An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture.

A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.

[Enhancement of the transforming activity of simian adenovirus S5 and of the DNA from human adenovirus type I by benzo(a)pyrene and TPA]. / Usilenie transformiruiushchei aktivnosti adenovirusa S5 obez'ian i DNK adenovirusa tipa I cheloveka benz(a)pirenom i TFA.

Benz(a)pyrene in a concentration of 2.5 micrograms/ml produced a 3.3-fold increase in the transforming activity of simian adenovirus S5 in primary cultures of rat kidney cells. Under analogous conditions TPA increased (2.1 times) the transforming activity of DNA of human adenovirus of type I (AdI). PX5 cells (cotransformed by S5 and benz(a)pyrene) induced tumours in 100% of syngeneic and 81% xenogeneic animals. PX4 cells (transformed by S5 only) induced tumours in 60% of syngeneic animals. Cells transformed by DNA AdI and DNA AdI + TPA did not induce tumours in animals.

[Antisense polynucleotides and prospects for their use in fighting viruses]. / Antismyslovye polinukleotidy i vozmozhnost' ikh ispol'zovaniia dlia bor'by s virusami.

Natural or synthetic anti-sense (as) polynucleotides complementary to distinct functional regions of mRNA (asRNA or asDNA) are able to inhibit the expression of any target gene. If certain viral mRNAs important for virus replication are targeted the inhibition of viral infection by asRNA or asDNA takes place. Inhibitory effects of complementary polynucleotides on gene activity in eukaryotic cells is due to the disturbance of translation of corresponding mRNAs as well as to the impairment of their splicing or transportation from the nuclei to cytoplasm. In prokaryotic cells, obviously, only the first factor is operating. The recombinant genes programming anti-viral asRNA can confer the resistance to the infection by other virus to the transformed cells. The resistance to viral infection observed in transgenic animals, expressing asRNA genes, may be considered as a new unnatural form of informational immunity.

[Prospects and achievements of genetic engineering in development of antiviral vaccines]. / Perspektivy i dostizheniia v sozdanii genno-inzhenernykh protivovirusnykh vaktsin.

Proceeding from the known data various theoretical and experimental approaches to the construction of gene-engineering vaccines are considered. Gene-engineering subunit vaccines of the first generation are based on isolation of the genes coding for the synthesis of full length capsid proteins with the main antigenic determinants and their subsequent expression in suitable recipient cells. Initial idea of the microbiological synthesis as the main way for production of any antiviral vaccines was not confirmed by the later development. Now for this type of vaccines eucaryotic systems are widely employed using the animal virus vectors and the animal cell cultures. Gene-engineering subunit vaccine of the second generation appears to be a chimeric protein with built-in antigenic determinants of different viruses and maximal immunogenicity in monomeric form. The last point reopens the perspective to use a microbiological synthesis for the production of antiviral vaccines. Besides that the chemically synthesized polypeptide antiviral vaccine will be used widely. In gene-engineering subunit vaccines of the third generation it is possible to use not the natural antigenic determinants which often are characterized by high level of the primary structure changes but artificial (non-natural) antigens, that are the capsid protein conservative regions which under natural conditions of infection or immunization do not induce the protective antiviral antibodies. The recombinant DNA technology in addition to subunit type vaccine allows to construct living vaccines which represent a DNA-containing attenuated virus with build-in natural or synthetic gene of the capsid or chimeric protein with antigenic determinants of another viral species.

[Possible mechanisms of the transforming and tumorigenic effects of DNA-containing animal viruses]. / O vozmozhnykh mekhanizmakh transformiruiushchego i tumorogennogo deistviia DNK-soderzhashchikh virusov zhivotnykh.

The literature devoted to oncogenic action of DNA-containing animal viruses and their role in the development of human neoplasias are reviewed. The regularities of persistence and expression of genetic material of DNA-containing viruses in transformed and tumor cells are comprehensively analyzed. The mechanisms of recombination of cellular and viral DNA during cell transformation as well as the specificity of integration of viral DNA into the host genome are considered. The functions and mechanisms of transforming and tumorigenic action of the products of oncogens of DNA-containing viruses of different groups are discussed. The data on the cell transformation by some DNA-containing viruses without oncogene expression are represented. The mechanism of cell transformation by DNA-containing viruses related to the activation of cellular oncogens is discussed.

[Calculating light scattering when determining "true absorption magnitudes" of virus particles]. / K voprosu ob uchete svetorasseianiia pri opredelenii "istinnykh velichin pogloshcheniia" virusnykh chastits.

Results of the study by V. I. Permogorov et al. (Molecular Biology, 1977, 11, 134--138) on the absence of a deficit of hypochromism in intraphage DNA are discussed. It is deduced that the conclusion of V. I. Permogorov and coworkers is erroneous since it is based on: 1) incorrect interpretation of the results of determination of lightscattering contribution to the absorption of intact phages; 2) not taking into account the lightscattering contribution in the case of disrupted phages; 3) underestimation of the value of hyperchromism of melting of phage DNAs.

[Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons]. / Strukturnyi i immunokhimicheskii analiz produktov proteoliza geksonov adenovirusov primatov.

Hexon capsomers of simian adenovirus sim16 (SA7) and of human adenoviruses h5 (Ad5) and h6 (Ad6) were proteolytically digested and the resulting products studied by SDS-polyacrylamide gel electrophoresis and by radioimmunoprecipitation analysis. The trypsinolysis of native SA7 hexon leads to a stable molecular "core" containing 4-5 fragment species of 10 to 65 kDa and resembling the intact capsomer in quarternary structure (trimer). Similar cores but consisting of smaller fragments (less than 40 kDa) were obtained after chymotryptic digestion of native SA7, Ad5 and Ad6 hexons. The chymotryptic hexon fragments were also held together in pseudotrimeric structures. The similarity of proteolytic hexon fragment patterns between different primate adenoviral hexons suggested a homology to exist in localisation of the exposed tryptic and chymotryptic cleavage sites in their respective hexon polypeptide chains. Papain caused a complete hydrolysis of native SA7 hexon (trimer) yielding small peptides, but at first stage of digestion a stable papain hexon core containing small fragments (less than 10 kDa) was observed. The tryptic SA7 hexon cores in native state retained their antigenicity in reactions with homo- and heterologous antibodies, but after core denaturation the resulting fragments had no antigenic activity of native capsomer. In contrast to the data previously published, chymotryptic cores of SA7, Ad5 and Ad6 hexons not only reacted with respective homologous antibodies but also retained (at least in part) cross-reactive antigenic determinants. The questions of formation and stability of native adenoviral hexon conformation are discussed as well as the possible nature of hexon antigenic determinants.

[Synthesis of adenovirus SA7 and Ad5 DNA in extracts from the nuclei of green monkey kidney cells]. / Sintez DNK adenovirusov SA7 i Ad5 v ékstraktakh iader kletok pochek zelenoi martyshki.

A soluble extract from the nuclei of green monkey kidney cells, infected with adenovirus SA7, carries out replication of SA7 DNA. It was observed, that in vitro DNA synthesis, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, proceeds by a semiconservative mechanism. The data obtained are compared with literary data on soluble enzyme systems from adenovirus-infected cells. The early factors provided by SA7 also supported replication of the DNA-protein complex, prepared from human adenovirus type 5, in nuclear extracts of monkey cells.

[Physical mapping of the genome of phage Cd]. / Fizicheskoe kartirovanie genoma faga Cd.

The effect of 7 specific endonucleases EcoRI, BamHI, BglII, SalI HindIII, XhoI, SmaI, on the genome of the phage Sd was studied. The molecular weight of the genome was estimated as 45.10(6). BamHI, EcoRI, HindIII, BglII, SmaI have altogether 15 sites of restriction SalI and XhoI do not hydrolyse the phage DNA. Fragmentation of the phage DNA in conditions of partial hydrolysis and simultaneous action of several enzymes have allowed to draw a physical man to the phage Sd DNA is more resistent to the action of restriction enzymes than DNAs from other phages. The mean size of Sd DNA fragments exceeds the statistical value almost by one order of magnitude.

[Optical rotatory dispersion by 5 viruses of the tobacco mosaic virus group and their components]. / Dispersiia opticheskogo vrashcheniia piati virusov gruppy virusa tabachnoi mozaiki i ikh komponentov.

Optical rotatory dispersion (ORD) spectra in 250 to 350 nm region were measured for preparations of five TMV-like viruses (TMV vulgare, HR and U2 strains of TMV dolihosenation mosaic virus and cucumber virus 4) and also for RNA and protein preparations of these viruses. The data obtained testify against the possibility that the double peak with maxima at 286 and 293 nm observed in ORD of all the five viruses is due to interaction of tryptophan residues in virus coat protein with the RNA of the virul particle. The spectra of intravirus RNA of the five viruses, calculated as the difference between ORD of the intact virus and of its coat protein, were found to differ significantly from each other and from ORD of free RNA. ORD spectra of hybrid viruses, reconstituted from RNA of one virus and coat protein of another, proved to be identical to the ORD of the virus, whose protein was used in reconstitution. We suppose that the difference in ORD of the intravirus RNA of the five viruses reflect differences of RNA-protein interactions in them.

[Effect of nuclease S1 on DNA of the highly oncogenic simian adenovirus SA7(C8)]. / Izuchenie deistviia nukleazy S1 na DNK vysokoonkogennogo adenovirusa obez'ian SA7(C8).

The effect at specific nuclease S1 on DNA and the complex viral DNA-terminal protein of the highly oncogenic simian adenovirus SA7(C8) was studied. It was shown that nuclease S1 did not digest the bound between DNA and terminal protein in the complex but residual amino acid(s) was cleaved out after digestion with pronase. The DNA obtained after nuclease S1 action could be ligated and its 5'-ends were phosphorylated by polynucleotide kinase.

[Bacteriophage P22 DNA structure in situ]. / Issledovanie struktury DNK in situ Bakteriofaga.

The structure of phage P22 DNA in situ was investigated by optical methods and by chemical modification with sodium bisulfite. On disruption of the phage particles by heating at 45 degrees a drop in absorbance at the 250 nm to 290 nm region was observed. At 260 nm this hypochromism was about 12%. CD spectra of intraphage DNA differed from that of free P22 DNA in the intensity as well as in the position of the positive band (lambda max 280 nm, delta epsilon max=1.3). In the intraphage DNA 21 per cent of cytosines reacted with sodium bisulfite. Cytosyl-amino acid products were found in the HClO4 and HCl hydrolysates of the modified phage. The main amino acid component of the product was identified as lysine. It was shown by means of gradient centrifugation and electron microscopy that the cytosyl-amino acid products result in the crosslinking of DNA to protein in the phage particles.

[Biological effects accompanying the microinjection of adenovirions, adenoviral and plasmid DNA into mammalian cells]. / Biologicheskie éffekty, soprovozhdaiushchie mikroin''ektsiiu adenovirionov, adenovirusnykh i plazmidnykh DNA v kletki mlekopitaiushchikh.

Microinjection of either type 1 human adenovirus, type SA7 monkey adenovirus virions or circular adenovirus DNA, obtained by the treatment of DNA-terminal protein complexes with glutaraldehyde, into nuclei of permissive cells results in the complete cycle of virus reproduction. Microinjection of neither linear native, condensed adenovirus DNA nor the DNA-terminal protein complexes under the same conditions initiates the adenovirus reproduction thought the synthesis of early and some late viral antigens is observed in the injected cells. Integration of injected adenovirus DNA into the cellular DNA occurs as far as 30 min after injection. Microinjection of either adenovirus DNA or its oncogene containing fragments into nuclei of semipermissive cells induces the transformation of these cells. In this case the time of the first appearance of transformation foci is decreased.

[Electron microscope maps of SA7 DNA melting]. / Elektronno-mikroskopicheskie karty plavleniia DNK SA7.

Electron microscopic denaturation maps corresponding to the first peaks of the differential melting curve of SA7 DNA were constructed by fixation of partly denatured molecules with glyoxal at temperatures within the melting range. These maps were oriented with respect to the functional map of the virus genome. The localization and the size of the most AT-rich SA7 DNA regions were determined.

[Synthesis of somatostatin in Escherichia coli cells: isolation and characteristics]. / Sintez somatostatina v kletkakh Escherichia coli. Vydelenie i kharakteristika.

A synthetic gene coding for somatostatin-14 (SST) was cloned in plasmid expression vectors in frame with the chloramphenicol acetyl transferase (CAT) gene, both genes being divided by a Met residue. The hybrid gene was expressed under the control of the CAT gene promoter (Pcat) or the tryptophan operon promoter (Ptrp). Them fused genes gave insoluble polypeptide products amounting from 5% of the total cellular protein under constitutive biosynthesis conditions (Pcat) to 30% upon induction (Ptrp). SST was liberated from the fused polypeptide by treatment with cyanogen bromide, purified to homogeneity by gel-filtration and reverse phase HPLC, and finally refolded by dilution and air oxidation. The renaturated recombinant SST showed the specific biological and immunological activities of the native peptide.

[Human hepatitis B virus and hepatocellular carcinoma]. / Virus gepatita B cheloveka i gepatotselliuliarnaia kartsinoma.

Hepatitis B virus (HBV) is the causative agent of hepatocellular carcinoma (HCC) in man. The HBV genome is a circular partially double-stranded DNA molecule of about 3.2 kb. The HBV genome contains four structural genes coding for the HBV envelope (HBsAg) and core (HBcAg/HBeAg) proteins, endogenous DNA-polymerase with the additional enzymatic activity of a reverse transcriptase and polypeptide X functioning as a trans-activator of cellular and viral genes. HBV DNA integration in the genomes of HCCs and hepatocytes of HBV carriers is an important evidence establishing a relationship between the HBV infection and the development of HCC. The mechanism of HBV DNA integration into the cellular genome and the possible role of integrated HBV DNA sequences in the malignant transformation of hepatocytes are discussed.

[A phased poly-linker for cloning and expression of genes]. / Faziruiushchii polilinker dliia klonirovaniia i ékspressii genov.

15 oligodeoxynucleotides were synthesized using the phosphotriether technique which were subsequently enzymatically ligated in polylinker with subsequent phasing of left and right sides. Based on the phased polylinker a series of vehicles for the gene cloning and expression was constructed. The vectors of pRK series contain all three variants of polylinker with the frame shift of the reading frame for 1, 2, or 3 nucleotides in both chains. The obtained polylinkers do not effect the enzymatic activity of the beta-galactosidase alpha-peptide. Structure of the phased polylinker was confirmed by Maxam and Gilbert's sequencing method.

[Polymerization of DNA fragments coding epitopes of the surface antigen of hepatitis B in Escherichia coli cells]. / Polimerizatsiia fragmentov DNK, kodiruiushchikh épitopy poverkhnostnogo antigena virusa gepatita B v kletkakh Escherichia coli.

Synthetic oligonucleotides coding for the amino acid residues 135-151 (A, B) and 93-109 (C) of the hepatitis B surface antigen (HBsAg) have been polymerized. The obtained polymers (AC)n and (BCAC)n were inserted into the beginning of the lacZ gene under the control of the chloramphenicol promoter or the right promoter PR of bacteriophage lambda. Stability of the obtained plasmids was investigated during transformation, cell growth and gene expression.

[Suppression of replication of swine parvoviral antisense RNA against the NS PPV gene in swine thyroid gland cells]. / Podavlenie replikatsii parvovirusa svinei antismyslovymi RNK protiv NS gena PPV v kletkakh shchitovidnoi zhelezy svin'i.

The possibility of suppression of porcine parvovirus (PPV) reproduction in the culture of thyroid gland cells of a swine that contain the integrated genes for asRNA against the nonstructural proteins of the virus has been studied. 10 cell lines with the asRNA genes have been obtained. The line with the maximal number of integrated gene copies was used to inflict with the parvovirus. The expression of asRNA in this cell line was shown to lead to 95% suppression of PPV replication as compared with the control cell line.

[Use of U3 promotor from the LTR region of bovine leukemia virus for expressing genes for antiviral antisense RNAs in cell cultures]. / Ispol'zovanie promotora U3 oblasti LTR virusa leikoza korov dlia èkspressii genov protivovirusnykh antismyslovykh RNK v kletochnykh kul'turakh.

The possibility was studied to use the U3 promoter from LTR region of bovine leukemia virus to control the expression of the antisense RNA genes directed against the BLV genome in cell cultures FLK-BLV, CC81, and HeLa. The genetic engineering constructions were obtained carrying the reporter beta-galactosidase gene and the genome of antisense RNA to R-U5 region of the viral genome under the control of the promoter. The functioning of the viral promoter was studied in three cell lines. Its specific activation and kinetics of inhibition of BLV virus reproduction in cell culture CC81 have been demonstrated. The maximal 75% level of the viral reproduction inhibition was achieved at threefold molar excess of the plasmid containing the antisense RNA gene over the pregenomic viral DNA.