Photochemrsrry und Phofohiolog).. 1977. Vol. 25, pp. 477-482.
Pcrgamon Press. Printed i n Great Britain
RADIATION ENHANCED REACTIVATION OF NUCLEAR REPLICATING MAMMALIAN VIRUSES* LARRYE. BOCKSTAHLER and C. DAVIDLYTLE Bureau of Radiological Health, Food and Drug Administration, Department of Health, Education, and Welfare, Rockville, MD 20852, U.S.A. (Received 4 August 1976; accepted 14 December 1976)
Abstract-When CV-1 monkey kidney cells were UV-irradiated (0-18 Jim’) or X-irradiated (0-10 krads) before infection with UV-irradiated simian adenovirus 7 (SA7) or simian virus 40 (SV40), increases in the infectivity of these nuclear replicating viruses as measured by plaque formation were observed. These radiation enhanced reactivations, UV enhanced reactivation (UVER) and X-ray enhanced reactivation (X-ray ER), occurred both when virus infection immediately followed irradiation of the cells (except for X-ray ER with SA7) and when virus infection was delayed until 3-5 days after cell irradiation. While there was little difference in the levels of reactivation of UV-irradiated SV40 between immediate and delayed infection, delayed infection resulted in higher levels of reactivation of SA7. X-ray enhanced reactivation of UV-irradiated Herpes simplex virus persisted for several days but did not increase. Thus, X-ray enhanced and UV enhanced reactivations of these mammalian viruses were relatively long-lived effects. Essentially no UVER or X-ray ER was found in CV-1 cells for either immediate or delayed infection with UV-irradiated vaccinia virus or poliovirus, both of which replicate in the cell cytoplasm. These results suggest UVER and X-ray ER in mammalian cells may be restricted to viruses which are replicated in the cell nucleus. INTRODUCTION
When monolayers of suitable host mammalian cells are ultraviolet (UV) irradiated prior to infection with UV-irradiated Herpes simplex virus, an enhancement in survival of this nuclear replicating virus is observed (Bockstahler and Lytle, 1970). This phenomenon, termed UV enhanced reactivation (UVER),? has been demonstrated for herpesvirus in cells of several mammalian species, including monkey (Bockstahler and Lytle, 1970), rat (Hellman et al., 1974), and human (Lytle et al., 1974). An increase in the amount of UVER has been observed when UV-irradiated cell monolayers were infected several days later with UVirradiated herpesvirus (Lytle et al., 1974; Bockstahler et al., 1976). The maximum amount of this long-lived ‘delayed UVER’ in CV-1 cells was usually several fold greater than the amount of UVER produced
* A preliminary report was presented at the Fifth International Congress of Radiation Research, Seattle, Washington, 1&20 July, 1974. ?We have previously used the terms ultraviolet reactivation (UVR) or Weigle reactivation (WR) to denote enhanced survival of UV-irradiated virus when the host mammalian cells were UV-irradiated, by analogy with the terminology used with bacterial systems. However, the analogy may no longer be tenable since the mechanism in E . coli is becoming more clearly defined and since little is known about the mechanism(s) in mammalian cells. Therefore, we shall use the more general term “enhanced reactivation” (ER) to denote enhanced virus survival. $Representative products and manufacturers are named for identification only and listing does not imply endorsement by the Public Health Service and the US. Department of Health, Education and Welfare.
when irradiated cells were infected immediately following cell irradiation (‘immediate UVER’). In certain cell strains, delayed UVER of herpesvirus has been found where immediate UVER was not observed: in marsupial (Potoroo) cells (Lytle and Benane, 1975) and in normal human and repair deficient human cells (Lytle et al., 1976). A similar enhancement in survival of UV-irradiated herpesvirus occurs when the virus is assayed in certain host cells pre-exposed to X-rays. This X-ray enhanced reactivation (X-ray ER)? has been demonstrated in monkey (Bockstahler and Lytle, 1971a; Bockstahler and Lytle, 1971b) and rat (Hellman et al., 1974) cells. The purpose of this study was to determine whether X-ray ER, like UVER, of herpesvirus is long-lived and to examine UVER and X-ray ER in CV-1 monkey kidney cells using several other mammalian viruses representative of different virus genera. The results show that (1) X-ray ER of herpesvirus resulted for delayed as well as immediate infection, (2) immediate andfor delayed UVER and X-ray ER occurred for the nuclear replicating viruses simian virus 40 and simian adenovirus 7, and (3) these reactivation phenomena were absent for the cytoplasmic replicating vaccinia virus and poliovirus. MATERIALS AND METHODS
Cells and culture conditions. CV-1 cells, an established line of African green monkey kidney cells, were propagated in Eagle’s minimal essential medium (Flow Laboratories, Inc., Rockville, MD 8. This medium was supplemented with a two-fold concentration of essential amino acids and
477
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LARRYE. B~CKSTAHLER and C. DAVIDLYTLE
vitamins, 10% fetal bovine serum, 4 mM L-glutamine and 100 units/m/ each of penicillin and streptomycin (Flow Laboratories, Inc.). Stock cultures of cells were grown at 37‘C in 75 cm2 plastic flasks (Falcon Plastics, Oxnard, CA) and were transferred with ATV solution (Madin and Darhy. 1958). Experimental cultures were grown at 37°C in 50 mm dia plastic Petri dishes (Falcon Plastics) for IJVER studies, and in 25 cm2 plastic flasks for X-ray ER studies. Subculturing and virus infections were performed in a vertical laminar flow biohazard hood (Baker Co., Biddeford, ME). Virus assuys. The macro-plaque strain of Herpes simplex virus (Herpesvirus horninitstType 1, a nuclear replicating, doublc-stranded DNA-containing virus, was used. The virus plaque assay procedure has been described (Bockstahler and Lytle, 1970). Strain WR of vaccinia virus, a cytoplasmic replicating, double-stranded DNA-containing poxvirus, was grown and plaque assayed (5 days) at 37°C by a standard agar overlay procedure used previously (Lytle et ul., 1972), except that the final overlay contained 0.01% neutral red. Mahoney virulent strain, type 3, of polio virus, a cytoplasmic replicating, single-stranded RNA-containing picornavirus, was grown and plaque assayed (4 days) at 34°C by the agar overlay procedure used for vaccinia virus. The oncogenic viruses SV40. a papovavirus, and SA7, an adenovirus, both nuclear replicating, double-stranded DNA-containing viruses, were plaque assayed in CV-1 monolayers by modification of the agar overlay procedure used for vaccinia virus. After removal of cell medium, virus suspension in Dulbecco’s phosphate buffered saline (1 mL) was inoculated with appropriate dilution onto each monolayer and incubated at 37°C with agitation for 90 min. Following virus adsorption, the inoculum was removed, and each monolayer was overlaid with 4 ml of supplemented MEM (see above) containing 0.9% agar. Seven days later and again at 14 days an additional 4 mC of the above agar overlay was added. The last agar overlay contained 0.01% neutral red. Plaques were read on days 15-17. Virus inocula were diluted to give 20-200 plaques per plastic assay dish or flask. Plaque numbers from two or three dishes (or flasks) were averaged for each assay point. Irradiation. Virus and confluent cell monolayers were irradiated separately. A germicidal lamp (General Electric G8T5, with emission principally at 254nm) was used as the source of UV radiation. Details of the irradiation technique and dosimetry have been described previously (Bockstahler and Lytle. 1970; Lytle, 1971; Bockstahler et a/., 1976). UV fluences to cell monolayers were within the range &18 J/m’. Higher fluences caused the monolayers to strip off in sheets from culture dishes within a day or two. The UV fluences given to each virus are listed in the figure legends. UV fluence values were selected which would result in virus survival of about 10-2-10-3. For X irradiation of cell monolayers, 250 kV (constant potential) X rays from a Westinghouse Coronado Therapeutic X-ray machine (0.33mm Cu half-value thickness) with no added filtration were used. The dose rate was 1.48krads per min. Monolayers of freshly confluent cells were irradiated in cell culture medium. Irradiated monolayers were inoculated with appropriate dilutions of viruses either within 0.5 h (immediate) or several days (delayed) following cell irradiation. For experiments with a time delay between cell irradiation and virus infection, three or more days were selected, since shorter times with these cells resulted in incomplete expression of 1976). For the time delayed reactivation (Bockstahler rf d,, delay experiments, the X-irradiated medium was replaced within 0.5 h after irradiation by fresh culture medium which was not changed until virus infection. For all experiments, virus was irradiated immediately prior to infection. Presentation of results. The quantitation of UVER was achieved by calculating the reactivation factor as pre-
viously described (Bockstahler ot a/.. 1976: Callet-Fauquet and Defais, 1972). Values of thc reactivation factor greater than 1 indicate UVER. For X-ray ER experiments a similar attempt to quantitate the increase of reactivation of UV-irradiated virus was made. For each reactivation study 2-6 replicate experiments were performed. Enhanced reactivation was found only for the nuclear replicating viruses. The data displayed in the figures show typical levels of enhanced reactivation for each virus and type of radiation. The maximum amount of enhanced reactivation fluctuated with different cell subculture lots in replicate experiments. For any one of the nuclear replicating viruses this variation in maximum enhanced reactivation ranged from 0.7 to 2.0 times the maximum value presented.
RESULTS
Ultraviolet enhanced reactiuation Immediate and delayed UVER of the nuclear replicating viruses SA7 and SV40 were investigated in the standard manner (Bockstahler and Lytle, 1970; Lytle and Benane, 1975), where assay of unirradiated and UV-irradiated virus by plaque formation on confluent monolayers of UV-irradiated cells was initiated with no delay and with a delay of several days following cell irradiation. The plaque-forming ability of unirradiated and UV-irradiated SA7 on CV-1 cells as a function of U V fluence t o cells is shown in Fig. la. When the cells were infected immediately (Day 0) following monolayer irradiation, their capacity to support unirradiated virus plaque formation increased for UV fluences above 4 Jim2. The relative plaque formation of UV-irradiated SA7 increased with U V fluence t o monolayers between 0 and about 10 Jm’. The reactivation factor curve for this experiment (Fig. l b ) indicates that immediate UVER was expressed for SA7 in CV-1 monolayers. The maximum amount of UVER obtained was about the same magnitude as that obtained for immediate UVER of herpesvirus in CV-1 cells (Bockstahler et al., 1976). The results for a delay of 4 days between cell irradiation and initiation of SA7 assay are also shown in Fig. 1. A substantial portion of the increase in cellular capacity for unirradiated virus growth observed with immediate infection was lost within 4 days (Fig. la). The relative plaque formation of UVirradiated SA7 increased for monolayers irradiated with U V fluences greater than 5 J/m’. The amount of delayed reactivation for fluences above 7 J/m’ was greater than that found for immediate reactivation (Fig. lb). U V enhanced reactivation of SV40 in CV-1 cells was investigated in the same manner (Fig. lc). The capacity of irradiated cells t o support plaque formation of unirradiated SV40 decreased as a function of U V fluence to the cells for immediate infection. Partial recovery t o the normal level of unirradiated cells was observed with delayed infection. For immediate infection the relative plaque formation of irradiated SV40 increased slightly with U V fluence t o monolayers and maximized at approximately 13J/m’. For
Radiation enhanced reactivation
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UV FLUENCE (J/m2) TO CELLS
Figure 1. Immediate and delayed expression of UV enhanced reactivation of simian adenovirus 7 and simian virus 40 in CV-1 cell monolayers: SA7, (a) and (b); SV40, (c) and (d). Relative plaque formation, (a) and (c), by unirradiated (0)and UV-irradiated (A) virus assayed immediately following UV irradiation of cell monolayers (open symbols) or assayed 4 days after monolayer irradiation (closed symbols). Reactivation factors (U),(b) and (d), were calculated from the data in (a) and (c), respectively. UV fluence to SA7 was 760 J/m2; SV40, 1,080J/mZ.
delayed infection, increased relative infectivity of virus X-ray enhanced reactivation was observed for irradiated monolayers with UV Preliminary investigation (Bockstahler and Lytle, fluences above 8 J/m2. The reactivation factor curves 1971b) indicated that X-ray ER of UV-irradiated her(Fig. Id) indicate UVER for SV40 with either imme- pesvirus in CV-1 cells persisted for at least 6 h. Figure diate or delayed infection. The highest values of im- 7 shows a three-day, time-dependent study of X-ray mediate and delayed UVER expressed were similar. ER with the same virus-host cell system. Confluent Thus, both immediate and delayed UVER were monolayers of X-irradiated cells were infected with expressed in CV-1 cells for the nuclear replicating vir- unirradiated and UV-irradiated virus immediately and uses SA7 and SV40, as well as that previously shown three days following cell irradiation (Fig. 3a). The for Herpes simplex virus (Bockstahler and Lytle, shapes of the X-ray response curves for immediate infection were typical of those observed previously 1970). Vaccinia virus and poliovirus were investigated in (Bockstahler and Lytle, 1971a): an absolute increase similar manner to determine whether UVER would in relative plaque forming ability of UV-irradiated occur with cytoplasmic replicating virus. Figure 2a virus as a function of X-ray dose to monolayers from shows the results of an attempt to find immediate 0 to 1 krad, and little effect of X-irradiation on the or delayed UVER with vaccinia virus in CV-1 cells. capacity of cells to support unirradiated virus plaque The reactivation factor curves (Fig. 2b) indicate no formation. The reactivation factor curve (Fig. 3b) expression of immediate or delayed UVER; the reac- showed that the highest level of immediate X-ray ER tivation factors decreased slightly with increasing UV represented about an order of magnitude increase in plaque forming ability. fluence to the cells. Investigation of possible UVER for poliovirus in With infection delayed 3 days (Fig. 3a), the capacity CV-1 cells is shown in Fig. 2c. Very little change was for unirradiated virus increased slightly for X-irraobserved in the relative plaque formation of either diated cells; the curve for irradiated virus was similar irradiated or unirradiated virus over the range of UV to that observed with immediate infection. Figure 3b fluences to monolayers examined. It is clear from the indicates that reactivation was still present at three reactivation factor curves (Fig. 2d) that very little or days, although the maximum quantity expressed was no UVER was expressed. reduced relative to that for immediate infection. Thus,
480
LARRYE. BOCKSTAHLER and C . DAVIDLYTLE
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UV FLUENCE (J/rn2) TO CELLS
Figure 2. Investigation of immediate and delayed expression of UV enhanced reactivation of vaccinia virus, (a) and (b), and poliovirus, (c) and (d), in CV-1 cell monolayers. Same symbols as in Fig. 1. UV fluence to vaccinia virus was 38 J/mZ; poliovirus, 147 J/m2. Delayed virus infection was 4 days after cell irradiation. X-ray ER of herpesvirus, like UVER, was relatively long-lived, but unlike UVER, the amount of reactivation did not increase with time after irradiation of the cells (Bockstahler et al., 1976). SA7 and SV40 viruses were examined in similar manner for the presence of X-ray ER in CV-1 cells. Figure 4a shows the results for SA7. For immediate
and delayed infection, X-irradiation of cells caused an increase in the relative plaque formation of both unirradiated and irradiated virus. The major increase occurred over the range &1 krad, as observed for irradiated herpesvirus. The reactivation factor curves (Fig. 4b), however, show that there was, at most, only a slight increase for immediate infection, but an ap-
---- -- ---DAY 3
HERPES
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X-RAY DOSE (krods) TO CELLS
Figure 3. Immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated Herpes simplex virus in CV-1 cell monolayers. Same symbols as in Fig. 1. UV.fluence to virus was 360 J/m2. Delayed virus infection was 3 days after cell irradiation.
48 1
Radiation enhanced reactivation
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Figure 4. Immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated SA7, (a) and (b), and SV40, (c) and (d), in CV-1 cell monolayers. Same symbols as in Fig. 1. UV fluence to SA7 was 740 J/mZ; SV40, 1,070J/mz. Delayed virus infection was 5 days after cell irradiation for SA7, 4 days for SV40.
Figure 5. Investigation of immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated vaccinia virus, (a) and (b), and poliovirus, (c) and (d), in CV-1 cell monolayers. Same symbols as Fig. 1. UV fluence to vaccinia virus was 38 J/m2; poliovirus, 147 J/m2. Delayed virus infection was 4 days after cell irradiation.
482
LARRYE. BOCKSTAHLER and C. DAVIDLYTLE
DISCUSSION
order of magnitude increase, depending on the type of virus and time (post cell-irradiation) of infection. In addition, it was shown that X-ray ER of UV-irradiated herpesvirus occurred for delayed infection. Little or no reactivation was exhibited following immediate or delayed infection of UV- or X-irradiated CV-1 cells by UV-irradiated poliovirus and vaccinia virus, two cytoplasmic replicating viruses. Thus, it can be concluded for the mammalian virus-CV-1 cell systems investigated that (1) the expression of UVER or X-ray ER is dependent on the type of virus, perhaps restricted to nuclear replicating viruses, suggesting that the responsible cellular process may be localized in the cell nucleus; (2) X-ray ER, like UVER, is a relatively long-lived phenomenon persisting for at least several days; and (3) UVER and X-ray ER appear to be related, in that, dependent upon the virus used, either both phenomena are expressed or both are absent in CV-1 cells. The reason for the increase in capacity of UV- or X-irradiated cells to support SA7 plaque formation is unknown . It is possible that cell irradiation inactivated some form of cellular inhibition of SA7 growth. The capacity increase found with UV-irradiated monolayers subsided to a considerable extent with delayed infection of SA7 and thus, in comparison with the UVER of irradiated virus, was not long-lived.
For SA7 and SV40, two DNA-containing nuclear replicating viruses representative of different virus genera, UVER and X-ray ER were readily demonstrated in CV-1 monolayers. The maximum amounts of UVER and X-ray ER expressed varied up to an
Acknowledgements-The authors acknowledge generous gifts of virus and cell stock from Drs. A. Hellman, M. Klagsbrun and R. Martin. We are grateful to J. Stafford, L. Hester, S . Adams, and J. Goddard for excellent technical assistance.
proximate order of magnitude increase for delayed infection. Therefore, the highest reactivation levels for both delayed X-ray ER and delayed UVER of SA7 (Fig. lb) were greater than the corresponding values for immediate reactivation. An investigation of X-ray ER of UV-irradiated SV40 is shown in Fig. 4c. There was negligible effect of X-radiation on cellular capacity for growth of unirradiated virus for immediate or delayed infection; for UV-irradiated virus there were increases in relative plaque formation in the cell X-ray dose range (r2 krad. The highest quantities of reactivation for immediate and delayed infection were approximately equal (Fig. 4d). Thus, X-ray ER in CV-1 cells was exhibited for the nuclear replicating viruses SA7 and SV40. Figure 5 shows experimental results for an investigation of X-ray ER in CV-1 cells with immediate and delayed (4 days) infection by UV-irradiated vaccinia virus and poliovirus. Neither immediate nor delayed X-ray ER was observed with vaccinia virus (Fig. 5b). The results for poliovirus showed no X-ray ER for immediate infection and little, if any, for delayed infection (Fig. 5d).
REFERENCES
Bockstahler, L. E. and C. D. Lytle (1970) Biochem. Biophys. Res. Commun. 41. 184-189. Bockstahler, L. E. and C. D. Lytle (1971a) J . Virol. 8. 601-602. Bockstahler, L. E. and C. D. Lytle (1971h) In First Europeari Biophysics Congress, Proceedings, Vol. I1 (Edited by E. Broda, A. Locker and H. Springer-Lederer).Vcrlag der Wiener Medizinischen Akademie, Vienna. Bockstahler, L. E., C. D. Lytle, J. E. Stafford and K. F. Haynes (1976) Mutation Res. 35, 189-198. Caillet-Fauquet, P. and M. Defais (1972) Mutation Res. 15. 353-355. Hellman, K. B., K. F. Haynes and L. E. Bockstahler (1974) Proc. Soc. ExptE. BioE. Mrd. 145. 255-262. Lytle, C. D. (1971) Int. J. Radiat. Biol. 19, 329-337. Lytle. C. D., S . A. Aaronson and E. Harvey (1972) Int. J . Radiat. Biol. 22, 159-165. Lytle, C. D., S. G. Benane and L. E. Bockstahler (1974) Photochem. Photobiol. 20. 91-94. Lytle, C. D. and S. G. Benane (1975) I n t . J. Radiat. Bid. 27, 487491. Lytle, C. D., R. S . Day, 111, K. B. Hellman and L. E. Bockstahler (1976) Mutation Res. 36. 257--264. Madin, S. H. and N. B. Darby, Jr. (1958) Proc. Soc. Exptl. Biol. Med. 98. 574-576.
Pcrgamon Press. Printed i n Great Britain
RADIATION ENHANCED REACTIVATION OF NUCLEAR REPLICATING MAMMALIAN VIRUSES* LARRYE. BOCKSTAHLER and C. DAVIDLYTLE Bureau of Radiological Health, Food and Drug Administration, Department of Health, Education, and Welfare, Rockville, MD 20852, U.S.A. (Received 4 August 1976; accepted 14 December 1976)
Abstract-When CV-1 monkey kidney cells were UV-irradiated (0-18 Jim’) or X-irradiated (0-10 krads) before infection with UV-irradiated simian adenovirus 7 (SA7) or simian virus 40 (SV40), increases in the infectivity of these nuclear replicating viruses as measured by plaque formation were observed. These radiation enhanced reactivations, UV enhanced reactivation (UVER) and X-ray enhanced reactivation (X-ray ER), occurred both when virus infection immediately followed irradiation of the cells (except for X-ray ER with SA7) and when virus infection was delayed until 3-5 days after cell irradiation. While there was little difference in the levels of reactivation of UV-irradiated SV40 between immediate and delayed infection, delayed infection resulted in higher levels of reactivation of SA7. X-ray enhanced reactivation of UV-irradiated Herpes simplex virus persisted for several days but did not increase. Thus, X-ray enhanced and UV enhanced reactivations of these mammalian viruses were relatively long-lived effects. Essentially no UVER or X-ray ER was found in CV-1 cells for either immediate or delayed infection with UV-irradiated vaccinia virus or poliovirus, both of which replicate in the cell cytoplasm. These results suggest UVER and X-ray ER in mammalian cells may be restricted to viruses which are replicated in the cell nucleus. INTRODUCTION
When monolayers of suitable host mammalian cells are ultraviolet (UV) irradiated prior to infection with UV-irradiated Herpes simplex virus, an enhancement in survival of this nuclear replicating virus is observed (Bockstahler and Lytle, 1970). This phenomenon, termed UV enhanced reactivation (UVER),? has been demonstrated for herpesvirus in cells of several mammalian species, including monkey (Bockstahler and Lytle, 1970), rat (Hellman et al., 1974), and human (Lytle et al., 1974). An increase in the amount of UVER has been observed when UV-irradiated cell monolayers were infected several days later with UVirradiated herpesvirus (Lytle et al., 1974; Bockstahler et al., 1976). The maximum amount of this long-lived ‘delayed UVER’ in CV-1 cells was usually several fold greater than the amount of UVER produced
* A preliminary report was presented at the Fifth International Congress of Radiation Research, Seattle, Washington, 1&20 July, 1974. ?We have previously used the terms ultraviolet reactivation (UVR) or Weigle reactivation (WR) to denote enhanced survival of UV-irradiated virus when the host mammalian cells were UV-irradiated, by analogy with the terminology used with bacterial systems. However, the analogy may no longer be tenable since the mechanism in E . coli is becoming more clearly defined and since little is known about the mechanism(s) in mammalian cells. Therefore, we shall use the more general term “enhanced reactivation” (ER) to denote enhanced virus survival. $Representative products and manufacturers are named for identification only and listing does not imply endorsement by the Public Health Service and the US. Department of Health, Education and Welfare.
when irradiated cells were infected immediately following cell irradiation (‘immediate UVER’). In certain cell strains, delayed UVER of herpesvirus has been found where immediate UVER was not observed: in marsupial (Potoroo) cells (Lytle and Benane, 1975) and in normal human and repair deficient human cells (Lytle et al., 1976). A similar enhancement in survival of UV-irradiated herpesvirus occurs when the virus is assayed in certain host cells pre-exposed to X-rays. This X-ray enhanced reactivation (X-ray ER)? has been demonstrated in monkey (Bockstahler and Lytle, 1971a; Bockstahler and Lytle, 1971b) and rat (Hellman et al., 1974) cells. The purpose of this study was to determine whether X-ray ER, like UVER, of herpesvirus is long-lived and to examine UVER and X-ray ER in CV-1 monkey kidney cells using several other mammalian viruses representative of different virus genera. The results show that (1) X-ray ER of herpesvirus resulted for delayed as well as immediate infection, (2) immediate andfor delayed UVER and X-ray ER occurred for the nuclear replicating viruses simian virus 40 and simian adenovirus 7, and (3) these reactivation phenomena were absent for the cytoplasmic replicating vaccinia virus and poliovirus. MATERIALS AND METHODS
Cells and culture conditions. CV-1 cells, an established line of African green monkey kidney cells, were propagated in Eagle’s minimal essential medium (Flow Laboratories, Inc., Rockville, MD 8. This medium was supplemented with a two-fold concentration of essential amino acids and
477
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LARRYE. B~CKSTAHLER and C. DAVIDLYTLE
vitamins, 10% fetal bovine serum, 4 mM L-glutamine and 100 units/m/ each of penicillin and streptomycin (Flow Laboratories, Inc.). Stock cultures of cells were grown at 37‘C in 75 cm2 plastic flasks (Falcon Plastics, Oxnard, CA) and were transferred with ATV solution (Madin and Darhy. 1958). Experimental cultures were grown at 37°C in 50 mm dia plastic Petri dishes (Falcon Plastics) for IJVER studies, and in 25 cm2 plastic flasks for X-ray ER studies. Subculturing and virus infections were performed in a vertical laminar flow biohazard hood (Baker Co., Biddeford, ME). Virus assuys. The macro-plaque strain of Herpes simplex virus (Herpesvirus horninitstType 1, a nuclear replicating, doublc-stranded DNA-containing virus, was used. The virus plaque assay procedure has been described (Bockstahler and Lytle, 1970). Strain WR of vaccinia virus, a cytoplasmic replicating, double-stranded DNA-containing poxvirus, was grown and plaque assayed (5 days) at 37°C by a standard agar overlay procedure used previously (Lytle et ul., 1972), except that the final overlay contained 0.01% neutral red. Mahoney virulent strain, type 3, of polio virus, a cytoplasmic replicating, single-stranded RNA-containing picornavirus, was grown and plaque assayed (4 days) at 34°C by the agar overlay procedure used for vaccinia virus. The oncogenic viruses SV40. a papovavirus, and SA7, an adenovirus, both nuclear replicating, double-stranded DNA-containing viruses, were plaque assayed in CV-1 monolayers by modification of the agar overlay procedure used for vaccinia virus. After removal of cell medium, virus suspension in Dulbecco’s phosphate buffered saline (1 mL) was inoculated with appropriate dilution onto each monolayer and incubated at 37°C with agitation for 90 min. Following virus adsorption, the inoculum was removed, and each monolayer was overlaid with 4 ml of supplemented MEM (see above) containing 0.9% agar. Seven days later and again at 14 days an additional 4 mC of the above agar overlay was added. The last agar overlay contained 0.01% neutral red. Plaques were read on days 15-17. Virus inocula were diluted to give 20-200 plaques per plastic assay dish or flask. Plaque numbers from two or three dishes (or flasks) were averaged for each assay point. Irradiation. Virus and confluent cell monolayers were irradiated separately. A germicidal lamp (General Electric G8T5, with emission principally at 254nm) was used as the source of UV radiation. Details of the irradiation technique and dosimetry have been described previously (Bockstahler and Lytle. 1970; Lytle, 1971; Bockstahler et a/., 1976). UV fluences to cell monolayers were within the range &18 J/m’. Higher fluences caused the monolayers to strip off in sheets from culture dishes within a day or two. The UV fluences given to each virus are listed in the figure legends. UV fluence values were selected which would result in virus survival of about 10-2-10-3. For X irradiation of cell monolayers, 250 kV (constant potential) X rays from a Westinghouse Coronado Therapeutic X-ray machine (0.33mm Cu half-value thickness) with no added filtration were used. The dose rate was 1.48krads per min. Monolayers of freshly confluent cells were irradiated in cell culture medium. Irradiated monolayers were inoculated with appropriate dilutions of viruses either within 0.5 h (immediate) or several days (delayed) following cell irradiation. For experiments with a time delay between cell irradiation and virus infection, three or more days were selected, since shorter times with these cells resulted in incomplete expression of 1976). For the time delayed reactivation (Bockstahler rf d,, delay experiments, the X-irradiated medium was replaced within 0.5 h after irradiation by fresh culture medium which was not changed until virus infection. For all experiments, virus was irradiated immediately prior to infection. Presentation of results. The quantitation of UVER was achieved by calculating the reactivation factor as pre-
viously described (Bockstahler ot a/.. 1976: Callet-Fauquet and Defais, 1972). Values of thc reactivation factor greater than 1 indicate UVER. For X-ray ER experiments a similar attempt to quantitate the increase of reactivation of UV-irradiated virus was made. For each reactivation study 2-6 replicate experiments were performed. Enhanced reactivation was found only for the nuclear replicating viruses. The data displayed in the figures show typical levels of enhanced reactivation for each virus and type of radiation. The maximum amount of enhanced reactivation fluctuated with different cell subculture lots in replicate experiments. For any one of the nuclear replicating viruses this variation in maximum enhanced reactivation ranged from 0.7 to 2.0 times the maximum value presented.
RESULTS
Ultraviolet enhanced reactiuation Immediate and delayed UVER of the nuclear replicating viruses SA7 and SV40 were investigated in the standard manner (Bockstahler and Lytle, 1970; Lytle and Benane, 1975), where assay of unirradiated and UV-irradiated virus by plaque formation on confluent monolayers of UV-irradiated cells was initiated with no delay and with a delay of several days following cell irradiation. The plaque-forming ability of unirradiated and UV-irradiated SA7 on CV-1 cells as a function of U V fluence t o cells is shown in Fig. la. When the cells were infected immediately (Day 0) following monolayer irradiation, their capacity to support unirradiated virus plaque formation increased for UV fluences above 4 Jim2. The relative plaque formation of UV-irradiated SA7 increased with U V fluence t o monolayers between 0 and about 10 Jm’. The reactivation factor curve for this experiment (Fig. l b ) indicates that immediate UVER was expressed for SA7 in CV-1 monolayers. The maximum amount of UVER obtained was about the same magnitude as that obtained for immediate UVER of herpesvirus in CV-1 cells (Bockstahler et al., 1976). The results for a delay of 4 days between cell irradiation and initiation of SA7 assay are also shown in Fig. 1. A substantial portion of the increase in cellular capacity for unirradiated virus growth observed with immediate infection was lost within 4 days (Fig. la). The relative plaque formation of UVirradiated SA7 increased for monolayers irradiated with U V fluences greater than 5 J/m’. The amount of delayed reactivation for fluences above 7 J/m’ was greater than that found for immediate reactivation (Fig. lb). U V enhanced reactivation of SV40 in CV-1 cells was investigated in the same manner (Fig. lc). The capacity of irradiated cells t o support plaque formation of unirradiated SV40 decreased as a function of U V fluence to the cells for immediate infection. Partial recovery t o the normal level of unirradiated cells was observed with delayed infection. For immediate infection the relative plaque formation of irradiated SV40 increased slightly with U V fluence t o monolayers and maximized at approximately 13J/m’. For
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Figure 1. Immediate and delayed expression of UV enhanced reactivation of simian adenovirus 7 and simian virus 40 in CV-1 cell monolayers: SA7, (a) and (b); SV40, (c) and (d). Relative plaque formation, (a) and (c), by unirradiated (0)and UV-irradiated (A) virus assayed immediately following UV irradiation of cell monolayers (open symbols) or assayed 4 days after monolayer irradiation (closed symbols). Reactivation factors (U),(b) and (d), were calculated from the data in (a) and (c), respectively. UV fluence to SA7 was 760 J/m2; SV40, 1,080J/mZ.
delayed infection, increased relative infectivity of virus X-ray enhanced reactivation was observed for irradiated monolayers with UV Preliminary investigation (Bockstahler and Lytle, fluences above 8 J/m2. The reactivation factor curves 1971b) indicated that X-ray ER of UV-irradiated her(Fig. Id) indicate UVER for SV40 with either imme- pesvirus in CV-1 cells persisted for at least 6 h. Figure diate or delayed infection. The highest values of im- 7 shows a three-day, time-dependent study of X-ray mediate and delayed UVER expressed were similar. ER with the same virus-host cell system. Confluent Thus, both immediate and delayed UVER were monolayers of X-irradiated cells were infected with expressed in CV-1 cells for the nuclear replicating vir- unirradiated and UV-irradiated virus immediately and uses SA7 and SV40, as well as that previously shown three days following cell irradiation (Fig. 3a). The for Herpes simplex virus (Bockstahler and Lytle, shapes of the X-ray response curves for immediate infection were typical of those observed previously 1970). Vaccinia virus and poliovirus were investigated in (Bockstahler and Lytle, 1971a): an absolute increase similar manner to determine whether UVER would in relative plaque forming ability of UV-irradiated occur with cytoplasmic replicating virus. Figure 2a virus as a function of X-ray dose to monolayers from shows the results of an attempt to find immediate 0 to 1 krad, and little effect of X-irradiation on the or delayed UVER with vaccinia virus in CV-1 cells. capacity of cells to support unirradiated virus plaque The reactivation factor curves (Fig. 2b) indicate no formation. The reactivation factor curve (Fig. 3b) expression of immediate or delayed UVER; the reac- showed that the highest level of immediate X-ray ER tivation factors decreased slightly with increasing UV represented about an order of magnitude increase in plaque forming ability. fluence to the cells. Investigation of possible UVER for poliovirus in With infection delayed 3 days (Fig. 3a), the capacity CV-1 cells is shown in Fig. 2c. Very little change was for unirradiated virus increased slightly for X-irraobserved in the relative plaque formation of either diated cells; the curve for irradiated virus was similar irradiated or unirradiated virus over the range of UV to that observed with immediate infection. Figure 3b fluences to monolayers examined. It is clear from the indicates that reactivation was still present at three reactivation factor curves (Fig. 2d) that very little or days, although the maximum quantity expressed was no UVER was expressed. reduced relative to that for immediate infection. Thus,
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Figure 2. Investigation of immediate and delayed expression of UV enhanced reactivation of vaccinia virus, (a) and (b), and poliovirus, (c) and (d), in CV-1 cell monolayers. Same symbols as in Fig. 1. UV fluence to vaccinia virus was 38 J/mZ; poliovirus, 147 J/m2. Delayed virus infection was 4 days after cell irradiation. X-ray ER of herpesvirus, like UVER, was relatively long-lived, but unlike UVER, the amount of reactivation did not increase with time after irradiation of the cells (Bockstahler et al., 1976). SA7 and SV40 viruses were examined in similar manner for the presence of X-ray ER in CV-1 cells. Figure 4a shows the results for SA7. For immediate
and delayed infection, X-irradiation of cells caused an increase in the relative plaque formation of both unirradiated and irradiated virus. The major increase occurred over the range &1 krad, as observed for irradiated herpesvirus. The reactivation factor curves (Fig. 4b), however, show that there was, at most, only a slight increase for immediate infection, but an ap-
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Figure 3. Immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated Herpes simplex virus in CV-1 cell monolayers. Same symbols as in Fig. 1. UV.fluence to virus was 360 J/m2. Delayed virus infection was 3 days after cell irradiation.
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Figure 4. Immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated SA7, (a) and (b), and SV40, (c) and (d), in CV-1 cell monolayers. Same symbols as in Fig. 1. UV fluence to SA7 was 740 J/mZ; SV40, 1,070J/mz. Delayed virus infection was 5 days after cell irradiation for SA7, 4 days for SV40.
Figure 5. Investigation of immediate and delayed expression of X-ray enhanced reactivation of UV-irradiated vaccinia virus, (a) and (b), and poliovirus, (c) and (d), in CV-1 cell monolayers. Same symbols as Fig. 1. UV fluence to vaccinia virus was 38 J/m2; poliovirus, 147 J/m2. Delayed virus infection was 4 days after cell irradiation.
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LARRYE. BOCKSTAHLER and C. DAVIDLYTLE
DISCUSSION
order of magnitude increase, depending on the type of virus and time (post cell-irradiation) of infection. In addition, it was shown that X-ray ER of UV-irradiated herpesvirus occurred for delayed infection. Little or no reactivation was exhibited following immediate or delayed infection of UV- or X-irradiated CV-1 cells by UV-irradiated poliovirus and vaccinia virus, two cytoplasmic replicating viruses. Thus, it can be concluded for the mammalian virus-CV-1 cell systems investigated that (1) the expression of UVER or X-ray ER is dependent on the type of virus, perhaps restricted to nuclear replicating viruses, suggesting that the responsible cellular process may be localized in the cell nucleus; (2) X-ray ER, like UVER, is a relatively long-lived phenomenon persisting for at least several days; and (3) UVER and X-ray ER appear to be related, in that, dependent upon the virus used, either both phenomena are expressed or both are absent in CV-1 cells. The reason for the increase in capacity of UV- or X-irradiated cells to support SA7 plaque formation is unknown . It is possible that cell irradiation inactivated some form of cellular inhibition of SA7 growth. The capacity increase found with UV-irradiated monolayers subsided to a considerable extent with delayed infection of SA7 and thus, in comparison with the UVER of irradiated virus, was not long-lived.
For SA7 and SV40, two DNA-containing nuclear replicating viruses representative of different virus genera, UVER and X-ray ER were readily demonstrated in CV-1 monolayers. The maximum amounts of UVER and X-ray ER expressed varied up to an
Acknowledgements-The authors acknowledge generous gifts of virus and cell stock from Drs. A. Hellman, M. Klagsbrun and R. Martin. We are grateful to J. Stafford, L. Hester, S . Adams, and J. Goddard for excellent technical assistance.
proximate order of magnitude increase for delayed infection. Therefore, the highest reactivation levels for both delayed X-ray ER and delayed UVER of SA7 (Fig. lb) were greater than the corresponding values for immediate reactivation. An investigation of X-ray ER of UV-irradiated SV40 is shown in Fig. 4c. There was negligible effect of X-radiation on cellular capacity for growth of unirradiated virus for immediate or delayed infection; for UV-irradiated virus there were increases in relative plaque formation in the cell X-ray dose range (r2 krad. The highest quantities of reactivation for immediate and delayed infection were approximately equal (Fig. 4d). Thus, X-ray ER in CV-1 cells was exhibited for the nuclear replicating viruses SA7 and SV40. Figure 5 shows experimental results for an investigation of X-ray ER in CV-1 cells with immediate and delayed (4 days) infection by UV-irradiated vaccinia virus and poliovirus. Neither immediate nor delayed X-ray ER was observed with vaccinia virus (Fig. 5b). The results for poliovirus showed no X-ray ER for immediate infection and little, if any, for delayed infection (Fig. 5d).
REFERENCES
Bockstahler, L. E. and C. D. Lytle (1970) Biochem. Biophys. Res. Commun. 41. 184-189. Bockstahler, L. E. and C. D. Lytle (1971a) J . Virol. 8. 601-602. Bockstahler, L. E. and C. D. Lytle (1971h) In First Europeari Biophysics Congress, Proceedings, Vol. I1 (Edited by E. Broda, A. Locker and H. Springer-Lederer).Vcrlag der Wiener Medizinischen Akademie, Vienna. Bockstahler, L. E., C. D. Lytle, J. E. Stafford and K. F. Haynes (1976) Mutation Res. 35, 189-198. Caillet-Fauquet, P. and M. Defais (1972) Mutation Res. 15. 353-355. Hellman, K. B., K. F. Haynes and L. E. Bockstahler (1974) Proc. Soc. ExptE. BioE. Mrd. 145. 255-262. Lytle, C. D. (1971) Int. J. Radiat. Biol. 19, 329-337. Lytle. C. D., S . A. Aaronson and E. Harvey (1972) Int. J . Radiat. Biol. 22, 159-165. Lytle, C. D., S. G. Benane and L. E. Bockstahler (1974) Photochem. Photobiol. 20. 91-94. Lytle, C. D. and S. G. Benane (1975) I n t . J. Radiat. Bid. 27, 487491. Lytle, C. D., R. S . Day, 111, K. B. Hellman and L. E. Bockstahler (1976) Mutation Res. 36. 257--264. Madin, S. H. and N. B. Darby, Jr. (1958) Proc. Soc. Exptl. Biol. Med. 98. 574-576.
