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PubMed1999

Activation-induced resistance of human macrophages to HIV-1 infection in vitro.

Cells of the monocyte/macrophage lineage are the first targets of HIV-1 in patients and also serve as reservoirs for the virus during the course of infection. We investigated the effects of cell activation on early events of HIV-1 infection of monocyte-derived macrophages. Addition of LPS, a potent stimulator of macrophages, at the time of infection stimulated entry of HIV-1 into monocyte-derived macrophages, as judged by accumulation of early products of RT, but inhibited the synthesis of late RT products and strongly repressed nuclear import of the viral DNA, resulting in protection from infection. This effect was mediated by the CD14 receptor and involved activation of the p38 mitogen-activated protein kinase pathway. Disruption of this signaling pathway using a specific inhibitor of the p38 mitogen-activated protein kinase (SB203580) restored HIV-1 infection in the presence of LPS. These results suggest a novel view of the role of macrophage activation in anti-HIV responses of the immune system.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1989

Chromosomal assignment of woodchuck hepatitis virus (WHV) DNA integration sites in a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10).

The chromosomal sites of woodchuck hepatitis virus (WHV) DNA integration were identified in a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10) by the in situ hybridization technique using 3H-labelled WHV whole genome (WHV 2) as a probe. The G-banded chromosome spreads from WH257GE10 were identified and diagrammed schematically according to their band patterns. WHV DNA was integrated into 2 sites: 33 region of the long arm of chromosome 6 (6q33) and 31 region of the long arm of chromosome 8 (8q31). Chromosomal sites of WHV DNA integration were stable during successive passage periods analyzed in vitro.

Animals↗

PubMed1994

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in the hepatocellular carcinoma cell line HCC36.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.

Base Sequence↗

PubMed1988

Co-amplification of integrated hepatitis B virus DNA and transforming gene hst-1 in a hepatocellular carcinoma.

Transforming activity was detected in a hepatocellular carcinoma (HCC) carrying four integrated hepatitis B virus (HBV) DNA. This transforming gene was identified as hst-1, that lies in chromosome 11, band q13.3. One of the integrated HBV DNAs was found to lie close to the hst-1, and the hst-1 and the integrated HBV DNA were found to be co-amplified. The region of the amplification was limited. A model has been proposed that correlates the viral integration, amplification and activation of an oncogene.

Carcinoma, Hepatocellular↗

PubMed2001

Analysis of integrated hepatitis B virus DNA and flanking cellular sequence by inverse polymerase chain reaction.

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral-cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral-cellular junctions.

Cloning, Molecular↗

PubMed1999

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Animals↗

PubMed1985

Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP.

Among recombinant phages carrying integrated hepatitis B virus (HBV) DNA sequences cloned from the human hepatoma-derived cell line huSP, one clone, lambda hu-489, revealed some unusual features. The 2.25-kilobase Eco D fragment from the insert of this clone hybridized to the HBV DNA probe only and its nucleotide sequence was determined. The viral sequence, as well as a cellular flanking sequence, showed extensive rearrangement accompanied by inverted repetition. The Eco D fragment contained HBV DNA from the 5'-end region of gene S to the middle of gene X, followed by a long cellular flanking sequence. Moreover, a part of gene X was found inversely repeated at the head of the same gene S in a head-to-head configuration truncated by the same cellular sequence. Therefore, the same junction sequence of viral DNA and the cellular sequence was found at two different sites in the Eco D fragment in opposite polarities.

Base Sequence↗

PubMed1980

Chromatin conformation of integrated Moloney leukemia virus DNA sequences in tissues of BALB/Mo mice and in virus-infected cell lines.

The technique of preferential DNase I digestion of transcriptionally active chromatin regions was used to study the structural organization of integrated Moloney murine leukemia virus (M-MuLV) proviral sequences in various cells carrying integrated viral genomes. BALB/Mo mice, which carry M-MuLV as an endogenous virus at a single Mendelian locus, were used to examine the genetically transmitted viral genome copy and additional M-MuLV sequences acquired somatically during leukemogenesis. It has been shown previously that M-MuLV genome expression in these mice is restricted to lymphatic target tissues. In young homozygous BALB/Mo mice carrying one M-MuLV genome copy per haploid mouse genome in all cells we found that the genetically transmitted viral genome copy was in a preferentially DNase I-sensitive conformation in lymphatic target tissues, whereas in nontarget tissues the same sequence was not preferentially DNase I sensitive. This suggests that the chromatin conformation and the transcriptional activity of the integrated proviral genome are related to and probably determined by the state of cellular differentiation. In target tissues from BALB/Mo mice examined at different ages and in different stages of leukemogenesis the majority of the new somatically acquired M-MuLV sequences were preferentially DNase I digestible. A very similar pattern of DNase I digestibility was observed in target tissues from BALB/c mice exogenously infected with M-MuLV. This shows that in these tissues somatically acquired proviral sequences integrate preferentially or exclusively at sites of the host genome in which they are in a transcriptionally active chromatin conformation. Alternatively, the chromatin structure of the respective host genome region may be changed after the integration of viral DNA. In nontarget tissues from BALB/Mo mice the M-MuLV-specific sequences remained DNase I resistant throughout the lives of the animals. A different pattern of DNase I digestibility was observed in virus-infected cell lines which had been produced by low-multiplicity infection, cloned, and selected for virus production. When cell lines harboring different numbers of M-MuLV proviral copies were examined, it was found that a minority of the proviral sequences (on the average only one M-MuLV genome copy per haploid mouse genome) were preferentially digestible by DNase I, independent of the total number of proviral genome copies present. This suggests that the chromatin conformation of newly acquired proviral sequences is influenced by the state of differentiation of the infected cell or the way infected cells are selected or both.

Aging↗

PubMed1988

Nucleotide sequence of integrated hepatitis B virus DNA and human flanking regions in the genome of the PLC/PRF/5 cell line.

Three fragments of cellular DNA containing integrated hepatitis B virus (HBV) sequences have been cloned from the genomic library of a PLC/PRF/5 cell line. The complete nucleotide sequences of the HBV DNA inserts have been determined, including the S gene and the cellular flanking DNA. Viral sequences were found to be fragmented and rearranged. The nucleotide sequences of the HBV-HBV and HBV-human DNA junctions in two of the clones were precisely the same as described by others for the analogous HBV-DNA inserts, providing direct evidence for the stability of HBV-DNA integration pattern and sequence in the genome of the PLC/PRF/5 line. Two clones contain the HBV surface antigen gene which is well conserved. According to the amino acid sequence it could be related to the adw subtype. HBV DNA and contiguous human sequences in HC217 clone are flanked by the cellular perfect inverted repeat of at least 3.5 kb. Similar sequences have been found in the genome of the original PLC/PRF/5 cell line and human placental DNA.

Amino Acid Sequence↗

PubMed2001

Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase.

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.

Binding Sites↗

PubMed1980

DNA of avian myeloblastosis-associated virus type 2 integrates at multiple sites in the chicken genome.

The cellular sites of integration of the avian myeloblastosis-associated virus type 2 (MAV-2) DNA have been examined by Southern blot analysis of cellular DNA from infected cloned and uncloned chicken embryonic fibroblasts. Provirus-cell juncture fragments were not detected in restriction enzyme digests of DNA from MAV-2-infected uncloned cells. However, each MAV-2-infected cell clone examined produced a unique set of junctive bands. Thse findings indicate that multiple sites of integration exists for MAV-2 proviruses in cellular DNA.

Animals↗

PubMed1983

Susceptibility of Rous sarcoma virus-specific sequences integrated into SR-C3H/He mouse ascites sarcoma cell chromatin to DNase I and DNase II.

The susceptibility of Rous sarcoma virus (RSV) genomes integrated in mouse ascites sarcoma cells (SR-C3H/He cells) to DNase I and DNase II was investigated. Approximately half of the viral sequences were sensitive to DNase I and DNase II when 17% and 7.4% of the chromatin DNA was rendered acid soluble, respectively. The results suggest that newly acquired exogenous proviral sequences are integrated into both transcriptionally active and inactive regions of chromatin in cells lacking related endogenous viral sequences.

Animals↗

PubMed2001

CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression.

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

Adenine Phosphoribosyltransferase↗

PubMedDate not supplied

Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization.

Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.

Animals↗

PubMed1995

Integration of hepatitis B virus and alteration of the 1p36 region found in cancerous tissue of primary hepatocellular carcinoma with viral replication evidenced only in noncancerous, cirrhotic tissue.

We have studied the genetic profile of the host genome and hepatitis B virus (HBV) in HBV-associated primary hepatocellular carcinoma (HCC). Comparative analyses of HCC cell line Hep 40 and the original biopsy specimens showed the episomal and replicating form of HBV only in the biopsy specimen from nontumor (NT) cirrhotic liver tissue, where a molecular change in the 1p36 region was detected (NT tissue showed a normal 46XY karyotype). In contrast, only integrated HBV was detected in HCC tumor (T) tissue and Hep 40 cells. Two HBV integration sites were identical in HCC tissue and the hyperdiploid Hep 40 cell line, where genetic alteration in the 1p36 region was identified. These data indicate that viral replication is ongoing only in NT cirrhotic-hyperplastic, chromosomally normal tissue with evidence for genetic instability. Only the tumor cell with altered genotype has virus integrated

Carcinoma, Hepatocellular↗

PubMed1984

Characterization of cellular sequences flanking an integrated bovine leukemia virus genome.

We cloned a 20.2-kilobase fragment, including the unique proviral integration site, from the tumor tissue DNA of a bovine leukemia virus-induced lymphoma (tumor 1351). This clone contained 5.5 kilobases of flanking sequences at either side of the full-length integrated viral genome. Hybridization analysis of both 3' and 5' cellular flanking regions provided no evidence for rearrangements at this locus in other tumors, RNA transcription from either flanking region, or homology to eight known onc genes.

Animals↗

PubMed2000

Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance.

Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HBX variants from HBV-related human hepatocellular carcinoma (HCC). These HBX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HBX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HBX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HBX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HBX.

Amino Acid Sequence↗

PubMed1979

Integration of different sarcoma virus genomes into host DNA: evidence against tandem arrangement and for shared integration sites.

Cellular DNA of 50--54 S was extracted from chicken embryo cells doubly infected with two different avian sarcoma viruses and was analyzed by the infectious DNA assay. Approximately 80--90% of the transformed foci that were induced by this DNA were found to give rise to one kind of avian sarcoma virus only, indicating that most proviral genomes are not integrated in tandem. When the two infecting viruses were varied with respect to multiplicity or time of infection, the initial infecting virus or the virus of higher multiplicity of infection was recovered at higher frequency in foci produced by the extracted DNA. This observation suggests that existence of common integration sites for different avian sarcoma viruses. Cells uniformly infected with avian leukosis virus could be transformed by superinfection with an avian sarcoma virus from a different envelope subgroup. Infectious DNA recovered from such cells contained 3--10 50% infectious dose (ID50) units of leukosis virus per microgram but only 0.3--0.4 ID50 of sarcoma virus. DNA from cells infected with sarcoma virus alone contained 3 sarcoma virus ID50 per microgram. These results suggest that, even though a second virus integrates with lower efficiency into preinfected cells, there is not a complete block of integration sites by the first virus.

Animals↗