Biomedical subjects
Publications and source records attributed to Jean Cohen.
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Paramecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes.
The potential of rotavirus 2/6-virus-like-particles (VLP2/6) for use as tracers in the marine environment was investigated. The stability of bovine rotavirus (strain RF) and VLP2/6 in natural seawater at 25 degrees C for six days was studied. ELISA and western blot methods were used to quantify the particles. The rates of decline of rotavirus particles and VLP2/6 were similar (approximately 0.5log(10) per day). Western blot analysis showed that the integrity of capsid proteins VP2 and VP6 was conserved during the incubation time. These results demonstrate that VLP2/6 particles have the same stability in seawater as rotavirus particles. Thus, VLP2/6 can be used as a tracer, which should be of particular value for studying the fate of rotavirus particles in the marine environment.
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Exocytotic mutants can be obtained in Paramecium that affect the organization of the fusion machinery, visible by electron microscopy. The site of action of the genes in the plasma membrane, cytosol or secretory compartment can easily be determined in such mutants. Functional complementation cloning of exocytotic mutants specifically affected in the secretory compartment, nd2-1 and nd169-1, reported here, and the previously studied nd7-1, led to the discovery of a set of novel proteins that display PSI and EGF domains, normally found in extracellular matrix proteins and involved in transmembrane signaling. The structure of one of these proteins, Nd2p, and of the product of a paralog found in the genome Nd22p, corresponds to that of type I membrane receptors, generally involved in protein and vesicle sorting. Our characterization suggests that the proteins we have identified are required to indicate the presence of a mature secretory vesicle to the plasma membrane, to prepare the machinery for fusion. We propose to name this novel subclass of receptors VEMIF, for Vesicular Extracellular-Matrix-like proteins Involved in preparing membrane Fusion.
Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20 degrees C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20 degrees C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.
Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.
Immunity to human group A rotavirus (RV), a major cause of viral gastroenteritis in infants, involves B lymphocytes that provide RV-specific antibodies. Additionally, some arguments suggest that naive B cells could be implicated in the first steps of the immune response against RV. The aim of our study was to analyze the interaction of VP6 and VP7 RV capsid proteins with human B cells depending on the immune status of the individual, i.e., naive or RV experienced. For this purpose, a two-color virus-like particle flow cytometry assay was devised to evaluate the blood B-lymphocyte reactivity to VP6 and VP7 proteins from healthy RV-exposed adults, recently infected infants, and neonates at birth. Both VP6 and VP7 interactions with B cells were mediated by surface immunoglobulins and probably by their Fab portions. VP7-reactive B lymphocytes were mainly detected from RV-experienced patients and almost exclusively in the CD27-positive memory cell fraction. Conversely, VP6-reactive B lymphocytes were detected at similar and high frequencies in adult, infant, and neonate samples. In adult samples, VP6 reacted with about 2% of the CD27-negative (CD27(neg)) naive B cells. These results demonstrated that the VP6 RV protein interacted with a large fraction of naive B lymphocytes from both adults and neonates. We propose that naive B cell-VP6 interaction might influence the strength and quality of the acquired immune response and should be considered for elaborating RV vaccine strategies.
In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.
The gene encoding the protein VP7 that induces the major neutralizing response has been sequenced from 34 human rotaviruses isolated from children with acute diarrhea in Montevideo (Uruguay) over a 4-year period (1996-1999). These sequences were analyzed and compared to representative corresponding sequences available on databases. In most years, serotype G1 was present as the single serotype, except in 1999 when serotypes G1 and G4 were present simultaneously. Two G1 VP7 lineages were identified. Serotype G2 was present in 1997. The G4 isolates are grouped with Argentine strains and emerged during 1998 in a recently defined sublineage. Neither serotype G3 nor the emerging serotype G9 were isolated during the study. Antigenic domains of isolates and of representative reference strains of each serotype were compared. Sequences of strains isolated during the same year, showed a high degree of homology among strains belonging to the same serotype.
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Rotavirus spike protein VP4 is implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. It is present at the plasma membrane and colocalizes with the cytoskeleton in infected cells. We looked for cellular partners responsible for the localization of VP4 by two-hybrid screening of a monkey CV1 cell cDNA library. In the screen we isolated repeatedly three cDNAs encoding either two isoforms (a and c) of Rab5 protein or the prenylated Rab acceptor (PRA1). The small GTPase Rab5 is a molecule regulating the vesicular traffic and the motility of early endosomes along microtubules. Rab5 interacts with a large number of effectors, in particular with PRA1. Interactions of VP4 with both partners, Rab5 and PRA1, were confirmed by coimmunoprecipitation from infected- or transfected-cell lysates. Interaction of Rab5 and PRA1 was restricted to free VP4, since neither triple-layered particles nor NSP4-VP4-VP7 heterotrimeric complexes could be coprecipitated. Site-directed and deletion mutants of VP4 were used to map a VP4 domain(s) interacting with Rab5 or PRA1. Of the 10 mutants tested, 2 interacted exclusively with a single partner. In contrast, the domain extending from amino acids 560 to 722 of VP4 is essential for both interactions. These results suggest that Rab5 and PRA1 may be involved in the localization and trafficking of VP4 in infected cells.
Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.
The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH(2)-terminal sequencing of the proteolytic fragments showed that they all contained the beta-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the alpha-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.
N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of approximately 30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-gamma-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation.
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There is no European view on gender selection. Conclusions of the ESHRE Ethics Task force are reported.