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Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.

Transformation potential of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) was quantitatively compared in a system selecting for biochemical transformants. The highest frequency of transformation was obtained with HSV-2 strain 333. The transformation rates for two strains of HSV-1 were lower and distinctly different from HSV-2 and from each other. The dose of inactivating ultraviolet light received by the virus and the multiplicity of infection proved to be critical components of the procedure. Clones of these biochemically transformed cells expressed HSV-specific antigens, altered cell morphology, and increased levels of thymidine kinase activity. Isolates of HSV-1 recovered from the same site 11 years apart yielded similar transforming frequencies. These results suggest that this assay should prove useful in further characterizing the biological properties of HSV.

The characteristics of the cytoskeleton of a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet (UV)-irradiated HSV were studied by indirect immunofluorescence using anti-actin IgG. Parental 3Y1 cells possessed well-developed actin filaments, while 3Y1 cells transformed by UV-irradiated HSV also retained well-developed actin filaments. Transformed cells were divided into 2 groups according to tumorigenicity in newborn Fischer rats; one had a strongly tumorigenic potential and the other a weakly tumorigenic potential. Tumor-derived cell lines exhibited a highly tumorigenic potential, and were also divided into 2 groups, one with well-developed actin filaments and the other without well-developed actin filaments. Our results suggested that transformation or tumor formation by HSV is a multi-step process and that morphological loss of actin filaments in the cells is not essential to the tumorigenic potential of the cells transformed by HSV.

C57 mice aged 8-10 weeks in groups of 50 each received vaginal cotton pellets soaked in lysates of HEp-2 cells, either mock-infected or infected with herpes simplex virus I, herpes simplex virus 2, and highly attenuated recombinant viruses 5 times a week for 89 to 114 weeks. An untreated group was also included. The mock-infected and some of the infected cell lysates were exposed to ultraviolet light at a dose sufficient to inactivate virus. Smears of exfoliated vaginal cells collected once a month and histopathologic sections of genital organs removed at autopsy were coded and examined blind for the presence of abnormal cells indicative of malignant changes and cervical cancer, respectively. Sera collected before termination of the study were tested blind for the presence of antibody to infected cell lysates and to purified herpes simplex virus glycoprotein B. The results were as follows: Over 74% of 826 mice examined at autopsy contained tumors at non-genital sites. The tumors were randomly distributed among the various groups. Gross genital abnormalities were less common in untreated animals than in mice receiving vaginal implants. The fraction of mice which developed cervical cancer diagnosed by histopathologic examination was small (7.2%) and not significantly different among various groups. There was no correlation between the presence of abnormal exfoliated cells indicative of early invasive or invasive cancer lesions and the histopathologically proven diagnosis of micro-invasive or invasive cervical cancer. The incidence and levels of antibody were highest in animals exposed to live virus; some mice exposed to inactivated virus also developed weak or moderately high antibody levels. The presence of antibodies did not correlate with the presence of histopathologically proven cervical cancer. The results do not support the ability of herpes simplex viruses to cause genital neoplasia in mice.

The variation of lactate dehydrogenase, peroxidase, acid and alkaline phosphatase isoenzymes was studied in VERO cells inoculated with infectant and UV-inactivated herpes simplex virus type 1 (HSV--1). Infectant HSV--1 induced quantitative and qualitative modifications in isoenzyme patterns within the first 4 hours post inoculation (p.i.). The modifications caused by the UV-inactivated HSV--1 were similar, but appeared 8 hours p.i. The possibility of using isoenzyme modifications as rapid, sensitive and specific biochemical tests for virus detection and differentiation between infectant and inactivated virus is discussed.

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblasts cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties.

Although there exists a desire to eliminate the possibility of cross-infection from microbial contaminated nitrous oxide nasal hoods, effective and practical methods of sterilization in a dental office are unsatisfactory. Microwaves have been used to sterilize certain contaminated dental instruments without damage. In this study nasal hoods contaminated with rhinovirus, parainfluenza virus, adenovirus, and herpes simplex virus were sterilized in a modified microwave oven. Ninety-five percent of the virus activity was destroyed after 1 minute of exposure of the contaminated nasal hoods to microwaves. By the end of 4 minutes, complete inactivation of all four viruses was found. Repeated exposure of the nasal hoods to microwaves resulted in no damage to their texture and flexibility. Microwave sterilization may potentially provide a simple and practical method of sterilizing nitrous oxide anesthesia equipment in a dental or medical practice.

Recombination frequencies for two sets of genetic markers of herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied and did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent.

The oncogenic properties of hamster embryo cells transformed by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and SV40 virus following photodynamic inactivation using neutral red were determined by subcutaneous inoculation into newborn Syrian hamsters. Cells transformed by all three viruses produced palpable tumors after different latent periods. Histopathological examination showed that HSV-2 tumors were fibrosarcomas and metastases were often seen in the lungs. HSV-2 primary tumors were reinoculated subcutaneously into weanling hamsters; they developed palpable tumors within 2 weeks. HSV-specific antigens were detected in the cytoplasm and/or on the surface of both the HSV-1 and HSV-2 tumor-cell cultures by the indirect immunofluorescence technique. The same method revealed SV40 tumor antigen in the nuclei of the SV40 tumor cells. Sera from HSV or SV40 tumor-bearing hamsters gave positive reactions when tested against HSV-infected hamster cells or SV40-infected monkey cells, respectively. These results demonstrate that herpes simplex virus and SV40, whose infectivity was lost following photodynamic inactivation, retained the virus genetic information necessary for transformation of normal cells to an oncogenic phenotype.

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.

A BALB/c continuous cell line, 238, was transformed following infection with UV-inactivated herpes simplex virus type 2 (HSV-2) was designated H238. Several cell lines were derived from single cells of H238 by cloning in soft agar. The clonal lines express cytoplasmic antigens by immunofluorescence when reacted with rabbit HSV-2 antiserum and mouse tumor-bearer serum. The cloned cell lines induce tumors in immunocompetent mice by the subcutaneous route with a 100% incidence at an inoculum of 10(6) cells. These properties are consistent with those of the H238 parent cell line. In contrast to the parent line, however, the cloned cell lines do not elicit HSV neutralizing antibody in the tumor-bearing host.

After nitrous acid mutagenesis of herpes simplex virus type 1 (HSV-1), a mutant, 1093, was isolated which, during productive infection, induced very low levels of thymidine kinase (tk). The mutant virus was found, after UV irradiation, to be unable to transform L cells lacking tk (Ltk-) to a tk+ phenotype as chararcterized by growth of the cells in a modified HAT-selective medium containing 1.6 X 10(-5) M thymidine. Cells transformed by wild-type virus grew vigorously under the same conditions. The mutant was able to transform Ltk- cells if the medium contained 10(-3) M thymidine. These transformed cells maintained their conditional character and would not grow in low concentrations of thymidine in selective medium. Therefore, this mutant is conditional on the thymidine concentration in the selection medium in its ability to transform Ltk- cells to a tk+ phenotype. The conditionally transformed cells could be supertransformed with wild-type UV-irradiated HSV-1 to a phenotype which would grow in low-thymidine selective medium. The frequency of supertransformation closely approximated the frequency of transformation of Ltk- cells by wild-type virus. Supertransformation at high frequency could not be effected by mutant 1093 or the tk- mutant B2006. These results indicate that the presence of HSV-1 genetic information in HSV-1-transformed cells does not preclude the acquisition by these cells of at least one additional HSV-1 gene, that for tk.

First generation progeny herpes simplex type 1 (HSV-1) virions grown in the presence of 5-iodo-2'-deoxyuridine (IdUrd) were irradiated with either 254 or 302 nm ultraviolet (u.v.) light. The kinetics of virus inactivation revealed decreased sensitivity of IdUrd-substituted virions to irradiation with 302 nm light under all conditions examined, and with 254 nm u.v. light when substituted and control virions were irradiated at equal particle concentrations. Comparison of virus survival after irradiation measured in Vero or Xeroderma Pigmentosum (complementation group A) cells indicated that cellular repair of ultraviolet-induced lesions was not a significant factor in the observed decrease in u.v. sensitivity. IdUrd substitution altered neither the formation of ultraviolet-induced thymidine photoproducts nor the ability of irradiated virions to express delayed early viral enzymes (thymidine kinase, DNA polymerase). It is suggested that nucleocapsid proteins or the highly ordered structure of IdUrd-substituted virions play a key role in u.v. desensitization, either by the formation of non-lethal photoproducts or by the prevention of the formation of DNA-uracilyl free radicals.

The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line.

The effect of irradiation with UV light on herpes simplex virus infection in the mouse ear was investigated. Irradiation 2 days before infection had no effect on the titre of virus in the skin 3 days after infection. In ears irradiated 24 hours or immediately before infection virus titres measured 3 days after infection were depressed. Irradiation 2 days after infection enhanced virus titres. The dose of irradiation used did not directly inactivate herpes simplex virus in the ear. The results are discussed with reference to theories of herpes simplex reactivation.

A transformed mouse cell line (H238) was obtained following the infection of 238 mouse cells with ultraviolet (UV) irradiation-inactivated herpes simplex virus type 2 (HSV-2). The transformed cells produced tumors with a 100% incidence within 8 weeks in 6-week-old syngeneic BALB/c mice at an inoculum of 1 times 10(6). Indirect immunofluorescence (IF) tests revealed the presence of HSV antigens in the transformed cells. Antibodies to HSV-2 were found in the sera of tumor-bearing animals by neutralization and IF techniques. Neither HSV-2 infectious virus nor viral antigens could be detected by the transfer of transformed-cell DNA into permissive cells.

The nucleoside analogue 1-beta-D-arabinofuranosylcytosine (ara-C) is incorporated into herpes simplex virus type 1 (HSV-1) DNA, and this correlates with inhibition of virus replication. The technique of Weigle-type reactivation (WR) was used to compare the ability of induced cellular DNA repair pathways to recognize or repair ara-C incorporated into HSV-1 DNA and ultraviolet (UV)-irradiated virus DNA (254 nm). Pretreatment of monkey cells with low-fluence UV irradiation, growth in cis-dichlorodiammineplatinum(II), or growth in ara-C followed by infection after a 24-hr incubation period resulted in enhanced survival of UV-irradiated HSV-1. Under the same experimental conditions, no reactivation of HSV-1 inactivated by growth in ara-C is observed. Comparisons between uninfected Vero cells exposed to UV irradiation (30 J/m2) or grown in 10(-6) M ara-C demonstrated repair replication in irradiated cells, whereas there was no evidence for DNA repair at various time intervals following removal of the nucleoside analogue. These observations suggest that, once ara-C is incorporated into HSV-1 or eukaryotic DNA, it is not recognized as a repairable lesion within the limits of the DNA repair assays used in these studies.

Dibutyryl cyclic AMP inhibits the increase of dThd and BrdUrd transport normally observed after infection with Herpesvirus hominis, type I and II. Incorporation is also reduced. Inhibition of uptake is non-competitive as analysed by the Lineweaver-Burk plot. Addition of this drug to infected cells also reduces the activity of the thymidine kinase (EC 2.7.1.75). Transport of dUrd, dCyd and dAdo is not reduced. 4--8 h after infection with thymidine kinase (+) herpes strains the level of cAMP increases. On infection with a thymidine kinase (-) virus, only a small elevation of cAMP can be shown. It was also found that early addition of actinomycin D or of cycloheximide prevents the increase of the cAMP level. This increase seems to depend on the activity of the herpes genome, because ultraviolet irradiation of infective particles destroys this ability.

Growth of HSV-1 in (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) results in incorporation of the analog into HSV-1 DNA. Employing the technique of Weigle-type reactivation (WR), HSV-1 inactivated by uv irradiation but not by growth in BVDU is a substrate for induced cellular repair pathways. Growth of mammalian cells in BVDU does not induce cellular repair pathways detected by WR. HSV-1 temperature sensitive (ts) mutants demonstrate an increased reversion frequency to the nonpermissive phenotype (ts+) following growth in a high concentration of BVDU. A ts mutant did not display an increased reversion frequency following growth in equally inhibitory concentrations of an HSV-1 polymerase inhibitor, aphidicolin. These findings suggest that BVDU incorporated into HSV-1 DNA may not be readily excised and may be a mutagenic lesion in viral DNA, although other explanations are possible.

Transcription of virus DNA was detectable in vitro in nuclei isolated from cells infected with wild type and bromodeoxyuridine (BUdR)-resistant herpes simplex virus type 1 but was suppressed in nuclei of cells infected with ultraviolet irradiated or defective virus or cells treated with BUdR during infection.

The sensitivity of interfering herpes simplex virus (HSV) particles to u.v.-irradiation was studied in a virus stock of HSV-1 strain ANG that contained an excess of interfering over infectious particles. Following u.v.-irradiation, samples of this virus stock were assayed for their plaque-forming capacity and their capacity to interfere with the replication of unirradiated standard virus. Depending on the assay conditions, interfering particles appeared to be less, equally, or more sensitive to u.v. light than infectious particles. It is concluded that interference is a gene function of interfering particles rather than being exerted directly by structural constituents of these particles.

Cultures of foetal lymphocytes were exposed to u.v.-irradiated herpes simplex virus (HSV). The cells responded with increased 6-3H-thymidine incorporation, the formation of clumps of enlarged lymphoblastoid cells and cell division. This response was first detected 3 to 4 days after exposure to virus material and was shown to be virus-dose dependent. The ability to stimulate foetal cells was considerably more u.v. resistant than infectivity. Two isolates of HSV type 2 (4663 and 37174), which had a high "transforming" ability, produced large numbers of non-infectious particles (particle: infectivity ratios in excess of 10(4)). The cells, which responded to u.v.-irradiated HSV with blastoid transformation, were associated with the non-E-rosetting (T-cell-depleted) subpopulation.

A hypothesis is proposed which states that multiple sclerosis (MS) is caused by herpes simplex virus type 2 (HSV-2) in persons lacking herpes simplex virus type 1(HSV-1) immunity. It is examined by comparing the epidemiology of these viruses with that of MS. The age at which HSV-2 infections are first detected, as well as the age of peak incidence are similar to those for MS. If age-specific HSV-1 immunity data from various places is compared, an inverse relationship of immunity levels with latitude is apparent, and may be related to the effects of ultraviolet irradiation on HSV-1 activation and transmission rates. Prior HSV-1 immunity, if related to subsequent risk of MS, would be protective. This hypothesis also appears to be consistent with the generalisations concerning effects of migration on subsequent rates of MS, and with certain other features of MS epidemiology.

Exponentially growing cells of the PtK-2 line (ATCC No. CCL56, from the marsupial Potorous tridactylus) require protein and RNA synthesis in a limited period following UV-radiation damage for optimal recovery as colony formers [Overberg et al. (1988) Mutat. Res. 194, 83-92]. Overall behavior suggests the operation of damage-induced recovery processes. The capacity of confluent cell monolayers for infection with unirradiated herpes simplex virus 1 (HSV-1) is sharply reduced by UV-irradiation. We have followed capacity changes in exponentially growing cells after irradiation and varying amounts of photoreactivation by means of an infectious center assay. These changes closely parallel changes of colony formation. Spontaneous recovery of capacity in the dark occurs over approximately the same time period that the UV sensitivity of colony formation depends on macromolecular synthesis. The effect of photoreactivation is complementary rather than additive to this recovery, suggesting that the dark recovery in this period concerns pyrimidine dimers in cell DNA.

An enhanced reactivation of ultraviolet-damaged (u.v. at 254 nm) unclear replicating double-stranded DNA viruses occurs when corresponding host cells are treated with radiation or carcinogens prior to infection. This phenomenon seems to be due to an induced DNA repair activity the nature of which is yet unknown. The u.v.-induced enhanced reactivation (ER) of u.v.-damaged herpes simplex virus (u.v. - HSV) was compared in dividing skin fibroblasts of 30 donors either normal or afflicted by genetic disorders, some of which confer a high risk for sunlight induced skin cancers. Cultures were exposed to a single dose of 1.0-25 J.m-2 from 0-60 h before infection with u.v.-HSV (at about 10-3 survival) and the rate of viral production was determined. ER was maximal for a 36 h time interval in all lines. The u.v. dose eliciting maximal ER was 15 J.m-2 in fibroblasts from normal donors, xeroderma pigmentosum (XP) heterozygotes, Mibelli's porokeratosis, diffused naevomatosis, Down's syndrome, xerodermoids, XP variants and epidermodysplasia verruciformis. However, in the latter 3 cases, ER was almost 10 times more pronounced than in the normal cases. The u.v. dose eliciting maximal ER was 0.1, 0.3 and 2 J.m-2 in excision deficient XP fibroblasts from groups A, D and C, respectively, 2.5 J.m-2 in 11961 fibroblasts and 5 J.m-2 in fibroblast lines from cockayne s syndrome.

Comparison of methods to inactivate lytic properties of herpes simplex viruses revealed that ultraviolet irradiation, photodynamic procedures, and heat all destroyed infectivity effectively. Ability to biochemically transform thymidine kinase deficient cells to an enzyme positive phenotype was retained after limited exposure to heat or ultraviolet light but appeared to be destroyed by photodynamic methods employing neutral red. Exposure to 56 degrees C quickly and effectively destroyed transforming activity with lower temperatures being less effective. The most reproducible transforming assays were obtained following inactivation by ultraviolet light. Cell cultures developed by this procedure were virus-free but retained ability to synthesize virus-specific antigens.

The transcriptional organization of the genome of herpes simplex virus type 1 was analyzed by measuring the sensitivity of viral polypeptide synthesis to UV irradiation of the infecting virus. Herpes simplex virus type 1 was irradiated with various doses of UV light and used to infect xeroderma pigmentosum fibroblasts. Immediate early transcription units were analyzed by having cycloheximide present throughout the period of infection, removing the drug at 8 h postinfection, and pulse-labeling proteins with [35S]methionine. Delayed early transcription units were analyzed in similar studies by having 9-beta-D-arabinofuranosyladenine present during the experiment to block replication of the input irradiated genome. The viral polypeptides were separated by gel electrophoresis and quantitated by densitometry of the gel autoradiograms. The following results were obtained. (i) The UV target sizes for the viral transcription units analyzed ranged from 1.44 to 5.65 kilobase pairs. This implies that the corresponding primary transcripts have minimum molecular weights ranging from 0.46 x 10(6). (ii) The genes for the four viral proteins, 165, 145, 116, and 71 (molecular weight x 10(3), exhibited UV target sizes that agree with their calculated gene size or measured mRNA size or both and thus must reside in promoter-adjacent positions. (iii) The transcription units for the remaining genes analyzed showed target sizes that range from 0.42 to 2.59 kilobase pairs greater than needed to encode the respective proteins. This probably is a reflection of their distances from promoters or the presence of intervening sequences or both. It further suggests that these genes are transcribed as precursor RNA molecules that are larger than their mRNA's. (iv) The results indicate that none of the immediate early genes analyzed can be cotranscribed, whereas some of the delayed early genes might be cotranscribed. No evidence was found for the existance of large, multigene transcription units.

Herpes simplex virus type 2 (HSV-2) strains inhibit the synthesis of both DNA and protein of the host cell more rapidly than HSV-1 strains. Several intertypic HSV-1 X HSV-2 recombinants and parental strains were examined for their ability to inhibit rapidly the synthesis of host protein and DNA. The two functions cosegregated in all of eight recombinants tested and are therefore controlled by the same gene or by different genes in the same region of the viral DNA.

Generalized vascular changes and diffused proliferation of reactive microglia were observed in an experimental model of HSV encephalitis of mice. The wide spread of these changes contrasted with the localized character of virus replication and the confined areas of damaged nervous tissue. The vascular and microglial changes were precocious in animals inoculated with concentrated virus suspension (10(5.5)LD50) while they appeared late in mice inoculated with diluted virus suspension (100 LD50). After inoculation with U.V. inactivated virus no changes were seen. The results obtained in this study suggest that the vascular and microglial modifications are not related to a direct cytopathic effect of the virus but dependent on the amount of virus present in the central nervous system and linked to the virus DNA.

Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 degrees C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 degrees C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 degrees C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 degrees C) before the shift but which cannot be made at 38 degrees C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.