Effect of D-Penicillamine on Poliovirus Replication In HeLa Cells

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1 JOURNAL OF VIROLOGY, Apr. 1974, p Copyright i 1974 American Society for Microbiology Vol. 13, No. 4 Printed in U.SA. Effect of D-Penicillamine on Poliovirus Replication In HeLa Cells PARVIN MERRYMAN, ISRAELI A. JAFFE, AND ELLIE EHRENFELD Department of Medicine, New York Medical College and the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York Received for publication 10 December 1973 A series of mercaptan compounds were studied with respect to their ability to inhibit the growth of poliovirus in cultured cells. Of the compounds tested only D-penicillamine possessed antiviral activity. There was no direct effect on the virus itself, nor were the processes of adsorption, penetration and uncoating, or virus-induced "shut-off" of host cell protein synthesis inhibited. At concentrations where there was no effect on host cell RNA or protein synthesis, D-penicillamine caused a marked inhibition of virus-specific RNA and protein synthesis. Although much reduced, the relative concentrations of single-stranded and double-stranded viral RNA synthesized in the presence of D-penicillamine was unchanged. Similarly, all apparent precursor and cleavage product proteins could be synthesized in the presence of the drug. The inhibitory effect was reversible, after a lag of 1.5 to 2 h after removal of the drug, and normal yields of virus could be obtained. The structural and functional properties of D-penicillamine are discussed in relation to requirements for anti-polioviral activity. D-Penicillamine (,S, d-dimethyl cysteine) is an amino acid isolated from penicillin which displays several biologic functions, including antivitamin B, activity (10), interference with collagen fibril formation (14), mixed disulfide formation with cysteine (4), and copper chelation (18). It has been shown to be highly effective in suppressing the clinical and laboratory manifestations of rheumatoid arthritis (9), and although no etiology has been proven for this disease, infection with a latent virus is considered as a possible antigenic stimulus (3). It has been reported that D-penicillamine is a potent inhibitor of poliovirus replication in cell cultures (7), although the drug exerted only marginal antiviral activity against coxsackie and Echo viruses, and none whatsoever against herpes simplex, vaccinia or vesicular stomatitis viruses. The L-isomer of penicillamine was ineffective against poliovirus (7). This study was undertaken to further define the mechanism of inhibition of D-penicillamine on poliovirus replication and to assess the antipolioviral effect of other mercaptan compounds. MATERIALS AND METHODS Cells and virus. KB cells were grown in monolayer and HeLa cells were grown either in monolayer or suspension culture in Eagle minimum essential medium supplemented with 7% calf serum and 2 mm glutamine. Experiments were performed with the Mahoney strain of poliovirus type 1 and the Sabin strain of poliovirus type 2. Virus was purified as described previously (17). Measurement of virus yield. Tissue culture plates (60 by 15 mm) were seeded with 1.5 x 106 HeLa or 10' KB cells and incubated for 72 h at 37 C in 5% CO2 to obtain confluent monolayers. Plates were then infected with 107 PFU of poliovirus for 1 h. Unadsorbed virus was removed by washing four times with Hanks balanced salt solution. All of the compounds to be tested were dissolved in Hanks solution buffered with Tris-hydrochloride, ph 7.3. A 3-ml amount of these solutions containing the test agents was added to duplicate plates, and cultures were incubated for 16 h at 37 C in 5% CO2. The fluid was then removed, centrifuged, and the supernatant was assayed for infectious particles by its ability to form plaques on HeLa cell monolayers. Measurement of RNA and protein synthesis. Suspension cultures of HeLa cells were washed with Earle solution and resuspended in serum-free medium at a concentration of 5 x 101 cells/ml. When virusspecific macromolecular synthesis was to be measured, 5 Ag of actinomycin D per ml was added, and the cultures were infected with purified poliovirus at a multiplicity of 200 PFU/cell. Calf serum was added 30 min postinfection (p.i.) to 5%. The accumulation of virus-specific RNA and protein was measured by the addition of [14C]uridine (0.1 ACi/ml, 498 mci/mmol, Amersham) at 30 min p.i., or of [14Clleucine (0.1 MuCi/ml, 312 mci/mmol, Schwartz/Mann) at 2 h p.i., 881

2 882 MERRYMAN, JAFFE, AND EHRENFELD J. V IROL. respectively. At appropriate times, 0.2-ml samples of the culture were removed and diluted with 2 ml of cold Earle solution. Samples were centrifuged, supernatants were discarded, and the cells were lysed in 1 ml of distilled water and then precipitated with 2 ml of cold 10% trichloroacetic acid. Samples were filtered on Whatman GF/C glass fiber filters, and the acidinsoluble material was analyzed for radioactivity in a Beckman scintillation spectrometer. Analysis of virus-specific RNA and mature virions. Infected, ["C ]uridine-labeled cells were harvested by centrifugation, washed with Earle solution, and incubated for 5 min at 0 C in hypotonic RSB (0.01 M Tris-Cl, ph 7.4, 0.01 M NaCl, M MgCl2). The cells were then disrupted in a Dounce homogenizer. Nuclei and cell debris were removed by centrifugation, and for analysis of RNA, the cytoplasmic extract was brought to 1c7( sodium dodecyl sulfate (SDS), heated for 1 min at 60 C, and layered over a linear 15 to 30% sucrose gradient prepared in NETS (0.01 M Tris-Cl, ph 7.4, 0.1 M NaCl M EDTA, 0.1% SDS). Gradients were centrifuged in the SW 27 rotor for 17 h at 22,000 rpm at 23 C. To measure the production of virions, cytoplasmic extracts were prepared as above, then brought to 0.5% deoxycholate (DOC) and centrifuged through 7 to 47% sucrose gradients prepared in RSB for 16 h at 16,000 rpm at 4 C in the SW 27 rotor. The contents of the gradients were analyzed for their absorption at 260 nm in a Gilford automatic recording spectrophotometer. Fractions (1 ml) were collected, and macromolecules were precipitated with 5% acid after the addition of 50,g of bovine albumin as carrier. Analysis of virus-specific proteins. Infected cells were incubated with [355 ]methionine (40 MCi/ml, approximately 1 Ci/mmol, New England Nuclear Corp.) under conditions described in the legend to Fig. 5. The cells were harvested by centrifugation, and washed with Earle solution, and cytoplasmic extracts were prepared as described above. Macromolecules were precipitated with 5% trichloroacetic acid, washed once with 1% acid and once with cold acetone, and collected by centrifugation at 10,000 x g for 30 min in the Sorvall RC2-B. The precipitates were dried and solubilized in a minimum volume (approximately 200 pliters for 2.5 x 107 infected cells) of 0.05 M Tris-Cl, ph 6.8, 0.1% SDS, 1% 3-mercaptoethanol, and 10%7 glycerol. The ph was adjusted to approximately 7 with NH4OH, and samples were heated for 3 min on a steam bath. Samples of 20,uliters were applied to slabs of a linear 7 to 30% polyacrylamide gel as described by Maizel (1, 13). Details of the slab gel apparatus and preparation are described by Studier (16). Electrophoresis was at 50 V for 18 h at room temperature. The gels were stained with Coomassie blue, and then dried onto a sheet of Whatman no. 1 filter paper for exposure on X-ray film. RESULTS Effect of D-penicillamine and other mercaptans on poliovirus, host cells, and virus replication. The presence of 100 gg of D-penicillamine (0.67 mm) per ml in the medium of cells infected with poliovirus reduced the virus yield approximately 100-fold (Table 1). Similar inhibitory effects were observed with poliovirus type 1 (Mahoney) and type 2 (Sabin) grown in HeLa or KB cells. The following mercaptan compounds at 0.67 mm were also examined for their ability to reduce poliovirus growth: L-Cysteine, ca-mercaptopropionyl-glycine, N-acetyl- L-cysteine, D-cysteine, and N-acetyl-D-penicillamine. None of these SH compounds showed a significant anti-poliovirus effect. Desoxypyridoxine-hydrochloride was also studied to determine a possible role for vitamine B8 antagonism in poliovirus inhibition, and it too was without activity. D-Penicillamine had no direct effect on poliovirus, since pretreatment of the virus for 2 h at room temperature prior to plating on HeLa cells did not reduce virus yield (Table 1). Virus adsorption to cells was not altered by the drug, nor was there any effect of D-penicillamine on the host cell so as to prevent its subsequent ability to support viral replication, since incubation of cell monolayers with drug for 2 h before infection or during the adsorption period did not affect total virus yield (Table 1). Examination of HeLa or KB cell monolayers after incubation for 17 h in medium containing D-penicillamine at concentrations up to 500 Ag/ml indicated that the drug was not cytotoxic. There was no detectable effect on cellular RNA or protein synthesis, as measured by uridine or leucine incorporation into acid-precipitable material, for periods up to 6 h (data no shown). Effect of D-penicillamine on poliovirus RNA synthesis. In poliovirus-infected HeLa cells, the addition of D-penicillamine to the medium resulted in marked inhibition of virus- TABLE 1. Effect of D-penicillamine on poliovirus replication in HeLa cells Treatment Virus yield 16 h p.i. (PFU/ml) Virus grown - D-penicillamine x D-penicillamine (100,ug/ml) x 106 Virus preincubated 2 h room temperature - D-pen x 10' + D-pen x 108 Cells preincubated 2 h 37 C - D-pen x D-pen x 108 Virus adsorbed to cells 1 h - D-pen x D-pen x 108

3 VOL. 13, 1974 ANTIVIRAL EFFECT OF D-PENICILLAMINE 883 specific RNA synthesis (Fig. 1). Since concentrations greater than 500 jig/ml produced no further inhibition, all subsequent experiments were performed at this concentration. The ability of D-penicillamine to inhibit viral RNA synthesis was studied as a function of time after infection. A similar degree of inhibition was obtained when D-penicillamine was added at the time of infection, 30 min or 1 h p.i. Addition of the drug at 2 and 3 h p.i. showed a marked reduction of its inhibitory effect, and there was virtually no activity detectable when it was added after 4 h p.i. (Fig. 2), although there is only minimal replication occurring at this time anyway. To ascertain whether there was any effect on the size or the relative amounts of virus-specific single- and double-stranded RNA synthesized in the presence of D-penicillamine, the inhibitor was added at submaximal concentration (250 gg/ml) to cultures incubated in the presence of [I4C ]uridine throughout the infectious cycle, and the cytoplasmic extract was analyzed on sucrose gradients. Figure 3 shows the sedimentation profile of virus-specific 35S singlestranded and 20S double-stranded RNA. Even though D-penicillamine caused a reduction in the total amount of viral RNA synthesis, it did not alter the relative concentrations of viral RNA species. Treatment of the extract with 10,gg of ribonuclease A per ml prior to sedimentation showed complete digestion of the 35S 4000 w] 3500 z a 3000 t V 2000 E 1500 O IY /\ NO D- PEN i! X os D-PEN/mi /2501 D-PEN/mI / ooos D-PEN/mI 5001 D-PEN/mI FIG. 1. Effect of D-penicillamine on poliovirusspecific RNA synthesis. HeLa cells (8 x 107) were infected with poliovirus and labeled with [IC ]uridine in the presence of actinomycin D. At 30 min p.i. the culture was divided into equal parts, and each was treated with D-penicillamine at the indicated concentration. Accumulation of ["4C]uridine into acidprecipitable material was analyzed as described in Materials and Methods IwJ 3500 z Z D t o NO D-PEN , 1/2,and HOUR FIG. 2. Inhibition of poliovirus-specific RNA synthesis by D-penicillamine at different times after infection. HeLa cells (5 x 107) were infected with poliovirus and labeled with ["4C uridine in the presence of actinomycin D. At the indicated times after infection, samples of the culture were removed and treated with D-penicillamine (500 Aig/ml). Incorporation of uridine into acid-insoluble material was measured. species, and greater than 90% resistance of the 20S species. Effect of D-penicillamine on virus-induced inhibition of host cell protein synthesis. Infection of HeLa cells with poliovirus results in a rapid and marked inhibition of cellular protein synthesis (15). Figure 4 shows that despite the failure of virus to replicate in cells in the presence of D-penicillamine, the virus-induced "shut off" of host cell protein synthesis occurred at the same rate as in infected, untreated cells. Effect of D-penicillamine on virus-specific protein synthesis. D-penicillamine markedly inhibited viral protein synthesis, as one would expect from the reduced yield of virus-specific RNA. An examination of the proteins synthesized in the presence of the drug, added either at the time of infection or 2.5 h p.i. (when virus-specific protein synthesis has already begun), showed that all of the normal precursor and cleavage product molecules could be produced (Fig. 5). (Precursor proteins are high molecular weight polypeptides migrating more slowly than viral structural proteins, designated VP 1-4.) Thus, no specific inhibition of posttranslational processing could be demonstrated. Reversibility of D-penicillamine inhibition. Experiments were performed to determine whether the inhibitory effect of D-penicillamine on virus-specific RNA and protein synthesis and on virus production were reversible. D- Penicillamine was added to the infected cell

4 884 MERRYMAN, JAFFE, AND EHRENFELD J. VIROL. z - 5w s oo _ V" ll&8~~~st18s /,.I 5-PEN 1I000.,& A A Is FRACTION NUMBER FIG. 3. Sedimentation analysis of virus-specific RNA synthesized in the presence and in the absence of D-penicillamine. HeLa cells (5 x 107) were infected with poliovirus and labeled with ['4C]uridine in the presence of actinomycin D. At 30 min p.i., D-penicillamine (250,g/ml) was added to half the culture, and incubation was continued for 5 h. Cytoplasmic extracts were prepared, treated with SDS, and centrifuged through sucrose gradients. w w Z L C 250C Q L 2 I VP 1 3 c:> _ci NO D-PEN /ml D-PEN FIG. 4. Inhibition of host cell protein synthesis by poliovirus infection in the presence and in the absence of D-penicillamine. HeLa cells (6 x 107) were infected with poliovirus in the presence of actinomycin D, and at 30 min p.i., half the culture was treated with D-penicillamine (500,g/ml). At the indicated times, 0.25 x 106 cells in 0.5 ml were removed and incubated with 0.5,C of ['4C]leucine for 10 min. Incorporation was stopped by dilution with water and precipitation with 5% trichloroacetic acid. Samples were analyzed for radioactivity. cultures 30 min p.i. and removed after 1 or 1.5 h by washing the cells two times and resuspending them in fresh medium. Figure 6A and B shows that both RNA and protein synthesis occurred after a lag period of 1.5 to 2 h after removal of the drug. Viral RNA synthesis after reversal occurred in a reproducible manner, 4AC = FIG. 5. Polyacrylamide gel analysis of poliovirusspecific proteins synthesized in the presence and in the absence of D-penicillamine. HeLa cells (7.5 x 107) were infected with poliovirus. At 30 min p.i., the culture was divided into three equal parts. One of these was used as control, and D-penicillamine (500 og/ml) was added to the other two at 30 min and 2.5 h p.i., respectively. All three cultures were given [35S]methionine for 2 h. The cells were harvested and prepared for analysis on polyacrylamide slab gels. Radioautograms were exposed for 4 days. The left hand column shows the poliovirus structural proteins obtained from ["C ]amino acid-labeled purified poliovirus. The second column represents virus-specific proteins in the cytoplasm of untreated infected cells. The third column shows proteins synthesized after treatment of infected cells with D-penicillamine at 2.5 h p.i.; the right hand column shows proteins synthesized after treatment with D-penicillamine at 30 min

5 5000 A w z C) oo o50c Ct 1000 a. Q M * * NO D- PEN - SOQ/ml D-PEN o-o 500r/ml D-PEN ADDED 30O REMOVED 908 w 2 w -1 U 0. UL 3oo M*O 200C JwOO HOOURS NO D- PEN 5001/ml D-PEN O-o SOOr/mi D-PEN ADDED 308 REMOVED 908 FIG. 6. Virus-specific RNA and protein synthesis after reversal of D-penicillamine. HeLa cells (7.5 x 107) were infected with poliovirus in the presence of actinomycin D. At 30 min p.i., two-thirds of the culture received D-penicillamine (500 jig/ml) for 1 h. In half of the treated culture, drug was removed by centrifuging and washing cells two times with Earle solution, and then resuspending them in fresh medium. The reversed, unreversed, and untreated cultures were each divided in half, and incorporation of [14C]uridine (A) or [14C]leucine (B) was measured for each case. 885

6 886 MERRYMAN, JAFFE, AND EHRENFELD J. VIROL. with biphasic kinetics (Fig. 6A). To insure that viral RNA and protein synthesized after removal of the drug could be assembled into completed virions, cytoplasmic extracts were sedimented through sucrose gradients and analyzed for the presence of 150S particles. The amount of virus present in the D-penicillamine treated and reversed culture was similar to that found in infected, nontreated controls when sufficient time was allowed for the lag after reversal (Fig. 7). DISCUSSION The inhibitory activity of D-penicillamine toward poliovirus replication does not involve the initial steps of adsorption, penetration, or uncoating of input virus. Maximal inhibition is observed even if the drug is added subsequent to 5000I 4000I 3500I w z C.) 3000I I CL) OOC0 50C 2 0 a a a L FRACTION * NO D-PEN NUMBER O-O 500 V/mI D-PEN ADDED 30' REMOVED 90' FIG. 7. Synthesis of virions after reversal of D-penicillamine. HeLa cells (5 x 107) were infected with poliovirus in the presence of actinomycin D. Half of the culture received D-penicillamine at 30 min p.i., and the drug was removed after I h as described in the legend to Fig. 6. Both cultures were labeled with ['4C]uridine at 90 min p.i. The control culture was harvested at 5.5 h p.i.; the D-penicillamine-treated, reversed culture was harvested at 6.5 h p.i. Cytoplasmic extracts were prepared, treated with DOC, and sedimented through sucrose gradients.

7 VOL. 13, 1974 ANTIVIRAL EFFECT OF D-PENICILLAMINE 887 these steps, and no inhibition of virus yield is observed if the drug is present only during these steps (Fig. 2 and Table 1). Since D-penicillamine exerts its maximal inhibitory effect when added prior to 1 or 1.5 h p.i., it is apparently affecting a step which is occurring during the early phase of virus infection rather than a later process such as virion assembly (2). This is supported by the fact that introduction of the drug after 2 h p.i. results in a marked reduction in its inhibitory capacity (Fig. 2). The reversibility of D-penicillamine inhibition (Fig. 6 and 7) indicates that there was no permanent inactivation of viral or cellular components required for virus replication. Although D-penicillamine reduced the cytopathic effect of poliovirus on the cell, it did not prevent the virus-induced inhibition of host cell protein synthesis (Fig. 4). In this respect it resembles other poliovirus inhibitors such as guanidine and HBB [2(a-hydroxybenzyl)benzimidazole (5, 6), in contrast to the thiopyrimidine, S-7, which does prevent virus-induced "shut off' of host cell protein synthesis (12). Although D-penicillamine resembles guanidine in its antiviral activity, this compound cannot replace guanidine for the growth of guanidine-dependent strains of poliovirus, and it inhibits both guanidine-sensitive and guanidine-resistant strains equally (7). Furthermore, the inhibitory effects of guanidine and D- penicillamine are additive when both inhibitors are used simultaneously (7). Other amino acid analogues such as fluorophenylalanine, ethionine, and canavanine have been shown to inhibit the cleavage of poliovirus precursor polypeptides to form product structural (and presumably nonstructural) proteins (8). D-Penicillamine could not be shown to have similar activity, since no accumulation of high molecular weight precursor polypeptides could be demonstrated in the presence of the drug (Fig. 5). The failure of a representative group of mercaptan compounds to inhibit poliovirus replication indicates that this property is not a nonspecific effect of sulfhydryl reducing agents, analagous to the disruption of disulfide bonds. The inhibitory activity can neither be attributed to its antipyridoxine or copper chelation properties, since the L-enantiomorph of penicillamine is the more potent vitamin B6 antagonist (11) and has comparable chelation activity, yet is ineffective against poliovirus replication (7). When the amino group of D-penicillamine is acetylated, anti-polioviral activity is lost. Thus, both the D configuration and a free amino group are structural requirements for the antiviral action. ACKNOWLEDGMENTS We thank Ethel Hurston for skillful technical assistance. This work was supported by the Maresi Fund Center Grant Program, New York Chapter of the Arthritis Foundation and the National Science Foundation, grant No. GB 18026, and by American Cancer grant VC-33. E.E. is supported by a Public Health Service Career Development Award no. IK 04A LITERATURE CITED 1. Baum, S. G., M. S. Horwitz, and J. V. Maizel Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 40. J. Virol. 10: Baltimore, D., M. Girard, and J. E. Darnell Aspects of the synthesis of poliovirus RNA and the formation of virus particles. Virology 29: Christian, C. L Rheumatoid arthritis etiologic considerations. Arthritis Rheum. 7: Crawhall, J. C., E. F. Scowen, and R. W. E. Watts Effects of penicillamine on cystinuria. Brit. Med. J. 1: Crowther, D. and J. L. Melnick Studies of the inhibitory action of guanidine on poliovirus multiplication in cell cultures. Virology 15: Eggers, H. J., and I. Tamm On the mechanism of selective inhibition of enterovirus by 2-(-hydroxybenzyl)-benzimidazole. Virology 18: Gessa, G. L., B. Loddo, G. Brotzu, M. L. Schiva, A. Tagliamante, A. B. G. Spanedda, and W. Ferrar Selective inhibition of poliovirus growth by D-penicillamine in vitro. Virology 30: Jacobson, M. F., and D. Baltimore Polypeptide cleavages in the formation of poliovirus proteins. Proc. Nat. Acad. Sci. U.S.A. 68: Jaffe, I. A The treatment of rheumatoid arthritis and necrotizing vasculitis with penicillamine. Arthritis Rheum. 13: Jaffe, I. A., K. Altman, and P. Merryman The antipyridoxine effect of penicillamine in man. J. Clin. Invest. 43: Kuchinskas, E. J., and J. du Vigneaud An increased vitamin B, requirement in the rat on a diet containing L-penicillamine. Arch. Biochem. Biophys. 68: La Colla, P., M. A. Marciolis, G. P. Meren, and B. Loddo Specific inhibition of poliovirus-induced blockade of cell protein synthesis by a thiopyrimidine derivative. J. Gen. Virol. 17: Maizel, J. V., Jr Polyacrylamide gel electrophoresis of viral proteins, p In K. Maramorosch and H. Kaprowski (ed.), Methods in virology, vol. 5. Academic Press Inc., New York. 14. Nimnio, M. G., and L. A. Batetta Collagen defect induced by penicillamine. Science 150: Penman, S., and D. Summers Effects on host cell metabolism following synchronosis infection with poliovirus. Virology 27: Studier, F. W Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79: Summers, D. F., J. V. Maizel, and J. E. Darnell Evidence for virus-specific noncapsid proteins in poliovirus infected HeLa cells. Proc. Nat. Acad. Sci. U.S.A. 54: Walshe, J. M. D Disturbance of amino acid metabolism following liver injury. Quart. J. Med. 22:

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