Vol. 22, No. 2

JOURNAL OF VIROLOGY, May 1977, p. 340-345 Copyright © 1977 American Society for Microbiology

Printed in U.S.A.

Mitochondrial DNA Synthesis in Adenovirus Type 2-Infected HeLa Cells PAUL B. FISHER' AND MARSHALL S. HORWITZ* Departments of Microbiology-Immunology,* Cell Biology, and Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication 17 November 1976

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. An asymmetrical model of bidirectional replication has been proposed for adenovirus DNA (5, 11, 12, 29, 31). This model of displacement replication is similar to models proposed for the replication of mitochondrial DNA (1, 17, 27). Adenovirus and mitochondrial DNA syntheses have also been found to be similar in their resistance to the effect of protein inhibitors (13, 30). Individual molecules of adenovirus DNA are both initiated and completely replicated in the presence of concentrations of cycloheximide, which inhibit up to 97% of HeLa cell protein synthesis (13). Similarly, mitochondrial DNA synthesis in HeLa cells is within 75% of normal for 45 min and approximately 50% of the rates of controls for an additional 3 h after addition of cycloheximide (30). In contrast, HeLa cell chromosomal DNA synthesis is reduced more than 90% within 10 min of the addition of cycloheximide (13). This dissociation between protein synthesis and DNA synthesis demonstrated by both mitochondria and adenovirus is not found in various other DNA viruses infecting eucaryotic cells. Inhibition of protein synthesis in cells infected with polyoma virus (3), simian virus 40 (SV40) (19), rabbit poxvirus (18), or pseudorabies virus (16) results in a rapid cessation of viral DNA synthesis. The effects of three DNA viruses, SV40 (21), polyoma (33), and herpes simplex type 1 (25, 26), on mitochondrial DNA synthesis have been studied. SV40 infection of confluent African green monkey kidney or mouse embryo 3T3 cultures results in a stimulation of both nuclear and mitochondrial DNA synthesis; in contrast, SV40 infection of BSC-1 cells does not stimulate nuclear or mitochondrial DNA syn-

thesis (21). A stimulatory effect on mitochondrial DNA synthesis also results when confluent 3T3 cultures are infected with the DNA tumor virus, polyoma (33). In type 1 herpes simplex-infected HeLa cells, chromosomal DNA synthesis is inhibited during 1 to 5 h postinfection (p.i.), whereas mitochondrial DNA synthesis was stimulated (26). Similarly, a herpes-induced enhancement of DNA synthesis in isolated HeLa mitochondria has been demonstrated (25). Because of the similar mechanism of displacement synthesis and resistance to protein inhibitors during the replication of both adenovirus and mitochondrial DNA, we have investigated the effect of adenovirus type 2 (Ad2) infection on mitochondrial DNA synthesis in HeLa cells.

MATERIALS AND METHODS Cells and virus. The sources of HeLa S3 cells and Ad2 have been previously described (22). Cells were grown in suspension culture with Eagle medium supplemented with 5% fetal calf serum. Cells were infected with purified virion at an input multiplicity of 4,000 particles/cell as previously described (10). Mock-infected cells were similarly washed and concentrated, but no virus was added. Isolation of mitochondria. Mitochondria were isolated from cells that were washed twice in Eagle medium without serum and suspended in hypotonic buffer consisting of 50 mM NaCl, 1.5 mM MgCl2, and 10 mM Tris-hydrochloride, pH 8.0 (9). The cells (5 x 106/ml) were ruptured by homogenization (16 strokes) with a Dounce homogenizer. Approximately 95% of the cells were disrupted, as monitored by phase-contrast microscopy. The cytoplasmic supernate was freed of nuclei and debris by three centrifugations at 2,000 rpm for 5 min in the international PRJ centrifuge. Mitochondria were sedi1 Present address: Institute of Cancer Research, Colummented from the supernate by centrifugation at bia University College of Physicians and Surgeons, New 10,000 rpm for 30 min in the angle 30 rotor of the York, NY 10032. 340

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Ad2 AND MITOCHONDRIAL DNA SYNTHESIS

Spinco ultracentrifuge (Beckman). The crude pellet was washed twice, in one-half the original volume, in 0.25 M sucrose, 1 mM EDTA, and 30 mM Trishydrochloride, pH 7.4 (9). The final mitochondrial pellet was suspended in 1 ml of 0.01 x SSC (1 x SSC is 0.15 M sodium chloride and 0.015 M sodium citrate). The DNA was purified by lysing the mitochondria in sodium dodecyl sulfate, adding Pronase, and extracting the aqueous phase twice with chloroformisoamyl alcohol, as described by Schildkraut and Maio (28). The RNase step included in their procedure (28) was omitted because Hela cell mitochondrial DNA contains a small number of ribonucleotides and is, therefore, nicked by RNase A and T,

(34).

Characterization of mitochondrial DNA. Mitochondrial DNA purified from Ad2-infected cultures or mock-infected HeLa cell controls was analyzed by sedimentation in neutral sucrose (12) and cesium chloride-ethidium bromide (CsCl-EtBr) (24) gradients. For analysis of DNA in neutral sucrose, a 0.5-ml sample of purified mitochondrial DNA was layered on a 16.5-ml gradient containing 5 to 20% neutral sucrose in 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA with a 0.5-ml cushion of 60% sucrose. Samples were centrifuged for 18 h at 24,000 rpm in the SW27.3 rotor of the L350 Beckman ultracentrifuge. Gradients were fractionated by pumping equal portions (approximately 0.6 ml) through a probe placed 1.5 cm above the bottom of the tube. Determination of acid-precipitable radioactivity was performed by methods previously reported (10). For CsCl-EtBr analysis of purified mitochondrial DNA, a 0.75-ml sample was added to 3.2 g of CsCl, 0.2 ml of EtBr solution (10 mg/ml), 4 1Lg of calf thymus DNA, and 2.25 ml of 0.Olx SSC. The refractive index was adjusted to 1.3888 (density, 1.580 g/cm8) with 0.01x SSC. Samples were then centrifuged in a Spinco angle 40 rotor at 36,000 rpm for 40 to 48 h, and 0. 1-ml fractions were collected by puncturing the bottom of the tube. Refractive indexes of every fifth sample were determined, and acid-precipitable material was quantitated. Band sedimentation of DNA on alkaline sucrose gradients. Whole cells were placed on alkaline sucrose gradients and sedimented at 24,000 rpm for 14 h with an SW27.3 rotor, as previously described (10). Gradients were fractionated, and the incorporated radioactivity in the fractions and the sonically treated pellet (2 ml) was quantitated. Under these conditions, cell DNA was pelleted into the cushion, and viral DNA appeared in the gradient. Radioactive labeling of DNA. Marker HeLa mitochondrial DNA was labeled with ['4C]thymidine by growing cells for 3 days in media containing 3 ,uCi of [I4C]thymidine (54 mCi/mmol) per 100 ml. Mitochondrial DNA from mock- and Ad2-infected cells was isolated from 1 x 108 to 1.5 x 108 cells grown for 4 h with 1.5 mCi of [3Hlthymidine (15 Ci/mmol) added at time 0, 4, 8, 12, 16, or 20 h p.i. Ad2-infected cells (1.25 x 106), used for quantitation of viral DNA at various times, were labeled with 25 ,ACi of [3H]thymidine (16 Ci/mmol) for 1 h followed by a 30min chase with unlabeled thymidine (10-s M).

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Reagents. [3H]thymidine (15 and 16 Ci/mmol) and ['4C]thymidine (54 Ci/mmol) were purchased from Schwarz BioResearch Inc.; CsCl (optical grade) was from Harshaw Chemical Co.; and EtBr B-grade was from Calbiochem.

RESULTS Neutral sucrose gradient analysis of mitochondrial DNA synthesis. Supercoiled (form I) and relaxed-circular (form II) mitochondrial DNA, which sediment at 37S and 26S, respectively (14), were separated from mock- and Ad2-infected HeLa cells by centrifugation in a 5 to 20% neutral sucrose gradient (Fig. 1). By using 14C-labeled uninfected cells mixed with 3H-labeled mock- or Ad2-infected cells, it was possible to quantitate mitochondrial DNA synthesis until 12 h p.i. Total mitochondrial DNA synthesis, monitored for 4-h periods, was found to be similar in 4-, 8-, and 12-h Ad2- or mockinfected cultures (Fig. 1 and Table 1). The use of neutral sucrose gradients to monitor mitochondrial DNA synthesis beyond 12 h p.i. was not possible, because large amounts of Ad2 DNA (31S) contaminated the mitochondrial region of gradients. We and others have found that most of the encapsidated adenoviral DNA leaks from the intact nuclei prepared by 0.5% Nonidet P-40 lysis of cells and is found in the cytoplasm (M. S. Horwitz, personal observation; reference 4). Unencapsidated viral DNA remains in the nucleus. Since there is approximately a 6-h lag between the onset of viral DNA synthesis and its encapsidation into virion, we were able to use the sucrose gradient method until 12 h p.i. The DNA in virions, which contaminated the cytoplasm, sedimented with the mitochondria at 10,000 x g for 30 min. Virion could not be separated from mitochondria by sedimentation through a 15 to 30% sucrose gradient at 24,000 rpm for 30 min (Fisher, personal observations). The inability to subsequently separate mitochondrial from adenovirus DNA on sucrose gradients necessitated the use of CsCl-EtBr equilibrium gradients to separate mitochondrial DNA from adenovirus DNA when the cells were infected for more than 12 h. CsCI-EtBr gradient analysis of mitochondrial DNA synthesis. Form I mitochondrial DNA was, therefore, quantitated in cells either mock or Ad2 infected from 4 to 24 h earlier (Fig. 2 and Table 1). Between 0 and 16 h, mitochondrial DNA synthesis in both mock- and Ad2-infected cultures was similar, whereas between 16 to 20 and 20 to 24 h, mitochondrial DNA synthesis in mock-infected cells was approximately twice that found in Ad2-infected cultures. This apparent decrease in mitochon-

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FIG. 1. Neutral sucrose gradient analysis of mitochondrial DNA synthesis in mock-infected and Ad2infected HeLa cells. (A) Mitochondrial DNA was isolated from a mixture of 108 mock-infected HeLa cells labeled with [3H]thymidine between 0 and 4 h and 5 x 107 [14C]thymidine-labeled HeLa cells (see text). A 0.5ml sample ofpurified DNA was run on a 5 to 20% neutral sucrose gradient for 18 h at 24,000 rpm. Gradients were fractionated, and acid-precipitable radioactivity from each fraction was determined. (B) Mitochondrial DNA was similarly isolated from a mixture of 108 Ad2-infected cells labeled with [3HWthymidine between 0 and 4 h and 5 x 107 [14C]thymidine-labeled HeLa cells. The purified DNA was run on neutral sucrose gradients as described above. (The peak of form I mitochondrial DNA is in fraction 8, and form II is in fraction 13. The peak of the Ad2 DNA marker (31S) sedimented in fraction 10. Sedimentation is from right to left in all gradients.) A

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FIG. 2. CsCl-EtBr gradient analysis of mitochondrial DNA synthesis in mock- and Ad2-infected HeLa cells. (A) Mitochondrial DNA was isolated from 108 [3Hthymidine-labeled mock-infected HeLa cells labeled for 4-h periods between 0 and 24 h p.i. The cells were mixed with 5 x 107 [14C]thymidine-labeled HeLa cells before purification of the mitochondria and the DNA. The samples were run on 4-ml CsCl-EtBr gradients for 48 h at 36,000 rpm. One hundred-microliter fractions were collected by puncturing the bottom of the tube, and acid-precipitable material was determined. Refractive indexes of every fifth sample were measured. The sample labeled from 0 to 4 h p.i. is shown. (The peak ofform I mitochondrial DNA is in fraction 10; other linear DNAs band in the upper fractions.) (B) Mitochondrial DNA was isolated from Ad2-infected cells and processed identically to (A). The sample labeled from 0 to 4 h p.i. is shown, and the results from other time points are summarized in Table 1. 342

TABLE 1. Mitochondrial DNA synthesis in Ad2infected HeLa cells

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0.90 0.96 0.98 0.97 12 to 16 0.46 c (0.93) 16 to 20 _ 0.49" (0.96) 20 to 24 a A total of 108 to 1.5 x 108 mock- or Ad2-infected HeLa cells were labeled with 1.5 mCi of [3H]thymidine (15 Ci/mmol) for 4-h periods. b Mitochondrial DNA synthesis was expressed as the ratio of [3H]thymidine-labeled mitochondrial DNA from adenovirus-infected cells divided by [3H]thymidine incorporated into HeLa mitochondrial DNA in uninfected cells. Both numbers were corrected for losses during processing by adding a constant amount of ['4C]thymidine-labeled HeLa cells to each sample. The "4C-labeled mitochondrial DNA served as a reference point for the recovery of purified DNA. Only form I DNA (37S in neutral sucrose or 1.595 g/cm3 in CsCl-EtBr gradients) was used for the calculations. Numbers reflected the average ratios of Ad2-infected to mock-infected cells from two experiments. c These numbers reflect the values obtained when the "control" was mock-infected cells. However, by 16 h after mock infection, the cell number had doubled in the uninfected cell culture but was essentially unchanged in Ad2-infected cells. If the values were either corrected for cell number or compared with cells mock infected for less than 16 h, the values shown in parentheses were obtained. 0 to 4 4 to 8 8 to 12

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drial DNA synthesis in the infected culture appears to result from an increased cell number in the uninfected controls that had divided by 16 to 20 h after mock infection. Due to the feeding schedule of our HeLa cells, they tend to become partially synchronized and divide over a 4- to 5-h period. This results in an abrupt rise in cell number rather than a gradual doubling over 18 to 24 h (Table 1). Thus, the amount of [3H]thymidine-labeled mitochondrial DNA during a 4-h pulse increases at 16 h in uninfected HeLa cells, but remains constant in adenovirus-infected cells. Figure 3 compares the species of DNA synthesized and associated with the mitochondrial pellet in adenovirus-infected and mock-infected HeLa cells from 16 to 20 h p.i. The mitochondrial DNAs from both cells are superimposable (fraction 12), but there is a shift of density in the nonmitochondrial DNA at the tops of these gradients. The density of the DNA from adenovirus-infected cells (fraction 31) is 6 mg/cm3 greater than that of uninfected cells. Thus, the

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FIG. 3. CsCl-EtBr gradient analysis of mitochondrial DNA synthesis in HeLa cells infected for 16 to 20 h. Mitochondrial DNA was isolated from a mixture of 108 Ad2-infected HeLa cells labeled with [3H]thymidine between 16 and 20 h p.i. and mixed with 5 x 107 [14C]thymidine-labeled uninfected HeLa cells. Samples were assayed on CsCl-EtBr gradients as described in Fig. 2 and the text.

greater density of native adenovirus DNA in comparison to HeLa DNA is maintained in the presence of ethidium bromide. Rates of viral, chromosomal, and mitochondrial DNA synthesis. Ad2 DNA can be separated from intact HeLa cell DNA as a consequence of differences in the sizes of these molecules (8). With alkaline sucrose gradients (10), Ad2 DNA is found in the gradient (34S), and HeLa cell DNA is largely found in the pellet (_77 to 80S). The effect of Ad2 infection on host chromosomal and mitochondrial DNA is shown in Fig. 4. Host DNA synthesis was markedly reduced by 10 h p.i. and continued to decline during the course of Ad2 infection, whereas Ad2 DNA was detected at 6 h p.i. and was at a maximum rate at 10 to 14 h p.i. In contrast to these changes, mitochondrial DNA synthesis remained at a constant rate throughout the course (0 to 24 h) of Ad2 infection (Fig. 4 and Table 1). DISCUSSION Mitochondrial DNA replication was investigated in adenovirus-infected cells because of several unusual similarities in the replication

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FIG. 4. Mitochondrial, Ad2, and host chromosomal DNA synthesis in Ad2-infected HeLa cells. Mitochondrial DNA synthesis was determined by both neutral sucrose and CsCl-EtBr gradient analysis between 0 and 24 h p.i. as described in the text and Table 1. Ad2 and total HeLa cell DNA synthesis after infection with Ad2 was determined by alkaline sucrose gradient analysis. 1.25 X 106 Ad2-infected cells at different times p.i. were labeled with 25 uCi [3H]thymidine for 1 h and chased with cold thymidine (10-5 M) for an additional 30 min. Cells were pelleted, suspended, and centrifuged as described in the text. One hundred percent synthesis for mitochondrial DNA refers to the radioactivity of mitochondrial DNA made in uninfected HeLa cells. The amount of radioactivity in HeLa cell chromosomal DNA between 4 and 8 h p.i. was the 100% reference point for both chromosomal and Ad2 DNA synthesized in the infected cells.

of these DNAs. Recently it has been demonstrated that synthesis of the linear adenovirus DNA begins at or near the left end for one strand and the right end for the complementary strand. Replication proceeds in the 5' to 3' direction on each of these strands (12). This mode of replication generates large regions of singlestranded DNA in a displacement reaction very similar to the D-loop displacement shown for mitochondrial DNA (27). In addition, both of these mammalian DNAs are peculiarly resistant to the effect of protein inhibitors, which rapidly shut down the synthesis of most other eucaryotic as well as procaryotic DNAs (13, 30). The inhibition of chromosomal DNA synthe-

sis in adenovirus-infected cells is not well understood (23); however, it may be secondary to the profound shutoff of host protein synthesis. Similar controls do not affect mitochondrial DNA synthesis in adenovirus-infected cells, although mitochondrial DNA synthesis is presumably dependent on enzymes synthesized on cytoplasmic polyribosomes. Perhaps the mitochondrial DNA is resistant to shutoff because each round of replication does not require the synthesis of proteins de novo, or the effect of adenovirus infection on chromosomal DNA may involve the inhibition of selected proteins and leaves the production of factors needed for mitochondrial DNA synthesis intact. Although adenovirus DNA is synthesized in the nucleus and mitochondrial DNA in the cytoplasm, it is possible that these two processes share some replicative enzymes. There is no evidence that adenovirus induces the synthesis of a new DNA polymerase (2, 15). However, the virion does code for its own DNA-binding protein (32). Since adenovirus infection inhibits not only the synthesis of chromosomal DNA but also that of SV40 (13) or vaccinia DNA (6) in cells coinfected with either of these viruses, the maintenance of mitochondrial DNA synthesis at normal levels in adenovirus-infected cells is also unique in this regard. In all of these experiments, the incorporation of [3H]thymidine into an appropriately characterized chromosomal, mitochondrial, or viral DNA is assumed to correlate with the rate of synthesis of that macromolecule. Since adenovirus genes do not code for a viral thymidine kinase (20) and the change in levels of thymidine kinase in exponentially growing uninfected cells varies less than 30% after infection (7), there is probably a good correlation between [3H]thymidine incorporation and DNA synthesis. Since chromosomal, mitochondrial, and viral DNA were determined under identical conditions in the cell, anomalies of labeling of mitochondrial DNA could only arise if it obtained [3H]TTP from a pool unique to the synthesis of that macromolecule. A separate thymidine kinase has been reported for mitochondrial DNA, but its enzymatic activity did not change after adenovirus infection (20). Since adenovirus-infected cells are notorious for the artifactual translocation of macromolecules upon cell fractionation (e.g., virion is isolated in the cytoplasm as mentioned above), we did not attempt to measure intramitochondrial pools of TTP after infection. It is furthermore unlikely that compensating artifacts of labeling in our experiments would combine to yield constant rates of [3H]thymidine incorporation into mitochondrial form I DNA for a 24-h period.

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ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant CA-11502 from the National Cancer Institute and American Cancer Society grant VC-201. M.S.H. is the recipient of a Public Health Service Career Development Award from the National Cancer Institute (1K04 CA35554) and the Irma T. Hirschl Trust Career Scientist Award. LITERATURE CITED 1. Berk, A. J., and D. A. Clayton. 1974. Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence. J. Mol. Biol. 86:801-824. 2. Bolden, A., J. Aucker, and A. Weissbach. 1975. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid. J. Virol. 16:1584-1592. 3. Branton, P. E., W. P. Cheevers, and R. Sheinin. 1970. The effect of cyloheximide on DNA synthesis in cells productively infected with polyoma virus. Virology 42:979-922. 4. Edvardsson, B., E. Everitt, H. Jornvall, L. Prage, and L. Philipson. 1976. Intermediates in adenovirus assembly. J. Virol. 19:533-547. 5. Ellens, D. J., J. S. Sussenbach, and H. S. Janz. 1974. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. 6. Giorno, R., and J. R. Kates. 1971. Mechanism of inhibition of vaccinia virus replication in adenovirus-infected HeLa cells. J. Virol. 7:208-213. 7. Green, M. 1962. Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27: 219-235. 8. Hodge, L. D., and M. D. Scharff. 1969. Effect of adenovirus on host cell DNA synthesis in synchronized cells. Virology 37:554-564. 9. Horak, I., H. G. Coon, and I. B. Dawid. 1974. Interspecific recombination of mitochondrial DNA molecules in hybrid somatic cells. Proc. Natl. Acad. Sci. U.S.A. 71:1828-1832. 10. Horwitz, M. S. 1971. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683. 11. Horwitz, M. S. 1974. Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13:10461054. 12. Horwitz, M. S. 1976. Bidirectional replication of adenovirus type 2 DNA. J. Virol. 18:307-315. 13. Horwitz, M. S., C. Brayton, and S. G. Baum. 1973. Synthesis of type 2 adenovirus DNA in the presence of cycloheximide. J. Virol. 11:544-551. 14. Hudson, B., and J. Vinograd. 1969. Sedimentation velocity properties of complex mitochondrial DNA. Nature (London) 221:332-337. 15. Ito, K., M. Arens, and M. Green. 1975. Isolation of DNA Polymerase -y from an adenovirus 2 DNA replication complex. J. Virol. 15:1507-1510. 16. Kaplan, A. S., and T. Ben-Porat. 1966. The replication of the double-stranded DNA of an animal virus during intracellular multiplication. Int. Congr. Microbiol. Symp. 9:463-482. 17. Kasamatsu, H., D. L. Robberson, and J. Vinograd. 1971. A novel closed-circular mitochondrial DNA

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with properties of a replicating intermediate. Proc. Natl. Acad. Sci. U.S.A. 68:2252-2257. Kates, J. R., and B. R. McAuslan. 1967. Relationship between protein synthesis and viral deoxyribonucleic acid synthesis. J. Virol. 1:110-114. Kit, S., T. Kurimura, R. A. de Torres, and D. R. Dubbs. 1969. Simian virus 40 deoxyribonucleic acid replication. I. Effect of cycloheximide on the replication of SV40 deoxyribonucleic acid in monkey kidney cells and in heterokaryons of SV40-transformed and susceptible cells. J. Virol. 3:25-32. Kit, S., W. C. Leung, G. Jorgensen, D. Trkula, and D. R. Dubbs. 1974. Subcellular localization and properties of thymidine kinase from adenovirus-infected cells. J. Gen. Virol. 24:281-292. Levine, A. J. 1971. Induction of mitochondrial DNA synthesis in monkey cells infected by simian virus 40 and (or) treated with calf serum. Proc. Natl. Acad. Sci. U.S.A. 68:717-720. Maizel, J. V., Jr., D. 0. White, and M. D. Scharff. 1968. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7a, and 12. Virology 36:115-125. Philipson, L., U. Pettersson, and U. Lindberg. 1975. Molecular biology of adenoviruses. In Virology monographs, vol. 14. Springer-Verlag, New York. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. Radsak, K., and M. Albring. 1974. Herpes simplex virus-induced enhancement of mitochondrial DNA synthesis in the absence of virus replication. J. Gen. Virol. 25:457-463. Radsak, K. D., and H. W. Freise. 1972. Stimulation of mitochondrial DNA synthesis in HeLa cells by herpex simplex virus (1). Life Sci. 11:717-724. Robberson, D. L., and D. A. Clayton. 1972. Replication of mitochondrial DNA in mouse L cells and their thymidine kinase-derivatives: displacement replication on a covalently-closed circular template. Proc. Natl. Acad. Sci. U.S.A. 69-.3810-3814. Schildkraut, C. L., and J. J. Maio. 1969. Fractions of HeLa DNA differing in their content of guanine and cytosine. J. Mol. Biol. 46:305-312. Schilling, R., B. Weingirtner, and E.-L. Winnacker. 1975. Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16:767-774. Storrie, B., and G. Attardi. 1972. Expression of the mitochondrial genome in HeLa cells. Xm. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis. J. Mol. Biol. 71:177-199. Tolun, A., and U. Pettersson. 1975. Termination sites for adenovirus type 2 DNA replication. J. Virol. 16:759-766. van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginberg. 1975. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15:348-354. Vesco, C., and C. Basilico. 1971. Induction of mitochondrial DNA synthesis by polyoma virus. Nature (London) 229:336-338. Wong-Staal, F., J. Mendelsohn, and M. Goulian. 1973. Ribonucleotides in closed circular mitochondrial DNA from HeLa cells. Biochem. Biophys. Res. Commun. 53:140-148.

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

Vol. 22, No. 2 JOURNAL OF VIROLOGY, May 1977, p. 340-345 Copyright © 1977 American Society for Microbiology Printed in U.S.A. Mitochondrial DNA Syn...
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