Studies of Molecular Mechanisms Underlying Cardioprotective Action of the ALM-802 Compound.

The mechanisms underlying cardioprotective activity of compound ALM-802 were studied in experiments on rats with chronic post-infarction heart failure. Real-time PCR showed that compound ALM-802 (daily intraperitoneal injections in a dose of 2 mg/kg for 28 days starting from day 91 after myocardial infarction modeling) restored the expression of genes encoding ß1- (p=0.00001) and ß2-adrenoreceptors (p=0.01) and type 2 ryanodine receptors (p=0.008) in the myocardium that was reduced in control animals. These effects can serve as the basis for the ability of the compound to reduce the intensity of remodeling and increase the inotropic function of the left heart ventricle shown earlier in this model.

[Effect of the antiviral drug Ingaviruin on intracellular transformations and import into the nucleus of influenza A virus nucleocapsid protein].

The paper presents the results of studying the effect of the antiviral drug Ingavirin on different stages of intracellular transformations of influenza A virus nucleocapsid protein (NP). Ingavirin 400-1000 microg/ml has been found to impair the biogenesis of influenza virus NP, to lower the efficiency of formation of conformationally mature compact NP oligomers, and to retard the migration of newly-synthesized NP from the cytoplasm to the nucleus. It is shown that there is an association of tritium-labeled Ingavirin with the nuclear membranes of MDCK cells. The investigations of the mechanisms of antiviral activity of Ingavirin are not only important for the characterization of this drug, but also promote the detection of potential targets to design novel antiviral agents.

[Relationship of nuclear import of influenza virus nucleoprotein polymers to their conformational status].

It has been earlier shown that in the cells infected with influenza virus, the molecules of nucleoprotein (NP) are polymers that differ in their conformational maturity and stability. The present investigation has studied the ability of different conformational forms of NP polymers to migrate into the nucleus. Conformationally mature compact NP oligomers are shown to predominantly import into the nucleus. In contrast, unstable, loose, and conformationally immature NP multimers accumulate in the cytoplasm and do not migrate into the nucleus. The present investigation is the first evidence for that that the conformational maturity of influenza virus NPs is essential for their nuclear traffic and, hence, for participation in the transcription and replication of viral genomes.

[Native and renatured oligomer-dependent epitopes of intracellular influenza virus nucleocapsid protein].

Intracellular NP oligomers have been shown to react with some anti-NP monoclonal antibodies (mAbs) in radio-immnoprecipitation, immunoblotting, and dot immunoassay. Soluble NP monomers obtained after thermal dissociation of NP oligomers are not recognized by mAbs unlike the NP monomers whose concentration increased by about 100-fold due to transfer to the nitrocellulose membrane after polyacrylamide gel electrophoresis. The findings demonstrated that in the intact NP oligomers there were epitopes determined by their quaternary structure. These oligomer-dependent epitopes may be renaturated in vitro under the conditions allowing for a concentration-dependent NP-NP association.

[Two types of NP-NP associations in influenza virus-infected cells].

Two types of NP-NP associations are shown to form in the influenza virus-infected cells. Early NP synthesis gives rise to NP associations stabilized by relatively weak bonds. These structures are designed as NP multimers. The high protease- and heat-sensitivities allow NP-multimers to be regarded as incompletely folded proteins. Post-translationally, NP-multimers transform to compact NP associations (NP oligomers) that are relatively highly heat-and protease-resistant. The NP-multimers untransformed to the folded compact NP-oligomers accumulate in the cells and partially degraded. Whether both types of NP-NP associations may be of significance is under discussion.

[Effect of Panavir on influenza A virus reproduction].

Mitogenic properties of panavir, as well as its effect on the grippe virus reproduction in cell systems in vitro and the effect on the survival of mice with the experimental grippe infection were studied. It was shown that panavir had no cytotoxic action whereas it was characterized by pronounced mitogenic activity and subsequently could be considered as a perspective immunomodulator. Under in vitro conditions with the use of relatively high doses for the cell contamination with the grippe virus, panavir lowered the virus production in the cell systems. When the contaminating doses were low, panavir inhibited the virus production detected at the early stages of the infection. In the in vivo studies on mice with the experimental grippe infection panavir showed antigrippe activity against both the romantadine resistant and the remantadine nonresistant populations of the grippe A virus.

[Temporary acquisition of intra-chain disulphide bonds by nucleocapsid protein of influenza virus].

The in vitro reducing agents were shown to promote the NP-NP association and to stabilize the NP oligomers, which dissociate when heated in non-reducing buffer. This confirms that non-covalent linkages in electrophoresis stabilize the influenza virus NP oligomers. The mobility of pulse-labeled and chased NPs in PAGE as well as their sensitivity to protease were investigated. The S-S bonds reduce at later stages of conformational maturation of NP; the disulfide-containing NP transforms itself into an NP free of S-S bonds with non-covalently linked NP-oligomers being subsequently formed. Presumably, the early disulfide-dependent stage in NP maturation is needed for the correct NP-NP association and for the protection of early monomeric NPs against protease action.

Transient disulfide bonds formation in conformational maturation of influenza virus nucleocapsid protein (NP).

It has been previously shown that influenza virus nucleocapsid protein (NP) forms homooligomers in vivo. Our analyses revealed that the reducing agent dithiothreitol (DTT) introduced in pulse labeling period prevented further formation of native NP-oligomers. The shortly pulse-labeled non-reduced newly synthesized NP possessed a relatively faster mobility in non-reducing PAGE and a higher resistance to protease than the reduced one. These data suggest that there is an early disulfide-dependent step in NP maturation and that the newly synthesized NP possesses the intrachain disulfide bonds. In contrast to the newly synthesized NP, the non-reduced chased NP possessed the same mobility in non-reducing PAGE and the same sensitivity to protease as the reduced NP. DTT introduced in the chase period did not prevent NP-oligomers formation and did not destabilize already formed NP-oligomers. This suggests that the chased NP monomers and NP-oligomers do not contain intrachain nor interchain disulfide bonds. It was also shown that the non-reduced newly synthesized NP could not form NP-NP complexes in vitro, and acquired such ability only after reducing. The possibility is discussed that there are several stages in the maturation of NP: the initial formation of intrachain disulfide-linked NP and conversion into disulfide-free NP, which forms non-covalently stabilized NP-oligomers. Early intrachain disulfide bonds may be necessary for the prevention of early spontaneous NP-NP association.

Extracellular truncated influenza virus nucleoprotein.

In the culture medium of MDCK cells infected with influenza A/Duck/Ukraine/1/63(H3N8) virus two kinds of virus nucleoprotein (NP) are detected: full-length 56 kDa NP and truncated 53 kDa NP. However, in infected cells 53 kDa NP may be detected only at short pulse and after 10 min chase it becomes nondetectable. The extracellular truncated 53 kDa NP is detected in free RNP, and not in the virions. Both extracellular free 53 and 56 kDa NP in the virions are completely oligomerized. Several data argue against the possibility of extracellular 53 kDa NP formation being a result of extracellular 56 kDa NP proteolytic degradation. Thus, the accumulation of extracellular 53 kDa NP takes place only in the course of infection, and the amount of 53 kDa NP is not increased during prolonged storage of cell-free culture medium at +37 degrees C. Moreover, all extracellular 56 kDa NP of A/Duck/Ukraine/1/63 influenza virus is present in the oligomeric form, and the latter, in contrast to the mononeric form, is highly resistant to proteases. The possibility is discussed that in the course of A/Duck/Ukraine/1/63 (H3N8) influenza virus infection a fraction of the synthesized 56 kDa monomeric NP undergoes the proteolytic cleavage in the infected cells before oligomerization and forms the 53 kDa NP. This 53 kDa NP is then oligomerized, enters the RNP and is quickly secreted from the cells.

Detection of viral antigens by solid phase radioimmunoassay on polyethylene film.

Polyethylene film, without any pretreatment, may serve as a solid phase (SP) for RIA. Viral antigens (HBsAg, and influenza virus) are detected by SP-RIA on the film with a sensitivity of about 2-3 ng/ml or 40-60 pg/assay. The use of polyethylene film allows one to record RIA autographically. The use of micro amounts of reagents and specimens tested is an added advantage. No special equipment is necessary, the method is inexpensive, easy to perform and may be used for mass screening.

Avian and human influenza a virus strains possess different intracellular nucleoprotein oligomerization efficiency.

We have previously shown (Prokudina-Kantorovich EN and Semenova NP, Virology 223, 51-56, 1996) that the nucleoprotein (NP) of influenza A virus forms in infected cells oligomers which in the presence of SDS and 2-mercaptoethanol (ME) as reducing agent are stable at room temperature (RT) and dissociate at 100 degrees C. Here we report that the efficiency of intracellular NP oligomerization depends on the host origin of influenza A virus strain. Thus, in the cells infected with avian influenza A virus strains the viral NP was almost completely oligomerized and only traces of monomeric NP were detected by polyacrylamide gel electrophoresis (PAGE) in unboiled samples. However, in the cells infected with human influenza A virus strains, besides oligomeric NP also a significant amount of non-oligomerized monomeric NP was detected in unboiled samples. In purified virions of avian and human strains the same difference in NP monomers/oligomers ratio was detected as in the infected cells. A reassortant having all internal protein genes from a human strain and the glycoprotein genes from an avian strain revealed the same intracellular pattern of NP monomers/oligomers ratio as its parental human virus. These findings suggest that the type of NP oligomerization is controlled by the NP gene. The possible connection between the accumulation of protease-sensitive monomeric NP in cells infected with a human influenza strain and the parallel accumulation of cleaved NP in these cells is discussed.

[Detection of influenza virus nucleoprotein on the surface of infected cells and in free nonvirion form]. / Vyiavlenie nukleoproteina virusa grippa na poverkhnosti zarazhennykh kletok i v svobodnoi vnevirionnoi forme.

The inner viral nucleoprotein synthesized de novo is shown to be exposed on the surface of the chicken embryo infected with influenza virus. The kinetics of the nucleoprotein located on the surface does not correlate with the kinetics of cell destruction. In culture or allantois virus containing liquids the large number of extracellular viral nucleoprotein prone to antinucleoprotein monoclonal antibodies was found. The accumulation of this nucleoprotein occurs in the period when cell destruction is absent, it is eliminated by the adsorption of the virus on erythrocytes or centrifugation at 70,000 g for 2 hours (20%) or by centrifugation at 10,000 g for 4 hours (50%).

[Intracellular "sorting" of highly marked (3H)-v-RNA during maturation of influenza virus]. / Vnutrikletochnaia "vybrakovka" vysokomechennykh [3H]-v-RNK pri sozrevanii virusa grippa.

In the presence of [3H]-nucleosides the high-labeled viral [3H]-RNAs are shown to be sorted out and not included into maturing viral particles in the cells infected by influenza virus. Finding of the "sorting out" mechanism came to be possible due to storing cells in the cold for a long time and thus permitting to accumulate [3H]-decays under the conditions of temporary interruption of infectious process. Most probably, the high-labeled [3H]-RNAs are not included into the maturing viral particles due to their radiolytic damages. The "sorting out" of high-labeled viral RNAs is evidently compensated by comparatively low-labeled RNAs from the redundant intracellular pool of v-RNAs.

[Complete oligomerization of the nucleoprotein and various strains of influenza virus]. / Polnaia oligomerizatsiia nukleoproteina u nekotorykh shtammov virusa grippa.

Previously we demonstrated that in the course of intracellular reproduction of WSN influenza virus strain, part of monomeric nucleoprotein (NP) undergo polymerization into dimers and trimers, which dissociate into monomers after boiling. Further studies showed that different strains of influenza virus are characterized by different degree of NP-oligomerization. Specifically, Duck/ Ukraine/63 (H3N8) and Seal Massacuhsets 1/80 (H7N7) NP monomers are completely transformed into oligomers. As a result of 40-min chase and of prolonged label exposure only NP-oligomers but not monomers can be detected in unboiled samples of infected cells or in virions. NP monomers of A/Duck/Ukraine strain are detectable in unboiled samples only after a short period of labeling. Influenza virus NP oligomers are more hydrophobic than NP monomers. Oligomers are hypothesized to be the native functionally important form of influenza virus NP.

[Polymeric forms of the influenza virus nucleoprotein]. / Polimernye formy nukleoproteina virusa grippa.

Two high-molecular-weight types of in vivo formed nucleoproteins (NP) capable of reacting with anti-NP monoclonal antibodies were detected in continuous MDCK cells and primary CEFs, along with the monomeric virus NP. High-molecular NP were stable at 37 degrees C, despite the presence of dodecyl sulfate and B-mercaptoethanol, but were converted to monomeric NP at 37 degrees C in the presence of 6 M urea, 1 M NaCl, and at acid pH. Hence, high-molecular-weight NP may be the polymeric forms of NP, which are probably stabilized by noncovalent bonds. The kinetics of NP-polymers formation suggests that NP-monomers are their precursors and NP-polymers are formed rapidly after monomeric NP molecules synthesis. Both forms of NP-polymers are detected in the sedimented and soluble fractions of not only cell lysate, but in the extracellular maintenance medium as well. NP-polymers are resistant to RNAse and are more resistant to protease than NP-monomers.

[Radioimmunological analysis of the antigenic variability of influenza virus nucleoprotein]. / Radioimmunologicheskii analiz antigennoi variabel'nosti nukleoproteina virusa grippa.

Influenza A virus ability to bind anti-NP monoclonal antibodies to two viral strains has been studied by radioimmunoassay on polyethylene film with the subsequent autoradiographic registration of results. Monoclonal antibodies were obtained to the viral strains differing in antigenic formula of outer glycoproteids and isolated at different time. The studied influenza viruses were divided into seven groups due to their ability to bind monoclonal antibodies. The absence of correlation between the antigenic properties of nucleoprotein and glycoproteids has been registered. Variability of some antigenic sites has been analyzed. The human epidemic strains of influenza virus are different in ability to bind monoclonal antibodies from the viral strains that are connected with animals in nature or laboratory practice.

[Detection of virus-specific protein synthesis in animal organs]. / Vyiavlenie virusspetsificheskikh belkovykh sintezov v organakh zhivotnykh.

Registration of intracellular protein syntheses in vivo was shown to be possible by inoculation of labeled amino acids directly into animal organs. In the lungs of intranasally infected mice there occurs inhibition of cellular protein syntheses against the background of which synthesis of virion proteins is recorded. In the brains of intracerebrally inoculated animals intracellular protein synthesis is enhanced. The recommended approach opens ways for investigations of the molecular mechanisms of the pathogenesis of viral infections as well as mechanisms of virulence.

[Virion protein synthesis in the lungs in experimental influenzal infection in animals]. / Sintez virionnykh belkov v legkikh pri éksperimental'noi grippoznoi infektsii u zhivotnykh.

Synthesis of virion proteins was found to occur in the lungs of mice 24 hours after intranasal infection of the animals with various influenza virus strains. A direct correlation between the intensity of this synthesis and the degree of virus virulence was established. It is assumed that avirulent influenza virus strains can undergo in the lungs of mice the early stages of virus reproduction including the synthesis of virion proteins whereas their capacity to form an infectious progeny and undergo several replication cycles is limited. In the lungs of rats which are not susceptible to influenza virus no synthesis of virion proteins was found which suggests that in this system the early stages of the parental virus expression is blocked. The "incomplete" virus in mouse lungs inhibited infectious virus production and decreased the intensity of virion protein synthesis.

[The effect of UV-radiation, heating and acidic pH on the antigenic activity of influenza virus nucleoproteins]. / Vliianie UF-oblucheniia, nagrevaniia i kislogo pH na antigennuiu aktivnost' nukleoproteina virusa grippa.

The capacity of influenza virus nucleoprotein (NP) to bind monoclonal antibodies upon virus treatment with factors affecting the conformation condition of the protein: ultraviolet irradiation, heating to 100 degrees C, acid pH, was studied. Upon the action of these factors NP epitopes demonstrated different time course of inactivation which suggests that they differ in their structural organization and, possibly, in their position in NP. From the possibility of formation in UV-irradiated virus of RNA-protein linkings, the UV-resistant epitope of NP is considered to be most distant from RNA as compared with other epitopes.

[Synthesis of influenza virus protein in the inhibition of cellular DNA transcription]. / Sintez belkov virusa grippa pri podavlenii transkriptsii kletochnoi DNK.

After preliminary UV-irradiation of cells as well as in the presence of actinomycin D the doses of both agents inhibiting influenza virus production are slightly lower than those inhibiting formation of viral proteins and considerably lower than those producing complete blocking of viral genome expression. These data demonstrate the existence of a cellular DNA-dependent factor the blocking of which prevents the formation of virus progeny without preventing primary transcription and formation of virion proteins. It is assumed that, apart from relatively small genes of cellular DNA participating in primary transcription of viral genome, some larger genes of the cell take part in some process of influenza virus reproduction.