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Studies on reovirus RNA
J. Mol.
Biol.
(1967) 29,1-17
Studies on Reovirus RNA I. Characterization A. R. BELLAMY, Departments
of Reovirus Genome RNA
LUCILLE XHAPIRO, J. T. AUGUST AND WOLFGANG K. JOKLIK
of Cell Biology
and Molecular Biology, Albert Einstein Bronx 61, N. Y. 10461, U.S.A.
(Received 28 March
College of Medicine
1967, and in revised form 22 May 1967)
Chemical treatment does not release the genome of reovirus in intact form. Irrespective of the procedure used, the same mixture of fragments is released with sedimentation coefficients of about 14, 12 and 10.5 s. On electrophoresis in polyacrylamide gels these size classes can be further resolved, the two former into two are double-stranded as components each, the last into three. These fragments judged by the following criteria: they exhibit a sharp melting profile with a T, dependent on the ionic concentration; they are resistant to ribonuclease, provided that the concentrations of both monovalent and divalent ions, as well as that of the enzyme, are suitably adjusted: their sedimentation behavior is independent of the ionic concentration over a wide range ; and their base composition displays equality of A and U, as well as of G and C. In addition to this mixture of double-stranded fragments there is released from virions an RNA of small size (2 to 3 s) which amounts to about 20% of the mass of the viral genome. The base composition of this RNA is 82% A, 13% U, 3% G and 3% C. This A-rich RNA is synthesized in step with the viral genome RNA. RNA molecules corresponding to intact viral genomes were not detected in infected cells at times when the viral genome was replicating. Irrespective of the method of breaking open the cells, or of the cell strain used, the same mixture of RNA fragments was obtained as was released from virions. These results suggest that the reovirus genome consists of a number of segments of double-stranded RNA; further, it would appear that the reovirus genome does not replicate as one intact molecule, but rather in the form of these same segments. The molecular weights of the L, M and S segments have been estimated by reference to three sets of data in the literature; the most likely values are 2.3 + 0.2 x 106, 1.3 + 0.2 x lo6 and 8 f 2 x 105. L, M and S segments are released from virions in the ratio of n : n : 1*5n. Owing to uncertainty concerning the size of the reovirus genome, the exact number of segments of which it is composed cannot be specified. The most likely value of m is 2, although a value of 3 or even 4 is not ruled out. The function of the A-rich material is not known. It is conceivable that it serves to link the double-stranded segments, giving rise to a structure which is so labile that it does not withstand any but the most gentle manipulation. However, the precise manner in which linking is accomplished, and the role, if any, of the Arich material in this process must await further work.
1. Introduction Reovirus RNA displays, under appropriate conditions, a sharp melting profile, resistance to ribonuclease and a base composition with approximately equimolar amounts of A and U on the one hand and G and C on the other (Gomatos $ Tamm, 1
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AND
JOKLIK
1963a,b). For these reasons, and because X-ray diffraction analysis lends support, it has been designated as double-stranded RNA (Langridge & Gomatos, 1963). However, there is as yet no proof that the genome of reovirus does, in fact, consist of two intact antiparallel complementary polynucleotide strands. RNA molecules corresponding to the entire genome have not yet been liberated from the virions. Very gentle dissociation of virions on protein monolayers yields structures about 5 p long (Granboulan & Niveleau, 1967). More recently, Vasquez & Kleinschmidt (1967) observed assemblies of filaments radiating from reovirus virions disrupted in situ on protein monolayers, the length of which added up to about 8 CL,and detected occasional long filaments up to 7.4 TVlong. However, when virions are disrupted prior to spreading on protein monolayers, a mixture of fragments with a tri-modal length distribution is obtained, with maxima at 1.1 k, O-6 TVand 0.35 TV(Dunnebacke & Kleinschmidt, 1967). One can summarize the current state of knowledge as follows : while the reovirus RNA genome appears to be highly base-paired, it is very fragile, suggesting the presence of regions where breakage occurs with great ease. In addition to the reovirus genome, viral messenger RNA is synthesized in infected cells. As would be expected, this messenger RNA is found in polyribosomes and hybridizes with viral genome RNA (Prevec & Graham, 1966; Shatkin & Rada, 1967). The object of the work reported in this paper was to attempt to isolate from reovirus virions, RNA molecules which would correspond to the entire genome and to examine their structure. However, irrespective of the procedure employed, no intact genome molecules were obtained. We found, as had Dunnebacke & Kleinschmidt, that the extracted genome RNA fragments fell into a size distribution with three maxima, which was highly reproducible and independent of the extraction procedure. These RNA fragments proved to be double-stranded according to a number of criteria, and were readily converted to the single-stranded state. Further, we found a hitherto unreported component of reovirus virions, namely a small RN-4 with a most unusual base composition (adenine content over 80%). In the succeeding paper, we report the characteristics of reovirus messenger RNA molecules and show that they too fall into a size distribution with three maxima; and further, we demonstrate by means of co-sedimentation and hybridization analyses that the reovirus genome is transcribed within the cell as discrete segments which correspond to the viral genome fragments which are released from virions. 2. Materials (a) Cells
Mouse
and Methods and
virus
L cells were
grown in spinner or monolayer culture in Eagle’s medium (Eagle, 1959) supplemented with 10% fetal calf serum. HeLa cells S3 were grown similarly in Eagle’s medium supplemented with 5% calf serum. BHK21 cells (kindly supplied by Dr Purnell Choppin of the Rockefeller University and Dr Howard Green of New York University) were grown in the medium described by MacPherson & Stoker (1962). The Dearing strain of reovirus type 3 was kindly supplied by Dr S. Dales, then of the Rockefeller University. (b) Mode of infection Cells grown to a population density of 5 to 7 x lo5 cells/ml. were harvested and concentrated by centrifugation to lo7 cells/ml. in Puck’s saline A (Marcus, Cieciura & Puck, 1956) 1964). Virus inoculum containing 20 mM-Mg2+ and 1% fetal calf serum (Becker & Joklik, (2000 virus particles/cell) was added and allowed to adsorb for 15 min at 37°C. Under these conditions about 50 O/oof thevirus inoculum is adsorbed. After the adsorption period the cells
CHARACTERIZATION
OF REOVIRUS
GENOME
RNA
3
were diluted to 3 x 105 cells/ml. in growth medium. Actinomycin D (0.5 pg/ml.) was added at this time in order to inhibit host but not viral RNA synthesis (Shatkin, 196%). For growth of virus stocks, cell monolayers were used since these produced a higher yield of virus than suspension cultures. Cells from spinner culture were transferred Do 22 cm x 9 cm Blake bottles and allowed to form confluent monolayers. The supernatant culture fluid was poured off and the cells in each bottle inoculated with 5 ml. of virus stock solution containing approximately IO8 p.f.u.t/ml. in Eagle’s medium without serum (added multiplicity of infection about 10 p.f.u./cell). After a 1-hr adsorption period, growth medium was added and the cells were incubated for 36 hr at 37°C. Under these conditions of virus growth, the bulk of the infective virus synthesized remained cellassociated and was not liberated into the culture medium. Infected cells were scraped or shaken free from the surface, collected by centrifugation, and generally stored at - 18°C prior to extraction of virus. (c) PurQkation
of virus
The cell pellet was resuspended in homogenization medium (0.01 M-Tris-HCl (pH S), 0.25 M-NaCl, 0.01 M-2-mercaptoethanol) (15 ml. for 60 Blake bottles) and blended for 30 set at half speed in a Virtis 45 homogenizer. The resulting homogenate was digested with cr-chymotrysin (25 pg/ml.) for 1 hr at room temperature, and then incubated for 1 hr at 0°C with 500 pg/ml. sodium deoxycholate. Cellular debris was sedimented from the resulting mixture (30,000 g, for 10 min) and the supernatant fluid decanted. The pellet was resuspended in a small volume of homogenization medium (5 ml.) and sonicated for 30 set with an MSE ultrasonic power unit; debris was sedimented once again and the supernatant fluid decanted. The combined supernatants were mixed with 0.5 vol. of Freon 113 (trichlorotrifluoroethane) and the mixture homogenized as above for 2 min. The mixture was centrifuged the virus to separate the phases (30,000 g, 10 min) and the upper aqueous phase containing was removed. The lower Freon phase and interphase were re-extracted with 5 ml. of homogenization buffer, sonicated for 30 set, and centrifuged. The combined Freon supernatants containing the virus were then sonicated and layered over 15 ml. preformed
I
I
I
Fraction no. FIG. 1. C&l equilibrium sedimentation of reovirus virions. Virus collected from a preparative 1.2 to 1.4 g/ cm3 density CsCl gradient was diluted with CsCl of p = 1.37 and centrifuged in the SW39 rotor of a Spinco model L ultracentrifuge for 48 hr at 38,000 rev./min. Fractions were collected by puncturing the bottom of the tube and counting drops. (-e-e-) Absorbancy at 260 mp; (- - 0 - - 0 --) infectivity; (-U-O-) density (determined from the refractive index, using the relation p = 10~86Olm- 13.4974 (Ifft, Voet & Vinograd, 1961). 7 The following abbreviations areused: p.f.u.,plaque-formingunits; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate. T,,, is the midpoint of the thermal transition curve. TYMV, turnip yellow mosaic virus; RSV, Rous sarcoma virus.
4
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linear CsCl density gradients prepared in 0.01 M-Tris-NaCl buffer, pH 8 (density 1.20 to 1.40 g/ems). Centrifugation was carried out at 24,000 rev./min for 2.25 hr in the SW25 rotor of the Spinco model L ultracentrifuge. The purified virus band, separated from contaminating cellular material and virus top component, was collected from the lower portion of the gradient. The resulting virus preparation was either further purified by rebanding (see Fig. I), or immediately dialyzed extensively against 1 x SSC (0.15 M-Nacl-0.015 M-sodium citrate, pH 7.4). Virus labeled with [32P]orthophosphate, C3H]uridine or [14C]uridine was purified in the same manner from cells treated iwith radioactive precursors during the course of infection. This purification procedure does not inactivate reovirus. From 50 to 100% of plaque-forming units in cell homogenates were recovered in bands of purified virus. (d) Plaque assay A modification of the procedures described by Gomatos, Tamm, Dales & Franklin (1962) was used. L cells were transferred from spinner culture to 12-cm Petri dishes (2 x lo6 cells/dish). When the cells had formed a confluent monolayer, the culture fluid was removed and 0.1 ml. of the appropriately diluted virus suspension was pipetted into the center of each dish. The virus was allowed to adsorb for 1 hr; then the monolayer was overlaid with 10 ml. of a mixture composed of equal parts of 1.2% agar and 2 x Eagle’s medium supplemented with serum. After 3 days incubation in an atmosphere of 5% COZ in air, the plates were fixed for 10 min with 10% formaldehyde and the agar overlay removed. The cell sheet was stained with crystal-violet and plaques were counted. Very recently it has been found that excellent plaques are also produced on BHK21 cells.
(e) Digestion of RNA with ribortuclease Ribonuclease-resistant RNA was detected on SDS-sucrose density-gradients by the method of Katz & Penman (1966). Fractions collected from sucrose density-gradients containing no SDS were diluted to 1.0 ml. with 1 x SSC! and digested with 10 pg/ml. RNase for 30 min at 37°C. The two methods gave similar results. The kinetics of degradation of reovirus RNA by heat or by RNase A was studied in the heated cell assembly of a Gilford recording spectrophotometer. (f) Dimethylsulphoxide Double-stranded Katz & Penman
reovirus (1966).
RNA
(g) Extraction
was rendered
and an&y&
treatment of RNA single
stranded
by the DMSO
method
of
of cytoplasmic RNA
Cytoplasmic fractions were analyzed by methods described elsewhere (Joklik & Becker, 1965). L cells were always disrupted in RSB (10 mM-NaCl-10 m&r-Tris-1.5 mM-Mgcl,, pH 7.4) containing NaCl rather than KC1 and were allowed to swell at 0°C for 15 min rather than 10 min before disruption by 12 strokes in a Dounce homogenizer. Cytoplasmic fractions were the supernatants after centrifuging at 800 g for 90 sec. For analysis of RNA by density-gradient centrifugation cytoplasmic fractions were made 1.0% with respect to SDS, 0.5 M with respect to urea and 0.1 M with respect toiodium acetate (pH 5) and then layered over 15 to 30% sucrose gradients containing SDS (Becker & Joklik, 1964). The presence of both urea and SDS, coupled with the lower pH of the mixture, ensured that whole virus in the cytoplasmic extract was completely solubilized. (h) Ext?+action and analysis of RNA from reovirus v&-ions Many different methods were tried; these are discussed below and summarized in Table 1. The standard method adopted for the preparation of RNA for zone sedimentation analysis was as follows: to a reovirus suspension in 1 x SSC was added SDS to a final concentration of 1 O/o,urea to 0.5 M and sodium acetate (pH 5) to 0.1 M. Themixture was warmed at 37°C for 5 min and was then ready for analysis on sucrose-SDS density-gradients. If reovirus RNA free from protein was desired, deproteinization was carried out by the method of Oda & Joklik (1967.
CHARACTERIZATION
OF REOVIRUS
GENOME
RNA
5
RNA used for studies on the kinetics of degradation by RNase was prepared by extraction of virus isolated from density gradients with SDS and phenol followed by ethanol precipitation. (i) Separation of single- and double-stranded RNA Virus-specific RNA extracted from the cytoplasm of infected cells is composed of a mixture of single- and double-stranded RNA species. These were separated as follows: RNA was deproteinized by the method of Oda & Joklik (1967), dissolved in 1 x SSC, mixed with an equal vol. of 4 M-Licl (Baltimore, 1966) and held overnight at 4°C. Singlestranded RNA precipitated by this treatment was removed. by centrifugation; doublestranded RNA was recovered from the supernatant fluid by precipitation with 2 vol. of ethanol. (j) Hybridization of cytopkwnic RNA with viral RNA The method of Weissman (1965) was used, except that ribonuclease digestion of the annealed product was carried out with 10 pg/ml. pancreatic RNase for 15 min at 37°C. (k) Preparation of the double-stranded RNA fragments by electrophoresis on polyacryla&de gels RNA isolated from virus was fractionated by electrophoresis through 2.4% polyacrylamide gels at 150 v for 10 hr (Loening, 1967). The pH 7.2 buffer system and fractionation techniques were those described by Summers, Maize1 & Darnell(1965), except that 20 mMEDTA was added to both gels and buffer, and gel fractionation was carried out in 1 x SSC. Pooled fractions were further homogenized in a Dounce homogenizer and the eluent freed from polyacrylamide particles by centrifugation. RNA was recovered free from major contaminants of unpolymerized acrylamide and sodium phosphate by precipitation as the cetyltrimethyl ammonium salt (Ralph & Bellamy, 1964). (1) Analysis of the base cowzposition of viral RNA Yeast RNA (200 pg) was added as carrier to fractions collected from density gradientst RNA was precipitated with TCA (final concentration 16%) and the tubes incubated at 0°C for 2 hr. The precipitated RNA was collected by centrifugation and washed twice with ethanol containing 10% potassium acetate (Halliman, Fleck & Mum-o, 1963) to remove TCA; then excess ethanol and water were removed by evaporation in an air stream. RNA was hydrolyzed in 1 N-KOH for 48 hr at 25°C. The hydrolysate (0.5 ml.) was passed through a column (10 cm x 05 cm) of Dowex 50-NH*+ cation exchange resin, and dried to remove ammonia. The resulting ribonucleotides were separated by thin layer eleotrophoresis in 0.25 M-ammonium acetate buffer (pH 3.5) (Bieleski, 1965) or by paper electrophoresis in pyridine-acetate buffer, pH 3.5. Radioactivity in areas localized by radioautography was measured directly, without prior elution, in a scintillation spectrometer. (m) Enzymes, chemicals and radioisotopes c+Chymotrypsin, pancreatic RN&se, RN&se A and micrococcal nuclease were products of the Worthington Biochemical Corporation, Freehold, N.J. Yeast RNA and pronase were obtained from Calbiochem, Los Angeles, Calif., and Nonidet P40 from The Shell Chemical Company, New York. [i4C]Poly A was purchased from Miles Laboratories, N.J. [14C]Uridine and [3H]uridme were products of the New England‘Nuclear Corporation. [32P]Orthophosphate was obtained from E. R. Squibb Ltd, New York.
3. Results (a) Size of RNA extracted from virions (i) Xediimentation analysis When reovirus RNA is liberated from virions by the addition of SDS to a final concentration of 1Oh,three peaks of RNA are discernible on density-gradient centrifugation. Typical absorbancy and radioactivity profiles are shown in Fig, 2. The sedimentation coefficients of the three peaks designated L, M and S are about 14,12 and lo.5 s
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SHAPIRO,
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IO Fraction
FIG. 2. Sucrose zone sedimentation
20
AND
JOKLIK
30
no.
analysis
of reovirus
RNA.
Reovirus labeled with 3zP and suspended in 1 x SSC was solubilized with SDS-urea, pH 5 (see Materials and Methods), layered onto a 15 to 30% SDS-sucrose density-gradient and centrifuged for 25 hr at 24,000 rev./min in the SW25 rotor of the Spinco model L ultracentrifuge. Absorbanoy was recorded with a Gilford recording spectrophotometer. ) Absorbancy at 260mp; (-O--O-) radioactivity. C------
respectively, using 16 s ribosomal RNA as marker. These values are virtually the same as those reported by Shatkin (19656) ; however, whereas Shatkin found the major component to be the middle peak, peak L invariably contained more material than the other two in our RNA preparations. In addition, if 32P-labeled virus is used, about 20% of the radioactivity is always found in the material at the top of the gradient, where most of the absorbancy, for SDS-dissociated virus preparations, is due to viral protein. This material, designated peak A, is discussed further below. From the sedimentation coefficient of reovirus virions (630 s) and their RNA content (la%, Gomatos & Tamm, 1963a) it can be calculated, making suitable assumptions, that the molecular weight of the reovirus genome should be about 107. It is not known what the sedimentation coefficient of a double-stranded RNA molecule of this size would be. However, since the sedimentation coefficient of the replicative form of the RNA of bacteriophage R17 (mol. wt 2.2 x 106) is 14.8 s (Franklin, 1967), and that of the replicative form of the RNA of poliovirus (4 x 106) is 20 s (Katz & Penman, 1966), one would expect a sedimentation coefficient of between 20 and 30 s for the intact genome of reovirus RNA. Considerable efforts were made to devise methods for releasing RNA molecules of such size from reovirus virions. It became apparent that there were three major factors to be considered: (a) The chemical treatment. Table 1 lists most of the methods tried for liberating intact reovirus RNA molecules. In no case was an RNA peak observed at a position corresponding to more than 14 s. When SDS was added to a suspension of reovirus virions at a very slow rate, it did appear on occasions, that the sedimentation coefficient of the largest of the three peaks was increased by 1 to 2 s ; but this was not reproducible, and not further investigated. (b) The age of the virus preparation. Haselkorn (1962) has reported that the intact viral genome cau only be extracted from freshly prepared turnip yellow mosaic virus: virus which has been stored for some time yields only RNA fragments. Similarly, Robinson, Pitkanen & Rubin (1965) found that it is essential to use
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
TABLE 1
Chemical treatments applied to reovirus in attempting to release intact viral genomes Treatmentt Reovirus in 1 x SSC or CsCl (as obtained from density-gradient) (i) Sodium perchlorate (5 M) plus EDTA (0.01 M) at 0, 20 or 50°C, with or without carrier ribosomal or yeast RNA (modifications of the method of Freifelder (1965)) Dialysis against sodium perchlorate (5 M) plus EDTA overnight at 4°C (ii) Urea (4 M or 8 M) at 0°C (iii) SDS: added over a period of 1 hr to a final concentration of 6.01% Added slowly over a period of 8 hr to a final concentration of 0.5% (iv) SDS plus urea (1% SDS, 0.5 M-USBaU) (2) Reovirus pre-treated with pronase (virus in 1 x SSC-60% sucrose; pronase heated at 85’C for 15 min, final concentration 1 mg/ml., incubation 15 mm at 2O’C) (i) As (i) above (ii) SDS: to 1% rapidly to 1% over a period of 30 min
s-Value of largest RNA molecule liberated
(I)
14 14 14
(sometimes
(sometimes to 1 y0 rapidly in the presence of high mol. wt DNA (1 mg/ml.) (iii) Urea: to 8 M slowly (iv) Pronase alone: 1 mg/ml. for 45 min at 37%
14 14 slightly 14
14 14 14 slightly
higher)
higher)
14 no lysis no lysis
t All these treatments were carried out in test tubes and the reaction mixtures were then transferred carefully onto density gradients. Many of the treatments were also applied to virus layered on density gradients. In no ease was any difference observed between the two methods. Over 90% of RNA was liberated in all cases except those marked “no lysis”.
freshly prepared Rous sarcoma virus preparations in order to obtain intact RSVRNA. It is conceivable that a similar situation holds for reovirus, although it is clearly not entirely analogous, since storage of TYMV and RSV is accompanied by loss of infectivity, whereas reovirus is stable. Nevertheless, RNA was extracted from reovirus immediately after its isolation from C&l gradients. The sedimentation profile of RNA from such virions was the same as that from virions which had been stored for some days. (c) The type of cell in which the virus was grown. Oda & Joklik (1967) found that vaccinia virus mRNA was degraded more rapidly in vitro by extracts of L cells than of HeLa cells. This raised the possibility that regions in the reovirus genome especially sensitive to enzymic attack might be less likely to survive intact in L cells than in HeLa cells, or indeed in other cells which also have a very low level of nuclease activity in their cytoplasm. Reovirus was therefore grown in HeLa cells and in BHK21 cells, the latter being chosen as they were the cells in which the reovirus was prepared which yielded long (about 5 p) RNA molecules on disruption in situ on electron microscope grids (Granboulon & Niveleau, 1967). However, the sedimentation profiles of RNA extracted from reovirus grown in HeLa or BHK21 cells were identical to that displayed by RNA extracted from reovirus grown in L cells.
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In conclusion, it has so far proved impossible to separate from reovirus virions RNA molecules corresponding to the intact genome. Irrespective of the method applied, density-gradient centrifugation analysis indicates that a mixture of fragments falling into three major size distributions is obtained. (ii) Polyacrylamide electrophoresis alzalysis The three classes of RNA molecules extractable from virions could also be-separated by means of polyacrylamide gel electrophoresis. In fact, the resolution achieved by this method greatly exceeds that of density:gradient centrifugation. Figure 3 shows 1
0
10
I
L’ .
20
30 Fraction
FIG. 3. Polyacrylamide
I
i
I
40
50
60
no.
gel electrophoresis
pattern of reovirus RNA.
3ZP-labeled reovirus RNA (separated from peak A material by preliminary zone sedimentation) was subjected to electrophoresis through a 2.4% polyacrylamide gel at 150~ for 10 hr. Migration was from left to right.
a typical pattern. The fastest moving material, shown in separate experiments to correspond to the slowest sedimenting peak S, was clearly resolvable into three components. The middle peak corresponds to peak M and the slowest peak to peak L. Each of these two peaks showed signs of resolving into two fractions on prolonged electrophoresis. (b) Size of re$icating
reovirus RNA
It may be argued that if the reovirus genome is one “molecule” with a number of built-in weak spots, breaking open the capsid may engender shearing forces sufficient to break the RNA. However, one would expect to find intact reovirus RNA within the cell prior to encapsidation. A search for such viral RNA was therefore undertaken. L cells infected with reovirus in the presence of actinomycin D (O-5 r.Lglml.) were pulse-labeled with [14C]uridine for 30 minutes from 1.2 to 125 hours after infection, the time at which the rate of viral genome replication was proceeding at its maximum (see Bellamy & Joklik, 1967, following paper). Cytoplasmic fractions were prepared as outlined in Materials and Methods, treated with SDS and centrifuged on sucroseSDS density gradients, Over 80% of the radioactivity incorporated was in material which was sensitive to ribonuclease : this RNA was identified as reovirus mRNA (see following paper). Material resistant to RNase was found in the region between 10 and 14 s only. A similar analysis was carried out using a method of analysis in which the oytoplasmic fractions are chilled prior to centrifugation in order to precipitate
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
9
most of the SDS, and the material is then centrifuged on sucrose-RSB gradients not containing SDS (McConkey & Hopkins, 1965). This method permits a more precise estimate of the sensitivity of RNA to digestion with RNase, but once again RNaseresistant RNA was only found in the 10 to 14 s region. Finally, the possibility remained that intact naked reovirus RNA was sheared during the breaking open of cells by Dounce homogenization. Infected cells were therefore opened by treatment with Nonidet P40 (0.5%) (O’Brien, 1964) and the cytoplasm so obtained analyzed by density-gradient centrifugation. Once again the only RNase-resistant RNA was in the 10 to 14 s region. This was true not only for the period from 12 to 12.5 hours after infection, but at all stages of the infection cycle when incorporation of label into double-stranded RNA could be detected. The conclusion from all this work is that if reovirus RNA replicates as an “intact” molecule of molecular weight 10 x 106, it is as prone to breakage when yet uncoated in the cytoplasm as in the virion. However, these results also strengthen the possibility that the reovirus genome does not replicate as an intact molecule but as a
I.0 50
60
70
80
Temperature
90
100
110
(“0
FIG. 4. The effect of ionic strength on the thermal transition of reovirus SSC; curve b, 0.1 x SSC; curve c, 1.0 x SSC; curve d, IO.0 x SW.
RNA.
Curve a., O-01 x
number of separate pieces, which are then processed by some maturation mechanism, which selects the correct assortment of individual pieces for encapsidation, possibly by some sort of loose binding. (c) Studies on the secondary structure of reovims
RNA
The following studies were carried out on PtiNA extracted from virions by means of SDS and phenol, followed by ethanol precipitation. The RNA was then dissolved in the appropriate solvent, as required. (i) Melting behavior Figure 4 presents data on the melting behavior of reovirus RNA at four different electrolyte concentrations. The ionic environment strongly influences the stability of the secondary structure. In 0.01 x ,0-l x , 1 x and 10 x SSC, the RNA exhibits sharp melting profiles with T, values of 75, 84, 96 and 104”C, respectively. These values are similar to those reported by Shatkin (19653). The maximum hyperchromic increase was about 34%.
10
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AND
40
60
JOKLIK
AAA-A-l----.11
L
20
0
Time (min) FIG. 5. The effect of ionic strength on the digestion of reovirus RNA with RNase A. 0.2 o,D.~~~ unit/ml. of RNA were incubated at 37°C with 1 pg/ml. RNase A. (a) Kinetics of digestion measured by the increase in absorbancy at 26Omp. (-O-O-) RNA iu 041 x SSC; (-O-O-) RNA in 0.1 x SSC; (-A--A-) RNA in 1.0 x SSC. (b) Kinetics of digestion measured by the release of acid-soluble radioactivity. (-O--e-) RNA in 1.0~ SSC. RNA in O-1 x SSC; (--O-O-) RNA in (5.15~ SSC; (-A-A-) Input: [32P]RNA, 1000 cts/min.
L) A- I-...--Ai-.-.-...-3:20 0
IO
Time (min)
FIG. 6. The effect of [Mg2+] on the digestion of reovirus RNA by RNase A. 0.2 o.D.~~~ unit/ml. of RNA in 0.01 x SSC vere incubated at 37°C with 1 pg/ml. RNase A. (-U-n-) No Mg2+ ; ;;-F,E~;~ 10-5~-Mg2+; (-.--.--) lo-%Mg2+; (-O-O-) lo-%Mg’+; (-A-A--)
CHARACTERIZATION (ii) Resistance
to hydrolysis
OF
REOVIRUS
CENOME
RNA
11
by RNase
The resistance of reovirus RNA to hydrolysis by pancreatic RNase A may be tested by measuring either hyperchromicity or production of acid-soluble radioactivity (Fig. 5(a) and (b)). At molarities greater than 0.1 M, reovirus RNA is resistant to RNase ; at lower molarities the RNA becomes progressively more susceptible to hydrolysis. At low ionic strengths (0.01 x SSC or less) the resistance of reovirus RNA to RNase is markedly increased by Mg2 + (Fig. 6). At ionic strengths which normally stabilize the RNA against a given level of RNase, higher concentrations of the enzyme cause complete hydrolysis (Pig. 7). Resistance to digestion by RNase thus depends, among other factors, on the concentration of monovalent ions, divalent ions and the concentration of enzyme. Further, digestion by RNase does not require extensive prior denaturation.
0
20
40
60
Time (mid
FIG. 7. The effect of enzyme concentration on the digestion of reovirus RNA by RNase A. 0.25 o.D.~~~ unit/ml. of RNA in 1 x SSC were incubated at 37°C with varying concentrations of RNase A. (-@----•--) lOOpg/ml.RNaseA; (-O-O-) lOpg/ml.RNaseA; (-A---A-) 1 pg/ml. RNase A.
(iii) Effect of electrolyte concentration
on the sedimentatiovb behavior
The sedimentation behavior of single-stranded RNA is strongly influenced by the ionic concentration. By contrast, the sedimentation properties of reovirus RNA are unaffected by ionic strength over the range from 0.01 x to 1 x SSC. Summarizing, these results are consistent with the notion that reovirus RNA fragments have a highly base-paired structure approaching double-strandedness. (d) Base composition
of reoviv-us RIVA
Table 2 presents the base compositions of (a) total viral RNA, (b) RNA in the pooled peaks L, M and S and (c) RNA in peak A isolated from the top of density gradients. The base composition of RNA within virions is far from .that expected for a doublestranded structure, since there is a large excess of A over TJ. The source of this excess A is the small molecular weight fraction, peak A, which has the extraordinary composition of 81.5 % A, 13 %U, and 3 % each of C and G. The base composition of the major
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BELLAMY,
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AND
JOKLIK
TABLE 2 Base composition Source of RNA preparation Whole virus Pooled peaks L plus M plus S Peak A
of reovirus RNA
c
A
G
u
16.9 21.9 3.2
31.7 27.5 81.3
17.5 21.9 2.8
27.9 28.7 12.7
G/C
W-J
1.035 I.00 -
1.35 0.96 6.4
portion of viral RNA (peaks L, M and S) indicates perfect base pairing, with A equal to U and G equal to C, and is similar to that reported for reovirus RNA by Gomatos & Tamm (1963c). The small molecular weight fraction, peak A, was further investigated. (a) The A-rich RNA is an internal viral constituent as it is still present after digestion of virions with micrococcal nuclease. Control experiments showed that this enzyme hydrolyzed poly A efficiently. (b) A-rich RNA is not confined to a special class of virus particle differing from the rest in density. Reovirus labeled with 32P was banded in CsCl and the virus peak divided into five fractions. The RNA from each fraction was then extracted and the ratio of A-rich RNA to 10 to 14 s viral RNA determined by appropriate densitygradient centrifugation analysis. The ratio was identical for all five fractions and was equal to 0.18 to O-20. TABLE 3 Estimation RNA L
of the molecular weight of L, .M and X pieces of reovirus RNA component M
2 x 106 2.3 x 106 1.25 x lo6 2.3 x 10s 1.45 x lo6 7 Using the value Franklin, 1966).
Method
S
From the mol. wt and s-value of R17 replioative form (Franklin, 1967) 7.3 x 105 From length measurement@ (Dunuebaoke & Kleinsohmidt, 1967) 1-O x 1C16 Studier (1965)
of 2.08
x
10” daltons/p
calculated
from
the data for
R17
(Granboulan
8r.
(c) The approximate size of the A-rich RNA was determined by density-gradient centrifugation. It sedimented more slowly than y-globulin and together with bovine serum albumin. Its sedimentation coefficient was approximately 2 s. Recent experiments have shown that it moves faster than 4 s tRNA on electrophoresis in polyacrylamide gels (Bellamy & Joklik, unpublished results). (d) A-rich RNA is synthesized in the cell in step with the rest of the viral genome. This was shown by pulse-labeling infected cells with 32P for four hours at various stages throughout the infection cycle and isolating the viral progeny at 24 hours after infection. The proportion of label in the A-rich material was constant. Actinomycin D (0.5 pg/ml.) was present in all these experiments from the time of infection.
Fraction
no,
FIG. 8. The effect of DMSO on reovirus RNA. Reovirus RNA (separated from peak A material by preliminary zone sedimentation) was treated with DMSO by the method of Katz t Penman (1966). RNA was then layered onto a 15 to 30% SDS-sucrose density-gradient and centrifuged for 18 hr at 24,000 rev./mm in rotor SW25 of the Spinco model L ultracentrifuge. (-e--e--) Total radioactivity; (-O--O-) RNase-resistant radioactivity.
0 fraction
IO
20
30
no.
FIG. 9. The effect of DMSO treatment on the three size classes of reovirus RNA. Reovirus RNA (separated from peak A material by preliminary zone sedimentation) was centrifuged on a 10 to 15% SDS-sucrose density-gradient for 20 hr at 24,000 rev./min in the SW25 rotor of the Spinco model L ultracentrifuge. One-twentieth of each fraction was counted to give the profile shown in (a). Fractions B, C, and D, corresponding to the three peaks of viral RNA, were then pooled as indicated and treated with DMSO. The DMSO-treated RNA was centrifuged on 15 to 30% SDS-sucrose density-gradients for 18 hr at 24,000 rev./min ( (b), (c) and (d) respectively).
14
BELLAMY,
(e) Conversion
SHAPIRO,
AUGUST
of reovirus RNA
AND
JOKLIK
to the single-stranded
state
Reovirus RNA was converted to single-stranded RNA by the method of Katz & Penman (1966). This method is known to convert the replicative form of poliovirus RNA (20 S) to a single-stranded form of RNA sedimenting with a sedimentation coefficient of 35 S, which is the sedimentation coefficient of native poliovirus RNA. This method therefore clearly dissociates double-stranded RNA molecules into components which behave like native single-stranded molecules. Figure 8 shows the sedimentation pattern given by reovirus RNA converted to the single-stranded condition. The three RNase-resistant peaks L, M and S (see Fig. 2) are replaced by three RNase-sensitive peaks designated peaks L’, M’ and S’, respectively. Peak L’ sedimented approximately midway between 28 s and 16 s ribosomal RNA; in polyacrylamide gel electropherograms it was located closer to 28 s than to 16 s ribosomal RNA markers. A sedimentation coefficient of 22 to 25 s was therefore assigned to it. Peak N’ almost coincided wit,h 16 s ribosomal RNA in density-gradients ; polyacrylamide gel electrophoresis revealed that it was slightly larger than 16 s ribosomal RNA, and a sedimentation coefficient of 16 to 18 s was assigned to it. Peak S’ had an approximate sedimentation coefficient of 14 s, and its behavior on electrophoresis was consistent with this estimate. These three species of single-stranded RNA correspond to the three RNase-sensitive fractions obtained by thermal denaturation of reovirus RNA (Iglewski & Franklin, 1967). To clarify the origin of these components, a density-gradient containing peaks L, M and S (Fig. 9(a) ) was subdivided as shown and each set of pooled fractions separately converted to the single-stranded form. Peak L gave rise to peak L’, peak M to peak M’ and peak S to peak S’ (Fig. 9 (b) to (d) ).
4. Discussion The reovirus genome is released from virions as a mixture of RNA fragments. The distribution of the size, and therefore of the length, of these fragments is highly reproducible and independent of the extraction procedure. The possibility that the reovirus genome is subject to breakage within the capsid was considered, but found to be unlikely for the following reasons : (1) the virus is not inactivated during isolation; (2) the virus does not lose infectivity on storage; and (3) the identical mixture of fragments was obtained from virions less than 24 hours old and others up to eight weeks old. Intact viral genome molecules were not found in cells in which viral RNA was being synthesized; double-stranded RNA molecules extracted from such cells fell into the same size distribution as those extracted from virions. This failure to extract larger molecules was not due to shearing during breaking of cells, since cell lysis by means of a non-ionic detergent yielded the same RNA fragments; nor was it due to nuclease activity in the cytoplasm, since the same result was obtained when infected BHK21 and HeLa cells were used, both of which have very little cytoplasmic nuclease activity. The individual RNA fragments appear to be double-stranded by all criteria applied : they exhibit a sharp melting profile with a T, dependent on the ionic strength, they are resistant to ribonuclease provided ionic strength, divalent cation concentration and enzyme concentration are suitably adjusted, their sedimentation behavior is independent of the ionic strength over a large range, and their base composition
CHARACTERIZATION
OF REOVIRUS
GENOME RNA
15
indicates equality of A and U, and G and C. These results confirm and extend those of previous workers (Gomatos $ Tamm, 1963a,b; Shatkin, 19655). It is not yet poesible to say whether the fragments are base-paired throughout their entire length, or whether there exist some short single-stranded regions at the ends. The fact that the same mixture of fragments is extracted from reovirus virions and from cells in which the virus is replicating, as well as the demonstration (see following paper) that these fragmen$s are the intracellular templates for messenger RNA transcription, suggests that the reovirus genome is composed of a linear array of tenuously linked segments. It thus becomes of interest to enquire into the number and type of segments which make up the reovirus genome. In order to do this, it is necessary to know the size of the various classes of segments, their relative frequency and t,he size of the viral genome. We will consider each of these in turn. (a) The size of the segments. The sedimentation coefficients of the three classes of reovirus RNA fragments are about 14,12 and 10.5 s (classes L, M and S). The approximate molecular weights of these classes of segments can be estimated as follows. (i) Class L segments may be compared with the replicative form of the RNA of phage R17, the sedimentation coefficient and molecular weight of which are known. The best estimate of the molecular weight of R17 RNA is 1.1 x lo6 (Sinha, Fujimara & Kaesberg, 1965). The molecular weight of its replicative form is therefore 2.2 x lo6 and the sedimentation coefficient of this double-stranded RNA is 14.8 s (Franklin, 1967). This sedimentation coefficient is very close to that of the L fragments of reovirus RNA (14 s) and the molecular weight of these fragments is therefore most probably close to 2 >( lo6 (Table 3). (ii) The molecular weight of double-stranded RNA molecules can be derived from their length. Dunnebacke & Kleinschmidt (1967) extracted RNA from reovirus with 2 M-NaClO, and examined it on protein monolayers by means of the electron microscope. They found a mixture of filaments displaying a trimodal pattern of length distributions, with peaks at 1.1, 0.6 and 0.35 p. This distribution is probably very similar to the one described in this paper. There are two sets of data relating the length of such double-stranded RNA molecules to their molecular weight. On the one hand, Langridge & Gomatos (1963) reported a translation value per nucleotide residue of about 3 A for reovirus RNA, corresponding to about 2.4 x lo6 daltons per p (Gomatos & Stoeckenius, 1964). On the other hand, Granboulan & Franklin (1966) found the replicative form of R17 RNA to be 1.06 p long. Since the molecular weight of this RNA is 2.2 x lo6 (Sinha et ccl., 1965; Franklin, 1967), this leads to a value of 2.08 x lo6 daltons per p. Since Langridge & Gomatos (1963) carefully pointed out that the translation value per nucleotide residue of 3 A can not yet be regarded as definitive, we shall, for the purposes of the discussion which follows, use the value of 2.08 x lo6 daltons per p, keeping in mind however the fact that there is evidence for a higher value. Equating the three classes of molecules in our distribution with the three size classes of filaments of Dunnebacke & Kleinschmidt, one arrives at the molecular weights shown in Table 3. (iii) Finally, one may apply the equation of Studier (1965) relating the sedimentation coefllcient of DKA to its molecular weight: S = 0.0882 x -U”.346, making the assumption (which most probably is not strictly true) that double-stranded RNA and DNA behave similarly. This equation yields the molecular weights shown in the last line of Table 3. Bearing in mind the fact that the sedimentation coefficients of L, M and S fragments are approximate, and averaging the three series of estimates just presented, one may assign the following approximate values to the molecular weights of L, M
16
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
and S segments of reovirus RNA : 2.3 -+ O-2 x 106, 1.3f0.2 x lo6 and 8f2 x 105. (b) The relative frequency of L, M and S segments. Dunnebacke & Kleinschmidt (1967) found the relative number of filaments in each of the three length distribution peaks, 1.1, O-6 and 0.35 p, to be in the ratio of 2 : 3 : 4 to 6. Our own estimate of the mass distribution in peaks L, M and S (based on the amount of radioactive isotope in peaks L, M and S, measured in polyacrylamide gel electropherograms in which these three classes of molecules are separated absolutely) is 2 : 1 : 1. This leads to the following estimate of the number of molecules in these three peaks based on the molecular weights derived above: 1-O : 0.89 : 1.44. The relative frequency of L, M and S molecules is thus most probably n : n : 15n. (0) The size of the reovirus genome. There is some uncertainty concerning the molecular weight of the reovirus genome. On the one hand, Gomatos & Tamm (1963a) derived a minimum value of 10.2 x lo6 daltons from an estimate of the molecular weight of the virion (derived from measurement of the sedimentation coeEicient and diffusion constant) and its percentage content of nucleic acid. On the other hand, Vasquez & Kleinschmidt (1967) have estimated the total length of the reovirus genome to be of the order of 8 p; using the value of 2.08 x lo6 daltons per ,LLderived above, this corresponds to a molecular weight of about 16.5 x 106. Further uncertainty is introduced by the presence of the single-stranded A-rich material, the contribution of which to the length of the reovirus genome is not known. The best estimate at this time is probably that the reovirus genome weighs not less than 12 and not more than 16 million daltons. This weight could be made up of L, M and S segments as follows: if n is equal to two, then the combined weight of the seven pieces would be 9.6 x 106; an extra 20% of A-rich material would bring the total molecular weight of reovirus RNA to 12 x 106. If n is equal to three, the combined weight of the double-stranded segments alone would be 14.4 x 106; if n is equal to four, the corresponding value is 19.2 x 106, a value irreconcilable with the molecular weight estimate of Gomatos & Tamm, but still compatible with a genome 8 TVlong, provided one uses the value of 2.39 x lo6 daltons per p (molecular weight 19.1 x 106). The A-rich RNA could then not contribute to the measured length of the viral genome. It is difficult, however, to accept a value as high as 19 x lo6 for the molecular weight of the reovirus genome. The most likely value of n is probably two, since after prolonged polyacrylamide gel electrophoresis peaks L and M resolve into two components each, and peak S into three. We thus favor a model in which the reovirus genome is composed of seven pieces of double-stranded RNA, namely two L plus two M plus three S segments. The significance and function of the large amount of A-rich RNA are not clear at the moment. It is tempting to speculate that this material serves to link together the double-stranded RNA fragments; indeed Gabor & Hotchkiss (1966) have suggested that A-rich regions may act as punctuation marks and breakage points in DNA. Further, since no intact genomes can be isolated from infected cells, it is possible that reovirus RNA replicates as a series of separate segments. This mode of replication would necessitate a mechanism for ensuring that each virion receives one of each of these segments, and a simple method of achieving this would be to join the segments in some way. However, the amount of A-rich material present seems far in excess of that required to join seven or even fourteen double-stranded RNA pieces, and its function may be quite different, Further work is clearly necessary before this question can be settled.
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
17
We would like to thank Dr J. V. Maize1 for helpful discussions concerning the electrophoresis of RNA in polyacrylamide gels. This investigation was supported by grants number AI-04913, AI-04163, GM-11936 and GM-11301 from the National Institutes of Health, grant number E-379 from the American Cancer Society, and grant number GB-5082 from the National Science Foundation. One of us (J.T.A.) is the recipient of an investigatorship from the Health Research Council of the City of New York (contract I-346) ; another (W.K.J.) is the recipient of a United States Public Health Service Research Career Award (no. l-K6-AI-22, 554). REFERENCES Baltimore, D. (1966). J. MOE. Biol. 18, 421. Becker, Y. & Joklik, W. K. (1964). Proc. Nut. Acad. Sci., Wash. 51, 577. Bellamy, A. R. & Joklik, W. K. (1967). J. Mol. Biol. 29, 19. Biochem. 12, 230. Bieleski, R. L. (1965). Analyt. Dunnebacke, T. & Kleinschmidt, A. (1967). 2. NatzLrf. 22b, 159. Eagle, H. (1959). Science, 130, 432. Franklin, R. M. (1967). J. Viral. 1, 64. Freifelder, D. (1966). Biochem. Biophys. Res. Comm. 18, 161. Gabor, M. & Hotohkiss, R. D. (1966). Proc. Nat. Acad. Sci., Wash. 56, 1441. Gomatos, P. J. & Stoeckenius, W. J. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1449. Gomatos, P. J. & Tamm, I. (1963a). Proc. Nat. Accd Sci., Wah. 49, 707. Gomatos, P. J. & Tamm, I. (19633). Science, 140, 997. Gomatos, P. J. & Tamm, I. (1963c). Proc. Nat. Acad. Sci., Wash. 50, 878. Gomatos, P. J., Tamm, I., Dales, S. & Franklin, R. M. (1962). Virology, 17, 441. Granboulan, N. & Franklin, R. M. (1966). J. Mol. BioZ. 22, 173. Granboulan, N. & Niveleau, A. (1967). J. Microbiologic, in the press. Hallinan, T., Fleck, A. & Munro, H. M. (1963). Biochim. biophys. Acta, 68, 131. Haselkorn, R. (1962). J. Mol. BioZ. 4, 357. Ifft, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phya. Chem. 65, 1138. Iglewski, W. J. & Franklin, R. M. (1967). J. Viral. 1, 302. Joklik, W. K. & Becker, Y. (1965). J. Mol. BioZ. 13, 496. Katz, L. & Penman, S. (1966). Biochem. Biophys. Res. Comm. 23, 557. Langridge, R. & Gomatos, P. J. (1963). Science, 141, 694. Loening, U. E. (1967). Biochem. J. 102, 251. MacPherson, I. A. & Stoker, M. G. P. (1962). Virology, 16, 147. McConkey, E. H. & Hopkins, J. W. (1965). J. Mol. BioZ. 14, 257. Marcus, P. I., Cieciura, S. J. & Puck, T. T. (1956). J. Ezp. Med. 104, 615. O’Brien, B. R. A. (1964). J. Cell. BioZ. 20, 521. Oda, K. & Joklik, W. K. (1967). J. Mol. BioZ. 27, 395. Prevec, L. & Graham, A. F. (1966). Science, 154, 522. Ralph, R. K. & Bellamy, A. R. (1964). Biochim. biophys. A&, 87, 9. Robinson, W. S., Pitkanen, A. & Rubin, H. (1965). Proc. Nat. Acad. Sk, Wmh. 54, 137. Shatkin, A. J. (1965a). Biochem. Biophys. Res. Comm. 19, 506. Shatkin, A. J. (1965b). Proc. Nat. Acad. Sci., Wash. 54, 1721. Shatkin, A. J. & Rada, B. (1967). J. Viral. 1, 24. Sinha, M. K., Fujimara, R. K. & Kaesburg, P. (1965). J. Mol. BioZ. 11, 64. Studier, F. W. (1965). J. Mol. BioZ. 11, 373. Summers, D. F., Maizel, J. V. & Darnell, J. E. (1965). Proc. Nut. Acad Sci., Wash. 54,505. Vasquee, C. & Kleinschmidt, A. (1967). Proc. Symp. Electron Microscope Society ofAmerica, Chicago (to be published). Weissman, C. (1965). Proc. Nat. Acad. Sk., Wash. 54, 202.
2
Biol.
(1967) 29,1-17
Studies on Reovirus RNA I. Characterization A. R. BELLAMY, Departments
of Reovirus Genome RNA
LUCILLE XHAPIRO, J. T. AUGUST AND WOLFGANG K. JOKLIK
of Cell Biology
and Molecular Biology, Albert Einstein Bronx 61, N. Y. 10461, U.S.A.
(Received 28 March
College of Medicine
1967, and in revised form 22 May 1967)
Chemical treatment does not release the genome of reovirus in intact form. Irrespective of the procedure used, the same mixture of fragments is released with sedimentation coefficients of about 14, 12 and 10.5 s. On electrophoresis in polyacrylamide gels these size classes can be further resolved, the two former into two are double-stranded as components each, the last into three. These fragments judged by the following criteria: they exhibit a sharp melting profile with a T, dependent on the ionic concentration; they are resistant to ribonuclease, provided that the concentrations of both monovalent and divalent ions, as well as that of the enzyme, are suitably adjusted: their sedimentation behavior is independent of the ionic concentration over a wide range ; and their base composition displays equality of A and U, as well as of G and C. In addition to this mixture of double-stranded fragments there is released from virions an RNA of small size (2 to 3 s) which amounts to about 20% of the mass of the viral genome. The base composition of this RNA is 82% A, 13% U, 3% G and 3% C. This A-rich RNA is synthesized in step with the viral genome RNA. RNA molecules corresponding to intact viral genomes were not detected in infected cells at times when the viral genome was replicating. Irrespective of the method of breaking open the cells, or of the cell strain used, the same mixture of RNA fragments was obtained as was released from virions. These results suggest that the reovirus genome consists of a number of segments of double-stranded RNA; further, it would appear that the reovirus genome does not replicate as one intact molecule, but rather in the form of these same segments. The molecular weights of the L, M and S segments have been estimated by reference to three sets of data in the literature; the most likely values are 2.3 + 0.2 x 106, 1.3 + 0.2 x lo6 and 8 f 2 x 105. L, M and S segments are released from virions in the ratio of n : n : 1*5n. Owing to uncertainty concerning the size of the reovirus genome, the exact number of segments of which it is composed cannot be specified. The most likely value of m is 2, although a value of 3 or even 4 is not ruled out. The function of the A-rich material is not known. It is conceivable that it serves to link the double-stranded segments, giving rise to a structure which is so labile that it does not withstand any but the most gentle manipulation. However, the precise manner in which linking is accomplished, and the role, if any, of the Arich material in this process must await further work.
1. Introduction Reovirus RNA displays, under appropriate conditions, a sharp melting profile, resistance to ribonuclease and a base composition with approximately equimolar amounts of A and U on the one hand and G and C on the other (Gomatos $ Tamm, 1
2
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
1963a,b). For these reasons, and because X-ray diffraction analysis lends support, it has been designated as double-stranded RNA (Langridge & Gomatos, 1963). However, there is as yet no proof that the genome of reovirus does, in fact, consist of two intact antiparallel complementary polynucleotide strands. RNA molecules corresponding to the entire genome have not yet been liberated from the virions. Very gentle dissociation of virions on protein monolayers yields structures about 5 p long (Granboulan & Niveleau, 1967). More recently, Vasquez & Kleinschmidt (1967) observed assemblies of filaments radiating from reovirus virions disrupted in situ on protein monolayers, the length of which added up to about 8 CL,and detected occasional long filaments up to 7.4 TVlong. However, when virions are disrupted prior to spreading on protein monolayers, a mixture of fragments with a tri-modal length distribution is obtained, with maxima at 1.1 k, O-6 TVand 0.35 TV(Dunnebacke & Kleinschmidt, 1967). One can summarize the current state of knowledge as follows : while the reovirus RNA genome appears to be highly base-paired, it is very fragile, suggesting the presence of regions where breakage occurs with great ease. In addition to the reovirus genome, viral messenger RNA is synthesized in infected cells. As would be expected, this messenger RNA is found in polyribosomes and hybridizes with viral genome RNA (Prevec & Graham, 1966; Shatkin & Rada, 1967). The object of the work reported in this paper was to attempt to isolate from reovirus virions, RNA molecules which would correspond to the entire genome and to examine their structure. However, irrespective of the procedure employed, no intact genome molecules were obtained. We found, as had Dunnebacke & Kleinschmidt, that the extracted genome RNA fragments fell into a size distribution with three maxima, which was highly reproducible and independent of the extraction procedure. These RNA fragments proved to be double-stranded according to a number of criteria, and were readily converted to the single-stranded state. Further, we found a hitherto unreported component of reovirus virions, namely a small RN-4 with a most unusual base composition (adenine content over 80%). In the succeeding paper, we report the characteristics of reovirus messenger RNA molecules and show that they too fall into a size distribution with three maxima; and further, we demonstrate by means of co-sedimentation and hybridization analyses that the reovirus genome is transcribed within the cell as discrete segments which correspond to the viral genome fragments which are released from virions. 2. Materials (a) Cells
Mouse
and Methods and
virus
L cells were
grown in spinner or monolayer culture in Eagle’s medium (Eagle, 1959) supplemented with 10% fetal calf serum. HeLa cells S3 were grown similarly in Eagle’s medium supplemented with 5% calf serum. BHK21 cells (kindly supplied by Dr Purnell Choppin of the Rockefeller University and Dr Howard Green of New York University) were grown in the medium described by MacPherson & Stoker (1962). The Dearing strain of reovirus type 3 was kindly supplied by Dr S. Dales, then of the Rockefeller University. (b) Mode of infection Cells grown to a population density of 5 to 7 x lo5 cells/ml. were harvested and concentrated by centrifugation to lo7 cells/ml. in Puck’s saline A (Marcus, Cieciura & Puck, 1956) 1964). Virus inoculum containing 20 mM-Mg2+ and 1% fetal calf serum (Becker & Joklik, (2000 virus particles/cell) was added and allowed to adsorb for 15 min at 37°C. Under these conditions about 50 O/oof thevirus inoculum is adsorbed. After the adsorption period the cells
CHARACTERIZATION
OF REOVIRUS
GENOME
RNA
3
were diluted to 3 x 105 cells/ml. in growth medium. Actinomycin D (0.5 pg/ml.) was added at this time in order to inhibit host but not viral RNA synthesis (Shatkin, 196%). For growth of virus stocks, cell monolayers were used since these produced a higher yield of virus than suspension cultures. Cells from spinner culture were transferred Do 22 cm x 9 cm Blake bottles and allowed to form confluent monolayers. The supernatant culture fluid was poured off and the cells in each bottle inoculated with 5 ml. of virus stock solution containing approximately IO8 p.f.u.t/ml. in Eagle’s medium without serum (added multiplicity of infection about 10 p.f.u./cell). After a 1-hr adsorption period, growth medium was added and the cells were incubated for 36 hr at 37°C. Under these conditions of virus growth, the bulk of the infective virus synthesized remained cellassociated and was not liberated into the culture medium. Infected cells were scraped or shaken free from the surface, collected by centrifugation, and generally stored at - 18°C prior to extraction of virus. (c) PurQkation
of virus
The cell pellet was resuspended in homogenization medium (0.01 M-Tris-HCl (pH S), 0.25 M-NaCl, 0.01 M-2-mercaptoethanol) (15 ml. for 60 Blake bottles) and blended for 30 set at half speed in a Virtis 45 homogenizer. The resulting homogenate was digested with cr-chymotrysin (25 pg/ml.) for 1 hr at room temperature, and then incubated for 1 hr at 0°C with 500 pg/ml. sodium deoxycholate. Cellular debris was sedimented from the resulting mixture (30,000 g, for 10 min) and the supernatant fluid decanted. The pellet was resuspended in a small volume of homogenization medium (5 ml.) and sonicated for 30 set with an MSE ultrasonic power unit; debris was sedimented once again and the supernatant fluid decanted. The combined supernatants were mixed with 0.5 vol. of Freon 113 (trichlorotrifluoroethane) and the mixture homogenized as above for 2 min. The mixture was centrifuged the virus to separate the phases (30,000 g, 10 min) and the upper aqueous phase containing was removed. The lower Freon phase and interphase were re-extracted with 5 ml. of homogenization buffer, sonicated for 30 set, and centrifuged. The combined Freon supernatants containing the virus were then sonicated and layered over 15 ml. preformed
I
I
I
Fraction no. FIG. 1. C&l equilibrium sedimentation of reovirus virions. Virus collected from a preparative 1.2 to 1.4 g/ cm3 density CsCl gradient was diluted with CsCl of p = 1.37 and centrifuged in the SW39 rotor of a Spinco model L ultracentrifuge for 48 hr at 38,000 rev./min. Fractions were collected by puncturing the bottom of the tube and counting drops. (-e-e-) Absorbancy at 260 mp; (- - 0 - - 0 --) infectivity; (-U-O-) density (determined from the refractive index, using the relation p = 10~86Olm- 13.4974 (Ifft, Voet & Vinograd, 1961). 7 The following abbreviations areused: p.f.u.,plaque-formingunits; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate. T,,, is the midpoint of the thermal transition curve. TYMV, turnip yellow mosaic virus; RSV, Rous sarcoma virus.
4
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
linear CsCl density gradients prepared in 0.01 M-Tris-NaCl buffer, pH 8 (density 1.20 to 1.40 g/ems). Centrifugation was carried out at 24,000 rev./min for 2.25 hr in the SW25 rotor of the Spinco model L ultracentrifuge. The purified virus band, separated from contaminating cellular material and virus top component, was collected from the lower portion of the gradient. The resulting virus preparation was either further purified by rebanding (see Fig. I), or immediately dialyzed extensively against 1 x SSC (0.15 M-Nacl-0.015 M-sodium citrate, pH 7.4). Virus labeled with [32P]orthophosphate, C3H]uridine or [14C]uridine was purified in the same manner from cells treated iwith radioactive precursors during the course of infection. This purification procedure does not inactivate reovirus. From 50 to 100% of plaque-forming units in cell homogenates were recovered in bands of purified virus. (d) Plaque assay A modification of the procedures described by Gomatos, Tamm, Dales & Franklin (1962) was used. L cells were transferred from spinner culture to 12-cm Petri dishes (2 x lo6 cells/dish). When the cells had formed a confluent monolayer, the culture fluid was removed and 0.1 ml. of the appropriately diluted virus suspension was pipetted into the center of each dish. The virus was allowed to adsorb for 1 hr; then the monolayer was overlaid with 10 ml. of a mixture composed of equal parts of 1.2% agar and 2 x Eagle’s medium supplemented with serum. After 3 days incubation in an atmosphere of 5% COZ in air, the plates were fixed for 10 min with 10% formaldehyde and the agar overlay removed. The cell sheet was stained with crystal-violet and plaques were counted. Very recently it has been found that excellent plaques are also produced on BHK21 cells.
(e) Digestion of RNA with ribortuclease Ribonuclease-resistant RNA was detected on SDS-sucrose density-gradients by the method of Katz & Penman (1966). Fractions collected from sucrose density-gradients containing no SDS were diluted to 1.0 ml. with 1 x SSC! and digested with 10 pg/ml. RNase for 30 min at 37°C. The two methods gave similar results. The kinetics of degradation of reovirus RNA by heat or by RNase A was studied in the heated cell assembly of a Gilford recording spectrophotometer. (f) Dimethylsulphoxide Double-stranded Katz & Penman
reovirus (1966).
RNA
(g) Extraction
was rendered
and an&y&
treatment of RNA single
stranded
by the DMSO
method
of
of cytoplasmic RNA
Cytoplasmic fractions were analyzed by methods described elsewhere (Joklik & Becker, 1965). L cells were always disrupted in RSB (10 mM-NaCl-10 m&r-Tris-1.5 mM-Mgcl,, pH 7.4) containing NaCl rather than KC1 and were allowed to swell at 0°C for 15 min rather than 10 min before disruption by 12 strokes in a Dounce homogenizer. Cytoplasmic fractions were the supernatants after centrifuging at 800 g for 90 sec. For analysis of RNA by density-gradient centrifugation cytoplasmic fractions were made 1.0% with respect to SDS, 0.5 M with respect to urea and 0.1 M with respect toiodium acetate (pH 5) and then layered over 15 to 30% sucrose gradients containing SDS (Becker & Joklik, 1964). The presence of both urea and SDS, coupled with the lower pH of the mixture, ensured that whole virus in the cytoplasmic extract was completely solubilized. (h) Ext?+action and analysis of RNA from reovirus v&-ions Many different methods were tried; these are discussed below and summarized in Table 1. The standard method adopted for the preparation of RNA for zone sedimentation analysis was as follows: to a reovirus suspension in 1 x SSC was added SDS to a final concentration of 1 O/o,urea to 0.5 M and sodium acetate (pH 5) to 0.1 M. Themixture was warmed at 37°C for 5 min and was then ready for analysis on sucrose-SDS density-gradients. If reovirus RNA free from protein was desired, deproteinization was carried out by the method of Oda & Joklik (1967.
CHARACTERIZATION
OF REOVIRUS
GENOME
RNA
5
RNA used for studies on the kinetics of degradation by RNase was prepared by extraction of virus isolated from density gradients with SDS and phenol followed by ethanol precipitation. (i) Separation of single- and double-stranded RNA Virus-specific RNA extracted from the cytoplasm of infected cells is composed of a mixture of single- and double-stranded RNA species. These were separated as follows: RNA was deproteinized by the method of Oda & Joklik (1967), dissolved in 1 x SSC, mixed with an equal vol. of 4 M-Licl (Baltimore, 1966) and held overnight at 4°C. Singlestranded RNA precipitated by this treatment was removed. by centrifugation; doublestranded RNA was recovered from the supernatant fluid by precipitation with 2 vol. of ethanol. (j) Hybridization of cytopkwnic RNA with viral RNA The method of Weissman (1965) was used, except that ribonuclease digestion of the annealed product was carried out with 10 pg/ml. pancreatic RNase for 15 min at 37°C. (k) Preparation of the double-stranded RNA fragments by electrophoresis on polyacryla&de gels RNA isolated from virus was fractionated by electrophoresis through 2.4% polyacrylamide gels at 150 v for 10 hr (Loening, 1967). The pH 7.2 buffer system and fractionation techniques were those described by Summers, Maize1 & Darnell(1965), except that 20 mMEDTA was added to both gels and buffer, and gel fractionation was carried out in 1 x SSC. Pooled fractions were further homogenized in a Dounce homogenizer and the eluent freed from polyacrylamide particles by centrifugation. RNA was recovered free from major contaminants of unpolymerized acrylamide and sodium phosphate by precipitation as the cetyltrimethyl ammonium salt (Ralph & Bellamy, 1964). (1) Analysis of the base cowzposition of viral RNA Yeast RNA (200 pg) was added as carrier to fractions collected from density gradientst RNA was precipitated with TCA (final concentration 16%) and the tubes incubated at 0°C for 2 hr. The precipitated RNA was collected by centrifugation and washed twice with ethanol containing 10% potassium acetate (Halliman, Fleck & Mum-o, 1963) to remove TCA; then excess ethanol and water were removed by evaporation in an air stream. RNA was hydrolyzed in 1 N-KOH for 48 hr at 25°C. The hydrolysate (0.5 ml.) was passed through a column (10 cm x 05 cm) of Dowex 50-NH*+ cation exchange resin, and dried to remove ammonia. The resulting ribonucleotides were separated by thin layer eleotrophoresis in 0.25 M-ammonium acetate buffer (pH 3.5) (Bieleski, 1965) or by paper electrophoresis in pyridine-acetate buffer, pH 3.5. Radioactivity in areas localized by radioautography was measured directly, without prior elution, in a scintillation spectrometer. (m) Enzymes, chemicals and radioisotopes c+Chymotrypsin, pancreatic RN&se, RN&se A and micrococcal nuclease were products of the Worthington Biochemical Corporation, Freehold, N.J. Yeast RNA and pronase were obtained from Calbiochem, Los Angeles, Calif., and Nonidet P40 from The Shell Chemical Company, New York. [i4C]Poly A was purchased from Miles Laboratories, N.J. [14C]Uridine and [3H]uridme were products of the New England‘Nuclear Corporation. [32P]Orthophosphate was obtained from E. R. Squibb Ltd, New York.
3. Results (a) Size of RNA extracted from virions (i) Xediimentation analysis When reovirus RNA is liberated from virions by the addition of SDS to a final concentration of 1Oh,three peaks of RNA are discernible on density-gradient centrifugation. Typical absorbancy and radioactivity profiles are shown in Fig, 2. The sedimentation coefficients of the three peaks designated L, M and S are about 14,12 and lo.5 s
6
BELLAMY,
0
SHAPIRO,
AUGUST
IO Fraction
FIG. 2. Sucrose zone sedimentation
20
AND
JOKLIK
30
no.
analysis
of reovirus
RNA.
Reovirus labeled with 3zP and suspended in 1 x SSC was solubilized with SDS-urea, pH 5 (see Materials and Methods), layered onto a 15 to 30% SDS-sucrose density-gradient and centrifuged for 25 hr at 24,000 rev./min in the SW25 rotor of the Spinco model L ultracentrifuge. Absorbanoy was recorded with a Gilford recording spectrophotometer. ) Absorbancy at 260mp; (-O--O-) radioactivity. C------
respectively, using 16 s ribosomal RNA as marker. These values are virtually the same as those reported by Shatkin (19656) ; however, whereas Shatkin found the major component to be the middle peak, peak L invariably contained more material than the other two in our RNA preparations. In addition, if 32P-labeled virus is used, about 20% of the radioactivity is always found in the material at the top of the gradient, where most of the absorbancy, for SDS-dissociated virus preparations, is due to viral protein. This material, designated peak A, is discussed further below. From the sedimentation coefficient of reovirus virions (630 s) and their RNA content (la%, Gomatos & Tamm, 1963a) it can be calculated, making suitable assumptions, that the molecular weight of the reovirus genome should be about 107. It is not known what the sedimentation coefficient of a double-stranded RNA molecule of this size would be. However, since the sedimentation coefficient of the replicative form of the RNA of bacteriophage R17 (mol. wt 2.2 x 106) is 14.8 s (Franklin, 1967), and that of the replicative form of the RNA of poliovirus (4 x 106) is 20 s (Katz & Penman, 1966), one would expect a sedimentation coefficient of between 20 and 30 s for the intact genome of reovirus RNA. Considerable efforts were made to devise methods for releasing RNA molecules of such size from reovirus virions. It became apparent that there were three major factors to be considered: (a) The chemical treatment. Table 1 lists most of the methods tried for liberating intact reovirus RNA molecules. In no case was an RNA peak observed at a position corresponding to more than 14 s. When SDS was added to a suspension of reovirus virions at a very slow rate, it did appear on occasions, that the sedimentation coefficient of the largest of the three peaks was increased by 1 to 2 s ; but this was not reproducible, and not further investigated. (b) The age of the virus preparation. Haselkorn (1962) has reported that the intact viral genome cau only be extracted from freshly prepared turnip yellow mosaic virus: virus which has been stored for some time yields only RNA fragments. Similarly, Robinson, Pitkanen & Rubin (1965) found that it is essential to use
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
TABLE 1
Chemical treatments applied to reovirus in attempting to release intact viral genomes Treatmentt Reovirus in 1 x SSC or CsCl (as obtained from density-gradient) (i) Sodium perchlorate (5 M) plus EDTA (0.01 M) at 0, 20 or 50°C, with or without carrier ribosomal or yeast RNA (modifications of the method of Freifelder (1965)) Dialysis against sodium perchlorate (5 M) plus EDTA overnight at 4°C (ii) Urea (4 M or 8 M) at 0°C (iii) SDS: added over a period of 1 hr to a final concentration of 6.01% Added slowly over a period of 8 hr to a final concentration of 0.5% (iv) SDS plus urea (1% SDS, 0.5 M-USBaU) (2) Reovirus pre-treated with pronase (virus in 1 x SSC-60% sucrose; pronase heated at 85’C for 15 min, final concentration 1 mg/ml., incubation 15 mm at 2O’C) (i) As (i) above (ii) SDS: to 1% rapidly to 1% over a period of 30 min
s-Value of largest RNA molecule liberated
(I)
14 14 14
(sometimes
(sometimes to 1 y0 rapidly in the presence of high mol. wt DNA (1 mg/ml.) (iii) Urea: to 8 M slowly (iv) Pronase alone: 1 mg/ml. for 45 min at 37%
14 14 slightly 14
14 14 14 slightly
higher)
higher)
14 no lysis no lysis
t All these treatments were carried out in test tubes and the reaction mixtures were then transferred carefully onto density gradients. Many of the treatments were also applied to virus layered on density gradients. In no ease was any difference observed between the two methods. Over 90% of RNA was liberated in all cases except those marked “no lysis”.
freshly prepared Rous sarcoma virus preparations in order to obtain intact RSVRNA. It is conceivable that a similar situation holds for reovirus, although it is clearly not entirely analogous, since storage of TYMV and RSV is accompanied by loss of infectivity, whereas reovirus is stable. Nevertheless, RNA was extracted from reovirus immediately after its isolation from C&l gradients. The sedimentation profile of RNA from such virions was the same as that from virions which had been stored for some days. (c) The type of cell in which the virus was grown. Oda & Joklik (1967) found that vaccinia virus mRNA was degraded more rapidly in vitro by extracts of L cells than of HeLa cells. This raised the possibility that regions in the reovirus genome especially sensitive to enzymic attack might be less likely to survive intact in L cells than in HeLa cells, or indeed in other cells which also have a very low level of nuclease activity in their cytoplasm. Reovirus was therefore grown in HeLa cells and in BHK21 cells, the latter being chosen as they were the cells in which the reovirus was prepared which yielded long (about 5 p) RNA molecules on disruption in situ on electron microscope grids (Granboulon & Niveleau, 1967). However, the sedimentation profiles of RNA extracted from reovirus grown in HeLa or BHK21 cells were identical to that displayed by RNA extracted from reovirus grown in L cells.
8
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
In conclusion, it has so far proved impossible to separate from reovirus virions RNA molecules corresponding to the intact genome. Irrespective of the method applied, density-gradient centrifugation analysis indicates that a mixture of fragments falling into three major size distributions is obtained. (ii) Polyacrylamide electrophoresis alzalysis The three classes of RNA molecules extractable from virions could also be-separated by means of polyacrylamide gel electrophoresis. In fact, the resolution achieved by this method greatly exceeds that of density:gradient centrifugation. Figure 3 shows 1
0
10
I
L’ .
20
30 Fraction
FIG. 3. Polyacrylamide
I
i
I
40
50
60
no.
gel electrophoresis
pattern of reovirus RNA.
3ZP-labeled reovirus RNA (separated from peak A material by preliminary zone sedimentation) was subjected to electrophoresis through a 2.4% polyacrylamide gel at 150~ for 10 hr. Migration was from left to right.
a typical pattern. The fastest moving material, shown in separate experiments to correspond to the slowest sedimenting peak S, was clearly resolvable into three components. The middle peak corresponds to peak M and the slowest peak to peak L. Each of these two peaks showed signs of resolving into two fractions on prolonged electrophoresis. (b) Size of re$icating
reovirus RNA
It may be argued that if the reovirus genome is one “molecule” with a number of built-in weak spots, breaking open the capsid may engender shearing forces sufficient to break the RNA. However, one would expect to find intact reovirus RNA within the cell prior to encapsidation. A search for such viral RNA was therefore undertaken. L cells infected with reovirus in the presence of actinomycin D (O-5 r.Lglml.) were pulse-labeled with [14C]uridine for 30 minutes from 1.2 to 125 hours after infection, the time at which the rate of viral genome replication was proceeding at its maximum (see Bellamy & Joklik, 1967, following paper). Cytoplasmic fractions were prepared as outlined in Materials and Methods, treated with SDS and centrifuged on sucroseSDS density gradients, Over 80% of the radioactivity incorporated was in material which was sensitive to ribonuclease : this RNA was identified as reovirus mRNA (see following paper). Material resistant to RNase was found in the region between 10 and 14 s only. A similar analysis was carried out using a method of analysis in which the oytoplasmic fractions are chilled prior to centrifugation in order to precipitate
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
9
most of the SDS, and the material is then centrifuged on sucrose-RSB gradients not containing SDS (McConkey & Hopkins, 1965). This method permits a more precise estimate of the sensitivity of RNA to digestion with RNase, but once again RNaseresistant RNA was only found in the 10 to 14 s region. Finally, the possibility remained that intact naked reovirus RNA was sheared during the breaking open of cells by Dounce homogenization. Infected cells were therefore opened by treatment with Nonidet P40 (0.5%) (O’Brien, 1964) and the cytoplasm so obtained analyzed by density-gradient centrifugation. Once again the only RNase-resistant RNA was in the 10 to 14 s region. This was true not only for the period from 12 to 12.5 hours after infection, but at all stages of the infection cycle when incorporation of label into double-stranded RNA could be detected. The conclusion from all this work is that if reovirus RNA replicates as an “intact” molecule of molecular weight 10 x 106, it is as prone to breakage when yet uncoated in the cytoplasm as in the virion. However, these results also strengthen the possibility that the reovirus genome does not replicate as an intact molecule but as a
I.0 50
60
70
80
Temperature
90
100
110
(“0
FIG. 4. The effect of ionic strength on the thermal transition of reovirus SSC; curve b, 0.1 x SSC; curve c, 1.0 x SSC; curve d, IO.0 x SW.
RNA.
Curve a., O-01 x
number of separate pieces, which are then processed by some maturation mechanism, which selects the correct assortment of individual pieces for encapsidation, possibly by some sort of loose binding. (c) Studies on the secondary structure of reovims
RNA
The following studies were carried out on PtiNA extracted from virions by means of SDS and phenol, followed by ethanol precipitation. The RNA was then dissolved in the appropriate solvent, as required. (i) Melting behavior Figure 4 presents data on the melting behavior of reovirus RNA at four different electrolyte concentrations. The ionic environment strongly influences the stability of the secondary structure. In 0.01 x ,0-l x , 1 x and 10 x SSC, the RNA exhibits sharp melting profiles with T, values of 75, 84, 96 and 104”C, respectively. These values are similar to those reported by Shatkin (19653). The maximum hyperchromic increase was about 34%.
10
BELLAMY,
SHAPIRO,
AUGUST
AND
40
60
JOKLIK
AAA-A-l----.11
L
20
0
Time (min) FIG. 5. The effect of ionic strength on the digestion of reovirus RNA with RNase A. 0.2 o,D.~~~ unit/ml. of RNA were incubated at 37°C with 1 pg/ml. RNase A. (a) Kinetics of digestion measured by the increase in absorbancy at 26Omp. (-O-O-) RNA iu 041 x SSC; (-O-O-) RNA in 0.1 x SSC; (-A--A-) RNA in 1.0 x SSC. (b) Kinetics of digestion measured by the release of acid-soluble radioactivity. (-O--e-) RNA in 1.0~ SSC. RNA in O-1 x SSC; (--O-O-) RNA in (5.15~ SSC; (-A-A-) Input: [32P]RNA, 1000 cts/min.
L) A- I-...--Ai-.-.-...-3:20 0
IO
Time (min)
FIG. 6. The effect of [Mg2+] on the digestion of reovirus RNA by RNase A. 0.2 o.D.~~~ unit/ml. of RNA in 0.01 x SSC vere incubated at 37°C with 1 pg/ml. RNase A. (-U-n-) No Mg2+ ; ;;-F,E~;~ 10-5~-Mg2+; (-.--.--) lo-%Mg2+; (-O-O-) lo-%Mg’+; (-A-A--)
CHARACTERIZATION (ii) Resistance
to hydrolysis
OF
REOVIRUS
CENOME
RNA
11
by RNase
The resistance of reovirus RNA to hydrolysis by pancreatic RNase A may be tested by measuring either hyperchromicity or production of acid-soluble radioactivity (Fig. 5(a) and (b)). At molarities greater than 0.1 M, reovirus RNA is resistant to RNase ; at lower molarities the RNA becomes progressively more susceptible to hydrolysis. At low ionic strengths (0.01 x SSC or less) the resistance of reovirus RNA to RNase is markedly increased by Mg2 + (Fig. 6). At ionic strengths which normally stabilize the RNA against a given level of RNase, higher concentrations of the enzyme cause complete hydrolysis (Pig. 7). Resistance to digestion by RNase thus depends, among other factors, on the concentration of monovalent ions, divalent ions and the concentration of enzyme. Further, digestion by RNase does not require extensive prior denaturation.
0
20
40
60
Time (mid
FIG. 7. The effect of enzyme concentration on the digestion of reovirus RNA by RNase A. 0.25 o.D.~~~ unit/ml. of RNA in 1 x SSC were incubated at 37°C with varying concentrations of RNase A. (-@----•--) lOOpg/ml.RNaseA; (-O-O-) lOpg/ml.RNaseA; (-A---A-) 1 pg/ml. RNase A.
(iii) Effect of electrolyte concentration
on the sedimentatiovb behavior
The sedimentation behavior of single-stranded RNA is strongly influenced by the ionic concentration. By contrast, the sedimentation properties of reovirus RNA are unaffected by ionic strength over the range from 0.01 x to 1 x SSC. Summarizing, these results are consistent with the notion that reovirus RNA fragments have a highly base-paired structure approaching double-strandedness. (d) Base composition
of reoviv-us RIVA
Table 2 presents the base compositions of (a) total viral RNA, (b) RNA in the pooled peaks L, M and S and (c) RNA in peak A isolated from the top of density gradients. The base composition of RNA within virions is far from .that expected for a doublestranded structure, since there is a large excess of A over TJ. The source of this excess A is the small molecular weight fraction, peak A, which has the extraordinary composition of 81.5 % A, 13 %U, and 3 % each of C and G. The base composition of the major
12
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
TABLE 2 Base composition Source of RNA preparation Whole virus Pooled peaks L plus M plus S Peak A
of reovirus RNA
c
A
G
u
16.9 21.9 3.2
31.7 27.5 81.3
17.5 21.9 2.8
27.9 28.7 12.7
G/C
W-J
1.035 I.00 -
1.35 0.96 6.4
portion of viral RNA (peaks L, M and S) indicates perfect base pairing, with A equal to U and G equal to C, and is similar to that reported for reovirus RNA by Gomatos & Tamm (1963c). The small molecular weight fraction, peak A, was further investigated. (a) The A-rich RNA is an internal viral constituent as it is still present after digestion of virions with micrococcal nuclease. Control experiments showed that this enzyme hydrolyzed poly A efficiently. (b) A-rich RNA is not confined to a special class of virus particle differing from the rest in density. Reovirus labeled with 32P was banded in CsCl and the virus peak divided into five fractions. The RNA from each fraction was then extracted and the ratio of A-rich RNA to 10 to 14 s viral RNA determined by appropriate densitygradient centrifugation analysis. The ratio was identical for all five fractions and was equal to 0.18 to O-20. TABLE 3 Estimation RNA L
of the molecular weight of L, .M and X pieces of reovirus RNA component M
2 x 106 2.3 x 106 1.25 x lo6 2.3 x 10s 1.45 x lo6 7 Using the value Franklin, 1966).
Method
S
From the mol. wt and s-value of R17 replioative form (Franklin, 1967) 7.3 x 105 From length measurement@ (Dunuebaoke & Kleinsohmidt, 1967) 1-O x 1C16 Studier (1965)
of 2.08
x
10” daltons/p
calculated
from
the data for
R17
(Granboulan
8r.
(c) The approximate size of the A-rich RNA was determined by density-gradient centrifugation. It sedimented more slowly than y-globulin and together with bovine serum albumin. Its sedimentation coefficient was approximately 2 s. Recent experiments have shown that it moves faster than 4 s tRNA on electrophoresis in polyacrylamide gels (Bellamy & Joklik, unpublished results). (d) A-rich RNA is synthesized in the cell in step with the rest of the viral genome. This was shown by pulse-labeling infected cells with 32P for four hours at various stages throughout the infection cycle and isolating the viral progeny at 24 hours after infection. The proportion of label in the A-rich material was constant. Actinomycin D (0.5 pg/ml.) was present in all these experiments from the time of infection.
Fraction
no,
FIG. 8. The effect of DMSO on reovirus RNA. Reovirus RNA (separated from peak A material by preliminary zone sedimentation) was treated with DMSO by the method of Katz t Penman (1966). RNA was then layered onto a 15 to 30% SDS-sucrose density-gradient and centrifuged for 18 hr at 24,000 rev./mm in rotor SW25 of the Spinco model L ultracentrifuge. (-e--e--) Total radioactivity; (-O--O-) RNase-resistant radioactivity.
0 fraction
IO
20
30
no.
FIG. 9. The effect of DMSO treatment on the three size classes of reovirus RNA. Reovirus RNA (separated from peak A material by preliminary zone sedimentation) was centrifuged on a 10 to 15% SDS-sucrose density-gradient for 20 hr at 24,000 rev./min in the SW25 rotor of the Spinco model L ultracentrifuge. One-twentieth of each fraction was counted to give the profile shown in (a). Fractions B, C, and D, corresponding to the three peaks of viral RNA, were then pooled as indicated and treated with DMSO. The DMSO-treated RNA was centrifuged on 15 to 30% SDS-sucrose density-gradients for 18 hr at 24,000 rev./min ( (b), (c) and (d) respectively).
14
BELLAMY,
(e) Conversion
SHAPIRO,
AUGUST
of reovirus RNA
AND
JOKLIK
to the single-stranded
state
Reovirus RNA was converted to single-stranded RNA by the method of Katz & Penman (1966). This method is known to convert the replicative form of poliovirus RNA (20 S) to a single-stranded form of RNA sedimenting with a sedimentation coefficient of 35 S, which is the sedimentation coefficient of native poliovirus RNA. This method therefore clearly dissociates double-stranded RNA molecules into components which behave like native single-stranded molecules. Figure 8 shows the sedimentation pattern given by reovirus RNA converted to the single-stranded condition. The three RNase-resistant peaks L, M and S (see Fig. 2) are replaced by three RNase-sensitive peaks designated peaks L’, M’ and S’, respectively. Peak L’ sedimented approximately midway between 28 s and 16 s ribosomal RNA; in polyacrylamide gel electropherograms it was located closer to 28 s than to 16 s ribosomal RNA markers. A sedimentation coefficient of 22 to 25 s was therefore assigned to it. Peak N’ almost coincided wit,h 16 s ribosomal RNA in density-gradients ; polyacrylamide gel electrophoresis revealed that it was slightly larger than 16 s ribosomal RNA, and a sedimentation coefficient of 16 to 18 s was assigned to it. Peak S’ had an approximate sedimentation coefficient of 14 s, and its behavior on electrophoresis was consistent with this estimate. These three species of single-stranded RNA correspond to the three RNase-sensitive fractions obtained by thermal denaturation of reovirus RNA (Iglewski & Franklin, 1967). To clarify the origin of these components, a density-gradient containing peaks L, M and S (Fig. 9(a) ) was subdivided as shown and each set of pooled fractions separately converted to the single-stranded form. Peak L gave rise to peak L’, peak M to peak M’ and peak S to peak S’ (Fig. 9 (b) to (d) ).
4. Discussion The reovirus genome is released from virions as a mixture of RNA fragments. The distribution of the size, and therefore of the length, of these fragments is highly reproducible and independent of the extraction procedure. The possibility that the reovirus genome is subject to breakage within the capsid was considered, but found to be unlikely for the following reasons : (1) the virus is not inactivated during isolation; (2) the virus does not lose infectivity on storage; and (3) the identical mixture of fragments was obtained from virions less than 24 hours old and others up to eight weeks old. Intact viral genome molecules were not found in cells in which viral RNA was being synthesized; double-stranded RNA molecules extracted from such cells fell into the same size distribution as those extracted from virions. This failure to extract larger molecules was not due to shearing during breaking of cells, since cell lysis by means of a non-ionic detergent yielded the same RNA fragments; nor was it due to nuclease activity in the cytoplasm, since the same result was obtained when infected BHK21 and HeLa cells were used, both of which have very little cytoplasmic nuclease activity. The individual RNA fragments appear to be double-stranded by all criteria applied : they exhibit a sharp melting profile with a T, dependent on the ionic strength, they are resistant to ribonuclease provided ionic strength, divalent cation concentration and enzyme concentration are suitably adjusted, their sedimentation behavior is independent of the ionic strength over a large range, and their base composition
CHARACTERIZATION
OF REOVIRUS
GENOME RNA
15
indicates equality of A and U, and G and C. These results confirm and extend those of previous workers (Gomatos $ Tamm, 1963a,b; Shatkin, 19655). It is not yet poesible to say whether the fragments are base-paired throughout their entire length, or whether there exist some short single-stranded regions at the ends. The fact that the same mixture of fragments is extracted from reovirus virions and from cells in which the virus is replicating, as well as the demonstration (see following paper) that these fragmen$s are the intracellular templates for messenger RNA transcription, suggests that the reovirus genome is composed of a linear array of tenuously linked segments. It thus becomes of interest to enquire into the number and type of segments which make up the reovirus genome. In order to do this, it is necessary to know the size of the various classes of segments, their relative frequency and t,he size of the viral genome. We will consider each of these in turn. (a) The size of the segments. The sedimentation coefficients of the three classes of reovirus RNA fragments are about 14,12 and 10.5 s (classes L, M and S). The approximate molecular weights of these classes of segments can be estimated as follows. (i) Class L segments may be compared with the replicative form of the RNA of phage R17, the sedimentation coefficient and molecular weight of which are known. The best estimate of the molecular weight of R17 RNA is 1.1 x lo6 (Sinha, Fujimara & Kaesberg, 1965). The molecular weight of its replicative form is therefore 2.2 x lo6 and the sedimentation coefficient of this double-stranded RNA is 14.8 s (Franklin, 1967). This sedimentation coefficient is very close to that of the L fragments of reovirus RNA (14 s) and the molecular weight of these fragments is therefore most probably close to 2 >( lo6 (Table 3). (ii) The molecular weight of double-stranded RNA molecules can be derived from their length. Dunnebacke & Kleinschmidt (1967) extracted RNA from reovirus with 2 M-NaClO, and examined it on protein monolayers by means of the electron microscope. They found a mixture of filaments displaying a trimodal pattern of length distributions, with peaks at 1.1, 0.6 and 0.35 p. This distribution is probably very similar to the one described in this paper. There are two sets of data relating the length of such double-stranded RNA molecules to their molecular weight. On the one hand, Langridge & Gomatos (1963) reported a translation value per nucleotide residue of about 3 A for reovirus RNA, corresponding to about 2.4 x lo6 daltons per p (Gomatos & Stoeckenius, 1964). On the other hand, Granboulan & Franklin (1966) found the replicative form of R17 RNA to be 1.06 p long. Since the molecular weight of this RNA is 2.2 x lo6 (Sinha et ccl., 1965; Franklin, 1967), this leads to a value of 2.08 x lo6 daltons per p. Since Langridge & Gomatos (1963) carefully pointed out that the translation value per nucleotide residue of 3 A can not yet be regarded as definitive, we shall, for the purposes of the discussion which follows, use the value of 2.08 x lo6 daltons per p, keeping in mind however the fact that there is evidence for a higher value. Equating the three classes of molecules in our distribution with the three size classes of filaments of Dunnebacke & Kleinschmidt, one arrives at the molecular weights shown in Table 3. (iii) Finally, one may apply the equation of Studier (1965) relating the sedimentation coefllcient of DKA to its molecular weight: S = 0.0882 x -U”.346, making the assumption (which most probably is not strictly true) that double-stranded RNA and DNA behave similarly. This equation yields the molecular weights shown in the last line of Table 3. Bearing in mind the fact that the sedimentation coefficients of L, M and S fragments are approximate, and averaging the three series of estimates just presented, one may assign the following approximate values to the molecular weights of L, M
16
BELLAMY,
SHAPIRO,
AUGUST
AND
JOKLIK
and S segments of reovirus RNA : 2.3 -+ O-2 x 106, 1.3f0.2 x lo6 and 8f2 x 105. (b) The relative frequency of L, M and S segments. Dunnebacke & Kleinschmidt (1967) found the relative number of filaments in each of the three length distribution peaks, 1.1, O-6 and 0.35 p, to be in the ratio of 2 : 3 : 4 to 6. Our own estimate of the mass distribution in peaks L, M and S (based on the amount of radioactive isotope in peaks L, M and S, measured in polyacrylamide gel electropherograms in which these three classes of molecules are separated absolutely) is 2 : 1 : 1. This leads to the following estimate of the number of molecules in these three peaks based on the molecular weights derived above: 1-O : 0.89 : 1.44. The relative frequency of L, M and S molecules is thus most probably n : n : 15n. (0) The size of the reovirus genome. There is some uncertainty concerning the molecular weight of the reovirus genome. On the one hand, Gomatos & Tamm (1963a) derived a minimum value of 10.2 x lo6 daltons from an estimate of the molecular weight of the virion (derived from measurement of the sedimentation coeEicient and diffusion constant) and its percentage content of nucleic acid. On the other hand, Vasquez & Kleinschmidt (1967) have estimated the total length of the reovirus genome to be of the order of 8 p; using the value of 2.08 x lo6 daltons per ,LLderived above, this corresponds to a molecular weight of about 16.5 x 106. Further uncertainty is introduced by the presence of the single-stranded A-rich material, the contribution of which to the length of the reovirus genome is not known. The best estimate at this time is probably that the reovirus genome weighs not less than 12 and not more than 16 million daltons. This weight could be made up of L, M and S segments as follows: if n is equal to two, then the combined weight of the seven pieces would be 9.6 x 106; an extra 20% of A-rich material would bring the total molecular weight of reovirus RNA to 12 x 106. If n is equal to three, the combined weight of the double-stranded segments alone would be 14.4 x 106; if n is equal to four, the corresponding value is 19.2 x 106, a value irreconcilable with the molecular weight estimate of Gomatos & Tamm, but still compatible with a genome 8 TVlong, provided one uses the value of 2.39 x lo6 daltons per p (molecular weight 19.1 x 106). The A-rich RNA could then not contribute to the measured length of the viral genome. It is difficult, however, to accept a value as high as 19 x lo6 for the molecular weight of the reovirus genome. The most likely value of n is probably two, since after prolonged polyacrylamide gel electrophoresis peaks L and M resolve into two components each, and peak S into three. We thus favor a model in which the reovirus genome is composed of seven pieces of double-stranded RNA, namely two L plus two M plus three S segments. The significance and function of the large amount of A-rich RNA are not clear at the moment. It is tempting to speculate that this material serves to link together the double-stranded RNA fragments; indeed Gabor & Hotchkiss (1966) have suggested that A-rich regions may act as punctuation marks and breakage points in DNA. Further, since no intact genomes can be isolated from infected cells, it is possible that reovirus RNA replicates as a series of separate segments. This mode of replication would necessitate a mechanism for ensuring that each virion receives one of each of these segments, and a simple method of achieving this would be to join the segments in some way. However, the amount of A-rich material present seems far in excess of that required to join seven or even fourteen double-stranded RNA pieces, and its function may be quite different, Further work is clearly necessary before this question can be settled.
CHARACTERIZATION
OF
REOVIRUS
GENOME
RNA
17
We would like to thank Dr J. V. Maize1 for helpful discussions concerning the electrophoresis of RNA in polyacrylamide gels. This investigation was supported by grants number AI-04913, AI-04163, GM-11936 and GM-11301 from the National Institutes of Health, grant number E-379 from the American Cancer Society, and grant number GB-5082 from the National Science Foundation. One of us (J.T.A.) is the recipient of an investigatorship from the Health Research Council of the City of New York (contract I-346) ; another (W.K.J.) is the recipient of a United States Public Health Service Research Career Award (no. l-K6-AI-22, 554). REFERENCES Baltimore, D. (1966). J. MOE. Biol. 18, 421. Becker, Y. & Joklik, W. K. (1964). Proc. Nut. Acad. Sci., Wash. 51, 577. Bellamy, A. R. & Joklik, W. K. (1967). J. Mol. Biol. 29, 19. Biochem. 12, 230. Bieleski, R. L. (1965). Analyt. Dunnebacke, T. & Kleinschmidt, A. (1967). 2. NatzLrf. 22b, 159. Eagle, H. (1959). Science, 130, 432. Franklin, R. M. (1967). J. Viral. 1, 64. Freifelder, D. (1966). Biochem. Biophys. Res. Comm. 18, 161. Gabor, M. & Hotohkiss, R. D. (1966). Proc. Nat. Acad. Sci., Wash. 56, 1441. Gomatos, P. J. & Stoeckenius, W. J. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1449. Gomatos, P. J. & Tamm, I. (1963a). Proc. Nat. Accd Sci., Wah. 49, 707. Gomatos, P. J. & Tamm, I. (19633). Science, 140, 997. Gomatos, P. J. & Tamm, I. (1963c). Proc. Nat. Acad. Sci., Wash. 50, 878. Gomatos, P. J., Tamm, I., Dales, S. & Franklin, R. M. (1962). Virology, 17, 441. Granboulan, N. & Franklin, R. M. (1966). J. Mol. BioZ. 22, 173. Granboulan, N. & Niveleau, A. (1967). J. Microbiologic, in the press. Hallinan, T., Fleck, A. & Munro, H. M. (1963). Biochim. biophys. Acta, 68, 131. Haselkorn, R. (1962). J. Mol. BioZ. 4, 357. Ifft, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phya. Chem. 65, 1138. Iglewski, W. J. & Franklin, R. M. (1967). J. Viral. 1, 302. Joklik, W. K. & Becker, Y. (1965). J. Mol. BioZ. 13, 496. Katz, L. & Penman, S. (1966). Biochem. Biophys. Res. Comm. 23, 557. Langridge, R. & Gomatos, P. J. (1963). Science, 141, 694. Loening, U. E. (1967). Biochem. J. 102, 251. MacPherson, I. A. & Stoker, M. G. P. (1962). Virology, 16, 147. McConkey, E. H. & Hopkins, J. W. (1965). J. Mol. BioZ. 14, 257. Marcus, P. I., Cieciura, S. J. & Puck, T. T. (1956). J. Ezp. Med. 104, 615. O’Brien, B. R. A. (1964). J. Cell. BioZ. 20, 521. Oda, K. & Joklik, W. K. (1967). J. Mol. BioZ. 27, 395. Prevec, L. & Graham, A. F. (1966). Science, 154, 522. Ralph, R. K. & Bellamy, A. R. (1964). Biochim. biophys. A&, 87, 9. Robinson, W. S., Pitkanen, A. & Rubin, H. (1965). Proc. Nat. Acad. Sk, Wmh. 54, 137. Shatkin, A. J. (1965a). Biochem. Biophys. Res. Comm. 19, 506. Shatkin, A. J. (1965b). Proc. Nat. Acad. Sci., Wash. 54, 1721. Shatkin, A. J. & Rada, B. (1967). J. Viral. 1, 24. Sinha, M. K., Fujimara, R. K. & Kaesburg, P. (1965). J. Mol. BioZ. 11, 64. Studier, F. W. (1965). J. Mol. BioZ. 11, 373. Summers, D. F., Maizel, J. V. & Darnell, J. E. (1965). Proc. Nut. Acad Sci., Wash. 54,505. Vasquee, C. & Kleinschmidt, A. (1967). Proc. Symp. Electron Microscope Society ofAmerica, Chicago (to be published). Weissman, C. (1965). Proc. Nat. Acad. Sk., Wash. 54, 202.
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