Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells

Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICdR-resistant mutants did not have a growth advantage over wild-type virus in irradiated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis?) became more mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not lead to higher mutagenesis in unirradiated cells. Thus the data did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic.     

Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts

Host-cell reactivation (HCR) of UV-irradiated herpes simplex virus type 2 (HSV-2), capacity of UV-irradiated cells to support HSV-2 plaque formation and UV-enhanced reactivation (UVER) of UV-irradiated HSV-2 were examined in fibroblasts from 4 patients with Cockayne syndrome (CS), 5 with xeroderma pigmentosum and 5 normals. All UV-survival curves for HSV-2 plaque formation showed 2 components. HCR was similar to normal for the XP variant strain and the 2 CS strains tested, but substantially reduced in the 4 excision-deficient XP strains. The capacity of UV-irradiated fibroblasts to support HSV-2 plaque formation was determined by UV-irradiating fibroblast monolayers with various doses of UV and 48 h later, infecting the monolayers with unirradiated HSV-2. The D37 values for the delayed-capacity curves so obtained were in the range 8.6-12.4 J/m2 for the normal strains, 2.8-3.2 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.3 and 1.5 for the XP excision-deficient strains tested. These results indicate that delayed capacity for HSV-2 plaque formation is a more sensitive assay than HCR in the detection of cellular DNA-repair deficiency for XP and CS. For the examination of UVER, fibroblasts were irradiated with various UV doses and subsequently infected with either unirradiated or UV-irradiated HSV and scored for plaque formation 2 days later. UVER expression was maximum when the delay between UV-irradiation of the cells and HSV infection was 48 h. The magnitude of UVER expression was also found to be dependent on the UV dose to the cells and increased with increasing UV dose to the virus. Using a UV dose to the virus resulting in a plaque survival of about 10(-2) on unirradiated cells, the the maximum UVER factor had a mean value of 1.3 for the normal strains following a dose of 15 J/m2 to the cells. Somewhat higher UVER values were found for all the patient strains tested and resulted from lower UV doses to the cells than for normal strains. Maximum UVER factors for the CS strains ranged from 2.2 to 3.3 at a dose of 5 J/m2 to the cells, for the XP excision-deficient strains; 2.1 to 2.6 at doses of 0.5 to 2.5 J/m2 to the cells and for the XP variant strain tested; 2.5 at UV dose of 10 J/m2 to the cells.     

The effect of cell irradiation on mutation in ultraviolet-irradiated and intact simian virus 40

The induction of phenotypic wild-type revertants in the progeny of an unirradiated or UV-irradiated temperature-sensitive late mutant of simian virus 40 was studied after low multiplicity passages in normal or UV-irradiated confluent monkey kidney cells. The production of wild-type revertants in the progeny of undamaged tsBC245 was followed by infecting the cells at distinct times after irradiation of the cells. Mutation frequencies reached a maximum when infection was delayed for 3--4 days after irradiation of the host cells, and declined gradually thereafter. Virus grown in unirradiated cells did not show such an alteration in mutation frequency. The temporarily higher mutation frequency of virus in UV-pretreated cells is due to a transient mutator activity operating in these cells rather than to an increased number of replications performed in UV-irradiated cells. A similar time course was found for the reactivation of UV-damaged SV40. This might suggest that reactivation and mutagenesis are manifestations of the same process. The yield of mutants due to irradiation of the virus alone was enhanced when infection was delayed for some days after the cells reached confluency; UV pretreatment of the host cells did not enhance the level of mutation obtained by UV irradiation of the virus.     

Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) on UV-irradiated monkey kidney CV-1P cells at 33° was increased over survival on unirradiated cells. During this process — called induced-virus reactivation — the progeny virus yielded by UV-irradiated cells had a much higher mutation frequency than did the progeny from unirradiated cells. Mutation rates were quantified by using phenotypic reversion towards wild-type growth of an early (tsA 58) or a late (tsB 201) temperature-sensitive SV40 mutant. Analysis of SV40 revertant genomes indicated that no detectable deletions or additions were resposible for the reversion process.These results suggest that enzymes from UV-irradiated cells are able to replicate UV-irradiated DNA by an error-prone mode of DNA repair. Induced virus reactivation and error-prone replication are probably one of the expressions of SOS functions in mammalian cells.     

Radiation enhanced reactivation of herpes simplex virus: effect of caffeine.

Ultaviolet enhanced (Weigle) reactivation of UV-irradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cell monolayers was decreased by caffeine. X-ray enhanced reactivation of UV-irradiated virus in X-irradiated monolayers (X-ray reactivation) and UV- or X-ray-inactivated capacity of the cells to support unirradiated virus plaque formation were unaffected by caffeine. The results suggest that a caffeine-sensitive process is necessary for the expression of Weigle reactivation for herpes virus. Since cafeine did not significantly affect X-ray reactivation, different mechanisms may be responsible for the expression of Weigle reactivation and X-ray reactivation.     

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in normal human fibroblasts

An enhanced reactivation (UVER) of U.V.-irradiated as well as of gamma-irradiated human adenovirus type 2 (Ad 2) was detected following infection of normal human fibroblasts which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad 2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Results obtained using 5 different normal fibroblast strains showed that irradiation of host monolayers with 10J/m2 immediately prior to infection gave a U.V. enhanced reactivation (UVER) factor +/- standard error equal to 3 . 1 +/- 1 . 2 for virus U.V.-irradiated with 1 . 2 x 10(3) J/m2, and 2 . 1 +/- 0 . 5 for virus gamma-irradiated with 2 x 10(4) Gy. For a fixed survival of about 5 . 9 x 10(-2) for irradiated virus, the efficiency of UVER for gamma-irradiated virus was about 0 . 18, slightly less than the value of about 0 . 24 obtained for U.V.-irradiated virus. The results of time course experiments indicated that while U.V.-irradiation of normal host monolayers prior to infection gave rise to an increased rate of Vag formation for infection by unirradiated Ad 2, U.V.-irradiation of the cells increased the proportion of cells able to repair U.V.-damaged virus as well as allowing an earlier onset and/or increased rate of synthesis of Vag from a U.V.-damaged template. Similar experiments involving gamma-ray enhanced reactivation (gamma-RER) of irradiated Ad 2 indicated that gamma-RER and UVER may operate, in part at least, by different mechanisms in normal human cells.     

Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C-treated host cells only at a low multiplicity of infection.

Treatment of monkey kidney cells with mitomycin C (MMC) 24 h prior to infection with UV-irradiated simian virus 40 (SV40) enhanced both virus survival and virus mutagenesis. The use of SV40 as a biological probe has been taken as an easy method to analyse SOS response of mammalian cells to the stress caused by DNA damage or inhibition of DNA replication. The mutation assay we used was based on the reversion from a temperature-sensitive phenotype (tsA58 mutant) to a wild-type phenotype. The optimal conditions for producing enhanced survival and mutagenesis in the virus progeny were determined with regard to the multiplicity of infection (MOI). Results showed that the level of enhanced mutagenesis observed for UV-irradiated virus grown in MMC-treated cells was an inverse function of the MOI, while enhanced survival was observed at nearly the same level regardless of the MOI. For the unirradiated virus, almost no increase in the mutation of virus progeny issued from MMC-treated cells was observed, while a small amount of enhanced virus survival was obtained. These results show that enhanced virus mutagenesis and enhanced virus survival can be dissociated under some experimental conditions. Enhanced virus mutagenesis, analogous to the error-prone replication of phages in SOS-induced bacteria, was observed, at least for SV40, only when DNA of both virus and host cells was damaged and when infection occurred with a small number of viral particles. We therefore hypothesize that an error-prone replication mode of UV-damaged templates is observed in induced monkey kidney cells.     

UV-reactivation,virus production and mutagenesis of SV40 in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) is higher in UV-irradiated than in non-irradiated monolayers of BSC-1 monkey cells. A similar reactivation is found when cells are infected with SV40-DNA, suggesting that reactivation acts on viral DNA. The enhanced reactivation of UV-irradiated SV40 and SV40-DNA is optimal when infection is delayed for 2–3 days after irradiation of the cells.UV-pretreated cells infected with SV40-DNA produce more virus than infected control cells; the time curve of this process is similar to that found for enhanced virus reactivation and suggests that facilitated virus production in UV-irradiated cells and enhanced virus reactivation might be manifestations of the same process.If the non-irradiated SV40 thermosensitive mutant BC245 is propagated in UV-irradiated BSC-1 cells the rate of back mutation to phenotypically wild-type is increased compared with that of the control. This suggests that an inducible error-prone system is functional in these cells. When the UV-irradiated tsBC245 is propagated in non-irradiated cells the reversion frequency is greatly enhanced, which suggests that either the introduction of UV-irradiated SV40-DNA is sufficient to induce an error-generating system, or that a constitutive error-prone mechanism is operative on this DNA.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts

Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

Reactivation and mutagenesis of herpes virus in 5-azacytidine-treated monkey kidney cells

Enhanced survival of UV-damaged herpes simplex virus and Simian virus 40 was investigated in CV-1 monkey cells treated with inhibitors of DNA methylation such as 5-azacytidine and ethionine. Survival of UV-irradiated virus was higher in treated cells than in untreated cells. Survival of herpes virus irradiated with 60Co gamma-ray was not enhanced in the treated cells. The frequency of forward mutation of herpes virus increased in 5-azacytidine-treated cells. Relative content of methylcytosine was reduced in the cells treated with 5-azacytidine. Therefore a mechanism similar to UV-enhanced reactivation of virus was operating in the cells with hypomethylated DNA.     

UV-Irradiation of related mouse hybrid cells: Similar increase in capacity to replicate intact minute-virus-of-mice but differential enhancement of survival of UV-irradiated virus

3 hybrid cell lines between mouse fibroblasts (A9) and mouse lymphoma cells (L5178YS) were compared with regard to the ability of UV-pre-irradiated cells to replicated intact (unirradiated) Minute-Virus-of-Mice (MVM) and to reactivate UV-irradiated MVM. UV irradiation of cells before virus infection enhanced their ability to plaque intact virus (Enhanced Capacity) to a similar extent in the 3 hybrid cell lines. However, pretreatment of cells with UV radiation enhanced the survival of UV-damaged virus (Enhanced Reactivation) in only 1 of these hybrids. The lack of detectable Enhanced Reactivation in the other hybrids without concomitant change in their Enhanced Capacity, suggets that these processes are at least partly independent. Virus survival in unirradiated cells was similar for all 3 hybrid cell lines, indicating that the absence of detectable Enhanced Reactivation in 2 of the hybrids was not due to the constitutive expression of this process, but might rather result from its loss or extinction. The expression of both Enhanced Capacity and Enhanced Reactivation requires synthesis of protein de novo shortly after cell irradiation.     

Relationship between enhanced reactivation and mutagenesis of u.v.-irradiated human cytomegalovirus in normal human cells.

The survival of u.v.-irradiated human cytomegalovirus (HCMV) on u.v.-irradiated human IAFP-1 cells was increased over that on unirradiated cells. Irradiated virus had a higher forward mutation frequency towards temperature sensitivity in irradiated than in unirradiated cells. Enhanced reactivation of u.v.-irradiated HCMV is thus mutagenic in normal human cells. This observation supports the possible induction of an error-prone mode of DNA repair in u.v.-irradiated mammalian cells.     

Enhanced reactivation and enhanced mutagenesis of herpes simplex virus in normal human and xeroderma pigmentosum cells.

Enhanced reactivation (ER) and enhanced mutagenesis (EM) of herpes simplex virus type 1 were studied simultaneously in UV-irradiated stationary cultures of diploid normal human and xeroderma pigmentosum (XP) fibroblasts. Mutagenesis was assayed with unirradiated herpes simplex virus type 1 as a probe in a forward mutation assay (resistance to iododeoxycytidine). Dose-response studies showed that ER increased with the UV dose given to the virus. Optimal reactivation levels were obtained when normal cells and XP variant cells were exposed to a UV dose of 8 J . m-2 and the virus was irradiated with 150 J . m-2. Repair-deficient XP cells of complementation groups A, C, and D showed optimal reactivation levels with a UV dose to the cells of 1.0 J . m-2 and a UV dose to the virus of 40 J . m-2. The time course of appearance of ER and EM was also studied, both in the normal and XP cells. In all cell types except the XP variant cells, EM followed similar kinetics of appearance as did ER. Maximal activities occurred when infection was delayed 1 or 2 days after cell treatment. In XP variant cells, however, maximal expression of the EM function was significantly delayed with respect to ER. The results indicate that ER and EM are transiently expressed in normal and repair-deficient XP cells. Although both phenomena may be triggered by the same cellular event, ER and EM appear to be separate processes that occur independently of each other.     

Rescue of Simian Virus 40 from Cell Lines Transformed at High and at Low Input Multiplicities by Unirradiated or Ultraviolet-irradiated Virus

The relation between simian virus 40 (SV40) input multiplicity during transformation of primary mouse kidney cultures and the subsequent rescue of SV40 from clonal lines of transformed cells has been studied. Primary mouse kidney cultures were transformed with unirradiated SV40 at input multiplicities varying from 0.06 to 200 plaque-forming units (PFU) /cell or with SV40 irradiated with ultraviolet (UV) light to a survival of 0.04 to 0.01. All of the transformed lines contained the intranuclear SV40 T antigen, but cell-free extracts prepared from the transformed cell lines failed to yield infectious virus when assayed on monkey kidney cell (CV-1) monolayers. After fusion with susceptible CV-1 cells induced by UV-inactivated Sendai, all of the lines transformed by unirradiated virus yielded infectious SV40. The frequency of induction and the incidence of successful trials did not depend on the multiplicity of infection. “Good” yielders were obtained from mouse kidney cells transformed at the low input multiplicity of 0.06 PFU /cell. In contrast, only 4 of 12 clonal lines transformed at moderately low input multiplicity, and none of the lines transformed at very low input multiplicity with UV-irradiated virus yielded infectious SV40. The four positive lines have been classified as “poor” or “rare” yielders.     

Enhanced mutagenesis parallels enhanced reactivation of herpes virus in a human cell line.

U.v. irradiation of human NB-E cells results in enhanced mutagenesis and enhanced reactivation of u.v.-irradiated H-1 virus grown in those cells ( Cornelis et al., 1982). This paper reports a similar study using herpes simplex virus (HSV) in NB-E cells. The mutation frequency of HSV (resistance of virus plaque formation to 40 micrograms/ml iododeoxycytidine ) increased approximately linearly with exposure of the virus to u.v. radiation. HSV grown in unirradiated cells gave a slope of 1.8 X 10(-5)m2/J, with 3.2 X 10(-5)m2/J for HSV grown in cells irradiated (3 J/m2) 24 h before infection. There was no evidence for mutagenesis of unirradiated virus by irradiated cells, as seen with H-1 virus. Enhanced reactivation of irradiated HSV in parallel cultures increased virus survival, manifested as a change in slope of the final component of the two-component survival curve from a D0 of 27 J/m2 in unirradiated cells to 45 J/m2 in irradiated cells. Thus, enhanced mutagenesis and enhanced reactivation occurred for irradiated HSV in NB-E cells. The difference in the enhanced mutagenesis of HSV (dependent on damaged DNA sites) and of H-1 virus (primarily independent of damaged DNA sites) is discussed in terms of differences in DNA polymerases.     

Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease

Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).