Accelerated Cytopathology in HeLa Cells Induced

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1 INFECTION AND IMMUNITY, Dec. 197, p Copyright 197 American Society for Microbiology Vol., No. Printed in U.S.A. Accelerated Cytopathology in HeLa Cells Induced by Reovirus and Cycloheximide PHILIP C. LOH, HERBERT K. OIE, AND R. M. T. RATNAYAKE Department of Microbiology, University of Hawaii, Honolulu, Hawaii Received for publication 1 August 197 The addition of the protein inhibitor cycloheximide, at concentrations which suppress virus replication, to HeLa cell monolayers infected with reovirus type results in the appearance of accelerated cytopathic effects (CPE). At high multiplicity of infection, CPE appeared after a lag period of to and increased progressively, until by 1 the entire monolayer had rounded and sloughed off. During this same period, both the uninfected cycloheximide-treated and untreated virusinfected controls exhibited no CPE. The phenomenon is affected by the kind of cell species employed and can be reversed if the antibiotic is removed within 1 after exposure. The evidence obtained tough studies in which specific metabolic inhibitors and direct biochemical analyses were used suggests that the accelerated CPE observed in cycloheximide-treated reovirus-infected cells is the consequence of the combined inhibition of the synthesis of both cellular protein and ribonucleic acid. The accelerated CPE is also induced in the antibiotic-treated cells by reovirus serotypes 1 and. It was previously reported that the addition of virus-inhibiting concentrations of the protein antagonist cycloheximide to HeLa cells infected with reovirus type resulted in an accelerated appearance of extensive cytopathic effects (CPE) on the monolayers (7). In contrast, parallel studies of normal cell cultures treated with the antibiotic and of infected cultures without cycloheximide revealed no morphological changes. The present communication describes some additional cytological and biochemical studies made on the interactions of cycloheximide, reoviruses, and HeLa cell cultures. In addition, the combined effect of the antibiotic and reovirus on cell cultures of other mammalian species was examined. MATERIALS AND METHODS Cells. The following mammalian cell cultures were grown as monolayers in Eagle's basal medium (EBM) supplemented with 1% fetal calf serum (FCS): HeLa (human carcinoma of the cervix), RA (an established line of human amnion cells), W1 (human embryonic lung, diploid), BSC-1 (an established line of green monkey kidney cells), AGMK (African green monkey kidney, primary), L (an established line of mouse fibroblast), MEF (mouse embryonic fibroblast, primary), and BHK-1 (an established line of baby hamster kidney cells). The basic experimental medium consisted of EBM plus.1% FCS. Viruses. Reovirus serotypes 1 (Lang), (D-5 Jones), and (Abney) were grown in HeLa cell cultures, and their titers were determined by the immunofluorescence method (15). Infection procedure. Each virus sample was allowed to adsorb to the cell monolayers for at 7 C with intermittent shaking. After adsorption, the residual virus was removed and the experimental medium was introduced. Assay of CPE. Cytopathology was recorded in the following manner:, suggestive morphological changes;, definite morphological changes;, more than 5% cell degeneration; and, complete cell degeneration. Analyses of nucleic acids and proteins. To determine the rate of synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein, the tritiated precursors uridine, thymidine, and leucine, respectively, were added to replicate cultures at zero time after infection. At various intervals after infection, cell cultures were washed twice with cold phosphatebuffered saline (PBS), ph 7., the cells were scraped off, and the amount of trichloroacetic acid-insoluble radioactivity incorporated was determined in a Packard liquid scintillation counter (9). Chemicals and radioactive precursors. Cycloheximide was purchased from Calbiochem, Los Angeles. Calif. H-uridine ( Ci/mmole), H-thymidine ( Ci/ mmole), H-leucine ( Ci/mmole), and H-labeled reconstituted protein hydrolysate were obtained from Schwarz BioResearch Inc., Van Nuys, Calif. 75 RESULTS Effect of cycloheximide and reovirus on HeLa cells. As was previously reported (7), the addition

2 7 LOH, OIE, AND RATNAYAKE INFEC. IMMUN. TABLE 1. Sample' Effect of cycloheximide and reovirus type infection on HeLa cells Appearance of cytopathologyb 5 1 Normal. AD Cy... V...- V AD V CY. - V Cy AD... - a V = reovirus type at an exposure multiplicity of 1 to 15 infectious units per cell. AD = actinomycin D at.5,ug per ml. Cy = cycloheximide at 1lg per ml. I Cytopathology was recorded in the following manner:, suggestive morphological changes;, definite morphological changes;, more than 5% cell degeneration; complete cell degeneration. of the antibiotic cycloheximide to monolayers of HeLa cells infected with reovirus type caused the appearance of early cytopathic alterations (Table 1). The early CPE was also observed when reovirus serotype 1 or was employed. Preincubation of reovirus type with its specific immune serum (1 at 7 C) before infection of the cycloheximide-treated HeLa cell cultures prevented the occurrence of CPE. Serial transfer of the supernatants from cell cultures exhibiting accelerated CPE to fresh monolayers did not result in the production of CPE. Examination by the direct immunofluorescence technique of the antibiotic-treated infected cells indicated the absence of the formation of any virus antigen. The acceleration of CPE was also observed in infected cultures when the protein antagonist puromycin was substituted for cycloheximide. Effect of cycloheximide on protein synthesis. Monolayers of HeLa cells were treated with various concentrations of cycloheximide for and min, and the synthesis of protein was followed by pulse-labeling for min with Hreconstituted protein hydrolysate. Incorporation of the radioactive amino acids into the trichloroacetic-insoluble precipitate was taken as a measure of protein synthesis. The results (Table ) indicated that protein synthesis was inhibited by 91% within min after addition of 1,ug of the antibiotic per ml. The inhibition was greater (95 %) with increasing lengths of exposure or when larger concentrations of the antibiotic were employed. It was previously reported that cycloheximide at concentrations as low as.5 Ag/ml completely suppressed viris production (7). Effect of virus concentration. Monolayers of HeLa cells were exposed to various multiplicities of reovirus type, and the cell cultures were treated with cycloheximide (1,g/ml). The acceleration of CPE was observed even in cell cultures exposed to reovirus at a multiplicity of 1 (Table ). At this multiplicity, the extent of CPE was small, and no effect was observed until after infection. In cell cultures receiving larger multiplicities of virus, the CPE appeared earlier ( postinfection), and by 1 the entire monolayer had sloughed off. The earlier appearance of CPE with larger virus doses suggests a need to infect the majority of cells at about the same time rather than a need for a high multiplicity of infection. Effect of different concentrations of cycloheximide. The addition of different concentrations of the antibiotic to reovirus-infected HeLa cells did not significantly alter the time of appearance of accelerated CPE. Cytopathic alterations were observed as early as postinfection and became more marked with time. Addition of cycloheximide at different intervals. To clarify further the role of the virus or its products in the causation of accelerated CPE, the effect of cycloheximide when added at different times during the growth cycle of the reovirus was examined. Monolayers of HeLa cells were infected with reovirus type (multiplicity of infection, 1 to 15). Cycloheximide (1 Ag/ml) was added to the cell cultures at various intervals after infection. The addition of the antibiotic at different TABLE. Effect of cycloheximide on protein synthesis in uninfected HeLa cells Cycloheximide Per cent inhibition of the incorporation of H-RPHa after min min pg/mi None (5)b (17) 1 91 (1) 95 (1) 9 (19) 95 (9) 9 (1) 9 () 97 () 97 (5) a Monolayers of HeLa cells were pulselabeled (.5 gci/ml) for min with H-reconstituted protein hydrolysate, and the amount of trichloroacetic acid-insoluble radioactivity incorporated was determined. bcounts per minute per microgram of protein.

3 VOL., 197 CPE BY REOVIRUS AND CYCLOHEXIMIDE 77 TABLE. Relationship of multiplicity of infection (MOI) and cytopathology in cycloheximidetreated HeLa cellsa MOI Appearance of cytopathology li i 1 _ 1 _ a Monolayers of HeLa cells were exposed to reovirus type at different multiplicities of infection. After an adsorption period of at 7 C, Eagle's basal medium plus.% fetal calf serum was added with or without cycloheximide. At various intervals after infection, the monolayers were examined for cytopathology. intervals after infection did not alter the time of appearance of accelerated CPE (Table ). Irrespective of the time of addition, all antibiotictreated infected cultures displayed a lag period of to before the appearance of CPE. In addition, the extent of CPE was similar in these cultures. The results suggest that there is no accumulation of any virus-induced product(s) which might further shorten the time of appearance of CPE. Furthermore, such a product(s), if made, is synthesized as late as postinfection. Effect of the removal of cycloheximide. To determine whether the phenomenon of accelerated CPE could be reversed, the effect of the removal of cycloheximide from antibiotictreated infected cell cultures was examined at various intervals after infection. The removal of cycloheximide from infected cultures after to min of exposure prevented the occurrence of accelerated CPE (Table 5). The reversal was less effective as the period of treatment was extended. In such cultures, the CPE was not as extensive as in the antibiotictreated infected-cell controls. These results again indicate the requirement of a lag period before the appearance of accelerated CPE. Effect on other mammalian cell species. The effect of cycloheximide and reovirus type infection on susceptible cell cultures derived from other animal species was examined (Table ). Two established cell lines of human and murine origins, RA (human amnion) and L (mouse) cells, were found to respond with accelerated CPE on infection and the addition of cycloheximide. In contrast, the human diploid cell line W1 (human embryonic lung), the established monkey cell line BSC-1 (African green monkey kidney), the primary African green monkey kidney cells, and the established hamster cell line BHK-1 (baby hamster kidney) did not produce the phenomenon. Cycloheximide was toxic for the primary mouse embryo cells even at concentrations of 5,ug/ml. It was concluded that the accelerated CPE phenomenon induced by cycloheximide and reoviruses is dependent upon the genetic state and origin of the cell. Reovirus infection and different metabolic antagonists. In an attempt to clarify this phenomenon of accelerated CPE, the effect of various TABLE. Effject of adding cycloheximide at differentt tinmes after intfection Appearance of cytopathology ^n;;^,,,a S h V Cy ( )... V Cy (1 )... V Cy ( )... V Cy ( )... V Cy ( )... V Cy ( )... V Cy ( ) Cy only... V Normal... I_ - a V=reovirus type at a multiplicity of infection of 1 to 15. Cy cycloheximide = at time of addition of cycloheximide is given in parentheses. 1,ug/ml. The,

4 Noma 7 LOH, OIE, ANDIRATNAYAKE INFEC. IMMUN. TABLE 5. Reversal of the effect of cycloheximide by metabolic inhibitors, either singly or in combination, on monolayers of uninfected and reo- removing it at various times after infection virus-infected HeLa cells was examined (Table Appearance of cytopathology 7). The inhibitors selected were those whose Conditions" mechanisms of action are fairly well defined. 1 1 Fluorophenylalanine (FPA), an analogue of V Cy (O min) _ phenylalanine, at concentrations ( _ Ag/ml) V Cy (min) _ that markedly suppress both protein synthesis V Cy ( min)... _- - - _ t and virus production was found, in combination V Cy (9 min) with the reovirus, to induce accelerated CPE. V Cy (1min) ± However, the degree of CPE observed was not V Cy ( min).. - as extensive as that seen with cycloheximide. It should be noted that FPA does not actually Normal... inhibit protein synthesis and can be incorporated into proteins. Cy.:::::. - V Cy ± When an antagonist of DNA synthesis, such as 5-fluoro-'-deoxyuridine (FUDR), was added a V = reovirus type at a multiplicity of infection of 1 to 15. Cy = cycloheximide at 1lg/ml. combination with cycloheximide, acceleration of to reovirus-infected cells, either by itself or in The time of removal of cycloheximide is given in CPE was observed only in cultures containing the parentheses. antibiotic. No CPE was observed in cell cultures TABLE. Effect of cycloheximide and reovirus type on other mammalian cell speciesa Appearance of cytopathology Origin Cellb Uninfected Infected Human Mo:key Mv;o use Hamster HeLa RA W1 BSC-1 AGMK L MEFc BHK-1._ i; a Each of the different cell cultures was treated as follows: (i) no treatment, (ii) with cycloheximide, (iii) with virus, (iv) with virus and cycloheximide. The multiplicity of infection was 1. The concentration of cycloheximide was,.g per ml. The basal Eagle's medium contained.5 of actinomycin D per ml and.1% fetal calf serum. RA = human amnion; W1 = human embryonic lung; BSC-1 = green monkey kidney; AGMK = African green monkey kidney; L = mouse fibroblast; MEF = mouse embryonic fibroblast; BHK-1 = baby hamster kidney. Cytotoxic to cycloheximide at 5 jag per ml.

5 VOL., 197 CPE BY REOVIRUS AND CYCLOHEXIMIDE 79 TABLE 7. Effect of different chemical inhibitors and reovirus type on HeLa cell cultures Cytopathology Sample' - 1 Control Virus Virus cycloheximide... - Cycloheximide (1.,Og/ml) FPA (,og/ml) _ - Virus FPA Virus FPA PA FUDR (1-M) _ Virus FUDR Virus FUDR cycloheximide...- FUDR cycloheximide _ AD (1jAg/ml) Virus AD...- Virus AD cycloheximide... - AD cycloheximide.. - a The different chemical inhibitors (FPA, fluorophenylalanine; PA, phenylalanine; FUDR, 5-fluoro-'-deoxyuridine; AD, actinomycin D) were added at zero time postinfection, and the HeLa cell monolayers were examined for morphological changes at intervals for a period of. Virus = reovirus type (multiplicity of infection, 1). receiving FUDR and cycloheximide. The concentration of FUDR used (1' CM) was sufficient to inhibit markedly the synthesis of cellular DNA (9). Although the addition of actinomycin D (1,ug/ml) by itself to uninfected cell cultures caused CPE to appear after 1 of treatment, its addition in combination with either the virus and cycloheximide or only cycloheximide produced accelerated CPE. Cell damage to almost the same extent was also observed when actinomycin D was added to infected cultures. The above results suggest that the phenomenon of accelerated CPE is probably the consequence of some early alterations in the synthesis of cellular RNA in the protein-inhibited cell and that cellular DNA may not be involved. Effect on RNA, DNA, and protein synthesis. Since degenerative changes in cell morphology can apparently be assumed to be due to severe disruptions of cell metabolism, the effect of cycloheximide and reovirus infection on the synthesis of RNA, DNA, and protein in HeLa cells was determined by following the rates of incorporation of the tritiated precursors uridine, thymidine, and leucine, respectively. Monolayers of HeLa cells were treated in the following manner: (i) untreated and uninfected; (ii) treated with cycloheximide (1 Ag/ml); (iii) infected with reovirus type (multiplicity of infection, 1); and (iv) treated with cycloheximide and infected. To each of the experimental cultures, the appropriate tritiated precursor (.5,uCi/ml) was added, and at various intervals after infection replicate cultures were removed and processed for the determination of the amount of trichloroacetic acid-precipitable radioactivity incorporated (see Materials and Methods). The addition of cycloheximide to uninfected HeLa cell cultures resulted in the almost immediate and complete inhibition of incorporation of H-leucine into protein (Fig. 1B). Under the same conditions, the synthetic activity of DNA was affected as early as after treatment and was inhibited by to 5%l at (Fig. 1C). The incorporation of H-uridine into RNA proceeded unaffected until after treatment, at which time a % inhibition of incorporation occurred (Fig. IA). As was found for the uninfected cultures, the addition of cycloheximide to reovirus-infected HeLa cells resulted in the immediate and complete suppression of the incorporation of labeled precursor into protein (Fig. B). At the concentration of the antibiotic used, virus production was completely inhibited. The incorporation of H-thymidine into the DNA of both the infected and the cycloheximide-treated infected cultures was found to be similar. Examination of the RNA activity of cycloheximide-treated infected cell cultures indicated that the incorporation of Huridine into RNA was suppressed as early as to 5 postinfection, and by the inhibition of RNA synthesis was 7%/o. These results again suggest the possible involvement of cellular RNA in the induction of accelerated CPE in cycloheximide-treated HeLa cells infected with reovirus. DISCUSSION CPE not associated with the production of infectious virus have been usually referred to as cytotoxic effects (1). The present data appear to fit the general criteria for a cytotoxic event in the following respects: lack of production of infectious virus, early onset of CPE, and existence

6 71 LOH, OIE, AND RATNAYAKE INFEC. IMMUN. 9 RNA 1 r PROTEIN DNA 7 A 1-11 B r) 1 x 5 1 _I QL () I TIME (hours) FIG. 1. Effect ofcycloheximide on the uptake of tritiated uridine, leucine, and thymidine into HeLa cells. Cycloheximide, at a concentration of 1,.g/ml, was added to monolayers of HeLa cells, and the cell cultures were incubated with each of the labeled precursor for the indicated time intervals. At the end of each interval, the cells were removed and assayedfor the amount of trichloroacetic acid-precipitable radioactivity incorporated. Symbols: () untreated culture; (a). cycloheximide-treated culture. 9 RNA j PROTEIN / I7 1 r DNA 1 1 F 7 A 1 1 F C 1F 1 5-1o 1 CL - 1 TIME (hours) FIG.. Effect ofcycloheximide on the uptake of tritiated uridine, leucine, and thymidine into HeLa cells infected with reovirus type (multiplicity of infection, 1). Cycloheximide, at a concentration of 1,ug/ml, was added to infected monolayers, and the cell cultures were incubated with each of the labeled precursors for the indicated time intervals. At the end of each interval, the cells were removed and assayed for the amount of trichloroacetic acidprecipitable radioactivity incorporated. Symbols: () infected culture; () infected culture plus cycloheximide.

7 VOL., 197 CPE BY REOVIRUS AND CYCLOHEXIMIDE 711 of a virus dose-response relationship. In regard to the last, the large virus doses employed are necessary to infect the majority of cells at about the same time rather than to the need for a high multiplicity of infection. Under certain conditions, several animal viruses have been reported to exhibit cytotoxicity for their susceptible host cells (1). To date, there have been only two reports concerning a toxicity phenomenon associated with reoviruses. The first involves strains of reovirus type isolated from patients with infectious hepatitis, which when propagated in the chorioallantoic sac or yolk sac of embryonated chick eggs were found to produce a hemolysin which could be separated from the virus by dialysis (B. T. Burlingham and A. P. McKee, Bacteriol. Proc., p. 11, 195). The second is the acquisition of the cytotoxic property by reovirus type after irradiation by ultraviolet light (). In contrast to the preceding systems, the present system requires the presence of a protein antagonist, such as cycloheximide in reovirus-infected cell cultures, for the induction of accelerated CPE. From the present data, cell death appears to be the consequence of severe alterations in the synthesis of both cellular RNA and protein. This is substantiated by the results obtained with the use of combinations of specific metabolic inhibitors. The addition of the RNA inhibitor actinomycin D, but not of the DNA antagonist FUDR, to cell cultures treated with cycloheximide results in the induction of accelerated CPE. Although infection of cells by reoviruses results in the suppression of cellular DNA synthesis (, 11), it is unlikely from the present evidence, obtained with the inhibitors FUDR and cycloheximide, that this class of macromolecules is involved. The possible involvement of RNA is unexpected, since RNA metabolism in reovirusinfected cells has been reported to remain essentially unaffected (, 5, 1). The occurrence of a persisting lag period of to before the onset of detectable CPE, even when high multiplicities of virus were employed, suggests the participation of some early event(s). The production of some viral components in several virus systems in which cytotoxic effects occur is not unknown (17). Reoviruses contain their own RNA transcriptase (1, 1), and the synthesis of viral messenger ribonucleic acids (mrna) in cycloheximidetreated L-cells infected with reovirus has been reported (1). Under such conditions, at least 7 of the 1 species of mrna were found to be transcribed early after infection. The probable involvement of the transcriptase or of any of these species of viral mrna, or of both, in the induction of the present phenomenon remains to be determined. _m l Although the mechanism of action for this synergistic effect of the reovirus and antibiotic on the cell remains to be defined, the possible involvement of a structural component(s) of the virus as an inducer of the CPE should be considered. The effect of this component(s) on the cell is only manifested when there is interference with the replication of the virus. This suggestion is supported by our recent report describing the induction of accelerated CPE in HeLa cells inoculated with ultraviolet-inactivated reovirus type (). In this system, cell death was also accompanied by suppression of the biosynthesis of cellular nucleic acids and proteins. Experiments with the adenovirus and vesicular stomatitis virus systems have provided the best evidence for the role of certain structural components of the virus in the disruption of cellular macromolecular synthesis and the subsequent loss of cell viability (, ). The response of the different cell species to cycloheximide and reovirus infection suggests that the variation in responses obtained under the present conditions may be a reflection of the ability of the cell to take up either the antibiotic or the virus. Although the yield of virus varied, the cell species selected were all capable of supporting the synthesis of reovirus (unpublished data). Variations in the response of different cell species to infection by the same virus are not unknown in other systems. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant Al 5 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Borsa, J., and A. F. Graham. 19. Reovirus: RNA polymerase activity in purified virions. Biochem. Biophys. Res. Commun. : Ensiminger, W. D., and I. Tamm Cellular DNA and protein synthesis in reovirus-infected L cells. Virology 9: Gomatos, P. J., and I. Tamm. 19. Macromolecular synthesis in reovirus-infected L cells. Biochim. Biophys. Acta 7: Huang, A. S., J. W. Greenwalt, and R. R. Wagner. 19. Defective T particles of vesicular stomatitis virus. I. Preparation, morphology, and some biologic properties. Virology : Kudo, H., and A. F. Graham Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 9: Levine, A. J., and H. S. Ginsberg Mechanism by which fiber antigen inhibits multiplication of type 5 adenovirus. J. Virol. 1: Loh, P. C., and J. R. Crowley Reovirus type -infection, cycloheximide, and cell death. Proc. Soc. Exp. Biol. Med. 15:17-19.

8 71 LOH, OIE, AND RATNAYAKE INFEC. IMMUN.. Loh, P. C., and H. K. Oie Growth characteristics of reovirus type : Ultraviolet light inactivated virion preparations and cell death. Arch. Gesamte Virusforsch. : Loh, P. C., and F. E. Payne Effect of 5-fluoro-'- deoxyuridine on the synthesis of vaccinia virus. Virology 5: Loh, P. C., and M. Soergel. 19. Growth characteristics of reovirus type : Actinomycin D and the preferential synthesis of viral RNA. Proc. Soc. Exp. Biol. Med. 1: Loh, P. C., and M. Soergel Macromolecular synthesis in cells infected with reovirus type and the effect of Ara-C. Nature (London) 1:-. 1. Pereira, H. G The cytopathic effect of animal viruses. Advan. Virus Res. : Shatkin, A. J Actinomycin and the differential synthesis of reovirus and L-cell RNA. Biochem. Biophys. Res. Commun. 19: Shatkin, A. J., and J. D. Sipe. 19. RNA polymerase activity in purified reoviruses. Proc. Nat. Acad. Sci. U.S.A. 1: Spendlove, R. S., E. H. Lennette, C.. Knight, and J. N. Chin. 19. Development of viral antigen and infectious virus in HeLa cells infected with reovirus. J. Immunol. 9: Watanabe, Y., S. Millward, and A. F. Graham. 19. Regulation of transcription of the reovirus genome. J. Mol. Biol. : Westwood, J. C. N. 19. Virus pathogenicity, p In W. Smith (ed.), Mechanisms of virus infection. Academic Press Inc., New York.