Confocal microscopy of cells implanted into tissue blocks: cell migration in long-term histocultures.

In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, them implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.

[The penetration of the Marburg virus into eukaryotic cells]. / Proniknovenie virusa Marburg v éukarioticheskie kletki.

The early stages of infection of Vero-E6 cell culture with Marburg virus, a member of filovirus family, highly pathogenic for man, were studied. Virus multiplication was completely or significantly inhibited by lysosomotropic agents (LTA) of two types: weak base (ammonium chloride) and ionophore monensin. The level of the inhibiting effect was proportional to LTA concentration and was maximal when the drug was introduced into the culture medium before virus inoculation. Complete inhibition of Marburg virus replication in Vero-E6 cells in the presence of 20 (30) mM ammonium chloride ("lysosomotropic blocking") was overcome by a short-time treatment of the cell culture with the virus adsorbed on it using a medium with a weak-acid pH (4.0-5.0). The results are discussed from the point of view of the mode of this virus penetration into eukaryotic cells.

The fusion of artificial lipid membranes induced by the synthetic arenavirus 'fusion peptide'.

The fusing activity of the synthetic 23 amino-acid fragment (fusion peptide, FP) of the fusion protein of the Lassa arenavirus membrane was tested in a model liposomal system. The resonance energy transfer between two fluorescent phospholipid probes was monitored in order to detect dioleoylphosphatidylcholine liposome fusion induced by the peptide. Fusion rates were compared at different pH values, ionic strength and calcium concentrations. FP demonstrated fusing activity at pH 4.5-5.5, indicating that the protonated form of the FP is the active one. A transmembrane proton-gradient induced by acidification was not relevant to the fusion process, since its elimination with nigericin did not affect the FP-mediated fusion. Both Ca2+ (8 mM) and the increase of the ionic strength (1 M NaCl) inhibited liposome fusion. The efficacy of liposome fusion depended also on the lipid-to-lipid ratio. Non-linear dependence was observed at a saturation ratio of 10 mol lipid per mol peptide. A model of 'side insertion' is suggested, describing FP interaction with the membrane.

[pH-dependent fusion of eukaryotic cells, caused by arenaviruses, pathogenic and non-pathogenic for humans]. / pH-zavisimoe sliianie éukarioticheskikh kletok, vyzyvaemoe patogennymii nepatogennymi dlia cheloveka arenavirusami.

The conditions necessary for fusion from inside (FFWI) of the BHK-21 cell culture affected by the Lassa and Mopeya arenaviruses were studied. The fusion was shown to occur only in the slightly acid medium and at lower pH meanings for the Mopeya virus, than for the Lassa virus.

[Preparative isolation of basic structural proteins of the influenza virus]. / Preparativnoe vydelenie osnovnykh strukturnykh belkov virusa grippa.

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.

A model for the study of the mechanism of a low pH-induced interaction of the virus fusion proteins and cell membranes.

A model is proposed for the study of molecular mechanisms of a low pH-induced interaction of fusion proteins of enveloped viruses and cell membranes. The model consists of large monolamellar liposomes containing ionophore nigericin in their membranes and ectodomains of fusion protein in their inner space. The process of interaction of the protein with the lipid bilayer is triggered by acidification of the liposomal constituents to the pH of fusion with the help of nigericin by adding citric acid to the outer medium. To visualize the protein structural reorganization, the tritium planigraphy was used. Comparison of the values of specific labelling of the proteins and distribution of radioactivity in individual amino acids in control (at neutral pH) and experimental liposome samples (at the pH of fusion) permits to realise the character of protein-membrane interaction. We have obtained the first results in the study of interaction of the bromelain-released soluble ectodomain of the HAXX molecule (BHA)--with the lipid membrane. The observed increase in the protein specific activity and selective increase in the specific activity of hydrophobic amino acids Ile, Phe and Tyr in experimental liposome samples as compared with the controls did not contradict to the conventional concept, that a hydrophobic N-terminus of HA2 subunit of hemagglutinin is responsible for its interaction with lipid membranes.

[The Mopeia virus induces pH-dependent "fusion from within" in a BHK-21 cell culture]. / Virus Mopeiia indutsiruet pH-zavisimoe "sliianie iznutri" kul'tury kletok BHK-21.

The capacity of BHK-21 cell culture to produce Mopeia virus (Arenaviridae family) to form syncytium upon acidification of the culture medium to pH 5.5 and lower was demonstrated. The cell fusion requires their active virus production: a virus titre in the culture medium must be at least 10(5) PEU/ml. The inhibition of virus multiplication with ammonium chloride as well as treatment of the cells before the medium acidification with immune serum reduced syncytium formation markedly. No cell fusion was observed upon acidification of the medium immediately after virus adsorption to cells. Thus, the observed cell fusion under the influence of the virus is an "internal fusion" and confirms our previous data on the endocytosis mode of arenavirus penetration into the cell.

[Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses]. / Sliianie iskusstvennykh lipidnykh membran indutsiruemoe sinteticheskim "peptidom sliianiia" arenavirusov.

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.

Prediction of arenavirus fusion peptides on the basis of computer analysis of envelope protein sequences.

Theoretical search and selection criteria for putative fusion peptides of enveloped viruses are proposed. Arenavirus fusion peptides are predicted on the basis of computer-assisted analysis of amino acid sequences of arenavirus envelope proteins and elements of their secondary and tertiary structure. Accordingly, two regions of GP2 surface protein from 5 viruses of Arenaviridae family have been detected with properties typical of fusion peptides of other enveloped viruses. One region, named peptide IV, located at the N-terminus of the GP2 protein, is followed by the other region or peptide V, more likely candidate for the arenavirus fusion peptide.

[Lysosomotropic agents inhibit the penetration of arenaviruses into a culture of BHK-21 and Vero cells]. / Lizosomotropnye agenty ingibiruiut proniknovenie arenavirusov v kul'turu kletok BHK-21 i Vero.

Lysosomotropic agents (NH4Cl, amantadine, chloroquine, monensin) which prevent acidification of intracellular vacuoles, when introduced into the culture medium before or during inoculation of cells (BHK-21, Vero) with arenaviruses inhibit reproduction of these viruses completely or significantly. Mozambique virus proved to be 10 times more sensitive to the effect of lysosomotropic agents than Pichinde and Lassa viruses. Thus, arenaviruses have a pH-dependent stage at the beginning of the reproduction cycle which is indirectly indicative of their penetration into cells by receptor endocytosis.

Early events in arenavirus replication are sensitive to lysosomotropic compounds.

Lysosomotropic compounds (ammonium chloride, chloroquine, amantadine, monensin) effectively inhibited the replication of Pichinde, Mopeia, and Lassa viruses in BHK-21 and Vero cells. The inhibitory effect was dependent upon the time of drug addition and was most effective when the drugs were added 1 h before the viral adsorption. The drugs had no direct effect on the infectious viruses nor on adsorption of the arenaviruses. These results suggest that the arenaviruses enter cells by adsorptive endocytosis with the participation of acidic intracellular vesicules.

[Isolation of influenza virus hemagglutinin and its separation into subunits by a stage-by-stage scheme for viral protein fractionation]. / Vydelenie gemaggliutinina virusa grippa i razdelenie ego na sub''edinits poétapnoi skhemoi fraktsionirovaniia virusnykh belkov.

The virus envelope protein, hemagglutinin, recovered by DTAB extraction of influenza virions, was purified to a high degree by the gel filtration high-performance liquid chromatography (GF HPLC). Then hemagglutinin was reduced and alkylated with iodoacetamide and the subunits were resolved by GF HPLC on the columns Spherogel TSK G 2000 SW and 3000 SW (600 X 7.5 mm ID).

Transfer of condensed viral DNA into eukaryotic cells using proteoliposomes.

High-molecular-weight viral DNAs have been packed into proteoliposomes prepared by reverse-phase evaporation followed by phospholipid membrane targeting by influenza virus glycoprotein bound to hydrophobic 'anchors'. DNA has been encapsulated in the form of spermine condensates--toroidal structures sized approx. 0.1 micron, resistant to ultrasound. The efficiency of entrapping into liposomes reached 30% for condensed DNA of Mr up to 3 X 10(7). Specific infectivity of simian virus 40 DNA and simian adenovirus DNA packed into such proteoliposomes was 50- to 100-fold higher than that shown by free DNA preparations under Ca.phosphate-precipitation conditions.

[Optimization of the transfer and incorporation of viral DNA into cells using liposomes conjugated with influenza virus glycoprotein]. / Optimizatsiia perenosa i vnedreniia v kletki virusnykh DNK s pomoshch'iu liposom, sviazannykh s glikoproteidami virusa grippa.

Liposomes connected with influenza viral glycoproteins increase by 1-2 orders the specific infectiousness of DNA from SV-40 or monkey adenovirus SA7 as compared with the one registered when the standard method of calcium precipitation is used.

[A rapid method of isolating phosphatidylcholine and phosphatidylethanolamine using high performance liquid chromatography]. / Uskorennyi metod vydeleniia fosfatidilkholina i fosfatidilétanolamina s ispol'zovaniem vysokoéffektivnoi zhidkostnoi khromatografii.

A modified procedure is described for isolation of lecithin and phosphatidyl ethanolamine by means of high pressure liquid chromatography, which enabled to decrease the purification period down to several hours and to use small volumes of eluents of simple composition. Extensive purification of the eluents was not required for separation of phospholipids on half-preparative scale.

[Electrophoretic purification of biologically active structural proteins of the simian adenovirus SA7 in the presence of sodium dodecyl sulfate]. / Elektroforeticheskaia ochistka biologicheski aktivnykh strukturnykh belkov adenovirusa obez'ian SA7 v prisutstvii dodetsilsul'fata natriia.

The modification of disc electrophoresis technique in polyacrylamide gel with sodium dodecylsulphate (SDS) has been elaborated for synchronous isolation of some structural proteins in biologically active form and in preparative quantities from adenoviruses. Virions of SA7 adenovirus were mildly dissociated in SDS solution at 20 degrees C and structural proteins were stained by fluorescamin. After separation the zones of proteins corresponding to the native capsomeres of hexon and protein IV as well as the zones of inner proteins V and VII have been identified as fluorescent at UV-irradiation, excised and extracted by SDS solution. After the removal of SDS by protein precipitation in acetone the preparations of hexon and IV reveal the quaternary structure of native capsomers and full spectrum of antigenic and immunogenic activities of native proteins. Preparations of inner proteins V and VII possess activity in condensing adenoviral DNA. The technique is usable for preparative purification of inner polypeptide VI SA7, as well as capsomers and inner proteins of other adenoviruses.

[The use of the cationic detergent dodecyltrimethylammonium bromide for the isolation of influenza virus glycoproteins with subsequent integration of the protein into liposome membrane]. / Ispol'zovanie kationnogo detergenta dodetsiltrimetilammonii bromida dlia vydeleniia glikoproteidov virusa grippa s posleduiushchim vstraivaniem belka v membranu liposom.

Glycoproteins were isolated from influenza virus with the use of cationic detergent dodecyltrimethylammoniumbromide and attached to preformed liposomes. Liposomal vesicles, thus, acquired their ability for hemagglutination and lysis of chicken erythrocytes. The possibility of using these liposomes for transfer of alien agents into eucaryotic cells is discussed.