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PubMed1997

The chimeric oncoproteins E2A-PBX1 and E2A-HLF are concentrated within spherical nuclear domains.

Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A and PBX1 as a result of t(1;19) in acute lymphoblastic leukemia. We evaluated subcellular localization patterns of E2A-PBX1 protein in transfected cells using immunofluorescence. Full-length E2A-PBX1 was exclusively nuclear and was concentrated in spherical domains denoted chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear fluorescence for wild-type E2A or PBX1 proteins was diffuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggested transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine failed to confirm altered transcriptional activity in relation to CODs. CODs also failed to co-localize with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP, or with foci of DNA replication. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impaired COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. We conclude that, as a 'gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis.

3T3 Cells↗
PubMed2006

Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells.

Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10(3)- to 10(4)-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system.

B-Lymphocytes↗
PubMed2001

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts.

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Animals↗
PubMed2003

A kinetic analysis of immune mediators in the lungs of mice infected with vaccinia virus and comparison with intradermal infection.

The early inflammatory response to a virus may be critical in restricting infection and in shaping the subsequent adaptive immune response. In this study we have examined the early inflammatory response of mice following infection with vaccinia virus (VV) strain Western Reserve (WR). Respiratory challenge of BALB/c mice with VV led to early virus replication in the lung and upper respiratory tract followed by dissemination of virus to other visceral organs and to the brain. The number of inflammatory cells, largely macrophages and T lymphocytes, recovered from bronchoalveolar lavage (BAL) fluid increased markedly during infection and coincided with the expression of CC chemokine ligands (CCL) 3, 2 and 11 and CXC chemokine ligands (CXCL) 1 and 2/3 in BAL. The peak of the inflammatory response occurred around day 10 and declined thereafter. The antiviral cytokines IFN-gamma and TNF-alpha, and the reactive nitrogen intermediate nitric oxide (NO), were also detected in BAL from VV-infected mice. A markedly different inflammatory response was observed after intradermal inoculation of WR into the ear pinnae of mice. Intradermal challenge was followed by highly localized virus replication and by a cellular influx, consisting largely of neutrophils and T lymphocytes, into the dermal compartment of the infected ear. Together these findings highlight differences in the pathogenesis and in the cellular inflammatory response to WR following intranasal and intradermal inoculation of mice.

Administration, Intranasal↗
PubMed1984

Fluorescence-activated cell sorting analysis of the induction and expression of acute thermal tolerance within the cell cycle.

We have examined the cell cycle specificity of 45.5 degrees heat-induced toxicity and the induction and expression of thermal tolerance. Ultrapure populations of G1-, S-, and G2-M-phase cells were obtained through sequential centrifugal elutriation and flow cytometric cell sorting of Hoechst 33342-stained cells. We found no interaction of Hoechst 33342 with hyperthermia under staining conditions that gave good cytometric resolution of DNA distributions. Single dose-response survival curves indicated that S phase was the most sensitive to 45.5 degrees hyperthermia (Do = 1.97, 1.26, and 1.95 min for G1, S, and G2-M, respectively). Both S and G2-M phases exhibited a decreased ability from G1 to accumulate sublethal heat lesions as evidenced by decreased heat survival curve shoulders (Dq) = 13.7, 9.51, and 8.39 min for G1, S, and G2-M, respectively). Thermal tolerance, as measured by the decreased inactivation slope of the split-dose treatment, could be induced and expressed in G1, S, and G2-M phases. However, both the magnitude and temporal expression of tolerance were dependent on the position of the cell within the cell cycle at the time of the initial heat treatment. S-phase cells exhibited slightly less thermal tolerance as compared to G1 cells given isosurvival thermal induction doses as measured by the split-dose inactivation rate constants (heated/control = 8.37 and 5.62 for G1 cells at 12 and 24 hr and 7.68 and 5.27 for S-phase cells at 12 and 28 hr). Also, split-dose survival curves for cells heated in G2-M indicated a near total inability to accumulate heat-induced sublethal damage. Simultaneous bivariate (90 degrees light scatter and DNA content) progression analysis of heated replicates indicated that tolerance could probably be expressed in those cells which moved into other cycle compartments following the initial heat treatment. For instance, G1-phase cells preheated for 20 min began progression into normally heat-sensitive S phase between 24 and 28 hr after the heat treatment. This corresponded to approximately the time of maximal thermal tolerance expression. [3H]Thymidine suicide experiments also indicated that the ultimately clonogenic cells began movement into S phase at or near the time of maximal tolerance. In this case then, tolerance expression appeared to supersede the S-phase acute heat sensitivity. Heated S-phase cells began progression into G2-M between 4 and 12 hr, which corresponded temporally to large amounts of tolerance expression4 +

Animals↗
PubMed1999

Assessing the replicative history of human T cells.

Upon encountering antigen, T cells clonally expand and differentiate into effector cells that directly or indirectly eliminate antigen-bearing pathogens. When renewed contact with the same pathogen occurs the immune response is mounted in a faster and more accurate way, a process that is referred to as immunological memory. The basis for T-cell memory is at least partially provided by an enhanced precursor frequency of antigen-specific T cells, and an increased responsiveness of primed T cells to activation signals. In contrast to B cells, which acquire mutations in the immunoglobulin genes after antigenic challenge, somatic markers are lacking that distinguish unprimed (or naive) from primed (encompassing memory and effector) T cells. Instead, differential expression of cell surface molecules on subsets of T cells and measures for replicative history can be used to obtain insight into the antigen-driven development of the T-cell compartment. Apart from fundamental issues addressing lineage relationships between naive, memory and effector T cells and the cellular basis for long-term T-cell memory, these types of studies have proved to be valuable in understanding T-cell reconstitution in situations of severe T-cell depletion, i.e., after chemotherapy, treatment with depleting CD4 monoclonal antibodies or during HIV infection.

Age Factors↗
PubMed1992

Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.

The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.

Antibodies, Monoclonal↗
PubMed1999

Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

3T3 Cells↗
PubMed1987

Changing spatial patterns of DNA replication in the developing wing of Drosophila.

Using an antibody against bromodeoxyuridine we have analyzed the distribution of S-phase nuclei in the wing disc of Drosophila as the larval disc transforms into the adult wing during metamorphosis. On the basis of the timing of replication three cell populations can be distinguished: the cells of the presumptive wing margin, the precursor cells of the longitudinal veins, and those of the intervein regions. In each of these populations the cell cycle is first arrested and later resumes at a specific time, so that at each developmental time point a characteristic spatial pattern of S-phase nuclei is seen. An interpretation of these changing patterns in terms of vein formation, compartments, and neural development is offered.

Animals↗
PubMed1999

Influenza C virus persistence depends on exceptional steps in viral RNA synthesis and transport.

The cell line MDCK-pi, which is persistently infected with a variant of influenza C/AnnArbor/1/50 virus (C/AA-pi), was studied as a long-term persistence model by means of a strand-specific in situ hybridization assay. As a typical feature of the persistence, we identified a continuous synthesis of antigenomic positive-strand RNA encoded by segment 7 (NS) during virus production. In contrast, infection with the parental wild-type virus led to a rapid reduction of antigenomic RNA as observed in the late period of replication particularly for RNA segment 7. Furthermore, the replication cycle of the persistent variant did not show the switch from early to late replication events followed by clearance of intracellular virus, but was regulated in terms of productive and nonproductive phases. Nonproductive phases were reversible and characterized by a low level of virus-specific RNA signals. In the productive phase, a difference in cytoplasmic RNA transport was detected for the two viruses: a marked cytoplasmic accumulation of negative- and positive-strand wild-type virus RNAs stood in contrast to a RNA localization in different cellular compartments for the persistent virus. Also, Vero cells infected with the C/AA-pi variant were restricted to a transient, non-persistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends on a distinctly modified and cell type-specific regulation of virus-specific RNA synthesis and transport.

Animals↗
PubMed1987

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells.

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. Active genes are transcribed at the nuclear matrix and during replication the loops are reeled through fixed matrix-associated replication complexes. We have investigated whether the repair of DNA damage also occurs in the nuclear matrix compartment. Biochemical analysis of confluent normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m-2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix either 30 min or 13 h after irradiation. Autoradiographic visualization of repair events in single DNA halo-matrix structures confirmed the biochemical observations. At a biologically more relevant dose of 5 J m-2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m-2) normal human fibroblasts post-u.v. incubated in the presence of hydroxyurea and arabinosylcytosine for 2 h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Different biochemical approaches revealed that most of the single-stranded regions are clustered, indicating that the repair process itself is non-random or that domains in the chromatin are repaired at different rates. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C). In XP-C cells these domains are localized near the attachment sites of DNA loops at the nuclear matrix. In contrast, xeroderma pigmentosum cells of complementation group D as well as Syrian hamster embryonic cells with limited excision-repair capacities, revealed a random distribution of repair events in DNA loops. The preferential repair of matrix-associated DNA in XP-C cells may be related partly to repair of transcriptionally active DNA and this may account for the ability of XP-C cells, in contrast to XP-D cells, to recover u.v.-inhibited synthesis of DNA and RNA.

Animals↗
PubMed2006

Possible compartmentalization of hepatitis C viral replication in the genital tract of HIV-1-coinfected women.

BACKGROUND: We estimated the prevalence of hepatitis C virus (HCV) in cervical cytobrush samples from HCV/human immunodeficiency virus (HIV)-coinfected women and analyzed the HCV quasi species in both cytobrush and plasma samples. Possible compartmentalization of viral quasi species in the genital tract and plasma was evaluated by comparison of genetic heterogeneity and use of phylogenetic analysis. METHODS: Paired plasma and cytobrush samples were obtained from 85 HCV/HIV-coinfected women. The presence of HCV in cytobrush samples was evaluated by reverse-transcription polymerase chain reaction of the 5' untranslated region. Viral quasi species were analyzed by cloning and sequencing the highly variable region-1 in 8 patients. RESULTS: HCV was detected in 27% of cytobrush samples. The composition of viral quasi species was different in the 2 body compartments at both the nucleotide and amino acid level. In fact, the mean complexity was significantly lower in cytobrush samples, and a similar trend was observed for the other parameters of heterogeneity. Phylogenetic analysis and amino acid alignment identified several viral variants that were unique to each body compartment. CONCLUSIONS: Our data suggest that the genital and plasma quasi species represent distinct subpopulations, which possibly reflects compartmentalized viral replication. Alternatively, cell carriers harboring viral quasi species in the genital tract that are distinct from those in plasma could transfer the virus through the barrier separating the 2 body sites.

5' Untranslated Regions↗
PubMed2005

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir.

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.

Anti-HIV Agents↗
PubMed2001

Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway.

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by L(d)-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial beta-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing beta-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.

Animals↗
PubMed1978

[DNA synthesis and mitotic division of myocytes of the ventricles, atria and conduction system of the heart during the myocardial development in mammals].

The degree of differentiation of various types of muscle and non-muscle cells that synthesize DNA in myocardia of mouse embryos and suckling rats was estimated by means of electron microscopic autoradiography with 3H-thymidine. No morphologically undifferentiated myoblasts were observed. DNA synthesizing capacity and mitotic activity are typical of numerous moderately differentiated myocytes in all myocardial compartments studied. As judged from proliferation kinetics studies, ventricular myocytes proliferate at embryonal stages and during the 1st postnatal week more actively than do atrial ones, specialized muscle cells from the conductive system replicating much less intensely as compared with both these kinds of myocytes. However, at the 2nd postnatal week, the withdrawal of myocytes from the mitotic cycle proceeds more rapidly in ventricles than in other heart compartments which results in a relatively more active proliferation of myocytes in atria and conductive system beginning from the end of the 2nd postnatal week. By the 17--18th days of the postnatal life, practically all the myocytes, irrespective of their topology, cease to proliferate. Nevertheless, even after this term, up to 0.1--0.5% of atrial and/or conductive system myocytes still go on entering periodically the mitotic cycle. The duration of S and G2 + 1/2 M periods is similar in both the ventricular and atrial myocytes of suckling rats. Probable causes and significance of the observed asynchrony of the myocyte proliferation rates in different heart compartments is discussed.

Animals↗
PubMed1983

Role of stromal and hematopoietic stem cells in Friend spleen focus forming virus effects in continuous bone marrow culture.

Replication of the Friend spleen focus forming virus (SFFV) in C3H/HeJ or C57BL/6J mouse continuous bone marrow cultures is associated with an increased cumulative production of pluripotential/hematopoietic stem cells (CFUs), granulocyte-macrophage progenitor cells (GM-CFUc), and total granulocytes, compared to uninfected or helper virus infected control cultures. The site(s) of action of the virus are not known. To determine whether viral effect(s) occurred in adherent stromal and/or non-adherent hematopoietic stem cells, purified cell populations, comprised exclusively of cells from each compartment, were separated from C57BL/6J (Fv-1bb) or C3H/HeJ (Fv-1nn) marrow cultures and were left uninfected or were infected with host range replication restricted B-tropic or N-tropic helper pseudotype viruses of SFFV respectively. Cell populations were then mixed to establish continuous hematopoiesis in allogeneic or syngeneic combinations. Virus host range restriction between compartments was maintained in allogeneic reconstituted cultures which showed active hematopoiesis for 16-17 weeks and no significant differences between: (1) virus infected and uninfected groups or (2) groups comprised of stromal cells from one or the other strain. Thus, these data indicate that the prolongation of hematopoiesis in undisturbed long-term marrow cultures by SFFV occurs through an interaction of adherent hematopoietic stem cells with the marrow stroma.

Animals↗
PubMed2000

Genetic susceptibility to intracellular infections: Nramp1, macrophage function and divalent cations transport.

Nramp1 is one of the few host resistance genes that have been characterized at the molecular level. Nramp1 is an integral membrane protein expressed in the lysosomal compartment of macrophages and is recruited to the membrane of bacterial phagosomes where it affects intracellular microbial replication. Nramp1 is part of a very large gene family conserved from bacteria and man that codes for transporters of divalent cations transporters. We propose that Nramp1 affects the intraphagosomal microbial replication by modulating divalent cations content in this organelle. Both mammalian and bacterial transporters may compete for the same substrate in the phagosomal space.

Animals↗
PubMed1995

Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I.

The human T-cell leukemia virus type I Tax protein trans-activates several cellular genes implicated in T-cell replication and activation. To investigate its leukemogenic potential, Tax was targeted to the mature T-lymphocyte compartment in transgenic mice by using the human granzyme B promoter. These mice developed large granular lymphocytic leukemia, demonstrating that expression of Tax in the lymphocyte compartment is sufficient for the development of leukemia. Furthermore, these observations suggest that human T-cell leukemia virus infection may be involved in the development of large granular lymphocytic leukemia.

Animals↗