VIROLOGY
The
11, 434-443
(1960)
Interaction
of Infectious Mammalian
Ribonucleic Cell Line
Acid
with
a
I. Relationship between the Osmotic Pressure of the Medium and the Production of Infectious Centers’ K. A. The
lt’istar
Institute
0.
ELLEM~
AND
J. S.
C~LTE~
of Anatonby/ and Biology, Philadelphia, Bccepted
March
Pennsylvania
18, 1960
A method for the assay of infectious viral ribonucleic acid (RNA) involving the formation of infectious centers in a suspended cell system is described. The efficiency with which RNA infects the cells is a function of the osmotic pressure of the medium in which cells and RNA interact. Maximum efficiency of infection is obtained in solutions of NaCl or of sucrose in physiological saline when the osmotic pressure of the diluent is 4.6-4.1 times that of physiological saline. Infection of cells by intact virus is virtually abolished in solutions of NaCl or sucrose that are optimal for cell-RNA interaction. INTRODUCTION
For quantitative studies of the interaction of an infectious viral ribonucleic acid (RNA) and a mammaliancell, a suspended,single-cell system is preferable to a monolayer, since under these conditions the statistics of discrete distributions can readily be applied and the cell population uniformly exposed to modifications of the environment in a precisely quantitated fashion. Mengo encephalomyelitis virus has been found to yield an RNA that is infectious when injected intracerebrally in mice (Colter et al., 1957), and to lyse Earle’s “L’‘-strain mouse fibroblast cells, a line which grows well on glassor in suspendedcell culture (Siminovitch and Graham, 1956; McLimans et al., 1957). The work described here concerns the effects of the osmolarity of the medium on the interaction of Mengo RNA or Mengo virus and L cells, in a suspended cell system. MATERIALS
AND
METHODS
virus. The virus employed was from two sources: tissue culture, and infected Ehrlich ascites tumor cells. Pools of Mengo
encephalomyelitis
1 Supported by grants from the National Institutes of Health (C-4534 and 2G-142 Cl), the American Cancer Society (E-89B), and the Samuel S. Fels Fund. 2 Travelling Fellow of the New South Wales State Cancer Council. 434
CELL-RNA
INTERACTIOh-.
I
435
t,issue culture virus were prepared by infecting suspended cultures of IA cells, harvesting the fluid after lysis (24-30 hours post infect-ion), and storing in aliquots at -70” C. Pools of infected tumor cells were also st,ored at’ -70”. Infectious RNA. Mengo ICJA was extracted from 10 % (w/v) suspensions of infected tumor cells by the phenol method, as previously described (Colt,er ct nl., 1957). RNA wa,s precipitated at 0” from an aqueous solution by the addition of solid sodium chloride t,o a collc:rnt’ratioii of I .\/. The precipitate was collected by celltrifugation and redissoh%l ill olre-quarter the original volume of 0.14 JI SuCl ill 0.02 Jf potassium phosphate buffer, pH 7.3 (0. L1 ~11 PBS). liolutioils prepared in t.hi.s manner were denignat’ed 1.10-I ILKA%. Aliquots, stowtl at - 70” C, ww stable for several months. Tissrtc cdtuw. Earle’s I,-strain mouse fibroblasts were growi iit suspended cult!ure in Ii:arle’s saline (without calcium and with 10 times the usual concentration of NazHI’04) cont,aining double the usual concent,ration of Eagle’s nutrients, and 10 ‘;L horse serum (2X Kagle’s in Earlr’s medium). For t,ht: preparation of monolayers, cells were collected by cent)rifugnt ioll of the spinner cultures and resuspended t’o a concentration of 10” cells per millilit,er in 2 times Eagle’s in Earle’s medium. Six-milliliter alicluots of the suspension were pip&ed into GO-mm pet,ri dishes, which wtw thcll incubated for 2-3 hours at 37” in an at,mospherr of 5 T; CO?. to permit the cells to set’tlc out, adhere t,o the glass, :ri~tl form mouolayers.” ‘I’itmtir~n of infmtivus &VA. The infectivity of the Mengo 1iNA preparatiolrs was measured by pipettiug 0. l-ml aliquots (in 0.WJI I%(-: or in phosphate-buffered KaC1 solutions of higher molarity) directly oilto t,wire-washed L-cell monolayers, or by incubation with 1, cells in suaprwsiou followed by an examinat,ion of the t’reat.ed cells for infect,ious centers. In the latt~er case, the method employed was as follows. Aliquot~s of a logarithmically growing suspended cell culture containing t)htx desired number of cells were centrifuged at :GO q for 5 minutes ill stoppered, 1%ml centrifuge t’ubes. The cells wcw washed once with 5 ml of Hanks’ balanced salt, solution and recentrifugrd. 1;ollowing a brief temperature equilibmtion at, X7’, t,he cells were suspended in a solution of RNA in the diluent to he tested and placed in a 37” water bath. After 1.5 minut,es’ incubation, during which the t,ubes were agitated manually every few minutes to keep t)he cells uniformly suspe~~ded, t>he wll-RN.\
436
ELLEM
AND
COLTER
mixture was diluted to 10 ml with 2X Eagle’s in Earle’s medium. The cells in this mixture were then titrated for infectious centers. With the higher concentrations of RNA, it was necessary to further dilute the mixtures before titration to reduce the plaque count to a readable number. When possible, the dilutions were adjusted to give 50-90 plaques per plate. Titration of infectious centers. Two 4.5-ml aliquots of each mixture were pipetted onto monolayers, which were incubated for 1 hour at 37” in an atmosphere of 5 % CO2 to permit the test cells to sediment and adhere to the indicator layer. The medium was then carefully removed and the monolayers covered with 4.5 ml of an agar overlay. The overlay consisted of 1% agar in Hanks’ balanced salt solution containing 0.1% yeast extract, 0.17 % bovine plasma albumin, 0.26 % sodium bicarbonate, 1:40,000 neutral red, double the usual concentration of Eagle’s nutrients, and penicillin and streptomycin. Incubation at 37” in an atmosphere of 5 % CO2 was continued. Plaques were counted on the second and third days. Osmotic pressure. The relative osmotic pressures of the saline and sucrose solutions employed were calculated from the expression, osmotic pressure = nm+ (RTWJlOOO V,), where n = number of moles of ions formed from 1 mole of electrolyte, m = moles of solute per kilogram of solvent (molality), and 4 = the osmotic coefficient. Since the term in parentheses is constant for both the NaCl and sucrose series, the value of nm4 was calculated for each of the KaCl and sucrose solutions, and expressed as a multiple of the nm$ value for 0.14 M PBS, which was given the arbitrary value of 1. The molalities were calculated using the specific gravity data from the International Critical Tables, and the osmotic coefficients were obtained from Robinson and Stokes (1955). RESULTS
Assay of Mengo RNA on Monolayers It was found that RNA solutions in 0.14 M PBS pipetted directly onto twice-washed monolayers produced plaques irregularly, and that the infectivity fell off rapidly and in a nonlinear fashion when the RNA was further diluted with 0.14 M PBS. When the RNA solutions applied to the monolayers were made 0.8 M with respect to NaCl, there was a marked increase in the number of plaques formed, and the number of plaques became inversely proportional to the dilution of RNA. Similar results have been reported previously for enterovirus RNA (Alexander et al., 1958; Holland et al., 1959). However, the L-cell monolayers were damaged by exposure to the hypertonic saline. Cell degeneration re-
CELL-RKA
INTEItAC!TIOS.
sulted in areas of poor plaque definition, tion in the estimated infectivity. Assay
qf Mengo
RNA
by Production
437
I
and caused considerable
of Infectious
vnria-
Centws
The technique of assaying Mengo RiYA by the production of infectious centers in a suspended cell system appears to have certain advantages over previously described met)hods. By avoiding exposure of t*he monolayer to the hypertonic solution necessary for efficient infection of t hc cells, t)he cellular degeneration accompanying such a manipulation is circumvented. As a result, t,he plaques are sharply defined and readily coumed. Furthermore, by flooding t)he indicator layer wit>ha suspension of infect,ed cells, a uniform dist,ribution of plaques is obtained. T’alidity of the q/stern. The pertinent charact,rristics of this m&hod of titrating infectious centers are listed below. 1. .I linear relationship exists between the number of plaques formed and the dilution of the cell suspension. Cells were incubated with a high concentration of R?;A. Dilutions of the treated cell suspension were made, and aliquots of these dilutions assayed for infectious cent,ers. Over the range 1)--100plaques per plate there was a st)rict, linear relat)ionship between plaque count and dilution of the cells. There was somedeparture from lirrearity when counts were higher than 100 per plate, due to ~OIIflueiw of plaques. 2. Dilution of the RKA-cell mixture with culture medium prevents any further cell-ICiA int,eraction. Also, R?;A in the absence of wlls, diluted wit.11 medium and pipetted onto monolayers, produced no plaques. That is, t*here is no carry-over of infectious RSX urlits to the monolayer, other than those which become stabilized by intcract.ion wit)h cells. 3. Incubation for 1 hour is adequate for the attachment of infect,ed rclls to the indicator monolayer. When fluids were carefully removed aft,er 1 hour and placed on fresh monolayers, no plaques were formed. 4. The tot’s1 number of cells put on the monolayer does not affect, the numhw of plaques formed from a standard number of infected cells. :Iddit)ion of increasing numbers of uninfected cells, up t)o a maximum of IO6 cells, produced no variation in the plaque counts greater than that expected on t,he basis of chance. Itfleet of the osmolarity of the diluent on RNA -I, ccl1 intrraction. The cffccts of hypert,onicit,y of the medium on ItS A-ccl1 int,rractSion in the suspendedcell system were examined. Representative data are shown in Table I. L4sthe molarit)y of the XaCl was increased from physiological,
438
ELLEM
AND
TABLE RELATIONSHIP INFECTIOUS
BETWEEN CENTERS
Molarity of sodium chloride in RNA diluent” 0.14 0.45 0.52 0.57 0.62 0.67 0.72 0.77 0.87
COLTER
1
SOLUTE CONCENTRATION FROM MENGO RIBONUCLEIC
Total number of infectious centersC 13 51 224 254 250 270 153 104 23
D Diluent also contained 0.02 b Diluent also contained 0.14 phate buffer, pH 7.3. i Developed by lo8 washed L of Mengo RNA in the indicated
AND ESTABLISHMENT ACID PREPARATIONS
Molarity of sucrose in RNA diluenP 0.0 0.4 0.6 0.7 0.8 0.9 1.0 1.2
-
OF
Total number of infectious centersc 13 191 317 381 237 107 45 29 -
M potassium phosphate buffer, pH 7.3. M sodium chloride and 0.02 M potassium
phos-
cells suspended in 1.0 ml of a 5 X 10m2 dilution diluent, after 15 minutes at 37”.
in 0.02 M phosphate buffer, there was a progressive increase in the number of infectious centers formed in a given volume of Mengo RNA, up to a maximum at 0.64 M NaCl. At molarities greater than 0.64, the number of infectious centers formed dropped sharply. To test whether the hypertonic sodium chloride solutions could be producing these effects primarily by dehydrating the cells, rather than by affecting the extracellular RNA, the effects of sucrose on the cellRNA interaction were examined. Cells and RNA were incubated in solutions of sucrose in 0.14 M PBS. The results of one such experiment are tabulated in Table 1. With increasing concentrations of sucrose in 0.14 M PBS, the number of infectious centers produced increased to a maximum at 0.7 M sucrose. At higher sucrose concentrations the number of infectious centers dropped precipitously. These data are presented in a somewhat different manner in Fig. 1. Here the number of infectious centers produced in L cell-RNA mixtures is plotted against the relative osmotic pressuresof the diluents in which cells and RNA were incubated. It can be seen that for both the sodium chloride and sucroseseries,the maximum number of infectious centers is formed when the osmotic pressure of the medium is 4.0-4.1 times that of physiological saline. The total number of plaque-forming units estab-
CELL-RNA
IXTERACTIOS.
I
I
0
I.0
2.0
RELATIVE
3.0 OSMOTIC
4.0
5.0
6.0
PRESSURE
70
80
OF DILUENT
FIG. 1. Relationship bet)ween the formation of infectious cent,ers in I, cellMengo RNA mixtures and t,he osmotic pressure of the diluent; 1Ofi rells incubated in 2.5 X 10F RNA for 15 minutes at 37” C. 0, Plaques produced by rells incubated with RNA in sucrose-PBS media; X, A, plaques produced 1)~ cells inruhated u-it h RNA in phosphate-buffered sodium chloride media.
lished in t’he optimal sucrose medium exceeds the number formed in t.he optimal sodium chloride solution by a f&or which has ranged, in an extensive series of experiments, from 1.5 to 2. Influence of sodium chloride and sucrose on dfengo virus-l cell interaction. It was of interest to see if intact Mengo virus behaved differently from its naked RNA in solutions of high osmolarity. The system used was the same as that used for the RSA, except that, after incubation of t,he cells with a standard virus suspension in the various media, the mixtures were diluted with 2X Eagle’s in Earle’s medium containing s&icient anti-Mengo gamma globulin4 to inactivate unabsorbed virus. The results are shown in Table 2. Any increase in XaCI concentration above 1 The Barbara
solution of anti-Mengo Hrownstein, The Wistar
gamma Institute,
globulin was Philadelphia,
kindly supplied l’ennsylvania.
by
Mrs.
440
ELLEM
AND
COLTER
TABLE INFLUENCE
OF SOLUTE
CONCENTRATION
Diluent of virus 0.14 0.37 0.47 0.57
M PBS” M PBS M PBS M PBS
0.0 M Sucroseb 0.3 M Sucrose 0.5 M Sucrose 0.7 M Sucrose
2
ON MENGO
VIRUS-L
Total infectious centersC
CELL
INTERACTION
70 Recovery
522 0 0 0
100 20.2 <0.2 <0.2
522 114 75 13
100 21.8 14.4 2.5
QXM PBS = XM NaCl in 0.02 M potassium phosphate buffer, pH 7.3 (PB). * XM sucrose = XM sucrose in 0.14 M NaCl in 0.02 M PB. c Obtained from 10” washed cells suspended in 1.0 ml of the virus preparation and exposed for 15 minutes at 37°C.
physiological virtually abolished cell-virus interaction, Although the effect of sucrosewas somewhat lessdramatic, increasing concentrations of this solute in the medium in which cells and virus were incubated sharply inhibited the formation of infectious centers. One may suspect that high electrolyte concentrations delay or prevent the initial virus-cell interaction, which has been shown in a number of cell-virus systems to be of an electrostatic nature (Puck et al., 1951; Bachtold et al., 1957; Levine and Sagik, 1956; Ishida and Ackermann, 1956). However, the sizable inhibition produced by sucrose, a nonelectrolyte, cannot so readily be explained on the basisof such a hypothesis. In an effort to determine whether adsorption or penetration of virus was affected, the following experiment was performed. Aliquots of L cells (containing lo6 cells) were incubated for 15 minutes at 37” with l-ml volumes of a standard virus preparation in 0.14 M PBS, 0.64 M PBS, and 0.7 M sucrosein 0.14 M PBS. Four sampleswere used for each of the three diluents. After incubation, the mixtures were centrifuged, the supernatants removed, and the amount of virus therein estimated by titration on L cell monolayers. Two cell samples from each group were suspendedin 2 X Eagle’s in Earle’s medium, and the remaining two sampleswere resuspendedin the same medium, containing antiMengo gamma globulin to inactivate virus which had attached to, but not yet penetrated, the cells. The number of infect’ious centers in the cell suspensions was measured by the usual method.
CELL-RNA
INTER.4CTIOS.
TABLE INTERA(*TION
OF MENGO
VIRI.S
AND
0.7
0.14
nf
I’BSb
Sl-CR~SE/l’~~‘L
13, 180 (!N .5) * 12,080 (x7.1)*
12.300 (66.6)* 65 (0.40)* 1,740 (12.6,*
6,180
0.7
M NW 11 Sll<‘/l’BS”
441
3
CELIS
ii/l
(33.4*
0.64
L
1
IN 0.11
1x,4x0
13.245 13,X40
M
I’M,
0.64
:\I PBS,
ANI)
4,x90
(26.4)* 2.x (0.0003 ) * 1111 (3 I*
39.7 3 .3 21.1
” I’ercentjage of total plaque-forming units recovered is indicated by askrisk. h 0.11 Molar sodium chloride-O.02 molar phosphate lmfier, pH 7.3. C 0.64 Molar sodium chloride-O.02 molar phosphate h~~ffrr, p1I 7.3. d 0.7 Molar sucrose in 0.14 M PBS. P Percentage of plaque-forming units adsorbed by ~11s which are not. wllnrrable to antiserum: (Value in rolumn V)/(V:tlue in column III) X 100.
The data are shown in Table 3. It is evident, t,hat’ the concenhrations of KaCl and sucrose which are optimal for establishing infectious centers from RSA preparations result, in almost completje inhibition of virus adsorpt’ion in the former, and in marked inhibition in the latter. I’enetration of adsorbed virus appears to he inhihit,ed somewhat by sucrose. In the case of SuCl (0.64 M), tbe fact that very few plaque-forming units are fixed by the cells makes it dificult~ to assess the significance of the apparent inhibition of penetration. Incubation of Mengo virus alone in 0.64 31 PBS or in 0.7 ,I1 sucrose in 0.11 M 1’BS produced a decrease of approximately 30 7: in the activity of the preparation as compared to t,hat of control samples incubated in 0.14 M PBS. This accounts for t,he lower recovery of t,ot:tl plaque-forming unit,s in t,hese two series. I )ISCl!SSI(
)S
The data suggest that t,he mode of action of media of high osmolarity is on the cell rather than on the RNA. Although high molecular weight RNA is insoluble in 1 M SaCl at 0” C (Colter and Brown, 1956), it is completely soluble in this solution at 37”. RNA is soluble in solutions of sucrose as concent’rated as 1.2 41. Hence, under the conditions of t,he reaction of cells and RKA, insolubility of RKA in the suspending medium can be excluded as part of t’he mechanism of the increased efficiency of
442
ELLEM
AND
COLTER
establishing infection. Preliminary studies of the uptake of P32-labeled RNA by L cells indicate that the cells do not absorb more RNA in the optimal sucrose or NaCl solutions than they do in 0.14 M PBS. The stability of RNA at 37” is essentially the same in 0.14 M PBS, 0.64 M PBS, and 0.7 d1 sucrose in 0.14 M PBS. These observations strongly suggest that the hypertonic saline and sucrose solutions act by modifying the cell in some way, thereby increasing its receptivity or stabilizing capacity for the infectious RNA. Cellular ribonucleases may be inhibited by the high intracellular electrolyte concentration produced by dehydration. Crystalline ribonuclease has been shown to be inhibited by solutions of high ionic strength (Kalnitsky et al., 1959). Another possible mechanism is suggested by the demonstration in this laboratory that the infectivity of Mengo RNA is abolished or sharply inhibited by several ribonuclease-free proteins when nucleic acid and protein are mixed in solutions of physiological ionic strength. At high ionic strengths, inhibition does not occur. It is possible that a hypertonic intracellular environment prevents nonspecific immobilization of the viral RNA by cytoplasmic proteins. ACKNOWLEDGMENT The edged.
skilled
technical
assistance
of Mrs.
Jeanne
M.
Kuhn
is gratefully
acknowl-
REFERENCES ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., and VAN DAMME, 0. (1958). Infectivity of ribonucleic acid from poliovirus in human cell monolayers. J. Exptl. Med. 108, 493-506. BACHTOLD, J. G., BUBEL, H. C., and GEBHARDT, L. P. (1957). The primary interaction of poliomyelitis virus with host cells of tissue culture origin. Virology 4, 582-589. COLTER, J. S., and BROWN, R. A. (1956). Preparation of nucleic acids from Ehrlich ascites tumor cells. Science 134, 1077-1078. COLTER, J. S., BIRD, H. H., MOYER, A. W., and BROWN, R. A. (1957). Infectivity of ribonucleic acid isolated from virus-infected tissues. Virology 4, 522-532. HOLLAND, J. J., MCLAREN, L. C., and SYVERTON, J. T. (1959). The mammalian cell-virus relationship. IV. Infection of naturally insusceptible cells with enterovirus ribonucleic acid. J. Exptl. Med. 110, 6580. ISHIDA, N., and ACKERMANN, W. W. (1956). Growth characteristics of influenza virus. Properties of the initial cell-virus complex. J. Exptl. Med. 104, 501-515. KALNITSKY, G., HUMMEL, J. P., and DIERKS, C. (1959). Some factors which affect the enzymatic digestion of ribonucleic acid. J. Biol. Chem. 234, 1512-1516. LEVINE, S., and SAGIK, B. P. (1956). The interactions of Newcastle disease virus (NDV) with chick embryo tissue culture cells: attachment and growth. Virology 2. 57-68.
W. F., DAWS, E. V., GLOVER, F. I,., and HAKE, G. W. (1957). The submerged culture of mammalian cells: the spinner culture. J. In~munol. ‘79. 428-433. PITCK, T. T., (;OREN, A., and CLIXE, J. (1951). The mechanism of virus :tttacl~ment t,o host cells. I. The role of ions in the primary reaction. J. Expfl. Med. 93, 65-88. I~OBISHON, I<. A., and STOKES, It. H. (1955). Electrolyte Solutiom; The Measrcrc-
MCLIMANB,
went
ad
Interpretation
of Conductance,
Ch.eruical
Potential
and
DiJusion
iu
Sol~tio,as of Simple Electrolytes. Academic Press, New York. Sra~r~'ovr~c:t!, I,., and GRAHAM, A. F. (1956). Significance of ribonucleic acid and deos~ri~)otlrlcleic acid turnover studies. J. Histochem. rind C’ytochern. 4, 50X-515.