion, and tested a number of unrelated animals from old world primate species. The presence of the ERV insertion in the orangutan, rhesus macaque and green monkey as well as its absence in gorillas and chimpanzees could be confirmed. In addition, the insertion was also detected in the baboon and the cynomolgus macaque whereas it was not found in a single gibbon. Among rhesus and cynomolgus macaques one individual was identified in each species only carrying short C4 genes demonstrating further structural heterogeneity in these species. Based on these findings we propose that the primigenial retroviral integration occurred prior to the radiation of old world primate species, and that both the long and the short forms of the C4 gene have existed side by side since then.

Animals↗
PubMed2003

Functional characterization of transgene integration patterns by halo fluorescence in situ hybridization: electroporation versus retroviral infection.

Gene transfer techniques are essential for bioengineering and gene therapy. Retroviral vectors constitute an important tool in this context as integrase-catalyzed genomic anchoring is one of the best defined results of nonhomologous recombination, occurring at loci with favorable expression properties. On the basis of a retroviral expression cassette, this study focuses on differences regarding the genomic targets after its transfer by either retroviral infection or electroporation. Fluorescence in situ hybridization (FISH) of clones generated by infection revealed for the majority an association of the transgene with the nuclear matrix while this was true only for a minority of single copy clones if the same construct was transferred by electroporation. Our results demonstrate that the process of integration is distinct for both gene transfer routes and confirm that retroviral survival strategies favor integration next to scaffold/matrix attachment regions. These results may be one key to explain recent consequences of gene therapy trials that have led to the deregulation of endogenous genes.

Animals↗
PubMed2002

Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRv1.

The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.

Amino Acid Sequence↗