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.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 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.     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.     

Characterization of a Temperature-Sensitive Fiber Mutant of Type 5 Adenovirus and Effect of the Mutation on Virion Assembly

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5°C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32°C, fiber polypeptides synthesized at 39.5°C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5°C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32°C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.     

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.     

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.     

Two "early" mRNA species in adenovirus type 2-transformed rat cells

mRNA isolated from adenovirus 2-infected HeLa cells at early times during the productive cycle and from two lines of adenovirus 2-transformed rat embryo cells (F17 and T2C4) was fractionated on sucrose gradients after disaggregation. Viral mRNA species were identified by hybridization across such gradients with the separated strands of restriction endonuclease fragments of 32P-labeled DNA known to be complementary to adeovirus 2 "early" and adenovirus 2-transformed cell mRNA. mRNA transcribed from the left-hand 14% of the adenovirus 2 genome was found to comprise two species, 16 to 17S and 20 to 21S: the same sized mRNA's were present both at early times during productive infection and in the two transformed rat cell lines. Direct comparison of the sequences present in these two mRNA species by additional saturation hybridizations suggests that they are not related to one another. Three additional regions of the adenovirus 2 genome, all of which are located in the right-hand 40% of the adenovirus 2 genome, are complementary to early mRNA sequences: each of these appears to specify one major mRNA species of about 22S. Thus, five major species of adenovirus type 2 early mRNA have been identified. Two of these, copied from the left-hand 14% of the viral genome, are also present in adenovirus 2-transformed rat cells.     

Initiation of Mammalian Viral Protein Synthesis

CULTURED human cells (KB) infected with human adenovirus type 2 (Ad 2) provide a model system for protein synthesis in mammalian cells. Adenovirus messenger RNA molecules are transcribed from nuclear viral DNA and transported to the cytoplasm for translation¹. Late after infection (18 h) 9–10 viral mRNA species with sedimentation values of 7S to 32S are present in polysomes (Parsons, Gardner and Green, in preparation) and specify eight viral structural proteins which account for 80–90% of the polypeptides synthesized in vivo^2–4. We now describe an in vitro cell-free system, derived from KB cells infected with Ad 2, which synthesizes 8–9 viral polypeptides and can initiate protein synthesis with a special class of yeast methionyl-tRNA. In vivo and in vitro experiments suggest that methionine is the initiator amino-acid for most, if not all, adenovirus structural proteins.     

Mitochondrial DNA synthesis in adenovirus type 2-infected HeLa cells.

Mitochondrial DNA synthesis in adenovirus type 2-infected HeLa cells was measured at various times from 0 to 24 h postinfection. Although viral infection effectively turned off host chromosomal DNA synthesis, mitochondrial DNA synthesis was not inhibited. These findings indicate a dissociation between the regulation of host and mitochondrial DNA synthesis after infection with adenovirus type 2.     

Alterations to controls of cellular DNA synthesis by adenovirus infection.

Human adenovirus type 5 and temperature-sensitive mutants ts36, ts37, and ts125 induced cellular DNA synthesis in quiescent rodent cells at both permissive and nonpermissive temperatures. Cellular DNA synthesis induced by adenovirus type 5 or by serum required protein synthesis for both initiation and continuation, whereas viral DNA synthesis was not dependent upon continued protein synthesis once it was initiated. Both cellular and viral DNA replication was induced in adenovirus type 5-infected cells in the presence of dibutyryl cyclic AMP at concentrations which inhibited induction by serum which suggested that some of the controls of DNA synthesis in serum-treated and virus-infected cells are different. After adenovirus infection of quiescent cells, there was a decrease in the number of cells with G1 DNA content and an increase in cells with G2 diploid and greater DNA contents. Thus, adenovirus type 5 induces a complete round of cellular DNA replication, but in some cells, it induces a second round without completion of a normal mitosis. These results suggest that adenovirus type 5 is able to alter cell growth cycle controls in a way which may be related to its ability to transform cells.     

Macromolecular Content of Inclusions Produced by a Canine Adenovirus

Early inclusions induced by a canine adenovirus in a canine cell line, appearing before the formation of infectious virus particles, were purified by differential centrifugation in sucrose followed by CsCl density gradient centrifugation. Chemical analysis of these inclusions revealed that they contained deoxyribonucleic acid (DNA), ribonucleic acid, and protein. On the basis of density gradient centrifugation, the DNA extracted from the inclusions was found to be viral DNA. Electron microscope autoradiography showed that these inclusions were the sites of DNA synthesis. In addition, association of DNA polymerase activity with the inclusions was detected by incorporation of radioactivity from (3)H-thymidine triphosphate into a DNA product. The in vitro product of the enzyme had a density equal to that of viral DNA rather than host DNA. The level of DNA polymerase activity in exponentially growing infected and uninfected whole cells was similar, but in cells in stationary phase the enzyme activity of infected cells was twice that in noninfected cells. Furthermore, nuclei isolated from infected cells showed a fourfold increase in DNA polymerase activity over the noninfected cells.     

Initiation rate of adenovirus DNA synthesis in infected cell

A method was developed to determine the rate of viral DNA synthesis initiation in adenovirus 2-infected cells. The initiation of DNA synthesis appeared as the rate-limiting step for accumulation of viral DNA. The multiplicity of infection slightly influenced the rate of synthesis of viral DNA, and only during the linear phase of viral DNA production. The initiation of DNA-synthesis was found to occur preferentially on newly synthesized DNA molecules. These kinetics data and the effect of novobiocin suggested that binding of viral DNA with some enzymatic complexes favored the replication of a minor, active class of adenovirus DNA molecules.     

Chemical and physical properties of adeno-associated satellite virus DNA produced during coinfection with herpes simplex virus

Infectious DNA from adeno-associated satellite virus (ASV) has been isolated from cells coinfected with a temperature-sensitive mutant of herpes simplex virus (HSV) type 1 in the absence of contaminating HSV DNA. This satellite virus DNA does not appear to differ in its physical, chemical and biological properties from DNA isolated directly from virions or from cells co-infected with adenovirus. The DNA is double-stranded with a buoyant density of 1.718 gm/cm³. It sediments at 16S in both neutral and alkaline sucrose gradients. Single-stranded DNA from alkaline sucrose gradients has a modal length of 1.5 μm and demonstrates evidence of internal redundancies in the electron microscope.     

Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4⁺ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.     

Adenovirus core protein synthesis in the absence of viral DNA synthesis late in infection.

The acid extraction of the adenovirus type 5 core proteins V, VII, and pVII (the precursor to VII) from infected cells and the subsequent electrophoresis on a 15% acrylamide-2.5 M urea-0.9 N acetic acid (pH 2.7) gel, revealed that peptide VII has a similar electrophoretic mobility to that of histone H1. The core proteins, which are coded by late adenovirus mRNA, continued to be synthesized late in infection when viral DNA synthesis was inhibited either by cytosine arabinoside in wild-type infections or by shifting adenovirus H5 ts 125-infected cells to the nonpermissive temperature (40 degree C). Only the initiation, not the continuation, of viral DNA replication was essential for core protein synthesis. The synthesis of viral core proteins continued for over 8 h after the cassation of DNA synthesis. This was in contrast to the rapid shutdown of cellular histone synthesis in the absence of cellular DNA synthesis.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

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 DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.