EFFECT OF p-fluorophenylalanine ON PSITTACOSIS VIRUS

Transcription

1 EFFECT OF p-fluorophenylalanine ON PSITTACOSIS VIRUS IN TISSUE CULTURES YOH TANAMI' AND MORRIS POLLARD2 Department of Preventive Medicine and Public Health, University of Texas Medical Branch, Galveston, Texas Received for publication May 15, 1961 ABSTRACT TANAMI, YOH (University of Texas Medical Branch, Galveston) AND MORRIS POLLARD. Effect of p-fluorophenylalanine on psittacosis virus in tissue cultures. J. Bacteriol. 83: The inhibitory effect of p-fluorophenylalanine (FPA) on maturation of psittacosis virus was investigated, with attention to the time sequence of viral protein synthesis. Extracellular virus particles were not inactivated by FPA at a concentration of 100 ug per ml, at which level it interfered with maturation of intracellular virus. NVhen FPA was added to infected tissue cultures earlier than 15 hr after infection, intracellular virus maturation was suppressed. However, when FPA was added after 15 hr, infective virus was produced, which indicates that the synthesis of a FPA-sensitive virus precursor (presumably viral protein) had already occurred. A latent ("dormant") infection of psittacosis virus, established in a medium deficient in phenylalanine and tyrosine, was also investigated. The influence of fluorinated pyrimidines on nucleic acid synthesis in cell cultures infected with psittacosis virus has been described (Pollard et al., 1960; Tanami, Pollard, and Starr, 1961). The present paper deals with the inhibitory effect of DL-p-fluorophenylalanine (FPA) on the replication of psittacosis virus, with attention to the time course of viral protein synthesis. The nature of the "dormant" state of psittacosis virus in nutritionally deficient tissues (Bader and Morgan, 1958; MIorgan et al., 1959; McCloskey and Morgan, 1961) was also investigated. I Present address: Department of Bacteriology, Faculty of Medicine, Shinshu University, Matsumoto, Japan. 2 Present address: LOBUND, University of Notre Dame, Notre Dame, Ind. MATERIALS AND METHODS Virus strain. An enhanced derivative (TTF) of strain TT psittacosis virus (Boney et al., 1952) was used. This virus has been propagated by serial passage through tissue cultures of McCoy cells. To eliminate phenylalanine (PA) from the virus stock fluid, virus was propagated in a medium which lacked both PA and tvrosine. As will be noted laber, McCoy cells growrn in nutritionally complete medium supported subsequent virus propagation even in the absence of such essential amino acids in the medium. After 30 passages in complete medium, virus stock representing the third passage in McCoy cells depleted of PA and tyrosine was stored at -50 C. The titer of the virus stock was 4 X 107 units per ml, as determined by particle count in tissue culture (Tanami et al., 1960). Cell strain. An established cell line (McICoy) derived from human synovial tissue was used (Fernandes, 1959). The cells were prepared in monolayer sheets on coverslips in Leighton tubes; each tube contained approximately 5 X 105 cells when inoculated with virus. Medium. The "complete medium" consisted of mixture 199 (Microbiological Associates, Bethesda, Md.) with 0.5% lactalbumin hydrolyzate, 5% heat-inactivated calf serum, and 0.1 mg of streptomycin per ml. The ph was adjusted to 7.2 with sterile sodium bicarbonate. The "deficient medium" contained all ingredients of mixture 199 except PA and tyrosine. It contained 1% heat-inactivated calf serum. FPA. FPA (California Corporation for Biochemical Research, Los Angeles, Calif.) was dissolved in distilled water (2 mg per ml), sterilized by Selas filtration, and stored at 4 C. It was further dissolved in nutrient medium (200,g per ml to 12.5,ug per ml) and tested for toxic effect on McCoy cells. Subsequent examination of cells indicated that a dosage of 100,ug of drug per 437

2 438 TANAMI AND POLLARD (VOL. 83 ml of nutrient medium could be tol erated by the cells. Therefore, all experiments were performed with 100,ug or less per ml. Virus assay. The "infected cell cosunt method" described by Weiss and Huang (1954) was adopted. The practicality and relia6bility of this method in our host-virus systern has been described (Tanami et al., 1960). 8 :E 7 $ m 6- \o I RESULTS Effect of FPA on extracellular psilttacosis virus. Virus stock, harvested from bottlee cultures of psittacosis-infected McCoy cells, wals centrifuged CONC. OF FPA (pg/m1) at 2,000 rev/min for 20 min, to eliminate cell debris and large viral aggregates. The supernatant FIG. 2. Inhibitory action of FPA on production of fluid was then centrifuged at 10,00C rev/min for TTF virus. Drug was added to the medium simultaneously with virus. Virus, produced in cells culti- 30 min, and virus in the sedimen t was resus- medium. vated in media which contain FPA at varying pended in a smaller volume of deficiient This concentrated virus suspensio: n (5 X 108 dosages, was assayed after incubation for 38 hr. viral units per ml) was diluted 1:1o with comor deficient grown in deficient medium. Symbol X represents virus produced in complete medium; open and solid circles represent virus plete medium, deficient medium, medium containing FPA at 100 jg per ml; each was then incubated at 35 C. At various time intervals, samples were diluted 1:20 and assayed action of FPA on maturation of the TTF virus for infective units in fresh tissue cultures. Sig- in McCoy cell cultures was delicately influenced ficant differences were not obsernved between by the nature of the nutrient medium, as well as inactivation in either the presence cdr absence of by the nutritional history of the host cell, as FPA (Fig. 1), indicating that matiure nonrepli- follows: When the infected McCoy cells were eating virus particles were insensitive to the maintained in complete medium, the inhibitory influence of FPA at the concentrnation tested. effect of added FPA on TTF virus multiplication The influence of FPA (100 Mg per ml) on rate was not remarkable (Fig. 2) because the complete of virus adsorption to McCoy ce-lls was not medium contained PA at a level of 50,g per ml. significant. On the other hand, when the McCoy cells were Inhibitory action of FPA on psittacosis virus cultivated for 2 days before TTF virus infection propagation as a function of dose. TIhe inhibitory in deficient medium, they thereafter did not support the replication of TTF virus when the infected cells were maintained in the same deficient medium. This was similar to the results.8 t.6 - reported by Morgan et al. (1959). If the nutrient.4 medium was replaced with complete medium - Vo I after Vt virus infection, normal viral development was demonstrable, as described below. However,.2 - McCoy cells grown on complete medium supplete medium ported full virus production, even if they were * In coml O In defic FPA ( TIME IN HOURS FIG. 1. Heat inactivation curves of fr virus particles at 35 C, showing rati4 o of original virus to cells that had been grown in the complete medium and thereafter incubated in virus titer with time. defi- 2 cient medium + maintained in the deficient medium after TTF 0 pg/mi) virus infection. This indicates that there may -r- be a sufficient residuum of PA and tyrosine in 4 5 the cells to support the propagation of TTF virus in the absence of such nutrients in the nutrient -ee psittacosis fluid. When FPA was added simultaneously with

3 1962] p-fluorophenylalanine ACTION ON PSITTACOSIS VIRUS 439 z NO. INFECTE 5_ CELLS PERIML ' ever, cells appeared to degenerate and were occasionally detached from the coverslips. The results of this experiment (Fig. 2) can be summarized as follows. (i) The production of infective virus in deficient medium was I.{ /inhibited completely by FPA at a concentration of 200 jg per ml. (ii) In the complete medium, which contained PA at 50,ug per ml, about 106 infective units per ml were produced, even in the presence of FPA at 200 jg per ml. (iii) There is ai, Z et an approximately linear dose-response relationship between analogue concentration in deficient 4' gl 4medium and log units of virus produced. This i, was similar to the effect of fluorinated pyrimidines on the same virus (Tanami et al., 1961) ,7 The developmental stages of psittacosis virus *CELL-ASSOCIATED VIRUS in infected cells, as observed microscopically,virus ADS,ORPTION with acridine orange fluorochrome, are a sequence I, of color changes of viral inclusions (Pollard et al., 1960; Starr et al., 1960). That is, non- HOURS AFTER INFECTION infectious initial bodies, which appeared around the 10th hr after infection in our virus-host FIG. 3. Single gro)wth cycle of cell-associated virus system, were stained red-orange (ribonucleic (open circles) and tthe "FPA-curve". Each point in acid the FPA-curve inm staining); mature virus particles, which the tained when the.icates final viral yield obfilled the cytoplasm within anaslogue was added at the time indi- 48 to 72 hr after.. s cated. Different synnbols in the FPA-curve indicate nfectlion, were stained green-yellow (deoxyriborent experiments. Symbols with nucleic acid staining). the results of diffe, downwards arrows i ndicate that the active virus units In preparations of EPA-treated virus-infected were below 10 per nxl. cell cultures, small aggregates of red-colored initial particles ("red balls") were observed in cient medium, in} iibitory action of added FPA the cytoplasm 48 hr after infection. This indicates on psittacosis viruis became apparent (Fig. 2). that, in the presence of FPA at 100M,g per ml, A number of mc)nolayer cell sheets propagated virus adsorbed and penetrated the cell wall but in the complete rniedium for 2 days were inocu- viral development was inhibited at the "red-ball lated with TTF viirus at a relatively high multi- stage". plicity of infection. Most of the cells were Effect of FPA added during the course of viral infected. After in cubation for 1 hr at 37 C, for development. The effect of time of addition of virus adsorption, the inoculum was decanted FPA on the course of psittacosis virus maturation and 1.5 ml of the deficient medium was added in deficient medium was investigated by adding to each tube, alc ng with varying amounts of FPA (100 Ag per ml) to the virus-infected cell FPA. These tube s were incubated at 37 C for cultures at various time intervals after infection. 38 hr, at which tlime virus maturation in ana- The treated cultures were incubated at 37 C for logue-free control ( cultures was virtually complete 38 hr after inoculation of virus. All cultures but without mas: sive cell destruction. All cell were then washed twice and 2 ml of complete cultures were then washed twice with mixture medium were added to each. Cultures were frozen 199, 2 ml of fresh complete medium were added and the infective virus units in each tube culture to each, and the iwhole culture was then frozen. were determined in fresh cell cultures. Virus The number of Nvirus units in each tube was titrations of the analogue-free cultures were made assayed by the ' 'infected cell count" method. periodically in the form of control one-step At FPA concentraltions of 100lg per ml or less, growth experiments. no significant cyrtotoxicity was observed. At In Fig. 3, the final yields of virus in the in- than 200,g per ml, how- hibited cultures were plotted on the time concentrations higrher axis

4 440 TANAMI AND POLLARD [VOL C- 5- I- C, 4- C " The deficient medium was replaced with complete medium at intervals indicated below, following virus infection. O hr (Control) 3hr 5hr 7 hr hr 'hr hr 1I I I g I I O HOURS AFTER THE ADDITION OF COMPLETE MEDIUM TO "DORMANT' VIRUS-CELL COMPLEXES FIG. 4. Active virus growth obtained by the addition of complete medium to the "latent" virus-cell complexes, which had been established in deficient medium. (horizontal) at the time of analogue addition. WThen FPA was added to the virus-infected cells during the latent noninfectious period (up to 15 hr after infection), virus production was suppressed. Infective virus were produced only when the analogue was added later than 15 hr. The "FPA-curve" (Fig. 3) rises at 2 hr prior to the virus-maturation curve, at almost the same rate and to the same ultimate yield of virus as in the control cultures. These results, however, do not necessarily mean that an earlier stage of the latent period is sensitive to FPA; if a later stage of the latent period is sensitive, viral maturation will be suppressed whether the earlier stage is sensitive or not. As noted before, microscopic examination revealed that viral development did proceed to the red-colored (RNA) inclusion stage in the presence of 100 log FPA per ml, thus suggesting that the early stages of replication may be relatively insensitive to the compound. Dormant state of psittacosis virus in deficient medium. Morgan et al. (1959) and McCloskey and Morgan (1961) have shown that sensitive tissue-culture cells do not support maturation of psittacosis virus if the cells were maintained on medium deficient in phenylalanine or tryptophan. The addition of nutritionally complete medium to such "dormant" (or "latent") virus induces resumption of the virus maturation process. Periodic addition of essential amino acids to the dormant virus provides a useful tool for analysis of the biological character of the dormant state. Experiments were carried out in the following manner. A number of McCoy cell cultures prepared on coverslips in Leighton tubes with deficient medium were inoculated with a large dose of TTF virus. After 1 hr at 37 C (for adsorption), cell cultures were washed twice with the deficient medium, replaced with 2 ml of the same medium, and incubated at 37 C. Under such conditions, infective viruses were not produced during the following 4 days. Daily observations, using acridine orange stain and fluorescence microscopy, were made on the virus-infected cell cultures. A few red-colored initial bodies, representing a step of normal viral development, appeared. The number of such "red balls" remained static until the missing essential amino acids were supplied. The biological activity of such dormant virus was investigated in the following manner. At varying intervals of time after infection of virus, the deficient medium of the virus-infected cultures was replaced with the complete medium,

5 1962] p-fluorophenylalanine ACTION ON PSITTACOSIS VIRUS 441 and the production of infective virus units was thereafter determined. A step-wise virus growth occurred after a shorter latent period of time than that required by the control cultures (Fig. 4). This indicates that viral development proceeded at a suboptimal rate during the first few hours after adsorption and penetration of virus into the cell, under nutritionally deficient conditions. Thereafter, the rate of viral activity decelerated with time and finally stopped midway in the latent period. This was reflected in the cytochemical observations described above. Thus, in our virus-host system, the status of dormant virus in the deficient medium corresponds to the noninfectious "red-ball" stage observed at an early stage of normal viral development. This preceded the development of viral protein. The above results indicate that an adequacy of free amino acids is required for multiplication of initial bodies containing ribonucleic acid and is a prerequisite for maturation of virus. DISCUSSION The process by which psittacosis virus replicates can be divided into several stages, in terms of sensitivity to FPA. Mature extracellular virus particles were resistant to the effects of the compound at a level of FPA (100,ug per ml) which suppresses maturation of intracellular virus. The initial stages of virus invasion (virus adsorption, penetration, and the first several hours after penetration) appeared to be relatively insensitive to the compound. However, FPA interfered with viral development during the second half of the latent period, 10 to 15 hr after infection. This drug-sensitive stage corresponds to a period during which noninfectious initial bodies multiply rapidly. Thereafter, developing virus became resistant to FPA again. When FPA (100 jig per ml) was added to cultures later than 15 hr after inoculation of virus, infective virus were produced. This indicates that certain FPA-sensitive viral components were already formed at that time. Although the nature of the FPA-sensitive viral factor was not identified, it may be the precursor of viral protein, because viral antigen begins to appear at this stage (Pollard et al., 1960; Starr et al., 1960). The above view is also compatible with previous experiments in which fluorinated pyrimidines inhibited the biosynthesis of viral nucleic acids and wvhich showed that the protein of psittacosis virus particles is synthesized as a final process of the latent period (Pollard et al., 1960; Tanami et al., 1961). It has been reported that the synthesis of hemagglutinin in encephalomyocarditis virus (Sanders, 1960), and of poliovirus protein (Darnell and Levintow, 1960) which is incorporated into particulate virus, occurred at the final stage of the latent period, just preceding virus maturation. The inhibitory pattern of FPA on psittacosis virus, as described here, resembles that obtained with poliovirus (Ackermann et al., 1954) and with fowl plague virus (Zimmermann and Schafer, 1960). This similarity suggests that the growth pattern of psittacosis virus may therefore relate it more closely to such true viruses, rather than to the rickettsiae or bacteria. However, interactions between psittacosis virus and the cell nucleus have not been observed. This is dissimilar to the action of such small viruses as herpes virus (Morgan et al., 1959) and fowl plague virus (Zimmermann and Schiifer, 1960), which require interaction between virus and cell nucleus to establish a mechanism by which the membrane substance of the virus particles is completed. ACKNOWLEDGMENT Supported in part by grant E2983 from the U. S. Public Health Service. LITERATURE CITED ACKERMANN, A. A., A. RABSON, AND H. KURTZ Growth characteristics of poliomyelitis virus in HeLa-cell cultures: lack of parallelism in cellular injury and virus increase. J. Exptl. Med. 100: BADER, J. P., AND H. R. MORGAN Latent viral infection of cells in tissue culture. VI. Role of amino acids, glutamate, and glucose in psittacosis virus propagation in L cells. J. Exptl. Med. 108: BONEY, W. A., JR., L. C. GRUMBLES, J. P. DELAPLANE, J. V. IRONS, AND T. D. SULLIVAN Another agent causing air sac involvement in turkeys. J. Am. Vet. Assoc. 121: DARNELL, J. E., AND L. LEVINTOW Poliovirus protein: Source of amino acids and time course of synthesis. J. Biol. Chem. 235: FERNANDES, M. V Irradiation of cells in tissue culture. VII. Studies on the susceptibility to blue tongue virus on radiation in-

6 442 TANAMI AND POLLARD [VOL. 83 duced giant cells in vitro. Z. Zellforsch. 50: MICCLOSKEY, R. V., AND H. R. MORGAN Latent viral infection of cells in tissue culture. VIII. Morphological observations of psittacosis virus in L cells. Proc. Soc. Exptl. Biol. Med. 106: MORGAN, C., H. M. ROSE, M. HOLDEN, AND E. P. JONES Electron microscopic observations on the development of herpes simplex virus. J. Exptl. Med. 110: POLLARD, M., T. J. STARR, R. W. MOORE, AND Y. TANAMI Cytochemical changes in human amnion cells infected with psittacosis virus. Nature 188:770. SANDERS, F. K Role of infective nucleic acid in the production of encephalomyocarditis virus. Nature 185: STARR, T. J., M. POLLARD, Y. TANAMI, AND R. W. MOORE Cytochemical studies with psittacosis virus by fluorescence microscopy. Texas Repts. Biol. Med. 18: TANAMI, Y., M. POLLARD, T. J. STARR, AND R. W. MOORE Quantitative interaction between psittacosis virus and human cells. Texas Repts. Biol. Med. 18: TANAMI, Y., M. POLLARD, AND T. J. STARR Replication pattern of psittacosis virus in a tissue culture system. Virology 15: WEISS, E., AND J S. HUANG The infected cell count method of titration of feline pneumonitis virus. J. Infectious Diseases 94: ZIMMERMANN, T., AND W. SCHAFER Effect of p-fluorophenylalanine on fowl plague virus multiplication. Virology 11: Downloaded from on May 12, 2018 by guest