Multiplicity Reactivation Research Articles

Bromodeoxyuridine-labeled bacteriophage T4D. I. Growth parameters and frequency of markers recovered from BUdR-labeled and from non-labeled phages studied in mass lysates.

T4D bacteriophages, grown on heavy Escherichia coli strain CR63 in a synthetic medium containing BUdR, FUdR and uracil, showed a uniform BUdR labeling as judged from visible light inactivation and from CsCl density gradients. A variable number of “dead” BUdR phages were found in different BUdR phage lysates by comparing plaque former titer with complementation titer. The “dead” BUdR phages had the same mean density in CsCl as plaque-forming BUdR phages from the same lysate, indicating that “dead” BUdR phages do not suffer from a large deficiency of DNA. The “dead” BUdR phages participated in multiplicity reactivation. Production of intracellular phages was delayed in BUdR experiments compared to control experiments, especially when both parents in a cross were BUdR labeled, and when BUdR phages were used for single infection. In all Type A crosses (one parent BUdR-labeled, one parent non-labeled) the recovery of markers was significantly lower from BUdR parents than from the corresponding non-labeled parents in the control crosses. In one Type B experiment (both parents BUdR-labeled) the frequency of recombinants was significantly higher than in all Type A experiments.

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA + or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD + allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.

Inactivation Kinetics of Viral Aggregates

A kinetic model is developed for the inactivation of viruses that exhibit a range of initial aggregate sizes. Aggregates are assumed to be more resistant to inactivation than single virions because (1) all virions within an aggregate must be inactivated before the aggregate as a whole is considered inactive; and (2) undamaged components of inactive virions within an aggregate may recombine to cause host-cell infection (multiplicity reactivation). Survival curves calculated from the model compare well with inactivation data published in a previous study. The analysis presented in this report provides an approach for estimating the contact time (or disinfection concentration) required to inactivate viral aggregates, and a framework for predicting virus survival in water-treatment systems and natural environments.

The genetics of the Luria-Latarjet effect in bacteriophage T4: evidence for the involvement of multiple DNA repair pathways.

The Luria-Latarjet effect is an increase in resistance of a virus to DNA damage during infection of a host. It has often been assumed to involve recombinational repair, but this has never been demonstrated experimentally. Using nine bacteriophage (phage) T4 mutants, I present evidence indicating that, for phage T4, the Luria-Latarjet effect is due to three repair pathways-excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR) (a second form of recombinational repair). The results also show that the Luria-Latarjet effect develops in two stages. The first stage starts soon after infection. Damage which occurs during the first stage can be repaired by excision repair or PRRR. The second stage appears to start after the first round of DNA replication is complete. DNA damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways. The results of this study support the hypothesis that recombinational repair has been selected to ensure that the progeny phage genomes which are packaged have minimum DNA damage. Since other viruses which infect bacterial, animal and plant cells show a Luria-Latarjet effect similar to that in phage T4, the conclusions from this study may have wide applicability.

Multiplicity reactivation and mutagenesis of trimethylpsoralen-damaged herpes virus in normal and Fanconi's anaemia cells

Fanconi's anaemia (FA) cells are hypersensitive to the lethal effect of DNA cross-linking compounds. Herpes simplex virus (HSV) has been used here as a probe to monitor in FA cells repair of psoralen damage of which cross-links are a part. The replication of HSV is impaired when its DNA contains covalently photobound psoralen molecules. In comparison to other psoralens, 4,5',8-trimethylpsoralen (4,5',8-TMP) is one of the most photoreactive psoralens and it forms a relatively high proportion of DNA interstrand cross-links. TMP-damaged HSV is efficiently reactivated by multiple infection in human fibroblasts. The extent of multiplicity reactivation is greater in cells from FA donors (five strains tested) than in normal cells (three strains). Mutagenesis studied in the viral thymidine kinase locus revealed that: (i) spontaneous viral mutation rate is lower in FA than in normal cells; and (ii) under conditions of multiple infection, the mutation rate is either greater (normal cells) or unchanged (FA cells) in the progeny from psoralen-damaged HSV compared to that from untreated virus. Taken together, these observations suggest that the pathway underlying multiplicity reactivation of psoralen-damaged HSV is error-free in FA cells relative to normal cells.

Molecular Mechanisms of Viral Inactivation by Water Disinfectants

Publisher Summary Hepatitis, gastroenteritis, and other diseases may be transmitted when viruses evade filtration and pass through water treatment plants. Treatment is required to ensure that those viruses, which successfully pass through the filtration process, become inactivated. The inactivation of bacteria is usually more easily accomplished than the inactivation of viruses. This may be because of the complexity of bacteria and the interaction between a bacterium and its environment. The complex interaction of viruses and disinfectants varies from one type of virus to another making the mode of inactivation unclear. The chapter discusses two-stage disinfection, which offers an efficient means of inactivation of viruses. In the two-stage approach to disinfection, an initial attack of capsid proteins alters the structural integrity of the virus. Second-stage disinfectants, which normally would be unable to diffuse across the intact viral capsid, are then able to reach their target sites easily and inactivate the RNA, thus reducing the likelihood of multiplicity reactivation.

Recombinational repair of hydrogen peroxide-induced damages in DNA of phage T4

Recently, hydrogen peroxide and its free-radical product, the hydroxyl radical (OH ·) have been identified as major sources of DNA damage in living organisms. They occur as ubiquitous metabolic by-products and, in humans, cause several thousand damages in a cell's DNA per day. They are thought to be a major source of DNA damage leading to aging and cancer in multicellular organisms. This raises two questions. First, what pathways are used in repair of DNA damages caused by H 2O 2 and OH·? Second, a new theory has been proposed that sexual reproduction (sex) evolved to promote repair of DNA in the germ line of organisms. If this theory is correct, then the type of repair specifically available during the sexual process should be able to deal with important natural lesions such as those produced by H 2O 2 and OH·. Does this occur? We examined repair of hydrogen peroxide damage to DNA, using a standard bacteriophage T4 test system in which sexual reproduction is either permitted or not permitted. Post-replication recombinational repair and denV-dependent excision repair are not dependent on sex. Both of these processes had little or no effect on lethal H 2O 2 damage. Also, an enzyme important in repair of H 2O 2-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H 2O 2 damage to the phage. However, multiplicity reactivation, a recombinational form of repair depending on the sexual interaction of two or more of the bacteriophage, was found to repair lethal H 2O 2 damages efficiently. Our results lend support to the repair hypothesis of sex. Also the homology-dependent recombinational repair utilized in the phage sexual process may be analogous to the homology-dependent recombination which is widespread in diploid eucaryotes. The recombinational repair pathway found in phage T4 may thus be a widely applicable model for repair of the ubiquitous DNA damage caused by endogenous oxidative reactions.

SOS-type functions in mammalian cells. Enhanced reactivation of UV-irradiated SV 40 in UV-irradiated CV-1 cells.

The reactivation of UV-irradiated SV 40 was measured in UV-irradiated CV-1 cells as a function of time between irradiation of the cells and infection. To avoid the possible bias of multiplicity reactivation and/or virus propagation, infection was quantitated in terms of V-antigen-positive nuclei at the end of the first lytic cycle. In irradiated cell cultures enhanced reactivation of SV 40 was observed, which indicates induction of DNA repair enzymes. Maximal reactivation was obtained when the time interval between irradiation and infection was 72 h. The UV-inducible DNA repair enzymes might represent elements of an SOS-type response in mammalian cells.

Multiplicity reactivation of bacteriophage T7 inactivated by methyl methanesulfonate.

Experiments were conducted to determine whether phage T7 treated with methyl methanesulfonate used multiplicity reactivation to repair alkylation lesions. This type of repair was found to be operative at high multiplicities in actively growing wild-type Escherichia coli B cells.

Gene 49 endonuclease VII is not essential for multiplicity reactivation of bacteriophage T4.

Endonuclease VII, the product of phage T4 gene 49, has been shown previously to resolve Holliday structures in vitro. Two different processes, genetic recombination and multiplicity reactivation are presumed to have Holliday structure intermediates. Other workers have shown that genetic recombination is reduced in a gene 49 mutant infection. However, in the present study, multiplicity reactivation of UV-irradiated ts or amber mutant phage defective in gene 49 was nearly identical to that of UV-irradiated wild-type phage T4. Thus endonuclease VII is not thought to be essential for multiplicity reactivation of phage T4.

Ultraviolet irradiation of murine cytomegalovirus.

Ultraviolet irradiation of murine cytomegalovirus (MCMV) caused a rapid dose-related decline in virus infectivity, manifested by virus antigen induction, and in virus production as measured by plaque formation and infectious centre assay. The virus survival curve was multi-component, suggesting host cell-assisted reactivation. Multiplicity reactivation and photoreactivation of MCMV were not observed in these experiments. Productive infection was more sensitive to u.v. irradiation than was virus antigen production, indicating differential inactivation of virus functions. The effects of u.v. irradiation were similar in most respects to those reported for human cytomegalovirus.

Repair of psoralen-induced crosslinks in cells multiply infected with SV40.

Experiments were conducted to study the relationship between the production of interstrand crosslinks by 4,5',8-trimethylpsoralen (psoralen) in simian virus 40 DNA and the ability of psoralen to inactivate the virus. Under conditions where only single viral particles enter a given host cell, approximately one crosslink was lethal to the virus and could not be repaired. In contrast, when multiple viral genomes infected a host cell, psoralen-induced crosslinks were repaired (multiplicity reactivation). A model is proposed for multiplicity reactivation which involves genetic recombination between damaged viral genomes.

Multiplicity reactivation of alkylating agent damaged herpes simplex virus (type I) in human cells

Herpes simplex virus type I (Strain KOS) is inactivated by treatment with MMS, MNNG and HN2 as determined by plaque assay on Vero cell monolayers, or by an infectious center assay with FS2 cells, a human foreskin fibroblast line. At a given dose of MMS and MNNG, survival of the virus was significantly higher at a multiplicity of infection of 1.0 PFU/cell compared to 0.01 PFU/cell. These results indicate that HSV-1 infected human cells are capable of repairing chemically induced lesions by way of multiplicity reactivation. No evidence for multiplicity reactivation with HN2-treated virus could be obtained, however.

Alteration of bacteriophage attachment capacity by near-UV irradiation.

Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either "all" or "none" of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H2O2. H2O2 (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.

Topoisomerase involvement in multiplicity reactivation of phage T4.

The products of phage T4 genes 39, 52 and probably 60 have been previously characterized as forming a type II DNA topoisomerase. Other evidence suggested that this topoisomerase promotes normal initiation of DNA replication, and that when it is defective its loss is partially compensated for by the host gyrase. We present evidence here that mutants defective in genes 39, 52 and 60 have reduced ability to carry out multiplicity reactivation (MR, a form of recombinational repair) of phage DNA damaged either by mitomycin C (MMC) or psoralen plus near-UV light (PUVA). We also observed that there is not extensive superhelicity in the intracellular phage DNA either in the presence or absence of the phage topoisomerase. This tends to rule out the possibility that the topoisomerase influences MR by controlling the general superhelicity of the phage DNA. The dependence of MR on topoisomerase could occur in several possible ways. However, we favor the explanation that the lesions are bypassed by a postreplication recombinational repair process that is influenced by the topoisomerase through its role in initiating replication.

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitive mutants of Newcastle disease virus.

Random UV irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the UV target sizes for the complementing abilities of mutants A1 and E1. After UV irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after UV irradiation, A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1 delta P, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of delta P in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high UV doses, A1 was able to complement E1 in a different, UV-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by UV-irradiation paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiation virions.

Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by epstein-barr virus (EBV)-transformed lymphoblastoid cell lines

The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockayne's syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockayne's syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivate uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

Repair of psoralen plus near ultraviolet light damage in bacteriophages T3 and T4.

Abstract— Repair of T3 and T4 DNA damaged by treatment with 8‐methoxypsoralen plus near UV (PNUV) has been investigated. It is shown that the excision repair mechanisms of the host cell can repair a substantial fraction of the psoralen‐DNA mono‐adducts in T3 DNA, but cannot by themselves repair crosslinks. In contrast neither the excision repair system of the host nor the phage coded v gene endonuclease is involved in the repair of psoralen adducts in T4 DNA. Multiplicity reactivation is effective in the recovery of T4 DNA containing psoralen‐DNA mono‐adducts, but is ineffective in the recovery of crosslinked phages. Comparisons of the lethality of PNUV treatment and the number of crosslinks induced in T4 DNA show clearly that mono‐adducts are lethal to this phage. Both T3 and T4, however, appear to effectively repair many mono‐adducts by postreplicational repair.

Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation.

A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA. (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates. A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.