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Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism
VIROLOGY
105, 490-500 (1980)
Evidence for Repair of Ultraviolet Light-Damaged Herpes Virus in Human Fibroblasts by a Recombination Mechanism JENNIFER
D. HALL, JULIE
D. FEATHERSTON,
AND
Department of Cellular and Developmental Biology, University of Arizona,
RYDIA E. ALMY Tucson, Arizona 85721
Accepted April 12, 1980 Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (UV) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each UV dose examined. These high survival levels of UV-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether UV light is recombinogenic for HSV-1, the effect of UV irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperaturesensitive mutants was also investigated. Increased recombination frequencies were observed after UV irradiation of the parental viruses, indicating that damaged sites on the HSV-1 genome stimulate genetic exchanges. This stimulation provides strong support for the model that genetic recombination between lethally damaged HSV-1 chromosomes can lead to the production of undamaged virus. Experiments similar to those described above were performed with repair-deficient (xeroderma pigmentosum, group A, and xeroderma pigmentosum variant) cells. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or UVstimulated viral recombination in herpes-infected cells. INTRODUCTION
Evidence that genetic recombination between damaged chromosomes is involved in repair of damaged viral DNA in bacteria has been provided by the observation that the survival of UV’-irradiated bacteriophages is greatly enhanced when more than one UV-damaged viral genome infects a given cell (Luria and Delbecco, 1949; Huskey, 1969). This process, called’multiplicity reactivation, is considered to depend upon genetic exchanges because it is not observed when both the host and infecting phages are deficient in genetic recombination (Huskey, 1969). Further evidence that genetic recombination is involved in multiplicity reactivation in bacteriophage-infected cells is provided by the observation that UV light stimulates genetic recombination between ’ Abbreviations: UV, ultraviolet; XP, xeroderma pigmentosum; HSV-1, herpes simplex virus type 1; DNA, deoxyribonucleic acid. 0042~0822/80/1204%11$02.00/0 Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
490
genetically marked bacteriophages (Jacob and Wollman, 1955; Epstein, 1958). Multiplicity reactivation in mammalian cells infected by UV-irradiated SV40 and adenovirus (Yamamoto and Shimojo, 1971), pox virus (Abel, 1962), and herpes simplex virus has been reported (Selsky et al., 1979). Evidence that multiplicity reactivation in animal viruses systems might also require recombination has been suggested by the observation that UV irradiation stimulates the production of SV40 recombinants (Dubbs et al., 1974). However, to date -the recombinogenic affects of UV light and multiplicity reactivation of UV-irradiated viruses have not been studied under the same experimental conditions and with the same virus and host cell strains. In the experiments reported here, we have sought evidence for a role for recombination in DNA repair by-studying both multiplicity reactivation and UV-stimulated recombination of herpes simplex virus type
REPAIR
OF UV-IRRADIATED
1 (HSV-1) in the same host cell lines. HSV-1 was selected for these experiments because of the availability of both viral and human host cell lines which might be defective in DNA repair and/or genetic recombination and could be tested for multiplicity reactivation. We have chosen first to examine these processes in host cells deficient in DNA repair because it is known that repair of UV-irradiated HSV-1 is less efficient in human xeroderma pigmentosum cells (Selsky and Greer, 1978; Selsky et al., 1979; this manuscript), and, therefore, excision repair of viral DNA appears to depend upon host cell functions. HSV-1 might also depend on host cell functions for multiplicity reactivation. The host lines studied include normal human fibroblasts and two cell lines derived from patients suffering from the human genetic disease, xeroderma pigmentosum (XP). The first of these lines is deficient in excision repair of UV-induced pyrimidine dimers, a repair process which involves nucleolytic removal of a single-stranded region of DNA containing the dimer followed by repair synthesis to fill the gap (Cleaver, 1974, 1978; Arlett and Lehmann, 1978; Setlow, 1978). The second cell line, from a group called XP variant, exhibits a defect in replication of UV-irradiated DNA (Lehmann et al., 1975). These latter cells were of particular interest because mutant strains of bacteria deficient in replication of irradiated DNA are also deficient in genetic recombination and multiplicity reactivation of certain bacteriophages (Smith and Meun, 1970; Huskey, 1969). We have demonstrated that multiplicity reactivation of UV-irradiated HSV-1 and UV-stimulated viral genetic recombination both occur in normal and in XP and XP variant human cells. These studies provide convincing evidence that recombination can participate in repair of viral DNA in human cells. MATERIALS
AND METHODS
Cell lines and virus stocks. The characteristics of the cells and virus strains used are described in Table 1. Cells were grown in Dulbecco’s modifled Eagle’s medium sup-
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plemented with fetal calf serum at 20% (human cells) or 10% (Vero cells) and incubated in an atmosphere of 10% CO, at 37”. Virus stocks were prepared on Vero cells at 37” (wild type) or 33” (mutant) by inoculating progeny from a single plaque onto cells at a multiplicity of infection of 0.01 plaque-forming units per cell. Virus stocks were harvested in culture medium containing 1% fetal calf serum and were maintained at -85”. Plaque assays. Virus titers were determined on human or Vero cells by infecting monolayers of cells growing on 60-mm plastic petri dishes for 1 hr with 1 ml of virus diluted in medium supplemented with 1% fetal calf serum. Four milliliters additional medium containing 0.5% human gamma globulin to inactivate unadsorbed virus was then added, and incubation was continued. When plaques appeared, medium was removed, cells were fixed with 3% glutaraldehyde, and stained with 2% methylene blue. UV inactivation. Virus stocks were diluted l/10 in phosphate-buffered saline and irradiated at ice temperature in a liquid layer of approximately 1 mm thickness. The virus suspension was swirled to ensure a uniform dose of irradiation. Irradiation was carried out using a General Electric germicidal lamp with an output maximum at 254 nm. The dose rate (2.5 J/m2/sec) was determined using a Blak-Ray Ultraviolet meter (Ultraviolet Products, Inc.). Infective center assay. Human fibroblasts (2 x 105)were inoculated from the same cell suspension onto a series of replica petri dishes and incubated. After 24 hr to allow attachment, the cell number from one plate was determined and the cells were infected at the desired multiplicity of infection with UV-irradiated or untreated virus in a volume of approximately 0.5 ml medium plus 1% fetal calf serum for 1 hr at 37”. Cells infected with irradiated virus received the same total number of viral particles (damaged plus residual undamaged) as cells infected with unirradiated virus. Subsequently, virus suspensions were removed and the cells washed three times with phosphate-buffered saline to remove unadsorbed virus. As a further precaution to inactivate unadsorbed virus, cells were covered with 5 ml medium supplemented with 1% fetal
TABLE 1 CELL LINES AND VIRUS STRAINS Cell lines Ability to replicate uv-irradiated DNA
Source
Genetic characteristics
Excision repair ability
CRL1220
American type culture collection (Rockville, MD)
Human skin fibroblasts from a normal l&year-old male
Presumably proficient
Presumably proficient
XP12BE or CRL1223
American type culture collection
Human skin fibroblasts from a 7-yearold female with xeroderma pigmentosum (XP complementation group A)
Deficient”
Presumably proficient
XPIBE or CRL1162
American type culture collection
Human skin fibroblasts from a 27-yearold male with xeroderma pigmentosum (XP variant)
Proficient”
Deficientb
Vero or CCL81
American type culture collection
Established cell line derived from African Green Monkey kidney cells
Presumably proficient
Presumably proficient
Line
HSV-1 strains
Line
Complementation groupC
Source
Genetic characteristics
Physical map position
Al6
I-l
Gift of P. Schaffer (Harvard University)
Temperature sensitive; DNA negative phenotype at nonpermissive temperatured
Maps near mutants C and Don genetic map’; C and D map at 0.395-0.418’
E6
I-5
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
0.12-0.22’
F1’7
I-6
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
Same complementation group as F18 which maps at 0.086-o. 103”
G8
I-7
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
Same complementation group as G3 which maps at 0.103-o. 186”
Originally obtained from S. Kit (Baylor College of Med)
Wild type
CL101
” Kleijer et al. (1973); Cleaver (1970). * Lehmann et al. (1975, 1977). c Nomenclature of Schaffer et al. (1978). d Benyesh-Melnick et al. (1974). c Parr-is et al. (1978). ’ P. Schaffer, personal communication. g Morse et al. (1977). * Parr-is et al. (1980). 492
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calf serum and 0.5% human gamma globulin and incubated for 1 hr at 37”. Cells were then washed two times with phosphate-buffered saline, trypsinized, and inoculated onto 60 mm petri dishes containing 4 ml medium supplemented with 10% fetal calf serum and 0.5% human gamma globulin. After 1 hr at 37” to allow attachment of the human cells, approximately lo6 uninfected Vero cells were added to each plate in 1 ml medium without gamma globulin, and incubation was continued. In experiments involving caffeine, 300 pg/ml caffeine was added to virus dilution medium, medium used for viral inactivation, and medium used to measure plaques produced by infective centers. When plaques becamevisible (2-3 days), plates were fixed and stained as described above. To calculate the average multiplicity of infection, the virus titers in virus suspensions removed from cells infected with unirradiated virus were compared to the virus titers of suspensions prior to infection. These titers were determined by a plaque assay on the same human cell line used for a given infective center assay. Adsorption of the input virus was generally found to be 60-80%. Similar tests for adsorption with UV-irradiated virus showed that UV irradiation did not alter adsorption levels (J. Hall, unpublished results). The amount of adsorbed virus was then compared to the number of cells per petri dish to determine the average multiplicity of infection. Recombination assays. Monolayer cultures of human fibroblasts in 35 mm petri dishes were infected with pairs of UV-irradiated or untreated temperature-sensitive viruses in a volume of about 0.5 ml. The same quantity of irradiated virus suspension, containing the same total number of viral particles (both UV-inactivated and residual active particles), was added to cells as that of the unirradiated virus suspension. After 1 hr at 37”, virus suspensions were removed and 1 ml medium supplemented with 1% fetal calf serum added to each plate. Plates were incubated for 24 hr at 33”, the permissive temperature for replication of these mutants. The cells and overlying medium were then harvested together and frozen at -85”. When ready to be assayed, virus suspensions were thawed, sonicated,
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VIRUS
and titered on Vero cells as described under “Plaque assays.” The total amount of virus (temperature sensitive plus temperature resistant) produced during infection of the human cells was determined at 33” (permissive temperature) and the number of recombinant (temperature resistant) virus was determined at 39” (nonpermissive temperature). Temperature-resistant plaques must have resulted from recombinant virus since under the conditions of this assay, less than low2 plaque-forming viruses per cell were added to the plates incubated at 39”. Therefore, mixed infection by two complementing viruses could account for only 0.50% of the plaques produced, assuming infection follows a Poisson distribution. The percent frequency of recombination (RF) was calculated as: RF = Virus titer at 39” x 200. Virus titer at 33 In each experiment control infections receiving only one mutant virus indicated that reversion of the viral mutants to a temperature-resistant phenotype was insignificant compared to the recombination frequencies measured (i.e., the fraction of temperature resistant virus obtained in single infections was s10m2% of that found in mixed infections). RESULTS
Repair of UV-Irradiated
Virus
The survivals of UV-damaged HEW-1 in human cells was measured either by a direct plaque assay on human fibroblasts (Fig. 1) or by an infective center assay in which human cells were first infected at a very low multiplicity of infection (to ensure single infection of cells) and then inoculated onto plates containing an excess of uninfected monkey kidney cells. Virus survival in this secondassaywas scored as the yield of plaqueproducing cells (Fig. 2). For UV doses up to 150 J/m2, the logarithm of the surviving fraction of virus in normal human fibroblasts (CRL1220) decreased as a linear function of UV dose, and similar survivals were obtained by either the direct plaque or infective center assay (compare Fig. 1 and
494
HALL, FEATHERSTON,
Fig. 2, panel A, lower curve). However, at doses above 150J/m2, the survival by direct plaque assay was higher than that expected by extrapolation of the upper, exponential portion of the survival curve (extrapolation shown by dotted line, Fig. 1). Under these conditions, it was likely that many cells were infected by two or more damaged viral genomes. Consequently, the increased survivals at high UV closesmight have resulted from high levels of virus production by multiply infected cells. To investigate whether multiply infected cells might repair UV-damaged virus more readily than singly infected cells, normal human fibroblasts were multiply infected with known quantities of UV-irradiated HSV-1 and assayed for their ability to produce plaques on monkey kidney cells as described above. Cells infected with irradiated virus received the same number of viral particles (damaged plus residual active particles) as those infected by unirracliatecl virus. The survival of virus in normal fibroblasts (CRL1220) infected at a multiplicity of infection of 3.3 plaque forming units per cell (virus titer determined prior to irradiation) is shown in Fig. 2, panel A, upper curve. It is quite apparent that cells multiply infected with UV-damaged virus were considerably more capable of producing virus than cells infected singly with 9 x 10d3plaque forming units per cell (compare upper and lower curves, Fig. 2, panel A). This increased ability to produce virus resulted in part from the increased chance of infection of the multiply infected cells by undamaged virus particles. Survivals based upon chance infection by undamaged virus particles (those capable of producing a plaque in singly infected cells) were calculated as described in the legend to Fig. 2 and are plotted in Fig. 2 as a dotted line. Since the experimentally measured survivals were substantially higher than those calculated by this procedure, the increased repair in multiply infected cells was more than could be attributed to chance infection by undamaged virus and presumably, therefore, involves interactions between damaged viral genomes. This phenomenon will be referred to as multiplicity reactivation be-
AND ALMY
UV
DOSE
(J/m’
)
FIG. 1. Survival of UV-irradiated herpes simplex virus on normal human fibroblasts. Normal cells (CRL1220) were infected with irradiated virus in a plaque assay as described in Materials and Methods and the surviving fractions of virus calculated. The linear portion of this survival curve was determined from the least-squares fit of an exponential curve to the data points. The dotted line indicates an extrapolation of this linear part of the survival curve (see text). Numbers in parentheses are the maximum multiplicities of infection at a given UV dose determined from the number of plaque-forming units (titer determined prior to irradiation) added per cell. The data represent a composite of three experiments.
cause this term has been used to describe similar effects in bacteria which were multiply infected with damaged bacteriophage. Repair deficient cells (XP and XP variant) were also infected with UV-damaged HSV+ 1 at both low and high multiplicities of infection and assayed, as described above, for the yield of virus-producing cells (Fig. 2, panels B and C). Repair of UV-irradiatea HSV-1 in singly infected cells was cleficientl in the XP cells, XPl2BE (Fig. 2, panel BI lower curve), which show reduced excision repair of pyrimicline climers, but not in XF variant cells, XP4BE (Fig. 2, panel C lower curve), which have normal excisior: capability. These results are in good agree ment with the results of direct plaque as says on these repair-deficient strains (Selsky and Greer, 1978; J. Hall, unpublished results). Caffeine has been previously
REPAIR OF UV-IRRADIATED
x P URIANT I
1
I
75
I30
25
(IO’,)
1
73
UV
DOSE
495
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I
I
1
7s
I30
NORMAL 1
73
I
IW
(J/m*)
FIG. 2. Survival of plaque formation by human fibroblasts infected with UV-irradiated herpes simplex virus. The infective center assay was performed as described in Materials and Methods. Human fibroblasts used in these experiments were (A) and (D) CRL1220 (normal), (B) XPl2BE (XP, group A), and (C) XP4BE (XP variant). The average multiplicities of infection (plaque-forming units per cell) for cells infected with mm-radiated virus are indicated in the figures. Cells infected with irradiated virus were infected with the same number of viral particles as cells infected with unirradiated virus. Plating efficiencies of infective centers were ~61%. Dotted lines represent theoretical survival curves for plaque formation by cells infected at the higher multiplicity in each graph, assuming that plaque formation occurred only by infection of cells with undamaged virus and that the presence in the same cell of viruses damaged by UV light did not interfere with the growth of undamaged virus. The equation from which these curves were generated is derived below, assuming a Poisson distribution for the multiplicity of infection by both damaged and undamaged virus particles. Theoretical values were calculated to an accuracy of 3%. For a multiplicity of infection n, the chance of an infected cell being infected by at least one undamaged virus is: 1 - (1 - S/So)“, where S/S, is the surviving fraction of infective centers determined experimentally for singly infected cells. At a given average multiplicity of infection, x (determined empirically as described in Materials and Methods), the probability that a given cell receives n virus particles is given by the Poisson term, Pn, where Pn = (z”ems)/n! The fraction of cells receiving 1 or more virus particles is (1 - emz). Consequently, the fraction of infected cells which received n virus particles is Pnl(1 - e-s). The fraction of infected cells which receive both n particles and an undamaged particle, and may therefore produce a plaque, Fn, is given byPn[l - (1 - S/S,)“]/[l - ems].Finally, the fraction of infected cells in the total population which can form plaques is 2 &, Fn. This fraction has been previously calculated by Luria and La&jet (1947) for bacteria infected with UV-irradiated bacteriophage, assuming that the logarithm of the survival of singly infected cells decreased as a linear function of UV dose (single hit kinetics). When our survival curves were linear, survival values for multiply infected cells based on formulas presented by Luria and Latarjet (1947) were in excellent agreement with those calculated by our method, but when our survival curves deviated from linearity, small differences were obtained (data not shown).
shown to reduce cellular survival in UVrradiated XP variant cells and to block the residual ability of these cells to replicate ;heir damaged DNA (Arlett et al., 1975; Lehmann et al., 1977). Addition of caffeine
to the infective center assay of singly infected XP variant cells, XP4BE, had no effect on the survival of plaque-forming ability (data not shown). Virus production from repair-deficient
496
HALL, FEATHERSTON,
cells infected with multiple UV-damaged herpes genomes indicated that multiplicity reactivation occurred in both XP and XP variant cells (Fig. 2, panels B and C, compare lower and upper curves) and in XP variant cells in the presence of caffeine (data not shown). In Fig. 3, the logarithms of virus survival in singly infected cells have been replotted against the logarithms of virus survival in multiply infected cells using the data for normal, XP, and XP variant cells from Fig. 2, panels A, B and C. Because the slopes of these curves are almost identical, it was concluded that XP and XP variant cells have the same capacity for multiplicity reactivation of UV-irradiated HSV-1 as do normal cells. Stocks of HSV-1 typically contain defective particles (which may carry incomplete genomes) in loo-fold excess over plaqueforming particles (Wagner et al., 1974). These particles might also be expected to contribute toward multiplicity reactivation of HSV-1. We have obtained preliminary evidence for such an involvement by demonstrating that multiplicity reactivation can be detected at lower multiplicities of infection than those employed in the above experiments. A determination of the amount of UV-irradiated virus suspension added to cells in the experiment in Fig. 1 indicated that the survival curve deviated from linearity at a point where cells were infected at multiplicities of infection slightly greater than 0.01 plaque-forming units per cell. In addition, an experiment was performed in which cells were infected with UV-damaged virus at an average multiplicity of infection of 0.17 plaque-forming units per cell and the survival of virus-producing cells compared to that measured for cells infected with 5 x lop3 plaque-forming units per cell. As shown in Fig. 2, panel D, the yield of virusproducing cells from cultures infected at the higher multiplicity was substantially greater than that observed for cells infected at the lower multiplicity. Therefore, significant multiplicity reactivation occurred in cells receiving an average of only 0.17 plaqueforming units per cell, despite the fact that only 8.3% of the infected cells received two or more plaque-forming particles, as calculated from a Poisson distribution.
AND ALMY
0.3 0. I 1.0 SURVIVINQ FRACTION IN MUJIPLY-WECYEO
CELLS
FIG. 3. Survival of plaque formation by human fibroblasts infected with UV-irradiated herpes simplex virus. Data from Fig. 2, panels A, B, and C are replotted to compare the survivals in singly infected cells with those in multiply infected cells. The curves were determined from the least-squares fit of an exponential curve to the data points. -O-, normal (CRL1220); -- 0 --, XP (XP12BE); -.- 0 -.-, XP variant (XP4BE).
W-Stimulated
Recombination
In bacteriophage-infected bacteria, multiplicity reactivation is generally considered to involve genetic recombination. In support of this mechanism, it has been observed that UV is recombinogenic for phage systems which show multiplicity reactivation and that recombination does not occur in the absence of a functional system for genetic recombination (see Introduction). In order to test whether UV light is recombinogenic for UV-irradiated HSV-1, genetic crosses between UV-irradiated HSV-1 mutants were performed and the recombination frequency determined as a function of UV dose. Cells were multiply infected with each of a pair of temperature sensitive HSV-1 mutants, and, following appropriate incubation times at the permissive temperature to allow viral growth
REPAIR OF UV-IRRADIATED
loo
50 UV
DOSE
0
( J/m*
497
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400
1
FIG. 4. Yield of temperature-resistant herpes virus recombinants in human fibroblasts infected with UV-irradiated temperature-sensitive mutants. Crosses between pairs of viral mutants were performed as described in Materials and Methods, and the recombination frequencies calculated. Spontaneous recombination frequencies were determined as the average of two separate infections in each experiment as shown by error bars in the figure. The results of two separate experiments for each cross are presented. (A) A16 x F17, only Al6 was irradiated, (B) E6 x G8, both viruses were irradiated at identical UV doses. Plaque-forming units (titers determined prior to irradiation) added per cell were: 8 (A16), 2 (F1’7), 5 (E6), 5 (G8).
and recombination, progeny virus was harvested and the fraction of temperature resistant recombinant virus determined. Since the herpes simplex genome undergoes frequent rearrangements during normal growth of the virus (Roizman, 1979), viral mutants were chosen which map exclusively in a genetically stable region of the genome in order to avoid any contribution of these rearrangements to the recombination frequencies measured. This region, designated U,, is at the left end of the herpes genome and is one of two regions of nonrepetitive DNA in the HSV-1 genome (Roizman, 1979). Temperature-sensitive mutants from different complementation groups were employed and the infection was carried out at the permissive temperature in order to minimize any effects of viral temperature sensitive functions on recombination. The data in Figure 4 show typical variations in recombination frequencies as a function of UV dose with two different sets of viral mutants: Al6 and F1’7 (panel A); E6 and GS (panel B). Only one of the two
virus parents was UV irradiated in the experiments in panel A, and both parents were UV irradiated in the experiments depicted in panel B. For each mutant pair and each irradiation procedure, the recombination frequencies increased with UV dose to a maximum value and then showed no further increase. These data provide evidence that UV light is recombinogenic for HSV-1. Moreover, this stimulation of recombination appears to be a general phenomenon because it was observed using two different sets of viral mutants. The fact that increased recombination frequencies were observed whether one parent or both were irradiated prior to infection suggests that UV damage in one genome was able to promote exchanges with a homologous region in a second undamaged genome. In order to identify conditions which might maximize the stimulation of recombination by UV light, crosses employing different multiplicities of infection of each of the two parent viruses were performed. The crosses shown in Fig. 4, panel B employed a multiplicity of infection of 5 plaque-
498
HALL,
FEATHERSTON,
forming units per cell of each parent. Reducing the multiplicity of infection of one parent relative to the other tended to reduce the spontaneous recombination frequency, presumably because such conditions reduce the chance of exchange events which can yield temperature-resistant progeny. Experiments employing such uneven multiplicities of infection are shown in Fig. 4, panel A. In these experiments, UVstimulated recombination could be readily detected. Crosses between viral mutants in XP (XPlZBE) and XP variant (XP4BE) cells were also conducted using the protocol described in Fig. 4, panel B. The data (Table 2) indicated that spontaneous viral recombination occurred at a level comparable to that observed with the normal cell line (CRL1220). In addition, the extent of stimulation of recombination by UV light was comparable to that observed for CRL1220 cells (Table 2). These results suggest that both viral recombination and UV-stimulated viral recombination do not require the repair functions deficient in these mutant host cells.
AND ALMY TABLE
2
RECOMBINATIONBETWEENHERPESTEMPERATURESENSITIVE MUTANTS (E6 AND Gs) IN HUMAN FIBROBLAST HOSTS"
Cell line
CRL1220 XPlPBE (XP) XP4BE (XP variant)
Relative increase in recombination frequency by virus irradiated with:
SpOMZW?OUS recombination frequency (%I
20 JhP
40Jim2
109 J/m2
10.3 8.5
1.7 1.4
1.8 2.0
1.3 1.5
13.0
1.5
1.9
1.6
' The protocol usedhasbeendescribedin Fig. 4, panel B. The data for CRL1220are also included in this figure. Spontaneous recombination frequencies were determined from two separate infections in each experiment and an average of these values was calculated.
cells may involve both host cell and virally coded functions. The involvement of host functions in multiplicity reactivation has been demonstrated by Selsky et al. (1979), who showed that repair of UV-irradiated herpes is reduced in a multiply infected Bloom’s Syndrome cell line. We have tested this same cell line for recombination between undamaged herpes temperature-sensitive mutants (J. Featherston and J. Hall, DISCUSSION unpublished data) and have obtained approximately normal viral recombination freWe have demonstrated that multiplicity reactivation of UV-irradiated HSV-1 and quencies. Consequently, the deficiency in UV-stimulated recombination of this virus multiplicity reactivation in this cell line is both occur in human cells. Others have dem- not due to a recombination defect, but rather onstrated multiplicity reactivation of UV- these cells may lack a DNA repair function irradiated animal viruses, including HSV-1 which acts on damaged DNA at a step prior and DasGupta and to genetic recombination in the multiplicity (see Introduction), Summers (1980) have independently shown reactivation pathway. Our studies with XP that UV light is recombinogenic for HSV-1. and XP variant cells indicate that UV-stimOur results have demonstrated that these ulated viral recombination and multiplictwo processes are both observed under the ity reactivation of UV-irradiated HSV-1 same experimental conditions and the same both occur in these repair-deficient cells. Either multiplicity-dependent repair and range of UV doses and are, therefore, likely viral recombination do not require the celto be functionally related. Taken together, these results provide evidence that recom- lular functions deficient in these host cells bination is an important mechanism for re- or herpes virus provides its own functions for these processes. pair of UV-irradiated herpes simplex virus. We propose two possible recombination The existence of a recombinational repair mechanism in animal virus-infected cells is mechanisms for multiplicity reactivation of UV-irradiated herpes virus. First, repair or made more plausible by the well-established role of recombination in repair of UV dam- replication of UV-damaged viral chromosomes could give rise to structures which age in bacteria and bacteriophage-infected are not able to undergo additional rounds of cells (see Introduction). replication unless they are modified by reMultiplicity reactivation and UV-stimucombination. For example, as is thought to lated recombination in herpes-infected
REPAIR OF UV-IRRADIATED
be the case in bacteria (Rupp and HowardFlanders, 1968), DNA containing UV-induced damage could be replicated leaving gaps in the progeny strands opposite the damaged sites. These discontinuities might be later filled by recombination events with sister chromosomes, as in bacteria (HowardFlanders et al., 1968; Rupp et al., 1971; Ganesan, 1974). Alternatively, replication might be blocked by DNA damage (as suggested by Sarasin and Hanawalt, 1980; Meneghini and Hanawalt, 1976; Park and Cleaver, 1979) and might not continue until recombination has occurred. Such repeated removal of replication blocks by a recombination mechanism would permit additional rounds of replication despite the continued presence of UV damage. A second possible mechanism for multiplicity reactivation might involve removal of damaged regions from viral DNA by a breakage and joining mechanism which reconstructs undamaged virus from the undamaged portions of irradiated genomes. How such a mechanism would recognize damaged as opposed to undamaged regions is a matter of speculation. It is additionally possible that host or viral recombination functions not normally present in herpes-infected cells might be induced by the presence of damaged viral DNA. These functions might promote multiplicity reactivation by increasing genetic recombination, thereby promoting greater activity of one of the mechanisms of exchange described above. In Escherichia coli, UV irradiation stimulates the production of recA protein (Gudas and Mount, 1977), a protein also required for genetic recombination (Low, 1968). There is a report of an inducible host cell function in monkey cells which enhances repair of UV-irradiated HSV-1 in singly infected cells (DasGupta and Summers, 1978), but no information is presently available about possible inducible functions which might be active in multiply infected cells. Although we favor recombination as the mechanism for multiplicity reactivation of herpes virus due to the stimulation of herpes recombination by UV irradiation, our data cannot totally exclude other mechanisms for multiplicity reactivation, such as complementation between damaged genomes. Such a mechanism would require that in
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cells infected by lethally damaged viruses, essential viral functions could be provided by several damaged genomes, thus allowing replication of at least some of these genomes. Our results are best accounted for by proposing that genetic recombination promotes repair of UV-irradiated HSV-1 in multiply infected cells. These results raise the possibility that recombination may also constitute a repair mechanism for the host cell. ACKNOWLEDGMENTS This work was supported by the National Cancer Institute Grant 5 ROl CA20429. The authors thank Dr. D. W. Mount for many helpful discussions, Karen Scherer for excellent technical assistance, and Dr. P. Schaffer for the generous gift of the herpes mutants. REFERENCES ABEL, P. (1962). Multiplicity reactivation and marker rescue with vaccinia virus. Virology 17, 511-519. ARLETT, C. F., HARCOURT,S. A., and BROUGHTON, B. C. (1975). The influence of caffeine on cell survival in excision-proficient and excision deficient xeroderma pigmentosum and normal human cell strains following ultraviolet-light irradiation. Mut. R&s. 33, 341-346. ARLETT, C. F., and LEHMANN, A. R. (1978). Human disorders showing increased sensitivity to the induction of genetic damage. Ann. Rev. Genet. 12, 95-115. BENYESH-MELNICK,M., SCHAFFER,P. A., COURTNEY, R. J., ESPARZA, J., and KIMURA, S. (1974). Viral gene functions expressed and detected by temperature sensitive mutants of herpes simplex virus. Cold Spring Harbor Symp. Quant. Biol. 34, 731-746. CLEAVER, J. E. (1974). Repair processes for photochemical damage in mammalian cells. Adv. Radiat. Biol. 4, l-75. CLEAVER, J. E. (1978). DNA repair and its coupling to DNA replication in eukaryotic cells. Biochim. Biophys. Acta 516, 489-516. DASGUFTA, U. B., and SUMMERS, W. C. (1978). Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammalian cells. Proc. Nat. Acad. Sci. U. S. A. 75, 2378-2381. DASGUPTA, U. B., and SUMMERS,W. C. (1980). Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus. Molec. Gen. Genet. (in press). DUBBS, D. R., RACHMELER, M., and KIT, S. (1974). Recombination between temperature-sensitive mutants of simian virus 40. Virology 57, 161-174. EPSTEIN, R. H. (1958). A study of multiplicity-re-
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activation in the bacteriophage T4. I. Genetic and functional analysis of T4D-K12( A)complexes. Virology 6,382-404.
GANESAN, A. K. (1974). Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12. J. Mol. Biol. 87, 103-119. GUDAS, L. J., and MOUNT, D. W. (1977). Identification of the recA (tif) gene product of Escherichia coli. Proc. Nat. Acad. Sci. U. S. A. 74,5280-5284. HOWARD-FLANDERS, P., RUPP, W. D., WILKINS, B. M., and COLE, R. S. (1968). DNA replication and recombination after UV irradiation. Cold Spring Harbor Symp. Quant. Biol. 33, 195-207. HUSKEY, R. J. (1969). Multiplicity reactivation as a test for recombination function. Science 164, 319-320. JACOB, F., and WOLLMAN, E.-L. (1955). Etude g&tique d’un bacteriophage temper6 d%scherichia coli. III. Effet du rayonnement ultraviolet sur la recombinaison gknktique. Ann. Inst. Pasteur, 88, 724-749.
KLEIJER, W. J., DE WEERD-KASTELEIN, E. A., SLUYTER, M. L., KEIJZER, W., DE WIT, J. and BOOTSMA, D. (1973). UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes. Mut. Res. 20, 417-428.
LEHMANN, A. R., KIRK-BELL, S., ARLE?T, C. F., HARCOURT, S., DE WEERD-KASTELEIN, E. A., KEIJZER, W., and HALLSMITH, P. (1977). Repair of ultraviolet light damage in a variety of human fibroblast cell strains. Can. Res. 37, 904-910. LEHMANN, A. R., KIRK-BELL, S., ARLETT, C. F., PATERSON,M. C., LOHMAN, P. H. M., DE WEERDKASTELEIN, E. A., and BOOTSMA, D. (1975). Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation. Proc. Nat. Acad. Sci. U. S. A. 72, 219-223. LOW, B. (1968). Formation of merodiploids in matings with a class of Ret-recipient strains of Escherichia coli K12. Proc. Nat. Acad. Sci. U. S. A. 60,160- 167. LURIA, S. C., and DULBECCO,R. (1949). Genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics 34, 93-125. LURIA, S. E., and LATARJET, R. (1947). Ultraviolet irradiation of bacteriophage during intracellular growth. J. Bacterial. 53, 149-163. MENEGHINI, R., and HANAWALT, P. (1976). TCendonuclease V-sensitive sites in DNA from UV-irradiated human cells. Biochim. Biophys. Acta 425, 428-437. MORSE, L. S., BUCHMAN, T. G., ROIZMAN, B., and SCHAFFER,P. A. (1977). Anatomy of herpes simplex virus DNA. IX. Apparent exclusion ofsome parental DNA arrangements in the generation of intertypic
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(HSV-1 x HSV-2) recombinants. J. Viral. 24, 231-248. PARK, S. D., and CLEAVER, J. E. (1979). Postreplication repair Questions of its definition and possible alteration in xeroderma pigmentosum cell strains. Proc. Nat. Acad. Sci. U. S. A. 76,3929-3931. PARRIS, D. S., COURTNEY, R. J., and SCHAFFER, P. A. (1978). Temperature-sensitive mutants of herpes simplex virus type 1 defective in transcriptional and post-transcriptional functions required for viral DNA synthesis. Virology 99, 177-186. PARRIS, D. S., DIXON, R. A. F., and SCHAFFER,P. A. (1980). Physical mapping of herpes simplex virus type 1 ts mutants by marker rescue: Correlation of the physical and genetic maps. Virology 100, 275287. ROIZMAN, B. (1979). The structure and isomerization of herpes simplex virus genomes. Cell 16,481-494. RUPP, W. D., and HOWARD-FLANDERS,P. (1968). Discontinuities in the DNA synthesized in an excision-defective strain of Eschmichia coli following ultraviolet irradiation. J. Mol. Biol. 31, 291-304. RUPP, W. D., WILDE, C. E., RENO, D. L., and HOWARD-FLANDERS,P. (1971). Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J. Mol. Biol. 61, 25-44. SARASIN, A. R., and HANAWALT, P. C. (1980). Replication of ultraviolet irradiated simian virus 40 in monkey kidney cells. J. Mol. Biol. 138, 299-319. SCHAFFER, P. A., CARTER, V. C., and TIMBURY, M. C. (1978). Collaborative complementation study of temperature sensitive mutants of herpes simplex virus types 1 and 2. J. Viral. 27, 490-504. SELSKY, C. A., and GREER, S. (1978). Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-l by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts. Mut. Res. 50, 395-405.
SELSKY, C. A., HENSEN, P., WEICHSELBAUM,R. R., and LITTLE, J. B. (1979). Defective reactivation of ultraviolet light-irradiated herpes virus by a Bloom’s Syndrome fibroblast strain. Can. Res. 39,3392-3396. SETLOW, R. B. (1978). Repair deficient human disorders and cancer. Nature (London) 271, 713-717. SMITH, K. C., and MEUN, D. H. C. (1970). Repair of radiation-induced damage in Eseherichia coli. I. Effects of ret mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51, 459-472. WAGNER,M., SKARE,J., and SUMMERS,W. C. (1974). Analysis of DNA of defective herpes simplex virus type 1 by restriction endonuclease cleavage and nucleic acid hybridization. Cold Spring Harbor Symp. Quant. Biol.
39, 683-685.
YAMAMOTO,H., and SHIMOJO,H. (1971). Multiplicity reactivation of human adenovirus type 12 and simian virus 40 irradiated by ultraviolet light. Viral. 45, 529-531.
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Evidence for Repair of Ultraviolet Light-Damaged Herpes Virus in Human Fibroblasts by a Recombination Mechanism JENNIFER
D. HALL, JULIE
D. FEATHERSTON,
AND
Department of Cellular and Developmental Biology, University of Arizona,
RYDIA E. ALMY Tucson, Arizona 85721
Accepted April 12, 1980 Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (UV) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each UV dose examined. These high survival levels of UV-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether UV light is recombinogenic for HSV-1, the effect of UV irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperaturesensitive mutants was also investigated. Increased recombination frequencies were observed after UV irradiation of the parental viruses, indicating that damaged sites on the HSV-1 genome stimulate genetic exchanges. This stimulation provides strong support for the model that genetic recombination between lethally damaged HSV-1 chromosomes can lead to the production of undamaged virus. Experiments similar to those described above were performed with repair-deficient (xeroderma pigmentosum, group A, and xeroderma pigmentosum variant) cells. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or UVstimulated viral recombination in herpes-infected cells. INTRODUCTION
Evidence that genetic recombination between damaged chromosomes is involved in repair of damaged viral DNA in bacteria has been provided by the observation that the survival of UV’-irradiated bacteriophages is greatly enhanced when more than one UV-damaged viral genome infects a given cell (Luria and Delbecco, 1949; Huskey, 1969). This process, called’multiplicity reactivation, is considered to depend upon genetic exchanges because it is not observed when both the host and infecting phages are deficient in genetic recombination (Huskey, 1969). Further evidence that genetic recombination is involved in multiplicity reactivation in bacteriophage-infected cells is provided by the observation that UV light stimulates genetic recombination between ’ Abbreviations: UV, ultraviolet; XP, xeroderma pigmentosum; HSV-1, herpes simplex virus type 1; DNA, deoxyribonucleic acid. 0042~0822/80/1204%11$02.00/0 Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
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genetically marked bacteriophages (Jacob and Wollman, 1955; Epstein, 1958). Multiplicity reactivation in mammalian cells infected by UV-irradiated SV40 and adenovirus (Yamamoto and Shimojo, 1971), pox virus (Abel, 1962), and herpes simplex virus has been reported (Selsky et al., 1979). Evidence that multiplicity reactivation in animal viruses systems might also require recombination has been suggested by the observation that UV irradiation stimulates the production of SV40 recombinants (Dubbs et al., 1974). However, to date -the recombinogenic affects of UV light and multiplicity reactivation of UV-irradiated viruses have not been studied under the same experimental conditions and with the same virus and host cell strains. In the experiments reported here, we have sought evidence for a role for recombination in DNA repair by-studying both multiplicity reactivation and UV-stimulated recombination of herpes simplex virus type
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1 (HSV-1) in the same host cell lines. HSV-1 was selected for these experiments because of the availability of both viral and human host cell lines which might be defective in DNA repair and/or genetic recombination and could be tested for multiplicity reactivation. We have chosen first to examine these processes in host cells deficient in DNA repair because it is known that repair of UV-irradiated HSV-1 is less efficient in human xeroderma pigmentosum cells (Selsky and Greer, 1978; Selsky et al., 1979; this manuscript), and, therefore, excision repair of viral DNA appears to depend upon host cell functions. HSV-1 might also depend on host cell functions for multiplicity reactivation. The host lines studied include normal human fibroblasts and two cell lines derived from patients suffering from the human genetic disease, xeroderma pigmentosum (XP). The first of these lines is deficient in excision repair of UV-induced pyrimidine dimers, a repair process which involves nucleolytic removal of a single-stranded region of DNA containing the dimer followed by repair synthesis to fill the gap (Cleaver, 1974, 1978; Arlett and Lehmann, 1978; Setlow, 1978). The second cell line, from a group called XP variant, exhibits a defect in replication of UV-irradiated DNA (Lehmann et al., 1975). These latter cells were of particular interest because mutant strains of bacteria deficient in replication of irradiated DNA are also deficient in genetic recombination and multiplicity reactivation of certain bacteriophages (Smith and Meun, 1970; Huskey, 1969). We have demonstrated that multiplicity reactivation of UV-irradiated HSV-1 and UV-stimulated viral genetic recombination both occur in normal and in XP and XP variant human cells. These studies provide convincing evidence that recombination can participate in repair of viral DNA in human cells. MATERIALS
AND METHODS
Cell lines and virus stocks. The characteristics of the cells and virus strains used are described in Table 1. Cells were grown in Dulbecco’s modifled Eagle’s medium sup-
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plemented with fetal calf serum at 20% (human cells) or 10% (Vero cells) and incubated in an atmosphere of 10% CO, at 37”. Virus stocks were prepared on Vero cells at 37” (wild type) or 33” (mutant) by inoculating progeny from a single plaque onto cells at a multiplicity of infection of 0.01 plaque-forming units per cell. Virus stocks were harvested in culture medium containing 1% fetal calf serum and were maintained at -85”. Plaque assays. Virus titers were determined on human or Vero cells by infecting monolayers of cells growing on 60-mm plastic petri dishes for 1 hr with 1 ml of virus diluted in medium supplemented with 1% fetal calf serum. Four milliliters additional medium containing 0.5% human gamma globulin to inactivate unadsorbed virus was then added, and incubation was continued. When plaques appeared, medium was removed, cells were fixed with 3% glutaraldehyde, and stained with 2% methylene blue. UV inactivation. Virus stocks were diluted l/10 in phosphate-buffered saline and irradiated at ice temperature in a liquid layer of approximately 1 mm thickness. The virus suspension was swirled to ensure a uniform dose of irradiation. Irradiation was carried out using a General Electric germicidal lamp with an output maximum at 254 nm. The dose rate (2.5 J/m2/sec) was determined using a Blak-Ray Ultraviolet meter (Ultraviolet Products, Inc.). Infective center assay. Human fibroblasts (2 x 105)were inoculated from the same cell suspension onto a series of replica petri dishes and incubated. After 24 hr to allow attachment, the cell number from one plate was determined and the cells were infected at the desired multiplicity of infection with UV-irradiated or untreated virus in a volume of approximately 0.5 ml medium plus 1% fetal calf serum for 1 hr at 37”. Cells infected with irradiated virus received the same total number of viral particles (damaged plus residual undamaged) as cells infected with unirradiated virus. Subsequently, virus suspensions were removed and the cells washed three times with phosphate-buffered saline to remove unadsorbed virus. As a further precaution to inactivate unadsorbed virus, cells were covered with 5 ml medium supplemented with 1% fetal
TABLE 1 CELL LINES AND VIRUS STRAINS Cell lines Ability to replicate uv-irradiated DNA
Source
Genetic characteristics
Excision repair ability
CRL1220
American type culture collection (Rockville, MD)
Human skin fibroblasts from a normal l&year-old male
Presumably proficient
Presumably proficient
XP12BE or CRL1223
American type culture collection
Human skin fibroblasts from a 7-yearold female with xeroderma pigmentosum (XP complementation group A)
Deficient”
Presumably proficient
XPIBE or CRL1162
American type culture collection
Human skin fibroblasts from a 27-yearold male with xeroderma pigmentosum (XP variant)
Proficient”
Deficientb
Vero or CCL81
American type culture collection
Established cell line derived from African Green Monkey kidney cells
Presumably proficient
Presumably proficient
Line
HSV-1 strains
Line
Complementation groupC
Source
Genetic characteristics
Physical map position
Al6
I-l
Gift of P. Schaffer (Harvard University)
Temperature sensitive; DNA negative phenotype at nonpermissive temperatured
Maps near mutants C and Don genetic map’; C and D map at 0.395-0.418’
E6
I-5
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
0.12-0.22’
F1’7
I-6
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
Same complementation group as F18 which maps at 0.086-o. 103”
G8
I-7
Gift of P. Schaffer
Temperature sensitive; proficient in DNA synthesis at nonpermissive temperatured
Same complementation group as G3 which maps at 0.103-o. 186”
Originally obtained from S. Kit (Baylor College of Med)
Wild type
CL101
” Kleijer et al. (1973); Cleaver (1970). * Lehmann et al. (1975, 1977). c Nomenclature of Schaffer et al. (1978). d Benyesh-Melnick et al. (1974). c Parr-is et al. (1978). ’ P. Schaffer, personal communication. g Morse et al. (1977). * Parr-is et al. (1980). 492
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calf serum and 0.5% human gamma globulin and incubated for 1 hr at 37”. Cells were then washed two times with phosphate-buffered saline, trypsinized, and inoculated onto 60 mm petri dishes containing 4 ml medium supplemented with 10% fetal calf serum and 0.5% human gamma globulin. After 1 hr at 37” to allow attachment of the human cells, approximately lo6 uninfected Vero cells were added to each plate in 1 ml medium without gamma globulin, and incubation was continued. In experiments involving caffeine, 300 pg/ml caffeine was added to virus dilution medium, medium used for viral inactivation, and medium used to measure plaques produced by infective centers. When plaques becamevisible (2-3 days), plates were fixed and stained as described above. To calculate the average multiplicity of infection, the virus titers in virus suspensions removed from cells infected with unirradiated virus were compared to the virus titers of suspensions prior to infection. These titers were determined by a plaque assay on the same human cell line used for a given infective center assay. Adsorption of the input virus was generally found to be 60-80%. Similar tests for adsorption with UV-irradiated virus showed that UV irradiation did not alter adsorption levels (J. Hall, unpublished results). The amount of adsorbed virus was then compared to the number of cells per petri dish to determine the average multiplicity of infection. Recombination assays. Monolayer cultures of human fibroblasts in 35 mm petri dishes were infected with pairs of UV-irradiated or untreated temperature-sensitive viruses in a volume of about 0.5 ml. The same quantity of irradiated virus suspension, containing the same total number of viral particles (both UV-inactivated and residual active particles), was added to cells as that of the unirradiated virus suspension. After 1 hr at 37”, virus suspensions were removed and 1 ml medium supplemented with 1% fetal calf serum added to each plate. Plates were incubated for 24 hr at 33”, the permissive temperature for replication of these mutants. The cells and overlying medium were then harvested together and frozen at -85”. When ready to be assayed, virus suspensions were thawed, sonicated,
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and titered on Vero cells as described under “Plaque assays.” The total amount of virus (temperature sensitive plus temperature resistant) produced during infection of the human cells was determined at 33” (permissive temperature) and the number of recombinant (temperature resistant) virus was determined at 39” (nonpermissive temperature). Temperature-resistant plaques must have resulted from recombinant virus since under the conditions of this assay, less than low2 plaque-forming viruses per cell were added to the plates incubated at 39”. Therefore, mixed infection by two complementing viruses could account for only 0.50% of the plaques produced, assuming infection follows a Poisson distribution. The percent frequency of recombination (RF) was calculated as: RF = Virus titer at 39” x 200. Virus titer at 33 In each experiment control infections receiving only one mutant virus indicated that reversion of the viral mutants to a temperature-resistant phenotype was insignificant compared to the recombination frequencies measured (i.e., the fraction of temperature resistant virus obtained in single infections was s10m2% of that found in mixed infections). RESULTS
Repair of UV-Irradiated
Virus
The survivals of UV-damaged HEW-1 in human cells was measured either by a direct plaque assay on human fibroblasts (Fig. 1) or by an infective center assay in which human cells were first infected at a very low multiplicity of infection (to ensure single infection of cells) and then inoculated onto plates containing an excess of uninfected monkey kidney cells. Virus survival in this secondassaywas scored as the yield of plaqueproducing cells (Fig. 2). For UV doses up to 150 J/m2, the logarithm of the surviving fraction of virus in normal human fibroblasts (CRL1220) decreased as a linear function of UV dose, and similar survivals were obtained by either the direct plaque or infective center assay (compare Fig. 1 and
494
HALL, FEATHERSTON,
Fig. 2, panel A, lower curve). However, at doses above 150J/m2, the survival by direct plaque assay was higher than that expected by extrapolation of the upper, exponential portion of the survival curve (extrapolation shown by dotted line, Fig. 1). Under these conditions, it was likely that many cells were infected by two or more damaged viral genomes. Consequently, the increased survivals at high UV closesmight have resulted from high levels of virus production by multiply infected cells. To investigate whether multiply infected cells might repair UV-damaged virus more readily than singly infected cells, normal human fibroblasts were multiply infected with known quantities of UV-irradiated HSV-1 and assayed for their ability to produce plaques on monkey kidney cells as described above. Cells infected with irradiated virus received the same number of viral particles (damaged plus residual active particles) as those infected by unirracliatecl virus. The survival of virus in normal fibroblasts (CRL1220) infected at a multiplicity of infection of 3.3 plaque forming units per cell (virus titer determined prior to irradiation) is shown in Fig. 2, panel A, upper curve. It is quite apparent that cells multiply infected with UV-damaged virus were considerably more capable of producing virus than cells infected singly with 9 x 10d3plaque forming units per cell (compare upper and lower curves, Fig. 2, panel A). This increased ability to produce virus resulted in part from the increased chance of infection of the multiply infected cells by undamaged virus particles. Survivals based upon chance infection by undamaged virus particles (those capable of producing a plaque in singly infected cells) were calculated as described in the legend to Fig. 2 and are plotted in Fig. 2 as a dotted line. Since the experimentally measured survivals were substantially higher than those calculated by this procedure, the increased repair in multiply infected cells was more than could be attributed to chance infection by undamaged virus and presumably, therefore, involves interactions between damaged viral genomes. This phenomenon will be referred to as multiplicity reactivation be-
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UV
DOSE
(J/m’
)
FIG. 1. Survival of UV-irradiated herpes simplex virus on normal human fibroblasts. Normal cells (CRL1220) were infected with irradiated virus in a plaque assay as described in Materials and Methods and the surviving fractions of virus calculated. The linear portion of this survival curve was determined from the least-squares fit of an exponential curve to the data points. The dotted line indicates an extrapolation of this linear part of the survival curve (see text). Numbers in parentheses are the maximum multiplicities of infection at a given UV dose determined from the number of plaque-forming units (titer determined prior to irradiation) added per cell. The data represent a composite of three experiments.
cause this term has been used to describe similar effects in bacteria which were multiply infected with damaged bacteriophage. Repair deficient cells (XP and XP variant) were also infected with UV-damaged HSV+ 1 at both low and high multiplicities of infection and assayed, as described above, for the yield of virus-producing cells (Fig. 2, panels B and C). Repair of UV-irradiatea HSV-1 in singly infected cells was cleficientl in the XP cells, XPl2BE (Fig. 2, panel BI lower curve), which show reduced excision repair of pyrimicline climers, but not in XF variant cells, XP4BE (Fig. 2, panel C lower curve), which have normal excisior: capability. These results are in good agree ment with the results of direct plaque as says on these repair-deficient strains (Selsky and Greer, 1978; J. Hall, unpublished results). Caffeine has been previously
REPAIR OF UV-IRRADIATED
x P URIANT I
1
I
75
I30
25
(IO’,)
1
73
UV
DOSE
495
HERPES VIRUS
I
I
1
7s
I30
NORMAL 1
73
I
IW
(J/m*)
FIG. 2. Survival of plaque formation by human fibroblasts infected with UV-irradiated herpes simplex virus. The infective center assay was performed as described in Materials and Methods. Human fibroblasts used in these experiments were (A) and (D) CRL1220 (normal), (B) XPl2BE (XP, group A), and (C) XP4BE (XP variant). The average multiplicities of infection (plaque-forming units per cell) for cells infected with mm-radiated virus are indicated in the figures. Cells infected with irradiated virus were infected with the same number of viral particles as cells infected with unirradiated virus. Plating efficiencies of infective centers were ~61%. Dotted lines represent theoretical survival curves for plaque formation by cells infected at the higher multiplicity in each graph, assuming that plaque formation occurred only by infection of cells with undamaged virus and that the presence in the same cell of viruses damaged by UV light did not interfere with the growth of undamaged virus. The equation from which these curves were generated is derived below, assuming a Poisson distribution for the multiplicity of infection by both damaged and undamaged virus particles. Theoretical values were calculated to an accuracy of 3%. For a multiplicity of infection n, the chance of an infected cell being infected by at least one undamaged virus is: 1 - (1 - S/So)“, where S/S, is the surviving fraction of infective centers determined experimentally for singly infected cells. At a given average multiplicity of infection, x (determined empirically as described in Materials and Methods), the probability that a given cell receives n virus particles is given by the Poisson term, Pn, where Pn = (z”ems)/n! The fraction of cells receiving 1 or more virus particles is (1 - emz). Consequently, the fraction of infected cells which received n virus particles is Pnl(1 - e-s). The fraction of infected cells which receive both n particles and an undamaged particle, and may therefore produce a plaque, Fn, is given byPn[l - (1 - S/S,)“]/[l - ems].Finally, the fraction of infected cells in the total population which can form plaques is 2 &, Fn. This fraction has been previously calculated by Luria and La&jet (1947) for bacteria infected with UV-irradiated bacteriophage, assuming that the logarithm of the survival of singly infected cells decreased as a linear function of UV dose (single hit kinetics). When our survival curves were linear, survival values for multiply infected cells based on formulas presented by Luria and Latarjet (1947) were in excellent agreement with those calculated by our method, but when our survival curves deviated from linearity, small differences were obtained (data not shown).
shown to reduce cellular survival in UVrradiated XP variant cells and to block the residual ability of these cells to replicate ;heir damaged DNA (Arlett et al., 1975; Lehmann et al., 1977). Addition of caffeine
to the infective center assay of singly infected XP variant cells, XP4BE, had no effect on the survival of plaque-forming ability (data not shown). Virus production from repair-deficient
496
HALL, FEATHERSTON,
cells infected with multiple UV-damaged herpes genomes indicated that multiplicity reactivation occurred in both XP and XP variant cells (Fig. 2, panels B and C, compare lower and upper curves) and in XP variant cells in the presence of caffeine (data not shown). In Fig. 3, the logarithms of virus survival in singly infected cells have been replotted against the logarithms of virus survival in multiply infected cells using the data for normal, XP, and XP variant cells from Fig. 2, panels A, B and C. Because the slopes of these curves are almost identical, it was concluded that XP and XP variant cells have the same capacity for multiplicity reactivation of UV-irradiated HSV-1 as do normal cells. Stocks of HSV-1 typically contain defective particles (which may carry incomplete genomes) in loo-fold excess over plaqueforming particles (Wagner et al., 1974). These particles might also be expected to contribute toward multiplicity reactivation of HSV-1. We have obtained preliminary evidence for such an involvement by demonstrating that multiplicity reactivation can be detected at lower multiplicities of infection than those employed in the above experiments. A determination of the amount of UV-irradiated virus suspension added to cells in the experiment in Fig. 1 indicated that the survival curve deviated from linearity at a point where cells were infected at multiplicities of infection slightly greater than 0.01 plaque-forming units per cell. In addition, an experiment was performed in which cells were infected with UV-damaged virus at an average multiplicity of infection of 0.17 plaque-forming units per cell and the survival of virus-producing cells compared to that measured for cells infected with 5 x lop3 plaque-forming units per cell. As shown in Fig. 2, panel D, the yield of virusproducing cells from cultures infected at the higher multiplicity was substantially greater than that observed for cells infected at the lower multiplicity. Therefore, significant multiplicity reactivation occurred in cells receiving an average of only 0.17 plaqueforming units per cell, despite the fact that only 8.3% of the infected cells received two or more plaque-forming particles, as calculated from a Poisson distribution.
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0.3 0. I 1.0 SURVIVINQ FRACTION IN MUJIPLY-WECYEO
CELLS
FIG. 3. Survival of plaque formation by human fibroblasts infected with UV-irradiated herpes simplex virus. Data from Fig. 2, panels A, B, and C are replotted to compare the survivals in singly infected cells with those in multiply infected cells. The curves were determined from the least-squares fit of an exponential curve to the data points. -O-, normal (CRL1220); -- 0 --, XP (XP12BE); -.- 0 -.-, XP variant (XP4BE).
W-Stimulated
Recombination
In bacteriophage-infected bacteria, multiplicity reactivation is generally considered to involve genetic recombination. In support of this mechanism, it has been observed that UV is recombinogenic for phage systems which show multiplicity reactivation and that recombination does not occur in the absence of a functional system for genetic recombination (see Introduction). In order to test whether UV light is recombinogenic for UV-irradiated HSV-1, genetic crosses between UV-irradiated HSV-1 mutants were performed and the recombination frequency determined as a function of UV dose. Cells were multiply infected with each of a pair of temperature sensitive HSV-1 mutants, and, following appropriate incubation times at the permissive temperature to allow viral growth
REPAIR OF UV-IRRADIATED
loo
50 UV
DOSE
0
( J/m*
497
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200
400
1
FIG. 4. Yield of temperature-resistant herpes virus recombinants in human fibroblasts infected with UV-irradiated temperature-sensitive mutants. Crosses between pairs of viral mutants were performed as described in Materials and Methods, and the recombination frequencies calculated. Spontaneous recombination frequencies were determined as the average of two separate infections in each experiment as shown by error bars in the figure. The results of two separate experiments for each cross are presented. (A) A16 x F17, only Al6 was irradiated, (B) E6 x G8, both viruses were irradiated at identical UV doses. Plaque-forming units (titers determined prior to irradiation) added per cell were: 8 (A16), 2 (F1’7), 5 (E6), 5 (G8).
and recombination, progeny virus was harvested and the fraction of temperature resistant recombinant virus determined. Since the herpes simplex genome undergoes frequent rearrangements during normal growth of the virus (Roizman, 1979), viral mutants were chosen which map exclusively in a genetically stable region of the genome in order to avoid any contribution of these rearrangements to the recombination frequencies measured. This region, designated U,, is at the left end of the herpes genome and is one of two regions of nonrepetitive DNA in the HSV-1 genome (Roizman, 1979). Temperature-sensitive mutants from different complementation groups were employed and the infection was carried out at the permissive temperature in order to minimize any effects of viral temperature sensitive functions on recombination. The data in Figure 4 show typical variations in recombination frequencies as a function of UV dose with two different sets of viral mutants: Al6 and F1’7 (panel A); E6 and GS (panel B). Only one of the two
virus parents was UV irradiated in the experiments in panel A, and both parents were UV irradiated in the experiments depicted in panel B. For each mutant pair and each irradiation procedure, the recombination frequencies increased with UV dose to a maximum value and then showed no further increase. These data provide evidence that UV light is recombinogenic for HSV-1. Moreover, this stimulation of recombination appears to be a general phenomenon because it was observed using two different sets of viral mutants. The fact that increased recombination frequencies were observed whether one parent or both were irradiated prior to infection suggests that UV damage in one genome was able to promote exchanges with a homologous region in a second undamaged genome. In order to identify conditions which might maximize the stimulation of recombination by UV light, crosses employing different multiplicities of infection of each of the two parent viruses were performed. The crosses shown in Fig. 4, panel B employed a multiplicity of infection of 5 plaque-
498
HALL,
FEATHERSTON,
forming units per cell of each parent. Reducing the multiplicity of infection of one parent relative to the other tended to reduce the spontaneous recombination frequency, presumably because such conditions reduce the chance of exchange events which can yield temperature-resistant progeny. Experiments employing such uneven multiplicities of infection are shown in Fig. 4, panel A. In these experiments, UVstimulated recombination could be readily detected. Crosses between viral mutants in XP (XPlZBE) and XP variant (XP4BE) cells were also conducted using the protocol described in Fig. 4, panel B. The data (Table 2) indicated that spontaneous viral recombination occurred at a level comparable to that observed with the normal cell line (CRL1220). In addition, the extent of stimulation of recombination by UV light was comparable to that observed for CRL1220 cells (Table 2). These results suggest that both viral recombination and UV-stimulated viral recombination do not require the repair functions deficient in these mutant host cells.
AND ALMY TABLE
2
RECOMBINATIONBETWEENHERPESTEMPERATURESENSITIVE MUTANTS (E6 AND Gs) IN HUMAN FIBROBLAST HOSTS"
Cell line
CRL1220 XPlPBE (XP) XP4BE (XP variant)
Relative increase in recombination frequency by virus irradiated with:
SpOMZW?OUS recombination frequency (%I
20 JhP
40Jim2
109 J/m2
10.3 8.5
1.7 1.4
1.8 2.0
1.3 1.5
13.0
1.5
1.9
1.6
' The protocol usedhasbeendescribedin Fig. 4, panel B. The data for CRL1220are also included in this figure. Spontaneous recombination frequencies were determined from two separate infections in each experiment and an average of these values was calculated.
cells may involve both host cell and virally coded functions. The involvement of host functions in multiplicity reactivation has been demonstrated by Selsky et al. (1979), who showed that repair of UV-irradiated herpes is reduced in a multiply infected Bloom’s Syndrome cell line. We have tested this same cell line for recombination between undamaged herpes temperature-sensitive mutants (J. Featherston and J. Hall, DISCUSSION unpublished data) and have obtained approximately normal viral recombination freWe have demonstrated that multiplicity reactivation of UV-irradiated HSV-1 and quencies. Consequently, the deficiency in UV-stimulated recombination of this virus multiplicity reactivation in this cell line is both occur in human cells. Others have dem- not due to a recombination defect, but rather onstrated multiplicity reactivation of UV- these cells may lack a DNA repair function irradiated animal viruses, including HSV-1 which acts on damaged DNA at a step prior and DasGupta and to genetic recombination in the multiplicity (see Introduction), Summers (1980) have independently shown reactivation pathway. Our studies with XP that UV light is recombinogenic for HSV-1. and XP variant cells indicate that UV-stimOur results have demonstrated that these ulated viral recombination and multiplictwo processes are both observed under the ity reactivation of UV-irradiated HSV-1 same experimental conditions and the same both occur in these repair-deficient cells. Either multiplicity-dependent repair and range of UV doses and are, therefore, likely viral recombination do not require the celto be functionally related. Taken together, these results provide evidence that recom- lular functions deficient in these host cells bination is an important mechanism for re- or herpes virus provides its own functions for these processes. pair of UV-irradiated herpes simplex virus. We propose two possible recombination The existence of a recombinational repair mechanism in animal virus-infected cells is mechanisms for multiplicity reactivation of UV-irradiated herpes virus. First, repair or made more plausible by the well-established role of recombination in repair of UV dam- replication of UV-damaged viral chromosomes could give rise to structures which age in bacteria and bacteriophage-infected are not able to undergo additional rounds of cells (see Introduction). replication unless they are modified by reMultiplicity reactivation and UV-stimucombination. For example, as is thought to lated recombination in herpes-infected
REPAIR OF UV-IRRADIATED
be the case in bacteria (Rupp and HowardFlanders, 1968), DNA containing UV-induced damage could be replicated leaving gaps in the progeny strands opposite the damaged sites. These discontinuities might be later filled by recombination events with sister chromosomes, as in bacteria (HowardFlanders et al., 1968; Rupp et al., 1971; Ganesan, 1974). Alternatively, replication might be blocked by DNA damage (as suggested by Sarasin and Hanawalt, 1980; Meneghini and Hanawalt, 1976; Park and Cleaver, 1979) and might not continue until recombination has occurred. Such repeated removal of replication blocks by a recombination mechanism would permit additional rounds of replication despite the continued presence of UV damage. A second possible mechanism for multiplicity reactivation might involve removal of damaged regions from viral DNA by a breakage and joining mechanism which reconstructs undamaged virus from the undamaged portions of irradiated genomes. How such a mechanism would recognize damaged as opposed to undamaged regions is a matter of speculation. It is additionally possible that host or viral recombination functions not normally present in herpes-infected cells might be induced by the presence of damaged viral DNA. These functions might promote multiplicity reactivation by increasing genetic recombination, thereby promoting greater activity of one of the mechanisms of exchange described above. In Escherichia coli, UV irradiation stimulates the production of recA protein (Gudas and Mount, 1977), a protein also required for genetic recombination (Low, 1968). There is a report of an inducible host cell function in monkey cells which enhances repair of UV-irradiated HSV-1 in singly infected cells (DasGupta and Summers, 1978), but no information is presently available about possible inducible functions which might be active in multiply infected cells. Although we favor recombination as the mechanism for multiplicity reactivation of herpes virus due to the stimulation of herpes recombination by UV irradiation, our data cannot totally exclude other mechanisms for multiplicity reactivation, such as complementation between damaged genomes. Such a mechanism would require that in
HERPES VIRUS
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cells infected by lethally damaged viruses, essential viral functions could be provided by several damaged genomes, thus allowing replication of at least some of these genomes. Our results are best accounted for by proposing that genetic recombination promotes repair of UV-irradiated HSV-1 in multiply infected cells. These results raise the possibility that recombination may also constitute a repair mechanism for the host cell. ACKNOWLEDGMENTS This work was supported by the National Cancer Institute Grant 5 ROl CA20429. The authors thank Dr. D. W. Mount for many helpful discussions, Karen Scherer for excellent technical assistance, and Dr. P. Schaffer for the generous gift of the herpes mutants. REFERENCES ABEL, P. (1962). Multiplicity reactivation and marker rescue with vaccinia virus. Virology 17, 511-519. ARLETT, C. F., HARCOURT,S. A., and BROUGHTON, B. C. (1975). The influence of caffeine on cell survival in excision-proficient and excision deficient xeroderma pigmentosum and normal human cell strains following ultraviolet-light irradiation. Mut. R&s. 33, 341-346. ARLETT, C. F., and LEHMANN, A. R. (1978). Human disorders showing increased sensitivity to the induction of genetic damage. Ann. Rev. Genet. 12, 95-115. BENYESH-MELNICK,M., SCHAFFER,P. A., COURTNEY, R. J., ESPARZA, J., and KIMURA, S. (1974). Viral gene functions expressed and detected by temperature sensitive mutants of herpes simplex virus. Cold Spring Harbor Symp. Quant. Biol. 34, 731-746. CLEAVER, J. E. (1974). Repair processes for photochemical damage in mammalian cells. Adv. Radiat. Biol. 4, l-75. CLEAVER, J. E. (1978). DNA repair and its coupling to DNA replication in eukaryotic cells. Biochim. Biophys. Acta 516, 489-516. DASGUFTA, U. B., and SUMMERS, W. C. (1978). Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammalian cells. Proc. Nat. Acad. Sci. U. S. A. 75, 2378-2381. DASGUPTA, U. B., and SUMMERS,W. C. (1980). Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus. Molec. Gen. Genet. (in press). DUBBS, D. R., RACHMELER, M., and KIT, S. (1974). Recombination between temperature-sensitive mutants of simian virus 40. Virology 57, 161-174. EPSTEIN, R. H. (1958). A study of multiplicity-re-
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