Transcription and Replication of Viral Deoxyribonucleic acid in Cells Coinfected with Adenovirus Types 2 and 12

The yield of infectious virus was determined for KB cells infected with both adenovirus types 2 (ad 2) and 12 (ad 12). It was found that the yield of the former was greatly reduced, whereas that of the latter was not affected significantly. The reduction in virus yield was accompanied by an inhibition of ad 2 virus-specific ribonucleic acid (RNA) and viral deoxyribonucleic acid (DNA) synthesis at various times after infection. On the other hand, the rate of synthesis of ad 12 virus-specific RNA and viral DNA was not inhibited, but advanced in time. The total amount of ad 12 viral DNA synthesized was not affected by coinfection with ad 2. These results suggest that ad 2 infection hastens the maturation of ad 12.     

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 and Transport of Virus-Specific Ribonucleic Acids in African Green Monkey Kidney Cells Abortively Infected with Type 2 Adenovirus

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.     

Mechanism of Viral Carcinogenesis by Deoxyribonucleic Acid Mammalian Viruses IV. Related Virus-specific Ribonucleic Acids in Tumor Cells Induced by “Highly” Oncogenic Adenovirus Types 12, 18, and 31

Formation of hybrids between viral deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) was used to detect virus-specific RNA in the nuclei and polyribosomes of transformed and tumor cells induced by "highly" oncogenic human adenovirus (Ad) types 12, 18, and 31. The presence of virus-specific RNA in the cell nucleus, and the inhibitory effect of actinomycin D on its synthesis, suggest that adenovirus-specific RNA is transcribed from a DNA template in the nucleus. Ad 12, 18, and 31 virus-specific RNA did not hybridize significantly with the DNA of the "weakly" oncogenic adenovirus group (Ad 3, 7, 11, 14, 16, and 21) or with that of nononcogenic Ad 2 and 4. Labeled RNA from Ad 12, 18, and 31 tumor cells hybridized with heterologous Ad 12, 18, and 31 DNA 30 to 60% as efficiently as with homologous DNA. Thus, common viral genes are transcribed in tumor cells induced by Ad 12, 18, and 31.     

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.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

Viral DNA Synthesis In Vitro with the Inclusions Isolated from Adenovirus 12-Infected Cells

A fraction defined as the inclusions was isolated by banding in CsCl gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18 S, 22 S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.     

Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

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.     

Deoxyribonucleic Acid Polymerases of Rous Sarcoma Virus: Kinetics of Deoxyribonucleic Acid Synthesis and Specificity of the Products

The deoxyribonucleic acid (DNA) polymerase(s) of Rous sarcoma virus synthesizes two principal products-single-stranded DNA in the form of a DNA:ribonucleic acid (RNA) hybrid and double-stranded DNA. All of the single-stranded product and 50% of the double-stranded product can be hybridized to 70S viral RNA. These results, in combination with data obtained by analysis of the kinetics of double-stranded DNA synthesis, indicate that the synthesis of double-stranded DNA is a sequel to the synthesis of single-stranded DNA and is dependent upon the latter for the provision of initial template.     

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.     

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.     

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.