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At least 1,225 records · Page 68Linked to original sources
PubMed2000

Hepatitis B virus-transfected Hep G2 cells demonstrate genetic alterations and de novo viral integration in cells replicating HBV.

Hepatitis B virus (HBV) is a major etiological factor associated with hepatocarcinogenesis, but its role in the transformation process remains unclear. We previously documented the accumulation of genetic alterations in a HBV-transfected cell line. In the present study, we addressed the effect of HBV and its replication on the genome and phenotype of the host cell. Parental HBV-free Hep G2 cells and two HBV-transfected variant lines Hep G2215 and Hep G2T14. 1, which do and do not replicate HBV, respectively, were used to monitor genetic alterations in conjunction with HBV profile in vitro and in vivo. Comparison of in vitro growth rates showed that Hep G2T14.1 cells grew more rapidly, while Hep G2215 cells, replicating HBV, grew slower than parental Hep G2 cells. Molecular analysis confirmed an HBV integration site (s) in both variants, and reverse trancriptase-polymerase chain reaction (RT-PCR) amplification documented expression of transcript for the HBX protein, which has recently been implicated in the compromised efficiency of cellular DNA repair. Tumorigenisity testing indicate a comparable rate of tumor formation in nude mice of both HBV-transfected variants, giving rise to tumors in 3 weeks; parental Hep G2 cells did not form tumors in nude mice. Tumor tissue from nude mice injected with Hep G2T14.1 cells showed no change in HBV status. However, a new HBV integration site was detected in tumor tissue from Hep G2215-injected mice. Two cell lines derived from the respective tumor tissue grew in vitro at rates compatible to those observe before passage in nude mice. The Hep G2215 tumor-derived line continued to replicate HBV, while HBV status remained unchanged in the Hep G2T14.1 tumor-derived line. Unique genetic alterations were detected in both transfected cell lines, and Hep G2215 cells particularly showed cellular mosaicism and clonal selection when analyzed after the passage in nude mice. Further genetic alterations were detected in tumor-derived cell lines. Interestingly, the de novo genetic alterations in the Hep G2215 cells, which maintain the ability to replicate HBV, included a new HBV integration site, several chromosome rearrangements and loss of heterozygosity (LOH) of one p53 allele. Western analyses of p21/Waf1 protein indicate an upregulation of the protein in cells that replicate HBV. Based on the combined data, we hypothesize that the genetic alterations in the cellular genome could also be generated as a function in the presence of HBV and HBV replication. Possible mechanisms that could be implicated in cumulative mutagenetic events are discussed.

Animals↗
PubMed1998

The effect of different immune responses on the evolution of virulent CXCR4-tropic HIV.

We use mathematical models to determine possible mechanisms contributing to the evolution and rise of virulent CXCR4-tropic HIV in vivo. The models predict that the ability of the virus to specialize on a given target cell type depends on the exact fitness landscape of the viral mutants. Because this fitness landscape varies between people, this may explain why the evolution of fully CXCR4-tropic strains only occurs in about 50% of infected patients. Assuming that CXCR4-tropic HIV may evolve, we investigate the effect of different immune responses on the rise of such virulent strains. If we assume that CXCR4-tropic HIV is more cytopathic than CCR5-tropic virus, virulent CXCR4-tropic mutants remain suppressed at low levels both in the absence of an immune response, and in the presence of responses that act on the virus before integration into the host genome. On the other hand, this difference in cytopathogenicity is reduced by the presence of immune responses acting on infected cells, allowing CXCR4-tropic HIV to coexist with the CCR5-tropic virus. These results may help to interpret experimental data and are discussed with reference to the literature.

HIV↗
PubMed2005

LFA-1 is a key determinant for preferential infection of memory CD4+ T cells by human immunodeficiency virus type 1.

Memory CD4+ T cells are considered a stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) and a barrier to eradication of this retroviral infection in patients under therapy. It has been shown that memory CD4+ T cells are preferentially infected with HIV-1, but the exact mechanism(s) responsible for this higher susceptibility remains obscure. Previous findings indicate that incorporation of host-derived intercellular adhesion molecule 1 (ICAM-1) in HIV-1 increases virus infectivity. To measure the putative involvement of virus-anchored ICAM-1 in the preferential infection of memory cells by HIV-1, quiescent and activated naive and memory T-cell subsets were exposed to isogenic virions either lacking or bearing ICAM-1. Memory CD4+ T cells were found to be more susceptible than naive CD4+ T cells to infection with ICAM-1-bearing virions, as exemplified by a more important virus replication, an increase in integrated viral DNA copies, and a more efficient entry process. Interactions between virus-associated host ICAM-1 and cell surface LFA-1 under a cluster formation seem to be responsible for the preferential HIV-1 infection of the memory cell subset. Altogether, these data shed light on a potential mechanism by which HIV-1 preferentially targets long-lived memory CD4+ T cells.

CD4-Positive T-Lymphocytes↗
PubMed2003

The ion channels coded by viruses.

Pathogenicity and virulence are multifactorial traits, depending on interaction of viruses with susceptible cells and organisms. The ion channels coded by viruses, viroporins, represent only one factor taking part in the cascade of interactions between virus and cell, leading to the entry of virus, replication and to profound changes in membrane permeability. The M2 protein from influenza A virus forms proton-selective, pH-regulated channel involved in regulating vesicular pH, a function important for the correct maturation of HA glycoprotein. The NB glycoprotein of influenza B viruses is an integral membrane protein with an ion channel activity. The CM2 protein of influenza C virus is an integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. The picornavirus 3A protein is involved in cell lysis and shows homology with other lytic proteins. Vpu is an oligomeric integral membrane protein encoded by HIV-1, which forms ion channels. The togavirus 6K protein shows structural similarities with other viroporins.

Animals↗
PubMed2000

Mechanisms of retrovirus-induced oncogenesis.

Retroviruses are implicated in a series of human and animal tumours such as leukaemias, mammary tumours or skin cancer. The mechanism that they use to induce tumour formation varies. Insertional mutagenesis is a common mechanism in rodent, feline and avian retroviruses, where the retrovirus integrates into the host genome and affects the transcription of the neighbouring genes. Cloning of these affected genes led to identification of a series of oncogenes that play a significant role in the induction of human neoplasms. Retrovirus insertion also serves as a model to identify collaborating oncogenes. Human retroviruses use different, more complex mechanisms contributing to oncogenesis. Studies of the propagation and induction mechanisms used by retroviruses have given insight to the understanding of oncogenesis.

Animals↗
PubMed2003

Integration efficiency of a hybrid adenoretroviral vector.

The frequency with which the hybrid vector AdLTR-luc mediates genomic integration [Nat. Biotech. 18 (2000) 176-180] is unknown. To address this, we constructed AdLTR-red, using the AdLTR-luc backbone and the enhanced red fluorescence protein cDNA. Kinetic studies showed that AdLTR-red and a conventional adenoviral vector, AdCMV-red, entered and transduced epithelial cells comparably. AdLTR-red integration frequency in vitro, i.e., the percentage of red clones after 10-12 doubling times from limiting dilutions, was 8.0% (36/450; at 67 particles/cell). With AdCMV-red, 0/549 clones were integration-positive. Seven days after AdLTR-luc or AdCMV-luc (10(11) particles) femoral vein administration to adult rats splenocytes were prepared, stimulated with concanavalin A, and examined by FISH. AdLTR-luc integration occurred in 5-11% of mitotic rat splenocytes, while no unequivocal integration was found with AdCMV-luc. These data provide the first quantitative evidence of the frequency for genomic integration with this hybrid vector.

Adenoviridae↗
PubMed2004

HIV-1 gene expression: lessons from provirus and non-integrated DNA.

Replication of HIV-1 involves a series of obligatory steps such as reverse transcription of the viral RNA genome into double-stranded DNA, and subsequent integration of the DNA into the human chromatin. Integration is an essential step for HIV-1 replication; yet the natural process of HIV-1 infection generates both integrated and high levels of non-integrated DNA. Although proviral DNA is the template for productive viral replication, the non-integrated DNA has been suggested to be active for limited viral gene synthesis. In this review, the regulation of viral gene expression from proviral DNA will be summarized and issues relating to non-integrated DNA as a template for transcription will be discussed, as will the possible function of pre-integration transcription in HIV-1 replication cycle.

Animals↗
PubMed1997

MuLV-insertional mutagenesis of c-myb and Mml1 in a murine model for promonocytic leukemia.

Analysis of retroviral integration sites in MuLV-induced promonocytic leukemias has determined that two genetic loci, c-myb and Mml1, can contribute to disease development but not in the same leukemia. Recent studies aimed at understanding the function of Myb in leukemia development have focused on the consequences of ectopic Myb expression on monocytic and granulocytic differentiation in vitro. In all instances Myb was shown to block growth arrest but not commitment to differentiation, a result which is consistent with observed effects of Myb in leukemia development. No effect of Myb protein truncation was observed in these studies although similar truncations are produced as a result of insertional mutagenesis. Common integration site, Mml1, was recently identified and mapped to mouse chromosome 10 within 1cM of c-myb. Despite its linkage to c-myb, Myb mRNA and protein expression appear to be unaffected in leukemias with Mml1 integrations.

Animals↗
PubMed2002

Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Adenoviridae↗
PubMed1996

Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias.

Retroviruses induce myeloid leukaemia in BXH-2 mice by the insertional mutation of cellular proto-oncogenes or tumour suppressor genes. Disease genes can thus be identified by proviral tagging through the identification of common viral integration sites in BXH-2 leukaemia. Here, we describe a new approach for proviral tagging that greatly facilitates the identification of BXH-2 leukaemia genes. Using this approach, we identify three genes whose expression is activated by proviral integration in BXH-2 leukaemias; Hoxa7, Hoxa9, and a Pbx1-related homeobox gene, Meis1. Proviral activation of Hoxa7 or Hoxa9 is strongly correlated with proviral activation of Meis1 implying that Hoxa7 and Hoxa9 cooperate with Meis1 in leukaemia formation. These studies provide the first genetic evidence that Pbx1-related genes cooperate with Hox genes in leukaemia formation and identify a number of new murine myeloid leukaemia genes.

Animals↗
PubMedDate not supplied

Adaptive virus detection using filament-coupled antibodies.

We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10(10) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.

Antigen-Antibody Complex↗
PubMed1994

CD30-positive lymphoma in human T-cell leukaemia virus type 1 carrier without monoclonal integration of HTLV-1.

CD30, Ki-1 antigen, an activated T-cell antigen, is a member of the nerve growth factor receptor family. This antigen is expressed on the lymphoma cells of some adult T-cell leukaemia/lymphoma (ATL/L) patients and some patients with Epstein-Barr virus infection. CD30-positive large cell cutaneous T-cell lymphomas occasionally integrate a defective HTLV-1 provirus. We describe here an HTLV-1 carrier who developed Ki-1 lymphoma with no evidence of monoclonal integration of the HTLV-1 proviral sequence.

Blotting, Southern↗
PubMed1980

Integration site(s) of herpes simplex virus type 1 thymidine kinase gene and regional assignment of the gene for aminoacylase-1 in human chromosomes.

To investigate the chromosomal sites of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in HSV-1-transformed human HeLa(BU25)/KOS 8-1 cells, the biochemically transformed cells were fused with TK-negative mouse LM(TK-) cells, and human-mouse somatic cell hybrid lines (LH81) were isolated using a HATG-ouabain selection system. The presence of HSV-1 TK activity in the hybrid lines was verified by disc polyacrylamide gel electrophoresis (PAGE) and by enzyme neutralization with type-specific rabbit anti-HSV-1 TK immunoglobulin. Karyotype analyses of several somatic cell hybrid clones using G-banding, Hoechst 33258 staining, and combined G-banding and Hoechst staining demonstrated that they retained only a few human chromosomes. A marker chromosome, M7, consisting of a chromosome 17 translocated to the short arm of 3, occurred in 25 of the 28 metaphases examined. Also chromosomes 8 and X were found in a minority of metaphases. Isozyme analyses showed that all 19 hybrid clones analyzed expressed human aminoacylase-1 (ACY1) and esterase D (ESD), markers for 3 and 13, respectively. Back-selection of somatic cell hybrid clones with 5-bromodeoxyuridine resulted in the isolation of several subclones lacking HSV-1 TK activity, human ACY1, human ESD, and the human chromosomes. These experiments suggest that the HSV-1 TK gene is associated with either M7 or a segment of 13, or both, in biochemically transformed HeLa(BU25)/KOS 8-1 cells. These experiments also permit localization of the ACY1 structural gene to the pter leads to p12 region of 3.

Amidohydrolases↗
PubMed2006

Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

BACKGROUND: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription. RESULTS: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease. CONCLUSION: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

Cyclin-Dependent Kinase 2↗
PubMed1983

A new theory on the origin and the nature of viruses.

The hypothetical model presented herein concerns the origin and nature of viruses. It advances the possibility of the appearance and existence of an organism lacking a cohesive morphological structure, that is: its subsystems are not in structural continuity. An attempt to delimit the concepts of life and organism and to integrate the viruses into this framework is made. Viruses are presented as organisms which pass in their ontogenetic cycle through two distinctive phenotypic phases: (1) the vegetative phase and (2) the phase of viral particle or nucleic acid. In the vegetative phase, considered herein to be the ontogenetically mature phase of viruses, their component molecules are dispersed within the host cell. In this phase the virus shows the major physiological properties of other organisms: metabolism, growth, and reproduction. Therefore, life is an effective presence. It is shown also, that in this phase so called "DNA viruses" have both nucleic acids: RNA as well as DNA. The virions are considered to be "spores" or reproductive forms of the virus, possessing life only as a potential property.

Models, Biological↗
PubMed1978

Viral gene expression in murine sarcoma virus(murine leukemia virus)-infected cells.

NIH 3T3 cells infected with Moloney murine sarcoma virus (murine leukemia virus) produce virions which contain about 99% murine sarcoma virus RNA and 1% murine leukemia virus RNA. This report describes experiments which measured intracellular concentrations of proviral DNA and RNA transcripts for each of the viruses. We found that three to four copies of proviral DNA from each virus were integrated into the cellular DNA. Measurements of RNA specific for each of the genomes by hybridization to specific cDNA reagents revealed a 10- to 15-fold difference in concentration in both nuclear and polysomal RNA fractions, with murine sarcoma virus RNA predominating in both cases. Unless there are major differences in stability between the two viral RNAs, our results suggest that transcriptional control is responsible for much of the difference in final levels of virus synthesis.

Cell Line↗
PubMed2002

Ahi-1, a novel gene encoding a modular protein with WD40-repeat and SH3 domains, is targeted by the Ahi-1 and Mis-2 provirus integrations.

The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3' end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers.

A semiquantitative estimation of human T-lymphotropic virus type I (HTLV-I) integration by peripheral blood mononuclear cells (PBMC) was performed. Genomic DNA samples derived from 134 HTLV-I carriers were subjected to 40 or 60 cycles of the polymerase chain reaction to amplify the pol region of HTLV-I. The HTLV-I genome was detected by dot hybridization using a 32P-labeled oligonucleotide probe for the pol region. The radioactivity of hybridized dot membranes was then counted with an RI Imaging System (Ambis Inc, San Diego, CA) and the HTLV-I genome dose was determined by comparison with standard curve for serially diluted HTLV-I genome-positive DNA. A wide range of variation of HTLV-I genome integration was observed. When the integrated genome dose was calculated as the number of HTLV-I copies per 100 PBMC, 7 carriers (5%) had more than 10 copies, 56 (42%) had 1 to 10 copies, 46 (34%) had 0.1 to 1 copy, and 24 (18%) had less than 0.1 copy. In one sample, the HTLV-I genome was undetectable, which may indicate that the integrated genome was present at less than 0.01 copies per 100 PBMC. Age- or sex-related variations in the distribution of individuals with different HTLV-I genome were rather limited. However, carriers with a high level of the HTLV-I genome were always more than 30 years old and were predominantly male (six of seven).

Aging↗