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Butyrate-induced reversal of herpes simplex virus restriction in neuroblastoma cells
VIROLOGY
155,584~592 (1986)
Butyrate-Induced Reversal of Herpes Simplex Virus Restriction in Neuroblastoma Cells RONALD J. ASH Biochemical
Sciences, AferreU Dow Research Institute, Cincinnati, Ohio &215
Received May 9, 1986;accepted September 2, 1986 The synthesis of herpes simplex virus (HSV) in mouse neuroblastoma cells (NB, clone 41A3) is restricted. There was a disappearance of infectious virus upon serial passage of infected cells. NB cells treated with sodium-n-butyrate for 24 hr before infection synthesized 200-2000 times more HSV than untreated cells. Infectious center assays demonstrated that the number of cells capable of producing HSV was increased as a result of butyrate pretreatment. Although host protein synthesis was inhibited by HSV infection, viralinduced protein and DNA syntheses were not detected in the absence of butyrate. Cycloheximide blocked the induction of permissiveness by butyrate suggesting that a protein(s) was responsible for allowing HSV synthesis in NB cells. Regulatable host factors involved o 19% Academic in HSV replication in neural cells can be studied in the system described. Press, Inc.
INTRODUCTION
The information for producing infectious herpes simplex virus (HSV) resides in neurons of latently infected individuals (Baringer, 1975; Stevens, 1978). Whether other cells are involved in harboring the HSV genome is not known. The initial stages of infection and biochemical events leading to latency are not clearly defined nor are mechanisms involved in reactivation of virus understood. For example, it is not known whether HSV undergoes a productive primary infection of neurons in which the viral genome ultimately resides (Klein, 1982,1985) or whether certain cells of the nervous system lack host factors essential for virus replication thereby resulting in the latent state. Alternatively, nerve cells may possessa regulatable inhibitor of virus synthesis. It is difficult to study herpesvirus replication and mechanisms involved in virus reactivation in neurons of intact animals. For this reason, several investigators have studied HSV replication in cultured cells of the nervous system. Rat and human fetal neurons were found to be permissive for HSV when cultured in vitro (Wigdahl et al, 1983, 1984). In some established cell 0042-6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
lines of neural origin, however, HSV was reported to replicate poorly. Thus, rat glial (C,), Cl300 mouse neuroblastoma (clone 41A3), and rat brain neuroma (B103) cells were nonpermissive or restrictive for HSV growth (Schwartz and Elizan, 19’73;Lancz and Zettlemoyer, 1976; Vahlne and Lycke, 1977; Adler et cd, 1978). Clone N115 of Cl300 mouse neuroblastoma, on the other hand, was capable of supporting the synthesis of certain HSV strains if cells were starved into a state in which DNA synthesis was negligible, i.e., approximating a differentiated state (Gerdes et cd., 1979). In attempts to “differentiate” mouse neuroblastoma cells in vitro it was reported that butyric acid was effective in inhibiting cell division (Schneider, 1976). Further, the morphological characteristics of the cells were altered so that neurite extensions were produced. The biochemical characteristics of the cells were also changed by butyrate; for example, enzymes involved in neurotransmitter metabolism increased. Butyrate-treated neuroblastoma cells may provide an in vitro model for investigating HSV-neural cell interaction. This report shows that treatment of mouse neuroblastoma cells with sodium butyrate before infection renders them permissive for HSV
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ment (2 min) and then added to an equal volume of cold 10% TCA. After standing at 4” overnight, precipitates were collected on glass fiber filters and washed eight times with cold 5% TCA (3 ml/wash), Each filter MATERIALS AND METHODS was counted in 15 ml Ready-Solv HP (Beckman Instruments, Inc.) and radioacCells. Cl300 mouse neuroblastoma (clones 41A3 and ZA) and rat glial (C,) cells tivity determined in a Beckman LS7800 were obtained from the American Type liquid scintillation spectrometer. Culture Collection and propagated in EaAnalysis of inj?ectedcell proteins. NB cells gle’s basal medium (EBM) supplemented in 35-mm wells were labeled at various with 5% fetal calf serum (FCS). Vero cells, times after infection in medium containing Q normal methionine concentration and 5 originally obtained from Microbiological Associates, Inc. (Bethesda, Md.) were &i/ml of r5S]methionine (New England Nuclear, 985 Ci/mmol). Cells were lysed grown in the same medium. viruses. The KOS and MP strains of with 2% SDS + 0.2% @mercaptoethanol; HSV-1 were prepared by inoculating Vero glycerol was added to 20% and samples cells at an input m.o.i. of 0.1 PFU/cell. In- were boiled for 2 min before electrophofected cultures were sonically disrupted at resis on an SDS-polyacrylamide (7.5%) 20-24 hr p.i. and stored at -70” after in- slab gel (Maizel, 1971). Gels were fixed (30% clusion of DMSO to 5% (v/v) to prevent methanol + 10% TCA + 10% acetic acid), freeze damage (Wallis and Melnick, 1968). impregnated with En3Hance (New England Vaccinia and vesicular stomatitis viruses Nuclear, Inc.), dried, and exposed to Kodak were propagated in the same manner. All X-omat AR film for 24 hr. viruses were assayed by the plaque method RESULTS on Vero cells with a methyl cellulose overlay (0.5% in EBM + 1% FCS). Infectious center assays. The number of Eflect of Butyrate on Uninfected NB Cells cells capable of producing HSV was deterThe effect of sodium butyrate on the mined as follows: infected cell monolayers growth of NB cells, clone 41A3, is presented (3 hr p.i.) were washed once with EBM, once in Fig. 1. A concentration of 2.5 mM buwith EBM containing hyperimmune anti- tyrate effectively stopped cell division but HSV serum, and once again with EBM. cellular morphology was altered to only a Washed cells were trypsinized, counted, limited degree with flattening of all cells diluted in EBM, and plated on Vero cell and the appearance of short neurites on 5monolayers in 35-mm wells. Three milli10% of the total. DNA synthesis was inliters EBM (+2% FCS) were added to each hibited by 90% in cells treated with 2.5 mM well and incubation continued at 36” for butyrate for 24 hr (Fig. 1). The ability of 48 hr. Cell sheets were fixed with 10% For- cells to synthesize RNA or protein were afmalin and stained with crystal violet (0.3%, fected to a lesser extent with protein synaqueous). thesis being maintained at about 70% of DNA, RNA, and protein synthesis. The the control value. Butyrate treatment also ability of cells to synthesize macromole- stimulated the specific activity of acetyl cules was determined by exposing cultures cholinesterase by about 50% (data not (35-mm wells) to EBM containing 1 &i/ shown). ml of rH]thymidine (ICN, 72 Ci/mmol), [3H]uridine (New England Nuclear, 45.9 Ci/ HSV Synthesis in Butyrate-Treated Cells mmol), or [3H]leucine (New England NuNB cells treated with butyrate for 24 hr clear, 50 Ci/mmol) for 30 min at 36”. In all experiments reported, the incorporation of before infection were able to synthesize radiolabeled compounds was linear for at HSV. Butyrate was removed at the time of least 60 min. Labeled cells were suspended infection in these experiments and incluin ice cold saline after brief trypsin treat- sion of butyrate in the medium during the
synthesis. A butyrate-induced protein(s) is apparently responsible for the alteration of permissiveness.
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IO I 2
4
6
8
IO
mM BUTYRATE 0
l 2.5mM 0 IOmM
I 0
1 24
L 48
I 72
HOURS
FIG. 1. Effect of sodium n-butyrate on macromolecule synthesis and cell growth. (A) NB cells were exposed to the indicated concentrations of butyrate for 24 hr before measuring the ability of cells to synthesize DNA, RNA, or protein as outlined under Materials and Methods. Incorporation into controls, expressed as DPM/mg protein, were DNA, 548332; RNA, 259993, protein, 82843. (B) NB cells were seeded in 35 mm wells for 24 hr prior to changing the medium to EBM + 5% fetal calf serum + butyrate at the concentrations shown on each curve. Cell counts were taken at daily intervals.
growth cycle as well did not affect the virus yield. The synthesis of two other viruses which replicate in NB cells, i.e., vaccinia and vesicular stomatitis, was unaffected by butyrate pretreatment. Single step growth curves of HSV-KOS and HSV-MP in NB cells f butyrate pretreatment are presented in Fig. 2. Progeny virus was detected at 8-10 hr p.i. in cells treated with 2.5 mM butyrate. The difference in virus titer between control and bu-
J. ASH
tyrate-treated cultures was about 400-fold for both HSV strains in the experiment shown. In over 20 experiments this difference in titer ranged from 200 to 2000. The reasons for this variation are not known. It was evident, however, that little, if any, virus was synthesized in the absence of butyrate treatment. HSV persisted in untreated cells for the duration of the experiment (Fig. 2). The fact that cell-associated virus titers immediately after the adsorption period were the same in control and butyratetreated cultures suggested that butyrate did not influence virus adsorption. This was confirmed by demonstrating that control and butyrate-treated cells adsorbed HSV at the same rate (data not shown). Enhancement of HSV synthesis by butyrate was not observed in C6rat glial cells although DNA synthesis was inhibited by 90% after fatty acid treatment. Clone 2A NB cells supported HSV synthesis and virus titers were increased lo-fold by butyrate. Clone 41A3 was used for the rest of the experiments reported here. Efect of Other Cmpounds sis in NB Cells
on HSVSynthe-
Fatty acids of different chain length were tested on NB cells as inducers of the HSV permissive state (Fig. 3). Butyrate was the most effective compound tested although propionate (C,) and valerate (C,) also
6
12
24 HOURS PI
FIG. 2. Influence of butyrate on HSV growth in NB cells. (o), NB cells treated with 2.5 mM butyrate for 24 hr before infection; (0), untreated cells. Multiplicities of infection were 1 PFWcell for HSV-KOS and 0.2 PFU/cell for HSV-MP.
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CELLS
.
0
2 NUMBER
3
4 OF CARBON
5
6
I
I
I
&
I
5
IO
50
1
100
MO1 (PFU/CELLl
ATOMS
FIG. 3. HSV synthesis in NB cells treated with short chain fatty acids. The concentrations of fatty acids were W, 2.5 mM; 0, 5 mM; 0, 10 mM. HSV m.o.i. = 1 PFWcell; virus titers were determined at 24 hr p.i.
stimulated HSV synthesis in NB cells. Acetate (Cz) and caproate (C,) were without effect at the same or higher concentrations. Treatment of NB cells with agents used in other cell systems for in vitro differentiation failed to alter responsiveness to HSV. For example, the following had no effect: 5-azacytidine (0.5-25 piIf), dimethyl sulfoxide (l-10 mM), papaverine (0.1-20
1000 5oo F l------
FIG. 5. Effect of multiplicity of infection with HSVKOS on infectious center formation in NB cells. 0, cells treated with 2.5 mM butyrate for 24 hr before infection; 0, untreated cells. Point not shown: untreated cells infected at m.o.i. of 0.12 gave 0.06% infectious centers.
pLM),or serum starvation for 24 hr. Butyrolactone and y-amino butyric acid were also ineffective compounds. A comparison of butyrate and dibutyryl cyclic AMP (B&CAMP) as stimulators of HSV synthesis in NB cells is presented in Fig. 4. BuzcAMP pretreatment of cells stimulated HSV synthesis but on a molar basis butyrate was far more effective. For example, at 2.5 mM butyrate the increase in virus yield over untreated cells was 500fold while at the same concentration of BuzcAMP the increase was 2-fold. At 5 mM BuzcAMP the HSV titer was increased only lo-fold over controls. Infectious Centers in HSV-Ir$ected
mM
FIG. 4. HSV-KOS synthesis in NB cells treated with butyrate or dibutyryl CAMP. 0, butyrate; 0, BugAMP. HSV m.o.i. = 1 PFU/cell.
NB Cells
The number of neuroblastoma cells capable of producing HSV was low and depended upon the multiplicity of infection (Fig. 5). Pretreatment of cultures with butyrate increased the number of cells registering as infectious centers. The percentage of infectious centers in the NB cell cultures was less than 50% even after butyrate treatment. This suggested heterogeneity in the cell population. To test this
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possibility, NB cells were cloned and grown from single cells. All of the 42 clones isolated were responsive to butyrate in supporting HSV synthesis. Some clones were more responsive to butyrate than others in terms of virus yield.
J. ASH A
B
C
D
E
F
G
H
Protein Synthesis in HSV-In&ected NB The ability of NB cells to synthesize protein at intervals after infection is shown in Fig. 6. The inhibition of protein synthesis occurred in both butyrate-treated and control cells to about the same extent although the rate of inhibition was slightly faster in butyrate-treated cells. The subsequent rise due to the synthesis of viral proteins was only observed in butyratetreated cells. SDS-gel electrophoresis of [35S]methionine-labeled proteins confirmed this (Fig. 7). Virus-induced polypeptides were not detected in NB cells without prior butyrate treatment. The inhibition of synthesis of prominent cellular proteins was also evident in butyrate-treated cells and to a greater extent than in untreated cultures. HSV DNA Synthesis CsCl density gradient analysis of DNA from HSV-infected cells indicated that 100
t I
I I I 12345678
I
1
I
I
I
HOURS PI FIG. 6. Protein synthesis in NB cells infected with HSV-KOS. 0, butyrate-treated; 0, untreated. Cells were labeled with [aH]leucine for 30 min at various times after HSV infection.
FIG. 7. SDS-PAGE of HSV-KOS-infected NB cells. Lanes A-D, untreated; lanes E-H, butyrate-treated. Labeling with $S]methionine was as follows: lanes A and E, uninfected cells, 2 hr label; lanes B and F, 2-4 hr p.i.; lanes C and G, 4-6 hr pi.; lanes D and H, 6-8 hr p.i. HSV input m.o.i. = 10 PFU/cell.
viral DNA was not detected unless cells had been treated with butyrate before infection. There appeared to be significant labeling of cellular DNA in infected, butyrate-treated cells. Since the inhibition of cellular DNA synthesis by butyrate is reversible (data not shown), material in the peak at the density of cell DNA may represent cellular DNA stimulated by butyrate removal. Alternatively, newly synthesized viral DNA bound to cellular sequences or defective viral DNA of low density may be in the cell DNA peak. If defective viral DNA was made, however, it was not packaged into virions. This conclusion is based on an experiment in which rH]thymidine was used to label DNA in infected cells from 2 to 24 hr p.i. Cellular extracts were treated with DNAase and centrifuged to equilibrium on potassium tartrate density gradients. Only butyratetreated cells contained DNAase-resistant rH]DNA which banded at the position of intact virions. Role of Protein Synthesis in Butyrate-lnduced Eflects on NB Cells The induction of the HSV permissive state by butyrate required protein synthe-
HSV TABLE
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100 r
1
EFFECT OF CYCLOHEXIMIDE ON BUTYRATE-INDUCED ENHANCEMENT OF HSV-KOS SYNTHESIS’
Compounds
Relative virus yield
PFU/ml
None Cycloheximide, 2.5 pM Butyrate, 2.5 mM Butyrate + Cycloheximide
7.20 x 5.66 x 1.48 x 1.63 X
106 106 10s lo6
1 0.8 205 2.3
a NB cells were incubated with EBM + 2% FCS containing 2.5 mM sodium butyrate, 2.5 MM cycloheximide, or both for 16 hr prior to infection with HSVKOS. Fresh medium without compounds was added and cultures were incubated for 24 hr at 36” before determining virus titers.
sis (Table 1). The presence of cycloheximide during butyrate treatment of NB cells abolished the stimulation of HSV replication. This suggested that butyrate induced the synthesis of cellular proteins required for HSV production in NB cells.
8 I
A
7B
C
5L 0
I 48
I 24
I 72
HOURS
FIG. 8. HSV-KOS synthesis in NE cells treated with butyrate after infection. Cells were infected with HSV (m.o.i. = 1) at 0 time. Butyrate (2.5 mM) was added to culture A immediately after virus adsorption and to culture B at 24 hr p.i. Culture C did not receive butyrate. Arrows indicate the addition of hutyrate to cultures A or B.
DAYS PI
FIG. 9. Loss of HSV in infected NB cells upon serial passage. NB cells were infected with HSV-KOS (m.o.i. = 1) or HSV-MP (m.o.i. = 1) on Day 0. At the points shown cells were passaged (split ratio 1:4) and the number of infectious centers determined.
Eflect of Butyrate Addition aJter HSV Infection
Since NB cells treated with butyrate before infection were capable of synthesizing HSV, experiments were designed to determine whether addition of butyrate after infection would influence the yield of HSV. The results of an experiment in which butyrate was added immediately after the adsorption period or at 24 hr p.i. is shown in Fig. 8. The addition of butyrate at the end of the adsorption period failed to influence the virus titer for 24 hr; thereafter, virus synthesis was stimulated. Similarly, virus titers increased lo-fold between 48 and 72 hr p.i. when butyrate was added at 24 hr p.i. Thus, butyrate was able to activate HSV synthesis when added after infection but time was required for the butyrate to exert its effect. Fate of HSV in Passaged NB Cells NB cells infected with HSV could be passaged at intervals and retain a normal growth pattern. The level of HSV and the number of cells capable of registering as infectious centers gradually decreased in such cultures (Fig. 9). The ability to detect infectious virus was lost in both HSV-KOS
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and HSV-MP-infected cells in the example shown. In some cases, NB cells infected with either strain of HSV could be passaged and virus detected at a low level (lo3 PFU/ml) for over a year. Reasons for the establishment of persistent infections or loss of infectious virus are not clear. These cell lines should be of value in future studies. DISCUSSION
The ability of n-butyrate to alter the permissiveness of cultured cells infected with HSV has not been reported previously. Butyrate was tested in various cells infected with adeno-, polyoma, SV40, and EB viruses with divergent results. For example, butyrate inhibits viral DNA synthesis in the following systems: adenovirus 2 in 3T3 cells (Iseki and Baserga, 1983), polyoma virus in mouse kidney cells (Wawra et a& 1981), and SV40 virus in BSC-1 cells (Daniell et a& 1982). In EB virus infected cells, on the other hand, butyrate activates early antigen synthesis in producer (P3HR-1) or nonproducer (Raji) cell lines (Luka et al, 1979; Saemundsen et aZ., 1980). EB virus DNA and virion synthesis was also activated in P3HR cells by butyrate. My results with HSV are similar to the EB virus studies in that butyrate exerted a positive influence on virus synthesis. Experiments on another herpesvirus, cytomegalovirus (CMV) were performed in human teratocarcinoma cells (clone NT2/B9) before and after retinoic acid-induced differentiation (Gonczol et al, 1984). The growth of CMV did not occur in the absence of retinoic acid treatment. Collectively, these studies suggest that the differentiated phenotype of certain cells may be essential for productive infection with herpesviruses. In this report the induction of HSV permissiveness in NB cells was not limited to butyrate. Propionate and valerate were also capable of stimulating HSV synthesis in NB cells although not to the same extent as butyrate (Fig. 3). Schneider (1976) found that the differentiation of NB cells was also optimal with butyrate but that propionate and valerate displayed modest activity. BuzcAMP was a relatively poor inducer of
J. ASH
the HSV permissive state in NB cells and was useful only at higher concentrations than butyrate (Fig. 4). The fact that papaverine, a compound which elevates intracellular CAMP, was unable to render NB cells productive for HSV also suggests that the effects of butyrate are not merely on the concentration of CAMP. It is possible that the stimulation of HSV synthesis by BuzcAMP was due to contaminating butyrate or liberation of butyrate from degraded Bu,cAMP (Kaukel and Hilz, 1972). The inability of NB cells to support HSV synthesis in the absence of butyrate was not due to activation of the interferon (IFN) system. Interferon assays on mouse L cells of supernatant fluids from NB cells infected with HSV were negative (data not shown). NB cells were able to produce IFN upon stimulation with poly 1:C and were responsive to mouse IFN added to the medium indicating that there was no defect in the IFN system in NB cells. Vahlne and Lycke (1978) also suggested that the restriction of HSV in their NB cells was not due to IFN production. Experiments in which NB cells were infected with HSV at different m.o.i. demonstrated that high multiplicities resulted in a relative increase in the number of cells capable of registering as infectious centers (Fig. 5). Vahlne and Lycke (1978) found that only 1.4% of NB cells infected with HSV, strain F, at a m.o.i. of 5 PFU/cell became infectious centers. This compares with my experiments in which a m.o.i. of 5 yielded 4.2% infectious centers. In a subsequent paper, Vahlne et al (1981) noted a further increase in the number of infectious centers as the m.o.i. of HSV was increased to a maximum of 10 PFU/cell. It is not possible to compare these results directly with mine since the percentage infectious centers cannot be determined from their data. In my hands, increasing the m.o.i. increased the number of virus-producing cells but even at an input m.o.i. of 50 PFU/cell less than 10% of the cells were capable of infectious center formation. Butyrate treatment greatly increased the number of infectious centers when compared to control cells infected at comparable m.o.i. (Fig. 5). The maximum number
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L (TK-) cells results in cell hybrids which of infectious centers obtained in butyratetreated cultures, however, was only 38% at are sensitive to HSV. Epstein et al. (1985) a m.o.i. of 5-10 PFU/cell. Increasing the suggested that the XC cells are nonperm.o.i. to 50 PFU/cell did not result in a missive because they lack an essential elehigher percentage of infectious centers. It ment required for virus synthesis rather should be noted that Vahlne and co-work- than having an endogenous inhibitor of HSV. In the present study, an inhibitor of ers (1978, 1981) did not attempt to differentiate their NB cells prior to HSV infec- HSV in NB cells has not been completely ruled out. It is possible that the protein(s) tion. Reasons for the m.o.i. response of HSV induced by butyrate acts by binding or desynthesis in NB cells are unknown but it stroying an inhibitor or by repressing its would appear that the host factor(s) in- synthesis. The fact that butyrate could be added as duced by butyrate can be replaced to some late as 24 hr p.i. and still enhance HSV extent by increasing the number of virions entering the cell. Vahlne et al. (1981) con- synthesis (Fig. 8) indicated that the nonreplicating HSV genomes were not rapidly cluded that NB cells had to be infected with more than one HSV particle to produce degraded in NB cells. When infected cells progeny virus. These authors suggested were treated with butyrate the HSV DNA that NB cells contained an inhibitor of HSV could be converted into a productive state. which could be overcome by increasing the Although input HSV inhibited cell protein number of HSV DNA copies per cell. While synthesis by about 50% (Fig. 6), butyrate there may be some validity to this idea, the rendered the cells capable of supporting fact that less than 50% of the cells in the viral protein synthesis. Further, the initial culture are capable of producing virus even inhibition of host protein synthesis did not at a very high m.o.i. may suggest other interfere with subsequent NB cell division mechanisms. Additionally, the role of HSV since untreated (i.e., no butyrate) infected defective particles, even in cloned virus cells could survive infection and be paspopulations, must be considered. Such par- saged at regular intervals. The ability of butyrate to alter the reticles may play a suppressive role at high striction of HSV in NB cells provides a multiplicities. Although the NB cell population may be simple system for studying the regulation heterogeneous in some ways, it is not di- of virus synthesis by host factors. It should verse with respect to the ability to support be possible to identify the required host factors in cultured nerve cells and relate HSV synthesis after butyrate treatment. Forty-two separate NB clones were all ca- these to cells of the intact animal. The induction of certain neuronal proteins by pable of producing HSV after butyrate. stress, for example, may be responsible for Since the maximal number of infectious centers is 38%) it is possible that NB cells activation of the latent HSV genome. must be in a particular stage of the cell ACKNOWLEDGMENTS cycle to be affected by butyrate. Alternatively, the generation of defective virions The author thanks G. D. Mayer for his encourageunder permissive conditions may influence ment and support of this project, Charles Meiser for the number of cells yielding HSV. technical help, and Sue Treadway for assistance in The experiment in which cycloheximide manuscript preparation. blocked the induction of HSV permissiveness in NB cells (Table 1) suggested that REFERENCES butyrate was responsible for production of R., GLORIOSO, J. C., and LEVINE, M. (1978). a host protein(s) necessary for HSV syn- ADLER, Infection by herpes simplex viruses and cells of thesis. Experiments with another nonpernervous system origin: Characterization of a nonmissive cell, XC, have indicated that some permissive interaction. J. Gen. Wol 39.9-20. cellular factor required for virus replicaBARINGER, J. R. (1975). Herpes simplex virus infection tion is lacking (Epstein et al, 1985). Thus, of nervous tissue in animals and man. Prog. Med ViroL 20, l-26. fusion of XC (HPRT-) cells with permissive
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DANIELL,E., BURG,J. L., and FEDOR,M. J. (1982).DNA SAEMUNDSEN, A. K., KALLIN, B., and KLEIN, G. (1980). and histone synthesis in butyrate-inhibitied BSCEffect of n-butyrate on cellular and viral DNA syn1 cells infected with SVM. firology 116.196-206. thesis in cells latently infected with Epstein-Barr EPSTEIN,A. L., JACQUEMONT, B., and PATET,J. (1985). virus. Virologg 107, 557-561. Susceptibility to herpes simplex virus type 1 infec- SCHNEIDER,F. H. (1976). Effects of sodium butyrate tion of non-permissive rat XC (HPRT-) X permison mouse neuroblastoma cells in culture. Biochem. sive mouse L (TK) hybrid cells. J. Gen viral 66, PhmmmmL 25,2309-2317. SCHWARTZ, J., and ELIZAN, T. S. (1973).Chronic herpes 1805-1809. GERDES,J. C., MARSDEN,H. S., COOK,M. L., and STEVsimplex virus infection. Arch Neur& 28.224230. STEVENS,J. G. (1978). Latent characteristics of seENS, J. G. (1979). Acute infection of differentiated neuroblastoma cells by latency-positive and latencylected herpesviruses. Adv. Cancer Res. 26,227~2.56. negative herpes simplex virus ts mutants. virdogy VAHLNE, A., and LYCKE, E. (1977). Herpes simplex 94,430~441. infection of mouse neuroblastoma cells. Proc Sot GONCZOL, E., ANDREW& P. W., and PLOTKIN, S. A. Exp. Bid Med 156,82-87. (1984). Cytomegalovirus replicates in differentiated VAHLNE, A., and LYCKE, E. (1978). Herpes simplex but not in undifferentiated human embryonal carinfection of in vitro cultured neuronal cells (Mouse cinoma cells. Science 224, 159-161. neuroblastoma Cl306 cells). J. Gen. I%oL 39,321ISEKI, S., and BASERGA,R. (1983). Effect of butyrate 332. on adenovirus infection in semipermissive cells. Vi- VAHLNE, A., NILHEDEN, E., and SVENNERHOLM,B. rology 124,188-191. (1981). Multiplicity activation of herpes simplex viKAUKEL, E., and HILZ, H. (1972). Permeation of dirus in mouse neuroblastoma (C1300)cells. Arch Vibutyryl CAMP into HeLa cells and its conversion roL 70,345-356. to monobutyryl CAMP. Biochem Biophys. Res. WALLIS, C., and MELNICK, J. L. (1968). Stabilization Commun 46,1011-1018. of enveloped viruses by dimethyl sulfoxide. .I. ViroL KLEIN, R. J. (1982). The pathogenesis of acute, latent 2,953-954. and recurrent herpes simplex virus infections. Arch WAWRA, E., POCICL,E., MULLNER,E., and WINTERSViroL 72,143-168. BERGER,E. (1981). Effect of sodium butyrate on inKLEIN, R. J. (1985). Problems of herpes simplex virus duction of cellular and viral DNA syntheses in latency. Antiviral Rec. Suppl l, lll-120. polyoma virus-infected mouse kidney cells. J. ViroL LANCZ,G. J., and ZETIUYOYER, T. L. (1976).Restricted 38,973-981. replication of herpes simplex virus in neural cells. WIGDAHL,B., SMITH,C. A., TRAGLIA,H. M., and RAPP, Proc. Sot Exp. Bid Meal 152.302-306. F. (1984). Herpes simplex virus latency in isolated LUKA, J., KALLIN, B., and KLEIN, G. (1979). Induction human neurons. Proc NatL Ad Sci. USA 81,6217of the Epstein-Barr virus (EBV) cycle in latently 6221. infected cells by n-butyrate. virology 94,228-231. WIGDAHL,B., ZIEGLER,R. J., SNEVE,M., and RAPP,F. MAIZEL, J. V. (1971). Polyacrylamide gel electropho(1983). Herpes simplex latency and reactivation in resis of viral protein. Methds Viral 5,179~246. isolated rat sensory neurons. fir127,159-167.
155,584~592 (1986)
Butyrate-Induced Reversal of Herpes Simplex Virus Restriction in Neuroblastoma Cells RONALD J. ASH Biochemical
Sciences, AferreU Dow Research Institute, Cincinnati, Ohio &215
Received May 9, 1986;accepted September 2, 1986 The synthesis of herpes simplex virus (HSV) in mouse neuroblastoma cells (NB, clone 41A3) is restricted. There was a disappearance of infectious virus upon serial passage of infected cells. NB cells treated with sodium-n-butyrate for 24 hr before infection synthesized 200-2000 times more HSV than untreated cells. Infectious center assays demonstrated that the number of cells capable of producing HSV was increased as a result of butyrate pretreatment. Although host protein synthesis was inhibited by HSV infection, viralinduced protein and DNA syntheses were not detected in the absence of butyrate. Cycloheximide blocked the induction of permissiveness by butyrate suggesting that a protein(s) was responsible for allowing HSV synthesis in NB cells. Regulatable host factors involved o 19% Academic in HSV replication in neural cells can be studied in the system described. Press, Inc.
INTRODUCTION
The information for producing infectious herpes simplex virus (HSV) resides in neurons of latently infected individuals (Baringer, 1975; Stevens, 1978). Whether other cells are involved in harboring the HSV genome is not known. The initial stages of infection and biochemical events leading to latency are not clearly defined nor are mechanisms involved in reactivation of virus understood. For example, it is not known whether HSV undergoes a productive primary infection of neurons in which the viral genome ultimately resides (Klein, 1982,1985) or whether certain cells of the nervous system lack host factors essential for virus replication thereby resulting in the latent state. Alternatively, nerve cells may possessa regulatable inhibitor of virus synthesis. It is difficult to study herpesvirus replication and mechanisms involved in virus reactivation in neurons of intact animals. For this reason, several investigators have studied HSV replication in cultured cells of the nervous system. Rat and human fetal neurons were found to be permissive for HSV when cultured in vitro (Wigdahl et al, 1983, 1984). In some established cell 0042-6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
lines of neural origin, however, HSV was reported to replicate poorly. Thus, rat glial (C,), Cl300 mouse neuroblastoma (clone 41A3), and rat brain neuroma (B103) cells were nonpermissive or restrictive for HSV growth (Schwartz and Elizan, 19’73;Lancz and Zettlemoyer, 1976; Vahlne and Lycke, 1977; Adler et cd, 1978). Clone N115 of Cl300 mouse neuroblastoma, on the other hand, was capable of supporting the synthesis of certain HSV strains if cells were starved into a state in which DNA synthesis was negligible, i.e., approximating a differentiated state (Gerdes et cd., 1979). In attempts to “differentiate” mouse neuroblastoma cells in vitro it was reported that butyric acid was effective in inhibiting cell division (Schneider, 1976). Further, the morphological characteristics of the cells were altered so that neurite extensions were produced. The biochemical characteristics of the cells were also changed by butyrate; for example, enzymes involved in neurotransmitter metabolism increased. Butyrate-treated neuroblastoma cells may provide an in vitro model for investigating HSV-neural cell interaction. This report shows that treatment of mouse neuroblastoma cells with sodium butyrate before infection renders them permissive for HSV
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ment (2 min) and then added to an equal volume of cold 10% TCA. After standing at 4” overnight, precipitates were collected on glass fiber filters and washed eight times with cold 5% TCA (3 ml/wash), Each filter MATERIALS AND METHODS was counted in 15 ml Ready-Solv HP (Beckman Instruments, Inc.) and radioacCells. Cl300 mouse neuroblastoma (clones 41A3 and ZA) and rat glial (C,) cells tivity determined in a Beckman LS7800 were obtained from the American Type liquid scintillation spectrometer. Culture Collection and propagated in EaAnalysis of inj?ectedcell proteins. NB cells gle’s basal medium (EBM) supplemented in 35-mm wells were labeled at various with 5% fetal calf serum (FCS). Vero cells, times after infection in medium containing Q normal methionine concentration and 5 originally obtained from Microbiological Associates, Inc. (Bethesda, Md.) were &i/ml of r5S]methionine (New England Nuclear, 985 Ci/mmol). Cells were lysed grown in the same medium. viruses. The KOS and MP strains of with 2% SDS + 0.2% @mercaptoethanol; HSV-1 were prepared by inoculating Vero glycerol was added to 20% and samples cells at an input m.o.i. of 0.1 PFU/cell. In- were boiled for 2 min before electrophofected cultures were sonically disrupted at resis on an SDS-polyacrylamide (7.5%) 20-24 hr p.i. and stored at -70” after in- slab gel (Maizel, 1971). Gels were fixed (30% clusion of DMSO to 5% (v/v) to prevent methanol + 10% TCA + 10% acetic acid), freeze damage (Wallis and Melnick, 1968). impregnated with En3Hance (New England Vaccinia and vesicular stomatitis viruses Nuclear, Inc.), dried, and exposed to Kodak were propagated in the same manner. All X-omat AR film for 24 hr. viruses were assayed by the plaque method RESULTS on Vero cells with a methyl cellulose overlay (0.5% in EBM + 1% FCS). Infectious center assays. The number of Eflect of Butyrate on Uninfected NB Cells cells capable of producing HSV was deterThe effect of sodium butyrate on the mined as follows: infected cell monolayers growth of NB cells, clone 41A3, is presented (3 hr p.i.) were washed once with EBM, once in Fig. 1. A concentration of 2.5 mM buwith EBM containing hyperimmune anti- tyrate effectively stopped cell division but HSV serum, and once again with EBM. cellular morphology was altered to only a Washed cells were trypsinized, counted, limited degree with flattening of all cells diluted in EBM, and plated on Vero cell and the appearance of short neurites on 5monolayers in 35-mm wells. Three milli10% of the total. DNA synthesis was inliters EBM (+2% FCS) were added to each hibited by 90% in cells treated with 2.5 mM well and incubation continued at 36” for butyrate for 24 hr (Fig. 1). The ability of 48 hr. Cell sheets were fixed with 10% For- cells to synthesize RNA or protein were afmalin and stained with crystal violet (0.3%, fected to a lesser extent with protein synaqueous). thesis being maintained at about 70% of DNA, RNA, and protein synthesis. The the control value. Butyrate treatment also ability of cells to synthesize macromole- stimulated the specific activity of acetyl cules was determined by exposing cultures cholinesterase by about 50% (data not (35-mm wells) to EBM containing 1 &i/ shown). ml of rH]thymidine (ICN, 72 Ci/mmol), [3H]uridine (New England Nuclear, 45.9 Ci/ HSV Synthesis in Butyrate-Treated Cells mmol), or [3H]leucine (New England NuNB cells treated with butyrate for 24 hr clear, 50 Ci/mmol) for 30 min at 36”. In all experiments reported, the incorporation of before infection were able to synthesize radiolabeled compounds was linear for at HSV. Butyrate was removed at the time of least 60 min. Labeled cells were suspended infection in these experiments and incluin ice cold saline after brief trypsin treat- sion of butyrate in the medium during the
synthesis. A butyrate-induced protein(s) is apparently responsible for the alteration of permissiveness.
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IO I 2
4
6
8
IO
mM BUTYRATE 0
l 2.5mM 0 IOmM
I 0
1 24
L 48
I 72
HOURS
FIG. 1. Effect of sodium n-butyrate on macromolecule synthesis and cell growth. (A) NB cells were exposed to the indicated concentrations of butyrate for 24 hr before measuring the ability of cells to synthesize DNA, RNA, or protein as outlined under Materials and Methods. Incorporation into controls, expressed as DPM/mg protein, were DNA, 548332; RNA, 259993, protein, 82843. (B) NB cells were seeded in 35 mm wells for 24 hr prior to changing the medium to EBM + 5% fetal calf serum + butyrate at the concentrations shown on each curve. Cell counts were taken at daily intervals.
growth cycle as well did not affect the virus yield. The synthesis of two other viruses which replicate in NB cells, i.e., vaccinia and vesicular stomatitis, was unaffected by butyrate pretreatment. Single step growth curves of HSV-KOS and HSV-MP in NB cells f butyrate pretreatment are presented in Fig. 2. Progeny virus was detected at 8-10 hr p.i. in cells treated with 2.5 mM butyrate. The difference in virus titer between control and bu-
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tyrate-treated cultures was about 400-fold for both HSV strains in the experiment shown. In over 20 experiments this difference in titer ranged from 200 to 2000. The reasons for this variation are not known. It was evident, however, that little, if any, virus was synthesized in the absence of butyrate treatment. HSV persisted in untreated cells for the duration of the experiment (Fig. 2). The fact that cell-associated virus titers immediately after the adsorption period were the same in control and butyratetreated cultures suggested that butyrate did not influence virus adsorption. This was confirmed by demonstrating that control and butyrate-treated cells adsorbed HSV at the same rate (data not shown). Enhancement of HSV synthesis by butyrate was not observed in C6rat glial cells although DNA synthesis was inhibited by 90% after fatty acid treatment. Clone 2A NB cells supported HSV synthesis and virus titers were increased lo-fold by butyrate. Clone 41A3 was used for the rest of the experiments reported here. Efect of Other Cmpounds sis in NB Cells
on HSVSynthe-
Fatty acids of different chain length were tested on NB cells as inducers of the HSV permissive state (Fig. 3). Butyrate was the most effective compound tested although propionate (C,) and valerate (C,) also
6
12
24 HOURS PI
FIG. 2. Influence of butyrate on HSV growth in NB cells. (o), NB cells treated with 2.5 mM butyrate for 24 hr before infection; (0), untreated cells. Multiplicities of infection were 1 PFWcell for HSV-KOS and 0.2 PFU/cell for HSV-MP.
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CELLS
.
0
2 NUMBER
3
4 OF CARBON
5
6
I
I
I
&
I
5
IO
50
1
100
MO1 (PFU/CELLl
ATOMS
FIG. 3. HSV synthesis in NB cells treated with short chain fatty acids. The concentrations of fatty acids were W, 2.5 mM; 0, 5 mM; 0, 10 mM. HSV m.o.i. = 1 PFWcell; virus titers were determined at 24 hr p.i.
stimulated HSV synthesis in NB cells. Acetate (Cz) and caproate (C,) were without effect at the same or higher concentrations. Treatment of NB cells with agents used in other cell systems for in vitro differentiation failed to alter responsiveness to HSV. For example, the following had no effect: 5-azacytidine (0.5-25 piIf), dimethyl sulfoxide (l-10 mM), papaverine (0.1-20
1000 5oo F l------
FIG. 5. Effect of multiplicity of infection with HSVKOS on infectious center formation in NB cells. 0, cells treated with 2.5 mM butyrate for 24 hr before infection; 0, untreated cells. Point not shown: untreated cells infected at m.o.i. of 0.12 gave 0.06% infectious centers.
pLM),or serum starvation for 24 hr. Butyrolactone and y-amino butyric acid were also ineffective compounds. A comparison of butyrate and dibutyryl cyclic AMP (B&CAMP) as stimulators of HSV synthesis in NB cells is presented in Fig. 4. BuzcAMP pretreatment of cells stimulated HSV synthesis but on a molar basis butyrate was far more effective. For example, at 2.5 mM butyrate the increase in virus yield over untreated cells was 500fold while at the same concentration of BuzcAMP the increase was 2-fold. At 5 mM BuzcAMP the HSV titer was increased only lo-fold over controls. Infectious Centers in HSV-Ir$ected
mM
FIG. 4. HSV-KOS synthesis in NB cells treated with butyrate or dibutyryl CAMP. 0, butyrate; 0, BugAMP. HSV m.o.i. = 1 PFU/cell.
NB Cells
The number of neuroblastoma cells capable of producing HSV was low and depended upon the multiplicity of infection (Fig. 5). Pretreatment of cultures with butyrate increased the number of cells registering as infectious centers. The percentage of infectious centers in the NB cell cultures was less than 50% even after butyrate treatment. This suggested heterogeneity in the cell population. To test this
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possibility, NB cells were cloned and grown from single cells. All of the 42 clones isolated were responsive to butyrate in supporting HSV synthesis. Some clones were more responsive to butyrate than others in terms of virus yield.
J. ASH A
B
C
D
E
F
G
H
Protein Synthesis in HSV-In&ected NB The ability of NB cells to synthesize protein at intervals after infection is shown in Fig. 6. The inhibition of protein synthesis occurred in both butyrate-treated and control cells to about the same extent although the rate of inhibition was slightly faster in butyrate-treated cells. The subsequent rise due to the synthesis of viral proteins was only observed in butyratetreated cells. SDS-gel electrophoresis of [35S]methionine-labeled proteins confirmed this (Fig. 7). Virus-induced polypeptides were not detected in NB cells without prior butyrate treatment. The inhibition of synthesis of prominent cellular proteins was also evident in butyrate-treated cells and to a greater extent than in untreated cultures. HSV DNA Synthesis CsCl density gradient analysis of DNA from HSV-infected cells indicated that 100
t I
I I I 12345678
I
1
I
I
I
HOURS PI FIG. 6. Protein synthesis in NB cells infected with HSV-KOS. 0, butyrate-treated; 0, untreated. Cells were labeled with [aH]leucine for 30 min at various times after HSV infection.
FIG. 7. SDS-PAGE of HSV-KOS-infected NB cells. Lanes A-D, untreated; lanes E-H, butyrate-treated. Labeling with $S]methionine was as follows: lanes A and E, uninfected cells, 2 hr label; lanes B and F, 2-4 hr p.i.; lanes C and G, 4-6 hr pi.; lanes D and H, 6-8 hr p.i. HSV input m.o.i. = 10 PFU/cell.
viral DNA was not detected unless cells had been treated with butyrate before infection. There appeared to be significant labeling of cellular DNA in infected, butyrate-treated cells. Since the inhibition of cellular DNA synthesis by butyrate is reversible (data not shown), material in the peak at the density of cell DNA may represent cellular DNA stimulated by butyrate removal. Alternatively, newly synthesized viral DNA bound to cellular sequences or defective viral DNA of low density may be in the cell DNA peak. If defective viral DNA was made, however, it was not packaged into virions. This conclusion is based on an experiment in which rH]thymidine was used to label DNA in infected cells from 2 to 24 hr p.i. Cellular extracts were treated with DNAase and centrifuged to equilibrium on potassium tartrate density gradients. Only butyratetreated cells contained DNAase-resistant rH]DNA which banded at the position of intact virions. Role of Protein Synthesis in Butyrate-lnduced Eflects on NB Cells The induction of the HSV permissive state by butyrate required protein synthe-
HSV TABLE
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100 r
1
EFFECT OF CYCLOHEXIMIDE ON BUTYRATE-INDUCED ENHANCEMENT OF HSV-KOS SYNTHESIS’
Compounds
Relative virus yield
PFU/ml
None Cycloheximide, 2.5 pM Butyrate, 2.5 mM Butyrate + Cycloheximide
7.20 x 5.66 x 1.48 x 1.63 X
106 106 10s lo6
1 0.8 205 2.3
a NB cells were incubated with EBM + 2% FCS containing 2.5 mM sodium butyrate, 2.5 MM cycloheximide, or both for 16 hr prior to infection with HSVKOS. Fresh medium without compounds was added and cultures were incubated for 24 hr at 36” before determining virus titers.
sis (Table 1). The presence of cycloheximide during butyrate treatment of NB cells abolished the stimulation of HSV replication. This suggested that butyrate induced the synthesis of cellular proteins required for HSV production in NB cells.
8 I
A
7B
C
5L 0
I 48
I 24
I 72
HOURS
FIG. 8. HSV-KOS synthesis in NE cells treated with butyrate after infection. Cells were infected with HSV (m.o.i. = 1) at 0 time. Butyrate (2.5 mM) was added to culture A immediately after virus adsorption and to culture B at 24 hr p.i. Culture C did not receive butyrate. Arrows indicate the addition of hutyrate to cultures A or B.
DAYS PI
FIG. 9. Loss of HSV in infected NB cells upon serial passage. NB cells were infected with HSV-KOS (m.o.i. = 1) or HSV-MP (m.o.i. = 1) on Day 0. At the points shown cells were passaged (split ratio 1:4) and the number of infectious centers determined.
Eflect of Butyrate Addition aJter HSV Infection
Since NB cells treated with butyrate before infection were capable of synthesizing HSV, experiments were designed to determine whether addition of butyrate after infection would influence the yield of HSV. The results of an experiment in which butyrate was added immediately after the adsorption period or at 24 hr p.i. is shown in Fig. 8. The addition of butyrate at the end of the adsorption period failed to influence the virus titer for 24 hr; thereafter, virus synthesis was stimulated. Similarly, virus titers increased lo-fold between 48 and 72 hr p.i. when butyrate was added at 24 hr p.i. Thus, butyrate was able to activate HSV synthesis when added after infection but time was required for the butyrate to exert its effect. Fate of HSV in Passaged NB Cells NB cells infected with HSV could be passaged at intervals and retain a normal growth pattern. The level of HSV and the number of cells capable of registering as infectious centers gradually decreased in such cultures (Fig. 9). The ability to detect infectious virus was lost in both HSV-KOS
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and HSV-MP-infected cells in the example shown. In some cases, NB cells infected with either strain of HSV could be passaged and virus detected at a low level (lo3 PFU/ml) for over a year. Reasons for the establishment of persistent infections or loss of infectious virus are not clear. These cell lines should be of value in future studies. DISCUSSION
The ability of n-butyrate to alter the permissiveness of cultured cells infected with HSV has not been reported previously. Butyrate was tested in various cells infected with adeno-, polyoma, SV40, and EB viruses with divergent results. For example, butyrate inhibits viral DNA synthesis in the following systems: adenovirus 2 in 3T3 cells (Iseki and Baserga, 1983), polyoma virus in mouse kidney cells (Wawra et a& 1981), and SV40 virus in BSC-1 cells (Daniell et a& 1982). In EB virus infected cells, on the other hand, butyrate activates early antigen synthesis in producer (P3HR-1) or nonproducer (Raji) cell lines (Luka et al, 1979; Saemundsen et aZ., 1980). EB virus DNA and virion synthesis was also activated in P3HR cells by butyrate. My results with HSV are similar to the EB virus studies in that butyrate exerted a positive influence on virus synthesis. Experiments on another herpesvirus, cytomegalovirus (CMV) were performed in human teratocarcinoma cells (clone NT2/B9) before and after retinoic acid-induced differentiation (Gonczol et al, 1984). The growth of CMV did not occur in the absence of retinoic acid treatment. Collectively, these studies suggest that the differentiated phenotype of certain cells may be essential for productive infection with herpesviruses. In this report the induction of HSV permissiveness in NB cells was not limited to butyrate. Propionate and valerate were also capable of stimulating HSV synthesis in NB cells although not to the same extent as butyrate (Fig. 3). Schneider (1976) found that the differentiation of NB cells was also optimal with butyrate but that propionate and valerate displayed modest activity. BuzcAMP was a relatively poor inducer of
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the HSV permissive state in NB cells and was useful only at higher concentrations than butyrate (Fig. 4). The fact that papaverine, a compound which elevates intracellular CAMP, was unable to render NB cells productive for HSV also suggests that the effects of butyrate are not merely on the concentration of CAMP. It is possible that the stimulation of HSV synthesis by BuzcAMP was due to contaminating butyrate or liberation of butyrate from degraded Bu,cAMP (Kaukel and Hilz, 1972). The inability of NB cells to support HSV synthesis in the absence of butyrate was not due to activation of the interferon (IFN) system. Interferon assays on mouse L cells of supernatant fluids from NB cells infected with HSV were negative (data not shown). NB cells were able to produce IFN upon stimulation with poly 1:C and were responsive to mouse IFN added to the medium indicating that there was no defect in the IFN system in NB cells. Vahlne and Lycke (1978) also suggested that the restriction of HSV in their NB cells was not due to IFN production. Experiments in which NB cells were infected with HSV at different m.o.i. demonstrated that high multiplicities resulted in a relative increase in the number of cells capable of registering as infectious centers (Fig. 5). Vahlne and Lycke (1978) found that only 1.4% of NB cells infected with HSV, strain F, at a m.o.i. of 5 PFU/cell became infectious centers. This compares with my experiments in which a m.o.i. of 5 yielded 4.2% infectious centers. In a subsequent paper, Vahlne et al (1981) noted a further increase in the number of infectious centers as the m.o.i. of HSV was increased to a maximum of 10 PFU/cell. It is not possible to compare these results directly with mine since the percentage infectious centers cannot be determined from their data. In my hands, increasing the m.o.i. increased the number of virus-producing cells but even at an input m.o.i. of 50 PFU/cell less than 10% of the cells were capable of infectious center formation. Butyrate treatment greatly increased the number of infectious centers when compared to control cells infected at comparable m.o.i. (Fig. 5). The maximum number
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591
L (TK-) cells results in cell hybrids which of infectious centers obtained in butyratetreated cultures, however, was only 38% at are sensitive to HSV. Epstein et al. (1985) a m.o.i. of 5-10 PFU/cell. Increasing the suggested that the XC cells are nonperm.o.i. to 50 PFU/cell did not result in a missive because they lack an essential elehigher percentage of infectious centers. It ment required for virus synthesis rather should be noted that Vahlne and co-work- than having an endogenous inhibitor of HSV. In the present study, an inhibitor of ers (1978, 1981) did not attempt to differentiate their NB cells prior to HSV infec- HSV in NB cells has not been completely ruled out. It is possible that the protein(s) tion. Reasons for the m.o.i. response of HSV induced by butyrate acts by binding or desynthesis in NB cells are unknown but it stroying an inhibitor or by repressing its would appear that the host factor(s) in- synthesis. The fact that butyrate could be added as duced by butyrate can be replaced to some late as 24 hr p.i. and still enhance HSV extent by increasing the number of virions entering the cell. Vahlne et al. (1981) con- synthesis (Fig. 8) indicated that the nonreplicating HSV genomes were not rapidly cluded that NB cells had to be infected with more than one HSV particle to produce degraded in NB cells. When infected cells progeny virus. These authors suggested were treated with butyrate the HSV DNA that NB cells contained an inhibitor of HSV could be converted into a productive state. which could be overcome by increasing the Although input HSV inhibited cell protein number of HSV DNA copies per cell. While synthesis by about 50% (Fig. 6), butyrate there may be some validity to this idea, the rendered the cells capable of supporting fact that less than 50% of the cells in the viral protein synthesis. Further, the initial culture are capable of producing virus even inhibition of host protein synthesis did not at a very high m.o.i. may suggest other interfere with subsequent NB cell division mechanisms. Additionally, the role of HSV since untreated (i.e., no butyrate) infected defective particles, even in cloned virus cells could survive infection and be paspopulations, must be considered. Such par- saged at regular intervals. The ability of butyrate to alter the reticles may play a suppressive role at high striction of HSV in NB cells provides a multiplicities. Although the NB cell population may be simple system for studying the regulation heterogeneous in some ways, it is not di- of virus synthesis by host factors. It should verse with respect to the ability to support be possible to identify the required host factors in cultured nerve cells and relate HSV synthesis after butyrate treatment. Forty-two separate NB clones were all ca- these to cells of the intact animal. The induction of certain neuronal proteins by pable of producing HSV after butyrate. stress, for example, may be responsible for Since the maximal number of infectious centers is 38%) it is possible that NB cells activation of the latent HSV genome. must be in a particular stage of the cell ACKNOWLEDGMENTS cycle to be affected by butyrate. Alternatively, the generation of defective virions The author thanks G. D. Mayer for his encourageunder permissive conditions may influence ment and support of this project, Charles Meiser for the number of cells yielding HSV. technical help, and Sue Treadway for assistance in The experiment in which cycloheximide manuscript preparation. blocked the induction of HSV permissiveness in NB cells (Table 1) suggested that REFERENCES butyrate was responsible for production of R., GLORIOSO, J. C., and LEVINE, M. (1978). a host protein(s) necessary for HSV syn- ADLER, Infection by herpes simplex viruses and cells of thesis. Experiments with another nonpernervous system origin: Characterization of a nonmissive cell, XC, have indicated that some permissive interaction. J. Gen. Wol 39.9-20. cellular factor required for virus replicaBARINGER, J. R. (1975). Herpes simplex virus infection tion is lacking (Epstein et al, 1985). Thus, of nervous tissue in animals and man. Prog. Med ViroL 20, l-26. fusion of XC (HPRT-) cells with permissive
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DANIELL,E., BURG,J. L., and FEDOR,M. J. (1982).DNA SAEMUNDSEN, A. K., KALLIN, B., and KLEIN, G. (1980). and histone synthesis in butyrate-inhibitied BSCEffect of n-butyrate on cellular and viral DNA syn1 cells infected with SVM. firology 116.196-206. thesis in cells latently infected with Epstein-Barr EPSTEIN,A. L., JACQUEMONT, B., and PATET,J. (1985). virus. Virologg 107, 557-561. Susceptibility to herpes simplex virus type 1 infec- SCHNEIDER,F. H. (1976). Effects of sodium butyrate tion of non-permissive rat XC (HPRT-) X permison mouse neuroblastoma cells in culture. Biochem. sive mouse L (TK) hybrid cells. J. Gen viral 66, PhmmmmL 25,2309-2317. SCHWARTZ, J., and ELIZAN, T. S. (1973).Chronic herpes 1805-1809. GERDES,J. C., MARSDEN,H. S., COOK,M. L., and STEVsimplex virus infection. Arch Neur& 28.224230. STEVENS,J. G. (1978). Latent characteristics of seENS, J. G. (1979). Acute infection of differentiated neuroblastoma cells by latency-positive and latencylected herpesviruses. Adv. Cancer Res. 26,227~2.56. negative herpes simplex virus ts mutants. virdogy VAHLNE, A., and LYCKE, E. (1977). Herpes simplex 94,430~441. infection of mouse neuroblastoma cells. Proc Sot GONCZOL, E., ANDREW& P. W., and PLOTKIN, S. A. Exp. Bid Med 156,82-87. (1984). Cytomegalovirus replicates in differentiated VAHLNE, A., and LYCKE, E. (1978). Herpes simplex but not in undifferentiated human embryonal carinfection of in vitro cultured neuronal cells (Mouse cinoma cells. Science 224, 159-161. neuroblastoma Cl306 cells). J. Gen. I%oL 39,321ISEKI, S., and BASERGA,R. (1983). Effect of butyrate 332. on adenovirus infection in semipermissive cells. Vi- VAHLNE, A., NILHEDEN, E., and SVENNERHOLM,B. rology 124,188-191. (1981). Multiplicity activation of herpes simplex viKAUKEL, E., and HILZ, H. (1972). Permeation of dirus in mouse neuroblastoma (C1300)cells. Arch Vibutyryl CAMP into HeLa cells and its conversion roL 70,345-356. to monobutyryl CAMP. Biochem Biophys. Res. WALLIS, C., and MELNICK, J. L. (1968). Stabilization Commun 46,1011-1018. of enveloped viruses by dimethyl sulfoxide. .I. ViroL KLEIN, R. J. (1982). The pathogenesis of acute, latent 2,953-954. and recurrent herpes simplex virus infections. Arch WAWRA, E., POCICL,E., MULLNER,E., and WINTERSViroL 72,143-168. BERGER,E. (1981). Effect of sodium butyrate on inKLEIN, R. J. (1985). Problems of herpes simplex virus duction of cellular and viral DNA syntheses in latency. Antiviral Rec. Suppl l, lll-120. polyoma virus-infected mouse kidney cells. J. ViroL LANCZ,G. J., and ZETIUYOYER, T. L. (1976).Restricted 38,973-981. replication of herpes simplex virus in neural cells. WIGDAHL,B., SMITH,C. A., TRAGLIA,H. M., and RAPP, Proc. Sot Exp. Bid Meal 152.302-306. F. (1984). Herpes simplex virus latency in isolated LUKA, J., KALLIN, B., and KLEIN, G. (1979). Induction human neurons. Proc NatL Ad Sci. USA 81,6217of the Epstein-Barr virus (EBV) cycle in latently 6221. infected cells by n-butyrate. virology 94,228-231. WIGDAHL,B., ZIEGLER,R. J., SNEVE,M., and RAPP,F. MAIZEL, J. V. (1971). Polyacrylamide gel electropho(1983). Herpes simplex latency and reactivation in resis of viral protein. Methds Viral 5,179~246. isolated rat sensory neurons. fir127,159-167.