[The effect of losing glycosylation sites near the receptor-binding region on the receptor phenotype of the human influenza virus H1N1]. / Vliianie utraty saitov glikozilirovaniia, raspolozhennykh vblizi retseptor-sviazyvaiushchego uchastka, na retseptornyi fenotip H1N1 virusa grippa cheloveka.

The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.

[Functional interactions of the influenza virus glycoproteins]. / Funktsional'noe vzaimodeistvie glikoproteinov virusa grippa.

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.

[Reduction of the functional match of influenza virus hemagglutinin and neuraminidase after reassortation of genes]. / Vosstanovlenie funktsional'nogo sootvetstviia gemaggliutinina i neiraminidazy virusa grippa posle reassortatsii genov.

Influenza virus A (FluA) reassortants with low-functional neuraminidase (NA) of subtype N1 and hemagglutinin (HA) of subtypes H2, H3, H4, and H13 display virion aggregation and accumulate to a lower titer because sialyl residues are not completely removed from virion components. Nonaggregating variants of FluA (H13N1) were shown to result from a mutation that reduces the HA affinity for sialyl substrates. Amino acid substitution K156E, which increases a negative charge at the edge of the receptor-binding pocket of HA large subunit (HA1), was revealed in two independent variants. This substitution was the only difference between HA1 of the original reassortant and one of its variants and, therefore, accounted for restoration of the functional match between HA and NA.

[Variants of influenza H1N1 virus, adapted to mouse lungs and inhibitors of mouse serum inhibitors differ by hemagglutinin characteristics and structure]. / Varianty virusa girppa A H1N1, adaptirovannye k legkim myshei i ingibitoram syvorotki krovi myshei, razlichaiutsia po svoistvam i strukture gemaggliutinina.

Influenza virus A/USSR/90/77 variants adapted to mouse blood serum (USSR/90-MS) and lungs (USSR/90-ML) resistant to beta-inhibitors of mouse serum differ by biological properties and hemagglutinin (HA) structure. One of glycosylation site (GS) located at the tip of HA spike near the receptor binding site is lost because of mutations in both variants: GS 158 (Asn158Asp substitution) in USSR/90-MS and GS131 (Asp131Asp substitution) in USSR/90-ML. Probably adaptation to mouse lungs and serum represents adaptation to different types of receptor molecules. From these data we conclude that mouse lungs and/or bronchi and serum contain different beta-like inhibitors. Presumably the inhibitors in the lungs contain some additional factors in comparison with the serum, adaptation to which allows a wider spectrum of virus resistance to homologous and to many other normal animal sera.

[Phylogenetic analysis based on groE shows the closest evolutionary relationship between mitochondria and Rickettsia]. / Osnovannyi na groE filogeneticheskii analiz pokazyvaet tesneishee évoliutsionnoe rodstvo mitokhondrii i Rickettsia.

The nucleotide sequence of the groE operon of Rickettsia prowazekii, the obligate intracellular parasite of eukaryotes, was determined. The alignment of DNA-inferred amino acid sequences of the Hsp10 and Hsp60 heat-shock proteins with bacterial and mitochondrial homologues revealed the presence within Hsp60 of signatures shared by mitochondria and rickettsiae. Phylogenetic analysis demonstrated that heat-shock proteins of R. prowazekii are the earliest and the least diverging homologues within the family Rickettsiaceae--a sister group to the monophyletic clade of mitochondria. These results are in good agreement with the data obtained when using other molecular chronometers and show the closest relationship between mitochondria and Rickettsia. The possible nature of obligate intracellular parasitism of rickettsiae has been considered on the basis of the assumption that they and mitochondria could have a common evolutionary origin.

Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs.

There is growing evidence that the receptor-binding characteristics of influenza viruses are affected by the host-dependent glycosylation of viral hemagglutinin (HA). To better understand these effects, we propagated two variants of the human influenza virus USSR/90/77 (which differed by the mutation Asn131 reversible Asp131 in the glycosylation sequon of their HA) in either embryonated chicken eggs or MDCK cell. Those variants were then compared for their ability to bind soluble receptor analogs and to attach to receptors represented on a solid phase. The carbohydrate chain at position 131 of the HA (CHO 131) interfered with virus binding to soluble Sia2-6Gal-containing macromolecular receptors, but had little or no effect on its binding to Sia2-3Gal-containing macromolecules. This specificity could be explained by the different orientation of the asialic parts of the 2-3-linked sialosides versus 2-6-linked sialosides with respect to the receptor-binding site (Eisen et al., 1997, Virology 232, 19-31). In the case of virus attachment to solid-phase immobilized receptors, MDCK-grown viruses bound substantially more weakly than their egg-grown counterparts to receptors of avian origin, whereas binding to mammalian cell membranes was only marginally affected by differences in host-specific glycosylation of the virus. Our data indicated that the effects of the carbohydrate side chain of HA on virus receptor-binding activity are dependent on both the cells in which the virus was grown and the nature of the cellular receptors or intercellular inhibitors to which the virus binds.

[Changes in its hemagglutinin during the adaptation of the influenza virus to mice and their role in the acquisition of virulent properties and resistance to serum inhibitors]. / Izmeneniia v gemaggliutinine pri adaptatsii virusa grippa k mysham i ikh rol' v priobretenii virulentnykh svoistv i ustoichivosti k syvorotochnym ingibitoram.

Passages of influenza A/USSR/90/77 virus in mouse lungs produced a virulent virus (18th passage) carrying two mutations in hemagglutinin (HA) Asn127-->Asp and Tre89-->Ala. Cloning of this virus revealed two avirulent clones in the population. The analysis of one virulent (clone 7p) and one avirulent (clone 31 np) clones showed them to have both above-mentioned substitutions in HA. Clone 7p replicated in mouse lungs much more effectively than clone 31np, the difference in titres being 2 log10 EID50. In another experiment, the adapted virus (15th passage) did not differ in its antigenic, virulent, and reproductive properties from clone 7p but had only one substitution in HA Asn127-->Asp. Consequently, adaptation to mice required at least one mutation in HA resulting in the loss of glycosylation site. However, acquirement of virulence requires additional factors affecting the level of virus reproduction. Examinations of a number of normal animal sera and erythrocytes showed the mutations in HA accompanying virus adaptation to mice led to the development of resistance to serum inhibitors and changes in the receptor-binding specificity. These properties may be also associated with the loss of glycosylation site in the receptor-binding region of hemagglutinin molecule.

[The mechanisms of the antiviral action of the bora-adamantane derivative preparation BG-12]. / Izuchenie mekhanizmov protivovirusnogo deistviia proizvodnogo boraadamantana preparata BG-12.

The effect of one of the derivatives of boraadamantane, preparation BG-12, on reproduction of influenza type A and B viruses was studied. This preparation was shown to inhibit multiplication of a wide range of influenza type A and B virus strains. It is important that BG-12 inhibits in cell culture the replication of a mutant of fowl plague virus A/FPV/Weibridge resistant to remantadine. BG-12 was found to exert a marked viricidal effect, to inhibit the hemolytic activity of both influenza A and influenza B viruses, but not the neuraminidase activity of these viruses. The synthesis of some virus-specific proteins was found to be disturbed in influenza B/Ann Arbor/86-infected cells in the presence of BG-12. A FPVBAR mutant of influenza A/FPV/Weibridge (H7N7) virus resistant to BG-12 was obtained. All the BG-12-resistant reassortants inherited genes 4 and 7 coding for hemagglutinin (HA) and membrane proteins (M) from FPVBAR mutant, respectively, and the remaining genes from BG-12-sensitive A/Krasnodar/101/59 (H2N2) virus. The sequencing of a region of the M gene encoding for the transmembrane protein M2 revealed the substitution of Ala30-Thre in this protein. Multiple attempts at generating a mutant of influenza type B virus resistant to BG-12 failed.

[Genetic variability of the human influenza virus during adaptation in mice]. / Geneticheskaia izmenchivost' virusa grippa cheloveka v protsesse adaptatsii k mysham.

After 12 passages of a mouse-nonpathogenic influenza A/USSR/90/77 virus in mouse lungs a pathogenic virus was obtained causing death of the animals at 4-7 days after intranasal inoculation. The genetic and structural analysis of the initial and pathogenic viruses performed by oligonucleotide mapping of individual virus genes demonstrated that in the course of adaptation to mice structural changes had occurred at least in 5 out of 8 genes of virus with the exception of the genes coding for matrix and nonstructural proteins. The greatest differences were found in the genes coding for surface glycoproteins: hemagglutinin and neuraminidase. The experimental results indicate an important functional role of surface glycoproteins of influenza virus, particularly hemagglutinin, in the process of adaptation and formation of the pathogenic properties of virus.