Association of the interferon-ß gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a YY1-dependent mechanism.

Nuclear architecture as well as gene nuclear positioning can modulate gene expression. In this work, we have analyzed the nuclear position of the interferon-ß (IFN-ß) locus, responsible for the establishment of the innate antiviral response, with respect to pericentromeric heterochromatin (PCH) in correlation with virus-induced IFN-ß gene expression. Experiments were carried out in two different cell types either non-infected (NI) or during the time course of three different viral infections. In NI cells, we showed a monoallelic IFN-ß promoter association with PCH that strongly decreased after viral infection. Dissociation of the IFN-ß locus away from these repressive regions preceded strong promoter transcriptional activation and was reversible within 12 h after infection. No dissociation was observed after infection with a virus that abnormally maintained the IFN-ß gene in a repressed state. Dissociation induced after virus infection specifically targeted the IFN-ß locus without affecting the general structure and nuclear distribution of PCH clusters. Using cell lines stably transfected with wild-type or mutated IFN-ß promoters, we identified the proximal region of the IFN-ß promoter containing YY1 DNA-binding sites as the region regulating IFN-ß promoter association with PCH before as well as during virus infection.

Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects.

Rift Valley fever virus (RVFV) is an emerging, highly pathogenic virus; RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemorrhagic fever in humans, as well as death, abortions, and fetal deformities in animals. RVFV nonstructural NSs protein, a major factor of the virulence, forms filamentous structures in the nuclei of infected cells. In order to further understand RVFV pathology, we investigated, by chromatin immunoprecipitation, immunofluorescence, fluorescence in situ hybridization, and confocal microscopy, the capacity of NSs to interact with the host genome. Our results demonstrate that even though cellular DNA is predominantly excluded from NSs filaments, NSs interacts with some specific DNA regions of the host genome such as clusters of pericentromeric gamma-satellite sequence. Targeting of these sequences by NSs was correlated with the induction of chromosome cohesion and segregation defects in RVFV-infected murine, as well as sheep cells. Using recombinant nonpathogenic virus rZHDeltaNSs210-230, expressing a NSs protein deleted of its region of interaction with cellular factor SAP30, we showed that the NSs-SAP30 interaction was essential for NSs to target pericentromeric sequences, as well as for induction of chromosome segregation defects. The effect of RVFV upon the inheritance of genetic information is discussed with respect to the pathology associated with fetal deformities and abortions, highlighting the main role played by cellular cofactor SAP30 on the establishment of NSs interactions with host DNA sequences and RVFV pathogenesis.

Cryoprecipitation of an anti-Pr2 monoclonal IgM cold agglutinin in the presence of GM3 ganglioside.

The mechanism of cryoprecipitation of a monoclonal IgM kappa cryoglobulin (Mou) with a cold agglutinin activity of Pr2 specificity has been studied. By immunodiffusion this cryoglobulin reacted (by its Fab' fragment) with micellar GM3, a ganglioside bearing the Pr2 antigenic determinant. In contrast to previous reports that indicated a possible temperature dependent self-association of IgM molecules via an immunological interaction leading to cold precipitation, we could not detect any affinity of this cryoglobulin for IgM when we used passive hemagglutination or an indirect enzyme-linked immunosorbent assay (ELISA). However, a GM3-like ganglioside could be extracted, by drastic methods, from the cryoglobulin studied at 22 degrees C, whereas no GM3 was extracted from two control cryoglobulins. Some minor gangliosides (representing less than 25% of total amount of bound gangliosides) were also extracted from Mou cryoglobulin and these gangliosides were shown to crossreact with GM3, as they specifically bind to Mou cryoglobulin by ELISA. After cryoprecipitation the serum still contained a monoclonal anti-Pr2 IgM kappa. A GM3-like ganglioside could be extracted from this purified IgM, and cryoprecipitability could be induced by the addition of a minute amount of micellar GM3. These results suggest that Mou cryoglobulin circulates as an immune complex and that cryoprecipitation may depend on unique IgM-GM3 (or IgM-GM3 cross-reacting gangliosides) complexes.

Evaluation of a Crimean-Congo hemorrhagic fever virus recombinant antigen expressed by Semliki Forest suicide virus for IgM and IgG antibody detection in human and animal sera collected in Iran.

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by ticks or by direct contact with infected blood. It causes severe, often fatal, hemorrhagic diseases in humans but infection in animals is asymptomatic. CCHFV can spread from person to person and has caused many nosocomial outbreaks. Because the virus is very pathogenic for humans it must be manipulated in a biosafety level 4 (BSL4) laboratory, rendering the production of antigen for serological diagnosis difficult. To replace the native antigen, we produced a recombinant nucleoprotein expressed in mammalian cells via the recombinant Semliki Forest alphavirus replicon and developed an indirect immunofluorescence assay (IFA) as well as an enzyme-linked immunosorbent assay (ELISA) by immunocapture to detect IgM and IgG in human and animal serum. Using these methods, we analyzed clinical samples from human patients and sera from domestic animals collected in Iran and we show that this novel antigen provides a novel, sensitive and specific tool for CCHF diagnosis.

Production of monoclonal antibodies against Rift Valley fever virus Application for rapid diagnosis tests (virus detection and ELISA) in human sera.

This paper describes the production and characterization of RVFV monoclonal antibodies. The characteristics of 32 out of 55 ELISA and/or IFA positive monoclonal antibodies were determined, including the RVFV components against which they are directed. One monoclonal antibody recognized the nucleoprotein, 15 the Gc and 16 the Gn. Among the latter ones, five monoclonal antibodies possess another specificity and recognized both Gn and either the nucleoprotein (four of them) or the NSs protein (one). To validate the use of these monoclonal antibodies for diagnosis tests, a pool of monoclonal antibodies reacting with the structural proteins was prepared and used successfully to detect RVFV from cell culture as well as viral antigen-antibody complex in ELISA.

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain.

The phases of simple systems involving one type of protein (lysozyme or cytochrome c) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperature than the one observed in the case of the phase with electrostatic interaction.

Immunochemical studies of polyspecific natural autoantibodies: charge, lipid reactivity, Fab'2 fragments activity and complement fixation.

Polyspecific natural autoantibodies (NAAb) are antibodies present in normal unimmunized animals and are able to react with very dissimilar antigens (Ag). To better delineate the characteristics of polyspecificity, we subjected monoclonal NAAb to four different immunochemical studies: (1) Two-dimensional gel electrophoresis performed on eight NAAb did not reveal any obvious relationship between charge and antigen specificities; (2) NAAb widely polyspecific on proteins and nucleic acid were reactive with lipids bearing either phosphate, sulfate or carboxyl polar groups; (3) pepsin digestion of polyspecific IgM NAAb yielded Fab'2 fragments which maintained their multireactivities, but exhibited a decrease in reactivity as compared to that seen with monospecific mAb (induced); (4) two different assays were used to analyse the complement fixation ability of IgM NAAb. While very weak or no complement fixation was observed with a classical complement fixation test (fluid phase), when a complement enzyme immunoassay was used where Ag is immobilized on a solid phase, polyspecific NAAb fixed reproducible and easily detectable amounts of complement.

Protection by phospholipids of Schistosoma mansoni schistosomula against the action of cytotoxic antibodies and complement.

Schistosoma mansoni schistosomula cultured in the presence of phospholipids showed a decreased sensitivity to the lethal complement-mediated action of anti-schistosome antibodies. Phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine had a protective action on the schistosomula transformed in vitro by passage through the skin or by a mechanical procedure. Phosphatidyl choline acted regardless of its fatty acid composition. Phosphatidyl serine and phosphatidic acid did not protect. Thus, it appears that phospholipids can play a role in parasite resistance to immune attack by cytotoxic antibodies and complement, and that this role is specific to certain phospholipid types.

Characterization of galanin and 5-HT1A receptor coupling to adenylyl cyclase in discrete regions of the rat brain.

We have studied the coupling of galanin and 5-HT1A receptors with adenylyl cyclase in the hypothalamus, the entorhinal cortex and the hippocampus of the rat brain. Furthermore, we have evaluated the effects of simultaneous activation of galanin and 5-HT1A receptors on adenylyl cyclase activity. Galanin-(1-29) and galanin-(1-15) showed a dose-dependent inhibitory effect on forskolin-stimulated adenylyl cyclase activity in the hypothalamus and entorhinal cortex. No clear effects were observed in the hippocampus. Neither galanin-(1-29) nor galanin-(1-15) had any effect on the basal activity of adenylyl cyclase in these regions. The selective 5-HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH-DPAT) induced a dose-dependent inhibition of forskolin stimulated adenylyl cyclase activity in the hippocampus and the entorhinal cortex. 5-HT induced an inhibition in the hypothalamus. In all regions the effects could be fully counteracted by methiothepin. 5-HT was shown to stimulate the basal activity of adenylyl cyclase in the hippocampus and the entorhinal cortex. The effects could be counteracted by methiothepin. When galanin-(1-29) and 5-HT/8-OH-DPAT were incubated simultaneously additive inhibitory effects, but no synergistic interactions, could be observed on the stimulated adenylyl cyclase activity. In conclusion, galanin and 5-HT1A receptors seem to be linked to different independent pools of G proteins, indicating that the previously demonstrated intramembrane interactions between galanin and 5-HT1A receptors involve a mechanism not directly related to adenylyl cyclase.

[The structure of biological membranes: localization of sulfoquinovosyldiglyceride in various membranes of chloroplasts using specific antibodies]. / Structure des membranes biologiques: localisation du sulfoquinovosyldiglycéride dans les diverses membranes des chloroplastes au moyen des anticorps spécifiques

The sulfoquinovosyl pole of at least a part of the sulfolipid molecule, in the envelope and the thylakoids of chloroplasts, is accessible to the specific antibody. Agglutination of the chloroplasts is due to the sulfolipid which is located on the envelope and uniformly distributed on the external surface. Reactivity of the sulfolipid located on the membrane of thylakoids, when exposed to the corresponding antibody, appears to be different from the sulfolipid reactivity of the envelope: the antibody fails to agglutinate the thylakoids. Thylakoids altered by osmotic chock are sensitized to agglutination. Immunoenzymatic studies followed by electron microscopy revealed the presence of the sulfolipid on the membrane of the various thylakoids. The local reactions did not appear to be continous.

Persistent infection of mammalian cells by Rift Valley fever virus.

Infection of mammalian cells with Rift Valley fever virus (RVFV) leads generally to the production of virus and cell death. In this paper we examined the fate of Vero cells infected with three strains of RVFV and observed that, while a large proportion of cells exhibited a clear cytopathic effect (CPE), a small but significant fraction did not undergo a lytic infection but was able to proliferate and establish a persistent infection. Several independent RVFV persistently infected cell lines have been established and passaged for more than 1 year after infection with a virulent strain (ZH548) and two attenuated strains (C13 and MP12). Although the viruses used for the primary infection were plaque-purified, we do not know whether defective-interfering particles were responsible for the establishment of the persistent infection. The persistently infected cells became resistant to superinfection with RVFV but not with other viruses and shed low amounts of infectious, lytic and non-lytic virus during a limited number of passages. In all the passages tested, the three genomic segments or related products were synthesized as well as the structural nucleoprotein N and glycoproteins G1 and G2. Abnormal defective RNAs were detected, migrating faster or slower than their respective counterparts. The faster-migrating RNAs were internally deleted, some of them possessing only the very terminal part of the 5' genomic end.

[Phospholipid antigens acquired by the young forms of Schistosoma mansoni]. / Acquisition d'antigènes phospholipidiques par les formes jeunes de Schistosoma mansoni.

Immunofluorescence studies provide evidence of cardiolipin fixation at the schistosomulum's surface, following incubation with liposomes (cardiolipin-lecithin or cardiolipin-lecithin added with cholesterol). Fixation occurs at 37 degrees C as well as at 0 degree C whether proteins were present or not. Several washes do not remove cardiolipin fixation.

The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice.

Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405-411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the Bunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.

1 alpha,25-dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells.

1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH)2D3 could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, we analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH)2D3 in a dose- and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH)2D3 was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH)2D3 regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.

Damage of liposomes in antibody-dependent cell-mediated cytotoxicity.

Lymphoid cells bearing Fc receptors are able to lyse antibody-coated animal target cells in an antibody-dependent cell-mediated cytotoxicity reaction. The results presented here show that liposomes consisting of sphingomyelin, cholesterol, and cardiolipid and coated by cardiolipidic antibodies could be destroyed by spleen or thymus cells. No alteration of liposomes was observed when normal rabbit serum was used or when the effector cell population was depleted of cells bearing Fc receptors. The lysis of antibody-coated liposomes by effector cells could be carried out in two steps. In the first step, the fixation of antibodies on the cardiolipidic antigens could lead to a reorganization of the liposomal membrane. In the second step, the effector Fc-receptor-bearing cells might amplify this alteration of the liposomes.

Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences.

Rift Valley fever virus (RVFV) is an arbovirus of the BUNYAVIRIDAE: family, causing recurrent disease outbreaks in Africa. Natural vertebrate hosts include cattle and humans. Several mosquito species belonging to the AEDES: and CULEX: genera act as vectors of this phlebovirus. To test whether pathogen-derived resistance against RVFV could be induced by expressing genomic sequences in mosquito cells, as has been shown for La Crosse and dengue 2 viruses, we generated various recombinant Semliki Forest viruses expressing the S segment (or its genes) in the genomic or antigenomic sense. Expression of the N but not the NSs gene interfered with the production of RVFV in mosquito cells and this phenomenon was RNA- but not protein-dependent. These results raise questions on the molecular mechanisms involved in virus resistance.

Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool.

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), micro -capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity.

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase beta 1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in beta 1 subunit expression occur in the absence of a detectable increase in expression of any of the three alpha subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the beta subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of beta subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the beta subunit to the plasma membrane may be independent of its binding to the alpha subunit.

The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein.

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the Bunyaviridae family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557-561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.