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Introduction. Kindia tick virus (KITV) is a novel segmented unclassified flavi-like virus of the Flaviviridae family. This virus is associated with ixodes ticks and is potentially pathogenic to humans. The main goal of this work was to search for structural motifs of viral polypeptides and to develop a 3D-structure for viral proteins of the flavi-like KITV. Materials and methods. The complete genome sequences for KITV, Zika, dengue, Japanese encephalitis, West Nile and yellow fever viruses were retrieved from GenBank. Bioinformatics analysis was performed using the different software packages. Results. Analysis of the KITV structural proteins showed that they have no analogues among currently known viral proteins. Spatial models of NS3 and NS5 KITV proteins have been obtained. These models had a high level of topological similarity to the tick-borne encephalitis and dengue viral proteins. The methyltransferase and RNA-dependent RNA-polymerase domains were found in the NS5 KITV. The latter was represented by fingers, palm and thumb subdomains, and motifs A-F. The helicase domain and its main structural motifs IVI were identified in NS3 KITV. However, the protease domain typical of NS3 flaviviruses was not detected. The highly conserved amino acid motives were detected in the NS3 and NS5 KITV. Also, eight amino acid substitutions characteristic of KITV/2018/1 and KITV/2018/2 were detected, five of them being localized in alpha-helix and three in loops of nonstructural proteins. Conclusion. Nonstructural proteins of KITV have structural and functional similarities with unsegmented flaviviruses. This confirms their possible evolutionary and taxonomic relationships.

Objective: isolation of coronavirus SARS-CoV-2 from clinical sample of patient with COVID-19 in Novosibirsk; obtaining a purified and inactivated viral antigen and study of its antigenic properties. Materials and methods: virus isolation was carried out in Vero cell culture from nasopharyngeal swab positive on SARS-CoV-2 RNA. The efficiency of SARSCoV-2 replication in cell culture was assessed on the appearance of cytopathic effect (CPE) and the presence of viral RNA in cultural medium with reverse transcription – polymerase chain reaction (RT-PCR). Purification, concentration and inactivation of the viral preparation were carried out according to standard methods. The purity of the purified preparation and the profile of viral proteins were determined by electrophoresis in 10% polyacrylamide gel (PAG) with the addition of sodium dodecyl sulfate (SDS). The presence and specificity of viral proteins were detected using COVID-19 convalescent’s sera with enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results: SARS-CoV-2/human/ RUS/Nsk-FRCFTM-1/2020 isolate was obtained after passage on Vero cells from a virus-containing clinical sample. A purified, concentrated, inactivated, whole-virion antigen was obtained. It contains three structural proteins: glycoprotein S (approximately 200 kDa), nucleoprotein N (48 kDa), and matrix protein M (20-25 kDa). All viral proteins were detected with serum antibodies of COVID-19 convalescents. Conclusion: SARS-CoV-2 coronavirus can be isolated in Vero cell culture. The antigenic specificity of the three structural viral proteins (S, N, and M) is preserved in the purified inactivated viral preparation. The inactivated whole-virion antigen of SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 isolate can be used to study the antigenic immunomodulating properties of viral proteins, to obtain immune sera of laboratory animals, and also as a component of test systems for the detection of specific antibodies with ELISA and immunoblotting.

A comparative analysis on search for amino acid sequences in viral proteins causing respiratory infections (or respiratory infections syndrome) homologous to amino acid sequences from some human immune proteins was performed. The following viruses were used for comparative computer analysis: coronavirus (SARS-CoV), serotype C subgroup adenovirus C (adenoid 71 strain), measles virus (ICHINOSE-BA strain), rubella (Therien strain) and respiratory syncytial (B1 strain) virus. The search for homologous sequences in viral and human immune proteins was carried out by computer comparison of 12 amino acid fragments, which were assigned as homologous at identity in ≥ 8 positions. The data obtained showed that viral proteins contained homologous motifs in several host immune proteins involved in regulating both the inflammatory response and immune response. Mechanistically, all viruses studied were characterized by sequences homologous to host immune proteins such as complement system proteins, integrins, apoptosis inhibitory proteins, interleukins, and toll-like receptors. Such cellular proteins are actively involved in regulating host inflammatory process and immune response formation. Upon that, a set of host immune proteins, to which homologous fragments were found in viral proteins, was individual for each virus. Interestingly, the largest amount of homologous fragments (up to 20) was mainly concentrated in viral proteins with polymerase and protease activity suggesting that these proteins apart to their major role were involved in production of viral nucleic acids and might participate in regulating host immune system. Envelope, internal and non-structural viral proteins, homologous fragments were detected in much smaller quantities (from 1 to 4). In addition, two fragments homologous to various motifs of the same cellular protein were detected in some viral proteins. Thus, the data obtained further support our understanding that signs of immune system disorders in viral infections can result from multi-layered processes associated with modulation of host innate and adaptive immune system, and open up new approaches to study interaction of viruses with host immune system and identify new functions of viral proteins.

Objectives: To identify homologous segments of human hemostatic and viral proteins and to assess the role of human hemostatic proteins in viral replication. Materials and Methods: The following viruses were chosen for comparison: influenza B (B/Astrakhan/2/2017), coronaviruses (Hcov229E and SARS-Co), type 1 adenovirus (adenoid 71), measles (ICHINOSE-BA) and rubella (Therien). The primary structures of viral proteins and 41 human hemostatic proteins were obtained from open–access www.ncbi.nlm.nih. gov and www.nextprot.org databases, respectively. Sequence homology was determined by comparing 12-amino-acid segments. Those sequences identical in ≥ 8 positions were considered homologous. Results: The analysis shows that viral proteins contain segments which mimic a number of human hemostatic proteins. Most of these segments, except those of adenovirus proteins, are homologous with coagulation factors. The increase in viral virulence, as in case of SARS-Co, correlates with an increased number of segments homologous with hemostatic proteins. Conclusion: Hemostasis plays an important role in viral replication and pathogenesis. The conclusion is consistent with the literature data about the relationship of hemostasis and inflammatory response to viral infections.

Antibodies (Ab), especially natural, display multiple specificity not only due to intrinsic conformational dynamics. With computational analysis the distribution of identical and homologous peptides has been studied in surface proteins from RNA and DNA viruses of widely distributed infections. It was established that each virus protein shared the fragments homologous to other virus proteins that allowed to propose the existence of the peptide continuum of the protein relationship (PCPR). Possible manifestations of PCPR are multiple reactivity and autoreactivity in Ab and therefore it is not possible to consider the immune methods of virus identification as high reliable because of crossing interactions. The PCPR excludes the existence of 100% specificity in immune tests for virus identification. Immunodiagnostic collisions may occur either in identification of virus itself or identification of Ab to viruses. Also PCPR may be responsible for heterologous immunity and consequently the infection associated with severe pathology. The comparative analysis of peptide relationship of H1N1 influenza virus nucleoprotein and human proteins found out, beyond early described its common motif with human hypocretin receptor 2, peptides homologous to those in melanotonin and glutamate receptors and three ion channels. It allows to propose that the sleep disorder narcolepsy associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic may be determined not only by Ab to the peptide motif common to influenza nucleoprotein and hypocretin receptor but also Ab to melanotonin and glutamate receptors and ion channels. Decreasing and even avoiding risks of complications from vaccination may be feasible by means of a computer analysis of vaccine proteins for the occurrence of epitopes homologous to the human protein those and particularly by an analysis of Ab profiles induced by vaccine using microarrays with the large number of human protein antigens.

The review considers the main molecular biological features of the COVID-19 causative agent, the SARS-CoV-2 virus: life cycle, viral cell penetration strategies, interactions of viral proteins with human proteins, cytopathic effects. We also analyze pathological conditions that occur both during the course of the COVID-19 disease and after virus elimination. A brief review of the biological activities of polysaccharides isolated from various sources is given, and possible molecular biological mechanisms of these activities are considered. Data analysis shows that polysaccharides are a class of biological molecules with wide potential for use in the treatment of both acute conditions in COVID-19 and post-COVID syndrome.

Introduction: Influenza A virus infection can lead to endothelial dysfunction (ED), including apoptosis of endothelial cells and modulation of endothelial factor activities. Affected biochemical factors may include those playing important roles in vascular homeostasis. However, the effect of this pathogen on the expression pattern of key endothelial factors is still unknown.The aim of this work was to study the expression of endothelial nitric oxide synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1, serpin E1) in the EA.hy926 endothelial cells., Research Objectives: to assess expression of eNOS and PAI-1 in endothelial cells infected with influenza virus A(H1N1)pdm09, and to identify homologous fragments in structure of viral proteins and endothelial factors., Material and Methods: Cells were infected with influenza virus A/St. Petersburg/48/16 (H1N1)pdm09 and analyzed in dynamics in 6, 12, 18, 24, 48, and 72 hrs post infection (hpi). Detection of endothelial factors expression levels was performed by immunocytochemical method (ICC) using antibodies for eNOS and PAI-1 while quantitative assessment of expression levels was carried out by program Nis-Elements F3.2 («Nikon», Japan). The search for homologous sequences between viral proteins and eNOS and PAI-1 was performed by computer comparison. Sequences were analyzed as fragments 12 amino acid residues (aar) in length., Results and Discussion: eNOS expression in infected cells had decreased to 7.9% by 6 hpi (control was taken as 100%) to 3.3% at 72 hpi. PAI-1 expression varied significantly over the course of the experiment: by 6 hpi it had decreased to 49.6%, and to 43.2% by 12 hpi. Later PAI-1 levels were: 116.3% (18 hpi); 18.9% (24 hpi); 23.5% (48 hpi), and 35% (72 hpi)., Conclusion: These results indicate that influenza A infection of endothelial cells causes a significant decrease in eNOS expression, while modulating PAI-1 one. The described phenomenon can be used in the further development of directions of pathogenetic therapy of vascular complications of infection caused by this pathogen.

It has been shown that the use of a special growth medium enriched with amino acids and an inhibitor of aminotransferases alpha-aminooxyacetic acid makes possible the selectivity of labeling of barstar with 15N-leucine and 15N-tryptophan. The system of selective labeling, which was previously optimized with respect to the time of introducing the label relative to the time of introducing the inductor IPTG and the inhibitor of cell polymerase rifampicin, was substantially refined by the use of the transamination inhibitor. The inhibition of aminotransferases enables one to completely eliminate the redistribution of the isotope, which is a necessary step in NMR studies even if the strongly metabolizable 15N-leucine is used. The suppression of the redistribution of the isotope by alpha-aminooxyacetic acid is a successful approach to preparation of any selectively labeled proteins in the T7 polymerase system.

The unified conception of the origin of eukaryotic cells has been proposed. In the author's opinion, evolutionary transformation of prokaryotic cell into eukaryotic cell took place 3.3-1.4 billion years ago and involved the next four stages: 1) the appearance of intracellular membranes due to prokaryotic cell plasmalemma invaginating into its cytoplasm; 2) the cell nucleus formation by the double sheet of intracellular membrane surrounding and sequestrating genetic material of the cell; 3) the appearance of cytoskeleton in parallel with mitotic spindle formation and gradual transition from prokaryotic way of cell division to mitosis; 4) the establishment of symbiosis between the evolving nucleated cell and prokaryotic microorganicsms that subsequently transform into mitochondria and chloroplasts. Apoptosis of cells of the present day multicellular eukaryotic organisms is supposed to be an evolutionary altered response of mitochondrian predecessors to the influence of factors, which are able to damage eukaryotic host cell. The initial biological significance of this reaction pertained to attempts of endosymbionts to leave the host cell as soon as possible, if the probability of its irreversible injury was very high, and by this to escape from their death. It is possible that numerous proteins, known as sensors or transducers of proapoptotic signals in Bcl-2--p53-dependent apoptotic pathway, were initially encoded by mitochondrial genome, whereas antiapoptotic factors and also components of receptor-mediated and granzyme B perforin dependent apoptotic pathways have cellular origin.

The review reflects results of studies on the molecular mechanism of phage T4 Dam DNA-methyltransferase action. The enzyme (T4Dam) catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to N6-adenine position in the palindromic recognition sequence GATC (EC 2.1.1.72). The enzyme subunit structure, substrate-binding and kinetic parameters for a wide range of native and modified oligonucleotide duplexes, as well as steady-state reaction kinetic scheme, included T4Dam isomerization to catalytically active form, are considered. The found mechanisms of DNA induced T4Dam dimerization, target base flipping, enzyme reorientation in an asymmetrically modified recognition sequence, effector action of reaction substrates and processive methylation of DNA substrates, containing more than one specific site, are discussed. The results obtained with T4Dam may be useful for understanding mechanisms of action of other homologous enzymes, most of all for specimens of numerous family of Dam DNA-methyltransferases.

Transcription of RNA molecules from synthetic DNA templates with T7 RNA polymerase is a common procedure for the preparation of long RNA molecules. However, enzymatic synthesis does not allow for site-specific incorporation of modified nucleotides. RNA synthesis by chemical methods on the other side can satisfy this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed to combine both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end we have chemically synthesised a fluorescein labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We show that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesised by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.

New methods of chemical and chemoenzymatic synthesis of nucleoside 5'-thiophosphates and 5'-alpha-thiotriphosphates are developed. The 5'-alpha-thiotriphosphates are used as substrates both in template-dependent enzymatic PCR synthesis and in a T7-RNA transcription polymerase system. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.

The 3D structural data for a number of protein-DNA complexes were used to analyze the regions of specific contact with the major groove of B-DNA double helix. The set included seven nonhomologous complexes featuring 12 DNA-binding domains of transcription factors and regulatory factors. The protein domains differed in structure, contained different motifs in the binding region, and broadly varied in chain length, from 30 to 200 residues. Protein-DNA interaction was assessed as hydrogen bonding between polar atoms and van der Waals contacts between nonpolar atoms. The binding sites were formed mainly through polar side chain interactions. On average, the recognition site comprises seven residues, six of them polar. The contact residues nearly always belong to a largest polar cluster of the protein. Thus one can think that the protein polar residue clusters play an important role in forming the protein-DNA recognition module.

The contacts between phosphate groups of promoter DNA an Lys or His of T7 RNA polmerase (Pol) in the Pol-promoter complex were studied with single- and double- stranded oligonucleotides, which corresponded to the T7 promoter consensus and contained activated phosphate groups at position +1, +2, or -14 relative to the transcription start. To obtain reactive groups, terminal phosphates were modified with N-oxybenzotriazole (HOBT), and internucleotide phosphates were repalced with a trisubstituted pyrophosphate (TSP). The resulting derivatives produced covalent complexes with T7 Pol. Covalent bonding involved His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in the case of TSB at position -14.

A model of the simplest epigene was constructed to have two alternative states, which proved to be stable and transmittable through Escherichia coli cell generations. A switch from one to another state was induced by thermal and chemical external signals. The results gave further insight into the dynamic mechanism of preservation, coding, and transmission of the genetic information.

IPolar interactions between protein and the major groove of double-stranded DNA have been analyzed on the basis of a structural study of the complexes formed by two transcription factors: phage lambda repressor and murine immunoglobulin transcription factor NF kappa B-p50. Two identical molecules of these two factors form two binding sites within two different parts of a single DNA molecule. This allows one to study formation of the recognition module by comparing the binding pattern of two different parts of the DNA operator. We have shown that formation of the DNA-binding sites for the three structurally different protein domains (one in repressor and two in the immunoglobulin factor) involves polar residues selected from the largest polar cluster on the surface of the protein molecule. It was also shown that the same polar residues bind with different points of DNA sites. This result provides understanding of the recognition module adaptation to varying nucleotide sequence of the operator sites and shows the way of binding site formation.

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPi analogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV-1 RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

The insulin-like growth factor I (IGF-I) is produced in the liver and is considered mediating the effect of the growth hormone (GH). However, a knock-out only in liver IGF-I slightly disturbs the growth and development of mice. Such mice develop insulin resistance of various organs, including muscles. A knock-out in the liver insulin gene also results in insulin resistance. Selective inactivation of the gene for glucokinase (a target of insulin) in pancreatic islets or in the liver suppresses insulin secretion in the pancreas.

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.

The epidemiological situation with cytomegalovirus infection in Siberia is still to be studied and serological diagnosis of human cytomegalovirus (HCMV) is not satisfactory. Two regions of HCMV genome (strain AD-169) have been examined for obtaining diagnostic reagents by expression cloning. Using polymerase chain reaction, fragments of gene of the immediate early protein (IE2) and of the region encoding for the entire hydrophilic part (1176 bp) of the large phospoprotein gene (pp150) have been obtained. Both fragments were cloned in bacterial vectors. Analysis of nucleotide sequence showed negligible substitutions in comparison with previously reported sequences for these genes.

Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.

The major immediate early gene of human cytomegalovirus has been cloned. The fragment of the immediate early protein containing 351 amino acids has been fused with cro-beta-galactosidase and expressed in Escherichia coli cells. The obtained recombinant protein reacts in immunoblotting with the antibodies in human sera from patients suffering from acute cytomegalovirus infection.

Monomeric DNA and RNA polymerases contain a number of conserved motifs. By means of random and oligonucleotide-directed mutagenesis the point mutants of bacteriophage T7 RNA polymerase containing the amino acid substitutions in motif B which is the part of the active site were obtained. The kinetic properties of the mutants were studied. The results obtained suggest that amino acid residues 636, 639 and 646 are involved in the binding of the initiating GTP, the selection of correct nucleoside triphosphate and interaction with the promoter, respectively.

A recombinant tandem of mouse/human immunoglobulin (Ig) genes was constructed and inserted in the plasmid pGEM1 under the control of the T7 RNA polymerase promoter. Constant Ig genes of murine origin were replaced with human genomic ones. Isotype IgG1 was changed for IgE. Lymphoid cell line Sp2/0 and CHO (chinese hamster ovary) cells were used for transfection. Both cell lines contained the semisynthetic gene of T7 RNA polymerase in their genomes and stably expressed this polymerase. Transfected clones of Sp2/0 and CHO cells synthesized k-light (25 kD) and epsilon-heavy (70 kD) chains of Ig and the corresponding mRNAs. Both lymphoid and nonlymphoid transfected cell lines synthesized functionally active antibodies interacting with the antigen (pig transferrin). Particular clones synthesized up to 150 ng/ml of chimeric antibodies. However, only lymphoid Sp2/0 cells were capable of efficient secretion. Nonlymphoid CHO cells were able to produce antibodies, but incapable of their efficient secretion.

The scanning calorimetry technique has been used to study the influence of amino acid substitutions on thermodynamic parameters of formation and stabilization of a cooperative structure of Cro repressor of bacteriophage lambda molecule. It is shown that substitutions 16Gln-Leu and 26Tyr-Asp enhances the protein molecule stability by 32 degrees C as compared with the wild-type protein. It is also demonstrated that the denaturation enthalpy of the mutated Cro differs slightly from that of the wild-type protein at the same temperature, while the effective enthalpy value is significantly lower. The analysis of excess heat capacity of the mutated protein shows that this complex function determined experimentally is approximated by two functions, which is indicative of the presence of two quasi-independent transitions. Thus, the stabilization and redistribution of intermolecular interactions evoked by the above substitutions leads to disintegration of the single cooperative repressor molecule in two interacting cooperative domains. The plausible mechanism of formation of such a domain structure is discussed on the basis of the available calorimetric data.

Chemico-enzymatic synthesis and cloning of genes coding viral proteins--precursors of p50 and gp120 in the eukaryotic vector system were carried out. The recombinant proteins obtained were shown to be used in immunochemical evaluation of AIDS virus antibodies in blood serum.

Earlier we developed an expression vector on the basis of bacteriophage M13 allowing the exposure of short peptides on the virion surface. It was used to obtain a recombinant phage carrying the antigenic determinant of HIVI gag protein p17. This phage was tested as immunogen in rabbits. It was shown by ELISA that Ig against the fusion phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor p55 on strips activated by the transfer of HIVI viral proteins. These data may be used in vaccine development.

Interactions of the bacteriophage T7 DNA-dependent RNA polymerase with three GTP analogs have been studied. All of the three analogs tested contained substituted naphthalenesulphamide groups and were shown to be under appropriate conditions irreversible covalent inhibitors of the enzyme, the modified enzyme possessing fluorescent properties. One of these analogs contained the reactive 2-bromoethyl phosphonate group and was shown to cause the loss of the enzyme affinity for polynucleotide templates. The other two modifiers which contained the azide reactive group did not alter the enzyme-template affinity, the polynucleotide binding leading to a notable increase of the enzyme fluorescence intensity. The latter two modifiers are supposed to be convenient for fluorescent labelling of the active site of RNA polymerase for enzyme-template binding studies.

A study was carried out on the influence of point mutations of the functional amino acid residues on the secondary and ternary structure of bacteriophage T7 RNA polymerase as well as on the activity of the enzyme. A change in residue 631 is accompanied by significant changes in secondary structure (alpha-->beta transition). The substitution Lys-172 Leu changes both the secondary and ternary structures whereas the deletion of residues 172-173 does not lead to such changes. Changes in residues 631-632 do not affect the ability of the enzyme to bind the promoter and/or the synthesis of a full-length transcript but disturbs phosphodiester bond formation.

Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides. The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower. Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter. DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts. L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes. The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.

The transient expression of the HIV-1 gag genes and a HIV-1 ++trans-activator protein (tat)-encoded was made in cultured CV-1 cells. In recombinant plasmids, the gag gene was under the control of HIV-1 ++trans-activator sequence (tar) and the tat gene was under the control of a 7.5-kd vaccinia promoter. Transactivation of gag gene expression, which was stimulated by a tat gene expression product, was observed in the presence of wild vaccinia virus. The transaction was immunologically evaluated from the binding to monoclonal anti-p17 and anti-p24 antibodies. The findings lead to the discussion whether the regulatory proteins of HIV-1 can express in vaccinia virus vectors.

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.

The method of electrophoresis in a single polyacrylamide gel plate was used for comparative study on virion polypeptides mobility in human influenza A and B virus strains. Molecular masses of individual polypeptides and their portion in the virion were determined. No variations in the migration speed of nucleoprotein (NP) and membrane protein (MP) were found in strains belonging to the same genus, but there were differences in the migration speed of these proteins in the genus A and genus B. Significant differences in migration and the content of glycoproteins, particularly of the heavy chain of hemagglutinin (HAI) in the genus A strain having different subtypes of hemagglutinin, including strains A/WS/33 (HONI), A/FM/1/47 (HINI), A/Sing/1/57 (H2N2), A/Port Chalmers/73 (H3N2), as well as in the genus B in strains B/Lee/40, B/Yamagata/1/73, and B/Johanesbourg/56. No differences in the mobility of glycoproteins in strains similar in the antigenic specificity of hemagglutinin and neuraminidase were found. On the basis of the comparative analysis of virion polypeptide electrophoregrams, three new strains on influenza A virus isolated in December, 1977, were identified as belonging to the species A (HINI).

Complexes of synthetic double-stranded polynucleotides and DNA with model peptides--L-Lys-L-Tyr-L-Lys and L-Lys-Gly-L-Lys have been investigated, using UV-spectroscopy. Polynucleotide complexes containing L-Lys-Gly-L-Lys were studied in order to consider the influence of lysyl residues on the polynucleotide melting temperature. It was shown, that L-Lys-L-Tyr-L-Lys lowers the melting temperature of all the polynucleotides studied, except poly(rI) . poly(C). The dependence of melting temperature of polynucleotide (DNA) . L-Lys-L-Tyr-L-Lys complexes upon the polynucleotide double helix form and GC-content has been detected. These effects have reflected the intercalation of peptide tyrosyl residues into one of the chains of the double-stranded polynucleotide. Correlation o the melting temperature dependences of polynucleotide complexes with gene 5 protein and L-Lys-L-Tyr-L-Lys upon polynucleotide double helix form and GC-content was found.