DNA repair replication in human embryonic lung cells infected with herpes simplex virus

VIROLOGY

82, 401-408

(1977)

DNA Repair Replication

in Human Embryonic Lung Cells Infected Herpes Simplex Virus

ARNIJLF K. LORENTZ,* KLAUS MUNK,*

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GHOLAMREZA

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* Znstitut fiir Virusforschung, Deutsches Krebsforschungszentrum, Zm Neuenheimer Feld 280, 6900 Heidelberg, and tznstitut fiir Medizinische Virologie, Universitit Heidelberg, Zm Neuenheimer, Germany Accepted June 2,1977 Some aspects of repair replication of cellular DNA in herpes simplex virus (HSVlinfected human embryonic lung (HEL) cells were investigated. The buoyant density of the radioactive DNA, after incorporation of radioactive bromodeoxyuridine, was used as the criterion of whether semiconservative or repair replication of the host DNA had occurred. The results showed: HSV-infected cells were able to repair ultra violetdamaged cellular DNA. The HSV-infected cells did not induce repair replication of cellular DNA until 14 hr after infection. Twenty-four hours after infection, however, at least 10 to 15% of the cellular DNA replication was of the repair type. The onset of inhibition of cellular DNA replication, aRer infection with HSV, was not concomitant with the onset of repair replication of cellular DNA. INTRODUCTION

after infection with HSV or after uv irraHerpes simplex virus (HSV) inhibits cel- diation, we used density labeling with brolular DNA synthesis progressively in the modeoxyuridine (BUdR). If repair replicacourse of the infectious cycle (Munk and tion proceeds in the presence of radioactive Sauer, 1963; Roizman and Roane, 1964; BUdR, the BUdR label will appear at the Russell et al., 1964). HSV infection also same density as normal DNA when the induces chromosomal breaks and uncoil- DNA is submitted to equilibrium density ing and fragmentation of chromosomes in centrifugation. The repaired regions are aneuploid and diploid cell lines (Hampar too small to produce a shift in buoyant and Ellison, 1961 and 1963; Stich et al., density (Pettijohn and Hanawalt, 1964; 1964; Rapp and Hsu, 1965). In addition, an Cleaver, 1969). Semiconservative replication, however, produces DNA with a denincrease in DNase activity in HSV-insity considerably greater than that of norfected cells has been reported by various investigators (Keir and Gold, 1963; Mc- mal DNA if a high enough concentration Auslan et al., 1965; Weissbach et al., 1973). of BUdR is used (Simon, 1961). We investigated (1) whether infection by MATERIALS AND METHODS HSV damages cellular DNA in a way that Virus. A large-plaque variant of HSV leads to repair replication (Rasmussen and Painter, 1964 and 1966; Kolber, 1974; type 1, strain ANG (Darai and Munk, Grossman et al., 1975); (2) whether HSV- 1976; Schroder et al., 1976) which had been infected cells are able to repair damaged cloned three times (Munk and Ludwig, DNA at various stages during the infec- 1972)was used for all experiments. A virus tious cycle; and (3) whether the onset of stock containing 6 x 106 plaque-forming inhibition of cellular DNA synthesis is units (PFU)/ml was prepared from HeLa concomitant with the onset of repair repli- cells that had been infected at a multipliccation of cellular DNA. ity of infection (m.0.i.) of 0.1 PFU/cell. In order to assay for repair replication Cells. Human embryonic lung (HEL) cells which were prepared as described 1 Author to whom reprint requests should be adpreviously (Darai and Munk, 1973) were dressed. 401 Copyright 0 1977 by Academic Press, Inc. All rigbta of reproduction in any form reserved.

ISSN

0042-6822

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MUNK,

used. This cell clone showed epithelial morphology and was found to be absolutely not of HeLa cell derivation as checked by Dr. Nelson-Rees (Naval Biosciences Laboratory, University of California, Berkeley). Cells were free of contamination with mycoplasma as proved by an assay using both agar and plates (Flow Laboratories, Bonn, Germany) and by analysis of labeled DNA in CsCl density gradients. Cells were grown in Eagle’s medium, containing a twofold concentration of amino acids and vitamins with 10% calf serum. Cultures in petri dishes were incubated in a 5% CO, atmosphere. Confluent cultures were used and confluence was determined by measuring growth kinetics of parallel cell cultures in each experiment. Buffers and reagents. Phosphatebuffered saline (PBS): 0.12 M NaCl, 0.018 M Na,HPO,, 0.0025 M KHPPOI, pH 7.2. Standard saline-citrate (SSC): 0.15 M NaCl, 0.015 M Na,-citrate, pH 7. RNase from bovine pancreas (Serva, Heidelberg, Germany) was incubated at 80” for 10 min to inactivate traces of DNase. Radiochemicals. 13H]Thymidine (specific activity: 25.6 Ci/mmol), [14Clthymidine (specific activity: 60.5 Ci/mmol), and 13HlBUdR (specific activity: 25.2 Ci/mmol) were obtained from NEN Chemicals, Boston, Mass.). Labeling of DNA. For analysis of total DNA synthesis, DNA was labeled by incubating cells for 1 hr with medium containing dialyzed calf serum and 5 pCi/ml of 13Hlthymidine. For demonstration of repair replication, DNA of cells was labeled by incubating cells with medium containing dialyzed calf serum and 10 pg/ ml of 13HlBUdR (Specific activity: 5 &iIpg) for 2 hr. Growth curves showed that HEL cells grow at a normal rate during the first 48 hr at this concentration, suggesting that semiconservative DNA replication is essentially uninhibited during this time. Ultraviolet-induced repair replication of DNA. To induce repair replication the medium of confluent cultures of HEL cells was removed and cells were irradiated with a uv source (Hanau NN 30/89, Hanau Quarzlampen, Hanau, Germany) at a distance of 50 cm. An irradiation time of 30

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set corresponded to 400 ergs/mm2 as measured by a uv-measuring cell. Analysis of DNA in CsCl density gradients. Cells were washed twice with PBS, scraped into a small volume of PBS, and pelleted by low speed centrifugation. The pellet was resuspended in buffer for extraction of DNA, sodium deoxycholate was added to a final concentration of l%, and the sample was incubated at 37” for 10 min. DNA was extracted by repeated shaking with a chloroform-isoamyl alcohol mixture, RNase treatment (50 pglml) for 1 hr at 37”, and ethanol precipitation as described previously (Darai et al., 1975). DNA was dissolved in 0.1 x SSC. Preparations had a A26,,/A280 ratio of 1.7-1.8. For demonstration of repair replication, 30 pg of DNA were centrifuged to equilibrium in a CsCl density gradient with an average density of 1.739 g/cm3 as described previously (Kieff et al., 1971; Darai et al., 1975). [ 14C]Thymidine-labeled HEL-DNA containing no BUdR was used as a marker. For analysis of total DNA synthesis, 10 pg of DNA were centrifuged to equilibrium in a CsCl density gradient with an average density of 1.707 g/cm3. The DNA in each fraction of the gradients was precipitated by the addition of 8-10 vol of 5% trichloroacetic acid and filtered onto membrane filters of pore size 45 pm (Schleicher and Schtill, Dassel, Germany). Radioactivity on the filters was measured by liquid scintillation counting. RESULTS

The purpose of the first set of experiments was to investigate whether HSV infection leads to repair replication of cellular DNA. Infected and mock-infected cells were labeled with [3HlBUdR at various times after infection and DNA was extracted and centrifuged in CsCl density gradients as described in Materials and Methods. A mock-infected culture irradiated with uv light at a dose of 400 ergs/ mm2 served as a control to demonstrate that it is possible to detect repair replication by the above technique. The results shown in Fig. la illustrate the gradient profile of DNA from a mockinfected culture that had been labeled 1 hr

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FIG. 1. DNA repair replication in HSV-infected HEL cells. Confluent monolayers of HEL cells were mock-infected or infected with a multiplicity of 5 PFLJ/cell immediately after reaching saturation density and were labeled with L3H]BUdR for 2 hr at various times after infection. DNA was extracted and submitted to density gradient centrifugation as described in Materials and Methods. [‘*ClThymidine-labeled cellular DNA of normal density was used as a marker. Density increases from right to left. (a) Mock-infected culture labeled 1 hr after the change of medium. (b) Mock-infected culture irradiated with uv light at a dose of 400 ergs/mm2 1 hr after the change of medium and labeled immediately after irradiation. (c) Infected culture labeled 14 hr postinfection. (d) Infected culture labeled 24 hr postinfection.

aRer the medium change, but had not been irradiated. The DNA synthesized during the labeling period bands primarily at a hybrid density and therefore repre-

sents the result of semiconservative replication. In the gradient profile of DNA extracted from the irradiated culture (Fig. lb), there

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is also a peak of newly synthesized DNA that bands at a hybrid density. In addition, however, there is a peak of radioactive, substituted DNA that bands at a density almost identical to that of DNA containing no BUdR. Hence, using the BUdRlabeling technique it is indeed possible to detect DNA that has undergone repair replication in this system. Figures lc and d illustrates gradient profiles of DNA from infected cultures. In each case the peak of DNA with the greatest buoyant density represents viral DNA. Viral DNA has a density which, when not labeled with BUdR, is about 0.028 g/cm3 greater than that of cellular DNA (Goodheart et al., 1968). Until 14 hr postinfection (p.i.) there is no induction of repair synthesis of cellular DNA (Fig. lc). At 24 hr p.i., however, at least 10 to 15% of the cellular DNA synthesis is of the repair type (Fig. Id). The shoulder of the peak of viral DNA, corresponding to DNA of slightly lower density than viral DNA in Fig. Id, could represent repair replication of viral DNA. In an in vitro system containing nuclei isolated from HSV-infected HEL cells, repair replication of viral DNA has indeed been described (Kolber, 1975). Since radioactivity incorporated into repaired viral DNA will partially superimpose the radioactivity incorporated into substituted cellular DNA, an accurate estimation of the fraction of cellular repair replication to total cellular DNA synthesis is difficult. It might be as much as 50%. The purpose of the second set of experiments was to investigate whether the rate of uv-induced repair replication in HEL cells is altered by infection with HSV. Both infected and uninfected cultures were irradiated with uv light at 2.4 x lo3 ergs/ mm2 at various times after infection and, immediately after uv irradiation, were labeled with 13HlBUdR. The uv dose employed induced a maximum of repair replication when doses from 400 to 4.0 x 103 ergs/mm* were tested (data not shown). DNA was extracted and centrifuged in CsCl density gradients as described. The results with unirradiated, uninfected cells labeled with [3H]BUdR are shown in Fig. la. The results with irradi-

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ated, uninfected cells are shown in Fig. 2. The uv dose (2.4 x 103ergs/mm2) employed can he seen to have induced repair replication and to have inhibited semiconservative replication almost completely (Fig. 2a). DNA from irradiated, infected cells gives the same gradient profile (Fig. 2b). Irradiated, infected cells show essentially the same amount of uv-induced repair replication over a 1-hr period as uninfected cells (Fig. 3a). To determine whether the inhibition of cellular DNA synthesis is concomitant with the appearance of repair replication of cellular DNA the rate of total DNA synthesis was measured at various times after infection. Cells were labeled with [3H]thymidine (5 &i/ml) for 1 hr and the DNA was extracted and banded in CsCl density gradients. The amount of total cellular DNA synthesis was calculated from profiles of density gradients containing 10 pg of DNA, integrating the area under the peak at the density of cellular DNA. Total cellular DNA synthesis in unirradiated, infected cells decreased rapidly after 5 hr p.i. (Fig. 3b) as has been described previously (Munk and Sauer, 1963; Roizman and Roane, 1964; Russell et al., 1964; Kaplan, 1973). This is well before the onset of repair replication. The initial increase in the rate of incorporation of L3Hlthymidine (up to 5 hr after infection) into cellular DNA may be due to an increase in thymidine utilization with constant or declining DNA synthesis. The subsequent decline of the rate of incorporation of L3Hlthymidine into cellular DNA, however, can be interpreted as a decline in DNA synthesis. Thymidine kinase activity does not show a corresponding decrease. The rate of incorporation of 13H]thymidine into cellular DNA is probably not an accurate measure of DNA synthesis, since infection with HSV leads to a loto 20-fold increase in intracellular thymidine kinase activity (Kit et al., 1963; Klemperer et al., 1967; Jamieson and Subak-Sharpe, 1974; Jamieson et al., 1974; Coppey and Nocentini, 1976; Coppey, 1977). The fact that no semiconservative viral DNA replication can be detected in infected, irradiated cells may be due to damage to the viral DNA template or to

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FIG. 2. DNA

repair replication in uv-irradiated, uninfected and infected HEL-cells. (a) Confluent monolayers of HEL cells were irradiated with uv light at a dose of 2.4 x lo3 ergs/mm* and density labeled with 13H1BUdR for 2 hr. DNA was extracted and submitted to density gradient analysis as described in Materials and Methods. (b) Confluent monolayers of HEL cells were infected with a multiplicity of 5 PFU/ cell, irradiated with uv light at a dose of 2.4 x 103 ergs/mm* 12 hr p.i., and density labeled with 13HlBUdR for 2 hr immediately after irradiation. DNA was extracted and submitted to density gradient analysis as described in Materials and Methods.

damage to the cell. It has been shown that viral DNA synthesis in CV-1 cells is reduced if the cells are uv irradiated prior to HSV infection (Coppey and Nocentini, 1976). DISCUSSION

The results of this study show the following. 1. Infection of cells with HSV did not induce repair replication of cellular DNA until 14 hr p.i. However, 24 hr p.i. at least lo-15% of the cellular DNA replication was of the repair type. Damage to chromosomes in HSV-infected cells occurs early in infection (Rapp and Hsu, 1965; Waubke et by al., 1968). The precise mechanism which the virus produces this damage is not understood. Infectious virus is required; virus inactivated by uv light, heat, or antibody does not produce lesions (Tanzer et al ., 1964; Rapp and Hsu, 1965). Viral

DNA synthesis does not seem to be necessary (Rapp and Hsu, 1965; Waubke et al., 19681, suggesting that chromosome damage is the result of an early viral function. In our experiments repair replication of cellular DNA occurred only late, not early, in infection. Ultraviolet-induced repair replication of cellular DNA proceeded at essentially the same rate in infected as in uninfected cells. Repair replication could be demonstrated within 2 hr after irradiation. There was no significant delay between the production of uv-induced damage and its repair. This suggests that chromosome lesions early in infection do not include damage to DNA that can be repaired by the enzyme system that repairs uv-induced damage to DNA. 2. HSV-infected cells were able to repair uv-damaged cellular DNA. The amount of uv-induced repair replication per 1-hr period remained unaltered until 26 hr p.i.

406

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FIG. 3. Semiconservative and uv-induced repair replication of DNA in HSV-infected HEL cells. (a) Confluent monolayers of HEL cells were infected at a multiplicity of 5 PFU/cell. At different times after infection the cells were irradiated with uv light at a dose of 2.4 x lo3 ergs/mm* and subsequently density labeled with [3H]BUdR. DNA was extracted and submitted to density gradient centrifugation as described in Materials and Methods. The radioactivity profile of the gradient was determined and the peak corresponding to cellular DNA of normal density was integrated. (b) Confluent monolayers of HEL cells were infected at a multiplicity of 5 PFU/cell. At different times after infection the cells were labeled with lSH]thymidine. DNA was extracted and submitted to density gradient centrifugation as described in Materials and Methods. The radioactivity profile of the gradient was determined and the peak corresponding to cellular DNA was integrated.

Similar results have been found for Mengo virus and Newcastle disease virus. Infection with both viruses inhibits semiconservative replication of cellular DNA soon aRer infection, whereas repair replication is uninhibited (Hand and Tamm, 1971, 1972a,b). Infection with HSV inhibits cellular protein synthesis (Ben-Porat and Kaplan, 1973). However, protein synthesis is not required for repair replication (Cleaver, 1969; Hand and Tamm, 1971, 1972a,b). The half-life preexisting enzymes for repair replication in uninfected mammalian cells has been shown to be at least 30 hr (Gautschi et al., 1973). Our results suggest that in HSV-infected cells this enzyme system is similarly stable. 3. Between 5 and 14 hr p.i. there was no repair replication of cellular DNA concomitant with the inhibition of semiconservative replication of cellular DNA. No repair replication of cellular DNA occurred in HSV-infected cells at a time when semi-

conservative replication of cellular DNA is progressively inhibited. The enzyme systern for uv-induced repair replication was functioning during this time. Therefore the inhibition of semiconservative replication of cellular DNA cannot be due to damage of this DNA that normally is repaired by the cellular system that repairs uvinduced damage in DNA. There is other evidence that the inhibition of cellular DNA replication is not due to degradation of the DNA. Viral DNA replication is necessary for the induction of chromosomal aberrations (Tanzer et al., 1964; Rapp and Hsu, 1965). Although uv-inactivated HSV does not produce changes in chromosome structure (Tanzer et al., 1964), it is found to inhibit cellular DNA synthesis (Newton, 1968). The inhibition of cellular DNA synthesis in cells infected with pseudorabies virus (a virus closely related to HSV) was interpreted as being due to a specific protein

DNA REPAIR IN HERE ‘ES-INFECTED

synthesized during infection acting in a histone-like fashion (Ben-Porat and Kaplan, 1965). ACKNOWLEDGMENTS We are very grateful to Dr. W. A. Nelson-Rees, Cell Culture Laboratory, University of California, Berkeley, for his thorough genetic analysis of our human embryonic lung cells. We would like to thank Mrs. Christa Kleinicke for excellent technical assistance, Dr. Allan Fried for critical comments, and Dr. Rolf M. Fliigel for helpful discussions.

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