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At least 325 records · Page 18Linked to original sources
PubMed2006

Human T-cell leukemia virus type I p30 nuclear/nucleolar retention is mediated through interactions with RNA and a constituent of the 60 S ribosomal subunit.

Human T-cell leukemia virus type I is the etiological agent of adult T-cell leukemia/lymphoma, an aggressive and fatal lymphoproliferative malignancy. The virus has evolved strategies to escape immune clearance by remaining latent in most infected cells in vivo. We demonstrated previously that virally encoded p30 protein is a potent post-transcriptional inhibitor of virus replication (Nicot, C., Dundr, M., Johnson, J. M., Fullen, J. R., Alonzo, N., Fukumoto, R., Princler, G. L., Derse, D., Misteli, T., and Franchini, G. (2004) Nat. Med. 10, 197-201). p30 is unable to shuttle out of the nucleus in heterokaryon assays, suggesting the existence of specific retention signals. Because suppression of virus replication relies on nuclear retention of the tax/rex mRNA by p30, determining the retention features of p30 will offer hints to break latency in infected cells and insights into new therapeutic approaches. In this study, we used live cell imaging technologies to study the kinetics of p30 and to delineate its retention signals and their function in virus replication. Notably, this is the first study to identify p30 nucleolar retention domains. Using mutants of p30 that localized in different cellular compartments, we show that post-transcriptional control of virus replication by p30 occurs in the nucleoplasm. We further demonstrate that p30 nuclear/nucleolar retention is dependent upon de novo RNA transcripts and interactions with components of the ribosomal machinery.

Amino Acid Sequence↗
PubMed2001

Exposure to low pH is not required for penetration of mosquito cells by Sindbis virus.

It is widely held that the penetration of cells by alphaviruses is dependent on exposure to the acid environment of an endosome. The alphavirus Sindbis virus replicates in both vertebrate and invertebrate cell cultures. We have found that exposure to an acid environment may not be required for infection of cells of the insect host. In this work, we investigated the effects of two agents (NH(4)Cl and chloroquine), which raise the pH of intracellular compartments (lysosomotropic weak bases) on the infection and replication of Sindbis virus in cells of the insect host Aedes albopictus. The results show that both of these agents increase the pH of endosomes, as indicated by protection against diphtheria toxin intoxication. NH(4)Cl blocked the production of infectious virus and blocked virus RNA synthesis when added prior to infection. Chloroquine, in contrast to its effect on vertebrate cells, had no inhibitory effect on infectious virus production in mosquito cells even when added prior to infection. Treatment with NH(4)Cl did not prevent the penetration of virus RNA into the cell cytoplasm or translation of the RNA to produce a precursor to virus nonstructural proteins. These data suggest that while these two drugs raise the pH of endosomes, they do not block insect cell penetration. These data support previous results published by our laboratory suggesting that exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses.

Aedes↗
PubMed2005

Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments.

Picornavirus infection of cells generally results in the production of membranous vesicles containing the viral proteins necessary for viral RNA synthesis. To determine whether foot-and-mouth disease virus (FMDV) infection induced similar structures, and which cellular components were involved, the subcellular distribution of FMDV proteins was compared with protein markers of cellular membrane compartments. Using immunofluorescence analysis and digital deconvolution, it was shown that FMDV structural and non-structural proteins co-localize to punctate structures in juxtanuclear virus assembly sites close to the Golgi complex. Significantly, viral protein 2C did not co-localize with marker proteins of the cis- or medial-Golgi compartments or trans-Golgi network. Furthermore, incubation of infected cells with brefeldin A caused a redistribution of Golgi proteins to the endoplasmic reticulum, but did not affect the distribution of 2C and, by inference, the integrity of the virus assembly site. Taken with the observation that 2C was membrane-associated, but failed to fractionate with Golgi markers on density gradients, it was possible to conclude that Golgi membranes were not a source of structures containing 2C. Further immunofluorescence analysis showed that 2C was also separate from marker proteins of the endoplasmic reticulum, endoplasmic reticulum intermediate compartment, endosomes and lysosomes. The results suggest that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication.

Animals↗
PubMed1998

Somatotopic nucleocortical projections to the multiple somatosensory cerebellar maps.

The cerebellum is organized in a series of parasagittal compartments: in C1-C3 and C2 compartments Purkinje cells receive climbing fibre afferents from the rostral part of the accessory olives, and project their axon to the nucleus interpositus anterior and posterior, respectively. Within these compartments electrophysiological studies have shown that the cutaneous input carried by climbing fibre afferents is topographically organized so as to design a map of peripheral body districts. The body map is replicated over the anterior lobe-pars intermedia and the paramedian lobule, and anatomical studies have indicated that the replication is partly due to the axonal branching of olivocerebellar neurons. The aim of this study was to analyse the presence of a somatotopic organization and of a branching pattern in the nucleocortical projections, in relation to the replicated body maps within C1-C3 and C2 compartments. By using double retrograde neuronal tracing we explored, in the cat, the topographic distribution of single- and double-labelled cells in the interposed nuclear subdivisions, after tracer injections into forelimb or hindlimb regions of the anterior lobe-pars intermedia, paramedian lobule and hemisphere (medial crus II). Most of the nucleocortical neurons were found in ipsilateral nucleus interpositus posterior, with smaller numbers in the ipsilateral nucleus interpositus anterior. Nucleocortical neurons projecting to forelimb- or hindlimb-related areas are completely segregated, the forelimb neurons being located laterally and the hindlimb neurons medially in the nucleus interpositus posterior. Within their respective domains both the forelimb and hindlimb populations projecting to the anterior lobe-pars intermedia are partly segregated from those projecting to the paramedian lobule, in that the two populations are slightly shifted along the dorsoventral axis of the nucleus. Although mostly different, some of the cells are common to the two forelimb populations, since they send axonal branches to the homologous areas of the anterior lobe and paramedian lobule. Contralateral fastigial or interposed nucleocortical projections are restricted to the anterior lobe-pars intermedia, and their neurons of origin are different from those that project to the ipsilateral cerebellar cortex: i.e. they are not a bilateral, but a separate contralateral component.

Afferent Pathways↗
PubMed1995

The ecology and pathology of Epstein-Barr virus.

Epstein-Barr virus achieves its ubiquitous and uniform epidemiological distribution by a dual strategy of latency to guarantee lifelong persistence and intermittent replication to guarantee transmission. These two functions appear to dictate residence in different cell types: latency in B lymphocytes and replication in epithelial cells. Both of these cell compartments are potential sites for EBV-associated malignancies.

Burkitt Lymphoma↗
PubMed2001

Unique aspects of mitochondrial biogenesis in trypanosomatids.

Mitochondrial biogenesis consists of the sum of all processes required for the formation of the mitochondrial membranes as well as the soluble compartments they contain. Furthermore, it includes the replication of the mitochondrial genome and correct segregation of the organelles during cell division. Mitochondrial proteins come from two sources, a limited but essential set of inner membrane proteins is encoded by the mitochondrial genome, whereas the large majority (90-95%) is derived from nucleus-encoded genes and are posttranslationally imported into the organelle. Trypanosomatids belong to the earliest diverging branches of the eukaryotic evolutionary tree which have mitochondria. This is reflected in the organisation of their mitochondrial DNA that consists of a network of two classes of topologically interlocked circular DNA molecules as well as many unique features in their mitochondrial biogenesis. The proteins encoded on the mitochondrial genome are conventional for a mitochondrial genome, their expression, however, involves a complex series of processes. Many genes represent incomplete open reading frames and their primary transcripts have to remodelled by RNA editing to convert them into translatable mRNAs. RNA editing is mediated by small mitochondria-encoded transcripts, the guide RNAs, and is in that form specific for trypanosomatids and closely related organisms. Mitochondrial translation is also unconventional. No tRNA genes are encoded on the mitochondrial genome. Instead, mitochondrial protein synthesis functions exclusively with imported cytosolic, eukaryotic-type tRNAs. The composition of mitochondrial ribosomes is also unusual in that they contain the smallest known rRNAs. They are about 30% shorter than the already much reduced rRNAs in human mitochondria. Furthermore, the topological organisation of the mitochondrial genome requires an elaborate replication machinery involving topoisomerases. Finally, some trypanosomatids have life cycle stages exhibiting very different mitochondrial activities and can therefore serve as a model system for the regulation of mitochondrial biogenesis.

Animals↗
PubMed2002

Reliable and reproducible LightCycler qPCR for HIV-1 DNA 2-LTR circles.

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.

DNA Restriction Enzymes↗
PubMed2000

Fluorescent labelling of intracellular bacteria in living host cells.

The fluorescent reagent, CellTracker, labels metabolically-active cells and was used here to label Chlamydia in vivo during their exponential phase of growth in infected cells. HeLa cells infected with C. psittaci were labelled with the CellTracker reagents between 15 and 48 h post-infection. The fluorescent label accumulated in the host-cell membrane compartment (inclusion) within which Chlamydia reside and replicate, and was also incorporated by the bacteria. Labelling with the CellTracker affected neither the growth nor the differentiation of the chlamydiae, and labelled chlamydiae isolated from infected cells were infectious. Our results demonstrate that the CellTracker could become a valuable tool for in vivo labelling of obligate intracellular parasites for which no genetic tools exist.

Bacterial Adhesion↗
PubMed1999

In vivo anti-HIV activity of (+)-calanolide A in the hollow fiber mouse model.

In vivo anti-HIV efficacy of (+)-calanolide A has been evaluated in a hollow fiber mouse model. It was demonstrated that the compound was capable of suppressing virus replication in two distinct and separate physiologic compartments (i.p. and s.c.) following oral or parenteral administration on a once- or twice-daily treatment schedule. A synergistic effect was observed for the combination of (+)-calanolide A and AZT.

Animals↗
PubMed2001

Longitudinal analysis of CD8(+) T-cell phenotype and IL-7, IL-15 and IL-16 mRNA expression in different tissues during primary simian immunodeficiency virus infection.

Infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) represents a useful model of HIV infection that offers the unique opportunity to investigate the very early modifications that affect CD8(+) T-lymphocyte subsets and related cytokines during lentiviral infection. Herein, three cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac 251. In fresh isolated mononuclear cells from blood, lymph node and bronchoalveolar lavage, we analyzed changes in the phenotype of CD8(+) T cells and we used reverse transcription-PCR to monitor the expression of IL-7, IL-15 and IL-16 mRNA. We demonstrated that an expansion of CD8(+)CD28(-) T cells occurs from the third week of infection on in the peripheral blood and in the lung, whereas CD8(+)CD28(+) T cells expand in the lymph nodes. Concomitantly, we evidenced mRNA modulations in IL-16, IL-15 and IL-7 expression in the three compartments studied. The containment of systemic viral replication was associated with an overexpression of IL-16 mRNA in the lung and in the peripheral blood. Given the immunomodulatory properties of IL-15 and IL-7 and the potential antiviral ability of IL-16, these perturbations could have important implications in early viral dissemination and HIV immunopathogenesis.

Animals↗
PubMed2000

How to establish a lasting relationship with your host: lessons learned from Mycobacterium spp.

Mycobacterium spp. enjoy an intracellular lifestyle that is fatal to most microorganisms. Bacilli persist and multiply within mononuclear phagocytes in the face of defences ranging from toxic oxygen and nitrogen radicals, acidic proteases and bactericidal peptides. Uptake of Mycobacterium by phagocytes results in the de novo formation of a phagosome, which is manipulated by the pathogen to accommodate its needs for intracellular survival and replication. The present review describes the intracellular compartment occupied by Mycobacterium spp. and presents current ideas on how mycobacteria may establish this niche, placing special emphasis on the involvement of mycobacterial cell wall lipids.

Animals↗
PubMed2001

Chronic benzodiazepine treatment of cells expressing recombinant GABA(A) receptors uncouples allosteric binding: studies on possible mechanisms.

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

Animals↗
PubMed2002

Discordant Human Immunodeficiency Virus Type 1 drug resistance mutations, including K103N, observed in cerebrospinal fluid and plasma.

Discordant resistance mutations were seen in human immunodeficiency virus type 1 (HIV-1) isolated from specimens of blood and cerebrospinal fluid (CSF) obtained from 3 of 6 patients. To our knowledge, this is the first report of HIV-1 isolated from CSF harboring the K103N mutation, which confers resistance to the nonnucleoside reverse-transcriptase inhibitors, and this finding may indicate that virus in the CSF replicates independently from virus in the blood compartment.

Amino Acid Substitution↗
PubMed2004

Adaptation of the Brucellae to their intracellular niche.

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.

Adaptation, Biological↗
PubMed1978

Membrane fusion as a mechanism of simian virus 40 entry into different cellular compartments.

Permissive and nonpermissive simian virus 40 (SV40)-infected cells were ultrastructurally analyzed. Viral particles were found in the cytoplasm, rough endoplasmic reticulum, nuclear envelope, lysosomes, and mitochondria. Upon entering the cell the virion obtains a tight membrane envelope. It seems to be either released from the envelope upon fusion with other membranes of the cell or aggregated into tubular membrane specializations upon fusion with other membrane-enveloped particles. Reconstructed morphological sequences and the finding of SV40 in different spaces of the cell suggest that entry of SV40 into the different compartments and eventually into the site of replication is facilitated by its capacity for being enveloped by a variety of membranes (notably the cell membrane and the nuclear membrane) and the sequential fusion and fission of these membranes.

Cell Line↗
PubMed1996

Direct interactions of coxsackievirus B3 with immune cells in the splenic compartment of mice susceptible or resistant to myocarditis.

In vitro replication of coxsackievirus B3 (CVB3) in cells of the immune system derived from uninfected adolescent A/J and C57BL/6J mice and replication of CVB3 in and association with immune cells from spleens of infected animals in vivo were assessed. Nonstimulated or mitogen-stimulated spleen cells were minimally permissive for viral replication during an 8-h period. Three days postinfection (p.i.), CVB3 RNA was localized in vivo to B cells and follicular dendritic cells of germinal centers in both A/J and C57BL/6J mice; however, extrafollicular localization was greater in C57BL/6J mice (P = 0.0054). Although the pattern of CVB3 RNA localization was different, the total load of infections virus (PFU per milligram of tissue) was not different. Splenic CVB3 titers (PFU per milligram of tissue) in both strains were maximal at day 3 or 4 p.i. and were back to baseline by day 7 p.i., with most infectious virus being non-cell associated. CVB3 titers (PFU per milligram of tissue) correlated directly with in situ hybridization positivity in splenic follicles and extrafollicular regions in both murine strains; however, follicular hybridization intensity was greater in A/J mice at day 5 p.i. (P = 0.021). Flow cytometric analysis demonstrated that 50.4% of total spleen cells positive for CVB3 antigen were B cells and 69.6% of positive splenic lymphocytes were B cells. Myocardial virus load in C57BL/6J mice was significantly lower than that in A/J mice at days 4 and 5 p.i. These data indicate that CVB3 replicates in murine splenocytes in vitro and in B cells and extrafollicular cells in vivo.

Animals↗
PubMed2002

Clinical implications of CNS penetration of antiretroviral drugs.

The CNS serves as an important sanctuary site for HIV replication. The presence of HIV in this compartment may contribute to neurological complications in individuals infected with HIV. Understanding the CNS penetration capabilities of available antiretroviral agents may help clinicians to design treatment regimens with neuroprotective effects. Although numerous clinical studies and anecdotal reports have examined CSF antiretroviral drug exposure as a marker of CNS penetration, understanding the clinical relevance of these findings is difficult. Challenges with study design and subject recruitment often limit the investigator's ability to collect comprehensive data. Upon review of available data, the antiretroviral agents zidovudine, stavudine, lamivudine, nevirapine, efavirenz and indinavir demonstrate consistent penetration into the CSF. Zidovudine-, stavudine-, lamivudine-, didanosine- and protease inhibitor-based regimens also appear to suppress CSF viraemia or improve HIV neurological disease. These agents may be appropriate candidates for neuroprotective antiretroviral treatment regimens. Despite these data, several unanswered questions about the CSF antiretroviral drug exposure-response relationship still remain. Prospective, controlled studies examining this relationship are needed before absolute clinical recommendations are founded.

Anti-HIV Agents↗
PubMed2000

HIV-1 can be recovered from a variety of cells including peripheral blood monocytes of patients receiving highly active antiretroviral therapy: a further obstacle to eradication.

During highly active antiretroviral therapy (HAART), HIV-1 can still persist in circulating, resting CD4+ T lymphocytes, lymph node mononuclear cells, and seminal cells of patients despite sustained suppression of plasma viremia to undetectable levels. Sanctuary sites where antiretroviral drug penetration is not optimal may allow local HIV-1 infection of cells within and passing through these tissues. Factors such as imperfect drug adherence due to complicated drug regimens may also result in tissue compartments with suboptimal drug concentrations allowing viral replication. We have examined blood monocytes from HIV-1-infected subjects being effectively treated with HAART to determine virus carriage in these cells. Monocytes were purified from peripheral blood of patients with plasma HIV-1 RNA below 50 copies/mL and who had maintained levels of plasma RNA below detection for 3 months or more. Replication-competent virus could be recovered from the majority of monocyte populations by co-culture with CD8-depleted, PHA-activated, peripheral blood mononuclear cells. Sequencing of the reverse transcriptase and protease genes of the recovered viruses did not reveal resistance to both reverse transcriptase and protease inhibitors. Continued new infection of this transitory, circulating population of cells even during prolonged, effective HAART most likely reflects ongoing, low-level HIV-1 replication within cellular reservoirs and sanctuary sites in the body.

Anti-HIV Agents↗