[Autoimmune response in HIV-infected patients is directed against CD4 domain 4].

Phage display epitope library technology and a novel computer algorithm have been used for the localization of CD4 epitopes specific for monoclonal antibody (mAb) T6 and autoimmune antibodies found in an HIV infected patient. Both predicted epitope clusters have been shown to overlap and to be localized within the domain 4 of CD4. They included Cys303, Glu304, Glu330, and Lys331 amino acids. Few amino acids predicted by the algorithm as the epitope residues and two residues that did not relate to the epitope were sequentially substituted for Ala. Further analysis of the mutated forms of sCD4 expressed in 293T cells transfected with the corresponding DNAs, supported the predicted localization of the mAb T6 epitope. The results demonstrate that the autoimmune response in HIV-infected patients is directed against domain D4 of sCD4.

Dependence of conformation of D3/D4 domains of human CD4 on glycosylation and membrane attachment.

Conformational dynamics of human T-helper cell receptor protein CD4 has been studied with the help of monoclonal antibody (mAb) T6. The mAb T6 discriminates between s- and m-forms of CD4 and recognizes a specific conformation of the soluble (s) form of CD4 including the first nine amino acids of CD4 transmembrane sequence. However, change of tryptophan for serine in position 2 in this sequence destabilizes the T6-type conformation. By enzymatic deglycosylation and deletions of glycosylation sites, we show that T6-type conformation depends on glycosylation in both sites (Asn271 and Asn300). We show also that the sugars are not involved in direct binding to the antibody but stabilize the D3/D4 local conformation. Deglycosylated forms of sCD4 in vivo acquire a specific conformation similar to the wild type sCD4, which however cannot be restored after denaturation/renaturation under conditions of non-reducing Western blot. This observation indicates that the correct protein folding needs chaperone assistance and cannot be achieved in vitro. Completely non-glycosylated sCD4 is synthesized and secreted into the growth medium. In the medium, this mutant appears to be unstable and aggregates during time. In a contrast to soluble CD4, mutations in glycosylation sites abrogate expression of membrane CD4, thus demonstrating a different secretion pathways for soluble and membrane proteins.

[Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display].

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.

[Anti-CD4 autoimmunity in HIV-infected persons in Russia].

The frequency of anti-CD4 antibodies was determined in the sera or plasma derived from the patients infected with HIV-1 belonging to different genetic subgroups. The anti-CD4-antibodies in a dilution of > or = 1:1000 were found in 14% of the patients infected with the gagA/envA virus characteristic for injectable drug users in East Europe. The frequency of autoimmune antibodies among the HIV-infected patients with envB virus was substantially less (4.4%). Competitive ELISA using monoclonal antibodies to different CD4 domains demonstrated that irrespective of the viral genotype, the autoimmune epitope is located within the D4 or D3/D4 domains of CD4 receptor.

[Determination of the level of antibodies to CD4 in serum of patients with malignant lymphomas and of HIV-infected individuals]. / Opredelenie urovnia antitel k CD4 v syvorotke krovi u bol'nykh zlokachestvennymi limfomami i u VICh-infitsirovannykh lits.

Hardphase ELISA was used to compare the blood sera of patients with malignant skin lymphomas and HIV infection and of healthy volunteers for autoimmune antibodies to CD4. Antibodies to CD4 were not detected in the volunteers (30 sera) and in the patients with malignant skin lymphomas (52 patients with different disease stages). The antibodies were found in 4 of 11 HIV cases.

[Inhibition of nuclear import of HIV-1 Vpr protein by Gag polyprotein in the process of virus-like particles formation]. / Ingibirovanie iadernogo importa belka VPR VICh-1 poliproteinom GAG v protsesse formirovaniia virusopodobnykh chastits.

For studies of intracellular Vpr transport and the effect of the HIV-1 Gag polyprotein on the process, a recombinant baculovirus strain was constructed, which directs the synthesis of Vpr fused with the baculovirus secretory polypeptide. During infection the majority of Vpr has been observed in the cell nuclear fraction. These data suggest that Vpr nucleophilic signal is more active than the secretory one. However, during Vpr and Gag co-expression in the baculovirus expression system, Vpr content in the nuclei is decreased, since this protein incorporates effectively into virus-like particles and forms stable complexes with Gag polyprotein. Presumably, the Vpr-Gag post-translational interactions are needed for the Vpr incorporation into virions and suppress the nuclear import of this protein.

[Interaction of gag polyprotein-precursors p55 and p48 with p160gag-pol during formation of virus-like particles of HIV-1 with recombinant strains of vaccinia virus]. / Vzaimodeistvie gag poliproteinov-predshestvennikov p55 i p48 s p160gag-pol pri formirovanii virusopodobnykh chastits VICh-1 rekombinantnymi shtammami virusa ospovaktsiny.

The polypeptide composition of HIV-I virus-like particles produced by CV-I cells during mono- and coinfection with recombinant vaccinia virus (rVV) strains containing the whole (p55) and carboxyterminal truncated (p48) gag genes and gag-pol sequence is studied. In monoinfection both the gag-strains actively produced virus-like particles consisting of non-processed p55Gag and p48Gag polyprotein without p6 domain. In case of a coinfection of the cells with one of these strains and the rVV producing p160Gag-Pol polyprotein the virus-like particles consisted of p24 protein and a negligible amount of non-processed Gag precursors. The share of p24 protein increased in proportion to the duration of coinfection and decreased with a reduction of multiplicity of infection with rVV carrying p160Gag-Pol. Hence, the absence of p6 domain does not influence the processing of Gag proteins during virus-like particles assembly and budding. In contrast to the natural systems of HIV-I development, in the rVV expression system the p6Gag domain virtually does not contribute to reactions between Gag and Gag-Pol precursors and to the particles' morphogenesis.

[Expression of human immunodeficiency virus gag antigens and formation of virus-like particles in a cell culture, infected with recombinant vaccinia virus strains (an electron-microscopic study)]. / Ekspressiia gag-antigenov virusa immunodefitsita cheloveka i obrazovanie virusopodobnykh chastits v kul'ture kletok, infitsirovannykh rekombinantnymi shtammami virusa ospovaktsiny (élektronno-mikroskopicheskoe issldovanie).

The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied. The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29). The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.

[Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens]. / Metody polucheniia i selektsii rekombinantov virusov ospovaktsiny, ékspressiruiushchikh chuzherodnye virusnye antigeny.

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.

[Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin]. / Konstruirovanie rekombinantnogo shtamma virusa ospovaktsiny, soderzhashchego ékspressiruemyi gen gemaggliutinina virusa grippa A.

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.

[Factors limiting the gene expression of Escherichia coli in the cells of bacilli]. / Faktory, ogranichivaiushchie ékspressiiu genov Escherichia coli v kletkakh batsill.

The barrier to the Escherichia coli gene expression in bacillar cells is caused both by differences in DNA regulatory elements involved in the interaction with RNA polymerase and ribosomes, and by the structure and function features of the very enzymes which carry out this interaction.

[Coupling of transcription and translation as the factor regulating the transcription of genes rpoBC in Escherichia coli cells]. / Sopriazhenie transkriptsii i transliatsii kak faktor reguliatsii transkriptsii genov rpoBC v kletkakh Escherichia coli.

The natural transcription polarity of proximal and distal elements of the rplJL-rpoBC operon is increased when translation is inhibited in Escherichia coli cells. It is shown that transcription uncoupling to translation terminates within EcoRI-2,6 fragment of the operon which contains the transcription attenuator. We suggest that transcription attenuation in the rplJL-rpoBC operon is regulated by the coupling of transcription to translation of the intergenic fragment which overlaps the attenuator sequence.

[Non-coordinated transcription of RNA polymerase beta,beta'-polypeptide genes and adjacent ribosomal protein genes in Escherichia coli cells]. / Nekoordinirovannaia transkriptsiia genov beta,beta'-polipeptidov RNK-polimerazy i primykaiushchikh genov ribosomnykh belkov v kletkakh Escherichia coli.

When E. coli protein synthesis was blocked by chloramphenicol (100 micrograms/ml) or by essential amino acid deprivation, the transcription rates of rplKAJL genes (the ones for L11, L4, L10 and L7/L12 ribosomal proteins) and adjacent rpoBC genes (genes for RNA polymerase beta- and beta'-polypeptides) have been non-coordinately changed. The level of the gene transcription rate was obtained from RNA--DNA hybridization assays with E. coli pulse-labelled RNA and pJC703 or pJC720 plasmid DNA. The transcription of ribosomal protein genes has been found to be uncoupled with translation and controlled by the allelic state of relA gene. Conversely, the effective transcription of proBC gene was relA independent and coupled with translation of the mRNA. Chloramphenicol-induced transcription polarity within rplKAJL-rpoBC chromosome region can be suppressed by 10 micrograms/ml rifampicin.

[Using a combined transcription-translation system for determining the role of cryptic plasmids of Bacillus thuringiensis]. / Ispol'zovanie sovmeshchenoi sistemy transkriptsii--transliatsii dlia vyiusneniia roli kripticheskikh plazmid Bacillus thuringiensis.

The possibility of entomocyde crystal protein synthesis was studied using a heterological cell-free system with Bacillus thuringiensis plasmid DNA as template. The high level of template activity is usual for Bac. thuringiensis plasmid DNA. Immunochemical studies of the in vitro synthesized polypeptides showed that Bac. thuringiensis plasmid DNA does not direct crystal protein synthesis.

[Synthesis of beta, beta'-subunits of RNA-polymerase in E. coli cells starved for an essential amino acid]. / Sintez beta, Beta'-sub'edinits RNK-polimerazy v kletkakh E. Coli pri defitsite neobkhodimoi aminokisloty.

The differential rate of RNA-polymerase beta,beta'-subunits synthesis in E. coli rel+ and rel- cells starved for an essential amino acid, i. e. leucine, is decreased. Inhibition of the rpo BC genes expression proceeds faster in the rel A strain. Guanosine tetraphosphate within the concentration range of 0,2--0,6 mM specifically inhibits lambda drifd18 DNA directed beta,beta'-subunits synthesis in vitro. rpo BC genes are believed to be subject to a weak "stringent control".

[Partial suppression of the effect of gene re1A in Fusr-mutant Escherichia coli K-12 cells]. / Chastichnaia supressiia deistviia gena re1A v kletkakh Fusr-mutantov Escherichia coli K-12.

Mutants of Escherichia coli K-12 re1A+-strain CP 78 resistant to fusidic acid (Fusr) were isolated and forms sensitive to high concentration of leucine (500 g/ml) were selected. When shifted down from nutrient broth to minimal medium M9 with supplemented glucose and required amino acids, these leucine-sensitive mutants continued RNA synthesis and demonstrated the prolonged lag-phase in contrast to the parent strain CP 78. Both properties are known to be characteristic of the Rel- strains. At the same time withdrawal of the required amino acids results in cessation of RNA synthesis in Fusr mutants, in the parent Rel+ strain. Thus, leucine-sensitive Fusr mutants show Rel- phenotype only upon amino acid starvation caused by shift down from nutrient broth to minimal medium.

[In vivo and in vitro studies of phage T4 lysozyme mRNA translation]. / Izuchenie transliatsii mRNK lizotsima faga T4 in vivo i in vitro.

Translation of phage T4 lysozyme mRNA is studied in vivo and in vitro. Polyribosomes, carrying growing lysozyme polypeptides, are found to be homogenous enough and to contain 6 ribosomes. Complete molecules of phage lysozyme, which possess an enzymatic activity and are similar to the native enzyme in its electrophoretic mobility in polyacrylamide gel, have been synthetized in vitro on RNA isolated from phage-infected cells. The efficiency of RNA translation in cell-free system is discussed on the model of synthesis of functionally active individual protein.