Adenovirus Deoxyribonucleic Acid Research Articles

The Insertion of Foreign DNA Into Mammalian Genomes and Its Consequences: A Concept in Oncogenesis

Publisher Summary This chapter examines the consequences of foreign deoxyribonucleic acid (DNA) insertion into established mammalian genomes. In mammalian cells, the ever-present repetitive DNA sequences possibly constitute a reservoir of insertion targets whose disturbance may have less immediate functional consequences. Among the consequences of the insertion of foreign DNA into the established genomes and of the transformed cell phenotype are the de novo methylation of the foreign DNA and alterations in the existing patterns of DNA methylation in the preexisting cellular genome. Diet-ingested foreign DNA could play an important role in eliciting mutagenic and oncogenic events in mammals. In light of these findings, the postulated cellular defense mechanism by methylation of integrated foreign DNA becomes plausible. The results of studies on the model of adenovirus DNA integration in mammalian cells suggested that the insertion of foreign DNA had to be rather pliable. There is a possibility that the insertion of foreign DNA into an established mammalian genome can lead to far-reaching alterations in the patterns of cellular DNA methylation and gene expression. The classical concept of insertional mutagenesis relates to damage caused to cellular functions or genes that are encoded at the sites of foreign (adenoviral) DNA insertion into the host genome.

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Involvement of both DNA polymerases α and γ in the replication of adenovirus deoxyribonucleic acid in vitro

Adenovirus (Ad) DNA replication complexes (RCs) were purified from nuclear extracts of Ad-infected cells by exclusion chromatography, followed by equilibrium centrifugation in discontinuous sucrose-heavy water gradients. The material, which was characterized as the site for synthesis of Ad DNA by its time course of labeling in vivo, was able to elongate, in vitro, viral DNA molecules, the synthesis of which had been initiated in vivo. Endogenous DNA polymerase activity in the Ad RCs was totally inhibited by dideoxythymidine triphosphate (ddTTP) at a ddTTP:dTTP ratio of about 5:100; this suggests that the functional enzyme involved was DNA polymerase γ. The activity of the RCs was also strongly inhibited by 50 μg/ml phosphonoacetic acid (PPAA), and by Ara CTP:dCTP ratios of about 5:1; this suggests that the functional enzyme involved in the elongation activity was DNA polymerase α. Sensitivity of Ad RC's endogenous activity toward aphidicolin was somewhat lower than that reported for purified DNA polymerase α, but definitely much higher than that reported for DNA polymerase γ. Direct determination of the DNA polymerases present in partially purified Ad RCs was done by sucrose gradient centrifugation followed by identification of the enzymes through the use of specific assays. The major DNA polymerase present in Ad RCs was DNA polymerase α. In addition, small amounts of DNA polymerase γ were detected. No DNA polymerase β appeared to be present. These data are consistent with the hypothesis that Ad5 DNA replication involves both DNA polymerases α and γ.

Adenovirus Deoxyribonucleic Acid Synthesis in a Soluble Fraction Isolated from Infected Human Cells

Adenovirus Deoxyribonucleic Acid Synthesis in a Soluble Fraction Isolated from Infected Human Cells

Adenovirus deoxyribonucleic acid replication. Characterization of the enzyme activities of a soluble replication system.

Adenovirus deoxyribonucleic acid replication. Characterization of the enzyme activities of a soluble replication system.

Adenovirus deoxyribonucleic acid replication. Isolation of a soluble replication system and analysis of the in vitro DNA product.

Adenovirus deoxyribonucleic acid replication. Isolation of a soluble replication system and analysis of the in vitro DNA product.

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.

Transcription of the adenovirus genome by an -amanitine-sensitive ribonucleic acid polymerase in HeLa cells.

The properties of the ribonucleic acid (RNA) polymerase activity which transcribes the major portion of the adenovirus genome were studied. Nuclei were prepared from infected cells and incubated in vitro. Virus-specific RNA was determined by hybridization to adenovirus deoxyribonucleic acid (DNA). Adenovirus DNA is transcribed principally by an activity which resembles closely polymerase II of the host cell. This activity is inhibited by alpha-amanitine and stimulated by (NH(4))(2)SO(4). Its product is high-molecular-weight heterogeneous RNA. The polymerase activity measured early in infection (3 to 5 hr) resembles that found late in infection (16 to 18 hr).

State of adenovirus 2 deoxyribonucleic acid in the nucleus and its mode of transcription: studies with isolated viral deoxyribonucleic acid-protein complexes and isolated nuclei.

Newly replicated adenovirus 2 deoxyribonucleic acid (DNA) can be isolated from the nucleus of HeLa cells by a gentle lysis procedure as a fairly homogeneous complex with a sedimentation of 73S. The viral DNA complex can be prepared completely free from host cell DNA. The viral complex is slightly active in ribonucleic acid (RNA) synthesis in vitro. Treatment of the complex with Pronase and sodium dodecyl sulfate converts the DNA to a form which sediments at 43S. Nuclei isolated from adeno-infected cells synthesize high-molecular-weight virus-specific RNA in vitro. Optimal RNA synthesis requires a divalent cation, preferentially manganese, and relatively high salt concentrations. The synthesis of virus-specific RNA by the isolated nuclei is strongly inhibited by low doses of alpha-amanitine. The latter experimental result is discussed in terms of the polymerase used to transcribe the adenovirus DNA in vivo.

Intracellular forms of adenovirus deoxyribonucleic acid. I. Evidence for a deoxyribonucleic acid-protein complex in baby hamster kidney cells infected with adenovirus type 12.

The total intracellular deoxyribonucleic acid (DNA) from baby hamster kidney cells abortively infected with (3)H-adenovirus type 12 was analyzed in dye-buoyant density gradients. Between 10 and 20% of the cell-associated radioactivity derived from viral DNA bands in a density position which is 0.043 to 0.085 g/cm(3) higher than that of viral DNA extracted from purified virions. The DNA in the high-density region (HP-fraction) is almost completely absent when DNA, ribonucleic acid (RNA) or protein synthesis is chemically inhibited in separate experiments. The HP-fraction is not found when the virus does not adsorb to and enter the cell. The DNA in the HP-fraction appears as early as 2 hr after inoculation. At 2 hr after infection, the HP-fraction is present both in the nucleus and the cytoplasm. This DNA hybridizes exclusively with viral DNA and sediments at approximately the same rate in both neutral and alkaline sucrose density gradients. Electron microscopy has revealed no circular DNA molecules in this fraction. Evidence indicates that the viral DNA in the HP-fraction exists in a complex with protein and possibly RNA. The protein component of the complex is resistant to enzymatic digestion, whereas the complex is susceptible to ribonuclease treatment. Digestion with deoxyribonuclease reduces the amount of DNA found in the HP-fraction. The structure and biological function of this complex are currently being investigated.

Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid.

Intermediates in the synthesis of adenovirus type 2 deoxyribonucleic acid (DNA) were studied in HeLa cells. Pieces of DNA smaller than the viral genome were demonstrated after labeling with (3)H-thymidine for 10 to 240 sec. Intermediates as small as the Okazaki fragments (8 to 10S) do not predominate at any of the above times. No detectable addition of nucleotides to parental genome could be shown, nor was there any breakdown of recently synthesized viral DNA. The DNA intermediates were of viral origin for they hybridized to viral DNA and were made at a stage of the cell cycle (G(2)) when host DNA is not synthesized.

Three size-classes of intracellular adenovirus deoxyribonucleic acid.

When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3S for adenovirus type 2 and at 29.0S for adenovirus type 12, (ii) viral DNA which sediments at about 18S, and (iii) viral DNA which sediments at >45S and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. II. Relationship of adenovirus 2 deoxyribonucleic acid and simian virus 40 deoxyribonucleic acid in the Ad2+ND genome.

A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2(+)ND(1) virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2(+)ND(1) molecule. From the results obtained, it was estimated that 1% of the Ad2(+)ND(1) DNA consists of SV40 nucleotide sequences.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. 3. Base composition, molecular weight, and conformation of the Ad2+ND genome.

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), differs from the defective Ad-SV40 hybrid populations previously described, in that this hybrid virus can replicate without the aid of nonhybrid adenovirus helper. Consequently, the deoxyribonucleic acid (DNA) from this virus, which can be obtained free of nonhybrid adenovirus DNA, is well suited for biophysical studies on Ad-SV40 hybrid DNA. Such studies have been performed and demonstrate Ad2(+)ND(1) DNA to have a buoyant density (1.715 g/cm(3)) and thermal denaturation profile (T(m) = 75.1 C) almost identical with nonhybrid Ad2 DNA. Furthermore, its molecular weight, as determined by analytical zone sedimentation and electron microscopy, was 22 x 10(6) to 25 x 10(6) daltons, which is also very similar to that determined for Ad2. Electron micrographs showed all of the hybrid molecules to be double-stranded and linear. By using this determination of the molecular weight of Ad2(+)ND(1) DNA and assuming that 1% of this molecule consists of covalently linked SV40 DNA (see companion paper), we calculate that the hybrid DNA molecule contains 220 x 10(3) to 250 x 10(3) daltons of SV40 DNA, or the equivalent of one-tenth of the SV40 genome.

Biochemical consequences of type 2 adenovirus and Simian virus 40 double infections of African green monkey kidney cells.

African green monkey kidney (AGMK) cells were nonpermissive hosts for type 2 adenovirus although the restriction was not complete; when only 3 plaque-forming units/cell was employed as the inoculum, the viral yield was about 0.1% of the maximum virus produced when simian virus 40 (SV40) enhanced adenovirus multiplication. The viral yield of cells infected only with type 2 adenovirus increased as the multiplicity of infection was increased. Type 2 adenovirus could infect almost all AGMK cells in culture; adenovirus-specific early proteins and DNA were synthesized in most cells, but small amounts of late proteins were made in relatively few cells. Even when cells were infected with both SV40 and adenovirus, only about 50% were permissive for synthesis of adenovirus capsid proteins. Approximately the same quantity of adenovirus deoxyribonucleic acid (DNA) was synthesized in the restricted as in the SV40-enhanced infection. However, in cells infected with SV40 and type 2 adenovirus, replication of SV40 DNA was blocked, multiplication of SV40 was accordingly inhibited, and synthesis of host DNA was not stimulated. To enhance propagation of type 2 adenovirus, synthesis of an early SV40 protein was essential; 50 mug of cycloheximide per ml prevented the SV40-induced enhancement of adenovirus multiplication, whereas 5 x 10(-6)m 5-fluoro-2-deoxyuridine did not abrogate the enhancing phenomenon.

Properties of Adenovirus Messenger Ribonucleic Acid Synthesized In Vitro

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 mug of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 mug of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).

Production of highly labeled adenoviruses.

A method is described for increasing the incorporation of radioactive thymidine into adenovirus deoxyribonucleic acid by the use of amethopterin. In addition, a modified procedure is presented for the preparation of highly purified adenoviruses. This procedure, which employs enzymatic digestion of cellular debris, obviates the necessity for fluorocarbon treatment of crude virus suspensions, and routinely provides excellent recovery of virus.

Role of adenovirus structural proteins in the cessation of host-cell biosynthetic functions.

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.