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.     

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.     

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.     

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.     

Apurinic sites cause mutations in simian virus 40

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.     

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.     

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.     

Enhanced induction of SV40 replication from transformed mammalian cells by fusion with UV-irradiated untransformed cells

DNA-damaging agents such as ultraviolet (UV) light are known to cause stimulation of virus replication in SV40-transformed hamster and human cells. The dose-response curves of UV-induced SV40 replication in transformed hamster cells resemble that obtained for UV-enhanced reactivation (ER) and UV-enhanced mutagenesis (EM) of SV40 or herpes viruses in mammalian cells. We have investigated whether UV-enhanced production of SV40 from transformed hamster (THK) and human (NB-E) cells belongs to the same category of conditional responses as ER and EM. To answer this question we have made use of the phenomenon that fusion of the SV40-transformed cells with monkey cells that are permissive to SV40 results in a considerable increase in the production of SV40 virus. When THK or NB-E cells were fused with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased and reached a maximum value of 2--3-fold when fusion was delayed for 24-48 h after irradiation. The kinetics of enhanced SV40 induction resembled that of ER and EM, suggesting that the UV-stimulated part of the induction represents one of the pleiotropic responses that are transiently induced in mammalian cells by DNA-damaging agents. Evidence is presented, showing that this transient effect can be induced only in cells that are permissive to SV40 replication. This suggests that the enhanced induction observed after fusion with irradiated monkey cells may be attributed to a transient increase in the activity of "permissiveness' factors. No enhanced induction was found when the THK or NB-E cells were fused with irradiated rodent cells, that are not or only slightly permissive to SV40 replication.     

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.     

Regulation of SV40-induced cell division and tumor antigen by dibutyryl adenosine 3′-5′-monophosphate

Simian virus 40 (SV40) induces cell division in microcultures of sparsely plated nongrowing mouse BALB/3T3 cells during acute infection at moderate multiplicities of infection (MOI = 10–100). The infected cells are killed when a MOI of 1,000 is used. SV40 tumor (T) antigen is synthesized in the infected cells, but viral DNA, virion antigen, and progeny virions are not synthesized (abortive infection). The addition of exogenous dibutyryl adenosine 3′-5′-monophosphate (dbcAMP) at the time of infection stimulates the SV40-induced cell division at all MOI and inhibits SV40-induced cell death at high MOI. The percentage of T antigen-positive cells, as monitored by immunofluorescence, is also increased by the addition of dbcAMP at the time of infection. This regulation of SV40-induced cell division and T antigen formation by exogenous dbcAMP occurs within the first 6 hr after infection at 37° C and is dependent upon both the MOI and the concentration of added dbcAMP. The addition of dbcAMP to productively infected TC7 monkey cells has little effect on the SV40-induced cell death or T antigen formation.     

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.     

Influence of SV40 Genome on the Replication of an Adenovirus-SV40 "Hybrid" Population.

Feldman, L. A. (Baylor University College of Medicine, Houston, Tex.), J. L. Melnick, and F. Rapp. Influence of SV40 genome on the replication of an adenovirus-SV40 "hybrid" population. J. Bacteriol. 90:778-782. 1965.-Replication of a type 7 adenovirus-SV40 hybrid population in primary African green monkey kidney cells was accompanied by the formation of SV40 tumor antigen, adenovirus antigens, and cytopathic changes characteristic of adenovirus infection. Prior infection of the cultures with SV40 stimulated replication of nonintegrated adenovirus 7 but did not enhance the replication of the hybrid virus. These results suggest that the population of the adenovirus-SV40 hybrid studied contains many particles carrying SV40 information. Replication of SV40 virus was not enhanced by co-infection with nonintegrated adenovirus 7 or with the adenovirus-SV40 hybrid. Cytosine arabinoside strongly inhibited replication of the adenovirus-SV40 hybrid population in African green monkey kidney cells. Enhanced replication of nonintegrated adenovirus 7 by SV40 was blocked by cytosine arabinoside; this block could be reversed by 2-deoxycytidine or deoxycytidine triphosphate.     

Enhanced reactivation and mutagenesis after transfection of carcinogen-treated monkey kidney cells with UV-irradiated simian virus 40 (SV40) DNA

Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.     

Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells

Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection

Background

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.

Results

The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.

Conclusions

(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.     

Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state.