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.

[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.

[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.

[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.

Non-compact nucleocapsid protein multimers in influenza-virus-infected cells.

We have previously shown that protease-resistant and highly immunoreactive compact NP oligomers, dissociating at +80 degrees C and possessing properties of folded proteins, are post-translationally formed in influenza-virus-infected cells. In this study we demonstrate that, in addition to compact NP oligomers, incompletely folded NP multimers are detected intracellularly by SDS/PAGE carried out under weak dissociating conditions. In cells infected with avian, human A(H2N2), and human A(H3N2) viruses, NP multimers are detected in the stacking gel of SDS/PAGE as retarded and loose structures dissociating at +50 degrees C. NP multimers are more sensitive to proteolysis than NP oligomers, but they are more resistant to proteolysis than NP monomers. In contrast to compact NP oligomers, NP multimers possess a weak immunoreactivity to some monoclonal antibodies. Pulse-chase experiments have shown that NP multimers appear at early stages of NP synthesis and are partially converted post-translationally into faster-migrating compact NP oligomers. In the course of infection, the excess NP multimers not converted into compact NP oligomers accumulate in cells and degrade. Under weak dissociating conditions, intracellular NP multimers are relatively stable in avian, human A(H2N2) and human A(H3N2) viruses and unstable in human A(H1N1) viruses, dissociating into monomers. NP multimers presumably serve to bring nascent unfolded NP molecules into close contact with each other for further oligomerization, to protect NP monomers from proteolysis, and to serve as intermediates in the posttranslational folding of NP.

[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.

Stability of intracellular influenza virus nucleocapsid protein oligomers.

Stability of A/Duck/Ukrainae/63 (H3N8) influenza virus intracellular NP oligomers was studied using reducing agents, denaturants, detergents, salts, various pH and a range of temperatures. The results obtained indicate that influenza virus NP oligomers are noncovalently stabilized, and NP subunits are not linked by disulfide bonds. NP oligomers are thermostable and SDS resistant. Urea and high ionic strength also do not dissociate avian influenza virus intracellular NP oligomers. However, NP oligomers are completely dissociated at pH < 5. The data obtained suggest that hydrophobic bonds together with the electrostatic interactions take part in the stabilization of compact conformation of influenza virus NP oligomers. It was also shown that intrachain disulfides revealed in nascent NPs are reduced in NP subunits of NP oligomers, and this probably contributes to the stability and compactness of the oligomers.

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.

[Differences in oligomerization of nucleocapsid protein of epidemic human influenza A(H1N1), A(H1N2) and B viruses]. / Razlichiia v oligomerizatsii nukleokapsidnogo belka épidemicheskikh virusov grippa cheloveka A(H1N1), A(H3N2) i B.

A comparative analysis of involving the nucleocapsid protein (NP) into shaping-up of SDS-resistant oligomers was carried out presently in circulating epidemic strains of human influenza, viruses A and B. The study results of viral isolates obtained from clinical samples and recent standard strains revealed that the involvement of NP in the SDS-resistant oligomers, which are different in various subtypes of influenza A viruses. According to this sign, the human viruses A(9H3N2) are close to the avian ones, in which, as proved by us previously, virtually the entire NP transforms itself into the oligomers resistant to SDS. About 10-20% of NP are involved in shaping-up the virus influenza A(H1N1) of SDS-resistant oligomers. No SDS-resistant NP-oligomers were detected in influenza of type B. It is suggested that the prevalence of human viruses A(H3N2) in NP-oligomers are the peculiarities of NP structure and of the presence of the PB1 protein from avian influenza virus.

[The formation of the influenza virus aggregates, enriched with hemagglutinin]. / Obrazovanie agregatov virusa grippa, obogashchennykh gemaggliutininom.

The formation of electrostatic aggregates was studied by analysis of two types of virus-containing liquids: initial warm liquid collected at temperature 37 degrees and the same liquid stored over the night at temperature 4 degrees C. The formation of virus aggregates was revealed at 4 degrees C. The aggregates formed at temperature 4 degrees C had a relatively high HA/NP ratio in comparison with unassociated virus analyzed at 37 degrees. HA-enriched aggregates were found in the precipitate formed under short-term high-speed centrifugation as well as in "heavy arm" of the virus profile in the saccharose gradient. Aggregates formed at 4 degrees C dissociated at 37 degrees. The ability to form aggregates is reversible and correlates with the virus concentration. It is shown also that virus containing liquid contains heterogenic structures with molecular weight under 2000 kD having HA involved in the forming aggregates enriching HA. Possible nature of low-molecular HA-containing structures involved in the aggregates and nature of relations stabilizing aggregates are discussed.

[Extracellular immunoreactive nucleoprotein of Influenza virus not related to the virion]. / Vnekletochnyi immunoreaktivnyi nukleoprotein virusa grippa, ne sviazannyi s virionami.

Extracellular immunoreactive virus NP is accumulated in virus-containing fluid in the course of A/Duck/Ukraine/1/63(H3N8) influenza virus infection. The major part of this extracellular NP is included in viral RNP and characterized by relatively low molecular weight: 53 kD vs. 56 kD of virion NP. Extracellular immunoreactive NP is oligomerized. Presumably, there is partially intracellular cleavage of NP with loss of hydrophobic determinants. Such truncated NP in RNP is highly hydrophilic and passes through cell membranes. These data prompt the diagnosis of influenza infection by detection of free immunoreactive NP in analyzed fluids.

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.

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.

[Dependence of oligomerization of influenza virus nucleoprotein on the species affiliation of the virus]. / Zavisimost' oligomerizatsii nukleoproteina virusa grippa ot vidovoi prinadlezhnosti virusa.

Comparison of human and avian influenza virus nucleoprotein (NP) oligomerization showed that the efficiency of NP oligomerization is different in influenza viruses of different origin. NP oligomerization is virtually complete in avian influenza viruses, while in human influenza viruses only part of monomeric NP is oligomerized. The authors discuss the utilization of NP oligomerization efficiency as a sign for identification of the origin of influenza virus.

[Formation of two types of a proteolytically cleaved nucleoprotein in cells, infected by influenza virus]. / Formirovanie dvukh tipov proteoliticheski rasshcheplennogo nukleoproteina v kletkakh, zarazhennykh virusom grippa.

Two types of proteolytically cleaved influenza virus nucleoprotein (NP) are formed in cells infected with influenza virus. One, cell membrane-associated cleaved NP (C-NP), is heterogeneous in size and during analysis in PAGE is localized in the 53 kDa and less zone. Its formation depends on the species appurtenance of the infective virus and occurs only in viruses with a low efficacy of NP oligomerization. C-NP is incapable of intracellular oligomerization. The other type of cleaved NP, extracellular free (F-NP), is most likely a secreted product of intracellular proteolysis of 56 kDa NP. Its molecular weight is about 53 kDa. Its formation does not depend on the species of infective influenza virus and it is capable of intracellular oligomerization.

[Role of certain factors in intracellular oligomerization of influenza virus nucleoproteins]. / Rol' nekotorykh faktorov v protsesse vnutrikletochnoi oligomerizatsii nukleoproteina virusa grippa.

Oligomerization of influenza virus nucleoprotein (NP) depends on the virus strain. NP monomers of viruses A/Duck/Ukraine/63 (H3N8) and A/Seal/Massachusets/1/80/ (H7N7) are oligomerized completely. The A/USSR/90/77 virus (H1N1) NP is characterized by just partial oligomerization, similarly as a reassortant containing surface protein genes of virus A/Duck/Ukraine and internal protein genes of A/USSR/90 virus. Hence, it is probable that NP gene controls the type of NP oligomerization. NP oligomerization is shown to depend on the temperature, the optimal t = 37 degrees C, but not on the type of cells or intracellular concentration of total NP. NP oligomers forming in vivo are believed to differ from NP oligomers formed in vitro described previously.

Intracellular oligomerization of influenza virus nucleoprotein.

It has previously been shown that the purified influenza virus nucleoprotein (NP) forms the oligomers in vitro in NP preparations obtained from virions (Wiley et al., 1977, Virology, 79, 446-448; Ruigrok and Baudin, 1995, J. Gen. Virol., 76, 1009-1014) and infected cells (Becht and Weiss, 1991, Behring Inst Mitt., Justus-Liebig Universitat, Giessen, 89, 1-11). We have shown in this report that boiling-sensitive NP oligomers (di- and trimers) are formed in vivo in the course of intracellular influenza virus replication. They are detected by PAGE about 10 min after monomeric 56-kDa NP molecules are synthesized. NP oligomers are formed by different strains of influenza virus in different cell lines. Some influenza virus strains are characterized by complete conversion of NP monomers into oligomers and others by only partial conversion. In the Triton X-114 phase partitioning system NP oligomers show more hydrophobicity than NP monomers. NP oligomers are detected in the sedimentable and soluble fractions of both cell lysate and extracellular medium. The possibility is discussed that oligomeric NP is a native and functionally significant form of influenza virus NP.