Replication of Adenovirus 12 Deoxyribonucleic Acid in Association with the Nuclear Membrane

Analysis of nuclei of adenovirus 12-infected cells revealed that viral DNA replicated in association with the nuclear membrane and that complete viral DNA was liberated from the nuclear membrane. Analysis of isolated nuclei in vitro showed that DNA polymerase activity increased in the nuclear membrane of adenovirus 12-infected cells without addition of primer DNA.     

Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with (3)H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The (3)H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral (3)H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.     

Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid

Highly purified, (32)P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of (32)P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified (32)P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.     

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.     

Effect of Poxvirus Infection on Host Cell Deoxyribonucleic Acid Synthesis

Deoxyribonucleic acid (DNA) synthesis was studied in poxvirus-infected cells by measuring (14)C-thymidine incorporation into viral and host cell DNA. A complete separation of the two species of DNA was achieved by combining the previously used "Dounce method" with a separation method based on different reannealing properties of viral and vertebrate DNA. Shortly after infection of HeLa cells with poxviruses, a burst of viral DNA synthesis occurred in the cytoplasm, but a rapid inhibition of host-cell DNA synthesis in the nucleus was observed. This inhibition of cellular DNA synthesis was also found if an accumulation of viral DNA was prevented. At high multiplicites, ultraviolet-irradiated virus inhibited host-cell DNA synthesis to the same extent as fully infectious poxvirus. Under the same conditions, heating at 60 C for 15 min caused a decrease in the ability of cowpox virus to inhibit host-cell DNA synthesis, but did not produce the same effect on vaccinia virus strain WR.     

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.     

Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

Nonproductive Infection and Induction of Cellular Deoxyribonucleic Acid Synthesis by Bovine Adenovirus Type 3 in a Contact-Inhibited Mouse Cell Line

Bovine adenovirus type 3 (BAV-3), which has been reported to produce tumors in newborn hamsters, induced cellular deoxyribonucleic acid (DNA) synthesis in a contact-inhibited mouse kidney cell line (C3H2K). In this system, the virus did not multiply, whereas virus-specific tumor antigen (T antigen) was detected in nearly all cells. Replication of viral DNA could not be detected by DNA-DNA hybridization on membrane filters. The cellular DNA synthesis induced by BAV-3 did occur in the absence of added serum. Extent of induction of cellular DNA synthesis was closely correlated with the multiplicity of infection. Cells activated to synthesize DNA in the serum-free medium by the virus infection progressed to cell division without noticeable cell killing.     

Host Cell Deoxyribonucleic Acid Polymerase I and the Support of T4 Bacteriophage Growth

Ultraviolet irradiation of Escherichia coli polA(-) cells reduces their capacity to support the growth of T4 phage. There is no additional loss of capacity observed in pol tsA(-)recA(-) double mutants at the nonpermissive temperature. The reversion frequency of a T4 rII mutant after ultraviolet irradiation is not changed by the absence of host deoxyribonucleic acid polymerase I.     

Temperature-Dependent Cellular Regulation in Induction of Cellular Deoxyribonucleic Acid Synthesis by Bovine Adenovirus Type 3

Induction of cellular deoxyribonucleic acid synthesis by infection with bovine adenovirus type 3 was examined in 7 clones of a mouse cell line. Cellular DNA synthesis was induced by infection both at 37C and at 41C in 5 clones. In the other 2 clones, however, cellular DNA synthesis was induced only at 41C and not at 37C. In a clone non-inducible at 37C, the incubation at 41C prior to infection resulted in induction of cellular DNA synthesis at 37C. The preincubation effect was not inhibited by cycloheximide during the incubation at 41C. In an other clone non-inducible at 37C, the preincubation effect was not observed. The existence of a temperature-dependent cellular factor(s) regulating the induction of cellular DNA synthesis was suggested.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

The chromosomes of a tryptophan(-), thymine(-) double auxotroph of Bacillus subtilis were uniformly aligned at the chromosome terminus by an amino acid starvation treatment. By subsequent incubations, the starved culture was rendered competent, while its state of synchronous chromosome arrest was maintained by thymine starvation. The competent, chromosome-arrested cells were transformed for three unlinked markers, located in two different chromosome regions. Shortly after addition of deoxyribonucleic acid, the cell walls were removed with lysozyme in a medium containing deoxyribonuclease and no thymine, and the protoplasted culture was assayed for single and double transformants. It was found that markers both near and distant from the terminus entered freely into the cell interior. There was no important difference in the relative frequency of entry of different markers between synchronously arrested cells and nonsynchronized control cultures. It is concluded that entry of a given marker into the cell interior can occur even if the replication site of the chromosome is stationary at a location distant from the locus of the resident homolog of the entering marker. A mechanism of donor deoxyribonucleic acid entry involving homology at the replication fork is excluded.     

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Disruption of Adenovirus Type 7 by Lithium Iodide Resulting in the Release of Viral Deoxyribonucleic Acid

Adenovirus type 7 exposed to solutions of LiI was progressively converted into slower sedimenting deoxyribonucleic acid (DNA)-containing particles, and, ultimately, under proper conditions, DNA free or almost free from protein was released from the virus. The degree of viral degradation was dependent on the time of treatment, on the temperature, and on the concentration of the reagent.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Penetration of Host Cell Membranes by Adenovirus 2

Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.     

Involvement of Protein and Ribonucleic Acid in the Association of Deoxyribonucleic Acid with Other Cell Components of Escherichia coli

Cells of Escherichia coli were labeled with precursors of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein, lysed with detergent, and examined by starch-block electrophoresis and CsCl density gradient centrifugation. A large amount of the DNA was seen to remain at positions of low electrophoretic mobility and light density along with tryptophan and arginine-containing proteins and some RNA. Addition of labeled, phenol-extracted DNA to unlabeled cells prior to lysis and electrophoresis showed that only a small amount of the DNA became associated during or after lysis. Sonic treatment of a lysate removed most of the DNA to a position of electrophoretic mobility and density similar to that of free DNA, whereas pronase and ribonuclease released only a part of the DNA. We concluded that binding of DNA to cell membranes or other cell components occurs in the cell prior to lysis and involves protein and probably a specific type of RNA.     

Segregation of Deoxyribonucleic Acid in Bacteria: Association of the Segregating Unit with the Cell Envelope

Cells of the gram-positive organism Lactobacillus acidophilus R-26 were labeled with ³H-thymine to measure the segregation of radioactive deoxyribonucleic acid (DNA) into daugher cells. Such cells were found to contain 8 conserved units of DNA which would correspond to two replicating chromosomes per cell. Fluorescent antibody (FA) against this organism was used to demonstrate that portions of the cell surface (2 to 4 units per cell) were conserved during growth and division. The permanent association of DNA with these conserved cell surface units was measured by combining autoradiography with FA techniques. DNA synthesized immediately before FA labeling was not associated with the fluorescent cell surface, whereas DNA synthesized a generation previously was. The results are consistent with a model in which DNA becomes permanently fixed to the cell surface when it is first used as a template.