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At least 613 records · Page 34Linked to original sources
PubMed1986

Integration of hepatitis B virus DNA in hepatocellular carcinoma.

Integration of hepatitis B virus (HBV) DNA into genomic DNA was investigated in 34 livers bearing hepatocellular carcinoma (HCC) by Southern blot hybridization using 32P-labeled, cloned and purified HBV DNA as a probe. Rehybridization of nitrocellulose paper with a probe containing only the cloning vector was performed after dehybridization to avoid possible false-positive results. Integrated HBV DNA was detected in all 9 hepatitis B surface antigen (HBsAg)-seropositive cases and 3 out of 25 (12%) HBsAg seronegative cases. The hybridization patterns of viral DNA were the same among several cancer nodules in two HCC cases with multiple liver tumors, indicating unicentric hepatocarcinogenesis in these two cases. These results, obtained with avoidance of false-positive results, showed that only a minority of HBsAg-seronegative HCC cases in Japan had demonstrable HBV DNA in the tumors studied by the Southern blot hybridization technique.

Carcinoma, Hepatocellular↗
PubMed1990

Detection of integrated hepatitis B virus DNA in hepatocellular carcinoma cell lines by nonradioactive in situ hybridization.

A sensitive and specific nonradioactive in situ hybridization method capable of detecting single-copy integrated hepatitis B virus (HBV) DNA sequences in hepatocellular carcinoma (HCC) cell lines was developed. In situ hybridization of biotinylated HBV (adr, adw) DNA probes with nine different human HCC cell lines were carried out in 96-well microtiter plates. Integration was detected in HCC cell lines HCCM, Hep3B, huH-1, huH-4, and PLC/PRF/5. Detection of single-copy HBV DNA sequences was also achieved in Hep3B and huH-4. HCC cell lines HepG2, HUH-6, HUH-7, Mahlavu, and the non-HCC control MCF-7, gave clear negative results. These results show a 100% correlation with those obtained by Southern blot hybridization assay. The results demonstrate that nonradioactive detection of single-copy integrated HBV DNA sequences in HCC cell lines is possible by the method described, which has potential application for viral genome analysis requiring in situ hybridization.

Carcinoma, Hepatocellular↗
PubMed1976

Host restriction of Friend leukemia virus: synthesis and integration of the provirus.

Host restriction of exogenous infection by murine leukemia viruses is controlled in vitro predominantly by the murine Fv-1 locus. The mechanism of this host restriction was investigated by comparing the early events in the replication of N-tropic versus B-tropic Friend leukemia virus in NIH 3T3 cells. These cells, which are Fv-1nn in type, are permissive for the N-tropic strain, but nonpermissive for the B-tropic strain, which replicates permissively in Balb/c cells. We have studied the synthesis, intracellular location, and molecular form of virus-specific DNA early in replication by means of molecular hybridization with a virus-specific DNA probe. Our results suggest that in the permissive infection viral DNA rapidly becomes integrated with cellular DNA. However, in the nonpermissive infection, although almost equal amounts of both positive and negative strand viral DNA are synthesized, integration of the provirus does not occur.

Cell Line↗
PubMed1990

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells.

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.

Animals↗
PubMed1994

Identification of integrated Epstein-Barr virus in nasopharyngeal carcinoma using pulse field gel electrophoresis.

Integrated Epstein-Barr virus (EBV) was found in 4 of 17 pathologically diagnosed, EBV-positive nasopharyngeal carcinoma (NPC) biopsy samples using pulse field gel electrophoresis (PFGE). The analyses were carried out after digestion of biopsy DNA with rare restriction enzymes such as PacI, which does not cleave EBV due to the absence of the recognition sequence, and ClaI. Employing this technique, EBV integration in IB4, through EcoI, and Namalwa through the terminal repeats, was confirmed. We also established that integration of EBV in AW Ramos was through the terminal repeats. The Raji cell line was also found to harbour integrated virus in addition to episomal DNA. However, the site of integration could not be confirmed, since as the Raji DNA appeared to be heavily methylated and could not be cleaved with CPG rare cutters. As in the Raji cell line, the DNA of EBV in nasopharyngeal-carcinoma (NPC) biopsies also appeared to be methylated.

Animals↗
PubMed1986

Cloning and analysis of integrated hepatitis B virus DNA of the adr subtype derived from a human primary liver cell carcinoma.

A 10 kb genomic DNA fragment derived from a human primary liver cell carcinoma (PLC) and containing integrated hepatitis B virus (HBV) DNA was cloned and analysed. Physical mapping showed the viral DNA to comprise a linear sequence of at least 2.8 kb (87%) of the HBV genome and to be of the adr subtype. Integration appeared to have occurred in the region of the viral genome spanning the cohesive ends. The cellular flanking DNA sequences to one side of the integrated viral DNA contained repeats of the Alu family. The finding of no apparent rearrangements of the integrated HBV DNA sequences in this clone is in contrast to the situation in the huSP and PLC/PRF/5 PLC cell lines in which the integrated viral DNA sequences are greatly rearranged and suggests that such rearrangements may be atypical of solid PLCs.

Base Sequence↗
PubMed2004

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

Acetoacetates↗
PubMed1993

Rous sarcoma virus genome stably integrated into mouse ascites sarcoma cells.

The organization of RSV genes present in the mouse ascites sarcoma cells was analysed. The transformed cells kept in vivo and in vitro for a long time continued to contain intact RSV genome and the cellular integration site was unchanged. RSV was recovered by transfection of chick embryo cells with these cellular DNA. As these cells, especially in ascites form, grow very rapidly and it is easy to obtain a large amount of cells at a time, they are useful for further study as RSV-transformants derived from mouse cells.

Animals↗
PubMed1999

Double-stranded linear duck hepatitis B virus (DHBV) stably integrates at a higher frequency than wild-type DHBV in LMH chicken hepatoma cells.

Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5' and 3' ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence.

Animals↗
PubMed2005

A three-dimensional model of the human immunodeficiency virus type 1 integration complex.

While the general features of HIV-1 integrase function are understood, there is still uncertainty about the composition of the integration complex and how integrase interacts with viral and host DNA. We propose an improved model of the integration complex based on current experimental evidence including a comparison with the homologous Tn5 transposase containing bound DNA and an analysis of DNA binding sites using Goodford's GRID. Our model comprises a pair of integrase dimers, two strands of DNA to represent the viral DNA ends and a strand of bent DNA representing the host chromosome. In our model, the terminal four base pairs of each of the viral DNA strands interact with the integrase dimer providing the active site, while bases one turn away interact with a flexible loop (residues 186-194) on the second integrase dimer. We propose that residues E152, Q148 and K156 are involved in the specific recognition of the conserved CA dinucleotide and that the active site mobile loop (residues 140-149) stabilises the integration complex by acting as a barrier to separate the two viral DNA ends. In addition, the residues responsible for DNA binding in our model show a high level of amino acid conservation.

Amino Acid Sequence↗
PubMed2001

Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA.

We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 x 10(5) cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for approximately 10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells.

Cell Line↗
PubMed1987

Integration of hepatitis B virus: analysis of unoccupied sites.

Hepatitis B virus (HBV) sequences integrated in the PLC/PRF/5 cell line (Alexander cells), which was derived from a human primary liver carcinoma, were previously extensively studied. Here we describe the analysis of the unoccupied sites of two linearly integrated forms of HBV DNA, AL-14 and AL-26, that were characterized previously. No major cellular DNA rearrangements were seen at the integration sites except for small deletions of host sequences: 2 kilobases of DNA in AL-14 and 17 base pairs (bp) in AL-26. The unoccupied site of AL-26 was found to be missing 182 bp, which previously mapped next to the right end of the integration sites of several independent clones. These were believed to be of cellular origin, but we show here that these 182 bp are in fact from unusual HBV sequences. Surprisingly, a region of this newly detected HBV DNA sequence is more homologous to that of woodchuck HBV DNA. Our analysis shows that the normal counterparts of both AL-14 and AL-26 contain minisatellite-like repetitive sequences. Based on the data presented here and our previous finding of HBV DNA integration at satellite sequences, we propose that genomic simple repetitive sequences are hot spots for HBV DNA integration.

Base Sequence↗
PubMed2003

Transcriptional suppression of in vitro-integrated human immunodeficiency virus type 1 does not correlate with proviral DNA methylation.

Persistence of human immunodeficiency virus type 1 (HIV-1) constitutes a major obstacle in the control of HIV-1 infection. Here we investigated whether the CpG methylation of the HIV-1 promoter can directly influence the expression of the HIV-1 genome and thereby contribute to the persistence and latency of HIV-1. The levels of CpG methylation in the promoter of HIV-1 were studied after bisulfite-induced modification of DNA in five Jurkat clonal cell lines transduced by an HIV-1 long terminal repeat (LTR)-driven retroviral vector and expressing enhanced green fluorescent protein (GFP) and in primary resting memory T cells challenged with HIV-1 or with an HIV-1-derived retroviral vector. Basal HIV-1 promoter activities were low or undetectable in three tested HIV-1 LTR-GFP clones, one of which encoded the Tat protein, and they reached medium or high levels in two other clones. The CpG dinucleotide that occurred in a latently infected clonal cell line 240 nucleotides upstream of the transcription start remained methylated after reactivation of HIV-1 transcription with 10 nM phorbol-12-myristate-13-acetate. In two clones showing a medium promoter activity and in resting memory T cells, the HIV-1 LTR was generally not methylated. Our results show that the methylation profiles of the HIV-1 LTR, including those present in latently infected cells, are low and do not correlate with the transcriptional activity. We suggest that, in a noncloned cellular population in which the HIV-1 proviruses are randomly integrated in the human genome, HIV-1 latency is imperfectly controlled by CpG methylation and is inherently accompanied by residual replication.

Base Sequence↗
PubMed2006

Adeno-associated virus site-specific integration is regulated by TRP-185.

Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3'-->5' DNA helicase activity.

Binding Sites↗
PubMed1989

The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species.

The respiratory syncytial virus (RSV) 1A protein was previously identified as a 7.5-kilodalton (kDa) nonglycosylated species that, on the basis of its predicted sequence determined from the sequence of its mRNA, contains a hydrophobic central domain that was suggestive of membrane interaction. Here, four major, structurally distinct intracellular species of the 1A protein were identified in cells infected by RSV or by a recombinant vaccinia virus expressing the 1A gene. The four species of 1A were: (i) the previously described, nonglycosylated 7.5-kDa species that appeared to be the full-length, unmodified 1A protein; (ii) a nonglycosylated 4.8-kDa species that was carboxy-coterminal with the 7.5-kDa species and might be generated by translational initiation at the second AUG in the sequence; (iii) a 13- to 15-kDa species that contained one or two N-linked carbohydrate side chains of the high-mannose type; and (iv) a 21- to 30-kDa glycosylated species that appeared to be generated from the 13- to 15-kDa species by further modification of the N-linked carbohydrate. All four forms of the 1A protein were synthesized and processed on intracellular membranes, and several lines of biochemical evidence showed that all four species were integral membrane proteins. Thus, the 1A protein is a third RSV integral membrane protein and is present as such in both glycosylated and nonglycosylated forms. With the use of antiserum raised against a synthetic peptide representing the C terminus of the 1A protein, indirect immunofluorescence showed that the 1A protein was expressed at the cell surface. Antibody-antigen complexes formed at the surface of intact infected cells were immunoprecipitated, showing that the 7.5-kDa, 13- to 15-kDa, and 21- to 30-kDa, but not the 4.8-kDa, species, were accessible to extracellular antibodies. Thus, the 1A protein is a candidate to be a viral surface antigen. The small size, gene map location integral membrane association, and cell surface expression of the 1A protein strongly suggested that it is a counterpart to the SH protein that has been described for simian virus type 5. We suggest that, in the future, the RSV 1A protein be given the same designation, namely, SH.

Amino Acid Sequence↗
PubMed1992

Generation of deletion mutants of simian immunodeficiency virus incapable of proviral integration.

Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN) domain of the pol gene. The resulting plasmid was transfected into HUT-78 and human rhabdomyosarcoma cells. In comparison with the parental plasmid DNA transfected in parallel, the deletion mutant was found to direct efficient production of virus in both cell systems. Viruses derived from wild-type and mutant proviral DNAs were also tested for their relative replicative abilities in HUT-78 and U937 cells, and the kinetics of virus production was found to vary between these two cell systems. Analysis of DNA from infected cell nuclei showed that the deletion mutant lacked the ability to integrate despite being able to produce infectious virus. Using the sensitive polymerase chain reaction technique, we have clearly demonstrated the absence of the IN domain in the deletion mutant after infection and replication in HUT-78 cells. Such mutants might form the basis for the development of an experimental live attenuated vaccine.

Base Sequence↗
PubMed1990

Integration of hepatitis B virus DNA and its implications for hepatocarcinogenesis.

Hepatitis B virus (HBV) DNA integrates into human hepatocyte DNA. We have gathered the available data on the structure of the integrants from human hepatocellular carcinomas, and classified them into those that seem to represent primary integrants and those that are the products of secondary rearrangements. By means of structural analyses of the possible primary integrants, we deduced that the replication intermediates of the viral genome are the preferred substrates for integration. The integrated HBV DNA and the target cellular DNA are invariably associated with deletions, possibly reflecting the substrate for, and the mechanism of, the integration reaction. The target cell DNA sequence, as well as the target site of integration in chromosomes, seems to be selected randomly, suggesting that HBV DNA integration should bring about random mutagenic effects. Several samples recovered from hepatocellular carcinomas show that the integrated HBV DNA can mediate secondary rearrangements of chromosomes, such as translocations, inversions, deletions and (possibly) amplifications. Thus, HBV DNA integration causes multiple mutagenic effects. We argue that during hepatitis infection, the tendency of rearrangement of hepatocyte chromosomes is combined with the forcible turnover of cells. This is a constantly operating system for the selection of cells that grow better than average cells, possibly involving important steps in multistaged hepatocarcinogeneses.

Carcinoma, Hepatocellular↗
PubMed2004

Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliability of waterborne virus detection.

The total culturable virus assay (TCVA) and an integrated cell culture-PCR (ICC-PCR) were compared in parallel to evaluate their detection reliability. Source, finished, and tap water samples from three drinking water treatment plant systems were analyzed by TCVA, and every cell culture dish was subsequently examined by reverse transcription (RT) multiplex PCR using enterovirus- and adenovirus-specific primers. Twenty-seven of 180 (15%) inoculated dishes exhibited cytopathic effects (CPE). Virus concentrations for source water ranged from 3.3 to 21.0 most probable numbers of infectious units (MPN) per 100 liters. No finished or tap water samples were positive. On the other hand, 38 (21%) of the dishes were positive in multiplex ICC-PCR. Virus concentrations ranged from 4.5 to 10.2 MPN/100 liters for source water and 0 to 0.9 MPN/100 liters for finished and tap water. In spite of its superior sensitivity, the ICC-PCR assay resulted in lower virus concentration values than the TCVA for two of the source water sites. Retest of the CPE-positive dishes using reovirus-specific RT-PCR revealed that 24 of the 27 (89%) dishes were also positive for reoviruses. These observations suggested that the detection reliability of ICC-PCR is restricted by the primer sets that are integrated in the reaction mixture. The observation of an uneven distribution of PCR-positive culture dishes in a given sample raises an additional caution that simple extrapolation of the ICC-PCR result from the analysis of a limited fraction of collected samples should be avoided to minimize possible over- and underestimation of the amount of virus.

Adenoviridae↗