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At least 631 records · Page 35Linked to original sources
PubMed1993

Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration.

An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.

Base Sequence↗
PubMed1986

Hepatitis B virus DNA in human hepatocellular carcinoma: is the integration of hepatitis B virus DNA really carcinogenic?

Hepatitis B virus-specific DNA sequences (HBVDNA) in the liver were examined in 19 patients with hepatocellular carcinoma (HCC), 5 patients with liver cirrhosis (LC) and without HCC, 3 patients with chronic hepatitis (CH), 2 patients with metastatic liver cancer (MLC), 1 patient with primary biliary cirrhosis (PBC) and 4 patients with normal liver (NL) by the Southern blot hybridization procedure. Integration of HBVDNA was found in all 4 HCC patients with serum HBsAg, of whom one patient had HBVDNA only in the non-tumor (cirrhotic) region. Integration of HBVDNA was also detected in 4 of 8 HCC patients without serum HBsAg but with serum HBV-related antibodies, and in 2 HBsAg-positive patients of 5 LC patients. All 3 HBsAg-positive CH patients had only extrachromosomal HBVDNA. No HBVDNA was detected in the other 21 patients. Although integration of HBVDNA was observed in HBsAg-positive HCC patients with a higher frequency, integrated HBVDNA could also be detected in non-tumor regions of HCC patients and cirrhotic livers without HCC. It was concluded from these observations that integration of HBVDNA was frequently associated with HCC but might not have a direct causal effect on hepatocarcinogenesis even in HBV carriers.

Adult↗
PubMed1992

Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites.

The integration (IN) protein encoded by human immunodeficiency virus directs the integration of viral DNA into host DNA. We have probed the DNA sites required for the function of IN protein by attaching adducts to model DNA substrates and assaying their effects on integration in vitro. These experiments reveal that modifications in a short region on both DNA strands at the ends of the viral DNA block IN protein function. Modification of the target DNA near the point of DNA strand transfer also blocks IN protein function. Further experiments suggest that distinct subsets of the identified interactions are important for separate steps in the integration process.

Autoradiography↗
PubMed1991

Integration of bovine leukaemia virus at all stages of enzootic bovine leukosis.

Integration of bovine leukaemia virus DNA was investigated at all stages of infection in cattle. We report here the detection of integrated proviral DNA in the majority of antibody positive animals without lymphocytosis. In all but one case virus was integrated at a number of different sites. Hybridization analysis failed to detect proviral sequences in animals shown to be BLV antibody-negative by the Agar Gel Immunodiffusion assay. The pattern of integration in leukocytes from animals with persistent lymphocytosis was similar to that in sero-positive animals without lymphocytosis in that multiple sites of integration were evident. As reported by others only one or a few sites of integration were detected in tumours from enzootic bovine leukosis animals. Tumours from different sites in individual animals were either monoclonal or oligoclonal.

Animals↗
PubMed1990

Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues.

Cellular DNAs from human livers chronically infected with hepatitis B virus (HBV) were analyzed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 of 16 chronically infected hepatic tissues, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from three chronically infected hepatic tissues, deletion of cellular flanking DNA in all cases and rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction in some cases were noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA is not a specific incident of hepatocellular carcinoma formation. Detailed analyses of various integrants bearing rearranged viral DNA failed to indicate any gross structural alteration in cellular DNA, except for a small deletion at the integration site, indicating that viral DNA rearrangement with inverted duplication possibly occurs before integration of HBV DNA. Based on nucleotide sequencing analyses of virus-virus junctions, a one- to three-nucleotide identity was found. A mechanism for this inverted duplication of HBV DNA is proposed in which illegitimate recombination between two complementary viral strands may take place by means of a nucleotide identity at the junction site in a weakly homologous region (patchy homology) on one side of adjoining viral sequences. For virus-cell junctions, the mechanism may be basically similar to that for virus-virus junctions.

Adolescent↗
PubMed1982

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks.

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.

Animals↗
PubMed2006

A high-throughput method for cloning and sequencing human immunodeficiency virus type 1 integration sites.

Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.

Cell Line↗
PubMed1994

Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene.

Human adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on chromosome 19q13.3-qter. We report that both transfected and infected AAV-vectors will target a foreign gene preferentially to AAVS1. Of 11 infected cell lines studied, nine (82%) showed integration of the neomycin resistance marker into AAVS1, which confirms the findings of previous investigations. We also show site-specific integration (nine out of 12 cell lines, 75%) following transfection of an AAV vector containing the neomycin gene. The ability of transfected AAV sequences to preferentially target and express selectable genes at a known site in the human genome extends the potential use of AAV as a vector in gene therapy.

Anti-Bacterial Agents↗
PubMed1990

Identification of integrated hepatitis B virus DNA sequences in human hepatocellular carcinomas in Korea.

The rate of HBV DNA integration into the genome of hepatocellular carcinoma (HCC) cells varies greatly among HCC tissues from different geographical districts. Because of the potential etiological significance of identifying HBV DNA integration in HCC tissues from Korea, we investigated 13 aseptically obtained HCC surgical specimens by Southern blot analysis. Among 13 HCCs, eight (61.5%) contained integrated HBV DNA. Of eight HBsAg-positive patients, six (75%) had integrated HBV DNA in their HCCs while two (40%) out of five HBsAg-negative patients had integrated HBV DNA. Those two HBsAg-negative patients with integrated HBV DNA were all anti-HBs-positive. However, it was not demonstrated in the patient without any HBV serological markers. These findings highly suggest a prominent etiologic role of HBV in the development of HCC. On the other hand, it also strongly favors a multifactorial etiology of human HCC in korea.

Adult↗
PubMed1997

The CM2 protein of influenza C virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins.

We have undertaken a characterization of the CM2 protein of influenza C virus. The CM2 coding region of RNA segment 6 (nucleotides 731-1147) was cloned from two strains of influenza C virus and expressed using the vaccinia virus-bacteriophage T7 RNA polymerase (vac-T7) system. An antiserum raised to a C-terminal peptide in the CM2 open reading frame recognized the CM2 protein in influenza C virus-infected cells and after vac-T7 expression of the CM2 open reading frame. CM2 is posttranslationally modified by addition of high-mannose carbohydrate chains (Mr approximately 18 kDa) and by further addition of polylactosaminoglycans (Mr approximately 21-35 kDa). The available data indicate that CM2 has a cleavable signal peptide at the N-terminus of the protein. Site-directed mutagenesis eliminated the single potential N-linked carbohydrate attachment site on CM2 and indicated that the protein has an NoutCin orientation in membranes. Nonreducing SDS-PAGE indicated that the protein was expressed as disulfide-linked dimers and tetramers. Cell surface biotinylation and indirect immunofluorescence showed the protein to be expressed at the cell surface. Elimination of the N-linked carbohydrate attachment site and addition of a C-terminal HA epitope tag did not adversely affect surface expression of CM2. The NoutCin membrane orientation of CM2, the size of the ectodomain and cytoplasmic tail of CM2, and its ability to form disulfide-linked oligomers are reminiscent of the structural properties of influenza A virus M2 and influenza B virus NB proteins.

Cloning, Molecular↗
PubMed2000

Loss of ATM function enhances recombinant adeno-associated virus transduction and integration through pathways similar to UV irradiation.

Ataxia telangiectasia is caused by a genetic defect in the ATM gene that results in altered cellular sensitivity to DNA-damaging agents such as gamma-irradiation. ATM deficiency is associated with an increased incidence of neurological disorders, immune deficiency, and cancer. In this report we demonstrate that recombinant adeno-associated virus (rAAV) gene transfer in ATM-deficient fibroblasts is significantly enhanced over normal fibroblast cell lines. This enhancement of rAAV transduction in AT cells is correlated with an increased abundance of circular form rAAV genomes, as well as a higher number of integrated head-to-tail concatamer proviral genomes. Studies evaluating AAV trafficking using Cy3-labeled virus suggest that a nuclear mechanism is responsible for increased rAAV transduction in AT cells, because binding, endocytosis, and nuclear trafficking of virus are unaffected by the AT phenotype. Additionally, the profile of rAAV transduction after UV irradiation is significantly blunted in AT cells, suggesting that the level of DNA repair enzymes normally associated with UV augmentation of viral transduction may already be maximally elevated. These results further expand our understanding of genes involved in rAAV transduction.

Ataxia Telangiectasia↗
PubMed2005

Integrated hepatitis C virus treatment: addressing comorbid substance use disorders and HIV infection.

OBJECTIVES: To examine hepatitis C virus (HCV) and HIV testing patterns within the Northwest Veterans Integrated Service Network (VISN 20). METHODS: Using a comprehensive VISN 20 database, we retrospectively reviewed medical records of 293,445 veterans. RESULTS: 32.8% of patients were tested for HCV, 5.5% were tested for HIV, and 4.3% were co-tested. Of those tested, 12.3% were HCV positive, 5.4% were HIV positive, and 1.6% were co-infected. 79.1% of HIV-positive patients were tested for HCV, 29.2% of whom tested positive. 34.8% of HCV-positive patients were tested for HIV, 4.9% of whom tested positive. Of those tested, HCV-positive patients were significantly more likely than HCV-negative patients to test positive for HIV; HIV-positive patients were no more likely to test positive for HCV than HIV-negative patients. HIV-positive patients with substance use disorders (SUD) were significantly more likely to test HCV positive than those without. Within the total sample, veterans with SUD were significantly more likely to be tested for both diseases and to test positive for HCV but not HIV. After controlling for other categories of SUD, veterans with a history of cocaine abuse compared with those without were at an increased risk of HIV infection and co-infection. CONCLUSION: 79.1% of HIV-positive but only 34.8% of HCV-positive veterans were co-tested, suggesting barriers to HIV testing may exist in VISN 20. Results also indicate that HCV-positive patients are at increased risk for HIV infection and that HIV-positive patients with SUD are at increased risk of HCV infection; routine co-testing for these patients is therefore warranted. Given significant co-infection rates, HCV and HIV screening and testing should be increasingly integrated. Increased infection rates among patients with SUD also warrant integration of HCV and HIV screening and testing into mental health and addiction programmes.

Adult↗
PubMed2003

Integration of hepatitis B virus containing mutations in the core promoter/X gene in patients with hepatocellular carcinoma.

UNLABELLED: Integration of hepatitis B virus is thought to be an essential step in hepatitis B virus associated hepatocarcinogenesis. Mutations at nucleotides 1762 and 1764 in the hepatitis B virus, within a sequence encoding both the core promoter gene and the X gene, have been found frequently in patients with hepatocellular carcinoma. However, integration of these mutant sequences has not been reported to date. METHODS: A 228-base pair segment of the hepatitis B virus core promoter gene was amplified from hepatocellular carcinomas and adjacent non-tumourous liver tissue by nested PCR and sequenced. Integration of hepatitis B virus into human genomic DNA was investigated using the 'genome walking' method. RESULTS: Point mutations were found in both hepatitis B virus nucleotides 1762 and 1764 in 8 of 14 hepatocellular carcinoma tissues (57%) and in 11 of 14 adjacent non-tumourous liver tissues (79%). Three patients were evaluated using the 'genome walking' method; all were found to have hepatitis B virus DNA integrated in their hepatocellular carcinoma (two patients) and/or in their non-tumourous liver tissue (three patients). Integration occurred in all tissues near host genomic sites that are prone to integration. Hepatitis B virus was integrated at or near the hepatitis B virus DR1 site in all samples, and all contained truncated X gene sequences that have been reported to be capable of producing fusion transcripts with transactivation potential. CONCLUSIONS: Integrated hepatitis B virus DNA containing core promoter mutations at nucleotides 1762 and 1764 was found in hepatocellular carcinoma and/or adjacent non-tumourous liver tissue of three patients. These findings leave open the possibility that insertional mutagenesis or transactivation by fusion transcripts resulting from hepatitis B virus integration could play a role in hepatocarcinogenesis in some patients.

Aged↗
PubMed2005

DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration.

Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.

Animals↗
PubMed2004

Adeno-associated virus site-specific integration and AAVS1 disruption.

Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.

Chromosome Mapping↗
PubMed2002

Regulatory mechanisms by which barrier-to-autointegration factor blocks autointegration and stimulates intermolecular integration of Moloney murine leukemia virus preintegration complexes.

Retroviral integration is mediated by a preintegration complex (PIC) which contains the viral DNA made by reverse transcription together with associated protein factors. Prior to association with target DNA, the PIC must avoid suicidal intramolecular integration of its viral DNA (autointegration). We have demonstrated that barrier-to-autointegration factor (BAF) blocks the autointegration of Moloney murine leukemia virus (MoMLV) PICs in vitro. In this study, we show that BAF is an authentic component of MoMLV. Analysis of the sedimentation properties of initial, salt-stripped, and BAF-reconstituted PICs reveals that the viral DNA within the PIC is reversibly compacted by BAF, consistent with the functional role of BAF in protecting the viral DNA from autointegration. Furthermore, we find that BAF can promote the association of PICs with target DNA. Thus, our data suggest that BAF plays critical roles in promoting preferential intermolecular integration by both blocking autointegration and stimulating the capture of target DNA.

3T3 Cells↗
PubMed1978

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and Hölzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.

Cell Line↗
PubMed1997

Host site selection for concerted integration by human immunodeficiency virus type-1 virions in vitro.

Host site selection for full-site integration by human immunodeficiency virus type-1 (HIV-1) intergrase (IN) from nonionic detergent-lysed virions was investigated. Linear retrovirus-like DNA (469 bp) possessing 3' OH recessed long terminal repeat termini was efficiently inserted by a bimolecular donor reaction into a supercoiled DNA target (2867 bp), producing the HIV-1 5-bp host site duplication. Sequence data were analyzed from 193 donor-target recombinants obtained from the linear 3.8-kb DNA product. The selection of host target sites appeared randomly distributed and was independent of lysis and assay conditions. The fidelity of the 5-bp duplications in comparison to other size duplications was highest (94%) with high-salt (300 mM NaCl) lysis of the virions and 60 mM NaCl for strand transfer using Mg2+ as the divalent cation. Base sequence analysis demonstrated some biases in the 5-bp duplications at the sites of strand transfer and at the immediate host sequences surrounding the duplications. In addition to the observed duplications, approximately 30% of the recombinants isolated from the linear 3.8-kb DNA product contained specific and repetitive small-size deletions. No deletions smaller that 17 bp were observed and the distance between the deletion sets had a periodicity of approximately 10 bp. The mechanisms involved in how HIV-1 IN produces the 5-bp duplications and the repetitive host site deletions are discussed.

Cells, Cultured↗