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Effect of ouabain on Sindbis virus replication in ouabain-sensitive and ouabain-resistant Aedes albopictus cells (Singh)
VIROLOGY
87,56-65
(1978)
Effect of Ouabain on Sindbis Virus Replication Ouabain-Resistant Aedes albopictus STEVEN J. MENTO
AND
in Ouabain-Sensitive Cells (Singh)‘, *
and
VICTOR STOLLAR3
Department of Microbiology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Accepted January 28, 1978 The effects of ouabain on Sindbis virus replication in cloned sublines of ouabain-sensitive (B&4) and -resistant (Ouab) Aedes albopictus cells have been studied. The addition of ouabain (0.1 mM) to BiC4 cells 1 hr after infection reduced the yield of infectious virus 501% fold. The same concentration had no effect on the yield from Ouas cells. Analysis of r3H]uridiie-labeled RNA by gel electrophoresis showed that ouabain markedly inhibited Sindbis virus-specific RNA synthesis in BiC4 cells but only partially inhibited host RNA synthesis. In Oua5 cells, ouabain had little effect on either host or viral RNA synthesis. A close correlation was observed between the inhibition of host protein synthesis by ouabain and the inhibition of virus replication. INTRODUCTION
MATERIALS
AND
METHODS
Many workers have reported the isolaCells and viruses. The primary chicken tion of cultured cell lines resistant to the embryo cells (CEF) have been described cardiotonic glycoside ouabain (Mayhew, previously (Stollar et aZ., 1976). The growth conditions, the determinations of efficiency 1972; Mankovitz et al., 1974; Baker et al, 1974; Rosenburg, 1975; Lever et al., 1976), of plating, and the isolation of the A. alboa specific inhibitor of the Na+/K+-activated pictus cell lines (Singh, 1967) resistant to ATPase of the plasma membrane (Glynn, BUdR (B& cells) and to BUdR and oua1964). We have recently described the iso- bain (Ouas cells) have recently been relation of mutants of the Aedes albopictus ported (Mento and Stollar, 1978). B&4 cells cell line with greatly increased resistance to were the parent cells from which Oua5 cells the cytotoxic effects of ouabain (Mento and were isolated. A. albopictus cells were grown and maintained after infection in E Stollar, 1978). In this report, we describe the effects of medium [Eagle’s medium (Eagle, 1959) ouabain on Sindbis virus replication in oua- supplemented with nonessential amino bain-sensitive and -resistant mosquito cells. acids and glutamine]. This medium, which In addition, we compare the effects of oua- contained 5 n-&f potassium, was routinely bain on host and viral RNA synthesis and supplemented with 10% fetal calf serum on host protein synthesis in the two cell (FCS) (undiluted FCS contained 12 mi%f types. potassium as determined by flame photometry). Therefore, the final concentration of ’ A preliminary report of this work was given at the potassium in the serum-supplemented E Meeting of the American Society for Microbiology, medium was about 6 mM. New Orleans, 1977. Stocks of Sindbis virus (SVSTD)were pre2 The work reported in this paper will be included pared as described elsewhere (Shenk and in the dissertation to be submitted by S.J.M. in partial Stollar, 1974). Titers of infectious virus fulfillment of the requirements for the Ph.D. degree in were assayed by plaque titration of CEF at microbiology awarded by the Graduate School of Rut34“ (Shenk et al, 1974). gers University. 3 Author addressed.
to whom requests for reprints
0042~6622/76/0871-0056$02.00/O Copyright 0 1978 by Academic Press, Inc. AII rights of reproduction in any form reserved.
Labeling and extraction of intracellular RNA. Monolayer cultures of A. albopictus
should be 58
OUABAIN
AND
SINDBIS
cells were mock-infected with phosphatebuffered saline (PBS) or infected with SVsTn at a m.o.i. of 10 PFU/cell. The virus was allowed to adsorb at 28”. After 1 hr, the inoculum was removed, fresh E medium (10% FCS) with or without ouabain was added, and the cells were incubated at 28”. When actinomycin D (AMD) (1 pg/ml) was used, it was added to infected plates or mock-infected plates 30 min prior to the addition of label. Labeling was with [53H]uridine (25 pCi/ml of medium, 12.5 Ci/mmol) from 4 to 9 hr after infection. RNA was prepared by phenol extraction, ether extraction, and ethanol precipitation (Stollar et al., 1972; Guild and Stollar, 1975). Gradient polyacrylamide slab gel electrophoresis was used to analyze the phenolextracted RNA. The gel buffers employed have been described previously (Simmons and Strauss, 1972; Guild and Stollar, 1975). Gradient gels were poured at 4” by adding equal volumes of buffered 2% acrylamide (no glycerol) and 10% buffered acrylamide (20% glycerol) to a Hoeffer gradient maker. The gradient was formed directly between the glass plates used in the electrophoresis apparatus. After the gel had been poured, a small volume of 2% a&amide was layered on the top of the gradient to accommodate the comb used to make sample wells. The gel was allowed to polymerize at 37” for at least 2 hr before the comb was removed. The gels were routinely run for 16 hr at a constant current setting (25 mA/gel). At the end of each run, the gels were processed for fluorography as described by Laskey and Mills (1975). Chemicals. Ouabain was obtained from Sigma Chemical Co. and stored at -20’ as a 5 mM stock solution in E medium without fetal calf serum. Radioisotopes. [5-3H]Uridine (12.5 Ci/ mmol) and [4,5-3H]leucine (46 Ci/mmol) were obtained from New England Nuclear Corporation, as was NEN Formula 949. RESULTS
The Effect of Ouabain Replication.
on Sindbis
Virus
In Fig. 1 we show a comparison of the
VIRUS
REPLICATION
59
FIG. 1. The effect of various concentrations of ouabain on Sindbis virus replication in BIG and Ouas cells. Cells were grown in E medium (10% FCS) for 3 days at 28” prior to infection. Cells, 5 X 105, in 35-mm tissue culture plates were infected with SVsm (m.o.i. = lo), and the virus was allowed to adsorb for 1 hr. The inoculum was removed, cells were washed twice with PBS containing Ca*+ and Mg’+, and fresh E medium with various concentrations of ouabain was added. After 24 hr of incubation at 28’, aliquota of medium were taken and later assayed for infectious virus by plaque assay on CEF. The 24-hr viral yield obtained from cells incubated without ouabain was 1.8 x lo9 PFU/ml for both B& and Ouaa cells. V-V, B&, cells; A-A, Ouas cells.
relative yields of Sindbis virus from untreated B1C4 and Ouas cells and from the same cells treated with various concentrations of ouabain. In BC4 cells, a lOO-fold reduction in virus yield was achieved at a ouabain concentration of less than 0.05 mil$ in Ouas cells a concentration of 5 mM was needed to produce a similar level of inhibition. We had demonstrated previously (Mento and Stellar, 1978) that ouabain significantly inhibited clonal growth of B1C4cells at concentrations greater than or equal to 0.005 mM, whereas clonal growth of Ouas cells was inhibited only at ouabain concentrations greater than 0.1 mM. Thus, these results indicate that the sensitivity of virus replication to ouabain roughly paralleled that of the host cells. Incubation of intact Sindbis virus with ouabain resulted in no loss of infectivity (data not shown). For determining which stage in virus growth was affected by ouabain, we added ouabain (final concentration of 0.1 mM) to the medium of B1C4cells at different times after infection (Fig. 2). The curve on the left plots the 12-hr viral yields from oua-
60
MENTO
AND
bain-treated cultures versus the time at which ouabain was added, and the curve on the right shows the standard virus growth curve in the absence of ouabain. The addition of 0.1 mM ouabain 1 hr after infection reduced the yield of virus almost UK&fold.
0.i
2
4
6
8
STOLLAR
When ouabain was added to infected cells at later times, its effect on viral yields was progressively reduced. For example, when ouabain was added at 4 hr after infection, the 12-hr viral yield was reduced only threefold. The demonstration that the inhibitory effect of ouabain diminishes rapidly when the drug is added later during infection suggests that ouabain predominantly affects some step early in virus replication. However, since ouabain was added after virus was adsorbed to cells, inhibition of virus replication must occur after this step. To see whether the inhibitory effect of ouabain was reversible, ouabain (0.1 mM) was added-to infected B1C4cells 1 hr after infection and then removed either 7 or 23 hr later. Table 1 shows that the inhibitory effect of ouabain on Sindbis virus replication (compare series A and B) was reversed when ouabain was removed after 7 hr (series C) as well as after 23 hr (series D) of treatment. These results suggest that input viral RNA was stable in ouabain-treated cells for at least 24 hr. Since the viral yields from cultures refed with ouabain-containing medium (series E) were very similar to yields from cells maintained continuously in ouabain without changing the medium (series B), it seems clear that the increase in viral yield (series C and D) was indeed due to the removal of ouabain rather than to the addition of fresh medium.
12
TIME AFTER INFECTION OiOURS)
FIG. 2. The effect of ouahain added at different times on Sindbis virus replication in B&4 cells. BG cells were grown, infected, and processed as described in the legend to Fig. 1, except that ouabain (final concentration, 0.1 mA2) was added to the medium at different times after infection. Shown are a standard virus growth curve in the absence of ouabain (0) and the 12-hr yields (X) from cells treated with ouabain at the indicated times after infection. TABLE
1
THE REVERSIBILITY OF THE EFFECT OF OUABAIN ON SINDBIS VIRUS REPLICATION IN B1C4 CELLS” Ouabain
Series
Yield (PFU/ml)
Added Removed (hr after infection) A B C D E
1 1 1 1
8 24 -
at indicated
times after infection
2 hr
8 hr
24 hr
36 hr
48 hr
5.4 x lo4 7.6 x lo4 -
1.9 x lob: 5.3 x 10’ 3.5 x ld -
6.8 x 107 3.3 x 105 3.3 x 104 2.5 x lo4
9 x 107 -
4 4 2 6.5 3.4
x los x lo5 x 108 x 10’ x 10”
a Exponentially growing BC, cells in wells of a 24-well plate (2 x lo5 cells/well of 16-mm diameter) were infected with SVsro (m.o.i. = 10). After 1 hr of adsorption at 28”, excess virus was removed and 1 ml of medium without or with ouabain (0.1 n-&f) was added to each well. At 8 and 24 hr after infection, the medium was removed from some of the ouabain-treated cells, the monolayers were washed twice with PBS containing Mg*+ and Ca*+, and fresh medium without ouabain was added. At the indicated times aliquots of medium were taken and assayed later for infectious virus by plaque titration on CEF. Series: A, untreated cultures; B, ouabaintreated cultures; C, ouabain-treated cultures with ouabain removed 7 hr after addition of ouabain; D, ouabaintreated cultures with ouabain removed 23 hr after addition of ouabain; E, ouabain-treated cultures that were refed with ouabain-containing medium 23 hr after the first addition of ouabain.
OUABAIN
AND
SINDBIS
VIRUS
REPLICATION
61
The Effect of Ouabain on RNA Synthesis in Uninfected and Infected Cells
The effects of ouabain on host and viral RNA synthesis were studied by measuring [3H]uridine incorporation into TCA-precipitable material. The conditions for labeling host and viral RNA were the same except that incorporation into viral RNA was measured in the presence of actinomycin D @MD). Host RNA synthesis was defined as incorporation into the total TCA-precipitable material in uninfected cells. Virusspecific RNA synthesis was defined as TCA-precipitable counts per minute (cpm) in AMD-treated infected cells minus the TCA-precipitable counts per minute in AMD-treated uninfected cells. These values for both host and viral RNA synthesis were expressed relative to values for infected or uninfected cells labeled without ouabain (arbitrarily set as equal to 1.0). As shown in Fig. 3, in Ouas cells both host and viral RNA synthesis were only slightly inhibited by ouabain (about twofold at 1 m.iW ouabain). In contrast, in B1C4 cells, host RNA synthesis was inhibited lo-fold and viral RNA synthesis was inhibited lOO-fold at ouabain concentrations greater than 0.05 l-r&. Because ouabain acts at the plasma membrane and has been shown to alter the transport of certain amino acids (Schultz and Curran, 1970; Shank and Smith, 1976), it was important to test whether our measurements of RNA synthesis were only a reflection of reduced r3H]uridine uptake. Exponentially growing cells were pretreated for 4 hr with ouabain (0.1 mM) and then labeled for 2 hr with [3H]uridine (1 &i/ml). Monolayers were washed twice with cold PBS, cells were dissolved in 0.2 N NaOH (60” for 15 min), and 0.1~ml aliquots were counted. The uptake of [3H]uridine was reduced less than 10% in the presence of ouabain in Ouas cells, but in BIG cells uptake was reduced fivefold. It is uncertain whether the reduction in r3H]uridine uptake by B&4 cells is due to a direct effect of ouabain on uridine transport or secondary to a reduction in RNA synthesis. In either case, it seems unlikely that the reduced uptake can account completely for the lOO-fold reduc-
WABAIN CONCENTRATIONImU)
FIG. 3. The relative [3H]uridine incorporation into uninfected and infected cells incubated with or without ouabain. Exponentially growing cells in 35-mm tissue culture plates (5 x lo5 cells/plate) were mock infected with PBS or infected with Svs~o (m.o.i. = 10). Virus was allowed to adsorb for 1 hr at 28“; the inoculum was removed and E medium (10% FCS) was added with or without ouabain. Actinomycin D (AMD) (1 pg/ml) was added to the medium of infected cells and to one set of mock-infected cells 0.5 hr prior to addition of label. Cells were labeled with [3H]uridine (1 @i/ml, 12.5 Ci/mmol) from 5 to 7 hr after infection. After labeling, the cells were washed twice with cold PBS and scraped into PBS. An equal volume of 15% TCA was added to each cell suspension, and all samples were kept at 4’ for 30 min. TCA-precipitable material was collected on Whatman GF/A filters, which were then washed with 10 ml of 5% TCA per filter. The filters were dried, added to scintillation vials containing 5 ml of NEN 949, and radioactivity was measured. Host RNA synthesis, open symbols and solid lines; viral RNA synthesis, lilled symbols-and dashed lines.
tion of viral RNA synthesis observed in ouabain-treated B1C4cells. Furthermore, in other experiments with BHK cells (data not shown), viral RNA synthesis was inhibited by ouabain even though no significant effect on uridine uptake could be demonstrated. Effects of Ouabain on the Synthesis of Various Species of Host and Viral RNA
After observing the differential effects of ouabain on host and viral RNA synthesis in B1C4 cells (Fig. 3), we tested the effect of ouabain on the synthesis of specific host and viral RNA species. Accordingly,
62
MENTO
AND
[3H]uridine-labeled RNAs extracted from uninfected or Sindbis virus-infected B1C4 and Ouas cells which had been incubated in the absence or presence of ouabain (0.1 mi%l’)were examined by gradient acrylamide slab gel electrophoresis (Fig. 4). The absence or presence of ouabain is indicated by (-) or (+). As with many insect cell types, the large ribosomal RNA (26 S) was unstable during phenol extraction (Greenberg, 1969; Stollar et al., 1974) and partially dissociated into bands migrating just above and below the 19 S ribosomal RNA. After ouabain treatment of uninfected B1C4 cells, the labeling of rRNA was inhibited lo-fold [as measured by densitometer scans (see also Fig. 3)], and a new species of RNA appeared that migrated between the 26 and 19 S rRNAs. In Ouas cells, neither the pattern of labeled RNA nor the absolute amount of label in rRNA (as measured by densitometer scans) was affected by treatment with ouabain. The major single-stranded (42 and 26 S) and double-stranded (22 S) viral RNA species labeled in AMD-treated infected B1C4 and Ouas cells are also shown in Fig. 4. In this gel system the 22 S RNA migrates between the 42 and 26 S viral RNAs. Un-
26S-
HOST
VIRUS
B1C4 OUA5
B,C4 OUA5
-425 -22S(dsRNA) - 265
19sAMDR RNA
FIG. 4. RNA species synthesized in uninfected and infected cells in the presence or absence of ouabain. Cells were mock infected or infected and labeled with [3H]uridine from 4 to 9 hr after infection as described under Materials and Methods. Extraction and gel electrophoresis were also described there. Ouabain (0.1 mM) was added 1 hr after infection. Infected cells were treated with actinomycin D 30 min before the addition of label. The absence or presence of ouabain is indicated by (-1 or (-t), respectively. The left- and right-hand panels show the effects of ouabain on the labeling of host and viral RNA species, respectively.
STOLLAR
infected as well as infected mosquito cells contained three host AMD-resistant (AMDR) RNA species which migrated beyond the 19 S rRNA. In BIG4 cells, no viral RNA species were detected when labeling was carried out in 0.1 m&f ouabain. In Ouas cells, the viral RNA species were labeled both in the absence and in the presence of ouabain, and the amount of labeled viral RNA was reduced only twofold (as measured by densitometer scans) in the treated cells as compared to the untreated cells. The sensitivity of the AMDR host RNA species to ouabain appeared to be very similar to that of the viral RNA species; the labeling of all three species was inhibited in the B1C4cells but not in the Ouas cells. Effects of Ouabain on Host Protein Synthesis Ledbetter and Lubin (1977) have shown that a primary effect of reduced intracellular K+ concentrations in ouabain-treated human fibroblasts is the inhibition of protein synthesis. Since an early inhibition of protein synthesis could also explain why viral RNA is not made, we examined the effects of ouabain on host protein synthesis (as measured by [3H]leucine incorporation into TCA-precipitable material) in B1C4 and Ouas cells. Labeling conditions were identical to those employed to measure host-specific C3H]uridine incorporation (see Fig. 3). Figure 5 shows that host protein synthesis was inhibited significantly more in B1C4 cells than in Ouas cells (80-90s inhibition vs 10% at a concentration of 0.05 mJ4 ouabain). The patterns of ouabain inhibition of host protein synthesis closely resemble the patterns of reduction in viral yields (Fig. 1) and of inhibition of viral RNA synthesis (Fig. 3) by ouabain. DISCUSSION
Ouabain binds specifically to the Na+/K+ ATPase of the plasma membrane, thereby inhibiting both the active transport of potassium into the cell and the active extrusion of sodium. This, in turn, leads in both mammalian cells (Shank and Smith, 1976) and mosquito cells (Mento and Stollar, unpublished observations) to the rapid equilibration by passive diffusion of the intra-
OUABAIN
AND
SINDBIS
FIG. 5. The relative r3H]leucine incorporation into uninfected cells incubated with or without ouabain. Uninfected exponentially growing cells were labeled as described in the legend to Fig. 3 except that [“HIleucine (1 pa/ml, 46 Ci/mmol) was substituted for [3H]uridine. V-V, B& cells; A-A, Ouas cells.
cellular and extracellular sodium and potassium concentrations. Ledbetter and Lubin (1977) have demonstrated that a primary effect of reduced potassium levels in ouabain-treated cells is the inhibition of protein synthesis. In this report we have determined the effects of ouabain (presumably mediated through alterations in intracellular ion concentrations) on Sindbis virus replication, on host and viral RNA synthesis, and on host protein synthesis in ouabain-sensitive and -resistant cells. Our data indicate that (1) ouabain inhibits Sindbis virus replication in ouabainsensitive (B&d) mosquito cells but to a much lesser extent in ouabain-resistant (Ouas) mosquito cells; (2) in B1C4cells, the sensitivity of viral RNA synthesis to ouabain is greater than that of host RNA synthesis (although the explanation for this observation is uncertain, it may reflect the fact that host RNA synthesis occurs in the nucleus and viral RNA synthesis in the cytoplasm); (3) at concentrations which inhibit viral replication in B1C4cells, ouabain also inhibits host protein synthesis; in Ouas cells, the same concentrations had little effect on either viral replication or host protein synthesis. Other experiments (not shown) have demonstrated that ouabain also inhibits the replication of Sindbis virus in BHK-21 cells. Since hamster cells are inherently more resistant to ouabain than are mosquito cells, as shown by the concentrations nec-
VIRUS
REPLICATION
63
essary to affect the plating efficiencies, it was necessary to use higher concentrations (5 vs 0.1 mM) to inhibit virus replication in BHK-21 cells. As in B&4 cells, the concentrations of ouabain which inhibited virus yields also reduced viral RNA synthesis. Interestingly, however, such concentrations either had no effect on host RNA synthesis or led to a mild (twofold) stimulation. The ability of ouabain to inhibit viral RNA synthesis is dependent on when the drug is added. When ouabain is added to B1C4 cells 1 hr after infection, viral RNA synthesis is totally inhibited, whereas upon later addition (4 hr after infection but well before the peak of viral RNA synthesis) viral RNA synthesis must proceed almost normally since viral yields are only slightly reduced. These data are consistent with the observations of Scheele and Pfefferkorn (1969), who demonstrated in Sindbis virusinfected chick embryo fibroblasts that viral RNA synthesis was blocked by inhibitors of protein synthesis only if the inhibitors (puromycin or cycloheximide) were added at 1 hr after infection. In their experiments, if viral RNA synthesis was allowed to proceed until 3 hr after infection, it was no longer dependent on concomitant protein synthesis. Our data suggest that ouabain also blocks the protein synthesis necessary for the initiation of viral RNA synthesis. The experiments of Scheele and Pfefferkorn (1969) differ from ours, however, in that although viral RNA synthesis continued when cycloheximide or puromycin was added at 3 hr after infection, progeny virus was not produced since the synthesis of viral structural proteins was blocked. In contrast, when ouabain was added to BIG cells 4 hr after infection, viral yields were only slightly reduced, implying that both viral RNA synthesis and the synthesis of viral structural proteins must have continued. One explanation that could account for the inhibition of viral RNA synthesis by ouabain would be that it inhibits the synthesis of viral RNA polymerase from parental (42 S) viral RNA. In that case, the time dependence of the inhibitory effects of ouabain would imply that ouabain-induced alterations of intracellular ion concentrations would have to have different effects on the
64
MENTO
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translation of 42 S viral RNA [which codes for nonstructural proteins including the viral RNA polymerase (Strauss and Strauss, 1977)] and 26 S viral RNA [which codes for the viral structural proteins (Strauss and Strauss, 1977)J. Alterations in ionic conditions have been shown to have differential effects on the translation of host mRNA and viral mRNA in cells infected with various cytocidal RNA viruses (Nuss et aZ., 1975; Han&n, 1976; Carrasco et al, 1976). From these data, Carrasco (1977) has suggested that cytocidal viruses may inhibit host protein synthesis by distorting the gradient of monovalent cations across the plasma membrane. The observations of Farnham and Epstein (1963) and of Egberts et al. (1977) describing virus-mediated alterations in the intracellular ionic environment support this idea. Our data on the effects of ouabain in Sindbis virus replication suggest that alterations in the intracellular ionic conditions may also play a role in regulating the translation of different classesof viral RNA. This idea could be tested using in vitro systems. ACKNOWLEDGMENTS
I
This investigation was supported by Grant No. AI
11299 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Cooperative Medical Science Program through Public Health Service Grant No. AI 05920, and by the Institutional National Research Service Award CA-09069 from the National Cancer Institute. REFERENCES
BAKER, R. M., BRUNETTE, D. M., MANKOVITZ, R., THOMPSON, L. H., WHITMORE,G. F., SIMINOVITCH, L., and TILL, J. E. (1974).Ouabain-resistant mutants of mouse and hamster cells in culture. Cell 1,9-21. CARRASCO, L. (1977). The inhibition of cell functions after viral infection. FEBS Lett. 76,11-15. CARFUSCO, L., and SMITH, A. E. (1976). Sodium ions and the shut-off of host cell protein synthesis by picornaviruses. Nature (London) 264,807-809. DAHL, J. L., and HOKIN, L. E. (1974). The sodiumpotassium adenosinetriphosphatase. Anna. Rev. B&hem. 43,327-356. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cuitures. Science 130, 432-437. EGBERTS,E., HACKETT, P. B., and TRAUB, P. (1977). Alteration of the intracellular energetic and ionic conditions by mengovirus infection of Ehrlich ascites tumor cells and its influence on protein synthesis in the midphase of infection. J. Virol. 22, 591-597.
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FARNHAM, A. E., and EPSTEIN, W. (1963). The influence of encephalomyocarditis (EMC) virus infection on potassium transport in L cells. Virology 21, 436-447. GLYNN, I. M. (1964). The action of cardiac glycosides on ion movements. Pharmacol. Rev. 16, 361-407. GREENBERG,J. R. (1969). Synthesis and properties of ribosomal RNA in Drosophila. J. Mol. Biol. 46, 85-98. GUILD, G. M., and STOLLAR, V. (1975). Defective interfering particles of Sindbis virus: III, Intracellular viral RNA species in chicken embryo cultures. Virology 67,24-41. HAMLYN, P. (1976). Cytoplasmic control of protein synthesis. Nature (London) 264,274-277. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ‘H and Y! in polyacrylamide gels by fluorography. Eur. J. B&hem. 66,335-341. LEDBETTER, M. L. S., and LUBIN, M. (1977). Control of protein synthesis in human fibroblasts by intracellular potassium. Exp. Cell. Res. 105, 223-236. LEVER, J. E., and SEEGMILLER,J. E. (1976). Guabainresistant human lymphoblastoid Linesaltered in the (Na+ + K+)-dependent ATPase membrane transport system. J. Cell Physiol. 88.343-352. MANKOVITZ, R., BUCHWALD, M., and BAKER, R. M. (1974). Isolation of ouabain-resistant human diploid fibroblasts. Cell 3,221-226. MAYHEW, E. (1972). Ion transport by ouabain-resistant and sensitive Ehrhch ascites carcinoma cells. J. Cell Physiol. 79,441. MENTO, S. J., and STOLLAR, V. (1978). Isolation and partial characterization of drug-resistant Aedes albopictus cells. Somatic Cell &net., 4, 179-191. Nuss, D. L., OPPERMAN, H., and KOCH, G. (1975). Selective blockage of initiation of host protein synthesis in RNA virus infected cells. Proc. Nat. Acad. Sci. U.S.A. 72, 1258-1262. ROSENBURG,J. (1975). Variant HeLa cells selected for their resistance to ouabain. J. Cell Physiol. 85,135. SCHEELE, C. M., and PFEFFERKORN,E. R. (1969). Inhibition of interjacent ribonucleic acid (26s) sxnthesis in cells infected by Sindbis virus. J. Virol. 4, 117-122. SCHULTZ,S., and CURRAN, P. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50.637-710. SHANK, B. B., and SMITH, N. Ea(1976). Regulation of cellular growth by sodium pump activity. J. Cell Physiol. 87,377-386. SHENK, T. E., KOSHELNYK, K. A., and STOLLAR, V. (1974). Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus. J. Viral. 13,439-447. SHENK, T. E., and STOLLAR, V. (1974). Defective interfering particles of Sindbis virus: I, Isolation and some chemical and biological properties. Virology 53,162-173. SIMMONS, D. T., and STRAUSS, J. H. (1972). Replication of Sindbis virus: I, Relative size and genetic
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content of 26s and 49s RNA. J. Mol. Biol. 71, 599-613. SINGH, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes ae-
gypti. Curr. Sci. 36,506-508. STOLLAR, V., PELEG, J., and SHENK, T. E. (1974). Temperature sensitivity of a Sindbis virus mutant isolated from persistently infected Aedes aegypti ceII culture. Intervirology 2,337~344. STOLLAR, V., SHENK, T. E., and STOLLAR, B. D. (1972). Double-stranded RNA in hamster, chick and
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mosquito cells infected with Sindbis virus. Virology 47.122-132. STOLLAR, V., STOLLAR, B. D., Koo, R., HARRAP, K. A., and SCHLESINGER, R. W. (1976). Siahc acid contents of Sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties. Virology 69, 104-115. STRAUSS, J. H., and STRAUSS, E. G. (1977). Togaviruses. In “The Molecular Biology of Animal Viruses” (D. P. Nayak, ed.), Vol. 1, pp. 111-166. Dekker, New York.
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(1978)
Effect of Ouabain on Sindbis Virus Replication Ouabain-Resistant Aedes albopictus STEVEN J. MENTO
AND
in Ouabain-Sensitive Cells (Singh)‘, *
and
VICTOR STOLLAR3
Department of Microbiology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Accepted January 28, 1978 The effects of ouabain on Sindbis virus replication in cloned sublines of ouabain-sensitive (B&4) and -resistant (Ouab) Aedes albopictus cells have been studied. The addition of ouabain (0.1 mM) to BiC4 cells 1 hr after infection reduced the yield of infectious virus 501% fold. The same concentration had no effect on the yield from Ouas cells. Analysis of r3H]uridiie-labeled RNA by gel electrophoresis showed that ouabain markedly inhibited Sindbis virus-specific RNA synthesis in BiC4 cells but only partially inhibited host RNA synthesis. In Oua5 cells, ouabain had little effect on either host or viral RNA synthesis. A close correlation was observed between the inhibition of host protein synthesis by ouabain and the inhibition of virus replication. INTRODUCTION
MATERIALS
AND
METHODS
Many workers have reported the isolaCells and viruses. The primary chicken tion of cultured cell lines resistant to the embryo cells (CEF) have been described cardiotonic glycoside ouabain (Mayhew, previously (Stollar et aZ., 1976). The growth conditions, the determinations of efficiency 1972; Mankovitz et al., 1974; Baker et al, 1974; Rosenburg, 1975; Lever et al., 1976), of plating, and the isolation of the A. alboa specific inhibitor of the Na+/K+-activated pictus cell lines (Singh, 1967) resistant to ATPase of the plasma membrane (Glynn, BUdR (B& cells) and to BUdR and oua1964). We have recently described the iso- bain (Ouas cells) have recently been relation of mutants of the Aedes albopictus ported (Mento and Stollar, 1978). B&4 cells cell line with greatly increased resistance to were the parent cells from which Oua5 cells the cytotoxic effects of ouabain (Mento and were isolated. A. albopictus cells were grown and maintained after infection in E Stollar, 1978). In this report, we describe the effects of medium [Eagle’s medium (Eagle, 1959) ouabain on Sindbis virus replication in oua- supplemented with nonessential amino bain-sensitive and -resistant mosquito cells. acids and glutamine]. This medium, which In addition, we compare the effects of oua- contained 5 n-&f potassium, was routinely bain on host and viral RNA synthesis and supplemented with 10% fetal calf serum on host protein synthesis in the two cell (FCS) (undiluted FCS contained 12 mi%f types. potassium as determined by flame photometry). Therefore, the final concentration of ’ A preliminary report of this work was given at the potassium in the serum-supplemented E Meeting of the American Society for Microbiology, medium was about 6 mM. New Orleans, 1977. Stocks of Sindbis virus (SVSTD)were pre2 The work reported in this paper will be included pared as described elsewhere (Shenk and in the dissertation to be submitted by S.J.M. in partial Stollar, 1974). Titers of infectious virus fulfillment of the requirements for the Ph.D. degree in were assayed by plaque titration of CEF at microbiology awarded by the Graduate School of Rut34“ (Shenk et al, 1974). gers University. 3 Author addressed.
to whom requests for reprints
0042~6622/76/0871-0056$02.00/O Copyright 0 1978 by Academic Press, Inc. AII rights of reproduction in any form reserved.
Labeling and extraction of intracellular RNA. Monolayer cultures of A. albopictus
should be 58
OUABAIN
AND
SINDBIS
cells were mock-infected with phosphatebuffered saline (PBS) or infected with SVsTn at a m.o.i. of 10 PFU/cell. The virus was allowed to adsorb at 28”. After 1 hr, the inoculum was removed, fresh E medium (10% FCS) with or without ouabain was added, and the cells were incubated at 28”. When actinomycin D (AMD) (1 pg/ml) was used, it was added to infected plates or mock-infected plates 30 min prior to the addition of label. Labeling was with [53H]uridine (25 pCi/ml of medium, 12.5 Ci/mmol) from 4 to 9 hr after infection. RNA was prepared by phenol extraction, ether extraction, and ethanol precipitation (Stollar et al., 1972; Guild and Stollar, 1975). Gradient polyacrylamide slab gel electrophoresis was used to analyze the phenolextracted RNA. The gel buffers employed have been described previously (Simmons and Strauss, 1972; Guild and Stollar, 1975). Gradient gels were poured at 4” by adding equal volumes of buffered 2% acrylamide (no glycerol) and 10% buffered acrylamide (20% glycerol) to a Hoeffer gradient maker. The gradient was formed directly between the glass plates used in the electrophoresis apparatus. After the gel had been poured, a small volume of 2% a&amide was layered on the top of the gradient to accommodate the comb used to make sample wells. The gel was allowed to polymerize at 37” for at least 2 hr before the comb was removed. The gels were routinely run for 16 hr at a constant current setting (25 mA/gel). At the end of each run, the gels were processed for fluorography as described by Laskey and Mills (1975). Chemicals. Ouabain was obtained from Sigma Chemical Co. and stored at -20’ as a 5 mM stock solution in E medium without fetal calf serum. Radioisotopes. [5-3H]Uridine (12.5 Ci/ mmol) and [4,5-3H]leucine (46 Ci/mmol) were obtained from New England Nuclear Corporation, as was NEN Formula 949. RESULTS
The Effect of Ouabain Replication.
on Sindbis
Virus
In Fig. 1 we show a comparison of the
VIRUS
REPLICATION
59
FIG. 1. The effect of various concentrations of ouabain on Sindbis virus replication in BIG and Ouas cells. Cells were grown in E medium (10% FCS) for 3 days at 28” prior to infection. Cells, 5 X 105, in 35-mm tissue culture plates were infected with SVsm (m.o.i. = lo), and the virus was allowed to adsorb for 1 hr. The inoculum was removed, cells were washed twice with PBS containing Ca*+ and Mg’+, and fresh E medium with various concentrations of ouabain was added. After 24 hr of incubation at 28’, aliquota of medium were taken and later assayed for infectious virus by plaque assay on CEF. The 24-hr viral yield obtained from cells incubated without ouabain was 1.8 x lo9 PFU/ml for both B& and Ouaa cells. V-V, B&, cells; A-A, Ouas cells.
relative yields of Sindbis virus from untreated B1C4 and Ouas cells and from the same cells treated with various concentrations of ouabain. In BC4 cells, a lOO-fold reduction in virus yield was achieved at a ouabain concentration of less than 0.05 mil$ in Ouas cells a concentration of 5 mM was needed to produce a similar level of inhibition. We had demonstrated previously (Mento and Stellar, 1978) that ouabain significantly inhibited clonal growth of B1C4cells at concentrations greater than or equal to 0.005 mM, whereas clonal growth of Ouas cells was inhibited only at ouabain concentrations greater than 0.1 mM. Thus, these results indicate that the sensitivity of virus replication to ouabain roughly paralleled that of the host cells. Incubation of intact Sindbis virus with ouabain resulted in no loss of infectivity (data not shown). For determining which stage in virus growth was affected by ouabain, we added ouabain (final concentration of 0.1 mM) to the medium of B1C4cells at different times after infection (Fig. 2). The curve on the left plots the 12-hr viral yields from oua-
60
MENTO
AND
bain-treated cultures versus the time at which ouabain was added, and the curve on the right shows the standard virus growth curve in the absence of ouabain. The addition of 0.1 mM ouabain 1 hr after infection reduced the yield of virus almost UK&fold.
0.i
2
4
6
8
STOLLAR
When ouabain was added to infected cells at later times, its effect on viral yields was progressively reduced. For example, when ouabain was added at 4 hr after infection, the 12-hr viral yield was reduced only threefold. The demonstration that the inhibitory effect of ouabain diminishes rapidly when the drug is added later during infection suggests that ouabain predominantly affects some step early in virus replication. However, since ouabain was added after virus was adsorbed to cells, inhibition of virus replication must occur after this step. To see whether the inhibitory effect of ouabain was reversible, ouabain (0.1 mM) was added-to infected B1C4cells 1 hr after infection and then removed either 7 or 23 hr later. Table 1 shows that the inhibitory effect of ouabain on Sindbis virus replication (compare series A and B) was reversed when ouabain was removed after 7 hr (series C) as well as after 23 hr (series D) of treatment. These results suggest that input viral RNA was stable in ouabain-treated cells for at least 24 hr. Since the viral yields from cultures refed with ouabain-containing medium (series E) were very similar to yields from cells maintained continuously in ouabain without changing the medium (series B), it seems clear that the increase in viral yield (series C and D) was indeed due to the removal of ouabain rather than to the addition of fresh medium.
12
TIME AFTER INFECTION OiOURS)
FIG. 2. The effect of ouahain added at different times on Sindbis virus replication in B&4 cells. BG cells were grown, infected, and processed as described in the legend to Fig. 1, except that ouabain (final concentration, 0.1 mA2) was added to the medium at different times after infection. Shown are a standard virus growth curve in the absence of ouabain (0) and the 12-hr yields (X) from cells treated with ouabain at the indicated times after infection. TABLE
1
THE REVERSIBILITY OF THE EFFECT OF OUABAIN ON SINDBIS VIRUS REPLICATION IN B1C4 CELLS” Ouabain
Series
Yield (PFU/ml)
Added Removed (hr after infection) A B C D E
1 1 1 1
8 24 -
at indicated
times after infection
2 hr
8 hr
24 hr
36 hr
48 hr
5.4 x lo4 7.6 x lo4 -
1.9 x lob: 5.3 x 10’ 3.5 x ld -
6.8 x 107 3.3 x 105 3.3 x 104 2.5 x lo4
9 x 107 -
4 4 2 6.5 3.4
x los x lo5 x 108 x 10’ x 10”
a Exponentially growing BC, cells in wells of a 24-well plate (2 x lo5 cells/well of 16-mm diameter) were infected with SVsro (m.o.i. = 10). After 1 hr of adsorption at 28”, excess virus was removed and 1 ml of medium without or with ouabain (0.1 n-&f) was added to each well. At 8 and 24 hr after infection, the medium was removed from some of the ouabain-treated cells, the monolayers were washed twice with PBS containing Mg*+ and Ca*+, and fresh medium without ouabain was added. At the indicated times aliquots of medium were taken and assayed later for infectious virus by plaque titration on CEF. Series: A, untreated cultures; B, ouabaintreated cultures; C, ouabain-treated cultures with ouabain removed 7 hr after addition of ouabain; D, ouabaintreated cultures with ouabain removed 23 hr after addition of ouabain; E, ouabain-treated cultures that were refed with ouabain-containing medium 23 hr after the first addition of ouabain.
OUABAIN
AND
SINDBIS
VIRUS
REPLICATION
61
The Effect of Ouabain on RNA Synthesis in Uninfected and Infected Cells
The effects of ouabain on host and viral RNA synthesis were studied by measuring [3H]uridine incorporation into TCA-precipitable material. The conditions for labeling host and viral RNA were the same except that incorporation into viral RNA was measured in the presence of actinomycin D @MD). Host RNA synthesis was defined as incorporation into the total TCA-precipitable material in uninfected cells. Virusspecific RNA synthesis was defined as TCA-precipitable counts per minute (cpm) in AMD-treated infected cells minus the TCA-precipitable counts per minute in AMD-treated uninfected cells. These values for both host and viral RNA synthesis were expressed relative to values for infected or uninfected cells labeled without ouabain (arbitrarily set as equal to 1.0). As shown in Fig. 3, in Ouas cells both host and viral RNA synthesis were only slightly inhibited by ouabain (about twofold at 1 m.iW ouabain). In contrast, in B1C4 cells, host RNA synthesis was inhibited lo-fold and viral RNA synthesis was inhibited lOO-fold at ouabain concentrations greater than 0.05 l-r&. Because ouabain acts at the plasma membrane and has been shown to alter the transport of certain amino acids (Schultz and Curran, 1970; Shank and Smith, 1976), it was important to test whether our measurements of RNA synthesis were only a reflection of reduced r3H]uridine uptake. Exponentially growing cells were pretreated for 4 hr with ouabain (0.1 mM) and then labeled for 2 hr with [3H]uridine (1 &i/ml). Monolayers were washed twice with cold PBS, cells were dissolved in 0.2 N NaOH (60” for 15 min), and 0.1~ml aliquots were counted. The uptake of [3H]uridine was reduced less than 10% in the presence of ouabain in Ouas cells, but in BIG cells uptake was reduced fivefold. It is uncertain whether the reduction in r3H]uridine uptake by B&4 cells is due to a direct effect of ouabain on uridine transport or secondary to a reduction in RNA synthesis. In either case, it seems unlikely that the reduced uptake can account completely for the lOO-fold reduc-
WABAIN CONCENTRATIONImU)
FIG. 3. The relative [3H]uridine incorporation into uninfected and infected cells incubated with or without ouabain. Exponentially growing cells in 35-mm tissue culture plates (5 x lo5 cells/plate) were mock infected with PBS or infected with Svs~o (m.o.i. = 10). Virus was allowed to adsorb for 1 hr at 28“; the inoculum was removed and E medium (10% FCS) was added with or without ouabain. Actinomycin D (AMD) (1 pg/ml) was added to the medium of infected cells and to one set of mock-infected cells 0.5 hr prior to addition of label. Cells were labeled with [3H]uridine (1 @i/ml, 12.5 Ci/mmol) from 5 to 7 hr after infection. After labeling, the cells were washed twice with cold PBS and scraped into PBS. An equal volume of 15% TCA was added to each cell suspension, and all samples were kept at 4’ for 30 min. TCA-precipitable material was collected on Whatman GF/A filters, which were then washed with 10 ml of 5% TCA per filter. The filters were dried, added to scintillation vials containing 5 ml of NEN 949, and radioactivity was measured. Host RNA synthesis, open symbols and solid lines; viral RNA synthesis, lilled symbols-and dashed lines.
tion of viral RNA synthesis observed in ouabain-treated B1C4cells. Furthermore, in other experiments with BHK cells (data not shown), viral RNA synthesis was inhibited by ouabain even though no significant effect on uridine uptake could be demonstrated. Effects of Ouabain on the Synthesis of Various Species of Host and Viral RNA
After observing the differential effects of ouabain on host and viral RNA synthesis in B1C4 cells (Fig. 3), we tested the effect of ouabain on the synthesis of specific host and viral RNA species. Accordingly,
62
MENTO
AND
[3H]uridine-labeled RNAs extracted from uninfected or Sindbis virus-infected B1C4 and Ouas cells which had been incubated in the absence or presence of ouabain (0.1 mi%l’)were examined by gradient acrylamide slab gel electrophoresis (Fig. 4). The absence or presence of ouabain is indicated by (-) or (+). As with many insect cell types, the large ribosomal RNA (26 S) was unstable during phenol extraction (Greenberg, 1969; Stollar et al., 1974) and partially dissociated into bands migrating just above and below the 19 S ribosomal RNA. After ouabain treatment of uninfected B1C4 cells, the labeling of rRNA was inhibited lo-fold [as measured by densitometer scans (see also Fig. 3)], and a new species of RNA appeared that migrated between the 26 and 19 S rRNAs. In Ouas cells, neither the pattern of labeled RNA nor the absolute amount of label in rRNA (as measured by densitometer scans) was affected by treatment with ouabain. The major single-stranded (42 and 26 S) and double-stranded (22 S) viral RNA species labeled in AMD-treated infected B1C4 and Ouas cells are also shown in Fig. 4. In this gel system the 22 S RNA migrates between the 42 and 26 S viral RNAs. Un-
26S-
HOST
VIRUS
B1C4 OUA5
B,C4 OUA5
-425 -22S(dsRNA) - 265
19sAMDR RNA
FIG. 4. RNA species synthesized in uninfected and infected cells in the presence or absence of ouabain. Cells were mock infected or infected and labeled with [3H]uridine from 4 to 9 hr after infection as described under Materials and Methods. Extraction and gel electrophoresis were also described there. Ouabain (0.1 mM) was added 1 hr after infection. Infected cells were treated with actinomycin D 30 min before the addition of label. The absence or presence of ouabain is indicated by (-1 or (-t), respectively. The left- and right-hand panels show the effects of ouabain on the labeling of host and viral RNA species, respectively.
STOLLAR
infected as well as infected mosquito cells contained three host AMD-resistant (AMDR) RNA species which migrated beyond the 19 S rRNA. In BIG4 cells, no viral RNA species were detected when labeling was carried out in 0.1 m&f ouabain. In Ouas cells, the viral RNA species were labeled both in the absence and in the presence of ouabain, and the amount of labeled viral RNA was reduced only twofold (as measured by densitometer scans) in the treated cells as compared to the untreated cells. The sensitivity of the AMDR host RNA species to ouabain appeared to be very similar to that of the viral RNA species; the labeling of all three species was inhibited in the B1C4cells but not in the Ouas cells. Effects of Ouabain on Host Protein Synthesis Ledbetter and Lubin (1977) have shown that a primary effect of reduced intracellular K+ concentrations in ouabain-treated human fibroblasts is the inhibition of protein synthesis. Since an early inhibition of protein synthesis could also explain why viral RNA is not made, we examined the effects of ouabain on host protein synthesis (as measured by [3H]leucine incorporation into TCA-precipitable material) in B1C4 and Ouas cells. Labeling conditions were identical to those employed to measure host-specific C3H]uridine incorporation (see Fig. 3). Figure 5 shows that host protein synthesis was inhibited significantly more in B1C4 cells than in Ouas cells (80-90s inhibition vs 10% at a concentration of 0.05 mJ4 ouabain). The patterns of ouabain inhibition of host protein synthesis closely resemble the patterns of reduction in viral yields (Fig. 1) and of inhibition of viral RNA synthesis (Fig. 3) by ouabain. DISCUSSION
Ouabain binds specifically to the Na+/K+ ATPase of the plasma membrane, thereby inhibiting both the active transport of potassium into the cell and the active extrusion of sodium. This, in turn, leads in both mammalian cells (Shank and Smith, 1976) and mosquito cells (Mento and Stollar, unpublished observations) to the rapid equilibration by passive diffusion of the intra-
OUABAIN
AND
SINDBIS
FIG. 5. The relative r3H]leucine incorporation into uninfected cells incubated with or without ouabain. Uninfected exponentially growing cells were labeled as described in the legend to Fig. 3 except that [“HIleucine (1 pa/ml, 46 Ci/mmol) was substituted for [3H]uridine. V-V, B& cells; A-A, Ouas cells.
cellular and extracellular sodium and potassium concentrations. Ledbetter and Lubin (1977) have demonstrated that a primary effect of reduced potassium levels in ouabain-treated cells is the inhibition of protein synthesis. In this report we have determined the effects of ouabain (presumably mediated through alterations in intracellular ion concentrations) on Sindbis virus replication, on host and viral RNA synthesis, and on host protein synthesis in ouabain-sensitive and -resistant cells. Our data indicate that (1) ouabain inhibits Sindbis virus replication in ouabainsensitive (B&d) mosquito cells but to a much lesser extent in ouabain-resistant (Ouas) mosquito cells; (2) in B1C4cells, the sensitivity of viral RNA synthesis to ouabain is greater than that of host RNA synthesis (although the explanation for this observation is uncertain, it may reflect the fact that host RNA synthesis occurs in the nucleus and viral RNA synthesis in the cytoplasm); (3) at concentrations which inhibit viral replication in B1C4cells, ouabain also inhibits host protein synthesis; in Ouas cells, the same concentrations had little effect on either viral replication or host protein synthesis. Other experiments (not shown) have demonstrated that ouabain also inhibits the replication of Sindbis virus in BHK-21 cells. Since hamster cells are inherently more resistant to ouabain than are mosquito cells, as shown by the concentrations nec-
VIRUS
REPLICATION
63
essary to affect the plating efficiencies, it was necessary to use higher concentrations (5 vs 0.1 mM) to inhibit virus replication in BHK-21 cells. As in B&4 cells, the concentrations of ouabain which inhibited virus yields also reduced viral RNA synthesis. Interestingly, however, such concentrations either had no effect on host RNA synthesis or led to a mild (twofold) stimulation. The ability of ouabain to inhibit viral RNA synthesis is dependent on when the drug is added. When ouabain is added to B1C4 cells 1 hr after infection, viral RNA synthesis is totally inhibited, whereas upon later addition (4 hr after infection but well before the peak of viral RNA synthesis) viral RNA synthesis must proceed almost normally since viral yields are only slightly reduced. These data are consistent with the observations of Scheele and Pfefferkorn (1969), who demonstrated in Sindbis virusinfected chick embryo fibroblasts that viral RNA synthesis was blocked by inhibitors of protein synthesis only if the inhibitors (puromycin or cycloheximide) were added at 1 hr after infection. In their experiments, if viral RNA synthesis was allowed to proceed until 3 hr after infection, it was no longer dependent on concomitant protein synthesis. Our data suggest that ouabain also blocks the protein synthesis necessary for the initiation of viral RNA synthesis. The experiments of Scheele and Pfefferkorn (1969) differ from ours, however, in that although viral RNA synthesis continued when cycloheximide or puromycin was added at 3 hr after infection, progeny virus was not produced since the synthesis of viral structural proteins was blocked. In contrast, when ouabain was added to BIG cells 4 hr after infection, viral yields were only slightly reduced, implying that both viral RNA synthesis and the synthesis of viral structural proteins must have continued. One explanation that could account for the inhibition of viral RNA synthesis by ouabain would be that it inhibits the synthesis of viral RNA polymerase from parental (42 S) viral RNA. In that case, the time dependence of the inhibitory effects of ouabain would imply that ouabain-induced alterations of intracellular ion concentrations would have to have different effects on the
64
MENTO
AND
translation of 42 S viral RNA [which codes for nonstructural proteins including the viral RNA polymerase (Strauss and Strauss, 1977)] and 26 S viral RNA [which codes for the viral structural proteins (Strauss and Strauss, 1977)J. Alterations in ionic conditions have been shown to have differential effects on the translation of host mRNA and viral mRNA in cells infected with various cytocidal RNA viruses (Nuss et aZ., 1975; Han&n, 1976; Carrasco et al, 1976). From these data, Carrasco (1977) has suggested that cytocidal viruses may inhibit host protein synthesis by distorting the gradient of monovalent cations across the plasma membrane. The observations of Farnham and Epstein (1963) and of Egberts et al. (1977) describing virus-mediated alterations in the intracellular ionic environment support this idea. Our data on the effects of ouabain in Sindbis virus replication suggest that alterations in the intracellular ionic conditions may also play a role in regulating the translation of different classesof viral RNA. This idea could be tested using in vitro systems. ACKNOWLEDGMENTS
I
This investigation was supported by Grant No. AI
11299 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Cooperative Medical Science Program through Public Health Service Grant No. AI 05920, and by the Institutional National Research Service Award CA-09069 from the National Cancer Institute. REFERENCES
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FARNHAM, A. E., and EPSTEIN, W. (1963). The influence of encephalomyocarditis (EMC) virus infection on potassium transport in L cells. Virology 21, 436-447. GLYNN, I. M. (1964). The action of cardiac glycosides on ion movements. Pharmacol. Rev. 16, 361-407. GREENBERG,J. R. (1969). Synthesis and properties of ribosomal RNA in Drosophila. J. Mol. Biol. 46, 85-98. GUILD, G. M., and STOLLAR, V. (1975). Defective interfering particles of Sindbis virus: III, Intracellular viral RNA species in chicken embryo cultures. Virology 67,24-41. HAMLYN, P. (1976). Cytoplasmic control of protein synthesis. Nature (London) 264,274-277. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ‘H and Y! in polyacrylamide gels by fluorography. Eur. J. B&hem. 66,335-341. LEDBETTER, M. L. S., and LUBIN, M. (1977). Control of protein synthesis in human fibroblasts by intracellular potassium. Exp. Cell. Res. 105, 223-236. LEVER, J. E., and SEEGMILLER,J. E. (1976). Guabainresistant human lymphoblastoid Linesaltered in the (Na+ + K+)-dependent ATPase membrane transport system. J. Cell Physiol. 88.343-352. MANKOVITZ, R., BUCHWALD, M., and BAKER, R. M. (1974). Isolation of ouabain-resistant human diploid fibroblasts. Cell 3,221-226. MAYHEW, E. (1972). Ion transport by ouabain-resistant and sensitive Ehrhch ascites carcinoma cells. J. Cell Physiol. 79,441. MENTO, S. J., and STOLLAR, V. (1978). Isolation and partial characterization of drug-resistant Aedes albopictus cells. Somatic Cell &net., 4, 179-191. Nuss, D. L., OPPERMAN, H., and KOCH, G. (1975). Selective blockage of initiation of host protein synthesis in RNA virus infected cells. Proc. Nat. Acad. Sci. U.S.A. 72, 1258-1262. ROSENBURG,J. (1975). Variant HeLa cells selected for their resistance to ouabain. J. Cell Physiol. 85,135. SCHEELE, C. M., and PFEFFERKORN,E. R. (1969). Inhibition of interjacent ribonucleic acid (26s) sxnthesis in cells infected by Sindbis virus. J. Virol. 4, 117-122. SCHULTZ,S., and CURRAN, P. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50.637-710. SHANK, B. B., and SMITH, N. Ea(1976). Regulation of cellular growth by sodium pump activity. J. Cell Physiol. 87,377-386. SHENK, T. E., KOSHELNYK, K. A., and STOLLAR, V. (1974). Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus. J. Viral. 13,439-447. SHENK, T. E., and STOLLAR, V. (1974). Defective interfering particles of Sindbis virus: I, Isolation and some chemical and biological properties. Virology 53,162-173. SIMMONS, D. T., and STRAUSS, J. H. (1972). Replication of Sindbis virus: I, Relative size and genetic
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content of 26s and 49s RNA. J. Mol. Biol. 71, 599-613. SINGH, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes ae-
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