Proc. Natl. Acad. Sci. USA Vol. 75, No. 5, pp. 2378-2381, May 1978
Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammalian cells (SOS/Weigle reactivation/DNA repair/oncogenesis/thymidine kinase)
UMA BANDYOPADHYAY DASGUPTA AND WILLIAM C. SUMMERS Departments of Therapeutic Radiology, Molecular Biophysics and Biochemistry, and Human Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06510
Communicated by Edward A. Adelberg, February 22,1978
ABSTRACT The survival of UV-irradiated herpes simplex virus on UV-irradiated Vero cells was increased over that on unirradiated cells. A time period between irradiation of the host cells and infection with virus was needed to achieve maximum reactivation. In parallel experiments in which the frequencies of occurrence of the forward mutation in the thymidine kinase gene of the virus were measured, growth of herpes simplex virus on UV-irradiated cells yielded progeny virus that had higher frequencies of TK- mutants than did progeny from infections of control cells. The time course of development of this mutagenic effect was the same as that for the development of the UV-reactivation capacity. Furthermore, development of the UV reactivation could be blocked by inhibition of protein synthesis. These results suggest that an "error-prone" inducible UV-reactivation phenomenon exists in mammalian cells.
inducibility of DNA repair processes in mammalian cells has been presented by d'Ambrosio and Setlow (11). It is not known with any certainty whether WR in mammalian cells is "error prone" and leads to increased mutagenesis. We have made use of the genetic system involving the thymidine kinase (TK) locus of herpes simplex virus type 1 (HSV-1) (12-14) and have carried out experiments similar to those described with X phage by Weigle. The results of these experiments confirm that WR occurs in mammalian cells (Vero line), that such reactivation is mutagenic, and that it appears to be inducible.
When bacterial cells are treated with agents such as UV radiation, mitomycin C, or nalidixic acid, DNA synthesis is interrupted and a new set of functions can be observed. These functions, which have been termed "SOS" functions by Radman (1), include UV reactivation, mutagenesis, and prophage induction. Weigle (2) observed that UV-irradiated bacteriophage showed increa'sed survival when plated on host cells that had been irradiated. This phenomenon is called UV reactivation, or, more recently in honor of Weigle, W reactivation (WR). He noted a higher frequency of mutant phage among the survivors grown on UV-irradiated host cells than among survivors grown on unirradiated cells. It has been proposed that the recovery pathway involved in WR is also mutagenic (3, 4). Genetic studies in Escherichia coli show that WR, UV mutagenesis, and induction of phage X are all closely related phenomena, and all depend on the functions of the recA and lex genes. The inducibility of two of these functions-i.e., mutagenesis (5) and prophage induction (6)-by various agents correlates well with their carcinogenicity. It is therefore of special interest to inquire as to the existence of an analogous system in mammalian cells. If we can define the molecular basis of this induction, ultimately in terms of gene and protein functions, we might expect to find a regulatory molecule or specific enzyme that is the final common link whose activity (or lack of activity) results in the spectrum of new phenotypes associated with malignancy. WR has been reported to occur in mammalian cells (7-10). Monkey (CV-1), rat (a tumor cell line), and human (KD and some xeroderma pigmentosum lines) cells all have been shown to exhibit WR when UV-irradiated nuclear replicating DNA viruses were plated on UV-irradiated hosts. Evidence for the
MATERIALS AND METHODS Cells and Virus. Herpes simplex virus type 1 (HSV-1), strain Cl 101, was obtained from S. Kit (Baylor College of Medicine). Vero cells (an African green monkey kidney line) were purchased from the American Type Culture Collection and grown in medium 199 supplemented with 5% fetal calf serum except where otherwise noted. Mouse LMTK- cells (a TK-deficient line of Earle's L cells) were obtained from J. Eisenstadt (Yale University) and were grown in modified Eagle's minimal essential medium supplemented with 5% fetal calf serum. All media were obtained from Grand Island Biological Co. Chemicals. Stock solutions of 5-bromodeoxyuridine (BrdUrd) (Sigma Co.) were made in phosphate-buffered saline, sterilized by filtration, and stored at -60°. Stock solutions of cycloheximide (Sigma Co.) were made in 95% ethanol. IJV Irradiation. The UV source was a General Electric germicidal lamp that had an output maximum at 254 nm. The dose was determined with a Latarjet dosimeter. Virus suspensions in minimal medium with 2% fetal calf serum [107 plaque-forming units (pfu)/ml] were irradiated with constant stirring. The depth of the liquid layer was about 1 mm. Freshly confluent monolayers of cells were washed with serum-free medium and then irradiated. Fresh medium with 5% serum was added immediately after irradiation. Virus Growth and Assay. Virus stocks of 108 pfu/ml were obtained by growth at low multiplicities of infection from progeny of a single plaque. The frequency of TK- virus mutants was tested after each passage and only stocks with low frequencies of TK- mutants (