Biomedical subjects
Publications and source records attributed to I G Kharitonenkov.
Isolation of virion RNA of influenza virus by the SDS-phenol method from subviral particles obtained from purified virions by treating them with a cation detergent, cetyl-methyl-ammonium bromide, increased RNA yields approximately 2-fold as compared with isolation of this RNA from whole virions.
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The method of circular dichroism was used to evaluate the conformation parameters of the secondary structure of the molecule of isolated influenza virus hemagglutinin H3. Comparison of the data obtained with the similar known parameters for hemagglutinin H2 has shown significant differences in the secondary structure, which are, probably, responsible, (at the level of a higher structural organization) for the variations in the antigenic specificity of hemagglutinins H2 and H3. Temperature and pH stability of alpha-spiral areas of the hemagglutinin H3 molecule was studied, and the direct participation of aromatic amino acid residues in formation of the alpha-spiral areas was shown. The native state of the hemagglutinin alpha-spiral areas may serve as a factor determining potential hemagglutination.
Influenza virus hemagglutinin of H3 type was isolated using the proteolytic enzyme bromlaine. The isolated hemagglutinin was shown to have no neuraminidase activity and to contain no admixtures of extraneous proteins. This antigen was used to immunize rabbits and the resulting antiserum had high antibody titers in HI tests. The method of precipitation with the serum of labeled viral proteins showed the serum to be monospecific and to contain antibody to the hemagglutinin alone.
Electron microscopic examinations of morphology of Sendai virus ribonucleoprotein (RNP) isolated from purified virions by two methods and simultaneous staining of the preparations with uranyl acetate and phosphotungstate acid (PTA) were carried out. The staining method was shown not to influence the kind of nucleoproteins which had been isolated from disrupted virions by sucrose density gradient centrifugation. With both strains RNP appeared as strongly helixed filaments. The staining method, however, strongly influenced morphology of RNP isolated from disrupted virions by equilibrium centrifugation in cesium chloride density gradient. After uranyl acetate staining RNP had an appearance of hard filaments whereas in the same preparation stained with PTA partially and completely unwound spirals of nucleocapsid were found alongside with strongly spiralized structures.
Radioimmunoassay were carried out with influenza A/Cambridge/46, A/FM/1/47, A/England/1/51, A/Denwer/1/57, and A/USSR/90/79 of the H1N1 subtype to determine the antigenic determinants of influenza A/USSR/90/77 virus hemagglutinin. The test system included 125I-labeled hemagglutinin of A/USSR/90/77 virus and homologous antiserum to this virus. The most marked antigenic similarity was found between A/USSR/90/77 and A/USSR/90/79 viruses. The A/Cambridge/46 and A/Denwer/1/57 viruses showed clear-cut differences in the antigenic determinants from the A/USSR/90/77 virus.
Sedimentation analysis in a lineal (5%--30%) sucrose concentration gradient of an RNA preparation isolated from a highly concentrated Sendai virus suspension revealed an additional peak with sedimentation coefficient 69S apart from previously known peaks of 46S (genome RNA), 30S and 18S (incomplete virus RNA). Electron microscopy methods showed the single-stranded molecules of 46S RNA to have a marked hairpin structure. Molecules of double-stranded 69S RNA have a specific structure: lineal parts alternate with areas in the form of rosettes. It is suggested that molecules of of 69S RNA are a product of incomplete hybridization between "plus" and "minus" complementary chains of virion RNA.
Using alpha- and beta-forms of the non-ionic detergent octyl glucoside the conditions have been developed for treatment of influenza virus and Sendai virus suspensions with the purpose of solubilization from virions of exclusively surface glycoproteins. Both alpha- and beta-forms of the detergent have been shown to have similar effectiveness for the recovery of chromatographically pure preparations of surface glycoproteins.
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Proteolytic modification of NP-protein which is a part of influenza virus ribonucleoprotein (RNP) was studied in vitro. NP-protein of influenza virus (mol. weight 56 000) was shown upon RNP treatment with bromeline to undergo proteolytic cleavage revealing 2 more polypeptides with molecular weights of 53 000 and 45 000 daltons. The same classes of NP-proteins were present in RNPs recovered from subviral units obtained by virus treatment with bromeline at buffer pH below 7. Electrophoresis in 12% polyacrylamide gel under reducing conditions of subvirus units obtained by treatment of virions with bromeline at the buffer pH below 7 also demonstrated proteolytic cleavage of M-protein present in them into fragments with molecular weights of 8 500-12 000 daltons. Similar results were obtained upon direct treatment with bromeline of M-protein isolated from virions.
The use of affinity column sepharose-tyrosine-sulfanilic acid permits one to obtain preparative amounts of pure hemagglutinin of the types H1, H2, H3 from the total amount of surface glycoproteins solubilized by octylglycoside from antigenically different strains of influenza A virus. The yield of hemagglutinin ranges from 30% to 84% depending on the virus strain.
Competitive radioimmunoassay in the homologous system using 125I-labeled neuraminidase (NA) of A/Japan/305/57 virus and antibodies to it showed that influenza viruses NA of subtypes H2N2 (A/Singapore/1/57 and A/Tokyo/3/67) and H3N2 (A/Aichi/2/68, A/England/42/72, A/Port Chalmers/1/73, and A/Texas/1/77) had undergone clear antigenic change despite the presence of Japan strain NA determinant. The use of a heterologous system (125I-NA of the Japan strain--antibodies to NA of England/42/72 strain) for the study of the intratype determinant showed the presence of a common determinant in all the strains under study, this determinant being represented dissimilarly, however. Neuraminidase of the Texas strain has a determinant differing from the intratype one, common for the Japan and England viruses.
Dissociation of influenza virus RNP under the effect of salt was studied. Separation of RNA and protein components of influenza virus RNP was shown to occur in a linear 15-30% sucrose concentration gradient containing 1.1 M NaCl. Upon RNP dissociation, protein-protein interactions between individual molecules of the structural protein were retained. The sedimentation coefficient of the protein component was 52S. The possibility of reassociation of the RNA-protein complex was studied. More complete reassociation was observed to occur in the presence of 0.1 M NaCl. The resulting RNA-protein complex morphologically is similar to the native RNP of influenza virus.
Circular dichroism was applied to the study of the structure of the insulin-transferrin complexes ("bound insulin") isolated from the blood sera of donors and patients suffering from diabetes mellitus of moderate severity. There proved to be a considerable (in comparison with the normal) reduction of the alpha-helix areas in the "bound insulin"molecule of the patients. A comparative study of the circular dichroism spectra in the area of absorption of aromatic amino acids permitted to suppose that the structural changes of the molecule of a complex isolated from the blood sera of patients could not be explained by alterations in the area of the aromatic amino acids.
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The CD spectra of Influenza virus RNA and RNP were examined in the ultraviolet region (220--320 nm) at different temperatures, NaCl and urea concentrations. The magnitude of the positive band for RNP decreases gradually upon increasing the temperature, indicating that rather weak interaction between RNA and protein occurs. However the temperature drastically affects the intensity of the negative band about 222 nm. In the temperature range where the protein melts the positive band at longer wavelengths shows a temperature dependence that is similar for RNP and free RNA. Addition of NaCl results in an increase in intensity and blue shift of the positive CD band of RNP. In the presence of 1 M NaCl the CD spectrum of RNP is very close to that of protein-free RNA. Urea concentrations up to 10 M has little effect on the CD spectrum of RNP. These results suggest that only ionic but not hydrophobic and hydrogen bondings are involved in the RNA-protein interaction in Influenza virus RNP.
A two-step method for isolation of HA2 polypeptide, the light chain of influenza virus hemagglutinin, from influenza virions is described. The method consists in obtaining a preparation of surface glycoproteins by means of their solubilization from virions with a cation detergent (cetyl-trimethyl-ammonium bromide) followed by fractionation of this preparation by preparative electrophoresis. Relative molecular weights of HA2 and other polypeptides comprising the virion have been determined. The method was used with a recombinant MRC-11 strain.
Synthesis of neuraminidase activity in an affinity column with an inhibitor para-amino-phenyloxamine acid (PAPOA), immobilized on sepharose via tripeptide "spacer" (glycyl-glycyl-tyrosine) is described as well as the conditions of PAPOA synthesis. The column has been tested for isolation of neuraminidase types N1 and N2 from various influenza virus strains. The strain specificity of the action of the column in neuraminidase isolation was demonstrated. Neuraminidase free from admixtures has been isolated from the A/PR8/34, A/FM/1/47 and recombinant X-7 strains.