The interaction of infectious ribonucleic acids with mammalian cells

VIROLOGY

16,

113-126 (1961)

The interaction of Infectious Acids with Mammalian III. Comparison Cell-Polio

of Infection and RNA Uptake in the HeLa RNA and L Cell-Mengo RNA Systems’

K. -4. 0. ELLEM” The Wistar

Institute

Ribonucleic Cells

of Anatomy Accepted

AND

J. s. COLTER

and Biology,

Philadelphia,

Pennsylvania

June 12, 1961

Studies concerning the optimal conditions for and the kinetics of the interaction of poliovirus (strain Mahoney) RNA and HeLa cells in the previously described suspended cell system are described. The maximum number of infectious centers are produced when cell-RNA mixtures are incubated for 8 minutes at 37” in 0.9 M sodium chloride-O.02 M phosphate buffer, pH 7.3. Sucrose solutions are much less efficient than saline solutions in promoting infectious center formation. However the optimal sucrose and saline solutions have identical osmotic pressures : 5.7 times that of physiological saline. The number of infectious centers produced in polio RNA-HeLa cell mixtures is directly proportional to the concentration of RNA and to the total number of cells employed. The reaction is strongly temperature dependent, with an apparent activation energy of 34,000 Cal/mole over the temperature range 6-37”. The sharp decrease in the number of infectious centers recovered when Mengo RSA-L cell and polio RNA-HeLa cell mixtures are incubated (at 37”) in their optimal sucrose and saline media for longer than 15 and 8 minutes, respectively, has been shown to be due to a sharp decline in the viability of the cells at about these times. The relationship was established by an examination of the survival of viral-induced infectious centers after incubation in solutions of elevated osmolarity. The uptake of RNA-P% by L and HeLa cells was investigated. The study revealed that, under the conditions used for the assay of viral RNA, the cells take up a minute percentage of the total RNA present. The results have been used to develop the premise that viral RN-4 preparations may contain at least as many infectious units as do the corresponding viral suspensions. The similarities and differences between the Mengo RNA-L cell and polio RNAHeLa cell systems are discussed, with particular emphasis on the different responses of the two cell types to ionic and nonionic hypertonic environments. Data regarding the thermal stability of Mengo RNA are presented. INTRODUCTION communica’tions (Ellem 1960a, b), a method was de-

In two previous

and Colter,

1The work reported by grants from the (E-89C), the Atomic (30-l) 25661, Training United States Public Samuel S. Fels Fund. ’ Travelling Fellow

here was supported in part American Cancer Society Energy Commission [A T Grant No. 2G-142 from the Health Service, and the of the Kew South Wales

scribed permitting the quantitative estimation of the number of infectious centers formed in a suspension of L cells exposed to Mengo ribonucleic acid (RNA). The effect of the osmolarity of the medium on the establishment of infectious centers, and the kinetics of the cell-RNA interaction, were Cancer Council; Wistar Institute trainee. Present Address : Department of Bacteriology, University of Sydney, N. S. W., Australia. 113

ELLEM

114

AND COLTER

documented. The results of similar studies of the poliovirus RNA-HeLa cell system are presented in this paper. In addition, data are presented which shed additional light on the mechanism whereby solutions of high osmolarity stimulate the infection of cells by viral RNA, and which provide a rational explanation for the apparent low infectivity of such preparations. MATERIALS

AND METHODS

Virus. The virulent Mahoney strain of type 1 poliovirus was obtained from Dr. H. Koprowski of the Wistar Institute. Viral pools were made by superinfecting HeLa cells in suspension and incubating them at a concentration of IO6 cells per milliliter for an additional 24 hours. At the end of this time, cell debris was removed by centrifugation and the supernatant was either stored at -60” or used for the isolation of RNA. Preparation of infectious polio RNA. Versene and sodium deoxycholate were added to the virus pool to give final concentrations of 1O-3 1M and 0.5%, respectively. Extractions with HzO-saturated phenol were then performed in the usual manner. No loss of activity was noted when these preparations were stored at -60” for many months. Culture of HeLa celZs. HeLa cells were grown in suspended cell culture in Earle’s saline (without calcium and with ten times the usual concentration of Na2HP04) containing double the usual concentration of Eagle’s nutrients, and 10% calf serum. For the preparation of monolayers, 60-mm petri dishes were seeded with 2 to 3 x lo6 HeLa cells (harvested from the spinner cultures and suspended in 2 x Eagle’s in Earle’s medium) 24-48 hours before use. Titration of Mahoney RNA. The titration of polio RNA was performed by the technique described for the titration of Mengo RNA (Ellem and Colter, 1960a), except that HeLa cells replaced L cells as the target cells and that HeLa monolayers were employed for the estimation of the number of infectious centers produced. Preparation of P3Vabeled RNA. Random-bred Swiss mice, bearing the Ehrlich ascites carcinoma, were injected intraperitoneahy on each of days 5, 6, and 7 of tu-

mor growth with approximately 50 PC of Ps2 (as orthophosphate j . Twenty-four hours after the third injection, the cells were collected and RNA was isolated from them by the phenol method (in the presence of Versene and deoxycholate at the concentrationas indicated above). The high molecular weight fraction was separated by precipitation at 0” from 1 M NaCl and was washed repeatedly in ice-cold 1 M NaCl to free it from traces of low molecular weight RNA and other phosphorus-containing material before being dissolved in phosphatebuffered physiological saline, pH 7.3, containing 1O-3 M Versene. The solution was stored, in aliquots, at -60” until used. RESULTS

The criteria which were used to establish the validity of this method of assay for the Mengo RNA-L cell system (Ellem and Colter, 1960a) were shown to hold true for the polio RNA-HeLa cell system as well. Effect of the Osmolarity of the Diluent RhTA-HeLa Cell Interaction

on

The relationship between the formation of infectious centers and the osmolarity of the medium in which HeLa cells and polio RNA were incubated is illustrated in Fig. 1. The curves were constructed from data obtained from four separate experiments in which the formation of infectious centers in solutions of phosphate-buffered NaCl (X M PBS) and in solutions of sucrose in phosphate-buffered physiological saline (X M &c/PBS) was measured. As was the case with the Mengo RNA4-L cell system, the number of infectious centers formed in both series increased with increasing tonicity up to a maximum, beyond which there was a rapid decrease in the number of infectious centers present. In either saline or sucrose, the maximum was found in the solution having an osmotic pressure 5.6-5.8 times that of physiological saline-that is, in 0.9 M PBS and in 0.98 M Sue/PBS. Figure 1 is constructed so that. the saline and sucrose curves are nearly coincident-to emphasize the closeness of their optima. This should not obscure the fact that, in contradistinction to the findings with the Mengo RNA-L cell system,

INFECTIOUS

20

MENGO

40

RELF)TIVE OSMOTIC

BND

6,O

POLIO

8,O

115

RNA

IO,0

PRESSURE OF DILUENT

FIG. 1. The effect of the osmotic pressure of the diluent (expressed as a multiple of the osmot,ic pressure of physiological saline) on the formation of infectious centers in polio RNg-HeLa cell mixtures. Incubation was at 37” for 8 minutes. l , Infectious centers formed in solutions of phosphate-buffered (0.02 Jf) NaCl solutions. 0, Infect,ious centers formed in solutions of sucrose in phosphate-buffered physiological saline.

the infectivity in saline was much higher than that in sucrose. At the optimal osmotic pressure there were 7.1 times as many infectious centers formed in saline as in sucrose. For this reason 0.90 M PBS was used as the diluent for all subsequent studies of HeLa cell-polio RNA interaction, Effect of Time of Incubation on the Formation of Infectious Centers The relationship between the number of infectious centers in HeLa cell-polio RNA mixtures and the time of incubation is shown graphically in Fig. 2. In both the opt’imal saline and sucrose diluents, the number increased rapidly to a maximum followed by a rapid decline when the incubation was cont,inued. In each series the maximum was reached with a shorter incubation period than was the case with the Mengo-RNA-L cell system,

The results of a more precise determination of the time at which the maximum number of infectious centers are present are shown in Fig. 3. It is apparent that t’he rate of formation of infectious centers is linear with respect to time for 8 and 6 minutes in the optimal saline and sucrose solutions, respectively. It will be noticed that, in both diluents, the intersection with the ordinate of the regression lines (fitted by the method of least squares) is nonzero. At zero minutes of incubation in the saline and sucrose series, 6.5 and 18.5:%, respectively, of the maximum number of infectious centers formed in these dilucnts were theoretically present. The saline nonzero baseline of infectivity is not significantly different from zero C,t= 0.22; 0.9 > p > 0.8), but the sucrose non-zero ordinate differs significantly from zero CIt = 9.10; p < 0.001).

116

ELLEM

AND

COLTER

Effect of Total Cell Number As was the case with L cells exposed to Mengo RNA, the number of infectious centers formed in a fixed volume of polio RNA solution was found to be directly proportional to the number of HeLa cells employed. This fact is illustrated by Fig. 4. Effect of Temperature on the Formation Infectious Centers

b lb -lNCUBRTlON

2b

j, TIME

4b 5b IN MINUTESp

$0

FIG. 2. Effect of time of incubation at 37” on the number of infectious centers in HeLa cell-polio RNA mixtures. l , Incubations in 0.90 M PBS. 0, Incubations in 0.98 M Sue/PBS.

of

The effect of temperature on the rate of formation of infectious centers in polio RNA-HeLa cell mixtures was examined. As with the Mengo RNA-L cell system, the process was found to be markedly temperature dependent. An “Arrhenius plot” of the data obtained from two separate experiments is shown in Fig. 5. The temperature response of the rate of the reaction appeared to be a continuous function between 6” and 37”. No discontinuity in the regression line, as was found at 24” in the Mengo R,NA-L cell system, was seen. From t’he slope of t,his line, fitted to all the data between 6” and 37”, the apparent activation energy was calculated to be 34,300 cal/mole. There appears to be a sharp maximum at 37”, above which there is a rapid decrease in the rat’e of the reaction. Stability

of Infectious

Viral

RNA

Early in these investigations it was found that infectious RNA was extremely

FIG. 3. Rate of formation of infectious centers in HeLa cell-RNA mixtures incubated at 37” in 0.90 M PBS ( o ) and in 0.98 III Sue/PBS (0).

Effect of Concentration

of

RNA

Koch, et al. (1960) demonstrated that the number of plaques produced by Mahoney polio RNA in monolayers of susceptible cells was directly proportional to the concentration of the nucleic acid. A direct proportionality was shown to exist between the concentration of polio RNA and the number of infectious centers produced in the suspended cell system used in these investigations.

FIG. 4. Proportionality between infectious centers formed in 1 ml and the total number of HeLa Incubation was for 8 minutes at PBS.

the number of of polio RNA cells employed. 37” in 0.90 M

INFECTIOUS

MENGO

labile to thermal inactivation (Colter and Ellem, 1960). Estimates of the half-life of Mengo RNA at 37” were in the range 12-30 minutes, with no consistent differences noted between RNA incubated in 0.14M PBS, 0.64&f PBS and 0.70M SW/PBS. On the hypothesis that the inactivation was caused by heavy-metal contamination of the preparations, Versene was added (final concentration = lo-” M) and was found to effect a marked stabilization of the RNA. In Fig. 6, the exponential decay of the infectivity of Mengo RNA at 37” in the presence of 109” M Versene is illustrated. Versene was added to the solution employed in the incubation of t’he RNA with cells as well as to those used in the isolation of the nucleic acid. The halflife of this preparation was found to be 23.01 hours, with a 95% confidence interval of 20.88-25.60 hours. It was felt that the most likely reason for the sharp optima seen in the curves relating infectious center format’ion to time

’! 7.0

2L

-

6.0 -

AND

POLIO

0

RNA

20

117

40

60

00

IO0

I20

HOURS FIG. 6. Thermal in 0.70 M SW/PBS sene at 37” (half-life

inactivation of Mengo RNA in the presence of 10m3M Ver= 23 hours).

of incubation for both the Mengo RNA-L cell and polio RNA-HeLa cell systems was a rapid loss of cell viability at about the time at which the optima occurred. This premise was examined in two ways : (1) by measuring the ability of cells to concentrate the vital stain, neutral red, aft’er incubation in solutions of high osmolarity, and (2) by estimating the number of L and HeLa cells, infected with Mengo and poliovirus, respectively, which retain the capacity to support viral replication after incubation in solutions of elevated osmot’ic pressure. The latter is perhaps a more appropriate measure of cell viability from the point of view of the present investigation. Concentration

Uptake

of

Neutral

Red by

L and HeLa Cells

32 -RECIPROCRL

33 3.4 OF ADZOLUTE

35 TEMPERATURE

36 (X103)

FIG. 5. Arrhenius plot relating the rate of formation of infectious centers in HeLa cell-polio RNA mixtures (expressed as logarithms to the base e) and the reciprocal of the absolute temperature. Incubations were carried out in the optimal saline solution.

Aliquots (usually 2 x 106) of washed L and HeLa cells were suspended in l-ml volumes of physiological saline or of the optimal saline and sucrose solutions. These solutions were 0.64 M PBS and 0.7 M Sue/ PBS in the case of the L cells, and 0.90 ,U PBS and 0.98 M Sue/PBS in the case of HeLa cells. After incubation at 37” for varying periods of time, the suspensions were diluted to 10 ml with 2~ Eagle’s in Earle’s medium containing neutral red at

118

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AND

a concentration of 1: 10,000. After an additional incubation of 5 minutes at 37”, the number of cells stained with the dye were counted. The results of several experiments are illustrated in Figs. 7 and 8. There was considerable variation among experiments, but several facts emerged clearly. L cells, incubated in 0.642M PBS, very rapidly lost their ability to concen-

COLTER

trate neutral red. In all but one experiment (Fig. 7) the loss of viable cells after 15 minutes’ exposure to this diluent was of the order of 95%. In sharp contrast, only 5-10% of the cells incubated in 0.7iM Sue/PBS for 15 minutes had lost their viability, based on this criterion. The picture obtained with the HeLa cells (Fig. 8) was similar in principle, but different in one important detail. Although the percentage of viable cells in the population incubated for 40 minutes in 0.90 M PBS was much lower (approximately 50%) than t,he percentage which remained viable after incubation in 0.98 M Sue/PBS (8590%)) there was virtually no difference in the viability of HeLa cells in saline and sucrose after 8-10 minutes. It is only after more prolonged incubation that the lethal effects of the saline become evident. Ability of Cells to Support Viral Replication after Incubation in Solutions of High Osmolarity

FIG. 7. The effect of incubation at 37” hf PBS ( l ), 0.7 M &c/PBS CO), and PBS (0) on L-cell viability, as measured ability of the cells to concentrate neutral tracellularly.

0 -TIME

IO

20

OF EXPOSURE

30 IN

in 0.64 0.14 M by the red in-

40

MINUTES-

FIG. 8. The effect of incubation at 37” in 0.90 M PBS (0) and 0.98 M Sue/PBS ( 0 ) on HeLa cell viability, as measured by the ability of the cells to concentrate neutral red intracellularly.

An appropriate number of L or HeLa cells, harvested in the logarithmic phase of growth from suspension cultures, were infected with Mengo virus or Mahoney poliovirus by exposing them to virus for 30 minutes at 37” at a multiplicity of 5:l (virus: cell). The cells were then centrifuged and washed with 2~ Eagle’s in Earle’s medium containing 10% horse serum to remove unabsorbed virus. They were then resuspended in 0.14 M PBS, distributed into aliquots of lo6 cells, and recentrifuged. The cell pellets were suspended in l-ml volumes of the optimal sucrose or saline solutions and incubated for varying periods of time at 37”; they were then diluted to 10 ml with tissue culture medium. The number of infectious centers in the treated suspensions were estimated by titration on monolayers of L or HeLa cells for Mengo virus and poliovirus, respectively. The results are summarized in Figs. 9 and 10. Figure 9 records the rapid loss of viability of L-cell infectious centers after 10-20 minutes in t.he optimal sucrose solution (0.7 M Sue/PBS) and illustrates the much greater lethal effects of the 0.64 M PBS, which produced an almost immediate, linear fall in viability from time zero. In these experi-

INFECTIOUS

MENGO

mcnts, the ratio, after 15 minutes, of viable cells remaining in sucrose: viable cells remaining in saline was 1.4-1.8. Considering only the optimal sucrose solution, there appears to be a reasonable correlation bet\veen the cells’ loss of ability to support viral replication and the sudden drop in the number of infectious centers formed in ,Ilengo RNA-L cell mixtures (see broken curve in Fig. 9). Data obtained from similar studies lvith poliovirus-infected HeLa cells again point out the difference between HeLa and L cells in their response to solutions of high osmolarity. It is clear from Fig. 10 that the effects of the optimal sucrose and saline solutions on the poliovirus-infected HeLa cell are virtually identical. In the experiment illustrated by t’his figure, only about 60% of the original infectious centers remained functional after 8 minutes incubation at 37” in either solution. LG-ect Measurement Cells

of RXA

Cptake

by

Direct measurements lvere made of the amount of RNA taken up by L and HeLa cells using P”‘-labeled RNA (RNA-P3”) prepared as described in hlaterials and Jlethods. Pellets containing 1Oi lvashed cells were suspended in 0.5 ml RNA-P3” dissolved in 0.14 M PBS, 0.64 M PBS or 0.70 M Sue/PBS (L cells), or in 0.14 fi1 PBS, 0.90 M PBS, or 0.98 M Sue/PBS (HeLa cells). The suspensions were incubated at 37” for varying periods of time after Ivhich they vvere diluted to 10 ml \vit,h 2X Eagle’s in Earle’s medium containing 10% horse serum. The cells were sedimented l)y centrifugation, washed once in 5 ml of tissue culture medium, and then dissolved in 0.5 ml of 1 X NaOH solution. The solutions mere transferred quantitatively to planchets and dried for counting. Aliquots of the original RNA-P32 solution were also clried in planchets to which had been added 0.5 ml volumes of 1 N NaOH to equalize the amount of inactive, radiation-absorbing material. Counting was done in a tracerlab gas-flow counter Lvith an automatic sample changer. All experimental samples were run in duplicate, and all counts Jvere made to at least 4000. Counts Ivere made in tripli-

AND POLIO RNA

0 TIME

IO 20 30 OF EXPOSURE LN MINUTES

FIG. 9. The effect of incubation at 37” in 0.70 M &K/PBS ( l ) and 0.64 M PBS (0) on the capacity of L ceils to support virus (Mengo) replication. The broken curve illustrates the relationship between the formation of infectious centers in Mengo RNB-L cell mixtures (in 0.7 M Sucj PBS) and the time of incubation.

0

10

20

30

MINUTES

FIG. 10. The effect of incubation at 37” in 0.98 M Sue/PBS (0) and 0.90 M PBS (0) on the capacity of HeLa cells to support virus (polio) replication.

cate and the mean values taken as the actual count. Preliminary experiments demonstrated that the amount of RNA-P32 taken up by

120

ELLEM

UPTAKE

Expt.

OF

TABLE 1 RNA-Pa2 BY

1 i

AND COLTER

I, CELLS”

Percentage of the total

RNA-Pz2 taken up by the cells in

IlO.

RNA dissolved i

16 IV

0.14 M PBS 0.14 M PBS

0.01 0.046) 0.107 0.183 0.0: 0.091 0.135 0.240

0.70 M Sue/ PBS

0.0 0.119 0.1451 0.198 I 0.0, 0.079; 0.147 0.243

I II

0.70 M

SW/

PBS I II

0.64 M PBS 0.64 M PBS

0.0 0.416 0.819 1.692 0.0 0.318 0.771 1.060

a Aliquots of 107L cells were incubated at 37°C in 0.5-ml volumes of the RrJA solutions. b The 0.5.ml volumes of the RNA-P%” solutions used in Experiments I and II contained 45,800 and 82,230 cpm, respectively.

the cells was directly proportional to the number of cells employed. They also showed that, owing to some manipulative inadequacies, a high blank value (2-3 times background in magnitude) was present. This was not due to rapid binding of RNA by the cells, since it was shown to be independent of the number of cells used and, in fact, to be just as high in tubes manipulated in the absence of cells. In the light of this observation, zero-time blanks were included in all subsequent studies, and all values cited in the following paragraphs have been corrected by deducting this value. The results of two uptake experiments with L cells are summarized in Table 1. Perhaps the most significant observation is that extremely small quantities of RNA are absorbed. After 15 minutes incubation, between 0.05 and 0.123 of the total amount of RNA-P32 had been absorbed by the cells from the 0.14 M PBS and 0.70 M Sue/PBS solutions, and between 0.32 and 0.42% of the total from the 0.64 M PBS. No significant difference existed between the rates of uptake in the 0.14 iK PBS and 0.70 M SW/ Pl3S media. The resuIts of a third uptake experiment

art’ illustrated in Fig. 11. The uptake of RXA-Ps2 in all media is linear with respect to time from the beginning of the incubation, but the most. striking feature is the difference in rates of uptake between cells in 0.64 M PBS and those in 0.14 M PBS or 0.70 M Sue/PBS. From the slopes of the regression lines, the rate of uptake in 0.64 M PBS was 6.7 times t’hat in 0.14 M PBS. An attempt was made to determine how much of the RNA-P”” retained by the cells after a 15-minute incubation period was acid soluble, and how much remained in a sufficiently polymerized state to be insoluble in cold 10% trichloroacetic acid (TCA). The amounts of P32 extracted in cold 10% ‘WA were too small to permit one to place any confidence in t,heir reality. It was quite clear that little or none of the RNA absorbed in 15 minutes at 37” from any of the three media was degraded to acid-soluble components. The uptake by H&a cells of RNA-Pa2 dissolved in 0.14 M PBS, 0.90 M PBS, and 0.98 2 Sue/PBS is shown in Fig. 12. As was the case with the L cell system, uptake

0 -TIME

15 30 OF INCUl38TION

4.5 IN MINUTES

60 -

FIG. 11. Uptake of RNA-P% by 10’ L cells from 0.5-ml volumes of solutions containing approximately 2 mg RN&t per milliliter. Uptake is expressed as a percentage of the total amount of RNA-P”’ in the mixtures. Incubations were at 37” in 0.64 izI PBS (O), 0.14 M PBS (01, 0.70 Al Sic/PBS (0).

INF’ECTIOUS

MENGO

I.8 -

AND POLIO RNA

121 0

/

Fro. 12. Uptake of RNA-P”’ by 10’ HeLa cells from 0.5ml volumes of solutions containing approximately 2 mg RNA per milliliter. Uptake is expressed as a percentage of the total amount of RNA-P”’ in the mixtures. Incubations were at 37” in 0.14 M PBS ( l ), 0.90 Al PBS (a), 0.98 M &r/PBS (0).

was very small from the two solutions of physiological ionic strength, while much more extensive binding of RNA-P32 occurred when the incubation was carried out in the hypertonic NaCl solution. In the hypertonic medium, the rate of uptake became constant after about 13 minutes, and from then on was 12 times the rate of uptake from 0.14 M PBS or 0.98 M Sue/PBS. It may be seen that the regression line for the 0.90 M PBS uptake data does not pass through the origin (zero time), a finding which indicates that, in this medium, the rate of uptake of RNA-P”’ by the cells during the first lo-15 minutes is lower than it is later. This observation, coupled with the finding that the viability of L ceils fell precipitously in hypertonic saline, suggested that the greater uptake in hypertonic saline might be due simply to cell death and loss of the permeability barrier of the cell surface. The binding of RNA-P32 by HeLa cells in 0.90 M PBS during a 30-minute incubation period was therefore examined in some detail, and a simultaneous record

made of the ability of the exposed HeLa cell population to concentrate neutral red. The results are illustrated in Fig. 13. Figure 13 is composed of three pairs of curves. One of each pair shows cell viability, and the other RNA-P”” uptake. In 0.14 M PBS (lowest pair of curves), cell viability remained high, and the rate of RNA uptake low-and constant-over the 30-minute incubation period. The other two pairs of curves illustrate the loss of viability of the cells and the parallel increase in the rate at which they absorb RNA-P”” when the incubations are carried out in 0.90 ill PBS. In addition, they emphasize that populations of HeLa cells examined at different times (and perhaps under slightly different physiological conditions) exhibit varying degrees of resistance to the toxic effects of the hypertonic environment. The HeLa cells used to obtain the upper pair of curves had an initial viability of only 90%. They were found to die more rapidly-and to bind RNA-P3’ at a faster rate-than did the sample of cells (initial viability = 99%)

122

ELLEM

AND COLTER

I MCUBATIOM FIG. 13. Correlation between the uptake of RNA-P” by HeLa cells and the loss of cell viability (measured by ability of the cells to concentrate neutral red). 10’ HeLa cells were incubated in 05-ml volumes of media. Solutions used in uptake studies contained about 2 rnz RN-4 Der milliliter. Onen svmbols = cell viability; closed symbols = RNA-P”” uptake; &0.14 M-PBS;& 0, O.&MPBS.

that were used to obtain the pair of curves that occupy the intermediate position in Fig. 13. In a more extensive series of such experiments, the variability in the resistance of HeLa cells to exposure to 0.90 M PBS was found to be considerable. In general, a lower resistance was found in those populations with the greater number of nonviable cells initially present, and always, there was a close correlation between the loss of cell viability and the increased rate of RNA-P”” uptake. A similar conclusion can be drawn in the case of L cells, since viability in 0.64 II& PBS fell immediately and nearly linearly, while the uptake of RNA-P32 appeared to proceed at a constant rate from time zero. The correlation between loss of cell viability and uptake of RNA was made clear only because of the greater resistance of HeLa cells to the toxic effects of hypertonic saline.

The outstanding differences between the HeLa cell-polio RNA and L cell-Mengo RNA systems appear to result from the different responses of the two cell types to ionic and nonionic hypertonic environment. These differences are illustrated by the data presented in Table 2. These results were obtained from a series of experiments with both systems (using different pools of viral RNA and performed over a period of several months) which were designed to examine the effects of tonicity on the formation of infectious centers. In the Mengo RNA-L cell system, 0.70 M Sue/PBS and 0.64 1M PBS were, on the average, 26 and 17 times as efficient as physiological saline in the formation of infectious centers, and about 1.7 times as many infectious centers were produced in the optimal sucrose as in the optimal saline solu-

INFECTIOUS

MENGO

AND

TABLE

POLIO

123

RNA

2

COMI’ARISON OF THE RELATIVE EFFICIENCIES OF PHYSIOLOGICAL SALINE AND OF HYPERTONIC SALINE AND SUCROSE SOLUTIONS IN PROMOTING THE FORMATION OF INFECTIOUS CENTERS IN MENGO RN,%-L CELL AND POLIO RNA-HELA CELL MIXTURES L cell-Mengo Expt. no.

RNA

HeLa-Mahoney

I.C. in sucrosea I.C. in PBS*

I.C. in salinec I.C. in PBS*

I.C. in sucrosea I.C. in salinec

I.C. in sucrosed I.C. in PBSb

I II III IV V

29.3 15.5 22.0 20.4 42.6

20.8 7.3 9.8 17.4 27.6

1.41 2.13 2.24 1.17 1.54

Mean

26.0

16.6

1.70

a/b Ratio c/b Ratio a/c Ratio dlb Ratio e!* Ratio e/d Ratio

of of of of of of

infectious infectious infectious infectious infectious infectious

centers centers centers centers centers centers

produced produced produced produced produced produced

in in in in in in

RNA%

I.C. in salinee I.C. in PBS*

I.C. in salinee I.C. sucrosed

3.14 2.55 3.62 6.83

34.8 12.0 13.0 54.1 -

11.10 4.70 3.59 7.95 -

4.04

28.5

6.84

0.7 M Sue/PBS to those produced in 0.14 A4 PBS. 0.64 M PBS to those produced in 0.14 M PBS. 0.7 M Sue/PBS to those produced in 0.64 k’ PBS. 0.98 M Sue/PBS to those produced in 0.14 ill PBS. 0.9 M PBS to those produced in 0.14 M PBS. 0.9 M PBS to those produced in 0.98 M Sue/PBS.

In the HeLa cell-polio RNA system, on t,he other hand, the optimal saline solution (0.90 M PBS) was 28 and 7 times as efficient as physiological saline and 0.98 M Sue/PBS, respectively, in establishing infectious centers. The HeLa cell is characterized by its inertness-in this system at least-toward sucrose. The optimal sucrose solution was only 4 times as efficient as was isotonic saline. We are unaware of any reports regarding the effects of sucrose on HeLa and L cells which might explain the difference in the responses of the two lines to hypertonic solutions of this substance. It may be seen from Table 2 that there was considerable variation in the efficiency of the hypertonic media in both systems. This was caused in part, by the variability in the response of cells incubated with viral RNA in an isotonic environment, which would cause fluctuations in a ratio whose numerator was constant. It was shown in an

tion.3

3The difference in potency between the sucrose and saline media was highly significant, (,t = 4.621; 0.001 < p < 0.01). Since the two values are correlated, the t test was based on the difference between them in each experiment, and the mean of the differences tested for the significance of its departure from zero.

earlier report (Ellem and Colter, 1960b) that the yield of infectious centers in L cellMengo RNA mixtures was reproducible when the incubations were carried out in 0.70 M Sue/PBS. Another contributing fact,or, particularly in the HeLa cell-polio RNA interaction, was the marked variability in the resistance to 0.90 M PBS of HeLa cell populations examined at different times. This is undoubtedly due to variations in the physiological condition of the cells, which in turn may be the result of irregularities in feeding schedule (and thus of growth rate) and to other, unrecognized factors. The fact that the HeLa cell is much more resistant to hypertonic saline than is the L cell, would explain the observation that 0.90 M PBS is more efficient in promoting the formation of infectious centers in HeLa cell-polio RNA mixtures t.han is the optimal saline solution (0.64 M PBS) in the L cellMengo RNA system. Despite the fact that HeLa and L cells respond differently, in quantitative efficiency, toward hypertonic sucrose, the same phenomenon with respect to osmotic pressure is observed in both systems. With each type, the optimal osmotic pressure for the

124

ELLEM

AND

formation of infectious centers is the same in both hypertonic sodium chloride and hypertonic sucrose solutions. The fact that it is higher for the HeLa (5.7 times isotonic) than for the L cell (4.1 times isotonic) seems to be due to the greater resistance of the former cell to this type of stress. The finding of an optimal osmotic pressure in both systems supports the suggestion made earlier (Ellem and Colter, 1960b), that the withdrawal of water from the cell, thus creating a hypertonic intracellular environment, is the important factor in increasing the efficiency of infection with RNA. The kinetics of formation of infectious centers is similar in both systems and corresponds to t,he expectations of classic kinetic theory. The differences seen between the two systems in the Arrhenius description of the response of the rate of formation of infectious centers to temperature changes may again reflect the greater resistance of the HeLa cell to hypertonic stress. Whereas the L cell-Jlengo RNA curve had a discontinuity at about 24”, with a decreased rate above this temperature, the HeLa cellpolio RNA curve was a continuous, linear TABLE

3

YIELUOF~NFECTIOUS RNA FROMVARIOUS MENGO VIRUS PREPARATIONS Virus pr;g:-

Virus titer (X 107)”

RNA titerC

RNA titer/ virus titer x 10-6

M

3.3 7.4

284 310

8.5 4.2

sc

52.5 1.8

2123 665

4.0 3.7

L

10.9

5797

53.1

(1M, S, and L are plaque-size variants of Mengo virus-to be the subject of a forthcoming publication. h Virus titer = plaque-forming units per milliliter. c RNA titer = infectious centers formed in 106 L cells incubated for 15 minutes at 37” in 1 ml of RNA solution (in 0.7 ,I! Euc/PBS). A correction was made to account for the dilution of the RNA preparation relative to the original virus suspension.

COLTER

function from 6” to 37”. The inimical reaction responsible for the break in the curve in the L cell system might be one responsible for cell death; it may come into play only at temperatures in excess of 24”. The basic similarity of the two systems is emphasized by the nearly identical, apparent activation energies of both at temperatures below 24” (34,000 cal/molej . With both cell types, a disparity was observed between estimates of cell viability (after incubation in hypertonic media) when ability to concentrate neutral red and the survival of viral-induced infectious centers were used as the criteria for viability. With both cell types, the curves relating the survival of infectious centers to the time of incubation in hypertonic media followed those illustrating the decline in infectious centers in cell-RNA mixtures. The curves describing neutral red staining -part,icularly those obtained from cells incubated in hypertonic sucrose solutionsdid not show a corresponding decline. The survival of infectious centers requires that the cell be viable, and metabolically active for at least 4-5 hours, for the production of new virus, The immediate testing of cell viability with neutral red may thus be a less useful guide in the prognosis of the survival of cells subjected to this stress. If it is assumed that the cell cannot distinguish between viral and high molecular weight cellular RNA (both have a molecular weight of about 2 x lo6 daltons) insofar as uptake is concerned, then an estimate of the true infectivity of the RNA preparations can be made. Since the uptake of RNA-P32 in hypertonic saline is spurious, the figures for the uptake by L cells in 0.14 IM PBS and 0.70 M Sue/PBS have been used. It was found that lo7 cells absorb only 0.054% of the RNA present in 0.5 ml of RNA solution in 15 minutes at 37”. Thus lo6 cells in 1 ml of solution (the standard proportions used for the assay of Mengo RNA) would absorb only l/10 x I/’ x 0.054%, or 0.0027 per cent of the total RNA present. To correct for this nonabsorption, the observed titers of Mengo RNA preparations should be multiplied by l/O.000027 = 3.7 X IO”. Because of the magnitude of

INFECTIOUS

MENGO

AND POLIO RNA

125

same order of magnitude as that calculated for Mengo RNA. It seems likely that the low infectivity of viral RNA preparations reported here and elsewhere in the literature can be accounted for by the low permeability of the cell membrane to this macromolecule. So specific mechanism of attachment and penetration appears to exist for the polynucleotide (Holland et nl., 1959). A similar conclusion has been reached by Holland et al. (1960) on the basis of studies of the uptake of P32-labeled polio RNA by HeLa cell monolayers. They found that although there was a negligible fixation of label by t.he monolayer, there was a large drop in the infectivity of the RNA which had been incubated on the monolayer. Of the various explana4 x 10”. tions for this observation, they favored the .4 series of extractions of RNA from virus pools were performed, and the titers of the idea that there was a specific uptake by the cell of the infectious RNA moieties, with RNA preparations and of t,he original virus the bulk of the RNA left unabsorbed. Such pools compared. It may be seen from Table selectivity seems less likely than an ex3 that the ratios of RNA infectivity to PFU planation based on the inactivation of RXA of virus varied from 4 x 1O-6 to 53 x during exposure to the cells and during lo-“. Thus the RNA titers fell short of givsubsequent manipulations. We have reing a 1: 1 ratio with viral plaque-forming of infecunits by a factor of from 2.5 x 1Oj to 1.9 X ported on the marked instability tious RNA preparations (Colter and Ellem. 10”. These values overlap the minimum 1960) and have found that Versene produces correction (3.7 x 104) based on the RNAP:j2 uptake studies, and fall within t,he a sixtyfold increase in the half-life of Mengo RNA preparations. range of the adjusted correction factors That Versene produces a stabilization of (8 x lo4 to 4 x 105) discussed in the prethe infectivity of TRW-RN,4 has been receding paragraph.” (19573. The The uptake of RNT-P32 by HeLa cells ported by Fraenkel-Conrat of the polynucleotide to heavy from 0.14 M PBS or 0.98 M Sue/PBS was lability metal-catalyzed degradations particularly of similar magnitude to that observed with pertinent to studies such as those reported I, cells. The mean uptake in four experiments was 0.034% of the total RNA in 0.5 by Norman and Veomett (1960)) who reported differences in the thermal stability m1/107 HeLa cells in 15 minutes. Therefore, and poliovirus RNA. Since a correction for the infectivity of polio of TMV-RNA RNA could be made which would be of the these workers made no mention of the use of chelating agents in their studies, it seems ’ These calculations ignore the particle : infecquite possible that the observed differences tivity ratio of Mengo virus which is believed to in thermal stability could be explained by be low (a. F. Graham, personal communication). different levels of heavy-metal contaminaIt is conceivable t,hat competent RNA could be tion in their RPI’A preparations. enclosed in a viral prot,ein shell which is unab!e

mechanical error in working with viscous solutions with high cell densities, it has not been feasible to reduce the volume of the RNA solution used in the suspended cell assay system to less than 0.5 ml. An increase in t’he number of cells used to lo8 or log is logistically impractical. The lower limit to which t,he ratio of the volume of RNA solution to the number of cells used can be reduced without producing a deviation from linearity in the relationship between cell number and infectious centers formed has not been reached. ,4 reduction of l/ to l/10 does not seem unreasonable, and to account for t’his factor, therefore, the multiplicand of the RNA titers should be further increased 2- to lo-fold: from 8 x lo4 to

to attach to cells. It has been shown that infertious RN.4 can be extracted from virus inactivated by specific antibody (Rappaport, 1959), or by heat and acid (Ada and Bnderson, 1959; Bachrach, 1959). It, is thus possible that the corrected RNB titer could exceed t,he actual plaque-forming titer of the virlls pool from which the RNA was isolated.

REFERENCES G. I,., and PIERSON, s. G. (1959). Yield of infective “ribonucleic acid” from impure Murray Valley encephalitis virus after different treatments. *Vatcue 183,799-800. BACHRACH, H. L. (1959). Foot and mouth disease virus. Stability of its ribonucleic acid C‘OW to iiDA,

ELLEM

AND COLTER

acid and to heat. Biochem. Biophys. Research Communs.1,356-60. COLTER,J. S., and ELLEM, K. A. 0. (1960). Biological activity of ribonucleic acid of viral origin, In “Cell Physiology of Neoplasia,” pp. 407432, 14th Annual Symposium on Fundamental Cancer Research, Houston, Texas. Univ. of Texas Press, Austin. ELLEM, K. A. O., and COLTER,J. S. (1960a). The interaction of infectious ribonucleic acid with a mammalian cell line. I. Relationship between the osmotic pressure of the medium and the 11, production of infectious centers. Virology 434-443. ELLEM, K. A. O., and COLTER,J. S. (1960b). The interaction of infectious ribonucleic acid with a mammalian cell line. II. Kinetics of the formation of infectious centers. Virology 12, 511520. FRAENKEL-COXRAT,H. (1957). The infectivity of tobacco mosaic virus nucleic acid. In “Cellular

Biology, Nucleic Acids and Viruses,” pp. 217227. New York Academy of Sciences. 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. 110,65-80. HOLLAND, J. J., HOYER, W. H., MCLAREN, L. C., and SYVERTON,J. T. (1960). Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. J. Exptl. Med. 112,821-839. KOCH, G., KOENIG,S., and ALEXANDER,H. E. (1960). Quantitative studies on the infectivity of ribonucleic acid from partially purified and highly purified poliovirus preparations. Virology 10, 329-343. NORMAN,A., and VEOMETT,R. C. (1960). Heat inactivation of poliovirus ribonucleic acid. Virology 12,136-139. RAPPAPORT,I. (1959). Recovery of infectious nucleic acid from tobacco mosaic virus inactivated by antibody. Nature 184, 1732-1733.