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Impairment of EMC viral infectivity by cell-associated ribonuclease
VIROLOGY
29, 596404
Impairment
(1966)
of EMC Viral ROBERT
Service de Biologie
Infectivity E. BASES2
Molkculaire
by Cell-Associated JOSEPH
AND
Institut
Gustave Roussy,
Accepted April
Ribonuclease’
HUPPERT Villejuif-Seine,
France
20, 1966
The fate of purified labeled infectious RNA of encephalomyocarditis virus in suspensions of mouse or hamster cells was studied in order to determine why only lO+ of the corresponding viral infectivity could be recovered. All the recoverable RNA infectivity became cell bound in about 1 minute, but more than 987, of the radioactivity of labeled 37 S EMC RNA was detected in the supernatant as degraded fragments. It was concluded that t,he loss in infectivity was mainly due to rapid degradation of the viral RNA by cell-associated ribonuclease. INTRODUCTION
sheimer, 1963). These facts suggest t’hat’ some cellular activity very strongly influences the outcome. Our results with the infectious ribonucleic acid (RNA) of encephalomyocarditis virus (EMC) help clarify the problem by demonstrating that mammalian cells very rapidly degrade viral RNA while simultaneously binding only a small portion intact. Ent’ry of this small portion is enhanced by hypertonic treat,ment of the cells. Since very rapid degradation of viral 01 cellular ribosomal RSA occurred in cell suspensions where free ribonucleases were virtually eliminated by bentonite (Fraenkel-Conrat el al., 1961), it is suggested t,hat cell-associated or bound ribonucleases are responsible in great degree for the RiYL4’s loss of biological acbivity.
It is well known that viral nucleic acids have far less infectious activity (lop3 to lo-+) than the animal or bacterial viruses from which they were ext’racted (Alexander et al., 1958; Ellem and Colter, 1960a; Holland et al., 1960a; Weil, 1961; FraenkelConrat, 1959; Hoskins, 1961; Wahl et al., 1960; Guthrie and Sinsheimer, 1960; Huppert et al., 1962; Guthrie and Sinsheimer, 1963). While this loss might occur at any stage between ext,raction of the nucleic acid and liberation of progeny virus, partial improvement in efficiency of infection has been obtained with mammalian cells by subjecting them to hypertonic treatment (Alexander et al. 1958), or by m0derat.e exposure to elevat,ed temperat,ures or to amines (Rloscarello, 1965). In order to introduce bacterial virus nucleic acids int,o cells, it is generally necessary t’o use spheroplasts instead of normal bacteria. Even so, the efficiency varies greatly wit,h changes in the manner in which the cells are treated (Huppert et al., 1962; Guthrie and Sin-
MATERIALS
AND
METHODS
Cells. Krebs,, mouse ascites t’umor cells were grown in Swiss mice and harvested 6 or 7 days after intraperitoneal inoculation of about 10’ cells. Aliquots of a large inoculum, stored at -7O”, were thawed at regular
1 Supported by D&gation G&&ale B la Recherche Scientifique et Technique, contrat no. 62 FR 054. 2 Recipient of special Fellowship 13,275 of the National Cancer Institute, U.S.P.H.S., and of PO Eleanor Roosevelt Cancer Research Foundation Fellowship. Present address: Albert Einstein College of Medicine, Bronx, New York 10461.
intervals mouse
to
avoid
continuous
mouse
to
passage. A hamster ascites tumor cell line, T-6, grown in suspension cultures in vitro, was also used. This cell line was sent to us by Dr. F. K. Sanders (Sanders and Burford, 1964), who isolated it in ascit,es form
596
from
polyoma
virus
transformed
CELL-ASSOCIATED
BHK-21 clone 13 cells, originally provided by Drs. J. MacPherson and RI. St’oker (MacPherson, 1963). T-6 cells grew in suspension cult,ures in modified Eagle’s medium (1959) with twice the usual concentration of amino acids and vitamins, 10 times the usual phosphate concentrat,ion, and no calcium except that provided by supplementation with calf serum t’o 10 70, and tryptose phosphate to 10 %. Generation t.imes were 16-19 hours under these condit,ions. T-6 cells in suspension cultures were easily dispersed t.o single cell suspensions by pipetting. ~+zM. EMC virus stocks (1 to 2 X log PFU/ml) were prepared using Krebs II cells as described by Sanders et al. (1958). Plaque assays were performed by the agar suspension method (Sanders et al., 1958). In this assay, virus and 1.5 lo8 KrebsII cells were mixed and plated in agar at, 0.8 % on an underlayer of 1.5 % agar in Earle’s saline. Two days’ incubation at 37” in a moist incubator in 5% CO2 yielded 5-10 mm plaques when the petri dishes were st,ained for 1 hour with 1: 10,000 neutral red. J’irus puri$cation. Virus stocks were purified using centrifugat,ion, trypsinizat,ion, and chromatography on calcium phosphate columns (Faulkner et al., 1961). 32P-labeled virus was subjected to an additional purification as described by Montagnier and Sanders (1963) in which virus eluted from an initial calcium phosphate column was treat~ed with 1% sodium deoxycholate to lyse cellular membrane material which was occasionally encountered using the original method. A second calcium phosphate column purification followed. Montagnier and Sanders’ modification yielded a single virus component as det’ermined in equilibrium density cent’rifugation studies. Kaighn et al. (1964) have recently provided an improved purification of E12C virus from cellular material using pyrophosphate and ribonuclease. While we cannot exclude contamination of our virus preparation wit,h tiny amounts of cellular RNA, sucrose gradient analysis of t’he RNA indicates that most of it is indeed viral. Phenol extraction. Bent,onite (FraenkelConrat et ab., 1961) at 1 mg/ml was added
RIBONUCLEASE
597
to column purified virus (in 0.3 M phosphate) before 4 extractions with equal volumes of water saturated phenol. The aqueous layer was extracted 6 times with ether and then bubbled with nitrogen to remove the ether. In some experiments viral RNA was precipitated wit,h 66% ethanol using Krebs,, cellular RI\‘A as a carrier. This material, designated RNA, was stored at -70”. Aliquots were usually diluted before use. All preparations were sensitive t,o 1 pg/ml crystalline RXAase (Worthingt,on) at, room temperature for 2 minut’es. RNA assay. The procedure of Mont’agnier and Sanders (1962) was used with a few minor changes. Cells were washed 2 or 3 times by centrifugation in phosphatebuffered saline (PBS) (Dulbecco and Vogt, 1954) and then once in PBS wit,hout calcium and magnesium, just before use. The cell pellet was stirred gently with an equal volume of distilled water and to this was immediat,ely added an equal volume of a 1: 1 mixture of RNA and 2.6 M sucrose in 0.5 M sodium phosphate. After 60 seconds, the resulting mixt,ure was diluted six-fold with Eagle’s medium. RiYAase at 1 pg/ml was present in t#he Eagle’s medium to destroy any residual RNA. Then 1.5 x lo8 Krebs cells were added and plating was performed as in the virus assay. Cell concentrations before adding t,he Eagle’s medium were about 1O*/ml. Infectivity varied linearly with Rr\‘A concent,ration over a hundredfold dilut,ion. For convenience, we have arbitrarily referred to cell-associated RNA as being on or outside cells if 1 pg/ml of RNAase abolished infectious activity in 2 minutes at, room temperat,ure, and we considered it inside cells if RlSAase could no longer interfere. Krebsn mouse ascites cells or hamster cells from cultures in vitro served equally well as infectious cent.ers but Krebs,, cells were used t,hroughout to provide the agar suspension layer (for practical reasons). EMC virus titrations made with different cell preparat,ions rarely varied more than t,wofold, but, greater fluct’uations in RNA titers were noted from one experiment to another. However, with any single batch of cells reproducibility was comparable with
598
BASES
AND
t.hat in virus titrations. RNA t.it,ers did not prove especially dependent on t.emperature above 15”. Radioactive labeled GYM. 32P-labeled virus was prepared and purified according t’o Montagnier and Sanders (1963). 14C-Uridinelabeled virus was prepared by adding 14Curidine, .50 PC (Schwarz, 251 microcuries/ micromole), to Krebs,, cells at, 2 X 107/m1 in Earle’s saline 1 hour aft,er infection atJ a multiplicity of 3. 14C-Cridine-labeled virus purification was performed by the method of Faulkner et al. (1961). 14C-Uridine-labeled RSA was precipitated wit.h ethanol wit,h 200 pg of Krebsn cell RNA as carrier. After centrifugation the RNA was deposited uniformly on the top of slowly rotating planchet.tes, dried, and countled in a Trarerlab flow count,er sufficiently long to achieve an accuracy of 5%:. (Loevinger and Berman, 1951), at 33 %I efficiency. Sucrose gradients. Linear 5-20 % sucrose gradients were prepared in 0.1%. bentonit,e, nZgC& 0.001 M in distilled water and were run at 39,000 rpm in t,he model L Spinco ult,racent.rifuge in the SW 39 head. RNAase. Crystalline pancreatic ribonuclease (Worthington) was used. RESULTS
Recovery of Viral RNA Uridine Labeled EMC
from Virus
Purified
W-
Infectious Rr\‘A prepared from purified virus was assayed repeatedly, but at best its infectivity was only 10m3 of the virus before phenol ext,raction. Since the small amounts of purified virus material precluded use of opt,ical densit.y measurements to detect physical losses or of alterations in t.he sedimentat,ion pr0pert.y of the RNA, we studied radioactive labeled virus instead. Purified 14C-uridine-labeled EMC virus rvas extracted by phenol (bentonite at 1 mg/ml) and examined for its radioactivity, infectivity, and sedimentation properties. Table 1 and Fig. 1 show that very little viral RIYA was lost. or degraded during extraction, but t,hat. its biological activit.y was great,ly impaired. The correspondence of 14C label and infectivit,y in the 37 S region of Fig. 1 are within t.he variation of the plaque assay.
HUPPERT TABLE
1
RECOVERY OF W-URIDINE RNA EMC VIRUS' Material Virus (column purified) RNA (aqueous phase) Interphase Ether Phenol
FROM
Total PFU
Total counts per minute
1.3 x 10’0
1.20 x
105
2.0 x
1.01 x
105
0.13 x
105
Not
106b
tested
a Radioactive virus was purified and concentrat.ed after Faulkner et al. (1961). 837& of the input radioactivity was recovered in the infectious RNA. See Fig. 1 for sucrose gradient analysis of this material. b Mean in three experiments.
Our result,s are t,hus in excellent agreement with those of Montagnier and Sanders (1963), who previously characterized ERIC RNA. We cannot entirely exclude some cont,aminat.ion of the 14C-uridine-labeled 37 S viral RNA with labeled cellular RNA because the virus purificat,ion of Faulkner et al. (1961) is not as complete as that of Kaighn et al (1964) or of Montagnier and Sanders (1963). Nevertheless, wc can safely assume that little cellular RXA was present. in the 37 S region since so lit,tle label was detect’ed in t,he fractions where Krebs cell RSA (added as a reference) was found by opticaal density measurements. Impaired
Infectivity
of 37 S EMC
RNA
Slight, structural alterations incurred during phenol ext,raction could have impaired infectivity without great.ly altering EMC! RN,4’s sedimentat’ion properties. This seems unlikely because repeated phenol extractions after the first two served only to eliminate residual intact virus and did not alter infectivit,y or sedimentat,ion prop erties of the Rn’A. In order to determine whet,her a special minor fraction of phenol-resistant, RXA species were being preferentially selected by cells during exposure to infectious RS’A, Krebs,, cells were infected with a sample of RNA used to obtain the data of Fig. 1 and
CELL-ASSOCIATED
RIBONUCLEASE
,iyy
0.250
Fraction
number
1. Sucrose gradient analysis of 14C-uridine labeled EMG RNA. A linear sucrose gradient (520y0) was prepared, and 0.4 ml of EMC RXA was added with 0.4 mg of total cellular RNB (Xrebsll) as carrier. Centrifugation in a Spinco SW-39 head at 4” for 210 minutes was at 39,000 rpm. Three drops per fraction were collected. Counts per fraction, 0; PFU per fraction, 0; optical density 200 FIG.
immediately reextracted (bentonite present at 1 mg/ml) and tikated. A further thousandfold or greater loss of infectivity was not,ed in cell-associated infectivity. This finding, repeated many times under varied conditions, failed to support the n&ion of a specia#l RNA species responsible for all the recoverable infectivity. We found no evidence that, the poor infectivit,y of EMC RNA could be due to insufficient, numbers of infectible cells in a population exposed to RKA. Experiments in which very few cells were exposed to concentrated purified EMC RNA showed that at least 3 5% of them could serve as infe&ous cent,ers. Under the usual assay condit,ioq where the highest RKA infectivity titers mere obtained, only 0.01% of the cells participa,ted as infectious centers. Since phenol-extracted EMC RXA appeared largely intact and showed no evidence for a special minor infectious fraction, and since the cells’ capacity to act as a host for EMC RNA was not exceeded (la,ter sections will show that brief contact’ of EMC RNA with cells in suspension left behind iitt,le or no detectable residual infectivity in the supernat’ant), it, seemed likely that contact with t’he cells used to
assay RNA was actually dest,roying most of it. Either cell-associat,ed or soluble ribonucleases might be responsible. Soluble ribonucleases seemed to be .&her well inhibited by bent,onite since little ioss of infectivity was observed when EItlC RKA was incubated for a few minutes with media in which cells had been suspended. Bentonite at 0.1 mg/ml was preser,t as in our usual assay mixture. Purt,hermore, RNA infect,ivity titers were equivalent whether bent,onite was present at, 0.84 mgjmi III 1.0 mg/ml, indicating t,hat excess amow~is of inhibit,or were present in the ~;wal RX A excluding bauble titrations and virtually ribonucleases from responsibility for ! he poor infectivity of EXIC RNA. Supports is also shown in the for this statement experiment of Fig. 2, where Only rdatkeiy slow inact.ivation of cell surface bound ILK14 infectivity was observed in t,he preser,ce of bentonit!e. In this experiment unlabeled EMC RN FL was first allowed to at,tach for 3 minutes fo lo8 I
600
BASES AND HUPPERT
MINUTES
FIG. 2. Loss of infectivity of cell-bound EMC RNA during delay of hypertonic treatment. At time 0, lo* Krebsrr cells per milliliter were treated with EMC RNA under isotonic conditions for 3 minutes with 0.1 mg/ml of bentonite present. They were then diluted in PBS, dispensed in 10 equal portions, and centrifuged to remove unbound RNA. The pellets were allowed to incubate at 37’ in a humid 5% CO2 incubator. At the indicated times, aliquots were plated for infectious centers with hypertonic treatment, 0, or with isotonic treatment, 0. The shaded interval corresponds to the time for absorption of RNA to the cells and subsequent centrifugation. See text for further details.
infectivity remained in the supernatant. The usual 10e4 of viral infectivity could be recovered if the centrifuged cells were promptly subjected to hypertonic treatment for 1 minute, followed by dilution in ribonuclease containing Eagles’ medium and plating for infectious centers, as in the standard assay. The first point of the upper curve represents this amount of infectivity. The first point of the lower curve indicates the much lower infectivity found if RNAtreated cells simply were kept under isotonic conditions, and then were held in Eagles’ medium with 1 pg of ribonuclease for 2 minutes and plated for infectious centers. Infectivity detected by subjecting other aliquots of the cells to hypertonic treatment at later times is represented by succeeding points of the upper curve. The lower curve indicates corresponding infectivity detected using purely isotonic conditions. Since other experiments showed that the original supernatant medium was free of infectivity and that 2 minutes’ exposure of RNA-treated cells to 1 pg/ml of ribonuclease completely prevented the greater recovery of infectivity due to hypertonic treatment, hypertonic treatment must have acted to promote entry of surface-bound RNA.
2500
FRACTION
NUMBER
FIG. 3. Sucrose gradient analysis of 32P-labeled EMC RNA. Conditions as described for Fig. 1. An aliquot similar to the above material was subjected to a a-minute exposure to 1 pg/ml of RNAase, which destroyed infectivity and caused all azP to sediment in the region of 4 S material. Counts per fraction 0-O; ODzso , O-----O.
CELL-ASSOCIATED
I
5
IO Fraction
RIBONUCLEASE
15
20
25
number
I3
0.200
32~
285
?
0 I
5
IO Fraction
15
20
25
number
FIG. 4. Sucrose gradient analysis of 32P-labeled EMC RNA before and aft,er exposure to Krebsra cells under hypertonic conditions. 37 S, 32P-labeled EMC RNA from a previous sucrose gradient purification contained 5700 cpm. It was mixed with 5 X lo* Krebsrr cells in 6 ml of hypertonic suspeilsion. After 1 minute at room temperature the cells were spun down and plated for infectious cellters; 1350 were found. The supernatnnt contained 5400 counts per minute and only 60 FFU when it was tested on fresh cells. Aiiquots of this material were diluted and mixed with cellular RI’JA. (B) A sucrose gradient analysis of this material after exposure to cells. (A) Analysis of the material before exposure to cells. Cent,rifugation as in Fig. 1. 32P, 0-0; optical density at 260 mp, 0 -----0.
“Entry”
is used in a somewhat
arbitrary
sense, as noted previousiy. Holding RSA-treated cells in PBS led Do progressively smaller increments of infectivity obtainable by hypertonic treatment. This is shown in the gradual decline of the upper curve of Fig. 2 and probably indicates degradation of surface-bound RNA by traces of free ribonuclease or cell mem-
brane ribonuclease acting before hypertonic keatments were applied. In either case, this loss of sudace-bound infectivity is much too slow to account pCor the thousand- or ten thousandfold loss of infectivity when RKA was exposed tc? ce% in the st’andard assay. The flatness of the lower curve of Fig. 2 verifies that there was no harmful etiect
BASES AND HUPPERT
602
0.600
2% Before exposure
to cells
‘“‘9 P *#’
I
5
‘.
‘,
b\
IO Fraction
18s
15
20
25
number
FIG. 5. Exposure of cellular RNA to KrebsII cells under isotonic conditions. 1.5 X 108Krebsn cells were centrifuged twice in PBS and once from PBS minus calcium and magnesium. To the pellet was added 0.25 mg of KrebsIr RNA in 0.5 ml with bentonite at a final concentration of 1 mg/ml. After 2 minutes at 25’ the cells were spun down and the supernatant immediately was subjected to sucrose gradient analysis. The figure compares an anlysis of this RNA before, 0 - - - - - 0, or after, m-0, exposure.
in keeping cells under isotonic conditions before assay, since no decline in infectious centers was observed. There was also no progressive increase in the number of infectious centers among these cells, showing that under isotonic conditions little of the intact surface-bound RXA which was still present on these cells early after attachment could enter unless hypertonic treatment was applied. Nevertheless, at least a hundred infectious centers per 10’ cells were established immediately. Taken together, these findings strongly suggested that cell-associated ribonucleases were indeed responsible for the failure of EMC RNA to fully express its infectivity and that there were at least three possible fates for infectious RNA on contact with cells: fixation followed by slow destruction, fixation and rapid entry, or rapid destruction, to which we now turn. Fate of 32P-labeled EMC RNA on Exposure to Cells 32P-Iabeled EMC RNA was prepared as described by MonCagnier and Sanders (1963). More than 60 % of the IabeI was
found in the 37 S region upon sucrose gradient analysis as seen in Fig. 3. This RNA was used without further purification except as noted. (Its radioactivity to infectivity ratio was between one and 10 cpm per PFU depending on the infectivity titers in the various experiments.) In a preliminary experiment, KrebsII cells at 108/ml were exposed to this labeled RNA. After the usual l-minute exposure, 1000 cpm were cell bound while all the rest of the input of 60,000 cpm were recovered in the supernatant; S800 PFU had become cell associated while only 400 PFU could be found in the supernatant when it was tested on fresh cells. Prolonged exposures in hypertonic medium did not increase the infectivity titers when lo8 cells/ml were exposed to RNA. Since about 98% of the radioactivity remained in the supernatant fluid, another experiment was performed to analyze the sedimentation properties of purified 37 S RNA after a similar exposure to 108 Krebs cells/ml in hypertonic sucrose. 37 S-labeled EMC RNA was isolated from appropriate fractions of a preliminary sucrose gradient,
CELL-ASSOCIATED
and was added to Krebs,, cells under hypertonic conditions. After 1 minute the cells were spun down, and an aliquot of the supernatant (with cellular RSA added as a reference) was analyzed in another sucrose gradient. Figure 4B shows that at least 90% of Lhe radioactivity was in the region of lower S values after exposure to cells while t,he sedimentation properties of the original purified material remained unchanged (Fig. 4A). Other experiments showed continued cellular incorporation of labeled RNA4 which had originally been degraded and left in the medium. Therefore, it, is likely that all t’he recoverable RNA infectivity corresponded to mu& less than 2 % of the original RSA. Since it appeared that cells in suspension degraded EMC RNA, a compnrat’ive experiment (Fig. 5) was performed in which 0.250 mg of cellular RNA from IirebsrI cells were exposed to 1.5 X 108 washed MrebsIr cells in I ml in t>he presence of 1 mg/ml bentonite for 2 minutes at room temperature under isotonic conditions. Sucrose gradient analysis of the supernatant revealed extensive degradation of the RXA. Similar results were obtained in hypertonic medium. This enzymatic activity also to be cell associated since PBS which had been in contact, wit,h cells under the above conditions had far less ribonuclease activity when similarly tested (results not shown), DPSCIXSION
37 S EMC RNA was rapidly degraded during contact wit,11 the cells used to assay its infectivity. This powerful ribonuclease activity appeared to be located at the cell surface, alhhough it is not known if the enzyme is merely adsorbed or actually is part of the cell membrane. Purified 32Pviral RKiZ (Figs. 4A and 4B) under hypertonic conditions or cellular RNA (Fig. 5) under isotonic conditions were both susceptible. Our dat,a indicate that cell-associated ribonuclease is largely responsible for the impaired infectivity of EMC viral RNA. Soluble ribonuclease is almost completely inhibited by bentonit’e and cannot account for the impaired infectivity (Fig. 2). Our results extend those of previous authors by examining both the infectivity as well as the sedimentation properties of
RIBONUCLEASE
603
isotope-labeled purified infectious RbA before and aft,er contact vAt,h tells. In thet;e experiment,s, infect,ious RKA (less than I pg/ml) was exposed to 108 cells/ml. Virtua!ly all infectivity became cell associated in about 1 minute, leaving little or no infeetivity in the supernatant although most of the isotope could be recovered there, now in the form of much lighter, degraded molecules (Figs. 4A, 4B). Our studies can be compared with the earlier ones of Ellem and Colter (i960s,b, 1961) and of Holland et ol. (1960a,b). The latter group described nearly complete loss of infectivity from isotope labeled infectious polio virus RNA solutions after exposure to cells. In concurrence with 0111 results, most of the radioactivity remained in the supernatant (Holla~nd el (EL, 196%). However; Nollend pi al. did not cxamtr:e the sedimentation charact,erist,ics of t,hc supernata& material and unfortunat,ely allowed relatively long exposure of 1U;4. to cells without substantial cont,rol of soluble ribonuclease. Thus, it was not pass:-. ble to decide whet,her, as suggested by Ellem and Colter (1961), most of the cells could not act, as a host for intact RXA ii% whether degradat)ion of t,he RKA COU% account for t,he thousandfold loss of viral infectivit’y. Our own studies implicate CP% associated ribonueleases in t-he impairment of ENC viral infectivity. Awareness of the existence of cell-associated ribonuclease activity recently 1~~s facilitated the isolation and characterization of avian myeloblastosis virus (AMY) R.SA. Rosenbergova et al. (1965) demonstrated intense ribonuclease activity associated wit,b purified AMV, a virus which cbaracterislitally bears celluiar membrane material iu conjunction with its capsid and whose intact~ RKA had aot been previously isolated. Elimination of t,he ribonuelease by treat-ment of the virus w&h proteolyt,ic enzymes (Huppert a,nd Semmel, 1965) led to efficient recovery of 70 S AILIV RNA on subsequenT, (Hupperh et al., 1966). luda (1965) also succeeded in isolating 70 S AXV RNA by employing a protein denaturing detergent, sodium dodecyl sulfate, in conjunct,ion with phenol. Effort,s directed at, circumventing ceil.associated ribonueleases, once their present
BASES
604
AND
is appreciated, might. lead to more efficient methods of introducing intact RNA into mammalian cells. The excellent assistance is gratefully acknowledged.
of Mlle.
Lute Gresland
REFERENCES ALEXANDER, H. E., KOCH, CT., MORGAN-MOUNTAIN, I., and VAN DA~V~IIE, 0. (1958). Infectivity of ribonucleic acid from poliovirus in human cell monolayers. J. Exptl. Med. 108,493-506. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99, 167-182. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130,432437. ELLEM, K. A. O., and COLTER, J. S. (1960a). The interaction of infectious ribonucleic acid with a mammalian cell line. Virology II, 434443. ELLEM, K. A. O., and COLTER, J. S. (1960b). The interaction of infectious ribonucleic acid with a mammalian cell line. Virology 12, 511-520. ELLEM, K. A. O., and COLTER, J. S. (1961). The interaction of infectious ribonucleic acids with mammalian cells. Vi’irology 15, 113-126. FAULKNER, P., MARTIN, E. M., SVED, S., VALENTINE, R. C., and WORK, T. S. (1961). Studies on protein and nucleic acid metabolism in virus infected mammalian cells. Biochem. J. 80, 597605. FRAENKEL-CONRAT, H. (1959). Chemical nature of the infectivity of tobacco mosaic virus. In “Perspectives in Virology” pp. 7-15 (M. PolIard, ed) Wiley, New York. FRAENKEL-CONRAT, H., SINGER, B., and TSUGITA, A. (1961). Purification of viral RNA by means of bentonite. Viiirology 14, 54-58. GUTHRIE, G. D., and SINSHEIMER, R. L. (1960). Infection of protoplasts of Escherichia coli by subviral particles of bacteriophage +X 174. J. Mol. BioZ. 2, 297-305. GUTHRIE, G. D., and SINSHEIMER, R. L. (1963). Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage 6,X 174. Biochim. Biophys. Acta 72, 290-297. HOLLAND, J. J., HOYER, B. H., MCLAREN, L. C., and SYVERTON, J. T. (19GOa). Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. J. Exptl. Med. 112, 821-839. HOLLAND, J. J., MCLAREN, L. C., HOYER, B. H., and SYVERTON, J. T. (1960b). Enteroviral ribonucleic acid. II. Biological, physical and chemical studies. J. Exptl. Med. 112, 841-864. HOSKINS, J. M. (1961). Induction of cell competence to infection by poliovirus ribonucleic
HUPPERT acid by treatment with molar sodium chloride. Brit. J. Exptl. Pathol. 42, 95-98. HUPPERT, J., and SEMMEL, M. (1965). Suppression de l’activite ribonucleasique par la pronase. Biochim. Biophys. Acta 108, 501-503. HUPPERT, J., WAHL, R., and I%VIERIQUE-BLUM, L. (1962). Conditions de l’infection par l’acide desoxyribonucleique du phage +X 174. Biochim. Biophys. Acta 55, 1822201. HUPPERT, J., HAREL, J., LACOUR, F., and HAREL, L. (1966). High molecular weight RNA from avian myeloblastosis virus. Cancer Res. 26, No. 7, in press. KAIGHN, M. E., MOSCARELLO, M. A., and FUERST, C. R. (1964). Purification of murine eneephalomyocarditis virus. Virology 23, 183-194. LOEVINGER, R., and BERMAN, M. (1951). Efficiency criteria in radioactive counting. Nucleonics 9, No. I, 26-39. MACPHERSON, J. (1963). Characteristics of a hamster cell clone transformed by polyoma virus. J. NatZ. Cancer Inst. 30, 795-815. MONTAGNIER, L., and SANDERS, F. K. (1962). Titrage de l’acide ribonucleique infectieux du virus de l’encephalomyocarditis de la Souris sur cellules d’ascites de Krebsir in vitro. Compt. Rend. Acad. Sci. 254, 2247-2249. MONTAGNIER, L., and SANDERS, F. K. (1963). Sedimentation properties of infective ribonucleic acid extracted from encephalomyocarditis virus. Nature 197, 1178-1181. MOSCARELLO, M. A. (1965). Extraction and assay of infectious ribonucleic acid from L cells infected with murine encephalomyocarditis virus. Virology 26, 687-693. ROSENBERGOVA, M., LACOUR, F., and HUPPERT, J. (1965). Mise en evidence d’une activite nucleasique associee au virus de la myeloblastose aviare lors de tentatives de purification de ce virus et de son acide ribonuclkque. Compt. Rend. Acad. Sci. 260, 5145-5148. ROBINSON, W. S., alld BALUDA, M. (1965). The
nucleic acid from avian myeloblastosis
virus.
Proc. Natl. Acad. Sci. U.S. 54, 1686-1692. SANDERS, F. B., and BURFORD, B. 0. (1964). Ascites tumors from BHK 21 cells transformed in vitro by polyoma virus. Nature 201, 786-789. SANDERS, F. K., HUPPERT, J., and HOSKINS, J. M. (1958). Replication of an animal virus. Symp. Sot. Exptl. BioZ. 12, 123-137. WAHL, R., HUPPERT, J., and ~MERIQUE-BLUM, L. (1960). Production de phages par des “protoplastes” bacteriens par des preparations d’acide d&oxyribonucl&ique. Compt. Rend. Acad. Sci. 250, 4227-4229. WEIL, R. (1961). A quantitative assay for a subviral infective agent related to polyoma virus. Virology 14, 46-53.
29, 596404
Impairment
(1966)
of EMC Viral ROBERT
Service de Biologie
Infectivity E. BASES2
Molkculaire
by Cell-Associated JOSEPH
AND
Institut
Gustave Roussy,
Accepted April
Ribonuclease’
HUPPERT Villejuif-Seine,
France
20, 1966
The fate of purified labeled infectious RNA of encephalomyocarditis virus in suspensions of mouse or hamster cells was studied in order to determine why only lO+ of the corresponding viral infectivity could be recovered. All the recoverable RNA infectivity became cell bound in about 1 minute, but more than 987, of the radioactivity of labeled 37 S EMC RNA was detected in the supernatant as degraded fragments. It was concluded that t,he loss in infectivity was mainly due to rapid degradation of the viral RNA by cell-associated ribonuclease. INTRODUCTION
sheimer, 1963). These facts suggest t’hat’ some cellular activity very strongly influences the outcome. Our results with the infectious ribonucleic acid (RNA) of encephalomyocarditis virus (EMC) help clarify the problem by demonstrating that mammalian cells very rapidly degrade viral RNA while simultaneously binding only a small portion intact. Ent’ry of this small portion is enhanced by hypertonic treat,ment of the cells. Since very rapid degradation of viral 01 cellular ribosomal RSA occurred in cell suspensions where free ribonucleases were virtually eliminated by bentonite (Fraenkel-Conrat el al., 1961), it is suggested t,hat cell-associated or bound ribonucleases are responsible in great degree for the RiYL4’s loss of biological acbivity.
It is well known that viral nucleic acids have far less infectious activity (lop3 to lo-+) than the animal or bacterial viruses from which they were ext’racted (Alexander et al., 1958; Ellem and Colter, 1960a; Holland et al., 1960a; Weil, 1961; FraenkelConrat, 1959; Hoskins, 1961; Wahl et al., 1960; Guthrie and Sinsheimer, 1960; Huppert et al., 1962; Guthrie and Sinsheimer, 1963). While this loss might occur at any stage between ext,raction of the nucleic acid and liberation of progeny virus, partial improvement in efficiency of infection has been obtained with mammalian cells by subjecting them to hypertonic treatment (Alexander et al. 1958), or by m0derat.e exposure to elevat,ed temperat,ures or to amines (Rloscarello, 1965). In order to introduce bacterial virus nucleic acids int,o cells, it is generally necessary t’o use spheroplasts instead of normal bacteria. Even so, the efficiency varies greatly wit,h changes in the manner in which the cells are treated (Huppert et al., 1962; Guthrie and Sin-
MATERIALS
AND
METHODS
Cells. Krebs,, mouse ascites t’umor cells were grown in Swiss mice and harvested 6 or 7 days after intraperitoneal inoculation of about 10’ cells. Aliquots of a large inoculum, stored at -7O”, were thawed at regular
1 Supported by D&gation G&&ale B la Recherche Scientifique et Technique, contrat no. 62 FR 054. 2 Recipient of special Fellowship 13,275 of the National Cancer Institute, U.S.P.H.S., and of PO Eleanor Roosevelt Cancer Research Foundation Fellowship. Present address: Albert Einstein College of Medicine, Bronx, New York 10461.
intervals mouse
to
avoid
continuous
mouse
to
passage. A hamster ascites tumor cell line, T-6, grown in suspension cultures in vitro, was also used. This cell line was sent to us by Dr. F. K. Sanders (Sanders and Burford, 1964), who isolated it in ascit,es form
596
from
polyoma
virus
transformed
CELL-ASSOCIATED
BHK-21 clone 13 cells, originally provided by Drs. J. MacPherson and RI. St’oker (MacPherson, 1963). T-6 cells grew in suspension cult,ures in modified Eagle’s medium (1959) with twice the usual concentration of amino acids and vitamins, 10 times the usual phosphate concentrat,ion, and no calcium except that provided by supplementation with calf serum t’o 10 70, and tryptose phosphate to 10 %. Generation t.imes were 16-19 hours under these condit,ions. T-6 cells in suspension cultures were easily dispersed t.o single cell suspensions by pipetting. ~+zM. EMC virus stocks (1 to 2 X log PFU/ml) were prepared using Krebs II cells as described by Sanders et al. (1958). Plaque assays were performed by the agar suspension method (Sanders et al., 1958). In this assay, virus and 1.5 lo8 KrebsII cells were mixed and plated in agar at, 0.8 % on an underlayer of 1.5 % agar in Earle’s saline. Two days’ incubation at 37” in a moist incubator in 5% CO2 yielded 5-10 mm plaques when the petri dishes were st,ained for 1 hour with 1: 10,000 neutral red. J’irus puri$cation. Virus stocks were purified using centrifugat,ion, trypsinizat,ion, and chromatography on calcium phosphate columns (Faulkner et al., 1961). 32P-labeled virus was subjected to an additional purification as described by Montagnier and Sanders (1963) in which virus eluted from an initial calcium phosphate column was treat~ed with 1% sodium deoxycholate to lyse cellular membrane material which was occasionally encountered using the original method. A second calcium phosphate column purification followed. Montagnier and Sanders’ modification yielded a single virus component as det’ermined in equilibrium density cent’rifugation studies. Kaighn et al. (1964) have recently provided an improved purification of E12C virus from cellular material using pyrophosphate and ribonuclease. While we cannot exclude contamination of our virus preparation wit,h tiny amounts of cellular RNA, sucrose gradient analysis of t’he RNA indicates that most of it is indeed viral. Phenol extraction. Bent,onite (FraenkelConrat et ab., 1961) at 1 mg/ml was added
RIBONUCLEASE
597
to column purified virus (in 0.3 M phosphate) before 4 extractions with equal volumes of water saturated phenol. The aqueous layer was extracted 6 times with ether and then bubbled with nitrogen to remove the ether. In some experiments viral RNA was precipitated wit,h 66% ethanol using Krebs,, cellular RI\‘A as a carrier. This material, designated RNA, was stored at -70”. Aliquots were usually diluted before use. All preparations were sensitive t,o 1 pg/ml crystalline RXAase (Worthingt,on) at, room temperature for 2 minut’es. RNA assay. The procedure of Mont’agnier and Sanders (1962) was used with a few minor changes. Cells were washed 2 or 3 times by centrifugation in phosphatebuffered saline (PBS) (Dulbecco and Vogt, 1954) and then once in PBS wit,hout calcium and magnesium, just before use. The cell pellet was stirred gently with an equal volume of distilled water and to this was immediat,ely added an equal volume of a 1: 1 mixture of RNA and 2.6 M sucrose in 0.5 M sodium phosphate. After 60 seconds, the resulting mixt,ure was diluted six-fold with Eagle’s medium. RiYAase at 1 pg/ml was present in t#he Eagle’s medium to destroy any residual RNA. Then 1.5 x lo8 Krebs cells were added and plating was performed as in the virus assay. Cell concentrations before adding t,he Eagle’s medium were about 1O*/ml. Infectivity varied linearly with Rr\‘A concent,ration over a hundredfold dilut,ion. For convenience, we have arbitrarily referred to cell-associated RNA as being on or outside cells if 1 pg/ml of RNAase abolished infectious activity in 2 minutes at, room temperat,ure, and we considered it inside cells if RlSAase could no longer interfere. Krebsn mouse ascites cells or hamster cells from cultures in vitro served equally well as infectious cent.ers but Krebs,, cells were used t,hroughout to provide the agar suspension layer (for practical reasons). EMC virus titrations made with different cell preparat,ions rarely varied more than t,wofold, but, greater fluct’uations in RNA titers were noted from one experiment to another. However, with any single batch of cells reproducibility was comparable with
598
BASES
AND
t.hat in virus titrations. RNA t.it,ers did not prove especially dependent on t.emperature above 15”. Radioactive labeled GYM. 32P-labeled virus was prepared and purified according t’o Montagnier and Sanders (1963). 14C-Uridinelabeled virus was prepared by adding 14Curidine, .50 PC (Schwarz, 251 microcuries/ micromole), to Krebs,, cells at, 2 X 107/m1 in Earle’s saline 1 hour aft,er infection atJ a multiplicity of 3. 14C-Cridine-labeled virus purification was performed by the method of Faulkner et al. (1961). 14C-Uridine-labeled RSA was precipitated wit.h ethanol wit,h 200 pg of Krebsn cell RNA as carrier. After centrifugation the RNA was deposited uniformly on the top of slowly rotating planchet.tes, dried, and countled in a Trarerlab flow count,er sufficiently long to achieve an accuracy of 5%:. (Loevinger and Berman, 1951), at 33 %I efficiency. Sucrose gradients. Linear 5-20 % sucrose gradients were prepared in 0.1%. bentonit,e, nZgC& 0.001 M in distilled water and were run at 39,000 rpm in t,he model L Spinco ult,racent.rifuge in the SW 39 head. RNAase. Crystalline pancreatic ribonuclease (Worthington) was used. RESULTS
Recovery of Viral RNA Uridine Labeled EMC
from Virus
Purified
W-
Infectious Rr\‘A prepared from purified virus was assayed repeatedly, but at best its infectivity was only 10m3 of the virus before phenol ext,raction. Since the small amounts of purified virus material precluded use of opt,ical densit.y measurements to detect physical losses or of alterations in t.he sedimentat,ion pr0pert.y of the RNA, we studied radioactive labeled virus instead. Purified 14C-uridine-labeled EMC virus rvas extracted by phenol (bentonite at 1 mg/ml) and examined for its radioactivity, infectivity, and sedimentation properties. Table 1 and Fig. 1 show that very little viral RIYA was lost. or degraded during extraction, but t,hat. its biological activit.y was great,ly impaired. The correspondence of 14C label and infectivit,y in the 37 S region of Fig. 1 are within t.he variation of the plaque assay.
HUPPERT TABLE
1
RECOVERY OF W-URIDINE RNA EMC VIRUS' Material Virus (column purified) RNA (aqueous phase) Interphase Ether Phenol
FROM
Total PFU
Total counts per minute
1.3 x 10’0
1.20 x
105
2.0 x
1.01 x
105
0.13 x
105
Not
106b
tested
a Radioactive virus was purified and concentrat.ed after Faulkner et al. (1961). 837& of the input radioactivity was recovered in the infectious RNA. See Fig. 1 for sucrose gradient analysis of this material. b Mean in three experiments.
Our result,s are t,hus in excellent agreement with those of Montagnier and Sanders (1963), who previously characterized ERIC RNA. We cannot entirely exclude some cont,aminat.ion of the 14C-uridine-labeled 37 S viral RNA with labeled cellular RNA because the virus purificat,ion of Faulkner et al. (1961) is not as complete as that of Kaighn et al (1964) or of Montagnier and Sanders (1963). Nevertheless, wc can safely assume that little cellular RXA was present. in the 37 S region since so lit,tle label was detect’ed in t,he fractions where Krebs cell RSA (added as a reference) was found by opticaal density measurements. Impaired
Infectivity
of 37 S EMC
RNA
Slight, structural alterations incurred during phenol ext,raction could have impaired infectivity without great.ly altering EMC! RN,4’s sedimentat’ion properties. This seems unlikely because repeated phenol extractions after the first two served only to eliminate residual intact virus and did not alter infectivit,y or sedimentat,ion prop erties of the Rn’A. In order to determine whet,her a special minor fraction of phenol-resistant, RXA species were being preferentially selected by cells during exposure to infectious RS’A, Krebs,, cells were infected with a sample of RNA used to obtain the data of Fig. 1 and
CELL-ASSOCIATED
RIBONUCLEASE
,iyy
0.250
Fraction
number
1. Sucrose gradient analysis of 14C-uridine labeled EMG RNA. A linear sucrose gradient (520y0) was prepared, and 0.4 ml of EMC RXA was added with 0.4 mg of total cellular RNB (Xrebsll) as carrier. Centrifugation in a Spinco SW-39 head at 4” for 210 minutes was at 39,000 rpm. Three drops per fraction were collected. Counts per fraction, 0; PFU per fraction, 0; optical density 200 FIG.
immediately reextracted (bentonite present at 1 mg/ml) and tikated. A further thousandfold or greater loss of infectivity was not,ed in cell-associated infectivity. This finding, repeated many times under varied conditions, failed to support the n&ion of a specia#l RNA species responsible for all the recoverable infectivity. We found no evidence that, the poor infectivit,y of EMC RNA could be due to insufficient, numbers of infectible cells in a population exposed to RKA. Experiments in which very few cells were exposed to concentrated purified EMC RNA showed that at least 3 5% of them could serve as infe&ous cent,ers. Under the usual assay condit,ioq where the highest RKA infectivity titers mere obtained, only 0.01% of the cells participa,ted as infectious centers. Since phenol-extracted EMC RXA appeared largely intact and showed no evidence for a special minor infectious fraction, and since the cells’ capacity to act as a host for EMC RNA was not exceeded (la,ter sections will show that brief contact’ of EMC RNA with cells in suspension left behind iitt,le or no detectable residual infectivity in the supernat’ant), it, seemed likely that contact with t’he cells used to
assay RNA was actually dest,roying most of it. Either cell-associat,ed or soluble ribonucleases might be responsible. Soluble ribonucleases seemed to be .&her well inhibited by bent,onite since little ioss of infectivity was observed when EItlC RKA was incubated for a few minutes with media in which cells had been suspended. Bentonite at 0.1 mg/ml was preser,t as in our usual assay mixture. Purt,hermore, RNA infect,ivity titers were equivalent whether bent,onite was present at, 0.84 mgjmi III 1.0 mg/ml, indicating t,hat excess amow~is of inhibit,or were present in the ~;wal RX A excluding bauble titrations and virtually ribonucleases from responsibility for ! he poor infectivity of EXIC RNA. Supports is also shown in the for this statement experiment of Fig. 2, where Only rdatkeiy slow inact.ivation of cell surface bound ILK14 infectivity was observed in t,he preser,ce of bentonit!e. In this experiment unlabeled EMC RN FL was first allowed to at,tach for 3 minutes fo lo8 I
600
BASES AND HUPPERT
MINUTES
FIG. 2. Loss of infectivity of cell-bound EMC RNA during delay of hypertonic treatment. At time 0, lo* Krebsrr cells per milliliter were treated with EMC RNA under isotonic conditions for 3 minutes with 0.1 mg/ml of bentonite present. They were then diluted in PBS, dispensed in 10 equal portions, and centrifuged to remove unbound RNA. The pellets were allowed to incubate at 37’ in a humid 5% CO2 incubator. At the indicated times, aliquots were plated for infectious centers with hypertonic treatment, 0, or with isotonic treatment, 0. The shaded interval corresponds to the time for absorption of RNA to the cells and subsequent centrifugation. See text for further details.
infectivity remained in the supernatant. The usual 10e4 of viral infectivity could be recovered if the centrifuged cells were promptly subjected to hypertonic treatment for 1 minute, followed by dilution in ribonuclease containing Eagles’ medium and plating for infectious centers, as in the standard assay. The first point of the upper curve represents this amount of infectivity. The first point of the lower curve indicates the much lower infectivity found if RNAtreated cells simply were kept under isotonic conditions, and then were held in Eagles’ medium with 1 pg of ribonuclease for 2 minutes and plated for infectious centers. Infectivity detected by subjecting other aliquots of the cells to hypertonic treatment at later times is represented by succeeding points of the upper curve. The lower curve indicates corresponding infectivity detected using purely isotonic conditions. Since other experiments showed that the original supernatant medium was free of infectivity and that 2 minutes’ exposure of RNA-treated cells to 1 pg/ml of ribonuclease completely prevented the greater recovery of infectivity due to hypertonic treatment, hypertonic treatment must have acted to promote entry of surface-bound RNA.
2500
FRACTION
NUMBER
FIG. 3. Sucrose gradient analysis of 32P-labeled EMC RNA. Conditions as described for Fig. 1. An aliquot similar to the above material was subjected to a a-minute exposure to 1 pg/ml of RNAase, which destroyed infectivity and caused all azP to sediment in the region of 4 S material. Counts per fraction 0-O; ODzso , O-----O.
CELL-ASSOCIATED
I
5
IO Fraction
RIBONUCLEASE
15
20
25
number
I3
0.200
32~
285
?
0 I
5
IO Fraction
15
20
25
number
FIG. 4. Sucrose gradient analysis of 32P-labeled EMC RNA before and aft,er exposure to Krebsra cells under hypertonic conditions. 37 S, 32P-labeled EMC RNA from a previous sucrose gradient purification contained 5700 cpm. It was mixed with 5 X lo* Krebsrr cells in 6 ml of hypertonic suspeilsion. After 1 minute at room temperature the cells were spun down and plated for infectious cellters; 1350 were found. The supernatnnt contained 5400 counts per minute and only 60 FFU when it was tested on fresh cells. Aiiquots of this material were diluted and mixed with cellular RI’JA. (B) A sucrose gradient analysis of this material after exposure to cells. (A) Analysis of the material before exposure to cells. Cent,rifugation as in Fig. 1. 32P, 0-0; optical density at 260 mp, 0 -----0.
“Entry”
is used in a somewhat
arbitrary
sense, as noted previousiy. Holding RSA-treated cells in PBS led Do progressively smaller increments of infectivity obtainable by hypertonic treatment. This is shown in the gradual decline of the upper curve of Fig. 2 and probably indicates degradation of surface-bound RNA by traces of free ribonuclease or cell mem-
brane ribonuclease acting before hypertonic keatments were applied. In either case, this loss of sudace-bound infectivity is much too slow to account pCor the thousand- or ten thousandfold loss of infectivity when RKA was exposed tc? ce% in the st’andard assay. The flatness of the lower curve of Fig. 2 verifies that there was no harmful etiect
BASES AND HUPPERT
602
0.600
2% Before exposure
to cells
‘“‘9 P *#’
I
5
‘.
‘,
b\
IO Fraction
18s
15
20
25
number
FIG. 5. Exposure of cellular RNA to KrebsII cells under isotonic conditions. 1.5 X 108Krebsn cells were centrifuged twice in PBS and once from PBS minus calcium and magnesium. To the pellet was added 0.25 mg of KrebsIr RNA in 0.5 ml with bentonite at a final concentration of 1 mg/ml. After 2 minutes at 25’ the cells were spun down and the supernatant immediately was subjected to sucrose gradient analysis. The figure compares an anlysis of this RNA before, 0 - - - - - 0, or after, m-0, exposure.
in keeping cells under isotonic conditions before assay, since no decline in infectious centers was observed. There was also no progressive increase in the number of infectious centers among these cells, showing that under isotonic conditions little of the intact surface-bound RXA which was still present on these cells early after attachment could enter unless hypertonic treatment was applied. Nevertheless, at least a hundred infectious centers per 10’ cells were established immediately. Taken together, these findings strongly suggested that cell-associated ribonucleases were indeed responsible for the failure of EMC RNA to fully express its infectivity and that there were at least three possible fates for infectious RNA on contact with cells: fixation followed by slow destruction, fixation and rapid entry, or rapid destruction, to which we now turn. Fate of 32P-labeled EMC RNA on Exposure to Cells 32P-Iabeled EMC RNA was prepared as described by MonCagnier and Sanders (1963). More than 60 % of the IabeI was
found in the 37 S region upon sucrose gradient analysis as seen in Fig. 3. This RNA was used without further purification except as noted. (Its radioactivity to infectivity ratio was between one and 10 cpm per PFU depending on the infectivity titers in the various experiments.) In a preliminary experiment, KrebsII cells at 108/ml were exposed to this labeled RNA. After the usual l-minute exposure, 1000 cpm were cell bound while all the rest of the input of 60,000 cpm were recovered in the supernatant; S800 PFU had become cell associated while only 400 PFU could be found in the supernatant when it was tested on fresh cells. Prolonged exposures in hypertonic medium did not increase the infectivity titers when lo8 cells/ml were exposed to RNA. Since about 98% of the radioactivity remained in the supernatant fluid, another experiment was performed to analyze the sedimentation properties of purified 37 S RNA after a similar exposure to 108 Krebs cells/ml in hypertonic sucrose. 37 S-labeled EMC RNA was isolated from appropriate fractions of a preliminary sucrose gradient,
CELL-ASSOCIATED
and was added to Krebs,, cells under hypertonic conditions. After 1 minute the cells were spun down, and an aliquot of the supernatant (with cellular RSA added as a reference) was analyzed in another sucrose gradient. Figure 4B shows that at least 90% of Lhe radioactivity was in the region of lower S values after exposure to cells while t,he sedimentation properties of the original purified material remained unchanged (Fig. 4A). Other experiments showed continued cellular incorporation of labeled RNA4 which had originally been degraded and left in the medium. Therefore, it, is likely that all t’he recoverable RNA infectivity corresponded to mu& less than 2 % of the original RSA. Since it appeared that cells in suspension degraded EMC RNA, a compnrat’ive experiment (Fig. 5) was performed in which 0.250 mg of cellular RNA from IirebsrI cells were exposed to 1.5 X 108 washed MrebsIr cells in I ml in t>he presence of 1 mg/ml bentonite for 2 minutes at room temperature under isotonic conditions. Sucrose gradient analysis of the supernatant revealed extensive degradation of the RXA. Similar results were obtained in hypertonic medium. This enzymatic activity also to be cell associated since PBS which had been in contact, wit,h cells under the above conditions had far less ribonuclease activity when similarly tested (results not shown), DPSCIXSION
37 S EMC RNA was rapidly degraded during contact wit,11 the cells used to assay its infectivity. This powerful ribonuclease activity appeared to be located at the cell surface, alhhough it is not known if the enzyme is merely adsorbed or actually is part of the cell membrane. Purified 32Pviral RKiZ (Figs. 4A and 4B) under hypertonic conditions or cellular RNA (Fig. 5) under isotonic conditions were both susceptible. Our dat,a indicate that cell-associated ribonuclease is largely responsible for the impaired infectivity of EMC viral RNA. Soluble ribonuclease is almost completely inhibited by bentonit’e and cannot account for the impaired infectivity (Fig. 2). Our results extend those of previous authors by examining both the infectivity as well as the sedimentation properties of
RIBONUCLEASE
603
isotope-labeled purified infectious RbA before and aft,er contact vAt,h tells. In thet;e experiment,s, infect,ious RKA (less than I pg/ml) was exposed to 108 cells/ml. Virtua!ly all infectivity became cell associated in about 1 minute, leaving little or no infeetivity in the supernatant although most of the isotope could be recovered there, now in the form of much lighter, degraded molecules (Figs. 4A, 4B). Our studies can be compared with the earlier ones of Ellem and Colter (i960s,b, 1961) and of Holland et ol. (1960a,b). The latter group described nearly complete loss of infectivity from isotope labeled infectious polio virus RNA solutions after exposure to cells. In concurrence with 0111 results, most of the radioactivity remained in the supernatant (Holla~nd el (EL, 196%). However; Nollend pi al. did not cxamtr:e the sedimentation charact,erist,ics of t,hc supernata& material and unfortunat,ely allowed relatively long exposure of 1U;4. to cells without substantial cont,rol of soluble ribonuclease. Thus, it was not pass:-. ble to decide whet,her, as suggested by Ellem and Colter (1961), most of the cells could not act, as a host for intact RXA ii% whether degradat)ion of t,he RKA COU% account for t,he thousandfold loss of viral infectivit’y. Our own studies implicate CP% associated ribonueleases in t-he impairment of ENC viral infectivity. Awareness of the existence of cell-associated ribonuclease activity recently 1~~s facilitated the isolation and characterization of avian myeloblastosis virus (AMY) R.SA. Rosenbergova et al. (1965) demonstrated intense ribonuclease activity associated wit,b purified AMV, a virus which cbaracterislitally bears celluiar membrane material iu conjunction with its capsid and whose intact~ RKA had aot been previously isolated. Elimination of t,he ribonuelease by treat-ment of the virus w&h proteolyt,ic enzymes (Huppert a,nd Semmel, 1965) led to efficient recovery of 70 S AILIV RNA on subsequenT, (Hupperh et al., 1966). luda (1965) also succeeded in isolating 70 S AXV RNA by employing a protein denaturing detergent, sodium dodecyl sulfate, in conjunct,ion with phenol. Effort,s directed at, circumventing ceil.associated ribonueleases, once their present
BASES
604
AND
is appreciated, might. lead to more efficient methods of introducing intact RNA into mammalian cells. The excellent assistance is gratefully acknowledged.
of Mlle.
Lute Gresland
REFERENCES ALEXANDER, H. E., KOCH, CT., MORGAN-MOUNTAIN, I., and VAN DA~V~IIE, 0. (1958). Infectivity of ribonucleic acid from poliovirus in human cell monolayers. J. Exptl. Med. 108,493-506. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99, 167-182. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130,432437. ELLEM, K. A. O., and COLTER, J. S. (1960a). The interaction of infectious ribonucleic acid with a mammalian cell line. Virology II, 434443. ELLEM, K. A. O., and COLTER, J. S. (1960b). The interaction of infectious ribonucleic acid with a mammalian cell line. Virology 12, 511-520. ELLEM, K. A. O., and COLTER, J. S. (1961). The interaction of infectious ribonucleic acids with mammalian cells. Vi’irology 15, 113-126. FAULKNER, P., MARTIN, E. M., SVED, S., VALENTINE, R. C., and WORK, T. S. (1961). Studies on protein and nucleic acid metabolism in virus infected mammalian cells. Biochem. J. 80, 597605. FRAENKEL-CONRAT, H. (1959). Chemical nature of the infectivity of tobacco mosaic virus. In “Perspectives in Virology” pp. 7-15 (M. PolIard, ed) Wiley, New York. FRAENKEL-CONRAT, H., SINGER, B., and TSUGITA, A. (1961). Purification of viral RNA by means of bentonite. Viiirology 14, 54-58. GUTHRIE, G. D., and SINSHEIMER, R. L. (1960). Infection of protoplasts of Escherichia coli by subviral particles of bacteriophage +X 174. J. Mol. BioZ. 2, 297-305. GUTHRIE, G. D., and SINSHEIMER, R. L. (1963). Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage 6,X 174. Biochim. Biophys. Acta 72, 290-297. HOLLAND, J. J., HOYER, B. H., MCLAREN, L. C., and SYVERTON, J. T. (19GOa). Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. J. Exptl. Med. 112, 821-839. HOLLAND, J. J., MCLAREN, L. C., HOYER, B. H., and SYVERTON, J. T. (1960b). Enteroviral ribonucleic acid. II. Biological, physical and chemical studies. J. Exptl. Med. 112, 841-864. HOSKINS, J. M. (1961). Induction of cell competence to infection by poliovirus ribonucleic
HUPPERT acid by treatment with molar sodium chloride. Brit. J. Exptl. Pathol. 42, 95-98. HUPPERT, J., and SEMMEL, M. (1965). Suppression de l’activite ribonucleasique par la pronase. Biochim. Biophys. Acta 108, 501-503. HUPPERT, J., WAHL, R., and I%VIERIQUE-BLUM, L. (1962). Conditions de l’infection par l’acide desoxyribonucleique du phage +X 174. Biochim. Biophys. Acta 55, 1822201. HUPPERT, J., HAREL, J., LACOUR, F., and HAREL, L. (1966). High molecular weight RNA from avian myeloblastosis virus. Cancer Res. 26, No. 7, in press. KAIGHN, M. E., MOSCARELLO, M. A., and FUERST, C. R. (1964). Purification of murine eneephalomyocarditis virus. Virology 23, 183-194. LOEVINGER, R., and BERMAN, M. (1951). Efficiency criteria in radioactive counting. Nucleonics 9, No. I, 26-39. MACPHERSON, J. (1963). Characteristics of a hamster cell clone transformed by polyoma virus. J. NatZ. Cancer Inst. 30, 795-815. MONTAGNIER, L., and SANDERS, F. K. (1962). Titrage de l’acide ribonucleique infectieux du virus de l’encephalomyocarditis de la Souris sur cellules d’ascites de Krebsir in vitro. Compt. Rend. Acad. Sci. 254, 2247-2249. MONTAGNIER, L., and SANDERS, F. K. (1963). Sedimentation properties of infective ribonucleic acid extracted from encephalomyocarditis virus. Nature 197, 1178-1181. MOSCARELLO, M. A. (1965). Extraction and assay of infectious ribonucleic acid from L cells infected with murine encephalomyocarditis virus. Virology 26, 687-693. ROSENBERGOVA, M., LACOUR, F., and HUPPERT, J. (1965). Mise en evidence d’une activite nucleasique associee au virus de la myeloblastose aviare lors de tentatives de purification de ce virus et de son acide ribonuclkque. Compt. Rend. Acad. Sci. 260, 5145-5148. ROBINSON, W. S., alld BALUDA, M. (1965). The
nucleic acid from avian myeloblastosis
virus.
Proc. Natl. Acad. Sci. U.S. 54, 1686-1692. SANDERS, F. B., and BURFORD, B. 0. (1964). Ascites tumors from BHK 21 cells transformed in vitro by polyoma virus. Nature 201, 786-789. SANDERS, F. K., HUPPERT, J., and HOSKINS, J. M. (1958). Replication of an animal virus. Symp. Sot. Exptl. BioZ. 12, 123-137. WAHL, R., HUPPERT, J., and ~MERIQUE-BLUM, L. (1960). Production de phages par des “protoplastes” bacteriens par des preparations d’acide d&oxyribonucl&ique. Compt. Rend. Acad. Sci. 250, 4227-4229. WEIL, R. (1961). A quantitative assay for a subviral infective agent related to polyoma virus. Virology 14, 46-53.