Identification of DNA polymerase(s) involved in the repair of viral and cellular DNA in herpes simplex virus type 2-infected cells

VIROLOGY

129,524-528 (1983)

Identification

of DNA Polymerase(s) Involved in the Repair of Viral and Cellular DNA in Herpes Simplex Virus Type 2-Infected Cells

YUKIHIRO NISHIYAMA,’ TATSUYA TSURUMI, HIIZU AOKI, AND KOICHIRO MAENO Laboratory

of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan Received April

26, 1983; accepted June 27, 1983

When human embryonic fibroblasts (HEF) were infected with herpes simplex virus type 2 (HSV-2), replicative viral DNA synthesis and some repair synthesis of cellular DNA were induced at the early stage of infection, but almost all DNA synthesis at the late stage of infection was derived from repair synthesis of cellular and viral DNA (Y. Nishiyama and F. Rapp, Virobgy 110,466-475,198l). In this study, we have assessed the effects of DNA polymerase inhibitors on repair DNA synthesis in HSV-2-infected HEF. Both viral and cellular DNA syntheses during the late stage of infection were extremely resistant to aphidicolin and phosphonoacetic acid but partially sensitive to high concentrations of l-@-D-arabinofuranosylcytosine, while replicative viral DNA synthesis during the early stage of infection was very sensitive to all of those inhibitors. The results suggest that neither HSV-induced DNA polymerase nor cellular DNA polymerase LYwas involved in the repair synthesis of viral and cellular DNA but that cellular DNA polymerase @was.

It seems that repair DNA synthesis in herpes simplex virus (HSV)-infected cells plays important roles in various aspects of virus-cell relationships; HSV and pseudorabies virus DNAs have single-stranded nicks and gaps which are repaired during replication (1-5) and some repair synthesis of host cell DNA, followed by stimulation of semiconservative DNA synthesis, is induced in HSV-2-infected cells under restrictive conditions for virus replication (6). Furthermore, it has been recently shown that partially inactivated HSV is mutagenic for the host cell genome and suggested that the induction of DNA damage after infection with HSV may be involved in the HSV-induced mutagenicity (7). A previous report (8) has shown that infection of human embryonic fibroblasts (HEF) with HSV-2 induces repair synthesis of cellular DNA early in infection and that almost all DNA synthesis during the late stage of infection resulted from repair synthesis of viral and cellular DNA. How1 To whom correspondence should be addressed.

and requests for reprints

0042-6822/83 $3.00 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form resewed.

ever, the enzymological basis of repair DNA synthesis in HSV-2-infected cells remained unclear. In the present study, we attempted the identification of DNA polymerase(s) involved in the repair of viral and cellular DNA. The results strongly suggested that both HSV DNA and host cell DNA were repaired by DNA polymerase B but not HSV-induced DNA polymerase or DNA polymerase (Y. HEF were prepared as described previously (9) and grown in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 100 units/ ml penicillin, and 100 pg/ml streptomycin. HSV-2 strain 186 was obtained from Dr. Fred Rapp, Pennsylvania State University College of Medicine, Pennsylvania, and the virus stock was prepared in HEF by inoculating at a low multiplicity (0.01 PFU/ cell). DNA analysis by CsCl density-gradient equilibrium centrifugation was performed as described previously (8). Permeabilization of cells was performed according to Berger et al. (10) and DNA synthesis in permeable cells was measured at 37” in the reaction mixture containing 524

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35 mM Tris-HCl (pH 8.0), 3 mM MgCla, 80 mMNaC1,3 mM ATP, 0.05 mM dATP, 0.05 mM dCTP, 0.05 mM dGTP, and 0.3 PM rH]dTTP. For measuring acid-insoluble radioactivity, macromolecules were precipitated with trichloroacetic acid and filtered onto Whatman GF/C filter discs for scintillation counting as described previously (8). [methyZ-3H]Thymidine (20 Ci/ mmol) and [methyZ-3H]thymidine 5’-triphosphate were purchased from New England Nuclear Corporation. Dideoxythymidine triphosphate (ddTTP) and ~-B-Darabinofuranosylcytosine (ara-C) were purchased from P-L Biochemicals. Phosphonoacetic acid (PAA) and aphidicolin were from Abbot Laboratories and Wako Pure Chemicals, respectively.

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Aphidicolin, a tetracyclic diterpenoid obtained from Cephalmprwium aphidicola, is known to inhibit selectively HSV-induced DNA polymerase as well as cellular DNA polymerase LYbut not cellular DNA polymerase B and y (11, 12). We used this drug to quantify the DNA synthesis which is independent of HSV-induced DNA polymerase and DNA polymerase (Y.Figure 1 shows the effect of aphidicolin on [3H]thymidine uptake in HSV-2-infected or mockinfected cells. The rate of aphidicolin-resistant DNA synthesis (as measured by incorporation of rH]thymidine during a 30min pulse) in infected cells rapidly increased until 5 hr postinfection (p.i.) and gradually increased through 6-12 hr p.i., while the overall rate of DNA synthesis

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INFECTION

FIG. 1. Effect of aphidicolin on [‘Hlthymidine incorporation in HSV-e-infected and mock-infected cells. Confluent monolayers of HEF were infected with HSV-2 (5 PFU/cell) and incubated with growth medium at 37’. HSV-e-infected (0, 0) or mock-infected (A, A) cells were pulse-labeled for 30 min with 10 &i/ml [sH]thymidine in the presence (0, A) or absence (0, A) of 5 pg/ml aphidicolin at variou,s intervals after infection.

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at 4 hr p.i. and that in mock-infected cells were highly sensitive to this drug. PAA, an inhibitor of HSV-induced DNA polymerase (13-1.9, could not inhibit the DNA synthesis at 18 hr p.i., either. Only high concentrations of ara-C suppressed the late DNA synthesis in infected cells, although it was much more resistant to this drug compared to the early DNA synthesis in infected cells or to that in mock-infected cells. To learn the effects of those inhibitors on viral and cellular DNA synthesis separately, the DNA synthesized at the late stage of infection was analyzed by CsCl density-gradient equilibrium centrifugation. As reported previously (8), two peaks of radioactivity were detected (Fig. 3A); small and large peaks represent repair synthesis of viral and cellular DNA, respectively, because their positions did not shift to much higher densities even when labeled in the presence of 10 pg/ml5-bro-

decreased transiently but increased to preinfection level by 2 hr p.i., reached its maximum level by 4-5 hr p.i., and then dropped by 7-8 hr p.i. At 12 hr p.i., more than 80% of DNA synthesis was resistant to 5 pug/ml aphidicolin, suggesting that most DNA synthesis at the late stage of infection was independent of HSV-induced DNA polymerase and cellular DNA polymerase (Y.Figure 2 shows the sensitivities of the DNA synthesis in HSV-Zinfected or mock-infected cells to PAA and ara-C in addition to aphidicolin. Since it has been shown that approximately 70 to 80% of DNA synthesis between 3 and 5 hr p.i. is derived from replicative viral DNA synthesis and that almost all DNA synthesis after 14 hr p.i. is the repair type (8), we examined the effects of those inhibitors at both early (4 hr p.i.) and late (18 hr p.i.) stages of infection. The DNA synthesis at 18 hr p.i. was completely resistant to 5 pg/ ml aphidicolin, while the DNA synthesis

(A) APHIDICOLIN l

=

(B) PAA

(C) AM-C

l

@q/ml)

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FIG. 2. Sensitivities of DNA synthesis of HSV-e-infected and mock-infected cells to aphidicolin (A), PAA (B), and ara-c (C). Confluent monolayers of HEF were infected with HSV-2 (5 PFU/cell), incubated with growth medium at 3’7, and labeled with 5 &i/ml [sH]thymidine for 30 min from 4-4.5 hr (A) or from 18-18.5 hr (0) after infection in the presence of various concentrations of each inhibitor. Mock-infected cells (0) were labeled from 4-4.5 hr after infection.

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1.14 1.72 "E 1.70 p tn 1.68

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FIG. 3. Effect of aphidicolin,

PAA, and ara-C on viral and cellular DNA synthesis at the late stage of infection. Confluent monolayers of HEF were infected with HSV-2 (5 PFU/cell) and labeled with 10 pCi/ml [‘Hlthymidine between 18 and 19 hr p.i. in the absence (A) or presence of 5 pg/ml aphidicolin (B), 100 pg/ml PAA (C), or 50 PM ara-C (D). Extracted DNA was analyzed by CsCl density-gradient equilibrium centrifugation as described previously (8).

modeoxyuridine and because both radioactive peaks were not suppressed by 5 mM hydroxyurea (data not shown). Neither viral nor cellular DNA synthesis at the late stage of infection was inhibited by 5 pg/ml aphidicolin (Fig. 3B) or 100 pg/ml PAA (Fig. 3C). However, 50 pM ara-C inhibited both viral and cellular DNA synthesis by about 60% (Fig. 3D). These results suggest that an aphidicolin-resistant, PAA-resistant DNA polymerase which was partially sensitive to ara-CTP was involved in the viral and cellular DNA synthesis at the late stage of infection. In further experiments, we used permeable cells (IO, 16) to learn the effects of ddTTP, a

selective inhibitor of DNA polymerase p and y, and N-ethylmaleimide, a sulfhydrylblocking agent, on the late DNA synthesis in infected cells, since both inhibitors could not be incorporated by nonpermeable cells. However, permeabilization of infected cells at 18 hr p.i. predominantly induced aphidicolin-sensitive DNA synthesis. HSV-induced DNA polymerase may play a principal role in the DNA synthesis of the permeable cells since this aphidicolin-sensitive DNA synthesis was considerably resistant to high concentrations of salt. It appears that treatment of cells with hypotonic buffer (permeabilization) changed the mode of DNA synthesis because of the de-

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struction of intracellular compartmentation. Therefore, we failed to assess the effects of these inhibitors on the late DNA synthesis in infected cells. Many investigators have shown that DNA polymerase p is involved in the process of eukaryotic DNA repair (X-19), while the participation of DNA polymerase (Yin repair synthesis is still under debate (,W,21). In HSV-Z-infected cells at the late stage of infection, the DNA synthesis was exclusively performed by a DNA polymerase(s) resistant to aphidicolin and PAA. The DNA synthesis, however, was partially inhibited by high concentrations of ara-C. These results indicate that neither cellular DNA polymerase (Ynor HSV-induced DNA polymerase was involved in the repair synthesis of viral and cellular DNA during the late stage of infection. It is suggested that DNA damage of viral and cellular DNA is repaired by host cell DNA polymerase ,L?, although we can not eliminate the possibility that an unknown P-like DNA polymerase or DNA polymerase y plays a role in the repair in infected cells. The biological import of repair synthesis of viral and cellular DNA in HSV-2-infected cells remains unclear. However, the following involvement of host cell DNA polymerase ,f3would be possible: (1) because of repair of nicks and gaps of the virion DNA, viral DNA replication could be begun, (2) because of repair of HSV-induced DNA damage in neurons, neurons could be recovered from primary infection and harbor the viral genome in the latent stage, and (3) because of the possible error-prone repair (22) of cellular DNA damage induced by HSV under restrictive conditions for virus growth, cells might suffer mutation and morphological transformation. ACKNOWLEDGMENTS We thank E. Iwata and T. Tsuruguchi for their technical assistance. We also thank Dr. S. Yoshida, Kasugai, Japan, for helpful discussion, and Dr. F.

Rapp, Hershey, Pennsylvania, for criticizing the manuscript. This work was supported in part by a Grant-in-Aid for cancer research from the Ministry of Education, Science, and Culture, Japan. REFERENCES 1. KIEFF, E. D., BACHENHEIMER, S. L., and ROIZMAN, B., J. ViroL 8, 125-132 (19’71). 2. WILKIE, N. M., J. Gen. ViroL 21, 453-467 (1973). 8. GORDIN, M., OLSHEVSKY, U., ROSENKRANZ, H. S., and BECKER, Y., Virology 55, 280-284 (19’73). 4. HYMAN, R. W., OAKES, J. E., and KUDLER, L., Virology 76, 286-294 (1977). 5. BEN-P• RAT, T., KAPLAN, A., STEHN, B., and RuBENSTEIN, A., I&o&y 69,547-560 (1976). 6. KUCERA, L. S., and EDWARDS, I., J. ViroL 29, 8390 (1979). 7. SCHLEHOFER, J. R., and ZUR HAUSEN, H., Virology 122,471-475 (1982). 8. NISHIYAMA, Y., and RAPP, F., viro&y 110, 466475 (1981). 9. NISHIYAMA, Y., MAENO, K., and YOSHIDA, S., Virology 124, 221-231 (1983). lo. BERGER, N. A., KUROHARA, K. K., PETZOLD, S. J., and SIKORSKI, G. W., B&hem. Biophys. Res. Commun 89, 218-225 (1979). 11. PEDRALI-NOY, G., and SPADARI, S., J. ViroL 36, 457-464 (1986). 12. IKEGAMI, S., TADUCHI, T., OHASHI, M., OCURO, M., NAGANO, H., and MANO, Y., Nature (Won) 275,458-460 (1978). 1.9. BOLDEN, A., ALJCKER, J., and WEISSBACH, A., J. ViroL 16, 15&1-1592 (1975). 14. HONESS, R. W., and WATSON, D. H., J. ViroL 21, 584-600 (1977). 15. KNOPF, K-W., Eur. J. Biochem. 98,231~244 (1979). 16. SEKI, S., and ODA, T., B&him. Biophys. Acta 606, 246-250 (1980). 17. COETZEE, M. L., CHOU, R., and OVE, P., Cancer Res. 38, 3621-3627 (1978). 18. HOBSCHER, U., KUENZLE, C. C., and SPADARI, S., Proc. Nat. Acuu! Sci. USA 76,2316-2320 (1979). 19. WASER, J., H~BSCHER, U., KUENZLE, C. C., and SPADARI, S., Eur. J. B&hem. 97,361-368 (1979). 20. HANAOKA, F., KATO, H., IKEGAMI, S., OHASHI, M., and YAMADA, M., Biochem. Biophys. Res. Cornmun. 87, 575-580 (1979). 21. SNYDER, R. D., and REGAN, J. D., Biochem. Biophys. Res. Commun 99, 1088-1094 (1981). 22. LOEB, L. A., SPRINGGATE, C. F., and BUTTULA, N., Cancer Res. 34, 2311-2321 (1974).