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.     

Infection of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex virus: study of capacity and Weigle reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.     

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.     

SCP1-dependent Weigle activation of the VP5 phage in Streptomyces coelicolor A3(2)

The kinetics of induction of the UV-irradiated bacteriophage VP5 (Weigle reactivation) in Streptomyces coelicolor A3(2) strains with and without plasmid was investigated. Chloramphenicol (CAF) inhibits Weigle reactivation (WR) in UF strains (SCP1 absent) but not in SCP1+ strains of IF fertility (free plasmid). CAF, moreover, inhibits protein synthesis in non-irradiated UF and IF strains. In UV-irradiated IF strains, on the other hand, protein synthesis takes place irrespective of CAF. Weigle reactivation appears to require protein synthesis: the SCP1 plasmid, by protecting protein synthesis from CAF inhibition in UV-irradiated strains, allows WR. The proteins synthesized after UV induction during the pre-incubation period were investigated and the results suggest that a new UV-induced protein, coded by a gene localized on the plasmid, interacts with the cellular SOS system.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

groE genes affect SOS repair in Escherichia coli.

Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.     

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.     

Lack of enhanced mutation of UV- and gamma-irradiated herpes virus in UV-irradiated CV-1 monkey cells

The frequency of forward mutation of unirradiated, UV-irradiated or gamma-irradiated herpes virus was determined after infecting UV-irradiated or unirradiated CV-1 monkey kidney cells, to investigate the correlation between UV-enhanced reactivation (UVER) and mutagenesis. UV-irradiation to cells had no effect on mutation frequency of irradiated virus even in the conditions in which UVER was maximally expressed for the survival of UV-irradiated virus.     

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.     

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.     

Correlation of increased nuclease activity with enhanced virus reactivation

An increase in nuctease activity, which degraded both unirradiated and ultraviolet (UV)-irradiated DNA, was observed in the extract of monkey Vero cells after irradiation with an appropriate amount of UV. In contrast, no increase was observed with mouse L cells. Neither DNA polymerases nor uracil-DNA glycosylase was enhanced but rather suppressed by UV irradiation in both cell lines. Cytological studies showed that, in Vero cells, the reactivation of UV-irradiated herpes simplex virus was markedly enhanced by irradiating cells with UV before infection. However, no enhancement was observed with L cells. These results suggest that an increase in nuclease activity may be one of underlying mechanisms for the enhanced reactivation of DNA viruses.     

X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio kappa phage

Vibrio cholerae lysogenic kappa phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated kappa phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m-2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D)--survival (S) curves could be empirically described by the equation S = exp[-(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained.     

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.     

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.     

Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101.

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.     

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.     

Reactivation of UV and γ-inactivated herpes virus in BHK cells

The DNA-repair capabilities of baby hamster kidney (BHK) cells were investigated by comparing the reactivation of irradiated herpes simplex virus type I (HSV1) in BHK cells with its reactivation in mouse fibroblasts and in normal and repairdeficient human diploid fibroblasts. BHK cells were found to have an intermediate ability to reactive UV-irradiated HSV1 (the viral D_o was 14 J/m²) relative to normal human fibroblasts (viral D_o = 19 J/m²) and xeroderma pigmentosum (XP) group A cells (viral D_o = 4.5 J/m²). With mouse L929 cells as the host, the response of the UV-irradiated virus was biphasic with D_os of 4.6 and 30 J/m² for the low- and high-dose components respectively. In contrast to the response following UV radiation, γ-irradiated HSV1 was similarly reactivated by BHK and normal human cells (the D_os for the irradiated virus in BHK and CRl 1106 were 55 and 51 krad, respectively, whereas xeroderma pigmentosum cells were slightly less efficient in the repair of γ-irradiated virus (D_o = 45 krad). UV irradiation of BHK host cells 0–48 h prior to infection enhanced the reactivation of UV-irradiated HSV.     

Absence of error-prone repair in a Vibrio species

The effect of various DNA-damaging agents on a Vibrio species was investigated. The organism was readily mutable by N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C but not by UV light. No Weigle reactivation of UV-irradiated alpha 3a phage was detected. These results suggest that an error-prone repair mechanism is lacking in this species.     

Inducible UV repair potential of Pseudomonas aeruginosa PAO

Pseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.     

The study of DNA-repair defects using [125I]iododeoxycytidine incorporation as an assay for the growth of herpes simplex virus

[125I]Iododeoxycytidine incorporation was used to measure herpes virus (HSV-1) DNA synthesis following specific DNA damage. Xeroderma pigmentosum fibroblasts were less able to replicate UV-irradiated viral DNA than were normal fibroblasts, indicating the necessity for excision repair for the survival of UV-irradiated virus. Because of its rapidity and ease of quantitation, this assay had advantages over standard viral mediated assays of DNA excision repair. It was possible to monitor viral replication as a function of the cellular cell cycle. Other genetic defects which have been proposed to reflect deficiencies in DNA-repair capacity were not detected by this assay. DNA-repair inhibitors, caffeine and 3-aminobenzamide, also did not show synergistic lethal effects on the replication of damaged viral DNA.