WROLOOY 26, 246-252 (1965)

Infectious RNA Synthesized in Monkey Kidney Tissue Culture Cells Infected with Poliovirus 1'2

R E T O E N G L E R AND OTIS T O L B E R T

Section of Virus Research, University of Kansas Medical Center, Kansas City, Kansas

Accepted February 16, 1965

Monkey kidney tissue culture cells infected with type i poliovirus contain not only mature virions, but also a considerable amount of unbound or free viral RNA. The presence of such an RNA in virus harvests was shown by several methods such as: (1) facilitation and t~Nase sensitivity; (2) stability to elevated temperatures (52°); (3) stability of infection in presence of polio antiserum; and (4) chromatography on Sephadex G200. Furthermore the density of this free RNA was determined and com- pared with the density of RNA prepared from virions (phenol method). Both RNA's had a density of 1.63 g/ml in Cs:SO4, suggesting a single-stranded structure for the free viral RNA. The appearance of free viral RNA in the growth cycle of poliovirus was studied. The results indicated that maximum yield of free viral RNA was ob- tained approximately 10-15 hours after the maximal amount of virions was synthe- sized.

INTRODUCTION

In a previous publication evidence was presented that monkey kidney tissue culture cells (MKTCs) infected with poliovirus syn- thesized not only mature polio virions, but also slow-sedimenting particles yielding in- feetious R N A upon treatment with phenol (Tolbert and Engler, 1963). Several authors have demonstrated and characterized virus- specific R N A in infected tissue culture cells. Montagnier and Sanders (1963) for example demonstrated an infectious RNA fraction in cells inoculated with encephalomyocarditis (EMC) virus which had several character- istics of double-stranded RNA. Earlier, Kubinski and Koch (1963) reported a virus- specific double-stranded R N A in HeLa cells infected with poliovirus, but they could find

1Part of these results was presented at the 64th annual meeting of the American Society for Microbiology, Washington, D. C. (May 3-7, 1964).

2 Aided in part by a grant from the National Foundation and supported in part by Public Health Research grant no. AI-06263 from the National Institute of Allergy and Infectious Diseases.

no evidence that this R N A fraction was infectious. Baltimore et at. (1964) demon- strated a double-stranded virus-specific R N A in poliovirus-infected HeLa ceils based on density determinations and ribonuclease (RNase) sensitivity; no statement was made as to whether this virus-specific R N A ex- tracted from cellular particulates was in- feetious or not, but Pons (1964) could show a moiety of infectivity associated with double-stranded polio RNA. Still another contribution to this topic was made by Fenwick (1963) showing that in E R K cells infected with poliovirus, radioactive phos- phorous (ps2) was incorporated only by an R N A with a sedimentation constant of 32-35 S, corresponding to ribosomal or single- stranded RNA; no double-stranded form of polio R N A was found to be marked with p32. All authors mentioned above used vari- ous extraction procedures to obtain the virus-specific RNA, and, where mentioned, double-stranded R N A forms appeared dur- ing the latter stage of virus multiplication.

During our experiments in which we demonstrated slow-sedimenting infectious

246

INFECTIOUS RNA 247

particles produced by polio-infected MKTCs, we were led to assume that these particles were free infectious viral RNA (f-v-RNA) ; free, meaning no extraction pro- cedure was necessary to reveal this infectious RNA. With materials and methods at our disposal we sought to prove the presence of this f-v-RNA and also to characterize it as single- or double-stranded RNA.

MATERIALS AND METHODS

Virus stocks. A plaque-purified strain of poliovirus type 1 (Brunhilde) was used. Stocks were prepared by infecting MKTCs gro~nt in 50-ram petri dishes with one parental stock designated Br PPlpr2 (plaque- purified, second rapid passage). A multi- plicity of infection of 1:10 was used. After maximum degeneration of the cell sheet had occurred (2-3 days), the tissue culture fluid containing virus and cell debris was har- vested by repeated freezing and thawing and stored at - 2 0 ° until further use. During the growth period of the MXTCs laetalbumin growth medium (LG) containing 4 % calf serum and during virus multiplication high- eystine altered Eagle's medium (heAEm) without serum (Dubes and Chapin, 1958) were used to maintain the cells. Resulting stocks were designated as Br PP1pr320 to Br PPlpr340.

Purified virions and phenol RNA (¢-RNA ) of purified virions. Suspensions of purified polio virions and phenol extracts thereof were prepared as follows. Aliquots of the above virus stocks were freed from cell debris by low speed centrifugation (2000 rpm for 20 minutes). The polio virions were collected in a pellet by high speed centrifu- gation in a Spinco model L centrifuge. The pellet was resuspended in phosphate-buffered saline (PBS), and the virions were centri- fuged a second time. Depending on the volumes involved, the 30 or 40 head was used. With a table and slide rule specially developed for this centrifuge (Giebler, 1958) the centrifugation times and speeds were adjusted in such a manner that only 70 % of the suspended virions were expected to be collected in the pellets, thus assuring a more complete separation of the virions, especially from smaller particles. Expected losses in tiger were corrected by resuspending the pellet in smaller volumes of PBS.

For the preparation of ¢-RNA from these purified virions, essentially the method of Gierer and Sehramm (1956) was used; one volume of virus suspension was shaken with one volume of water-saturated phenol for 10 minutes at room temperature, the water phase was separated from the phenol, the phenol dissolved in the water was extracted with ether three consecutive times, and the remaining ether was removed by bubbling nitrogen through the solution.

Plaque assay of the virus and infectious viral RNA. Plaque-forming units (PFU) of suspensions of virions were determined using a method described by Dulbecco and Vogt (1954). MKTCs grown to a complete cell sheet in 50-ram petri dishes were washed twice with 4 ml of cold PBS and inoculated with 0.3 ml of the virus suspension. After an adsorption period of 30-50 minutes at 37 ° the ceils were overlaid with hcAEm con- gaining 0.9 % agar and were maintained in a continuous C02 flow incubator at 36 4- 1 °. Three days were allowed for plaque develop- ment. After this time plaques were made visible by staining the surviving cells with neutral red.

Plaque formation with viral RNA on MKTCs requires facilitation. The procedure of inoculation of MKTCs with infectious RNA was described by Dubes and Klingler (1961). Briefly, the MKTCs were depleted of calcium by washing them 4 times with Ca++-free PBS, allowing them a depletion period of 30 minutes at 37 ° between the second and third wash. To the inoculum containing the infectious RNA, kaolin (Fisher Scientific Company) was added as facilitator at a final concentration of 0.25 % (w/v). The previously Ca++-depleted cell sheet was then inoculated with 0.3 ml of this suspension, followed by an adsorption period of 30-50 minutes at 37 ° . After that period the cells were overlaid and plaques were developed as described above.

The facilitation method used here was compared with other methods which enhance the infectivity of polio RNA (H. Rouhandeh, in press). RNA titers, obtained with various methods (facilitator, MgSO4, historic) were equal using MKTC as assay system; how- ever, the kaolin-facilitation requires an end point dilution titration to obtain accurate RNA tigers, because at higher RNA concen-

248 ENGLER AND TOLBERT

trations the relation of P F U to concentration is not proportional (Dubes and Klingler, 1961).

Partial removal of virions from poliovirus stock solution (preparation of supernatant). For experiments with the f-v-RNA, the bulk of mature virions had to be removed fl'om virus stocks. This was done by differential centrifugation as previously described (Tol- bert and Engler, 1963). Virus stocks with an original virus titer of about 10 ~ or 107 P F U / m l were depleted of their virus content by a factor of 10 ~ or 104 by two consecutive centrifugations in a preparative Spinco model L centrifuge. Supernatants of the second centrifugation step, usually contain- ing the equivalent of 102 to 104 P F U mature virions per milliliter, were collected and stored at - 2 0 ° until further use. Virus stocks partially freed of virions as described above are referred to as supernatant or as supernatant fluid.

TABLE 1 FACILITATION AND RNAsE LABILITY OF

FREE VIRAL R N A IN SUPERNATANT a

Addit ions to inocu-

lum b

Number of plaques produced on 11 individual petri dishes Total

None 15, 24, 17, 19, 24, 17, 209 19, 20, 12, 17, 25

Kaolin c 29, 29, 33, 29, 31, 28, 349 31, 29, 34, 39, 37

Kaolin e 21, 20, 21, 25, 21, 23, 222 RNase ~ 14, 18, 16, 21, 22

a Supernatant prepared by centrifugation from a virus stock originally containing 107 PFU/ml was further diluted 100-fold bringing it to a final titer of 101.s PFU/ml. After aliquots of this dilu- tion were submitted to the three different treat- ments, 0.3 ml was inoculated onto each petri dish.

b All inocula were incubated for 30 minutes at 37 ° to allow the RNase sufficient reaction time.

Final concentration 0.25% (w/v). d Final concentration 20 gg/ml.

RESULTS

Facilitatable and RNase-Labile Infectivity of Poliovirus Harvests

The facilitation procedure described under Materials and Methods offers an excellent means of distinguishing mature virions from viral RNA. The efficiency of plaque for- mation (cop) by virions is not affected by the addition of kaolin, whereas the ability of viral RNA to cause infection, and hence plaques, is greatly enhanced (Dubes and Klingler, 1961). Supernatant was prepared (see Materials and Methods) and was further diluted by a factor of 102; this together with the depletion by centrifugation reduced the original titer of the virus stock (107 PFU/ml ) to 10 l's PFU/ml . Three aliquots of this diluted supernatant were incubated at 37 ° for 30 minutes: one contained no additions (other than PBS, substituting for additions made to other aliquots), one contained facilitator (0.25 % kaolin, final dilution), and one contained facilitator and RNase (20 #g/ml, final dilution). A group of 10-12 petri dishes each was inoculated. The results of a typical experiment are listed in Table 1. The differences as shown by plaque numbers were tested by use of a statistical table, based on comparison of the Poisson distri-

button of two populations (Lorenz, 1962). The number of plaques obtained with the facilitated inoculum was significantly higher than the plaque number produced by the nonfacilitated or RNase-treated inoculum. The plaque numbers of the nonfacilitated and RNase-treated inoculum, on the other hand, were not statistically different. This proved conclusively that the inoculum con- rained infectious particles tha t were facili- ratable and RNase labile, thus having two properties identical to those of ¢-RNA.

Temperature-Sensitive and Resistant In- fectious Particles in Poliovirus Harvests

The infectivity of poliovirus is destroyed rapidly at temperatures of 50-55 ° (Graham, 1959) whereas viral RNA retains most of its infectivity at these temperatures (Koch, 1960). These facts further helped to dis- tinguish virions from f-v-RNA in poliovirus harvests. The supernatant was incubated for various periods of time at 52 °. This temper- ature does not release infectious RN A from the virion, but simply incapacitates it to infect cells (Dubes et al., 1964). Samples ob- tained at intervals as well as prior to the inactivation were tested for infectivity by plaque assay on MKTCs, in presence and

INFECTIOUS RNA 249

TABLE 2 COMPARISON OF THE S T A B I L I T Y OF F R E E

VIRAL RNA, PItENOL-RNA AND VIRIONS AT 52 ° FOR V A R I O U S T I M E S

Average plaque number per petri dish Inac t iva tion time Su~ernatant a Supernatant a Phenol- Virions c

(min.) v . . not RNAb facilitated famhtated facilitated facilitated

0 > 50 > 50 ca 50 > 50 5 33 3 ca 50 2

15 36 0 ca 50 0 30 35 0 ca 50 0

Supernatant was prepared from a virus stock (107 PFU/ml) by centrifugation. This supernatant containing 103 PFU/ml was used undiluted for the inactivation experiment, thus giving overlapping plaques (expressed as >50) without heat treat- ment.

b Phenol RNA having a ti ter of 1.5 X 10 ~ PFU/ ml was diluted tenfold with PBS and submitted to heat treatment.

A suspension of purified virions with a pre- calculated t i ter of 103 PFU/ml was prepared and heat inactivated; >50 meaning overlapping plaques.

Facil i tat ion procedure was as described in Materials and Methods. 0.25% kaolin (w/v) was contained in the inoculum, and the MKTC's were depleted of Ca ++ before inoculation. In the non- facilitated inoculum the addition of kaolin was substituted with PBS, and the cell sheets were not Ca ++ depleted.

absence of fac i l i t a to r . F o r compar i son , pur i - fied v i r ions and ¢ - R N A were sub jec t ed to iden t i ca l t e m p e r a t u r e s for the same in t e rva l s of t ime. I n a c t i v a t e d v i r ion samples were inocu la t ed us ing f a c i l i t a t i o n - - t h i s as a con- t ro l to p rove t h a t f ac i l i t a t ion d id no t enhance the infec t ion of suscep t ib le cells b y v i r ions which were d e n a t u r e d a t 52 °. T h e resul t s o b t a i n e d are shown in T a b l e 2. T r e a t m e n t a t e l eva ted t e m p e r a t u r e r ende red the pur i - fied v i r ions comple t e ly uninfee t ious while the i n f ec t i v i t y of the ¢ - R N A was no t affected. T h e s u p e r n a t a n t , as expec ted , showed a r a t h e r h igh in fec t iv i t y t i t e r before h e a t t r e a t m e n t ; however , a f t e r 15 minu t e s of t r e a t m e n t no in fec t iv i t y was d e m o n s t r a b l e w i t h o u t fac i l i t a t ion , whereas the a l iquo t f rom the same sample upon fac i l i t a t ion y ie lded infect ious pa r t i c l es whose t i t e r was no t m a r k e d l y changed a f te r an add i t i ona l 15 minu te s of h e a t t r e a t m e n t . Here again , an

infec t ious pa r t i c l e b e h a v i n g l ike ¢ - R N A unde r the influence of h e a t was demon- s t r a t ed . Se lec t ive h e a t d e n a t u r a t i o n of t he v i r ion was l a t e r used r o u t i n e l y as a m e t h o d of qu ick ly revea l ing the presence of f - v - R N A in s u p e r n a t a n t fluids.

A n t i s e r u m - R e s i s t a n t and Sensi t ive In fec t ious Part ic les i n Pol iov irus Harvests

Pol iov i rus t r e a t e d wi th i ts a n t i s e r u m can- no t infect suscept ib le cells; however , if f ac i l i t a to r is a d d e d to the a n t i b o d y - v i r u s complex, infect ion a n d p l aque f o r m a t i o n are poss ible (Dubes et al., 1964; Engler , un- pub l i shed d a t a ) ; y e t the eop of the f ac i l i t a t ed a n t i b o d y - v i r u s complex is r educed b y a fac tor of a b o u t 103 as c o m p a r e d w i th the cop of v i rus in the absence of a n t i b o d y . There fo re in a s u p e r n a t a n t f luid con ta in ing 103.7 P F U / m l a comple te inh ib i t ion of the v i rus pal ' t ic les b y a n t i s e r u m is possible, and a n y f - v - R N A would stil l be infect ious in the presence of. an t ibodies . S u p e r n a t a n t was i n c u b a t e d wi th pol io t y p e 1 a n t i s e r u m a t a final d i lu t ion of 1:100 for 20 m i n u t e s a t 37 ° . Af t e r th is i ncuba t ion per iod, the mix tu r e was inocu la t ed on M K T C s in the presence

TABLE 3

I N S E N S I T I V I T Y OF F R E E V I R A L RNA TO

T R E A T M E N T WITH POLIO A N T I S E R U M

Content of inoculated supernatant, Log10 titer/ml

Antipolio serum b 2.7 Facil i tator c

Antipolio serum b Facil i tator ~ 0 RNase d

a Supernatant was prepared as usual from a virus stock containing 107 PFU/ml; after the centrifugation steps a residual infectivity of 103.7 PFU/ml was still present.

Inocula were incubated 30 minutes at 37 ° before they were plated on the MKTC cell sheet. After an adsorption period of 30 minutes at 37 ° onto the MKTC's the inoeulum was removed and the cell sheet was washed 3 times with 4 ml of cold PBS to remove any residual antiserum, which possibly could have interfered with plaque development.

b Final coneentration 1 : 100. c Final concentration 0.25% (w/v). d Final concentration 20 ug/ml.

250 ENGLER AND TOLBERT

of facilitator. As a control, RNase was added to an identical mixture (RNase final concentration, 20 ~g/ml). This was done to show that the iIffectious particles resisting antibody treatment were also RNase labile and that the plaques were not formed by facilitated virus-antibody complexes. The results obtained by treating supernatants with antiserum or antiserum plus RNase are listed in Table 3. Antiserum treatment alone did not inhibit the infectivity com- pletely, but antiserum and RNase destroyed all infectivity. This again showed that super- natants contained both virions that were susceptible to antibodies and f-v-RNA whose infectivity was inhibited not by antiserum, but by RNase.

Chromatography of Supernatant on Sephadex G200

We have found that polio virions and ¢~-RNA of poliovirus could be separated on Sephadex G200 (Bell a n d Engler, 1964). Based on this finding an attempt was made to learn something about the structure of the f-v-RNA, because a different chromato- graphic behavior of ¢-RNA and f-v-RNA on Sephadex G200 could be reasonably inter- preted as a variation of the molecular con- figuration. The procedure used has been described by Bell and Engler (1964). When supernatant was chromatographed on Sephadex G200 two peaks of infectivity were e]uted, one after the void volume of the column containing the virions and the other having a Kd value of 0.41. This latter fraction was RNase labile and contained f-v-RNA. No significant difference however was found between the Kd value of the f-v-RNA and the Kd value of 0.43 shown for the ¢-RNA.

Centrifugation of Supernatant and ¢-RNA in a Cesium Sulfate (Cs2S04) Gradient

The densities of two picornavirus RNA's are reported to be 1.69 g/ml for polio RNA (Baltimore et al., 1964) and 1.63 g/ml for EMC RNA (Montagnier and Sanders, 1963). The respective double-stranded forms re- ported by these authors have densities of 1.65 g/ml (polio) and 1.57 g/ml (EMC). Supernatant and ¢-RNA were both centri-

fuged in Cs~S04 density gradients. The starting solution was brought to a density of 1.58 g/ml by the addition of Cs2SO4. After 55 hours of centrifugation at 35,000 rpm in a SW-39 head in a model L Spinco, 15-20 fractions were collected and the number of PFU in each fraction was determined. The average density of the fractions was de- termined by measuring their refractive in- dexes and with the help of an experimentally determined conversion curve relating the refractive index of a Cs2S04 solution to its density. The fraetionation was done in such a way that one would have been able to distinguish differences in densities of 0.04 g/ml or more; closer fraetionation proved to be impractical for the infectivity test. Re- sults derived from five experiments showed that the maximum infectivity was accumu- lated at an average density of 1.63 g/ml. The ¢-RNA as well as the f-v-RNA had this identical density. Virions remaining in the supernatant were collected in the top fraction, which always had a density of more than 1.32 g/ml (the density of the virion). Thus the densities of ~b-RNA and f-v-RNA did not differ significantly, a find- ing that lends support to the conclusion that f-v-RNA consists of single-stranded polio RNA. However, the two types of infectious particles (virion and f-v-RNA) contained in the supernatant were clearly separated in the Cs2SO4 density gradient.

Appearance of f-v-RNA during the Course of Virus Multiplication

MKTCs infected with poliovirus were harvested together with the ~i ml of hcAEm culture medium per plate at various times after infection by freezing and thawing, and the plaque-forming virus units per milliliter of these harvests were determined. The harvests were partially freed from polio virions (see preparation of supernatants de- scribed in Materials and Methods). The re- maining virions were then inactivated at 52 ° for 65 minutes, after which the harvests were tested for the presence of f-v-RNA. The synthesis of poliovirions and f-v-l~NA are shown in Fig. 1, expressed as percentage of maximun~ yield. The maximum yield of 107 virions per milliliter was reached at least 8

INFECTIOUS RNA 251

E3 _J LU

I '- Z

O 0C

0 .

,oo ~ - ®

5 0

0

/ /

/ /

/ /

/ /

II, /

/ /

/ /

2~

I I I I I I I 1 I 0 2 0 3 0 4 0 5 0 6 0 7 0 S O

H O U R S A F T E R I N F E C T I O N

Fro. 1. Comparison of t ime course of the synthesis of polio virions and f-v-RNA. 0 - - - - 0 Synthesis of virions expressed as percentage of maximum yield (maximal yield representing 10 7 PFU/ml ) . • . . . . . • Synthesis of f-v-RNA expressed as l~ereentage of maximum yield (maximal yield repre- senting 103 PFU/ml).

hours after infection whereas at this point no f-v-RNA was detected. An additional 16 hours of incubation was required for the maximum yield of f-v-RNA. The maximum yield of f-v-RNA was 103 PFU/ml . In a representative experiment shown in Fig. 1 a high multiplicity of infection (ca. 10) was used, assuring a single cycle of infection. In experiments using lower multiplicities similar results were obtained, except that a longer time course was necessary to obtain maxi- mum yield of virions and f-v-RNA, but the sequence in which these two infectious units appeared was identical.

The possibility existed that the f-v-RNA was a degradation product of mature virions caused by prolonged incubation at 37 ° . To rule this out, a harvest collected at a time when 100 % of the virions, but no f-v-RNA, was synthesized (8 hours, Fig. 1) was incu- bated for 24 and 48 additional hours at 37 ° . After the additional incubation the harvest was again tested for f-v-RNA, but no f-v- RNA could be detected, a result indicating

tha t the f-v-RXA was not simply a product of degradation of vMons, but was produced by the cells in the course of virus synthesis.

DISCUSSION

The results presented show that M K T C s infected with poliovirus not only released mature virions, but also an infectious particle we called free viral RNA (f-v-tlNA). No extraction procedure was necessary to reveal this f-v-RNA. I t sedimented very slowly; it was destroyed by RNase, and its infectivity was not inhibited by poliovirus antiserum. Fm'thermore, its density and chromato- graphic properties on Sephadex G200 were identical to phenol-extracted RNA. ~iinor contamination with cellular or viral proteins could not be excluded; however, for all practical purposes this RNA released into the supernatant by the distintegrating cells can be considered to be unattached to any structure or carrier of any significance and at least as "free" as phenol-extracted RNA. The maximal amount of f-v-RNA synthe-

252 ENGLER AND TOLBERT

sized in infected cells expressed as P F U / m l is 103 in comparison to 107 infectious virions per milliliter. But considering the fact that infectious R N A has a very low efficiency of infection, the titer of 103 P F U / m l of f-v- R N A represents a rather large number of R N A molecules.

The role of the f -v-RNA during virus multiplication may be considered from a hypothetical viewpoint. The only fact re- vealed in this s tudy is that the f -v-RNA appears several hours after the maximum yield of mature virions was obtained. Pen- man et al. (1964) described so-called virus- synthesizing bodies (VSB) which synthesize viral R N A and protein and from which a double-stranded R N A could be extracted. If their assumption that all viral components are synthesized by the VSB's is correct, the f -v-RNA must also have its origin in these particles. The f -v-RNA could then represent an excess of single-stranded viral R N A pro- duced, which was no longer incorporated into virions; or it could be the result of a breakdown of the VSB, possibly the double- stranded virus-specific RNA. However the results presented here cannot reveal the f -v-RNA's origin because infectivity has been used entirely as criterion of its presence. I t would be of interest to make an a t tempt to separate the complementary strands of the double-stranded virus-specific R N A and to determine whether the f -v-RNA consists of one or possibly a mixture of both strands.

ACKNOWLEDGMENTS

The authors are indebted to Dr. H. A. Wenner for his helpful advice and suggestions, and to Drs. M. T. Found and H. Rouhandeh for critical reading of the manuscript. We also wish to express our thanks to Mr. W. C. Bell for the careful and apt performance of the chromatographic tests.

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