395
Mutation Research, 50 (1978) 395-405 @ Elsevier/North-Holland Biomedical Press
HOST-CELL REACTIVATION OF UV-IRRADIATED AND CHEMICALLYTREATED HERPES SIMPLEX VIRUS-l BY XERODERMA PIGMENTOSUM, XP HETEROZYGOTES AND NORMAL SKIN FIBROBLASTS
CLIFFORD
A. SELSKY
a** and SHELDON
Departments of Microbiology a, Biochemistry School of Medicine, Miami, Fla. (U.S.A.) (Received (Revision (Accepted
16 August 1977) received 25 November 2 December 1977)
GREER
b
b and Oncology b, University of Miami
1977)
Summary The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 pg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.
* Present Health,
address: Department of Physiology, Laboratory of Radiobiology, Harvard School Boston. Mass 02115 (U.S.A.). To whom reprint requests should be addressed.
DMSO, dimethylsulfoxide; HN2. acetylaminofluorene: XP. xeroderma pigmentosum.
Abbreviations:
nitrogen
mustard;
NAcO-AAF,
of Public
N-acetoxy-P-
396 Introduction Host-cell reactivation of ultraviolet-irradiated and chemically treated mammalian viruses has been demonstrated by comparing the inactivation rates of treated virus in excision-proficient normal cells and excision-defective xeroderma pigmentosum cells (XP) [1,8,9,11,12,16,18]. XP cells are presumably deficient in an ultraviolet light-induced cyclobutane pyrimidine dimer repair endonuclease activity [5,22]. This deficiency probably accounts for the increased sensitivity to the lethal action of ultraviolet radiation [4]. This excision defect of XP cells is also correlated with increased sensitivity to the lethal action of certain chemicals t h a t in normal cells induce "long patch repair" which results in the incorporation of 100--300 nucleotides per excised lesion in a mechanism similar to the repair of cyclobutane pyrimidine dimers [19]. Some representative compounds of this type are N-acetoxy-2-acetylaminofluorene [NAcO-AFF] and the polycyclic aromatic hydrocarbon 7-bromomethylbenz[a]anthracene [19]. If XP cells are treated with these agents, they demonstrate a defective excision-repair response relative to the response seen in normal cells treated with the same agents or compared to XP cells treated with chemicals that induce a "short p a t c h " or "X-ray-like repair" [ 19]. If the cellular enzymes that carry out the host-cell reactivation of damaged virus have similar affinities for viral and'cellular DNA lesions, then the sensitivity of a mammalian virus treated with damaging agents, should parallel the sensitivity of cells treated with the same agents. Day [10] has shown a correlation between the reactivation of ultraviolet-irradiated human adenovirus type 2 and the relative a m o u n t of UV-induced unscheduled DNA synthesis [18] in XP cells from different genetic complementation groups. The exception to this correlation are group D XP cells which demonstrate the least a m o u n t of reactivation of UV-treated adenovirus but the highest relative rate of UV-induced UDS. He has also shown that the XP variant, which has normal UV-induced UDS rates but is defective in the kinetics of postreplication repair, shows lower levels of reactivation than do normal cells [11]. In order to further characterize the XP phenotype, we have compared the survival of UV-irradiated herpes simplex virus I strain MP with the survival of N-acetoxy-2-acetylaminofluorene-treated virus in normal human skin fibroblasts, XP cells from the different genetic complementation groups, an XP variant and in two presumed XP heterozygotes. In addition, we have also compared the survival of NAcO-AAF-treated HSV-1 MP with the survival of nitrogen mustard (HN2) treated virus in complementation group D XP cells. Materials and methods The cells utilized in these studies were obtained from the American Type Culture Collection, Rockville, MD, with the exception of two normal human skin fibroblast strains which were kindly supplied by Dr. Theodore Malinin of the Department of Surgery at the University of Miami School of Medicine. Cells were routinely grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% v/v fetal bovine serum obtained either from Grand Island Biological Company or International Scientific Industries,
397 Cary, IL, penicillin 100 units/ml, streptomycin 100 pg/ml, fungizone 0.25 pg/ ml, and sodium bicarbonate 1200 mg/1. Cells were passaged every fifth day and fed on the third day following transfer. Herpes simplex virus type 1 strain MP was obtained from the American Type Culture Collection, Rockville, MD. Stock virus was grown in HEp-2 cells initially infected at a multiplicity of 0.05--0.1. Growth flasks were incubated at 37°C for 72 h. Virus was harvested by rapid freezing and slow thawing of the contents of each vessel three times followed by sonication of the resulting suspension for 60 sec in a Ultramet III sonicator, Buehler Ltd., Evanston, IL. Nitrogen mustard (HN2) was obtained from Aldrich Chemicals, Milwaukee, WI. N-Acetoxy-2-acetylaminofluorene [NAcO-AAF] was kindly supplied by Dr. James Miller of the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI. HN2 solutions were prepared by dissolving the chemical in either unsupplemented growth medium or in calcium--magnesium-free phosphate-buffered saline. HN2 solutions were neutralized by mixing with an excess volume of 10% sodium thiosulfate. NAcO-AAF solutions were prepared by dissolving the chemical in a mixture of e t h a n o l : dimethylsulfoxide (DMSO), 9 : 1. Dilutions were prepared in this mixture prior to addition to any aqueous phase since in the presence of water, this compound has an extremely short half-life hydrolysis. Neutralization of NAcO-AAF solutions and vessels in contact with this p o t e n t carcinogen was accomplished by mixing the solution with concentrated sulfuric acid. HN2 treatment of virus. Stock virus aliquots were prewarmed to 37°C. 0.1 volumes of HN2 yielding the final desired concentration was added to the indicated stock virus aliquot. Virus was treated at 37°C for 16 min. Following treatment at 37°C, an equal volume of 1% sodium thiosulfate was added to each aliquot to neutralize any residual unreacted HN2 remaining in solution. Each treatment tube was then aliquoted in 1.0 ml volume, quick frozen in a dry ice--ethanol bath and stored at --70 ° C. NAcO-AAF treatment of virus. Treatment of virus with NAcO-AAF was accomplished in the same manner as described above for HN2 treatment with the following exceptions. NAcO-AAF solutions were diluted 1 : 100 into virus aliquots yielding the final desired concentration. Following incubation at 37°C for 15 min residual unreacted NAcO-AAF was neutralized by adding an equal volume of fetal bovine serum. Each treatment tube was then aliquoted, frozen and stored as described for HN2 treatment. All treatment procedures were carried out under strict aseptic conditions. Ultraviolet (UV) irradiation of virus. In a number of experiments, the reactivation of UV-inactivated virus was studied. The source of ultraviolet light was an 8-W General Electric germicidal lamp whose major o u t p u t was near 254 nm. The lamp was calibrated with a Blak-Ray Ultraviolet meter, model J-225, Ultraviolet Products Inc., San Gabriel, CA. The height of the lamp was adjusted to give an incidence dose rate of 10 ergs/mm2/sec. Stock virus was diluted i n u n supplemented growth medium and aliquoted into 35-mm sterile tissue culture dishes to a depth of approximately 2 mm. Each dish was centered under the light source and exposed to the radiation for the required a m o u n t of time to deliver the desired dose at the incidence dose described. The virus suspension
398 was continuously agitated during irradiation to ensure maximum exposure of the virus. The irradiated suspension was immediately diluted into unsupplemented growth medium for titration of infectious virus and the rest of the undiluted suspension was quick frozen in a dry ice--ethanol bath and stored at --70 ° C. Inactivation curves and routine titrations of virus stocks were accomplished utilizing a modification of the plaque assay described by Roizman and Roane [21] for herpes simplex virus. 0.1 ml of a serial dilution of the virus was innoculated in triplicate onto newly confluent monolayers of the cells being studied for virus reactivation. Cell monolayers were prepared by passing the cells at a ratio of 1 : 2 in supplemented growth medium containing 3700 mg/1 sodium bicarbonate into Linbro multidish disposo trays, FB-16-24-TC, Linbro Scientific Co., New Haven, CT. The monolayers were grown to confluency before using them for titration. Virus was adsorbed to the cells for 2 h at room temperature with intermittant rocking of the dishes to aid in the dispersion of the virus suspension over the entire monolayer. Following adsorption, the monolayers were overlayed with 2 ml of assay medium. Assay medium consisted of growth medium supplemented with fetal bovine serum 1% v/v, antibiotics as described previously and pooled human immune serum globulin 0.03% w/v obtained from Cutter Laboratories, Berkeley, CA, or from Merck, Sharpe and Dohme, Merck and Co., West Point, PA. The immune serum globulin was included in the medium to inhibit secondary plaque formation. Assay dishes were incubated at 37°C in a humidified atmosphere of 10% CO2 and 90% air for 72 h. The medium was then poured off the cells and each dish was rinsed with 0.9% w/v NaC1 prior to staining of the monolayers with crystal violet. Virus plaques were counted with the aid of a dissecting microscope. In all cases, the number of plaque-forming units/ml represents the mean number of plaques of triplicate determinations multiplied by the dilution factor. Each experiment was repeated three times. The figures represent one of the triplicate experiments performed. In all cases, the triplicate experiments showed qualitatively similar results. In a number of experiments the effect of caffeine on the survival of treated virus was studied. Caffeine obtained from Sigma Chemical Company was prepared as a sterile 2% w/v solution. Aliquots of this stock solution were added to the assay medium to yield the desired final concentration of caffeine. In these experiments, the caffeine was present during the entire post-infection assay incubation. Results
Survival of UV-irradiated HSV-1 MP. The survival of UV-irradiated virus in xeroderma pigmentosum skin fibrobla~ts from complementation groups B, C and D and in an XP variant relative to the survival of virus in normal skin fibroblasts is shown in Fig. 1. The relative ability of XP cells from the different complementation groups to reactivate UV-irradiated virus was found to be D < C B with the XP variant demonstrating virus survival on the order of that seen in one of the two normal fibroblasts utilized in this study. Survival o f NAcO-AAF-treated HSV-1 MP. NAcO-AAF-treated virus was
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Fig. 2. Survival o f H S V - 1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f N A c O - A A F for 1 5 rain at 3 7 ° C . V i r u s survival w a s a s s a y e d in t h e f o l l o w i n g cells: C R L 1 1 2 1 (m), n o r m a l ; C R L 1 2 2 4 (e), n o r m a l ; C R L 1 1 6 2 ( × ) , XP variant; C R L 1 2 2 3 (o), XP c o m p l e m e n t a t i o n g r o u p A ; C R L 1 1 9 9 (•), XP c o m p l e m e n t a t i o n g r o u p B; C R L 1 1 6 6 (D), XP c o m p l e m e n t a t i o n g r o u p C; C R L 1 1 5 7 (A), X P c o m p l e m e n t a t i o n g r o u p D.
assayed for survival in XP skin fibroblasts from complementation groups A--D, in an XP variant strain and in two normal skin fibroblast strains. The inactivation curves for treated virus in these cells is shown in Fig. 2. The relative reactivation ability of these cells for NAcO-AAF-treated virus is D ~ A < B < C. As seen for UV-irradiated virus survival, the survival of NAcO-AAF-treated HSV-1 MP in the XP variant is on the order of the survival seen for virus treated with this chemical in the normal human skin fibroblasts. Survival of UV-irradiated and NAcO-AAF-treated HSV-1 MP in the presence of caffeine. Caffeine has been shown to potentiate the lethal action of alkylating agents and UV light in rodent cells [24]. It has been proposed that this potentiation of lethality is a result of the inhibitory effect of caffeine on the postreplication-repair function of these cells [13,14]. Caffeine, however, has been shown to have no effect on the postreplication-repair ability of human cells except for the XP variant cell strains which probably owe their inherent UV sensitivity to a defective postreplication-repair function [ 13,15 ]. Caffeine in the post-infection assay medium at concentrations of 2 and 4 mM had no effect on the inactivation kinetics of NAcO-AAF-treated HSV-1 MP in a normal and three XP cell strains from different genetic complementation groups (data not shown). Fig. 3 shows the effect of 4 mM caffeine in the postinfection assay medium on the survival of both UV-irradiated and NAcO-AAFtreated virus in the XP variant cell strain. For either type of inactivating treatment, caffeine had no significant effect on the inactivation kinetics of the treated virus.
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Fig. 3a. S u r v i v a l o f N A c O - A A F t r e a t e d H S V - 1 MP in C R L 1 1 6 2 X P v a r i a n t s k i n f i b r o b l a s t s . V i r u s t r e a t e d f o r 15 rain at 3 7 ° C w i t h 25 # g / m l a n d 50 /~g/ml. N A c O - A A F w a s a s s a y e d in t h e p r e s e n c e C~) a n d a b s e n c e (e) of 4 mM caffeine. Fig. 3b. S u r v i v a l o f U V - i r r a d i a t e d H S V - 1 MP in C R L 1 1 6 2 XP v a r i a n t s k i n f i b r o b l a s t s . V i r u s s u r v i v a l a s s a y e d in t h e p r e s e n c e ( e ) a n d a b s e n c e ( o ) o f 4 m M c a f f e i n e .
Survival o f NAcO-AAF-treated HSV-1 MP in two X P cell strains and their filial heterozygotes. Fig. 4a demonstrates the survival o f virus treated with up to 100 pg N A c O - A A F / m l in a group A and a group B XP cell strain. It is evident that relative to the normal skin fibroblast cell strains, there is deficient reactivation of the treated virus in the XP cell strains. Fig. 4b demonstrates the survival of the same virus in two normal skin fibroblast cell strains and in two strains which are presumed heterozygotes for xeroderma pigmentosum. These strains were derived from the mothers of the XP patients whose cells were utilized as viral hosts in 4a. The inactivation kinetics of the treated virus in the XP heterozygotes are the same as that seen for survival of the virus in the normal cell strains.
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Fig. 4a. S u r v i v a l o f H S V - 1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f N A c O - A A F f o r 15 m i n at 3 7 ° C in n o r m a l and x e r o d e r m a P i g m e n t o s u m skin fibroblasts: C R L 1222 ($), normal; C R L 1121 (i), normal; C R L 1199 (o), XP e o m p l e m e n t a t i o n g r o u p B; C R L 1 2 2 3 (~), X P e o m p l e m e n t a t i o n g r o u p A. Fig. 4b. S u r v i v a l o f N A e O - A A F t r e a t e d H S V - 1 MP in t w o n o r m a l a n d t w o p r e s u m e d X P h e t e r o z y g o u s s k i n f i b r o b l a s t cell strains. T r e a t m e n t w a s as d e s c r i b e d f o r Fig. 4 a : C R L 1 2 2 2 ( e ) , n o r m a l ; C R L 1 1 2 1 ( i ) , n o r m a l ; C R L 1 1 9 8 (o), X P h e t e r o x y g o t e - m o t h e r o f XP p a t i e n t Po Co; C R L 1 2 5 4 (~), XP h e t e r o z y g o t e - m o t h e r o f XP p a t i e n t C R L 1 2 2 3 .
401
Survival of UV-irradiated, NAcO-AAF-treated and HN2-treated HSV-1 MP. Survival of virus treated with UV light, the UV-like chemical NAcO-AAF or the classical alkylating agent HN2 was assayed in normal fibroblasts and in xeroderma pigmentosum genetic complementation group D cells, which demonstrate the least amount of reactivation of UV-irradiated adenovirus and UVirradiated or NAcO-AAF-treated HSV-1 MP. Both UV treatment and reaction of the virus with NAcO-AAF results in lower virus survival in the XP cells relative to the inactivation kinetics seen in the normal cells, (Figs. 5a, 5b). In contrast, treatment of the virus with HN2 results in equivalent survival of the viruS in both the XP genetic complementation group D cells and the normal skin fibroblasts (Fig. 5c). It should be noted that there is a difference in the survival of UV-treated virus between the normal fibroblasts used in the experiment described in Fig. 1 and the normal fibroblasts utilized in the experiment described in Fig. 5a. A similar difference in survival of NAcO-AAF-treated virus on these two normal strains was also observed.
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Fig. 5b. Survival o f HSV-1 MP t r e a t e d w i t h N A c O - A A F in n o r m a l ( e ) a n d C R L 1 2 0 0 (o) XP e o m p l e m e n t a t i o n g r o u p D skin f i b r o b l a s t s . Fig. 5c. Survival o f HSV-1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f H N 2 for 15 rain at 3 7 ° C in n o r m a l ( e ) a n d C R L 1 2 0 0 (o) X P c o m p l e m e n t a t i o n g r o u p D skin fibroblasts.
402 Discussion Treatment of HSV-1 MP with the ultimate carcinogen and UV-like chemical NAcO-AAF results in lower survival of the virus in XP cells from the genetic complementation groups than in normal cells. The XP variant studied reactivated NAcO-AAF-treated virus to the same extent as the normal cells utilized for comparison. NAcO-AAF-treated virus survival in XP cells from the different genetic complementation groups is qualitatively similar to survival seen for UV-irradiated herpes virus. For virus treated with either agent, survival in the XP variant was similar to survival in normal fibroblasts. The similar relative order of the reactivation of UV and NAcO-AAF-treated virus in the various XP cells indicate that the reactivation of virus damaged by either agent is accomplished by the same repair pathway or pathways with common intermediate steps. Caffeine had no effect on the inactivation kinetics of NAcO-AAF-treated or UV-irradiated virus in the XP variant. This finding will be discussed subsequently in relation to the findings of Day. The relative ability of xeroderma pigmentosum cells from the different genetic complementation groups to reactivate UV-irradiated herpes simplex virus type 1 strain MP was found to be qualitatively similar to that of the hostcell reactivation of UV-inactivated human adenovirus type 2, except for our finding that group C cells were slightly lower in reactivation ability than group B cells [10]. Day showed group B cells to have a lower reactivation ability for UVirradiated Adeno 2 than group C cells [10]. Our results correlate with the findings of Abrahams and Van der Eb, that group B cells reactivated UV-irradiated SV40 DNA better than group C cells [2]. This relationship did not correlate for the XP variant cell strain studied. We f o u n d no detectable difference in the ability of the XP variant and normal skin fibroblasts to reactivate UV-damaged herpes virus. Day demonstrated that this variant was defective in the repair of ultraviolet-irradiated adenovirus. Abrahams and Van der Eb also demonstrated defective reactivation of UVirradiated SV40 DNA in an XP variant [2]. Such inherent differences in the reactivation of these viruses in the XP variant could be the result of a number of mechanisms: (1) a difference in the number of repairable lesions in the viral genome might result from differences in the chemical content and physical structure of the viral DNA molecules; (2) the repair of similar lesions in the two viruses may be accomplished by different mechanisms one of which is defective in the XP variant, or (3) the replication of the viruses in the XP variant is sufficiently different to account for the observed differences in survival. The first possibility was considered the least likely since the survival of both UV-irradiated herpes and adenovirus was qualitatively similar in cells derived from the classical XP patients. The second and third possibilities seemed more likely since it has been shown that the XP variant is defective in the kinetics of postreplication repair. The XP variant cells so far studied have a caffeine-sensitive repair function and caffeine has been shown to potentiate the lethality of a number of DNA-damaging agents in rodent cells which in culture rely primarily on postreplication repair to reverse UV-induced DNA damage [23]. The presence of caffeine in the postinfection assay medium should potentiate the
403 lethality of UV-irradiated herpes virus if it were repaired to a measurable extent by caffeine-sensitive postreplication repair. Caffeine had no effect on the kinetics of inactivation of UV-irradiated herpes virus in the XP variant. This result supports the idea that UV-irradiated herpes virus is reactivated to little if any extent by a caffeine-sensitive mechanism. This premise is supported by the unpublished findings of Lytle, who also observed no difference between the survival of UV-irradiated herpes simplex virus strain MP in XP variants and normal human fibroblasts. In addition, the results of the correlative studies by Takebe et al. [23] also support this contention. These workers have shown that the survival of UV-irradiated herpes virus in mouse L cells, which rely primarily on postreplication repair, was on the same order as that seen for survival in classical XP cells, even though the colony-forming ability of UV-treated L cells was the same as for UV-irradiated normal human cells. Together these results support the implication that UV-irradiated herpes virus is reactivated by a caffeineinsensitive repair process while UV-irradiated adenovirus is reactivated by a combination of caffeine sensitive and insensitive repair functions. Caffeine-sensitive postreplication repair is presumably mediated by a hostDNA polymerase. Perhaps this cellular enzyme has low affinity for herpes virus DNA, resulting in negligible host-mediated postreplication repair for damaged HSV DNA. A herpes-encoded DNA polymerase may play a role in reactivation of UV or NAcO-AAF-treated virus by a caffeine-insensitive mechanism that could account for the equivalent survival of damaged herpes virus in the two normal and the xeroderma pigmentosum variant cell strains studied. Day has found that caffeine in his system potentiates the lethality of UV-treated adenovirus in the XP variant [7]. Even though this result supports the above hypothesis, it should be noted that he also found that caffeine potentiated the lethality of this virus in normal human cells b u t not in cells derived from patients suffering from classical xeroderma pigmentosum. Arlett et al. [ 3], however, has found that caffeine potentiates the lethal action of UV light on XP variant cells or cells from XP complementation group A b u t not in normal human cells. In any event, the physical characterization of the reactivation process for inactivated herpes virus would allow for the correlation of the specific repair functions with a biological endpoint; virus survival. When the survival of HN2-treated virus in XP cells from genetic complementation group D was compared to survival in normal human skin fibroblasts, no difference in the inactivation kinetics was observed. The lack of differential survival indicates that either HN2-treated virus is not repaired in human cells or the repair defect which confers sensitivity of XP cells to ultraviolet radiation or "UV"-like chemicals play little if any role in the recovery of nitrogen mustard-treated virus. Since mustard adducts have been shown to be excised from the DNA of human cells [6,20], it is not likely that nitrogen mustard-treated HSV-1 MP is reactivated to the same extent in both group D XP cells and normal human-skin fibroblasts. This may be a consequence of the introduction of nonenzymatic single-strand breaks by mustard treatment which could compensate for defective endonuclease activity. At the level of damage induced in these experiments by the treatment o f virus with NAcO-AAF, there was no apparent defect in the ability of XP heterozygotes to reactivate the infecting virus. This result does n o t necessarily
404 indicate that XP heterozygotes are normal for the reactivation of NAcO-AAFdamaged HSV-1 MP. It is possible that the level of damage in the viral genome in these experiments was not sufficient to detect a possible rate-limiting reduction of repair-enzyme levels in the heterozygotes studied. The correlation between the reactivation of UV-irradiated virus and NAcOAAF-treated virus indicates that herpes virus inactivated by either of these agents is a good system for detecting repair defects in human cells. The inherent difference in the reactivation of herpes virus, adenovirus and SV40 virus may indicate that they may be specific probes for studying different cellular DNA-repair processes.
Acknowledgements This work was supported by P.H.S. grant CA14395 from the National Cancer Institute to the Comprehensive Cancer Center for the State of Florida at the University of Miami. The authors gratefully acknowledge the advice and criticisms of Dr. Kenneth Kraemer during the course of this work and also the helpful discussions with Kenneth Carlson and Michael Dobersen.
References 1 A a r o n s o n , S,A., a n d L y t l e , C.D., D e c r e a s e d h o s t cell r e a c t i v a t i o n of i r r a d i a t e d S V 4 0 virus in xerod e r m a p i g m e n t o s u m , Nature (London), 228 (1970) 359. 2 A b r a h a m s , P.J., a n d A.J. v a n d e r E b , Host-cell r e a c t i v a t i o n o f u l t r a v i o l e t - I r r a d i a t e d S V 4 0 D N A in five c o m p l e m e n t a t i o n g r o u p s of x e r o d e r m a p i g m e n t o s u m , M u t a t i o n Res. 3 5 ( 1 9 7 6 ) 1 3 - - 2 2 . 3 A r l e t t , C.F., S.A., H a r c o u r t a n d B.C. B r o u g h t o n , T h e i n f l u e n c e o f c a f f e i n e on cell-survival in excisionp r o f i c i e n t a n d e x c i s i o n - d e f i c i e n t x e r o d e r m a p i g m e n t o s u m cells a n d n o r m a l h u m a n cell strains following u l t r a v i o l e t - l i g h t i r r a d i a t i o n , M u t a t i o n Res., 33 ( 1 9 7 5 ) 3 4 1 - - 3 4 6 . 4 Cleaver, J.E., D N A r e p a i r a n d r a d i a t i o n s e n s i t i v i t y in h u m a n ( x e r o d e r m a p i g m e n t o s u m ) cells, Int. J. R a d i a t . Biol., 18 ( 1 9 7 0 ) 5 5 7 - - 5 6 5 , 5 Cleaver, J . E . , X e r o d e r m a p i g m e n t o s u m : a h u m a n disease in w h i c h an initial stage of D N A r e p a i r is d e f e c t i v e , Proc. Natl. A c a d . Sci. ( U . S . A . ) , 63 ( 1 9 6 9 ) 4 2 8 - - 4 3 5 . 6 C r a t h o n , A . R . , a n d J,J, R o b e r t s , M e c h a n i s m of the c y t o t o x i c a c t i o n or a l k y l a t i n g a g e n t s in m a m m a han cells a n d e v i d e n c e f o r t h e r e m o v a l o f a l k y l a t e d g r o u p s f r o m d e o x y r i b o n u c l e i c acid, N a t u r e ( L o n d o n ) , 211 ( 1 9 6 6 ) 1 5 0 - - 1 5 3 . 7 D a y I I I , R.S., Caffeine i n h i b i t i o n of t h e r e p a i r of u l t r a v i o l e t - i r r a d i a t e d a d e n o v i r u s in h u m a n cells, M u t a t i o n Res., 33 ( 1 9 7 5 ) 3 2 1 - - 3 2 6 . 8 D a y I I I , R.S., Cellular r e a c t i v a t i o n o f u l t r a v i o l e t - i r r a d i a t e d h u m a n a d e n o v i r u s 2 in n o r m a l a n d x e r o d e r m a p i g m e n t o s u m f i b r o b l a s t s , P h o t o c h e m . P h o t o b i o l , , 19 ( 1 9 7 4 ) 9 - - 1 3 . 9 D a y I I I , R.S,, H u m a n cells r e p a i r D N A d a m a g e d b y n i t r o u s acid, M u t a t i o n Res., 27 ( 1 9 7 5 ) 4 0 7 - - 4 0 9 . 10 D a y I I I , R.S., Studies o n r e p a i r of a d e n o v i r u s 2 b y h u m a n f i b r o b l a s t s using n o r m a l , x e r o d e r m a p i g m e n t o s u m a n d x e r o d e r m a p i g m e n t o s u m h e t e r o z y g o u s strains, C a n c e r Res., 34 ( 1 9 6 5 - - 1 9 7 0 . 11 D a y I I I , R.S., X e r o d e r m a p i g m e n t o s u m v a r i a n t s h a v e d e c r e a s e d r e p a i r of u l t r a v i o l e t - d a m a g e d D N A , N a t u r e ( L o n d o n ) , 253 ( 1 9 7 5 ) 7 4 8 - - 7 4 9 . 12 D a y I I I , R.S., A.S. G i u f f r i d a a n d C.W. D i n g m a n , R e p a i r b y h u m a n cells of a d e n o v i r u s 2 d a m a g e d b y p s o r a l e n plus n e a r u l t r a v i o l e t light t r e a t m e n t , M u t a t i o n Res., 33 ( 1 9 7 5 ) 3 1 1 - - 3 2 0 . 13 L e h m a n n , A . R . , E f f e c t s o f c a f f e i n e o n D N A s y n t h e s i s in m a m m a l i a n cells, B i o p h y s . J., 12 ( 1 9 7 2 ) 1316. 14 L e h m a n n , A . R . , a n d S. Kirk-Bell, E f f e c t s o f c a f f e i n e a n d t h e o p h y l l i n e on D N A S y n t h e s i s in unirrad i a t e d a n d U V - i r r a d i a t e d m a m m a h a n cells, M u t a t i o n Res., 26 ( 1 9 7 4 ) 7 3 - - 8 2 . 15 L e h m a n n , A . R . , S. Kirk-Bell, C.F. A r l e t t , M.C. P a t e r s o n , P.H.M. L o h m a n , E.A. de W e e r d - K a s t e l e i n a n d D. B o o t s m a , X e r o d e r m a p i g m e n t o s u m cells w i t h n o r m a l levels of excision r e p a i r have a d e f e c t in D N A s y n t h e s i s a f t e r U V - i r r a d i a t i o n , Proc. Natl. A c a d . Sci. ( U . S . A . ) , 72 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 . 16 L y t l e , C.D., S.A. A a r o n s o n a n d E. H a r v e y , Host-cell r e a c t i v a t i o n in m a m m a l i a n cells, II. Survival of h e r p e s s i m p l e x virus a n d vaecinia virus in n o r m a l h u m a n a n d x e r o d e r m a p i g m e n t o s u m cells, Int. J. R a d i a t . Biol., 22 ( 1 9 7 2 ) 1 5 9 - - 1 6 5 .
405 17 R a b s o n , A.S., S.A. T y r t e l l a n d F. Legallaes, G r o w t h o f u l t r a v i o l e t - d a m a g e d h e r p e s virus in x e r o d e r m a p i g m e n t o s u m , P r o c . S o c . E x p t l . Biol. M e d . , 1 3 2 ( 1 9 6 9 ) 8 0 2 . 18 Rasmussen, R.E., and R.B. Painter, Evidence for repair of ultra-violet damaged deoxyribonucleic acid in c u l t u r e d m a m m a l i a n cells, N a t u r e ( L o n d o n ) , 2 0 3 ( 1 9 6 4 ) 1 3 6 0 - - 1 3 6 2 . 19 R e a g a n , J . D . , a n d R . B . S e t l o w , T w o f o r m s o f r e p a i r in t h e D N A o f h u m a n cells d a m a g e d b y c h e m i c a l c a r c i n o g e n s a n d m u t a g e n s , C a n c e r Res., 3 4 ( 1 9 7 4 ) 3 3 1 8 - - 3 3 2 5 . 2 0 R e i d , B.D., a n d I.G. W a l k e r , T h e r e s p o n s e o f m a m m a l i a n cells t o a l k y l a t i n g a g e n t s , O n t h e m e c h a n i s m o f t h e r e m o v a l of s u l f u r - m u s t a r d - i n d u c e d c r o s s l i n k s , B i o c h i m . B i o p h y s . A c t a , 1 7 9 ( 1 9 6 9 ) 1 7 9 - - 1 8 8 . 21 R o i z m a n , B., a n d P.R. R o a n e , A p h y s i c a l d i f f e r e n c e b e t w e e n t w o s t r a i n s o f h e r p e s s i m p l e x v i r u s a p p a r e n t o n s e d i m e n t a t i o n in c a e s i u m c h l o r i d e , V i r o l o g y , 1 5 ( 1 9 6 1 ) 75. 2 2 S e t l o w , R . B . , J . D . R e a g a n , J. G e r m a n , a n d W.L. C a r r i e r , E v i d e n c e t h a t x e r o d e r m a p i g m e n t o s u m cells d o n o t p e r f o r m t h e first s t e p in t h e r e p a i r o f u l t r a v i o l e t - d a m a g e t o t h e i r D N A , P r o c . Natl. A c a d . Sci. (U.S.A.), 64 (1969) 1035--1041. 2 3 T a k e b e , J., S. Nil, M.D. Ishii a n d H. U t s u m i , C o m p a r a t i v e s t u d i e s o f h o s t - c e l l r e a c t i v a t i o n , c o l o n y forming ability and excision repair after UV-irradiation of xeroderma pigmentosum, normal human a n d s o m e o t h e r m a m m a l i a n cells, M u t a t i o n R e s . , 2 5 ( 1 9 7 4 ) 3 8 3 - - 3 9 0 . 2 4 W a l k e r , I.G., a n d B.D. R e i d , C a f f e i n e p o t e n t i a t i o n o f t h e l e t h a l a c t i o n o f a l k y l a t i n g a g e n t s o n L-cells, M u t a t i o n Res., 1 2 ( 1 9 7 1 ) 1 0 1 - - 1 0 4 .
Mutation Research, 50 (1978) 395-405 @ Elsevier/North-Holland Biomedical Press
HOST-CELL REACTIVATION OF UV-IRRADIATED AND CHEMICALLYTREATED HERPES SIMPLEX VIRUS-l BY XERODERMA PIGMENTOSUM, XP HETEROZYGOTES AND NORMAL SKIN FIBROBLASTS
CLIFFORD
A. SELSKY
a** and SHELDON
Departments of Microbiology a, Biochemistry School of Medicine, Miami, Fla. (U.S.A.) (Received (Revision (Accepted
16 August 1977) received 25 November 2 December 1977)
GREER
b
b and Oncology b, University of Miami
1977)
Summary The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 pg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.
* Present Health,
address: Department of Physiology, Laboratory of Radiobiology, Harvard School Boston. Mass 02115 (U.S.A.). To whom reprint requests should be addressed.
DMSO, dimethylsulfoxide; HN2. acetylaminofluorene: XP. xeroderma pigmentosum.
Abbreviations:
nitrogen
mustard;
NAcO-AAF,
of Public
N-acetoxy-P-
396 Introduction Host-cell reactivation of ultraviolet-irradiated and chemically treated mammalian viruses has been demonstrated by comparing the inactivation rates of treated virus in excision-proficient normal cells and excision-defective xeroderma pigmentosum cells (XP) [1,8,9,11,12,16,18]. XP cells are presumably deficient in an ultraviolet light-induced cyclobutane pyrimidine dimer repair endonuclease activity [5,22]. This deficiency probably accounts for the increased sensitivity to the lethal action of ultraviolet radiation [4]. This excision defect of XP cells is also correlated with increased sensitivity to the lethal action of certain chemicals t h a t in normal cells induce "long patch repair" which results in the incorporation of 100--300 nucleotides per excised lesion in a mechanism similar to the repair of cyclobutane pyrimidine dimers [19]. Some representative compounds of this type are N-acetoxy-2-acetylaminofluorene [NAcO-AFF] and the polycyclic aromatic hydrocarbon 7-bromomethylbenz[a]anthracene [19]. If XP cells are treated with these agents, they demonstrate a defective excision-repair response relative to the response seen in normal cells treated with the same agents or compared to XP cells treated with chemicals that induce a "short p a t c h " or "X-ray-like repair" [ 19]. If the cellular enzymes that carry out the host-cell reactivation of damaged virus have similar affinities for viral and'cellular DNA lesions, then the sensitivity of a mammalian virus treated with damaging agents, should parallel the sensitivity of cells treated with the same agents. Day [10] has shown a correlation between the reactivation of ultraviolet-irradiated human adenovirus type 2 and the relative a m o u n t of UV-induced unscheduled DNA synthesis [18] in XP cells from different genetic complementation groups. The exception to this correlation are group D XP cells which demonstrate the least a m o u n t of reactivation of UV-treated adenovirus but the highest relative rate of UV-induced UDS. He has also shown that the XP variant, which has normal UV-induced UDS rates but is defective in the kinetics of postreplication repair, shows lower levels of reactivation than do normal cells [11]. In order to further characterize the XP phenotype, we have compared the survival of UV-irradiated herpes simplex virus I strain MP with the survival of N-acetoxy-2-acetylaminofluorene-treated virus in normal human skin fibroblasts, XP cells from the different genetic complementation groups, an XP variant and in two presumed XP heterozygotes. In addition, we have also compared the survival of NAcO-AAF-treated HSV-1 MP with the survival of nitrogen mustard (HN2) treated virus in complementation group D XP cells. Materials and methods The cells utilized in these studies were obtained from the American Type Culture Collection, Rockville, MD, with the exception of two normal human skin fibroblast strains which were kindly supplied by Dr. Theodore Malinin of the Department of Surgery at the University of Miami School of Medicine. Cells were routinely grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% v/v fetal bovine serum obtained either from Grand Island Biological Company or International Scientific Industries,
397 Cary, IL, penicillin 100 units/ml, streptomycin 100 pg/ml, fungizone 0.25 pg/ ml, and sodium bicarbonate 1200 mg/1. Cells were passaged every fifth day and fed on the third day following transfer. Herpes simplex virus type 1 strain MP was obtained from the American Type Culture Collection, Rockville, MD. Stock virus was grown in HEp-2 cells initially infected at a multiplicity of 0.05--0.1. Growth flasks were incubated at 37°C for 72 h. Virus was harvested by rapid freezing and slow thawing of the contents of each vessel three times followed by sonication of the resulting suspension for 60 sec in a Ultramet III sonicator, Buehler Ltd., Evanston, IL. Nitrogen mustard (HN2) was obtained from Aldrich Chemicals, Milwaukee, WI. N-Acetoxy-2-acetylaminofluorene [NAcO-AAF] was kindly supplied by Dr. James Miller of the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI. HN2 solutions were prepared by dissolving the chemical in either unsupplemented growth medium or in calcium--magnesium-free phosphate-buffered saline. HN2 solutions were neutralized by mixing with an excess volume of 10% sodium thiosulfate. NAcO-AAF solutions were prepared by dissolving the chemical in a mixture of e t h a n o l : dimethylsulfoxide (DMSO), 9 : 1. Dilutions were prepared in this mixture prior to addition to any aqueous phase since in the presence of water, this compound has an extremely short half-life hydrolysis. Neutralization of NAcO-AAF solutions and vessels in contact with this p o t e n t carcinogen was accomplished by mixing the solution with concentrated sulfuric acid. HN2 treatment of virus. Stock virus aliquots were prewarmed to 37°C. 0.1 volumes of HN2 yielding the final desired concentration was added to the indicated stock virus aliquot. Virus was treated at 37°C for 16 min. Following treatment at 37°C, an equal volume of 1% sodium thiosulfate was added to each aliquot to neutralize any residual unreacted HN2 remaining in solution. Each treatment tube was then aliquoted in 1.0 ml volume, quick frozen in a dry ice--ethanol bath and stored at --70 ° C. NAcO-AAF treatment of virus. Treatment of virus with NAcO-AAF was accomplished in the same manner as described above for HN2 treatment with the following exceptions. NAcO-AAF solutions were diluted 1 : 100 into virus aliquots yielding the final desired concentration. Following incubation at 37°C for 15 min residual unreacted NAcO-AAF was neutralized by adding an equal volume of fetal bovine serum. Each treatment tube was then aliquoted, frozen and stored as described for HN2 treatment. All treatment procedures were carried out under strict aseptic conditions. Ultraviolet (UV) irradiation of virus. In a number of experiments, the reactivation of UV-inactivated virus was studied. The source of ultraviolet light was an 8-W General Electric germicidal lamp whose major o u t p u t was near 254 nm. The lamp was calibrated with a Blak-Ray Ultraviolet meter, model J-225, Ultraviolet Products Inc., San Gabriel, CA. The height of the lamp was adjusted to give an incidence dose rate of 10 ergs/mm2/sec. Stock virus was diluted i n u n supplemented growth medium and aliquoted into 35-mm sterile tissue culture dishes to a depth of approximately 2 mm. Each dish was centered under the light source and exposed to the radiation for the required a m o u n t of time to deliver the desired dose at the incidence dose described. The virus suspension
398 was continuously agitated during irradiation to ensure maximum exposure of the virus. The irradiated suspension was immediately diluted into unsupplemented growth medium for titration of infectious virus and the rest of the undiluted suspension was quick frozen in a dry ice--ethanol bath and stored at --70 ° C. Inactivation curves and routine titrations of virus stocks were accomplished utilizing a modification of the plaque assay described by Roizman and Roane [21] for herpes simplex virus. 0.1 ml of a serial dilution of the virus was innoculated in triplicate onto newly confluent monolayers of the cells being studied for virus reactivation. Cell monolayers were prepared by passing the cells at a ratio of 1 : 2 in supplemented growth medium containing 3700 mg/1 sodium bicarbonate into Linbro multidish disposo trays, FB-16-24-TC, Linbro Scientific Co., New Haven, CT. The monolayers were grown to confluency before using them for titration. Virus was adsorbed to the cells for 2 h at room temperature with intermittant rocking of the dishes to aid in the dispersion of the virus suspension over the entire monolayer. Following adsorption, the monolayers were overlayed with 2 ml of assay medium. Assay medium consisted of growth medium supplemented with fetal bovine serum 1% v/v, antibiotics as described previously and pooled human immune serum globulin 0.03% w/v obtained from Cutter Laboratories, Berkeley, CA, or from Merck, Sharpe and Dohme, Merck and Co., West Point, PA. The immune serum globulin was included in the medium to inhibit secondary plaque formation. Assay dishes were incubated at 37°C in a humidified atmosphere of 10% CO2 and 90% air for 72 h. The medium was then poured off the cells and each dish was rinsed with 0.9% w/v NaC1 prior to staining of the monolayers with crystal violet. Virus plaques were counted with the aid of a dissecting microscope. In all cases, the number of plaque-forming units/ml represents the mean number of plaques of triplicate determinations multiplied by the dilution factor. Each experiment was repeated three times. The figures represent one of the triplicate experiments performed. In all cases, the triplicate experiments showed qualitatively similar results. In a number of experiments the effect of caffeine on the survival of treated virus was studied. Caffeine obtained from Sigma Chemical Company was prepared as a sterile 2% w/v solution. Aliquots of this stock solution were added to the assay medium to yield the desired final concentration of caffeine. In these experiments, the caffeine was present during the entire post-infection assay incubation. Results
Survival of UV-irradiated HSV-1 MP. The survival of UV-irradiated virus in xeroderma pigmentosum skin fibrobla~ts from complementation groups B, C and D and in an XP variant relative to the survival of virus in normal skin fibroblasts is shown in Fig. 1. The relative ability of XP cells from the different complementation groups to reactivate UV-irradiated virus was found to be D < C B with the XP variant demonstrating virus survival on the order of that seen in one of the two normal fibroblasts utilized in this study. Survival o f NAcO-AAF-treated HSV-1 MP. NAcO-AAF-treated virus was
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Fig. 2. Survival o f H S V - 1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f N A c O - A A F for 1 5 rain at 3 7 ° C . V i r u s survival w a s a s s a y e d in t h e f o l l o w i n g cells: C R L 1 1 2 1 (m), n o r m a l ; C R L 1 2 2 4 (e), n o r m a l ; C R L 1 1 6 2 ( × ) , XP variant; C R L 1 2 2 3 (o), XP c o m p l e m e n t a t i o n g r o u p A ; C R L 1 1 9 9 (•), XP c o m p l e m e n t a t i o n g r o u p B; C R L 1 1 6 6 (D), XP c o m p l e m e n t a t i o n g r o u p C; C R L 1 1 5 7 (A), X P c o m p l e m e n t a t i o n g r o u p D.
assayed for survival in XP skin fibroblasts from complementation groups A--D, in an XP variant strain and in two normal skin fibroblast strains. The inactivation curves for treated virus in these cells is shown in Fig. 2. The relative reactivation ability of these cells for NAcO-AAF-treated virus is D ~ A < B < C. As seen for UV-irradiated virus survival, the survival of NAcO-AAF-treated HSV-1 MP in the XP variant is on the order of the survival seen for virus treated with this chemical in the normal human skin fibroblasts. Survival of UV-irradiated and NAcO-AAF-treated HSV-1 MP in the presence of caffeine. Caffeine has been shown to potentiate the lethal action of alkylating agents and UV light in rodent cells [24]. It has been proposed that this potentiation of lethality is a result of the inhibitory effect of caffeine on the postreplication-repair function of these cells [13,14]. Caffeine, however, has been shown to have no effect on the postreplication-repair ability of human cells except for the XP variant cell strains which probably owe their inherent UV sensitivity to a defective postreplication-repair function [ 13,15 ]. Caffeine in the post-infection assay medium at concentrations of 2 and 4 mM had no effect on the inactivation kinetics of NAcO-AAF-treated HSV-1 MP in a normal and three XP cell strains from different genetic complementation groups (data not shown). Fig. 3 shows the effect of 4 mM caffeine in the postinfection assay medium on the survival of both UV-irradiated and NAcO-AAFtreated virus in the XP variant cell strain. For either type of inactivating treatment, caffeine had no significant effect on the inactivation kinetics of the treated virus.
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Survival o f NAcO-AAF-treated HSV-1 MP in two X P cell strains and their filial heterozygotes. Fig. 4a demonstrates the survival o f virus treated with up to 100 pg N A c O - A A F / m l in a group A and a group B XP cell strain. It is evident that relative to the normal skin fibroblast cell strains, there is deficient reactivation of the treated virus in the XP cell strains. Fig. 4b demonstrates the survival of the same virus in two normal skin fibroblast cell strains and in two strains which are presumed heterozygotes for xeroderma pigmentosum. These strains were derived from the mothers of the XP patients whose cells were utilized as viral hosts in 4a. The inactivation kinetics of the treated virus in the XP heterozygotes are the same as that seen for survival of the virus in the normal cell strains.
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Fig. 4a. S u r v i v a l o f H S V - 1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f N A c O - A A F f o r 15 m i n at 3 7 ° C in n o r m a l and x e r o d e r m a P i g m e n t o s u m skin fibroblasts: C R L 1222 ($), normal; C R L 1121 (i), normal; C R L 1199 (o), XP e o m p l e m e n t a t i o n g r o u p B; C R L 1 2 2 3 (~), X P e o m p l e m e n t a t i o n g r o u p A. Fig. 4b. S u r v i v a l o f N A e O - A A F t r e a t e d H S V - 1 MP in t w o n o r m a l a n d t w o p r e s u m e d X P h e t e r o z y g o u s s k i n f i b r o b l a s t cell strains. T r e a t m e n t w a s as d e s c r i b e d f o r Fig. 4 a : C R L 1 2 2 2 ( e ) , n o r m a l ; C R L 1 1 2 1 ( i ) , n o r m a l ; C R L 1 1 9 8 (o), X P h e t e r o x y g o t e - m o t h e r o f XP p a t i e n t Po Co; C R L 1 2 5 4 (~), XP h e t e r o z y g o t e - m o t h e r o f XP p a t i e n t C R L 1 2 2 3 .
401
Survival of UV-irradiated, NAcO-AAF-treated and HN2-treated HSV-1 MP. Survival of virus treated with UV light, the UV-like chemical NAcO-AAF or the classical alkylating agent HN2 was assayed in normal fibroblasts and in xeroderma pigmentosum genetic complementation group D cells, which demonstrate the least amount of reactivation of UV-irradiated adenovirus and UVirradiated or NAcO-AAF-treated HSV-1 MP. Both UV treatment and reaction of the virus with NAcO-AAF results in lower virus survival in the XP cells relative to the inactivation kinetics seen in the normal cells, (Figs. 5a, 5b). In contrast, treatment of the virus with HN2 results in equivalent survival of the viruS in both the XP genetic complementation group D cells and the normal skin fibroblasts (Fig. 5c). It should be noted that there is a difference in the survival of UV-treated virus between the normal fibroblasts used in the experiment described in Fig. 1 and the normal fibroblasts utilized in the experiment described in Fig. 5a. A similar difference in survival of NAcO-AAF-treated virus on these two normal strains was also observed.
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Fig. 5a. Survival of UV-i~adiated H S V - 1 M P gToup D.
in normal skin (e) and in C R L
1 2 0 0 (o) complementation
Fig. 5b. Survival o f HSV-1 MP t r e a t e d w i t h N A c O - A A F in n o r m a l ( e ) a n d C R L 1 2 0 0 (o) XP e o m p l e m e n t a t i o n g r o u p D skin f i b r o b l a s t s . Fig. 5c. Survival o f HSV-1 MP t r e a t e d w i t h i n c r e a s i n g d o s e s o f H N 2 for 15 rain at 3 7 ° C in n o r m a l ( e ) a n d C R L 1 2 0 0 (o) X P c o m p l e m e n t a t i o n g r o u p D skin fibroblasts.
402 Discussion Treatment of HSV-1 MP with the ultimate carcinogen and UV-like chemical NAcO-AAF results in lower survival of the virus in XP cells from the genetic complementation groups than in normal cells. The XP variant studied reactivated NAcO-AAF-treated virus to the same extent as the normal cells utilized for comparison. NAcO-AAF-treated virus survival in XP cells from the different genetic complementation groups is qualitatively similar to survival seen for UV-irradiated herpes virus. For virus treated with either agent, survival in the XP variant was similar to survival in normal fibroblasts. The similar relative order of the reactivation of UV and NAcO-AAF-treated virus in the various XP cells indicate that the reactivation of virus damaged by either agent is accomplished by the same repair pathway or pathways with common intermediate steps. Caffeine had no effect on the inactivation kinetics of NAcO-AAF-treated or UV-irradiated virus in the XP variant. This finding will be discussed subsequently in relation to the findings of Day. The relative ability of xeroderma pigmentosum cells from the different genetic complementation groups to reactivate UV-irradiated herpes simplex virus type 1 strain MP was found to be qualitatively similar to that of the hostcell reactivation of UV-inactivated human adenovirus type 2, except for our finding that group C cells were slightly lower in reactivation ability than group B cells [10]. Day showed group B cells to have a lower reactivation ability for UVirradiated Adeno 2 than group C cells [10]. Our results correlate with the findings of Abrahams and Van der Eb, that group B cells reactivated UV-irradiated SV40 DNA better than group C cells [2]. This relationship did not correlate for the XP variant cell strain studied. We f o u n d no detectable difference in the ability of the XP variant and normal skin fibroblasts to reactivate UV-damaged herpes virus. Day demonstrated that this variant was defective in the repair of ultraviolet-irradiated adenovirus. Abrahams and Van der Eb also demonstrated defective reactivation of UVirradiated SV40 DNA in an XP variant [2]. Such inherent differences in the reactivation of these viruses in the XP variant could be the result of a number of mechanisms: (1) a difference in the number of repairable lesions in the viral genome might result from differences in the chemical content and physical structure of the viral DNA molecules; (2) the repair of similar lesions in the two viruses may be accomplished by different mechanisms one of which is defective in the XP variant, or (3) the replication of the viruses in the XP variant is sufficiently different to account for the observed differences in survival. The first possibility was considered the least likely since the survival of both UV-irradiated herpes and adenovirus was qualitatively similar in cells derived from the classical XP patients. The second and third possibilities seemed more likely since it has been shown that the XP variant is defective in the kinetics of postreplication repair. The XP variant cells so far studied have a caffeine-sensitive repair function and caffeine has been shown to potentiate the lethality of a number of DNA-damaging agents in rodent cells which in culture rely primarily on postreplication repair to reverse UV-induced DNA damage [23]. The presence of caffeine in the postinfection assay medium should potentiate the
403 lethality of UV-irradiated herpes virus if it were repaired to a measurable extent by caffeine-sensitive postreplication repair. Caffeine had no effect on the kinetics of inactivation of UV-irradiated herpes virus in the XP variant. This result supports the idea that UV-irradiated herpes virus is reactivated to little if any extent by a caffeine-sensitive mechanism. This premise is supported by the unpublished findings of Lytle, who also observed no difference between the survival of UV-irradiated herpes simplex virus strain MP in XP variants and normal human fibroblasts. In addition, the results of the correlative studies by Takebe et al. [23] also support this contention. These workers have shown that the survival of UV-irradiated herpes virus in mouse L cells, which rely primarily on postreplication repair, was on the same order as that seen for survival in classical XP cells, even though the colony-forming ability of UV-treated L cells was the same as for UV-irradiated normal human cells. Together these results support the implication that UV-irradiated herpes virus is reactivated by a caffeineinsensitive repair process while UV-irradiated adenovirus is reactivated by a combination of caffeine sensitive and insensitive repair functions. Caffeine-sensitive postreplication repair is presumably mediated by a hostDNA polymerase. Perhaps this cellular enzyme has low affinity for herpes virus DNA, resulting in negligible host-mediated postreplication repair for damaged HSV DNA. A herpes-encoded DNA polymerase may play a role in reactivation of UV or NAcO-AAF-treated virus by a caffeine-insensitive mechanism that could account for the equivalent survival of damaged herpes virus in the two normal and the xeroderma pigmentosum variant cell strains studied. Day has found that caffeine in his system potentiates the lethality of UV-treated adenovirus in the XP variant [7]. Even though this result supports the above hypothesis, it should be noted that he also found that caffeine potentiated the lethality of this virus in normal human cells b u t not in cells derived from patients suffering from classical xeroderma pigmentosum. Arlett et al. [ 3], however, has found that caffeine potentiates the lethal action of UV light on XP variant cells or cells from XP complementation group A b u t not in normal human cells. In any event, the physical characterization of the reactivation process for inactivated herpes virus would allow for the correlation of the specific repair functions with a biological endpoint; virus survival. When the survival of HN2-treated virus in XP cells from genetic complementation group D was compared to survival in normal human skin fibroblasts, no difference in the inactivation kinetics was observed. The lack of differential survival indicates that either HN2-treated virus is not repaired in human cells or the repair defect which confers sensitivity of XP cells to ultraviolet radiation or "UV"-like chemicals play little if any role in the recovery of nitrogen mustard-treated virus. Since mustard adducts have been shown to be excised from the DNA of human cells [6,20], it is not likely that nitrogen mustard-treated HSV-1 MP is reactivated to the same extent in both group D XP cells and normal human-skin fibroblasts. This may be a consequence of the introduction of nonenzymatic single-strand breaks by mustard treatment which could compensate for defective endonuclease activity. At the level of damage induced in these experiments by the treatment o f virus with NAcO-AAF, there was no apparent defect in the ability of XP heterozygotes to reactivate the infecting virus. This result does n o t necessarily
404 indicate that XP heterozygotes are normal for the reactivation of NAcO-AAFdamaged HSV-1 MP. It is possible that the level of damage in the viral genome in these experiments was not sufficient to detect a possible rate-limiting reduction of repair-enzyme levels in the heterozygotes studied. The correlation between the reactivation of UV-irradiated virus and NAcOAAF-treated virus indicates that herpes virus inactivated by either of these agents is a good system for detecting repair defects in human cells. The inherent difference in the reactivation of herpes virus, adenovirus and SV40 virus may indicate that they may be specific probes for studying different cellular DNA-repair processes.
Acknowledgements This work was supported by P.H.S. grant CA14395 from the National Cancer Institute to the Comprehensive Cancer Center for the State of Florida at the University of Miami. The authors gratefully acknowledge the advice and criticisms of Dr. Kenneth Kraemer during the course of this work and also the helpful discussions with Kenneth Carlson and Michael Dobersen.
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