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PubMed1999

Inhibition of histone deacetylation augments dihydrotestosterone induction of androgen receptor levels: an explanation for trichostatin A effects on androgen-induced chromatin remodeling and transcription of the mouse mammary tumor virus promoter.

The integrated mouse mammary tumor virus (MMTV) promoter has provided an excellent model system with which to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The hormone response element (HRE) of this promoter is positioned on a phased nucleosome which becomes remodeled in response to steroids. One possible mechanism of chromatin remodeling by steroid receptors could involve recruitment of coactivators which alter the histone acetylation status of the HRE nucleosome. To examine how the androgen receptor (AR) influences transcription and chromatin remodeling and to assess whether changes in histone acetylation are involved in these effects, we determined whether the specific histone deacetylase inhibitor trichostatin A (TSA) influenced basal- and androgen-mediated transcriptional activation of the integrated MMTV promoter in the mouse L-cell fibroblast cell line 29+. These cells harbor the MMTV promoter integrated in the genome and express only one steroid hormone receptor subtype, i.e., the AR. Surprisingly, we found that treatment of the cells with TSA alone had virtually no effect on transcription and chromatin remodeling of the MMTV promoter nor on AR levels. However, pretreatment with TSA augmented the DHT effects on all three parameters. These results suggest that histone acetylation changes at the MMTV B nucleosome per se are not alone sufficient to induce chromatin remodeling and subsequent induction of MMTV transcription. Rather, the histone deacetylase inhibitor TSA exerts a portion of its effect on MMTV chromatin remodeling and transcriptional activation indirectly through increases in AR levels.

Acetylation↗

PubMed1987

Basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus G protein have been replaced by those of the influenza virus hemagglutinin.

Two integral membrane proteins, influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein, are transported to and accumulated on the apical and basolateral surfaces, respectively, of the plasma membrane of polarized epithelial cells. We have used chimeric constructions to identify the domains of HA and G proteins which contain the signals for polarized transport. Previously, we have shown that a chimeric protein containing the cleavable leader and the ectodomain of HA fused to the anchoring and cytoplasmic domains of G is transported to the apical surface of polarized MDCK cells (McQueen, N.L., Nayak, D.P., Stephens, E.B., and Compans, R.W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9318-9322). In this report we show that a chimera containing the cleavable leader and ectodomain of G fused to the anchoring and cytoplasmic domains of HA is transported to the basolateral surface of polarized cells. Another chimera which contains the leader sequence of G fused to leader minus HA is transported to the apical surface of polarized cells. These results taken together suggest that the signals for the polarized transport of HA and G proteins may reside in their ectodomains.

Amino Acid Sequence↗

PubMed1983

Human T-cell leukemia-lymphoma virus (HTLV): cloning of an integrated defective provirus and flanking cellular sequences.

Human T-cell leukemia-lymphoma virus (HTLV) is the first unequivocal human retrovirus. Seroepidemiological and virus isolation studies indicate that HTLV is etiologically associated with a subtype of adult T-cell malignancy. We have molecularly cloned approximately 1 kilobase of sequences derived from the 5' and 3' termini of the HTLV genome. Use of these clones as probes allowed isolation of a 9.8-kilobase EcoRI fragment from a genomic DNA library of an HTLV-infected neoplastic T-cell line (CR). Analysis of this clone revealed the presence of cellular sequences flanking approximately 5 kilobases of viral sequences including one long terminal repeat sequence. The 5' and 3' clones, as well as subclones derived from different regions of the genomic clone, were used as probes to compare integrated proviruses and viral RNA expression in different HLTV-infected neoplastic T cell lines. The results indicate that the infected cells are of clonal origin with respect to the virus integration sites and they express multiple viral mRNA species including a 35S RNA.

Cell Line↗

PubMed1993

A Burkitt lymphoma cell line with integrated Epstein-Barr virus at a stable chromosome modification site.

Fluorescence in situ hybridization (FISH), Southern, and slot blotting were used to detect Epstein-Barr virus (EBV) DNA and RNA sequences in a Burkitt's lymphoma (BL) cell line derived from a North American patient (NAB-2). FISH analysis after hybridization with a BamHI "V" region of EBV showed that NAB-2 cells have EBV genome integrated at a single site on the short arm of chromosome 2(p13). Single hybridization signals were detected at homologous sites on both chromatoids and nuclei. Furthermore, hybridization of intact nuclei without formamide denaturation and heat allowed the detection of single specific viral RNA transcripts visible as "tracks" or "traces." Southern blot analysis confirmed the integration of EBV genome into the host DNA. Quantification of slot blot hybridization revealed that NAB-2 cells have on average one copy of EBV per cell. Virus insertion into chromosomal DNA caused a stable modification site expressed as a distinctive achromatic region adjacent to the band 2p13. The chromatid lesion at the site of EBV integration involving a recombinogenic and fragile site may have contributed to the development of the NAB-2 BL.

Blotting, Southern↗

PubMed1976

Effect of Fv-1 gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses.

The amounts of unintegrated murine leukemia virus-specific DNA detected by molecular hybridization in extracts of Fv-ln/n (strains NIH/3T3, SIM) or Fv-lb/b (strains JLS-V9, SIM.R) mouse cells after infection with N- or B-tropic viruses were found to be the same in both permissive and resistant cells. Therefore, formation of DNA products from the viral RNA template does not appear to be grossly affected by the Fv-l gene product. Integration of virus-specific DNA into chromosomal cellular DNA was assayed by hybridization of radioactive complementary DNA to DNA from infected cells. With either NIH/3T3 or SIM.R cells infected with N- or B-tropic viruses, integration of proviral DNA could be detected in permissive cells but not in nonpermissive cells. The Fv-l gene product therefore appears to prevent integration of proviral DNA.

Animals↗

PubMed1992

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.

Base Sequence↗

PubMed1986

Selective integration of avian leukosis virus in different hematopoietic tissues.

Hematopoietic tissues obtained from avian leukosis virus (ALV)-infected Hyline SC chickens were analyzed for the presence of integrated viral DNA sequences. Cells were prepared from bone marrow, bursa, spleen, thymus, and peripheral blood. Following the removal of erythrocytes, cellular DNAs from each of these tissues were examined by Southern analysis. During the first few weeks of infection, DNA from the bone marrow contained as many as 0.5 copies of viral DNA per haploid genome. Cells from the bursa and peripheral blood contained between 0.05 and 0.15 copies per haploid genome. In contrast, neither splenic nor thymic DNA contained significant levels of viral DNA sequences even though infected birds developed titers of circulating virus between 10(5) and 10(6) IU/ml of plasma. DNA prepared from erythrocytes isolated from the peripheral blood of these birds contained approximately 0.4 copies of integrated viral sequences per haploid genome at 2 weeks after infection. Despite greater levels of integrated sequences in other tissues, by 9 weeks after infection viral sequences were detected only in DNA from bursal lymphocytes. Cells prepared from the spleen and thymus of infected birds were also examined for their size distribution, their internal complexity and their surface expression of immunoglobulin. None of the populations examined differed from normal, uninfected control preparations. These results suggest that ALV infection occurs primarily in the bone marrow and that the different tissues of the hematopoietic system are selectively infected. Further, these results indicate that ALV infection persists longer in bursal lymphocytes than in other hematopoietic tissues. Previous studies have demonstrated that the lymphoid tumors that develop in white leghorn chickens following ALV infection are bursal-dependent B-cell lymphomas that express immunoglobulin M. The observations presented in this communication offer, in part, an explanation for the restricted phenotype of the lymphoid tumor that develops in the SC chicken. Further, the data suggest an explanation for the bursal-dependent nature of the ALV-induced lymphoma.

Animals↗

PubMed1993

Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient viral replication in growth-arrested T cells.

Human immunodeficiency virus type 1 (HIV-1) replicates efficiently in nonproliferating monocytes and macrophages but not in resting primary T lymphocytes. To determine the contribution of cell division to the HIV-1 replicative cycle in T cells, we evaluated HIV-1 expression, integration of proviral DNA, and production of infectious progeny virus in C8166 T-lymphoid cells blocked in cell division by treatment with either mitomycin, a DNA cross-linker, or aphidicolin, a DNA polymerase alpha inhibitor. The arrest of cell division was confirmed by assay of [3H]thymidine uptake; the nondividing cells remained viable for at least 3 days after treatment. HIV-1 was expressed and replicated equally well in nondividing and dividing C8166 cells, as judged by the comparison of the levels of p24 core antigens in culture supernatants, the proportion of cells expressing HIV-1 specific antigens, the pattern and quantity of HIV-1 DNA present in the extrachromosomal and total cellular DNA fractions, and the biological activity of progeny viruses. A polymerase chain reaction-based viral DNA integration assay indicated that HIV-1 provirus was integrated in C8166 cells treated with either of the two inhibitors of cell division. Similar results were obtained by using growth-arrested Jurkat T-lymphoid cells. We conclude that cell division and cellular DNA synthesis are not required for efficient HIV-1 expression in T cells.

Aphidicolin↗

PubMed1980

Integration of hepatitis B virus sequences and their expression in a human hepatoma cell.

Hepatitis derived from hepatitis B virus (HBV) infection is endemic throughout the world, but it is particularly prevalent in Asia and Africa. In these areas, demographic studies show a strong coincidence between HBV infection (assayed by HBV antigenic markers) and the incidence of primary liver cancer. On these grounds, a causal link between HBV infection and primary hepatocellular cancer has been proposed. Recently, a human hepatoma cell line (PLC/PRF/5; Alexander cells) has been shown to produce hepatitis B surface antigen (HBsAg). We show here that the Alexander cell line contains at least six (four complete and two partial) hepatitis B viral genomes integrated into high molecular weight host DNA. An analysis using specific probes to fragments of the HBV genome suggests that integration of the virus in most cases occurs at the nicked cohesive end region of the virus. Expression of viral sequences using Northern blots demonstrates the presence of RNA transcripts specific for the surface antigen sequences of HBV DNA and the absence of detectable transcripts corresponding to the hepatitis B core antigen.

Carcinoma, Hepatocellular↗

PubMed2006

Integration site choice of a feline immunodeficiency virus vector.

We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a "bendable" structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus.

Animals↗

PubMed1974

Differences between the integration of avian myeloblastosis virus DNA in leukemic cells and of endogenous viral DNA in normal chicken cells.

The nature of integrated viral DNA in normal and leukemic chicken cells has been studied by sequential nucleic acid hybridization procedures that localize the viral specific DNA in cellular DNA regions differing in reiteration frequency. First, DNA.DNA reassociation was employed to fractionate cellular DNA sequences according to their reiteration frequencies. Next, the DNA in each fraction was denatured, immobilized on nitrocellulose filters, and then hybridized with viral [(3)H]RNA. In normal cells, endogenous viral DNA appears to be associated with cell sequences reiterated 1200 times, and each integration unit appears to have a maximal size approximately equivalent to the 35S RNA subunit of the virion. In infected cells, additional viral sequences are found which reassociate as if they integrated adjacent to unique cellular DNA, or in tandem with endogenous viral DNA.

Animals↗

PubMed2000

Effects of antiandrogens on chromatin remodeling and transcription of the integrated mouse mammary tumor virus promoter.

Inhibition of the ligand-activated androgen receptor (AR) by antiandrogens plays an important role in the treatment of various hyperandrogenic disorders including prostate cancer. However, the molecular mechanisms of antiandrogen activity in vivo remain unclear. In this study we analyzed the effects of cyproterone acetate (CPA), flutamide (F), and hydroxyflutamide (OHF) on transcriptional activation and chromatin remodeling of the genomically integrated mouse mammary tumor virus (MMTV) promoter. This promoter has provided an excellent model system to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The MMTV hormone response element is positioned on a phased nucleosome, which becomes remodeled in response to steroids. We utilized this model system in mouse L-cell fibroblasts that contain a stably integrated MMTV promoter. In these cells, dihydrotestosterone (DHT) induced a large increase of AR protein levels that correlated with transcriptional activation and chromatin remodeling of the MMTV promoter. Coadministration of DHT and CPA or DHT and OHF in these cells inhibited the increase of AR levels, which resulted in a strong blockage of transcriptional activation and chromatin remodeling of the MMTV promoter. In contrast, F had no significant influence on these activities. We conclude that a major portion of the antiandrogenic effects of CPA and OHF in vivo are mediated by the reduction of AR levels.

Androgen Antagonists↗

PubMed1993

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate.

Amino Acid Sequence↗

PubMed1981

Purification of measles virus glycoproteins and their integration into artificial lipid membranes.

We report a simple method for the isolation of the measles virus glycoproteins, and their subsequent incorporation into artificial lipid bilayers. The two viral glycoproteins, HA and F, were isolated in preparative amounts from disrupted purified virus by lentil lectin affinity chromatography. The proteins were reconstituted into single bilayer lipid vesicles by: (i) exchanging the non-dialysable detergent Nonidet P40 (NP40) for a dialysable one, octylglucoside, while the proteins were immobilized on the lectin column and (ii) co-dialysis of the eluted glycoproteins in octylglucoside with phosphatidylcholine. The resultant 'virosomes' had visible 'spikes' and possessed haemagglutinating activity. These measles virosomes should provide a useful reagent for studying immune responses to measles virus, independent of the immunosuppressive effects of the whole virus.

Centrifugation, Density Gradient↗

PubMed2005

Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis.

Growing evidence demonstrates that hepatitis B virus (HBV) integration and resulting insertional mutagenesis play an important role in cell growth or maintenance in hepatocellular carcinomas (HCCs). To determine if HBV integration occurs and affects cellular genes at such a stage of infection, we analysed viral-host junctions in chronic hepatitis tissues without HCC using PCR amplification with primers specific to human Alu-repeat and HBV. We obtained 42 independent viral-host junctions from six patients examined and identified chromosomal locations for 20 of the 42 junctions. In six clones, each integration apparently affected a single gene. These six candidate genes included one known tumor suppressor gene, three human homologs of drosophila genes that are critical for organ development, one putative oncogene and one recently found chemokine. Our data, together with previously reported HBV integrants in HCCs, suggested preferential HBV integration into chromosome 3 (P = 0.022). Our virus-tagging approach provided (a) firm evidence of HBV integration in hepatocytes at an early stage of chronic infection and (b) revealed cellular genes possibly affected by HBV integration and potentially involved in early steps of the process leading to carcinogenesis.

Biopsy↗

PubMed2026

Genomic and transcriptomic features of HBV integration in treatment-naïve, HBeAg-positive children with chronic HBV infection.

BACKGROUND: Hepatitis B virus (HBV) integration represents a major obstacle to curing HBV; however, the landscape of HBV integration and local immune response to transcriptionally active viral integration in children with chronic HBV infection remain unclear. Herein, we aimed to elucidate this landscape in this population. METHODS: Genomic analyses using a probe-based capture strategy were performed on 18 children and 28 adults with chronic HBV infection. Spatial transcriptomics (ST) was performed on 12 children from our cohort and 3 adults from a public database. FINDINGS: All patients were hepatitis B e antigen (HBeAg)-positive and treatment-naïve. Genomically, children exhibited significantly lower clonal expansion level of HBV-integrated hepatocytes than adults, despite comparable unique breakpoint counts. After adjusting for confounding variables, age was identified as an independent risk factor for total frequency of unique integration breakpoints (b = 3.22, P = 0.005). Spatially, ST revealed that spots with transcriptionally active viral integration exhibited a sparse distribution and accounted for a low proportion of all spots in children. Notably, at these spots, children showed reduced adaptive immune cells (e.g., CD8+ T cells) but increased innate components (myeloid cells, Kupffer cells, activated dendritic cells) and APC co-stimulation, whereas adults exhibited a uniform reduction of immune cell populations. INTERPRETATION: Compared with adults, children exhibit lower clonal expansion of HBV-integrated hepatocytes and distinct immune profiles in response to transcriptionally active viral integration, offering new insights into their differing clinical course. FUNDING: Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education).

Humans↗

PubMed1977

Integration of genomes on infectious viruses.

The article contains a review relating to the integration of genomes of infectious viruses in Eukaryotic cells. Data are presented that were obtained in the study of tissue cultures chronically infected with viruses, in the study of pathologic and ecologic processes. Based on the evidence accumulated, some theoretical and applied aspects of the problem are discussed.

Cell Line↗

PubMed2006

Novel pathway of human immunodeficiency virus type 1 uptake and release in astrocytes.

Astrocytes persistently infected with HIV-1 can transmit virus to CD4+ cells, suggesting that astrocytes may be a source of viral persistence and dissemination in the brain. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. HIV-1 was observed in vesicle-like structures. Unspliced genomic RNA and extrachromosomal HIV-1 DNA were detected in astrocytes, with levels declining over time. The extrachromosomal viral DNA was not de novo reverse transcribed in astrocytes but most likely the products of intravirion reverse transcription present in the virus inoculum. Integrated HIV-1 DNA was not detected in assays sensitive to detect 2 integrated copies of provirus. However, the majority of astrocyte cultures released infectious virus that could be transmitted to CD4+ cells. Our findings suggest a novel pathway of HIV-1 uptake and release in astrocytes that does not necessarily require virus replication, which may contribute to persistence and spread of HIV-1 in the brain.

Astrocytes↗