Comparison of replicase localization in different types of mouse hepatitis virus (MHV)-infected cells.
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Animals↗
Biomedical subjects
D Egger
Publications and source records attributed to D Egger.
Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex.
Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.
Animals↗
Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.
HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.
Cytoplasmic Vesicles↗
A cytopathic and a cell culture adapted hepatitis A virus strain differ in cell killing but not in intracellular membrane rearrangements.
Aside from a common gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype, the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Nevertheless, several cp HAV strains have been isolated during the past decade. In FRhK-4 cells infected with HM175/24a, a fast-growing cp strain, increasing amounts of viral RNA, detected by fluorescence in situ hybridization, indicated viral RNA replication. An ultrastructural analysis of the infected cells revealed a tubular-vesicular network in close proximity to the rough endoplasmic reticulum. Infection of the same cell type with a cell culture adapted (cc) strain, HM175/P35, divulged membrane alterations indistinguishable from the network induced by the cp strain. The overall appearance of the tubular-vesicular network resembles membrane alterations induced by other picornaviruses. However, the shape of the vesicle-like structures is rather oblong and tubular and, thus, seems to be specific for HAV. By electron microscopic immunocytochemistry (IEM), proteins 2B and 2C were found exclusively on the membranes of the network. Proteins expressed from the open reading frame of the cc HAV variant or 2B proteins originating from HM175 cp, cc, or the wt strain expressed in the absence of other HAV proteins induced membrane alterations resembling those seen in HAV-infected cells. The induction of similar structures suggests that protein 2B is involved in the rearrangement of cellular membranes. In all cases, IEM demonstrated that the 2B protein was closely associated with altered membranes. The extent of membrane changes did not seem to increase for both the cp strain and the cc strain during the infectious cycle. Late in the infection and shortly before the culture died off, a large number of cells infected with HM175/24a showed typical signs of apoptosis, whereas the cc strain did not induce cell killing in the same type of cells. Therefore, we conclude that cell death in HM175/24a-infected cells is induced by apoptosis rather than by cytopathology.
Apoptosis↗
Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle.
Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first approximately 2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.
Apoptosis↗
Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis.
Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.
Coated Vesicles↗
Characterisation and mitochondrial localisation of AUH, an AU-specific RNA-binding enoyl-CoA hydratase.
AU-rich elements function as instability elements which direct rapid mRNA degradation. AUH protein exhibits an AU-specific RNA-binding property and an intrinsic enoyl-CoA hydratase activity and may therefore function to link mRNA decay to metabolic processes (. Proc. Natl. Acad. Sci. USA 92, 2051-2055). The sequence encoding the murine protein, muAUH, was established by cloning, and the corresponding polypeptide predicted to have a molecular mass of 37kDa. As shown for the human protein, muAUH is expressed in a 32kDa form and there is 94% homology between the two species. Recombinant muAUH was shown to be an RNA-binding enoyl-CoA hydratase. All murine cells studied contained a single AUH transcript of approx. 1.7kb and an investigation of tissue-specific expression revealed highest levels in kidney, skeletal muscle, heart, liver and spleen. It was further determined, using immunoelectron microscopy, that AUH is located in the mitochondria of mouse cells.
Amino Acid Sequence↗
Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.
Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.
Fluorescein-5-isothiocyanate↗
Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.
The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.
Amino Acid Chloromethyl Ketones↗
Induction of leukotriene production by bleomycin and asparaginase in mast cells in vitro and in patients in vivo.
Bleomycin and asparaginase are widely used antineoplastic agents which may induce allergic or inflammatory side-effects. Mast cells are implicated as effector cells in allergic and inflammatory responses. The aim of this study was to establish whether bleomycin or asparaginase modulate leukotriene production in vitro and in vivo. Leukotriene C4 (LTC4) production by murine bone marrow-derived mast cells (BMMC) was determined by radioimmunoassay (RIA). Leukotriene production in patients was assessed by determining leukotriene E4 and N-acetyl-leukotriene E4 in urine by means of combined HPLC and RIA. Bleomycin induced an up to 2.1-fold increase in LTC4 production both in unstimulated and in calcium ionophore-stimulated mast cells. In 3 of 7 patients treated with bleomycin, a greater than 2-fold increase in endogenous leukotriene production was observed. This effect was associated with febrile responses and was most pronounced in a patient who developed an Adult Respiratory Distress Syndrome (ARDS). Asparaginase increased leukotriene production up to 10-fold in stimulated but not in unstimulated BMMC. In a patient who developed an anaphylactic reaction after treatment with asparaginase, a pronounced increase in urinary leukotriene concentration was observed. In contrast to bleomycin or asparaginase, a number of other cytostatic agents did not significantly change leukotriene production by BMMC. Our data indicate that some of the inflammatory and allergic side-effects of bleomycin and asparaginase could be mediated by leukotrienes, a possible source of which may be mast cells.
Adult↗
Colloidal gold staining and immunoprobing on the same western blot.
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Animals↗
Electron microscopic-silver enhancement for double labeling with antibodies raised in the same species.
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Animals↗
Pre-treatment of peripheral blood progenitor cells with macrophage inflammatory protein-1alpha induces prolonged survival of early progenitor cells over 6 weeks of long-term culture.
It has previously been shown that a combination of macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin (IL)-3 maintained human bone marrow (BM)-derived long-term culture-initiating cells (LTC-IC) for at least 8 weeks in vitro. We investigated colony- and cobblestone area-formation potential of peripheral blood progenitor cells (PBPC) at week 6 of long-term culture (LTC) in the absence of exogenous MIP-1alpha. but using cells which had been pre-incubated in the presence of MIP-1alpha for 40 h in liquid culture. The recovery of colony-forming cells (CFC) and cobblestone-area-forming cells (CAFC) after pre-incubation of PBPC with MIP-1alpha was up to threefold higher (P<0.05, n=5) than that of untreated controls. These results demonstrate that short-term pre-treatment of PBPC with MIP-1alpha induces long-lasting survival effects on early PBPC-derived progenitors in vitro.
Cell Survival↗
Intracellular localization of poliovirus plus- and minus-strand RNA visualized by strand-specific fluorescent In situ hybridization.
The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay. In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were visualized by fluorescent in situ hybridization (FISH) with strand-specific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA. The FISH experiments showed minus-strand RNA to be present in distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes. At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.
Cell Line↗
Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine.
The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent Km values are the same for PC and LPC, namely 4.9 mM. Vmax is 82.5 and 10.4 nmol h(-1) mg(-1) for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.
Animals↗
Induction of intracellular membrane rearrangements by HAV proteins 2C and 2BC.
Hepatitis A virus (HAV) is distinguished from other picornaviruses by its slow and relatively poor, noncytopathic growth in cultures of mammalian cells. The 2C and 2BC proteins of HAV have been implicated in the determination of virus growth in cultured cells. The homologous proteins from other picornaviruses, such as poliovirus, have been demonstrated to exhibit multiple activities, such as RNA binding, nucleotide binding and NTPase, and membrane binding and reorganization. At least some of these activities are required for viral RNA replication. We report here that HAV 2C and 2BC proteins, like their poliovirus counterparts, can induce rearrangement of intracellular membranes and directly or indirectly interact with membranes. Therefore, the inefficient replication properties of HAV are not consequences of the inherent ability of 2C (2BC) to interact with membranes. The effect of 2C (2BC) protein sequences derived from a cell culture-adapted (cc) strain of HAV was compared with that of corresponding protein sequences from either a wild-type (wt) strain of HAV or a faster replicating cytopathic (cp) strain. The analysis demonstrated that mutations acquired in wt virus during adaptation to cell culture do not change dramatically either the ability of these proteins to associate with membranes and induce membrane alterations or the specific architecture of the induced membrane structures. On the other hand, 2C, but not 2BC, protein from the cp strain of HAV induced different membrane structures.
Gene Expression Regulation↗
Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells.
Poliovirus protein 2C is a 329-amino acid-protein that is essential for viral RNA synthesis and may perform multiple functions. In infected cells, it is associated with virus-specific membrane vesicles. Recombinant 2C protein expressed in transfected cells has been shown to associate with and induce rearrangement of the intracellular membrane network. This study was designed to map the determinants of membrane binding and rearrangement in the 2C protein. Computer-assisted analysis of the protein sequence led to a prediction that the protein folds into a structure composed of three domains. Expression plasmids that encode each or combinations of these predicted domains were used to examine the abilities of the partial protein sequences to associate with intracellular membranes and to induce rearrangement of these membranes in HeLa cells. Biochemical fractionation procedures suggested that the N-terminal region of the protein was required for membrane association. Electron microscopic and immunoelectron microscopic observation showed that both the N- and C-terminal regions, but not the central portion, of 2C protein interact with intracellular membranes and induce major changes in their morphology. The central portion, when fused to the N-terminal region, altered the specific membrane architecture induced by the N-terminal region, giving rise to vesicles resembling those observed during poliovirus infection.
Binding Sites↗
Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis.
Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RI) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA. At 30 degrees C, the vesicles reassociate into rosettes synthesizing plus-strand RNA in the RI. The in vitro transcriptional activities of rosettes and vesicles kept separated by high dilution were assessed by an RNase protection assay. The synthesis of the first 178 nucleotides at the 5' end of the plus strand was considered to reflect initiation, and the detection of a 530-nucleotide fragment in the P2 genomic region was considered to reflect elongation. It could be shown that the initiation and elongation of plus strands on individual vesicles are comparable to those in rosettes, with initiation proceeding in de novo-assembled initiation complexes. By use of detergent treatment it was found that initiation, but not elongation, is dependent on vesicular membranes.
Humans↗