Pliulocliwii,sfry aiid Pltotohioloyy. 1976. Vol. 23, pp. 331-336.

Pergamon Press. Printed in Circa1 Britain

HOST CELL REACTIVATION IN MAMMALTAN CELLS-V. PHOTOREACTIVATION STUDTES WTTH HERPES VlRUS IN MARSUPIAL AND HUMAN CELLS C. D. LYTLE,S. G. BENANE*and J. E. STAFFORD Food and Drug Administration, Bureau of Radiological Health, Division of Biological Effects, U.S. Public Health Service, Department of Health, Education and Welfare, Rockville, MD 20852, U.S.A. (Received 2 M a y 1975: trccepted 24 Noremher 1975, Abstract-The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovar! cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes birus in Potoroo cells indicated a high le\el of “dark” host cell reacthation. No photoreactivation was found for UV-irradiated taccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting .;( > 600nm) and human cells with normal repair and with cells deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreactivating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps. obtained from L. E. Bockstahler of this laboratory). Virus growth and plaque assay procedures have been pretiously Reacthation of an ultraviolet(UV)-irradiated mam- described (Lytle et al., 1972b; Lytle, 1971). Following infecmalian tirus may be accomplished by any of sekeral tion cultures were incubated at 37”C, including all prodifferent repair processes, including excision repair cedures involving photoreactivation. Potoroo (Potorous tritloctylis-rat kangaroo) ovarian (Aaronson and Lytle, 19701, a caffeine sensitive repair follicle cells (obtained from Dr. K. T. S. Yao, Bureau of process (Lytle, 1972a), Weigle reactivation (Bock- Radiological Health, Rockville, MD) were grown in \tahler and Lytle, 1970), and photoreactivation (Pfef- Medium-199 (Microbiological Associates, Inc., Bethesda, ferkorn ef al., 1965). Photoreactivation of UV-irra- MD) supplemented with lo?,, newborn calf serum (Colordiated birus was first demonstrated by Dulbecco ado Serum Company Laboratories, Denver, Colorado). kidney cells (PtK2-American Type Culture Col(1949) with T bacteriophages. This phenomenon has Potoroo lection-CCL 56) were grown in MEM in Earle’s BSS with been shown to be host cell dependent in bacteria glutanline, and lo:/” fetal calf serum (Flow Laboratories, (Harm and Hillebrandt, 1962) and can only reactivate Rockbilk, MD). Human skin fibroblasts from a normal a damaged virus after it has infected its host cell (Dul- individual (KD) and a xeroderma pigmentosum patient (XP12BE) (obtained from R. S. Day, National Cancer Inbecco, 1949). stitute, Bethesda, MD) were grown in MEM supplemented This paper reports a characterization of photoreac- to contain twice the normal levels of amino acids and vitati\ ation of UV-irradiated herpes +nplex virus in mins, plus lo”, fetal calf serum. The XP cells lack host mammalian (marsupial) cells, which was similar to cell reactivation and do not remove thymine dimers that reported for herpes viruses in other animal (Cleaver and Trosko, 1970; Setlow et ol., 1969). Cultures were grown in 25 cmz disposable plastic T-flasks (Falcon (avian) cells (Pfefferkorn et al., 1965; Pfefferkorn er Plastics). All media contained penicillin-streptomycin. u/., 1966: Pfefferkorn and Coady, 1968). Attempts to Irratliution techniques. The procedure for irradiating detect photoreactikation of herpe< virus in human virus suspensions with germicidal UV (254 nm) has been cells failed, in contrast to a recent report of photo- previously described (Lytle, 1971). For photoreactivation the cell monolayer was illureactivation of herpes virus in the same cells. minated from below with either a bank of six 15W near-UV lamps (Sylvania Blacklite blue FISTS-BLB) or two standard 15W fluorescent visible light lamps (WestingMATERIALS AND METHODS house Daylight F15T8/D). A 5 mm thick glass plate was Virua tint/ cells. The viruses were herpes simplex birus, placed over the lights to provide a heat and short UV MP strain, and taccinia virus, strain WR (the latter wavelength filter. The flasks were placed on 5 mm thick clear Plexiglass at the necessary distance above the lamps *Present address: Environmental Protection Agency, to provide the desired illumination intensity. The near UV exposure rate was measured with a near National Environmental Research Center, Research UV dosimeter (Ultraviolet Products, Inc., San Gabriel, Triangle Park, NC 2771 1, U.S.A. 33 I INTRODUCTION

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C . D. LYTLE.S. G. BENANEand J. E. STAFFORD

CA). Maximum exposure rate available was about 15 W/m2. No far UV was detected. Illumination from the visible light source was 15 W/m’ as measured by the UDT-40A Opto-Meter (United Detector Technology, Santa Monica, CA). Work with marsupial cells was performed in an unlighted biohazard hood in a room lit with an incandescent lamp (3.4 W/m’ at the work surface, 80% of which had wavelengths greater than 600 nm). The low level of potentially photoreactivating illumination during the experimental procedures was not sufficient to provide photoreactivation in the control cultures. At all other times the control cultures were kept in the dark. For experiments with human cells special precautions were taken to prevent potential photoreactivation during experimental procedures in the dark control cultures. This study used red lightin& consisting of a Sylvania 15OW red outdoor flood lamp filtered with red Plexiglass acrylic sheet (Rohm & Haas Co., Philadelphia, PA) O D (optical density) of 1.0 at 608 nm and 2.0 at 593 nm) to give only wavelengths above 600 nm, in place of the incandescent lamp noted above as the sole source of light in the work rnnm RESULTS

Photoreactioation of plaque formation by UV-irradiated herpes uirus in Potoroo cells

Photoreactivation was readily demonstrated for UV-irradiated herpes virus in Potoroo ovary cells. Survival enhancement for different near UV exposures to infected cells is shown in Fig. 1 for irradiated and unirradiated virus. The maximum photoreactivated survival was at least 6-10 fold higher than the dark incubated control and was obtained with approximately 2 h exposure to near UV at 3 W/m’ when the illumination commenced immediately after the virus adsorption period. Furthermore, the illuI

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Near UVexposure, m i n

Figure 2. Relative plaque formation on Potoroo kidney cells by UV-exposed (340 J/m2) herpes virus for different photoreactivating light exposures at different exposure rates. Open symbols designate photoreactivating treatments which commenced at 1 h after the adsorption period; closed symbols for photoreactivating treatment completed immediately before virus infection. Exposure rates for near UV: 0-1.9 W/m2, V-3.8 W/m2, A-7.6 W/m2, 0 - 1 2 3 W/mz; visible light4-15 W/m2.

mination had no effect on plaque formation by unirradiated virus. The effect of different exposure rates of photoreactivating light on virus plaque formation was also studied. The data in Fig. 2 demonstrate that photoreactivation by near UV increased the irradiated virus survival in Potoroo kidney cells 3-6 fold. The highest exposure rate provided the greatest rate of photoreactivation, indicating that 12.5 W/m2 of near UV may not be a saturating intensity. Visible light at 15 W/m’ provided a photoreactivation level roughly equivalent to 2.5-3.0 W/m’ of near UV. Near UV illumination of cell cultures for 60 min immediately before infection had no effect on subsequent plaque formation (Fig. 2). Thus photoprotection (enhanced virus survival by illumination of the host cell before virus infection) was not observed in this virus-host cell system. These data demonstrate photoreactivation of UVirradiated herpes simplex virus in marsupial cells.

(1,

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-

Time during virus latent period of maximum photoreactiration efectireness

t

0

al

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Near U V e x p o s u r e ,

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Figure I . Relative plaque formation for UV-exposed (A, 0,150 Jim2) and control (0)herpes virus for different near U V exposures (3.0 W/m2) which commenced immediately after virus infection of Potoroo ovary cells.

Y O J/m’;

The effectiveness of photoreactivation may be limited by competitive factors which eliminate or reduce the effect of UV damage; e.g. dark (excision) repair may eliminate photoreactivable lesions (pyrimidine diners) or viral DNA synthesis may reduce the lethal effect of the UV damage. The time after virus infection when photoreactivation was most effective was determined. Thirty minute illumination of infected cultures commencing at different times after virus absorption

Photoreactibation of herpes virus in mammalian cells

0

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T i m e a f t e r i n f e c t i o n of lightexposure, h

Figure 3. Relative plaque formation (normalized to dark control) for UV-exposed (235 J/mz) herpes virus in Potoroo ovary cells when illuminated with photoreactivating light (near UV light at 5.7 W/mz) for 30 min commencing at different times after virus adsorption period. was used. Illumination with near UV produced greatest photoreactivation during the first 3 h after the 1:-h virus adsorption period (Fig. 3). Thus most of the viruses were ready to be photoreactivated by the end of the adsorption period. The time of maximum photoreactivability and the decrease in photoreactivability with time in our system were similar to the results reported for the pseudorabies virus-chick embryo system (Pfefferkorn et'al., 1966).

t

0.02 0.OlL 0

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I I00

I I I 200 300 UVdose, J / m 2 I

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I 400

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Figure 5. Survival curves of UV-exposed herpes virus in Potoroo kidney cells incubated in the dark (0)or exposed lor 2 h immediately following infection to near UV light (0) at 8.4 W/mZ or visible light (0)at 15 Wj'm'.

105 J/mz, respectively. in kidney cells. These values are several times greater than those found with human XP cells (Lytle et a!., 1972), implying that dark host cell reactivation occurred in both Potoroo cell lines. Incubation under conditions which provided maximum photoreactivation yielded survival curves having essentially only one component with e-' doses Suroiturl curves for herpes virus of about 84 J/m2 in ovary cells and 135 J/mz in kidSurvival curves were determined for UV-irradiated ney cells. herpes virus in Potoroo cells. Dark incubation yielded A quantitative measure of the extent of repair by two component survival curves (Figs. 4 and 5) typical photoreactivation may be obtained, since photoreactifor this virus in other mammalian cells (Lytle, 1971). vation increased the survival of UV-irradiated virus. The UV fluences which gave an average of one inacti- Higher survival can be correlated with a lower effecvating hit per virus (e-' fluence) for the first and tive UV exposure to the virus using the survival curve second components were about 11 Jim2 and 60 J/m2, for dark incubation. The fractional decrease from the respectikely. in ovary cells and about 40 J/m2 and original UV exposure to the effective one with maximum photoreactivation is called the dose reduction factor. This quantity was calculated from the data in Figs. 4 and 5 and is presented in Table 1. The dose reduction factor for UV-irradiated herpes virus was 0.7-0.8 in the Potoroo ovary cells and 0.5-0.7 in the kidney cells. These values are similar to the dose reduction (0.4-0.5) reported for survival of single Potoroo kidney cells (Todd et a/., 1973). Lack of photoreact ivution for racciniu rirus

UVdose,

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Figure 4. Survival curves for UV-exposed herpes virus in Potoroo ovary cells incubated in the dark (-%) or exposed for 2 h immediately following infection to near UV light (A) at 2.8 W/m2.

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.

Vaccinia virus replicates in the host cell's cytoplasm, rather than in the nucleus as does herpes virus. Photoreactivation was attempted to determine whether it can occur for the virus in the cytoplasm. The survival curves in Fig. 6 show very little difference between virus survival after dark incubation and following photoreactivating treatment. Thus there was little, if any, photoreactivation of UV-irradiated \accinia virus in Potoroo cells. The e-l fluence for vaccinia virus survival was about 11 J/mz, in reasonable agreement with the

C. D. LYTLE,S. G. BENANEand J. E. STAFFORD

334

Table 1. Dose reduction factor for photoreactivated herpes virus in Potoroo cells Effective

original Host Cells

UV Exposure t o Virus, Dd (J/rn2)

UV Exposure a f t e r Maximum Photoreactivation, Dp,(J/m2)

Dose Reduction Factor, Dd-Dpr

Dd Ovary

30 60 90 120 150 180

6 13

.80 .78 .78 .77 .77 .73

20

27 35 48 Avg.

.77

Kidney

50 100 150 200

250 300 350

16 50 63 75 100 135 170

.68 .50 .58 .63 .60 .55 .52

Avn.

Lalues (15 J/m2) previously reported for vaccinia virus in human cells (Lytle et al., 1972). Lack of photorecicrivation of herpes Virus in human cells

Preliminary experiments in our laboratory with several different human skin fibroblasts and herpes virus using a protocol similar to that used with the Potoroo cells showed no evidence of photoreactivation *or photoprotection (unpublished). Pfefferkorn and Cosidy (1968) reported no photoreactivation for

UVdose. J/m2

Figure 6 . Survival curves of UV-exposed vaccinia virus in Potoroo kidney cells incubated in the dark (0)or exposed for 2 h immediately following infection to near U V light (A) at 6.0 W/m2.

.58

pseudorabies virus in rabbit kidney cells although photoreactivation did occur for that virus in chick embryo cells. However, since the action spectrum for photoreactivation in vitro with photoreactivating enzyme from human leukocytes and fibroblasts extends from 300 to 600 nm (Sutherland et al., 1974; Sutherland and Sutherland, 1975), normal laboratory lighting may have provided enough illumination to photoreactivate the dark control during the experimental procedures. Therefore, a further study with only red lighting present during the experimental operations (see Materials and Methods) was conducted using normal skin fibroblasts (KD) and XP fibroblasts. Wagner et al. (1975) have reported that the level of photoreactivating enzyme activity in the XP cells was 36% that of normal human fibroblasts and that illumination from a yellow incandescent lamp was sufficient to photoreactivate UV-irradiated herpes simplex virus in the X P cells. The data in Fig. 7 show that neither photoreactivating light source (near UV or visible fluorescent lamps) enhanced survival of UV-irradiated virus. Therefore, in this study photoreactivation did not play a role in the survival of UV-irradiated herpes virus in human cells. The virus survivals found here were similar to those found independently for the same cells (Takebe et al., in preparation) and similar to that previously reported for other normal and XP cells (Lytle et al., 1972). DISCUSSION

The data presented in this paper demonstrate light dependent recovery for a UV-irradiated human virus in Potoroo cell\. Since Potoroo cells are known to

Photoreactivation of herpes virus in mammalian cells

0

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Exposure to photoreactivating light,

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Figure 7. Lack of photoreactivation of plaque formation by control and UV-irradiated herpes simplex virus on cultured skin fibroblasts from a normal individual (a,b) and a xeroderma pigmentosum patient (c,d). Experimental conditions: BLB lamp, 6.3 W/m2 (A); Daylight lamp, 16 W/m2 ( 0 ) :unirradiated virus (as); virus UV fluence and surviving fraction (b) 44 J/m2, 0.030 and (d) 20 J/m2, 0.018. Exposure to photoreactivating light commenced at I h after the virus adsorption period for the 1/2 and I h samples and immediately after for the 2 and 4 hr samples.

possess enzymatic photoreactivating activity (Cook and Regan, 1969), it is assumed that direct enzymatic photoreactivation was responsible. Photoprotection was not observed. That photoreactivation was found in Potoroo cells, but not in human cells demonstrated that PR is controlled by the host mammalian cell. Exposure of the confluent cell monolayers for long times to near UV or visible light from the photoreactivating lamps had no effect on subsequent infection and plaque formation by unirradiated virus, although Todd et a/. (1973) have reported that black light was lethal to individual Potoroo cells and Wang (1975) reported that 'visible' fluorescent light was lethal to individual human cells. This difference is probably because the ability of confluent monolayers of mammalian cells to support herpes virus plaque formation is much more resistant to near UV radiation than survival of cells irradiated individually (unpublished observation with monkey kidney cells). The level of photoreactivation for UV-irradiated herpes simplex virus by Potoroo cells was similar to that reported for two herpes-type viruses, pseudorabies and herpes simplex, in chick embryo cells (Pfefferkorn et al., 1965: Pfefferkorn et ul., 1966). The dose reduction factors for virus survival were similar in those cells (0.5) (Pfefferkorn et ol., 1966) and were alw h i l a r to the value reported for uninfected Potoroo cell survival (Todd et d.,1973). Also similar was the finding that no photoreactivation was demonstrable with vaccinia virus in chick embryo cells (Pfefferkorn et ul., 1966). Since photoreactivation does occur for at least one cytoplasmic replicating virus, frog virus 3, in chick embryo cells (Pfefferkorn and Boyle, 1972), the lack of photoreactivation for vac-

cinia virus may be due to different availabilities of photoreactivating enzyme to different viruses. The fluorescent lamps emitting primarily near UV radiation provided more photoreactivation than those emitting primarily visible radiation, suggesting that radiation between 300-400 nm was most effective at photoreactivation in Potoroo cells. This is different from the photoreactivating enzyme of human cells where the effective wavelengths are 300-600 nm (Sutherland et al., 1974; Sutherland and Sutherland, 1975). The dose reduction factor for herpes virus in the two Potoroo cells was about 0.7. Since pyrimidine dimers in the DNA are the substrate for photoreactivation (Setlow and Setlow, 1963), this implies that at least 0.7 of the UV inactivation of herpes virus (assayed in the dark) is caused by pyrimidine dimers. Further, although Potoroo cells have at least as much dark host cell reactivation as human cells, they have been reported to have much less excision repair (Buhl et a/., 1974). Thus a repair mechanism other than excision may provide most of the dark host cell reactivation for UV-irradiated herpes virus in Potoroo cells. In bacteria, dark host cell reactivation and photoreactivation overlap in function because they repair the same photoproducts (Rupert and Harm, 1965). The data reported here indicate that a similar overlap occurred in Potoroo cells; i.e. the kidney cells yielded more dark host cell reactivation than the ovary cells, thereby allowing lower maximum photoreactivation in the kidney cells. However, in our study with human cells photoreactivation was not found in cells which have a law level

336

C . D. LYTLE,S. G. BENANEand J. E. STAFFORD

of dark host cell reactivation, although these cells have photoreactivating activity when measured biochemically. Furthermore, Wagner et al. (1975) have reported photoreactivation of herpes simplex virus in the same cells. Several factors might account for the conflicting results: different strains of virus, different tissue culture conditions, different times after infection for exposure to photoreactivating light, different sources of photoreactivating light. We believe that the difference in sources of photoreactivating light was most important. While fluorescent lamps emit significant amounts of near ultraviolet radiation, incandescent lamps emit much of their energy as infrared radiation. Damage by near UV wavelengths from the fluorescent lamps may negate the photorepair (H Harm, personal communication). On the other hand,

infrared radiation emitted by the incandescent lamp may lead to elevated temperatures which could result in higher relative survival, interpretable as photoreactivation (Lytle, 197213).Further investigation is necessary to determine the actual cause of the different results. It has been shown in this study, however, that light from two types of fluorescent lamps can lead to photoreactivation of UV-irradiated herpes simplex virus in Potoroo cells but not in human cells. Acknowledgments-We gratefully acknowledge the generous gifts of virus or cell cultures from R. S. Day, K. T. S. Yao and L. E. Bockstahler. We thank R. S. Day. H. Harm, and B. M. Sutherland for many helpful discussions and R. s. Day, L. E. Bockstahler, F. A. Andersen, K. B. Hellman, and W. M. Leach for critical review of the manuscript.

REFERENCES

Aaronson, S. A., and C. D. Lytle (1970) Nature 228, 359-361. Bockstahler, L. E., and C. D. Lytle (1970) Biochem. Biophys. Res. Commun. 41, 184-187. Buhl, S. N.. R. B. Setlow and J. D. Regan (1974) Biophys. J . 14, 791-803. Cleaver, J. E. (1966) Biochem. Biophys. Res. Commun. 24. 569-576. Cleaver, J. E., and J. E. Trosko (1970) Photochem. Photobid 11, 547-550. Cook, J . S., and J. D. Regan (1969) Nurure 223, 106fS1067. Dulbecco, R. (19491 Nurure 163, 949-950. Harm. W., and B. Hillebrandt (1962) Phorochem. Phorobio/. 1, 271. Krishnan. D., and R. B. Painter (1973) Mutation Res. 17. 213-222. Lytle, C. D. (1971) Infernat. J . Radiation B i d . 19, 329-337. Lytle, C. D. (1972a) Infernut. J . Rudiution Biol. 22, 167-174. Lqtle, C. D. (1972b) Inrernur. J . Radiation Biol. 22, 175-177. Lytle, C. D., S. A. Aaronson and E. Harvey (1972) Internor. J . Rrrdiurion B i d . 22, 159-165. Lytle, C. D., .and S. G. Benane (1974) Internut. J . Rtrdiation Bid. 26, 133-141. Pfeflerkorn, E. R., and M. K. Boyle (1972) J . Virol. 9, 474478. Pfefferkorn, E. R., B. W. Burge and H. M. Coady (1966) J . Brrctid. 92, 856-861. Pfefferkorn, E. R., and H. M. Coadq (1968) J . Virol. 2, 474-479. Pfefferkorn, E. R., C. Rutstein and B. W. Burge (1965) J . Virol. 27, 457-459. Rupert, C. S., and W . Harm (1965) Ado. Radicrrion Biol. 2, 1-81, Setlow, R. B., J. D. Regan, J. German and W. L. Carrier (1969) Proc. Narl. Acad. Sci. U.S. 64. 1035-1 041. Setlow, J. K., and R. B. Setlow (1963) Nuture 197, 56Ck562. Sutherland, B. M. (1974) Nurure 248, 109-112. Sutherland, B. M., P. Runge and J. C . Sutherland (1974) Biochem. 13, 471M715. Sutherland, J. C., and B. M. Sutherland (1975) Biophys. J . 15, 435-440. Todd, P., C. B. Schroy and M. R. Lebed (1973) Photochern. Photobiol. 18, 433-436. Wagner, E. K., M. Rice and B. M. Sutherland (1975) Narure 254, 627-628. Wang, R. J. (1975) Phurochem. Photobiol. 21, 373-375.