Blocked Herpes Simplex Virus Type 2-Specific DNA - Infection and ...

Vol. 41, No. 2

INFECTION AND IMMUNITY, Aug. 1983, p. 584-590 0019-9567/83/080584-07$02.00/0 Copyright 0 1983, American Society for Microbiology

Blocked Herpes Simplex Virus Type 2-Specific DNA Synthesis in Simian Virus 40-Transformed Hamster Cells Permissive for Herpes Simplex Virus Type 1 DAVID A. CHERESHlt* AND HAROLD HAINES2 Departments of Microbiology' and Pathology,2 University of Miami School of Medicine, Miami, Florida 33136 Received 13 July 1982/Accepted 10 May 1983

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2-infected LL-1 cells demonstrated a block in HSV-2specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSVspecific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.

Progress towards understanding the basis of nonproductive herpes simplex virus (HSV) infections has been hampered by a lack of in vitro model cell systems available for study. In this report, we characterize a nonproductive infection of HSV type 2 (HSV-2) in a simian virus 40transformed cell line, LL-1. Although LL-1 cells are relatively nonproductive for HSV-2, they allow replication of HSV-1. Previous work in our laboratory has demonstrated that HSV-2infected LL-1 cells show a 3- to 4-log reduction in PFU as compared with HSV-1-infected cells at 24 h postinfection. This finding was documented with three different strains of HSV-1 and HSV-2. Moreover, this reduction in viral output is not due to virus-induced interferon production or an inability of HSV-2 to adsorb to the LL-1 cell surface (H. Haines, S. Barmack, D. Cheresh, and D. Bronson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, S280, p. 286). Nonproductive herpesvirus infections in certain cell lines have been studied because of the ability of this virus to establish latency in vivo (5, 13, 24) and promote neoplastic transformation under in vitro culture conditions (9, 10, 21, 22, 23, 25). One source of HSV nonproductive t Present address: Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037.

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infections comes from HSV-infected cells which have been biologically or physically altered. Photodynamic treatment of HSV decreases its lytic function in normally permissive cells, resulting in an enhancement of its transforming capacity (2, 15, 16). Jariwalla and co-workers demonstrated that certain fragments of HSV-2 DNA transformed Syrian hamster embryo cells yet could not promote development of complete viral progeny (15). In another study, evidence was presented for two distinct transforming regions responsible for morphological transformation in HSV-1 and HSV-2 DNA (21). Camacho and Spear demonstrated the transformation of hamster embryo fibroblasts (HEF) cells by a specific fragment of the HSV genome (4). In addition, it has been shown that proteins encoded by a fragment of HSV DNA have transforming activity in primary rat kidney (PRK) and NIH 3T3 cells (12). The results of these studies suggest that the transforming capacity of HSV may be related to virus-induced gene products which can interrupt or alter viral genetic information responsible for productive infection. Another source of in vitro nonproductive HSV model cell systems comes from the study of previously transformed cells. Docherty et al. described a hamster cell line, HDC-22, that is transformed by chemically inactivated HSV and

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shows resistance to HSV-2 but allows replication of HSV-1 (8). Later studies revealed that the block occurs at or just before HSV-2 DNA synthesis (7). It was found that the block in HSV lytic function occurs after viral adsorption, sometime early in the HSV replicative cycle. Hamster and human cells transformed by simian virus 40 also show an increased resistance to HSV as compared with nontransformed control cells (27), suggesting that the simian virus 40 genome may interfere with a productive event(s) of the HSV replicative cycle. In this paper, we investigated the possible mechanism of inhibition of HSV-2 in the LL-1 cell line. Our results indicate that HSV-2 is capable of entering the cell and carrying out various early virus-specific functions but is incapable of synthesizing DNA and subsequent structural proteins necessary for the late stages of HSV maturation. MATERIALS AND METHODS Cells and cell propagation. LL-1 cells were originally obtained from the laboratory of M. Michael Sigel, University of South Carolina, Columbia, S.C. They were derived from tumors induced by direct inoculation of simian virus 40 into LHC/lak inbred golden Syrian hamsters. For routine propagation, LL-1 cells were seeded with Eagle basal essential medium supplemented with 10%o fetal calf serum, 10%o tryptose phosphate broth, 0.075% NaHCO3, and 0.3 mg of glutamine, 100 U of penicillin, 100 mg of streptomycin, 1 g of gentamicin, and 1 mg of amphotericin B per ml. Basal essential maintenance medium contains 5% fetal calf serum, glutamine, NaHCO3, and antibiotics. LL-1 cells were passaged more than 100 times and were shown to be highly tumorigenic when injected back into Syrian hamsters. HEF, PRK, and baby hamster kidney (BHK-21) cells were passaged at regular intervals and nourished with Eagle minimum essential medium supplemented with 5% fetal calf serum, 0.075% NaHCO3, and 0.3 mg of glutamine, 100 U of penicillin, 100 mg of streptomycin, and 1 mg of amphotericin B per ml (minimal essential maintenance medium). Preparation of PRK and HEF ceJls. PRK cells were prepared from kidneys removed from 14-day-old rabbits, whereas HEF cells were prepared from the trunks of whole embryos removed from pregnant hamsters at 10 to 12 days of gestation. The primary cells were prepared as previously described (11). Viruses. HSV-1 strain Dorr and HSV-2 strain 1% were originally obtained from William Rawls, McMaster University, Hamilton, Ontario, Canada. Virus stocks were grown in PRK cells and stored in 2-ml samples at -70°C. LL-1 cells infected with HSV-1 or HSV-2. Confluent monolayers of LL-1 cells in 60-mm-diameter dishes were infected with 0.1 ml of HSV-1 or HSV-2 at a multiplicity of infection of 1. The virus was allowed to adsorb for 1 h at 37°C, after which the cells were covered with 5 ml of basal essential maintenance medium and incubated for 24 h at 370C in an atmosphere of 5% CO2. The infected cells were frozen and

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thawed three times to release virus progeny, which was titered by assay on PRK cells as described below. Plaque assay. HSV-1 and HSV-2 were serially diluted in minimal essential maintenance medium at 4°C, and 0.1 ml of virus was allowed to adsorb for 90 min to confluent PRK monolayers seeded in 60-mm-diameter dishes. After adsorption, infected monolayers were covered with 5 ml of 5% methylcellulose in minimal essential maintenance medium. The infected cell cultures were incubated for 72 h at 37°C in an atmosphere of 5% CO2. At 72 h postinfection, the methylcellulose was poured off, and the monolayers were washed once with phosphate-buffered saline (pH 7.4). The cells were stained with 0.2% crystal violet solution (containing 95% ethanol and 1% NH4 oxalate in water) for 60 s, after which the excess stain was removed, rinsed with tap water, and dried and plaques were counted. HSV DNA polymerase assay. (i) Enzyme extraction. Monolayers of LL-1 cells were grown to confluency in 100-mm-diameter dishes (four dishes per extract) and infected with virus at a multiplicity of infection of 10 or mock infected. At 1 h postinfection, cells were covered with basal essential medium containing 1% fetal calf serum and incubated at 37°C for an additional 5 h. Cells were scraped off the dishes and resuspended (4 x 107 cells) in 2 ml of a buffer consisting of 50 mM Trishydrochloride (pH 7.5) with 1.7 M KCI, 0.5 mg of bovine serum albumin per ml, 0.5 mM dithiothreitol, 0.2% Nonidet P-40, and 20% glycerol. The cell suspension was disrupted in a model W140 sonicator (Branson Instruments Co., Stamford, Conn.) at 14 W for 45 s. The sonic extract was centrifuged at 650 x g for 20 min at 4°C to pellet cellular debris. Each extract was then dialyzed for 4 h at 4°C against 250 ml to remove the KCI. (H) Polymerase assay. The polymerase assay used was a modification of that originally described by Weissbach et al. (28). The reaction mixture contained 0.05 ml of cell extract added to 0.10 ml of 100 mM Trishydrochloride (pH 8.2) containing 100 g of bovine serum albumin per ml, 0.1 mM each dCTP, dGTP, dTTP, and dATP, 0.2 mM [3H]TTP (50 mCi/mM), 4 mM MgCl, 62.5 mg of activated salmon sperm DNA per ml, and 1 mM 2-mercaptoethanol. Increasing concentrations of KCI were prepared in 4 mM 2-mercaptoethanol and added to the reaction mixture to reach a final concentration of 0 to 150 mM. The reaction mixture was maintained at 4°C until all components were added, after which incubation was carried out at 37°C for 30 min and terminated by adding 2 ml of 5% trichloroacetic acid to each reaction tube for 15 min. The precipitate was collected on Whatman G F/C filter paper disks, rinsed with 95% ethanol, and allowed to dry. The filters were added to 5 ml of a scintillation cocktail containing 0.4% 2,5-diphenyloxazole and 0.01% 1,4-bis-2-(5-phenylorazolyl)benzene in toluene, and radioactivity was enumerated in an LS-100 C liquid scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). TK assay. (i) Preparation of cell extracts. For the thymidine kinase (TK) assay, LL-1 cells (5 x 107) were infected with virus at a multiplicity of infection of 5 or mock infected. At 8 h postinfection, cells were scraped off the dish and centrifuged at 500 x g for 10 min at 4°C. The cells were resuspended in 40 ml of 0.9%o NaCl and centrifuged. Cell pellets were resuspended (5 x 107 ceUls per ml) in a buffer consisting of

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25 mM Tris-hydrochloride (pH 8.0) containing 3 mM 2mercaptoethanol, 0.2 mM thymidine, and 10% glycerol and disrupted by sonication with a Branson model W140 Sonicator at 14 W for 45 s. The sonic extract was then centrifuged at 1,000 x g at 4°C for 30 min. The supernatant fluid was used as the enzyme source and stored at -70°C. Protein determinations were done by the method of Lowry et al. (19) with bovine serum albumin as a standard. (ii) Kinase assay. The kinase assay used was a modification of that described by Doberson et al. (6). Reaction mixtures consisted of the cell extract (0.05 ml) added to 0.05 ml of 50 mM Tris-hydrochloride buffer (pH 7.2) containing 5 mM ATP, 5 mM MgCl2, 10 mM NaF, and [3H]thymidine (21 M, 23 Ci/mmol). Separate reaction mixtures were established for each time point. Incubations were carried out at 37°C, and the reaction was terminated by placing the reaction tubes in a boiling water bath for 1 min. Samples (0.05 ml) of each reaction mixture were applied to 2.4-cmdiameter disks of Whatman DE-81 chromatography paper. The disks were washed four times with 1 mM ammonium formate and rinsed twice with 95% ethanol. The duration of each wash was 5 min. The wash volume was approximately 20 ml per disk, and the disks were continually swirled during washing. The disks were dried, added to 5 ml of a scintillation cocktail containing 0.5% 2,5-diphenyloxazole and 0.01% 1,4-bis-2-(5-phenylorazolyl)benzene in toluene, and counted in a Beckman LS-100 C liquid scintillation counter. TK activity was determined by the amount of [3H]thymidine, which became phosphorylated to [3H]TMP. Neutralization of HSV-2 TK by immunoglobulin G from sera of immunized rabbits. Rabbit anti HSV-2 TK was kindly provided by Saul Kit, Baylor College of Medicine, Houston, Tex. Normal rabbit serum was obtained from a nonimmunized animal. Neutralization of TK activity was performed by mixing equal volumes of antisera diluted 1:4 to extract diluted 1:4. Control mixtures contained normal rabbit serum instead of immune sera. All extracts were preincubated with appropriate dilutions of antisera at 4°C for 45 min. After preincubation, 0.05 ml of each mixture was then used in the kinase assay described above. HSV DNA analysis. Monolayers of LL-1 and control (HEF or BHK-21) cells were grown in 35-cm2 tissue culture flasks (5 x 106 cells per flask), inoculated with virus at a multiplicity of infection of 5, and allowed to adsorb for 1 h. After adsorption, virus-treated and control monolayers were washed once with phosphate-buffered saline (pH 7.4) and covered with 2 ml of basal essential medium supplemented with 2% fetal calf serum and antibiotics. At this time, the cultures were pulsed with 5 ,uCi of [3H]thymidine (23 ,uCi/mmol) in a 0.1-ml volume and incubated at 37°C in a humidified 5% CO2 incubator for 23 h. Replicate control cultures were prepared to determine virus titers at the end of the incubation period. After incubation, all cell cultures were frozen and thawed to lyse cells. Appropriate lysates were combined and digested for 24 h at 370C in 3 ml of 1.0% Sarkosyl ML 30, 0.02 M EDTA, and 1% heat-inactivated pronase (B grade; Calbiochem-Behring Corp., San Diego, Calif.) in 0.015 sodium citrate (pH 7.3). A sample (0.2 ml) of each digestion mixture was added to cellulose nitrate tubes containing 3.8 ml of cesium chloride in 0.015 M sodium

citrate (density, 1.745 g/cm3). Density gradients were established by centrifugation at 100,000 x g for 60 h at 20°C in a Sorvall AH-650 rotor. Five-drop fractions were collected onto Whatman filter paper disks by bottom puncture of the centrifuge tubes. Acid-insoluble material was precipitated by three washes with 5% trichloroacetic acid at room temperature. The filters were dehydrated by rinsing in acetone. After drying, the filters were placed in 10 ml of Aquasol scintillation fluid, and counts per minute were enumerated in a model 3320 liquid scintillation counter (Packard Instrument Co., Inc., Rockville, Md.). Every tenth fraction was collected in a glass tube for density determination with a refractometer (Bausch & Lomb,

Inc., Rochester, N.Y.). Indirect immunofluorescent detection of structural antigens in HSV-infected LL-1 cells. Immunofluorescent labeling of HSV-infected LL-1 cells was carried out by a modification of the indirect immunofluorescence procedure used by Levine et al. (18). LL-1 or BHK-21 cells were grown to confluency on glass cover slips, infected with HSV-1 or HSV-2 at a multiplicity of infection of 5, and subsequently incubated for 20 h at 37°C in a 5% CO2 atmosphere. The cells were fixed in cold acetone for 1 min and rehydrated with phosphate-buffered saline (pH 7.4). The cover slips were

overlaid with 0.1 ml of HSV antibody produced in rabbits by immunization with purified intact virions (diluted 1:64) and incubated for 30 min at 37°C. The cells were gently rinsed three times in phosphatebuffered saline (pH 7.4) and stained with 0.1 ml of goat anti-rabbit gamma globulin conjugated with fluorescein isothiocyanate (Hyland Laboratories, Costa Mesa, Calif.) at a dilution of 1:8 for 30 min at 37°C. The cells were washed three times. Controls consisted of mockinfected cells or infected cells treated with nonimmune rabbit serum instead of antibody. The fluorescence was monitored with a Leitz UV microscope equipped with epifluorescence.

RESULTS LL-1 cells infected with HSV-1 or HSV-2. LL-1 cells were infected with HSV-1 or HSV-2 for 24 h, and infectious virus production was monitored by plaque assay on PRK cells (Table 1). There was approximately a 3-log reduction in PFU of HSV-2 compared with HSV-1. LL-1 cells infected with HSV-2 could be passaged at least 12 times with minimal virus output (106 PFU/ml of culture fluid). HSV DNA polymerase activity. To determine whether early HSV-2-directed protein synthesis was inhibited, the HSV-specific DNA polymerase activity was measbred in LL-1 cells infected with HSV-1 or HSV-2. The DNA polymerase activity of HSV has been shown to be stimulated in the presence of high salt (KCl) concentration, whereas ongoing host cell DNA polymerase is not stimulated by the same KCI concentration (28). The results of a representative experiment demonstrating HSV DNA polymerase activity in the presence of KCl are shown in Fig. 1. The results demonstrated marked stimulation of HSV DNA polymerase activity in LL-1 cells. The apparent difference in HSV-1 and HSV-2 polymerase activities at 150 mM KCl may reflect a slight difference in sensitivity to high salt. Mock-infected LL-1 cells showed only minimal DNA polymerase in the presence of all KCl concentrations tested. HSV TK activity. HSV-specific TK activity was monitored in infected LL-1 cells. Both HSV-1 and HSV-2 showed TK activity in LL-1 cells (Fig. 2A and B, respectively). Preincubation of infected cell extracts with HSV-2 TK antibody caused approximately 60% neutralization of HSV-2 TK. There was some crossreactivity between the TK activity of HSV-1 and HSV-2, since HSV-2 TK antibody showed a 10% neutralization of HSV-1 TK activity (Fig. 2A). Mock-infected LL-1 cells showed a base level of TK activity which was unaffected by HSV-2 TK antibody. DNA analysis. HSV-1 and HSV-2-infected LL-1 cells were monitored for the presence of virus and host cell DNA synthesis to determine whether the inability of HSV-2 to replicate in LL-1 cells was due to reduced virus-specific DNA synthesis. Infected cells were incubated in the presence of [3H]thymidine for 23 h and cell lysates were analyzed for the presence of virus and host cell DNA by CsCl2 density gradient centrifugation (Fig. 3). Gradient profiles from BHK-21 cell cultures productively infected with HSV-1 and HEF cell cultures productively infected with HSV-2 are shown in Fig. 3D and E, respectively. Cellular DNA resolved at a density at 1.695 g/ml, and the virus peak appeared at a density of 1.725 g/ml. HSV-1-infected LL-1 cells (Fig. 3A) showed a relatively small but measurable virus peak, whereas HSV-2-infected LL-1 cells showed no viral DNA (3B). Mock-infected BHK-21 and LL-1 cells showed the characteris-

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mM KCI FIG. 1. HSV DNA polymerase activity in infected LL-1 cells. LL-1 cells were infected with HSV-1 (0) or HSV-2 (0) or mock infected (A\) and harvested at 6 h postinfection. Infected cell extracts were incubated in the presence of dCTP, dGTP, dATP, [3H]TTP, and salmon sperm DNA. Polymerase activity was measured by the amount of [3H]TTP incorporated into trichloroacetic acid-precipitated DNA, which was collected on Whatman OF/C filter paper disks, and measured in the presence of various KCl concentrations (0 to 150 mM). The counts per minute of each filter disk was enumerated in a liquid scintillation counter. The total cellular protein in each reaction mixture was 0.09 mg.

tic host cell peak at 1.695 gIml (Fig. 3C and F, respectively). Although HSV-2 infection did not lead to the synthesis of virus-specific DNA, it was able to inhibit ongoing LL-1 DNA synthesis to levels comparable to that of productively infected control cultures (Table 2). These results suggest that virus-induced suppression of host DNA occurs independently from HSV-2-specific DNA synthesis in infected LL-1 cells. Detection of HSV structural antigens in infected LL-1 cells. Indirect immunofluorescence was carried out to determine whether HSV-2 structural proteins appeared in infected LL-1 cells. LL-1 cells were infected with HSV-2 or HSV-1, fixed, incubated in the presence of rabbit HSV antibody, and subsequently stained with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. In HSV-1-infected LL-1 and BHK-21 cells, nuclear and cytoplasmic staining was present in greater than 90%o of the cells. However, HSV-2-infected LL-1 cells showed 5 to 15% of the cells with faint nuclear staining only. The

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FIG. 2. HSV TK activity in infected LL-1 cells. LL-1 cells were infected with HSV-1 or HSV-2 or mock infected and harvested at 8 h postinfection. Infected cell extracts were preincubated in rabbit HSV-2 TK antibody or normal rabbit serum. The TK assay was carried out for 0, 5, 15, and 25 min at 37°C. TK activity was measured by the amount of [3H]thymidine that became phosphorylated to [3H]TMP and adhered to a DE-81 filter disk. The counts per minute of each filter disk were enumerated in a liquid scintillation counter. The total cellular protein in each reaction mixture was 0.23 mg. (A) HSV-1 TK activity in cells preincubated in normal rabbit serum (E) or in rabbit HSV-2 TK antibody (U). (B) HSV-2 TK activity in cells preincubated in normal rabbit serum (0) or in rabbit HSV-2 TK antibody (0). A, Host TK activity.

remaining cells were unstained, suggesting that HSV-2 structural protein synthesis was markedly reduced as compared with LL-1 cells productively infected with HSV-1. Mock-infected controls and infected LL-1 cells treated with nonimmune rabbit serum showed no fluorescent staining in the presence of fluorescein isothiocyanate-conjugated anti-rabbit antibody. DISCUSSION

Experiments were carried out to characterize a nonproductive infection of HSV-2 in the simian virus 40-transformed hamster cell line LL-1. LL-1 cells are a unique example of a cell culture system permissive for HSV-1 yet relatively nonpermissive for HSV-2 (Haines et al., abstract). Initial experiments by Haines et al. demonstrated that the nonpermissive HSV-2 infection was not due to interferon production or an inability of the virus to adsorb to the LL-1 cell surface (Haines et al., 1979 ASM Meet, S280). Furthermore, HSV-2 could be carried in the LL-1 cell line for at least 12 passages with minimal cytopathology. Although HSV-1 and -2 are quite similar morphologically, biochemically, and antigenically,

they show approximately 46% homology between their genomes (17). Thus, HSV-1 and HSV-2 have much the same host range. There have been very few cell types capable of supporting the growth of one virus type and not the other. Chicken embryo cells have been shown to be permissive for HSV-2 but restrictive for HSV-1 (20). A hamster cell line transformed by chemically inactivated HSV showed resistance to HSV-2 but allowed some replication of HSV1 (7). Thus, the LL-1 cell line is one of the few in vitro cell models capable of restricting the growth of HSV-2 but not that of HSV-1. In this study, we have found that early various HSV-2-specific functions appear to occur at or near permissive levels in infected LL-1 cells, as measured by HSV TK and DNA polymerase activity detected in cell extracts of HSV-2-infected LL-1 cells. TK and DNA polymerase are excellent viral markers to examine since they can be discerned from ongoing host background activity. They are also markers that reveal early HSV nonstructural protein synthesis. Adler and co-workers characterized an HSV nonproductive system in a chemically transformed rat nerve cell. They showed evidence of early viral function by demonstrating the presence of HSV TK and DNA polymerase. HSV

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DNA synthesis, a latter replicative step, was

blocked in these cells (1). In a subsequent study, it was shown that HSV was capable of establishing a persistent infection in these cells of neuronal origin (18). Furthermore, it has been shown that HSV TK activity appears in HSV-infected mouse trigeminal ganglia (26). Thus, early events in the HSV replicative cycle may be taking place in certain cells even though production of infectious progeny does not occur. HSV DNA synthesis marks the transition from early to late viral function and serves as a prerequisite to many of the late viral structural proteins (14). Although some early HSV-2 function was apparent in infected LL-1 cells, (TK and DNA polymerase activity) we were unable

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FRACTION NO. FIG. 3. DNA analysis of HSV-infected cells. LL-1, BHK-21, or HEF cells were infected with HSV-1 or HSV-2. The infections were carried out for 24 h in the presence of [3H]thymidine. Each peak represents the counts per minute of [3H]thymidine incorporation into the trichloroacetic acid-insoluble fraction. The viral and host cell DNA was separated by density centrifugation in cesium chloride. Five-drop fractions were collected onto filter paper disks from a bottom puncture of the gradient tube. Every tenth fraction was collected for density determination. (A) HSV-1-infected LL-1 cells; (B) HSV-2-infected LL-1 cells; (C) mock-infected LL-1 cells; (D) HSV-1-infected BHK21 cells; (E) HSV-2-infected HEF cells; (F) mockinfected BHK-21 cells.

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TABLE 2. Levels of LL-1 and BHK cell DNA suppression induced by HSV infectiona Host DNA peak % DNA Cell and virus incorporation DN suppression (cpm x 103) BHK Uninfected 70 20 71 HSV-2 LL-1 Uninfected 60 13 78 HSV-1 HSV-2 17 72 a For experimental details, see Materials and Methods.

to detect any HSV-specific DNA synthesis. HSV-2 was capable of inhibiting ongoing LL-1 cell DNA synthesis to levels comparable to that in productive HSV infection, suggesting that viral DNA synthesis and shut-down of host cell DNA synthesis are separate events. Thus, it appears that HSV-2 is capable of infecting LL-1 cells and carrying out some early virus-specific functions. The nonproductive infection apparently involves a block at or before HSV-2specific DNA synthesis, which is a middle replicative step. Indirect immunofluorescence experiments indicated that late structural HSV-2 protein synthesis was inhibited in infected LL-1 cells. The few cells that were stained (5 to 15%) showed primarily faint nuclear staining, suggesting that late maturation steps that occur in the cytoplasm were not taking place. Therefore, middle to late virus-specific events which involve HSV DNA synthesis and subsequent late protein synthesis are inhibited in LL-1 cells, thus accounting for nonproductive viral infection. The small percentage of nuclear stained LL-1 cells may be due to heterogeneity in LL-1 cell populations which contain cells marginally permissive for HSV-2. Understanding of nonproductive HSV infection may shed light on its ability to induce malignant transformation in vivo. Burns and Murray demonstrated that mice with herpetic lip lesions exhibited papillomas and squamous cell carcinoma when exposed to UV light and tetradecanoyl-phorbolacetate, whereas mice without herpetic lesions showed no altered histological appearance (3). Thus, by altering viral or host cell function by UV light and a tumor promoter, it was possible to induce a virus-related malignant transformation in vivo. Recently, much study has been devoted to HSV and its nonproductive capabilities. To better understand the phenomenon of latency and HSV oncogenic potential, further attempts

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should be made to characterize nonproductive HSV infections in vitro. The LL-1 cell system is an excellent example of such a system, since it has a built-in positive control (HSV-1 permissivity). Further studies such as these may help to illucidate the mechanism(s) of host-virus interaction responsible for virus-induced transformation and latency commonly associated with HSV infections. ACKNOWLEDGMENTS This work was supported by Public Health Service grant R01CA17544 from the National Institutes of Health. LITERATURE CITED 1. Adler, R., J. C. Glorloso, and M. Levine. 1978. Infection of herpes simplex virus and cells of nervous system origin: characterization of a non-permissive interaction. J. Gen. Virol. 39:9020. 2. Boyd, A. L., and T. W. Orme. 1975. Transformation of mouse cells after infection with ultraviolet irradiationinactivated herpes simplex type 2. Int. J. Cancer 16:526538. 3. Burns, J. C., and B. K. Murray. 1981. Conversion of herpetic lesions to malignancy by ultraviolet exposure and promoter application. J. Gen. Virol. 55:305-313. 4. Camacho, A., and P. G. Spear. 1978. Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome. Cell 15:993-1002. 5. Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection. Infect. Immun. 7:272288. 6. Dobersen, M. J., M. Jerkofsky, and S. Greer. 1976. Enzymatic basis for the selective inhibition of varicellazoster virus by 5-halogenated analogues of deoxycytidine. J. Virol. 20:478-486. 7. Docherty, J. J., R. A. Mantyjarri, and F. Rapp. 1972. Mechanism of the restricted growth of herpes simplex .virus type 2 in a hamster cell line. J. Gen. Virol. 16:255264. 8. Docherty, J. J., F. J. O'Neil, and F. Rapp. 1971. Differential susceptibility to herpes simplex viruses of hamster cell lines established after exposure to chemically inactivated herpes virus. J. Gen. Virol. 13:377-384. 9. Duff, R., and F. Rapp. 1971. Oncogenic transformation of hamster cells after exposure to irradiated herpes simplex virus type 2. Nature (London) 233:48-50. 10. Duff, R., and F. Rapp. 1971. Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultraviolet-irradiated herpes simplex virus type 2. J. Virol. 8:469-477. 11. Gallagher, J. G. 1973. Preparation of primary cultures, p. 102-105. In P. F. Kruse and M. K. Patterson (ed.), Tissue culture, methods and applications. Academic Press, Inc., New York.

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