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PubMed2005

Intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle.

Herpes virions are complex particles that consist of more than 30 different virally encoded proteins. The molecular basis of how this complicated structure is assembled is only recently beginning to emerge. After replication in the host cell nucleus viral DNA is incorporated into preformed capsids which leave the nucleus by budding at the inner nuclear membrane resulting in the formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment including the acquisition of more than 15 tegument and more than 10 envelope (glyco)proteins occurs by budding into Golgi-derived vesicles. Mature virions are released after fusion of the vesicle membrane with the plasma membrane of the cell. Thus, herpesvirus morphogenesis requires a sequence of envelopment--de-envelopment--re-envelopment processes which are distinct not only in the subcellular compartments in which they occur but also in the viral proteins involved. This review summarizes recent advances in our understanding of the complex protein-protein interactions involved in herpesvirus assembly and egress.

Animals↗
PubMed1999

Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0.

Herpes simplex virus type 1 is capable of inhibiting host cell DNA synthesis following lytic infection. However, the mechanism and nature of potential effects on cell cycle progression have not been described. In this report, we characterize the dysregulation of the cell cycle following infection with the replication-incompetent virus d106, where immediate-early gene expression is restricted to infected-cell polypeptide 0 (ICP0) and the expression of all other viral genes is dramatically reduced or is not observed. Infection with d106 resulted in the accumulation of cells in both the G(1)/S and G(2)/M compartments, consistent with cell cycle arrest at both checkpoints. The isogenic variant d109, which does not express any viral proteins, failed to induce this phenotype, suggesting that the expression of ICP0 is crucial for cell cycle arrest. Analysis of global cellular gene expression patterns following infection with d106 and d109 revealed that a relatively small subset of cellular genes were induced as a consequence of ICP0 expression. A number of these genes induced in the presence of ICP0 are classically considered p53-responsive genes, including p21, gadd45, and mdm-2. However, infection with d106 of cells with both alleles of p53 deleted resulted in the same cell cycle arrest phenotype and similar cellular gene expression patterns, suggesting that the expression of ICP0 results in cell cycle arrest potentially via p53-dependent and p53-independent mechanisms. In addition, it was found that the effects of infection with d106 on viral and cellular gene expression were similar to the effects observed following treatment of cells with the histone deacetylase inhibitor trichostatin A.

Animals↗
PubMed2003

Brucella stationary-phase gene expression and virulence.

The capacity of the Brucella spp. to establish and maintain long-term residence in the phagosomal compartment of host macrophages is critical to their ability to produce chronic infections in their mammalian hosts. The RNA binding protein host factor I (HF-I) encoded by the hfq gene is required for the efficient translation of the stationary-phase sigma factor RpoS in many bacteria, and a Brucella abortus hfq mutant displays a phenotype in vitro, which suggests that it has a generalized defect in stationary-phase physiology. The inability of the B. abortus hfq mutant to survive and replicate in a wild-type manner in cultured murine macrophages, and the profound attenuation displayed by this strain and its B. melitensis counterpart in experimentally infected animals indicate that stationary-phase physiology plays an essential role in the capacity of the brucellae to establish and maintain long-term intracellular residence in host macrophages. The nature of the Brucella HF-I-regulated genes that have been identified to date suggests that the corresponding gene products contribute to the remarkable capacity of the brucellae to resist the harsh environmental conditions they encounter during their prolonged residence in the phagosomal compartment.

Animals↗
PubMed1990

Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins.

Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to "model", in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and "panning". Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of dedifferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha2-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.

Animals↗
PubMed1988

Variation in crypt size and its influence on the analysis of epithelial cell proliferation in the intestinal crypt.

The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed.

Animals↗
PubMed2003

Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences.

The order Nidovirales, which includes the arteriviruses and coronaviruses, incorporate a cytoplasmic replication scheme; however, the nucleocapsid (N) protein of several members of this group localizes to the nucleolus suggesting that viral proteins influence nuclear processes during replication. The relatively small, 123 amino acid, N protein of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, presents an ideal model system for investigating the properties and mechanism of N protein nucleolar localization. The PRRSV N protein is found in both cytoplasmic and nucleolar compartments during infection and after transfection of gene constructs that express N-enhanced green fluorescent protein (EGFP) fusion proteins. Experiments using oligopeptides, truncated polypeptides and amino acid-substituted proteins have identified several domains within PRRSV N protein that participate in nucleo-cytoplasmic shuttling, including a cryptic nuclear localization signal (NLS) called NLS-1, a functional NLS (NLS-2), a nucleolar localization sequence (NoLS), as well as a possible nuclear export signal (NES). The purpose of this paper is to review our current understanding of PRRSV N protein shuttling and propose a shuttling scheme regulated by RNA binding and post-translational modification.

Active Transport, Cell Nucleus↗
PubMed1989

The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.

This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones.

Animals↗
PubMed1996

A quantitative morphometric study of the kinetics of tissue regeneration after administration of cisplatin.

The effect of the anti-neoplastic drug cisplatin was investigated on several rat tissues using a novel computerized morphometric image analysis system. The rats were sacrificed in pre-determined intervals, ranging from 1 to 36 days after drug administration, and their liver, kidneys and external ears were sampled and sectioned. Tritiated thymidine was injected into each rat 1 h prior to sacrifice to enable autoradiographical explorations. All sections were histomorphometrically studied by a computerized system. The kidneys of the experimental group revealed increased tubular diameters, especially in the corticomedular region. In the ears, decreases of the mean epithelial thickness, in the mean number of nuclei and in the mean nuclear surface were observed. The thickness of the connective tissue also decreased significantly by the end of the first week and returned to its normal size later on. No changes were detected in the ear cartilage. In the liver, no morphometrical differences were noticed in the hepatocyte density, Kupffer cell density or nuclear area. The nuclei of the hepatocytes and Kupffer cells retained a constant ratio to the total number of cells throughout the whole experiment. Liver autoradiography revealed that the hepatocyte and Kupffer cell labelling indexes after cisplatin administration were significantly higher than those of the control group, indicating enhanced replication of cells and increased synthesis of DNA, which in turn is accumulated in the nuclei and turn the cells into polyploids. In addition, the mean radius of the liver acini after cisplatin was diminished, followed by a significant reduction in the size of the progenitor compartment. It is concluded that while the kidneys are the organs most affected by cisplatin, the progenitor compartment of the liver is heavily affected as well. Computerized morphometry was demonstrated as a highly reproducible and accurate quantitative method for evaluation of histopathological changes in various mammalian tissues.

Animals↗
PubMed1996

Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages.

The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection. However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly. Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice. Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro. Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene. These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine.

Animals↗
PubMed1986

A new approach to the evolution of the blastic crisis from chronic myelocytic leukemia: dynamic interplay of cellular alterations and a changing microenvironment.

The mechanisms responsible for the massive hyperplasia and for the blastic crisis in chronic myelocytic leukemia are poorly understood. The most generally accepted hypothesis proposes that this progression is due to the development of genetic instability in the leukemic cells. In particular, the two phases of the disease are believed to reflect different, discrete genetic events. Such events remain undefined as yet, and the causal significance of observed genetic aberrations is not clear. An alternative hypothesis is presented here. It is assumed that the feedback interactions adjust the relative probabilities of maturation and replication of the 'committed' as well as the pluripotent cells, and further that mitotic cells at all stages possess considerable phenotypic adaptability; in particular their self-renewal capacity can vary in response to changes in the cellular composition of the tissue even within a conventionally defined compartment. On this basis, it is shown that chronic leukemia can arise and evolve into the blastic crisis from a progressive decline in a single clonal characteristic--inducibility to maturation. It is shown, with the help of mathematical considerations, how an initial hereditable event in an early hemopoietic cell can cause a disturbance of the tissue which feeds back onto the individual members of the clone, resulting in a cascade of dynamic changes which can lead to blast cell dominance.

Cell Cycle↗
PubMed1998

Micronucleus induction in somatic cells of mice as evaluated after 1,3-butadiene inhalation.

The effect of different 1,3-butadiene (BD) inhalation doses, 130, 250, and 500 ppm, on somatic cells of mice was studied. Two different cell populations with diverse replicative and differentiative activities, namely splenocytes and peripheral blood reticulocytes, were examined and micronucleus (MN) frequencies were estimated. In splenocytes, different postinhalation time intervals were studied with regard to MN induction and characterisation. BD was found to be clastogenic by inducing increased micronucleus frequencies in both cell compartments and also to induce cytotoxicity at the highest level of exposure. In mouse splenocytes, BD has also shown a weak aneugenic effect at a short time interval after the exposure. Postinhalation time influences the induction of chromosome damage in stimulated splenocytes treated in vivo, since MN frequency decreases with time; in addition, BD has shown its aneugenic and cytotoxic potential only at 2 days after exposure.

Animals↗
PubMed2003

Mucosal humoral immune response to hepatitis C virus E1/E2 surface glycoproteins and HCV shedding in saliva and cervicovaginal fluids from chronically HCV-infected patients.

BACKGROUND/AIMS: We herein focused on identifying biological factors possibly involved in non-parenteral transmission of hepatitis C virus (HCV), such as HCV excretion patterns and antibody-based immunity to the virus in saliva and/or cervicovaginal secretions (CVS). METHODS: Paired blood, saliva and cervicovaginal lavage samples were obtained from HCV-RNA plasma-positive hemoglobin (Hb) antigen and HIV-seronegative, HCV-seropositive males (n=13) and females (n=21). HCV-specific antibodies were detected by ELISA in paired samples, and HCV-RNA was detected in cell-free and cell-associated body fluids. RESULTS: Antibodies to E1 HCV surface glycoprotein of the IgG and IgA isotypes showed similar, but less pronounced, profiles as IgG and IgA to E2. HCV-specific IgG and IgA in mucosal fluids likely originated predominantly from the systemic compartment, because HCV-specific mucosal immunoglobulins involved primarily monomeric antibodies, including monomeric IgA, and because their specific activities for HCV antigens in corporeal fluids were similar to those in paired serum (Se). Viral shedding in saliva or CVS was restricted to cell-associated, non-replicating strand((+)) HCV-RNA in 42% (12 out of 28) of saliva and in 19% (four out of 21) of cervicovaginal fluids. CONCLUSIONS: The association in body fluids of HCV-specific IgG, and to a lesser extent IgA, directed to E1/E2 surface glycoproteins (which may block critical steps of virus-cell interactions), of undetectable free viral RNA, and of occasional non-replicating cell-associated HCV, suggests a resulting poor infectivity of saliva or cervicovaginal fluid in chronically HCV-infected individuals. Taken together, these observations provide the basis for the low risk of non-parenteral transmission of HCV infection.

Adult↗
PubMed2003

Two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity.

The receptor "priming" model for entry of the retrovirus avian sarcoma and leukosis virus (ASLV) predicts that upon binding cell surface receptors, virions are endocytosed and trafficked to acidic endosomes where fusion occurs. To test this model directly, we have now followed subgroup A ASLV (ASLV-A) virions entering cells via either the transmembrane (TVA950) or glycophosphatidylinositol (GPI)-anchored (TVA800) forms of the cellular receptor. Our results suggest that viruses entering via these two forms of receptor are subjected to different intracellular fates, perhaps due to use of different endocytic trafficking pathways to access acidic fusion compartments. Kinetic analyses demonstrated that virus bound to TVA800 was taken up from the cell surface more slowly but then trafficked to the site of fusion more quickly than that entering via TVA950. Furthermore, transiently arresting virions within putative fusion compartments with NH4Cl led to a substantially greater decrease in the infectivity of virions using TVA950 than with those using TVA800. The increased infectivity of virions using TVA800 correlated with the localization of this receptor to lipid rafts, since this effect was abolished by pharmacological disruption of lipid rafts. Together these results suggest that, in the presence of NH4Cl, virus bound to the GPI-anchored receptor may utilize a lipid raft-dependent pathway to accumulate within a fusion compartment where it is more stable than if it enters via the transmembrane receptor. The TVA800/ASLV-A system should prove useful for the molecular analysis of lipid raft-dependent endocytosis and may provide a tool for the biochemical dissection of the poorly understood uncoating step of retroviral replication.

Alpharetrovirus↗
PubMed1986

Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen.

Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.

Amino Acid Sequence↗
PubMed2003

CD8 T cells and aging.

Aging of the immune system, or "immunosenescence," is associated with both a marked reduction in responsiveness as well as functional dysregulation. These changes have been implicated in the increased morbidity and mortality of the elderly population from infectious disease, and may also play a role in autoimmunity and cancer. Though marginal alterations in B lymphocytes are apparent, the dramatic decline in humoral and cell-mediated responses is predominantly the consequence of alterations in the T-cell compartment. The effect of aging on CD4 cell function has been extensively summarized elsewhere. This review, therefore, focuses on the CD8 T-cell subset. Age-related changes in thymic function and involution, cellular homeostasis and lifespan, population shifts, T-cell activation, the process of replicative senescence, and oligoclonal expansions are discussed in terms of their effect on CD8 T cells. Age-associated alterations in CD4 T cells and antigen-presenting cells are mentioned insofar as these cells affect CD8 T-cell activation and function. Distinct patterns of immunosenescence in humans and mice are also noted.

Aging↗
PubMed2002

Cell proliferation during blastema formation in the regenerating teleost fin.

Epimorphic regeneration in teleost fins occurs through the establishment of a balanced growth state in which a blastema gives rise to all the mesenchymal cells, whereas definite areas of the epidermis proliferate leading to its extension, thus, allowing the enlargement of the whole structure. This type of regeneration involves specific mechanisms that temporally and spatially regulate cell proliferation. To understand how the blastema is formed and how this growth situation is set up, we investigated cell proliferation patterns in the regenerating fin of the goldfish Carassius auratus from the time of amputation to that of blastema formation by using proliferating cell nuclear antigen immunostaining and bromodeoxyuridine labeling. Wound closure and apical epidermal cap formation took place by epidermal migration and re-arrangement, without the contribution of cell proliferation. As soon as the apical cap had formed, the epidermis started to proliferate at its lateral surfaces, in which all layers maintained cycling for the duration of the studied process. The distal epidermal cap, on the contrary, presented very few cycling cells, and its cytoarchitecture was indicative of continuous remodeling due to ray growth. The basal layer of this epidermal cap showed a typical morphology and remained nonproliferative whilst in contact with the proliferating blastema. Proliferation in the mesenchymal compartment of the ray started far from the amputation plane. Subsequently, cycling cells approached that location, until they formed the blastema in contact with the apical epidermal cap. Differences observed between the epidermis and mesenchyma, regarding activation of the cell cycle and the establishment of proliferative patterns, suggest that differential mechanisms regulate cell proliferation in each of these compartments during the initial stages of regeneration.

Amputation, Surgical↗
PubMed1982

A B-lymphocyte-specific high-turnover protein: constitutive expression in resting B cells and induction of synthesis in proliferating cells.

A basic B-cell-specific protein of 35,000 daltons (XM1) is expressed at a high rate--comparable to actin--in resting splenic B lymphocytes of various species. Splenic or thymic T cells or fibroblasts do not express this protein. Upon polyclonal stimulation of B lymphocytes or in proliferating B-lymphocyte-derived cell lines, the synthesis of XM1 is switched off, but it is reinduced by agents that arrest replication, such as mitomycin C, hydroxyurea, ultraviolet light or gamma irradiation. Induction of XM1 after mutagen treatment is fast; the synthesis is regulated on the level of transcription or RNA processing. XM1 is turned over with half-life of less than 2 hr. XM1 is glycosylated. From compartment analysis and Triton solubilization it appears that XM1 is bound to the nuclear membrane.

Animals↗
PubMed2005

IgA-coated particles of Hepatitis A virus are translocalized antivectorially from the apical to the basolateral site of polarized epithelial cells via the polymeric immunoglobulin receptor.

Although Hepatitis A virus (HAV) is transmitted by the faecal-oral route, its target for replication is the liver. Little is known of its interactions with cells of the gastrointestinal tract, and it is not known by which mechanisms HAV crosses the intestinal epithelium. In this study, it is shown that HAV associated with IgA is translocated from the apical to the basolateral compartment of polarized epithelial cells via the polymeric immunoglobulin receptor by IgA-mediated reverse transcytosis. The relevance of this mechanism, by which HAV-IgA complexes may overcome the intestinal barrier and contribute to infections of the liver, results from the fact that HAV-IgA complexes are infectious for hepatocytes and that significant amounts of intestinal HAV-IgA are present during acute infections, which are also partly transmitted. Besides supporting the primary infection, this mechanism may play a role in relapsing infections by establishing an enterohepatic cycle for HAV.

Antigen-Antibody Complex↗