VIROLOGY
64, 96-105 (1975)
Reovirus
Genome
RNA Segments:
S. MUTHUKRISHNAN Department
of Cell Biology,
AND
Roche Institute Accepted
Resistance AARON
of Molecular
to S, Nuclease
J. SHATKIN
Biology,
Nutley,
New Jersey 07110
October 14, 1974
Reovirion genome RNA was purified by sedimentation in a sucrose-formamide gradient after treatment with 50% formamide under nondenaturing conditions for genome RNA. Both the 5’- and 3’-termini of purified double-stranded (ds) RNA were completely resistant to digestion by the single-stranded specific nuclease from Aspergillus oryzae, S,. In contrast, the unpaired 3’-terminal dinucleotide, CPC-~H, in tRNA was completely hydrolyzed by the enzyme. Reovirus ds RNA was sensitive to T, and pancreatic RNases to a small extent, even in the presence of 0.3 M NaCl. Ds RNA did not act as a primer/template for avian myeloblastosis virus (AMV) reverse transcriptase. The results indicate that reovirus ds genome RNA does not contain S,-sensitive single-stranded regions.
The arenaviruses, influenza virus, oncornaviruses, and reoviruses all contain segmented RNA genomes (Shatkin, 1974a). One of the intriguing features of these viruses is the mechanism by which the viral genome replicates to yield progeny virions containing the correct assortment of RNA molecules. In an effort to understand this process more completely, we have been studying the structure of the reovirus genome which consists of 10 double-stranded (ds) RNA segments (Shatkin et al., 1968). All 10 segments are required for reovirus infectivity since particles containing only nine are defective (Nonoyama et al., 1970). Furthermore, 10 viral proteins can be detected in productively infected cells (Both et al., 1975). One attractive hypothesis is that progeny genomes in infected cells are copied from a linked array of single-stranded or double-stranded RNA segments that are precisely ordered within parental subviral particles (Ward and Shatkin, 1972; Zweerink, 1974; Acs et al., 1971). Several possible modes of linkage of the 10 segments have been previously considered: (I) contiguous genome segments have
hydrogen-bonded base-pairs form (“Sticky-end” model,) (Millward and Nonoyama, 1970); (II) the segments are perfect duplexes, but terminal sequences of adjacent segments are identical and thus permit intersegment base-pairing of their strands (“street intersection” model ,Y) (Banerjee and Grece, 1971); (III) segments are aligned by viral proteins or other “linker” molecules, with or without base-pairing as in (I) or (II) (Ward and Shatkin, 1972; Zweerink, 1974). We have attempted to differentiate among these models by analyzing the terminal sequences of reovirus RNA (Miura et al., 1974) and by testing for the presence of “sticky ends” in the ds segments. It was previously suggested on the basis of studies with phosphomonoesterase and phosphodie&erase that short, purine-rich, singlestranded tails are present in the 5’ ends of reovirus RNA (Millward and Nonoyama, 1970). Since the enzyme S, from Aspergillus oryae degrades without base preference the single-stranded portions of DNA/ DNA, RNA/DNA, or RNA/RNA hybrid molecules (Ando, 1966; Sutton, 1971; Shishido and Ando, 1972), we have used it to test reovirus genome RNA for the presence
complementary
of single-stranded
INTRODUCTION
single-stranded
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
tails
that
regions.
REOVIRUS MATERIALS
AND
GENOME
METHODS
Cells and Virus Mouse L cells and BHK-21 cells were grown in suspension culture in Eagle’s medium containing 5% fetal calf serum. Cells were infected with reovirus type 3 Dearing strain at a multiplicity of infection of 10 PFU/cell. Virus was extracted with fluorocarbon and purified by isopycnic sedimentation in CsCl as described previously (Banerjee and Shatkin, 1971). For preparation of 32P-labeled ds RNA, cells were resuspended after virus adsorption in phosphate-free medium containing 0.25 pglml of actinomycin and [32P]phosphoric acid (20 pCi/ml) was added 2 hr later. After 36 hr at 37:: the cells were harvested and virus purified. Preparation
of Radioactive
RNA
32P-Labeled purified virions were extracted with phenol and the RNA separated from virion oligonucleotides by Sephadex G-100 gel filtration (Banerjee and Shatkin, 1971). Ds RNA was 3H-labeled at the 3’ termini by periodate oxidation and reduction with [3H]borohydride and repurified by gel filtration (Banerjee and Grece, 1971). Denatured genome RNA was similarly 3H-labeled in the 3’-penultimate residues. Purified RNA was denatured in 90% dimethyl sulfoxide at 37” for 30 min and precipitated by addition of 2 vol of ethanol in 0.15 M KC1 at -20”. The RNA was oxidized with periodate and sequentially treated with P-aniline, alkaline phosphatase, periodate, and [ 3H lborohydride (Hunt, 1970). The 3H-labeled RNA was repurified by gel filtration. Deadenylated 3’-cytidine 3H-labeled E. coli tRNA was kindly provided by Dr. David Ward, Yale University. It was prepared by removing the 3’-terminal CpA by two cycles of oxidation, p-elimination, and phosphatase digestion. The RNA was purified by gel filtration, dialyzed exhaustively, concentrated, and incubated with [3H]CTP and nucleotidyl transferase. The 3H-labeled tRNA contained 3 x lo5 cpm/ was OD,,,, and all of the radioactivity
97
FRAGMENTS
released as cytidine by digestion with 0.3 N KOH for 18 hr at 37” indicating that one 3’-terminal [3H]pC had been added per tRNA molecule during the transferase reaction. Purification of ds RNA in Formamide-Sucrose Ds RNA dissolved at a concentration of 1 mg/ml in 20 mM Tris buffer, pH 7.9, 1 mM EDTA, 1 x SSC (0.15 M NaCl, 0.015 M Na citrate) containing 50% formamide was incubated at 37’ for 1 hr and layered onto a 15-30% sucrose gradient in the same buffer-formamide mixture. After centrifugation for 8 hr (60,000 rpm. SW-65, 20”), fractions of 0.16 ml were collected and counted either by Cerenkov radiation (““P) or in Aquasol ( 3H) (New England Nuclear). The peak fractions containing the ds RNA were pooled as shown in Fig. 1 and precipitated by adding 2 vol of ethanol at -20”. Nuclease Digestions Ds RNA (ca. 100 pug) was digested with S, nuclease (10 ~1 of the DEAE-cellulose fraction of Sutton, kindly supplied by J. Sambrook, Cold Spring Harbor Laboratory) for 2 hr at 45” in 30 mM potassium acetate buffer, pH 4.5, 0.3 M NaCl, lo-‘M Zn2+ in a final volume of 0.2 ml. The reaction was stopped by chilling and adjusting the EDTA concentration to 10 mM. Digestion of ds RNA (0.5 mg/ml) with T, RNase (250 units/ml) or pancreatic RNase (1:20/w/w) was in 20 mM Tris buffer, pH 7.4, 1 mM EDTA, 0.3 M NaCl, at 37” for 2 hr. Reverse Transcriptase
Assay
Purified avian myeloblastosis virus (AMV) (2.2 mg/ml, kindly provided by A. Weissbach, Roche Institute) was pretreated with 0.4% NP-40 in 5 mM Tris buffer, pH 8, for 10 min at O”, and 0.02 ml of the treated preparation was added to a standard incubation mixture in a final volume of 0.15 ml containing 50 mM Tris buffer, pH 8, 7 mM MgCl,, 80 mM KCl, 0.5 mM dithiothreitol, 40 kg bovine serum
98
MUTHUKRISHNAN
albumin, four deoxyribonucleoside triphosphates (10 &f each, sp act = 1.6 Ci/ mmole), and the indicated primer/templates. Incubation at 37” was stopped after 20 min by chilling, adding 0.1 ml of 0.2 A4 sodium pyrophosphate, 1 ml HzO, and one-fifth volume of 12% perchloric acid. The acid precipitate was collected by centrifugation, dissolved in 0.2 N KOH, and reprecipitated. The acid-precipitable material was finally dissolved in 0.5 ml Protosol and counted in 4.5 ml toluene-based scintillation fluid acidified with acetic acid. Synthesis of Reovirus RNA in Vitro
Single-Stranded
Reovirus single-stranded RNA was synthesized with a-s*P-labeled ribonucleoside triphosphates in a standard reaction mixture of 0.5 ml containing 70 mM Tris buffer, pH 8, 7.5 mM Mg2+, 50 mM KCl, 2.5 mM cyclohexylammonium phosphoenolpyruvate, 2 mM of each of the four ribonucleoside triphosphates (except the s-32P-labeled one), 70 pg Macaloid, 90 pg chymotrypsin, 210 pg purified reovirus. After 30 min at 45”, 2.5 units of pyruvate kinase and the 32P-labeled nucleoside triphosphate were added and the incubation continued for the specified times. The RNA products were extracted with phenol and purified by gel filtration. The size of the products was determined by velocity sedimentation in 15-30% sucrose gradients. Radioactive
Compounds
and Enzymes
Pancreatic RNase (RASE) and crystalline a-chymotrypsin were from Worthington Biochemical Corp. T, RNase (Sankyo) was obtained from Calbiochem. (Y-“PLabeled ribonucleoside triphosphates and carrier-free [a2P]phosphoric acid were purchased from New England Nuclear. Potassium [3H]borohydride was from Amersham/Searle, and [3H]deoxyribonucleoside from obtained were triphosphates SchwarzlMann. RESULTS
Resistance of ds RNA Digestion 32P-Labeled reovirus isolated from purified
to S, Nuclease genome RNA was virions by phenol
AND SHATKIN
extraction and gel filtration. RNA purified by this procedure may contain residual single-stranded oligonucleotides (Bellamy et al., 1967) or noncovalently bound nascent chains of viral mRNA produced on the ds RNA template by the virion-associated transcriptase (Borsa and Graham, 1968; Shatkin and Sipe, 1968) and trapped during virus maturation. Therefore, the RNA was purified further by incubation at 37” for 1 hr in Tris buffer containing 1 x SSC and 50% formamide, and sedimented in a sucrose gradient containing 50% formamide (see Methods). Since 1% formamide lowers the Tm of nucleic acids by 0.7” (McConaughy et al., 1969), full-length reovirus RNA segments (Tm in 1 x SSC = 95”) (Shatkin, 1965; Bellamy et al., 1967) remain double stranded (ds) under these conditions. However, nonspecific, shorter, and less-stable hybrids would be denatured and separated from the ds RNA during the velocity sedimentation in sucrose. The peak of ds RNA that was collected from the gradient consisted exclusively of the 10 genome segments (Shatkin et al., 1968) when analyzed by polyacrylamide gel electrophoresis (Fig. 1 inset). RNA purified in this way was used in all the subsequent experiments. To test for the presence of singlestranded regions in the purified ds RNA, its sensitivity to S, nuclease digestion was examined. Two aliquots of 32P-labeled ds RNA, each 2.5 x lo6 cpm, were incubated with and without the enzyme for 2 hr at 45” in 0.3 M NaCl, conditions which favor digestion of single-stranded RNA. The mixtures were then analyzed by velocity sedimentation in 5-25% sucrose gradients. As shown in Fig. 2, no radioactivity was present at the top of either gradient in the fractions that would be expected to contain nucleotides released by S 1digestion. Under these conditions, the degradation of as little as 0.05% of the ds RNA, i.e., an average of one nucleotide per strand, would have released 1250 cpm. 5’ Termini of ds RNA. To test for the presence of intact 5’ termini in the S, nuclease-treated ds RNA, the enzymedigested segments were hydrolyzed with KOH, and the products were analyzed by
REOVIRUS
GENOME
FRAGMENTS
99
tides (0.18 M NaCl) 0.13% of the radioactivity, the expected amount of 5’ termini. 3’ Termini of ds RNA. The 3’ termini of reovirus ds RNA were similarly tested for resistance to removal by S, nuclease digestion. Ds RNA was extracted from virions, separated by gel filtration, and labeled at the 3’-terminal cytidine with [3H]borohydride as described previously (Banerjee and Grece, 1971). The 3H-labeled RNA was then purified by formamide treatment and centrifugation in sucrose-formamide gradients. Treatment of the purified 3’-3Hlabeled RNA with S, nuclease yielded 7% of the radioactivity at the top of the gradient as compared to 4% of the RNA incubated without enzyme (Fig. 4). The released radioactivity contained no 3Hlabeled cytidine trialcohol derivative and remained at the origin when analyzed by paper chromatography (De Wachter and Fiers, 1967). The results indicate that the I I I 0 5 IO 15 20 25 30 35 3’-termini are protected from S, nuclease FRACTION NUMBER by base-pairing. FIG. 1. Sedimentation of 32P-labeled reovirus ge- digestion, presumably Denatured RNA. To ensure that the S, nome RNA in a sucrose gradient containing formamide. Ds RNA purified by gel filtration was treated with nuclease is active against single-stranded 50% formamide as described in the Methods section reovirus RNA under the conditions of diand layered onto a 15%30% sucrose gradient in 50%’ gestion employed, 32P-labeled genome formamide, 1 x SSC, 1 mM EDTA, and 20 mM RNA was denatured with dimethyl sulfoxTris buffer, pH 7.9. Centrifugation was for 8 hr at ide and then digested with S,. More than 60K (SW-65) at 20”. Fractions were collected from the 50% of the radioactivity was acid soluble bottom and radioactivity in each fraction was deterafter 1 hr, and all the RNA was converted mined by Cerenkov counting. The samples indicated to low-molecular-weight material that sedby the bracket were combined, precipitated in ethaimented near the top of a sucrose gradient nol, and analyzed by electrophoresis in a 5% poly(Fig. 5). acrylamide gel (Shatkin et al., 1968) (inset).
chromatography on DEAE-cellulose in 7 M urea (Banerjee and Shatkin, 1971). As seen in Fig. 3, most of the radioactivity elutes at 0.08 M NaCl in the position of mononucleotides. The alkaline digests of both untreated RNA (Fig. 3A) and S,treated RNA (Fig. 3B) contain in the elution position of the 5’-terminal nucleo-
FRACTION
NUMBER
FIG. 2. Sedimentation of SzP-labeled ds RNA after S, nuclease digestion. Ds RNA was analyzed on a 5’%-25% sucrose gradient after incubation for 2 hr at 45°C in 0.3 M NaCl, 1 mM EDTA, 30 mM NaAc buffer, pH 4.5, with (0- -0) and without (O---O) S, nuclease. After centrifugation for 7 hr at 60,000 rpm (SW-45) at O”, 0.2.ml fractions were collected. Fractions l-10 were counted in Aquasol and 11-24 by Cerenkov radiation.
100
MUTHUKRISHNAN
n
I DO-
AND SHATKIN I
A
I
I
M NoCl - 0.3 50 -
FRACTION NUMBER FIG. 3. DEAE-cellulose chromatography of KOH digests of S,-treated and untreated ds RNA. The RNA in fractions lo-23 of Fig. 2 was pooled and precipitated with ethanol. The pellets of untreated (A) and S,-treated RNA (B) were dissolved in 0.3 N KOH, incubated at 37” for 18 hr, neutralized (Banerjee and Shatkin, 1971), and loaded onto 1 x 20.cm columns of DEAE-cellulose. The oligonucleotides were eluted with a gradient of 0.0-0.3 M NaCl containing 7 M urea and 50 mMTris, pH 8.0. Fractions of 1.8 ml were collected and counted in 9.5 ml 2-methyoxyethanol (methyl cellosolve) and 7.5 ml spectrofluor-toluene. The arrow indicates the elution position of ppGp (Banerjee and Shatkin, 1971).
0
2
4
6
s IO FRACTION
I2 14 NUMBER
16
IS
20
22
substrate. tRNA containing 3’-terminal . ..CpC - sH was prepared as described in the Methods section. It was treated with S, nuclease under the conditions described in the Methods section. As shown in Table 1, more than 50% of the 3’-terminal SHlabeled residues were hydrolyzed within 30 min. Similar findings were obtained with tRNA containing 3’[3H]CCA. Thus, S, nuclease can recognize the 3’-terminal unpaired bases in tRNA when the singlestranded tail is only two residues in length.
FIG. 4. Sedimentation of 3’-3H-labeled ds RNA after S, nuclease digestion. 3’-JH-labeled ds RNA (15 pg, 60,000 cpm) was incubated with and without S, nuclease and analyzed by sedimentation in 5-25% sucrose as in Fig. 2. The fractions collected from the gradient were counted in Aquasol. Incubation with (0- -0) and without (04) S, nuclease.
Action of Pancreatic RNA
3’-Termini of tRNA. As a test for the ability of S, nuclease to hydrolyze a short single-stranded tail in double-stranded RNA, tRNA which contains an unpaired 3’-terminal . ..CpCpA extending from a double-stranded stem was used as a model
The results indicate that both the 5’ and 3’ ends of reovirus genome RNA are resistant to the single-strand specific nuclease, S1, and suggest the absence of singlestranded tails in the RNA segments. The resistance of formamide-sucrose purified ds RNA to pancreatic and T, RNases is less
and T, RNases on ds
REOVIRUS
GENOME
101
FRAGMENTS
tively. Thus, even under conditions of relatively high ionic strength, the 3’ termini (and possibly 5’ termini as well) of ds RNA are sensitive to T, and pancreatic RNase digestion. Inactivity of ds RNA as FrimerlTemplate for AMV Reverse Transcriptuse
0
2
4
6
8
10 12 14 16 FRACTION NUMBER
18
20
22
24
FIG. 5. Analysis of denatured 3T-labeled reovirus genome RNA after digestion with S, nuclease. Ds RNA dissolved in 10 mM Tris buffer, pH 7.4, 1 mM EDTA was denatured by adding 9 vol of dimethyl sulfoxide and incubating at 37’ for 30 min. The denatured RNA, recovered by ethanol precipitation, was digested with S, nuclease and sedimented in sucrose as in Fig. 2 TABLE S, NUCLEASE
HYDROLYSIS
I OF 3’.TERMINAL
C FROM
tRNA” Time (min)
wm Acid-soluble
cpmb in pC
0
0
0
15 30 60 120
5,287 8,048 10,376 11,780
3,996 8,274 9,798 13,038
“The tRNA (14,000 cpm) was digested with S, nuclease as described in the Methods section. The reactions were stopped by chilling and addition of an equal volume of 10% trichloroacetic acid. The acidsoluble material was collected after filtration through a Millipore filter, extracted with ether to remove the acid, evaporated to dryness, and analyzed by highvoltage paper electrophoresis at pH 3.5. b A portion (-25%) of the released radioactivity migrated with authentic C, and the values reported are the sum of the radioactivity in pC plus C.
complete, however. Treatment of 32Plabeled ds RNA with either RNase in 0.3 A4 salt yielded 0.25% of the total radioactivity as low-molecular-weight material (Fig. 6A, fractions l-7). At least a portion of this material can be accounted for as 3’-terminal nucleotides from the results shown in Fig. 6B. Incubation of 3’-SH-labeled ds RNA with T, or pancreatic RNase released 35% and 100% of the radioactivity, respec-
Duplex structures containing 5’ singlestranded regions function as primer/ternplate for avian sarcoma virus reverse transcriptase (Baltimore and Smoler, 1971); the virion-associated enzyme adds deoxyribonucleotides to the 3’-end of a 4 S RNA primer that is base-paired with 35 S RNA genome subunits (Faras et al., 1973). Purified ds RNA was tested for primer/template activity with AMV reverse transcriptase as described in the Methods section Incorporation of 3H-labeled deoxynucleotides into acid-insoluble products was 0.03, 0.04, and 2.3 pmoles for reaction mixtures containing only endogenous primer/template, 20 pg of reovirus ds RNA,
I &-
i -
b 40 I x
A
f
” 20 : 0
4
8 FRACTION
12
16
20
24
NUMBER
FIG. 6. Sedimentation of pancreatic and T, RNase digestion products of ds RNA. Ds RNA labeled uniformly with [32P]phosphate (A) or at the 3’-termini with [SH]borohydride (B) was incubated with pancreatic RNase or T, RNase or no enzyme as described in the Methods section. The incubation mixtures were analyzed in 5%-25% sucrose gradients as in Fig. 2. Control (O--O), pancreatic RNase (.A), T, RNase (A-A).
102
MUTHUKRISHNAN
AND SHATKIN
and 10 pg of activated salmon sperm DNA (Fry and Weissbach, 1973), respectively. Complementarity of Terminal quences in ds RNA
I
I
I
KOH
----G’
I
I
U’
C’
---T---l---T
A’ ;
Base Se-
Since it has been reported that the 5’ termini contain G (Banerjee and Shatkin, 1971; Miura et al., 1974) and the 3’ ends are C (Banerjee and Shatkin, 1971) in reovirus RNA, it was of interest to determine if the penultimate bases are also complementary. To characterize the 3’-penultimate residues, ds RNA labeled at the 3’-termini by [SH]borohydride reduction was denatured and digested with T, or pancreatic RNase. T, RNase digestion will yield the 3H-labeled trialcohol derivative of cytidine (C’) from RNA containing G as the 3’penultimate residue. Similarly, pancreatic RNase will yield C’ from RNA containing a 3’-penultimate pyrimidine. The digestion products were analyzed by paper chromatography (Fig. 7). The total amount of ‘H-labeled 3’ termini was determined by analysis of KOH digests of the RNA. After T, RNase treatment, 45% of the total amount of 3’ ends based on the KOH value were recovered as C’, consistent with the 3’ sequence . ..GCOH in one strand. Pancreatic RNase treatment released the remaining 3’ termini as C’, and all the 3’ ends were released with a mixture of the two RNases. The complementary strand thus has a pyrimidine next to the 3’ cytidine. The same results were obtained when individual ds RNA segments were analyzed, consistent with . ..GCOH and . ..PyrCou at the 3’ ends of the two strands in each duplex. The 3’-penultimate residues were further identified by the stepwise procedures described by Hunt (1970). They included: (1) periodate oxidation of the denatured reovirus genome RNA; (2) P-elimination with aniline; (3) alkaline phosphatase digestion; with periodate; (5) (4) reoxidation [SH]borohydride reduction; (6) KOH hydrolysis of the 3H-labeled RNA; and (7) paper chromatography of the digest. As shown in Fig. 8, the 3’-penultimate nucleotides are G and U. If the 3’ and 5’-termini of reovirus ds RNA are base-paired, the 5’ sequences for
t
$ "
T, + Pam. RNose
1
20 IO 5 0 20 IO 5
-0
4
a
12
I6
io
24
ie
i2
DISTANCE MIGRATED (cm)
FIG. 7. Paper chromatography of the digestion products of 3’-3H-labeled reovirus RNA. Denatured 3’-8H-labeled RNA (30,000 cpm each) was digested with 0.3 N KOH for 18 hr at 37”; T, RNase (500 units/ml), and pancreatic RNase (20 rg/ml) for 18 hr at 37” in 10 mA4 Tris buffer, pH 7.4, and 1 mM EDTA; pancreatic RNase (100 pg/ml, 30 min, 37”) or T, RNase (500 units/ml, 18 hr, 37’). At the end of the incubations, the KOH hydrolysate was neutralized by passage through Dowex 50-H+ (Banerjee and Shatkin, 1971). A mixture of the four authentic trialcohol derivatives of cytidine (C’), uridine (U’), adenosine (A’), and guanosine (G’) (12 ~(g each) was added to the four digested RNA samples which were then analyzed by descending chromatography (De Wachter and Fiers, 1967) on Whatman No. 1 paper in a solvent system consisting of n-butanol: isobutyric acid: H,O:NH,OH (30:15:10:1). the two complementary strands would be GC... and GA... . Reovirus ds RNA is synthesized via single-stranded genome transcripts that are converted subsequently to genome RNA (Schonberg et al., 1971). Consequently, the single-stranded RNA corresponds to one of the strands of the duplex segments. We, therefore, determined the 5’-penultimate base of the single-stranded RNA by phosphate transfer experiments. Three single-stranded RNA preparations were synthesized in vitro by
REOVIRUS 401
1
’
GENOME
I
103
FRAGMENTS DISCUSSION
Model I for reovirus genome assembly requires that the ds RNA segments contain single-stranded tails. The single-strand L$P\d ii i[ specific nuclease, S1, would be expected to degrade these regions. No S,-sensitive ma0 4 i Ii I6 0 24 2s 32 36 terial was detected in ds RNA that had DISTANCE MIGRATED (4 been purified by sedimentation in forFIG. 8. Paper chromatography of the KOH digesmamide-sucrose under conditions detion products of reovirus RNA ‘H-labeled in the signed to remove noncovalently-associated 3’-penultimate positions. Denatured RNA was labeled with [3H]borohydride after removing the 3’ nucleic acids. S, nuclease degrades uncytidine as described in the Methods section. The paired nucleotide sequences without base RNA was digested with 0.3 N KOH and analyzed as preference by both exo- and endonudescribed in the legend of Fig. 7. cleolytic cleavage (Ando, 1966; Sutton, 1971; Shishido and Ando, 1972). In addiincubating the virion-associated RNA po- tion, the enzyme hydrolyzed the unpaired, lymerase (Borsa and Graham, 1968; Shat- 3’-terminal dinucleotide in tRNA, indicatkin and Sipe, 1968) with [LY-~~P]UTP, ing that it can recognize short single-CTP, or -ATP as the labeled precursor. stranded tails in RNA under the conditions The product RNAs were purified by gel used in these experiments. We thus should filtration and shown to consist of the three have detected single-stranded tails of two size classes of mRNA (1, m and s, Fig. 10 or more residues in reovirus genome RNA, inset) as reported previously (Banerjee and including purine-rich sequences (Millward Shatkin, 1970; Skehel and Joklik, 1969; and Nonoyama, 1970). The amount of ds Levin et al., 1970; Bellamy and Joklik, [32P]RNA used for S, digestion in our 1967). The RNAs were hydrolyzed with experiments was sufficient to detect the KOH which results in the transfer of the phosphate from the penultimate nucleotide to the 5’ nucleotide. Chromatography on DEAE-cellulose was used to separate the 5’-terminal residues (-5 charge) from the mononucleotides (-2 charge) (Banerjee and Shatkin, 1971). When [(Y-~~P]ATP or [N-~~P]UTP were the radioactive precursors, no radioactivity eluted in the position of -5 charge at 0.18 M NaCl (Fig. 9). The minor peaks eluting at 0.11 M NaCl in the position of -3 net negative charge are probably resistant dinucleotides (Lane and Butler, 1959). When [cx-~‘P]CTP was used, 0.2% of the radioactivity in the KOH digest eluted at 0.18 M NaCl in the position of FRACTION NUMBER 5’-termini (Fig. 10). Thus, the in vitro FIG. 9. DEAE-cellulose chromatography of KOH single-stranded RNA corresponding to the digests of reovirus mRNA synthesized in uitro with + strand of the duplex (Hay and Joklik, [(Y-~~P]ATP and [~x-~~P]UTP as the radioactive pre1971) contains the 5’ sequence GC... which cursors. Single-stranded RNA was synthesized under is complementary to one of the 3’ se- standard conditions as described in the Methods quences, . ..GCOH. The other 3’ sequence, section with (A) [w~~P]UTP (0.2 mM, sp act = 0.5 . ..UCOH. is complementary to the minus Ci/mmole) or (B) [u-~*P]ATP (0.2 mM, sp act _ 1.2 strand 5’ sequence, GAU... (Mirua et al., Ci/mmole) as the radioactive precursors. The purified 1974; Chow, N. L., and Shatkin, A. J., RNA was digested with KOH, and the digests were unpublished results). analyzed as described in the legend to Fig. 3.
64, 96-105 (1975)
Reovirus
Genome
RNA Segments:
S. MUTHUKRISHNAN Department
of Cell Biology,
AND
Roche Institute Accepted
Resistance AARON
of Molecular
to S, Nuclease
J. SHATKIN
Biology,
Nutley,
New Jersey 07110
October 14, 1974
Reovirion genome RNA was purified by sedimentation in a sucrose-formamide gradient after treatment with 50% formamide under nondenaturing conditions for genome RNA. Both the 5’- and 3’-termini of purified double-stranded (ds) RNA were completely resistant to digestion by the single-stranded specific nuclease from Aspergillus oryzae, S,. In contrast, the unpaired 3’-terminal dinucleotide, CPC-~H, in tRNA was completely hydrolyzed by the enzyme. Reovirus ds RNA was sensitive to T, and pancreatic RNases to a small extent, even in the presence of 0.3 M NaCl. Ds RNA did not act as a primer/template for avian myeloblastosis virus (AMV) reverse transcriptase. The results indicate that reovirus ds genome RNA does not contain S,-sensitive single-stranded regions.
The arenaviruses, influenza virus, oncornaviruses, and reoviruses all contain segmented RNA genomes (Shatkin, 1974a). One of the intriguing features of these viruses is the mechanism by which the viral genome replicates to yield progeny virions containing the correct assortment of RNA molecules. In an effort to understand this process more completely, we have been studying the structure of the reovirus genome which consists of 10 double-stranded (ds) RNA segments (Shatkin et al., 1968). All 10 segments are required for reovirus infectivity since particles containing only nine are defective (Nonoyama et al., 1970). Furthermore, 10 viral proteins can be detected in productively infected cells (Both et al., 1975). One attractive hypothesis is that progeny genomes in infected cells are copied from a linked array of single-stranded or double-stranded RNA segments that are precisely ordered within parental subviral particles (Ward and Shatkin, 1972; Zweerink, 1974; Acs et al., 1971). Several possible modes of linkage of the 10 segments have been previously considered: (I) contiguous genome segments have
hydrogen-bonded base-pairs form (“Sticky-end” model,) (Millward and Nonoyama, 1970); (II) the segments are perfect duplexes, but terminal sequences of adjacent segments are identical and thus permit intersegment base-pairing of their strands (“street intersection” model ,Y) (Banerjee and Grece, 1971); (III) segments are aligned by viral proteins or other “linker” molecules, with or without base-pairing as in (I) or (II) (Ward and Shatkin, 1972; Zweerink, 1974). We have attempted to differentiate among these models by analyzing the terminal sequences of reovirus RNA (Miura et al., 1974) and by testing for the presence of “sticky ends” in the ds segments. It was previously suggested on the basis of studies with phosphomonoesterase and phosphodie&erase that short, purine-rich, singlestranded tails are present in the 5’ ends of reovirus RNA (Millward and Nonoyama, 1970). Since the enzyme S, from Aspergillus oryae degrades without base preference the single-stranded portions of DNA/ DNA, RNA/DNA, or RNA/RNA hybrid molecules (Ando, 1966; Sutton, 1971; Shishido and Ando, 1972), we have used it to test reovirus genome RNA for the presence
complementary
of single-stranded
INTRODUCTION
single-stranded
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
tails
that
regions.
REOVIRUS MATERIALS
AND
GENOME
METHODS
Cells and Virus Mouse L cells and BHK-21 cells were grown in suspension culture in Eagle’s medium containing 5% fetal calf serum. Cells were infected with reovirus type 3 Dearing strain at a multiplicity of infection of 10 PFU/cell. Virus was extracted with fluorocarbon and purified by isopycnic sedimentation in CsCl as described previously (Banerjee and Shatkin, 1971). For preparation of 32P-labeled ds RNA, cells were resuspended after virus adsorption in phosphate-free medium containing 0.25 pglml of actinomycin and [32P]phosphoric acid (20 pCi/ml) was added 2 hr later. After 36 hr at 37:: the cells were harvested and virus purified. Preparation
of Radioactive
RNA
32P-Labeled purified virions were extracted with phenol and the RNA separated from virion oligonucleotides by Sephadex G-100 gel filtration (Banerjee and Shatkin, 1971). Ds RNA was 3H-labeled at the 3’ termini by periodate oxidation and reduction with [3H]borohydride and repurified by gel filtration (Banerjee and Grece, 1971). Denatured genome RNA was similarly 3H-labeled in the 3’-penultimate residues. Purified RNA was denatured in 90% dimethyl sulfoxide at 37” for 30 min and precipitated by addition of 2 vol of ethanol in 0.15 M KC1 at -20”. The RNA was oxidized with periodate and sequentially treated with P-aniline, alkaline phosphatase, periodate, and [ 3H lborohydride (Hunt, 1970). The 3H-labeled RNA was repurified by gel filtration. Deadenylated 3’-cytidine 3H-labeled E. coli tRNA was kindly provided by Dr. David Ward, Yale University. It was prepared by removing the 3’-terminal CpA by two cycles of oxidation, p-elimination, and phosphatase digestion. The RNA was purified by gel filtration, dialyzed exhaustively, concentrated, and incubated with [3H]CTP and nucleotidyl transferase. The 3H-labeled tRNA contained 3 x lo5 cpm/ was OD,,,, and all of the radioactivity
97
FRAGMENTS
released as cytidine by digestion with 0.3 N KOH for 18 hr at 37” indicating that one 3’-terminal [3H]pC had been added per tRNA molecule during the transferase reaction. Purification of ds RNA in Formamide-Sucrose Ds RNA dissolved at a concentration of 1 mg/ml in 20 mM Tris buffer, pH 7.9, 1 mM EDTA, 1 x SSC (0.15 M NaCl, 0.015 M Na citrate) containing 50% formamide was incubated at 37’ for 1 hr and layered onto a 15-30% sucrose gradient in the same buffer-formamide mixture. After centrifugation for 8 hr (60,000 rpm. SW-65, 20”), fractions of 0.16 ml were collected and counted either by Cerenkov radiation (““P) or in Aquasol ( 3H) (New England Nuclear). The peak fractions containing the ds RNA were pooled as shown in Fig. 1 and precipitated by adding 2 vol of ethanol at -20”. Nuclease Digestions Ds RNA (ca. 100 pug) was digested with S, nuclease (10 ~1 of the DEAE-cellulose fraction of Sutton, kindly supplied by J. Sambrook, Cold Spring Harbor Laboratory) for 2 hr at 45” in 30 mM potassium acetate buffer, pH 4.5, 0.3 M NaCl, lo-‘M Zn2+ in a final volume of 0.2 ml. The reaction was stopped by chilling and adjusting the EDTA concentration to 10 mM. Digestion of ds RNA (0.5 mg/ml) with T, RNase (250 units/ml) or pancreatic RNase (1:20/w/w) was in 20 mM Tris buffer, pH 7.4, 1 mM EDTA, 0.3 M NaCl, at 37” for 2 hr. Reverse Transcriptase
Assay
Purified avian myeloblastosis virus (AMV) (2.2 mg/ml, kindly provided by A. Weissbach, Roche Institute) was pretreated with 0.4% NP-40 in 5 mM Tris buffer, pH 8, for 10 min at O”, and 0.02 ml of the treated preparation was added to a standard incubation mixture in a final volume of 0.15 ml containing 50 mM Tris buffer, pH 8, 7 mM MgCl,, 80 mM KCl, 0.5 mM dithiothreitol, 40 kg bovine serum
98
MUTHUKRISHNAN
albumin, four deoxyribonucleoside triphosphates (10 &f each, sp act = 1.6 Ci/ mmole), and the indicated primer/templates. Incubation at 37” was stopped after 20 min by chilling, adding 0.1 ml of 0.2 A4 sodium pyrophosphate, 1 ml HzO, and one-fifth volume of 12% perchloric acid. The acid precipitate was collected by centrifugation, dissolved in 0.2 N KOH, and reprecipitated. The acid-precipitable material was finally dissolved in 0.5 ml Protosol and counted in 4.5 ml toluene-based scintillation fluid acidified with acetic acid. Synthesis of Reovirus RNA in Vitro
Single-Stranded
Reovirus single-stranded RNA was synthesized with a-s*P-labeled ribonucleoside triphosphates in a standard reaction mixture of 0.5 ml containing 70 mM Tris buffer, pH 8, 7.5 mM Mg2+, 50 mM KCl, 2.5 mM cyclohexylammonium phosphoenolpyruvate, 2 mM of each of the four ribonucleoside triphosphates (except the s-32P-labeled one), 70 pg Macaloid, 90 pg chymotrypsin, 210 pg purified reovirus. After 30 min at 45”, 2.5 units of pyruvate kinase and the 32P-labeled nucleoside triphosphate were added and the incubation continued for the specified times. The RNA products were extracted with phenol and purified by gel filtration. The size of the products was determined by velocity sedimentation in 15-30% sucrose gradients. Radioactive
Compounds
and Enzymes
Pancreatic RNase (RASE) and crystalline a-chymotrypsin were from Worthington Biochemical Corp. T, RNase (Sankyo) was obtained from Calbiochem. (Y-“PLabeled ribonucleoside triphosphates and carrier-free [a2P]phosphoric acid were purchased from New England Nuclear. Potassium [3H]borohydride was from Amersham/Searle, and [3H]deoxyribonucleoside from obtained were triphosphates SchwarzlMann. RESULTS
Resistance of ds RNA Digestion 32P-Labeled reovirus isolated from purified
to S, Nuclease genome RNA was virions by phenol
AND SHATKIN
extraction and gel filtration. RNA purified by this procedure may contain residual single-stranded oligonucleotides (Bellamy et al., 1967) or noncovalently bound nascent chains of viral mRNA produced on the ds RNA template by the virion-associated transcriptase (Borsa and Graham, 1968; Shatkin and Sipe, 1968) and trapped during virus maturation. Therefore, the RNA was purified further by incubation at 37” for 1 hr in Tris buffer containing 1 x SSC and 50% formamide, and sedimented in a sucrose gradient containing 50% formamide (see Methods). Since 1% formamide lowers the Tm of nucleic acids by 0.7” (McConaughy et al., 1969), full-length reovirus RNA segments (Tm in 1 x SSC = 95”) (Shatkin, 1965; Bellamy et al., 1967) remain double stranded (ds) under these conditions. However, nonspecific, shorter, and less-stable hybrids would be denatured and separated from the ds RNA during the velocity sedimentation in sucrose. The peak of ds RNA that was collected from the gradient consisted exclusively of the 10 genome segments (Shatkin et al., 1968) when analyzed by polyacrylamide gel electrophoresis (Fig. 1 inset). RNA purified in this way was used in all the subsequent experiments. To test for the presence of singlestranded regions in the purified ds RNA, its sensitivity to S, nuclease digestion was examined. Two aliquots of 32P-labeled ds RNA, each 2.5 x lo6 cpm, were incubated with and without the enzyme for 2 hr at 45” in 0.3 M NaCl, conditions which favor digestion of single-stranded RNA. The mixtures were then analyzed by velocity sedimentation in 5-25% sucrose gradients. As shown in Fig. 2, no radioactivity was present at the top of either gradient in the fractions that would be expected to contain nucleotides released by S 1digestion. Under these conditions, the degradation of as little as 0.05% of the ds RNA, i.e., an average of one nucleotide per strand, would have released 1250 cpm. 5’ Termini of ds RNA. To test for the presence of intact 5’ termini in the S, nuclease-treated ds RNA, the enzymedigested segments were hydrolyzed with KOH, and the products were analyzed by
REOVIRUS
GENOME
FRAGMENTS
99
tides (0.18 M NaCl) 0.13% of the radioactivity, the expected amount of 5’ termini. 3’ Termini of ds RNA. The 3’ termini of reovirus ds RNA were similarly tested for resistance to removal by S, nuclease digestion. Ds RNA was extracted from virions, separated by gel filtration, and labeled at the 3’-terminal cytidine with [3H]borohydride as described previously (Banerjee and Grece, 1971). The 3H-labeled RNA was then purified by formamide treatment and centrifugation in sucrose-formamide gradients. Treatment of the purified 3’-3Hlabeled RNA with S, nuclease yielded 7% of the radioactivity at the top of the gradient as compared to 4% of the RNA incubated without enzyme (Fig. 4). The released radioactivity contained no 3Hlabeled cytidine trialcohol derivative and remained at the origin when analyzed by paper chromatography (De Wachter and Fiers, 1967). The results indicate that the I I I 0 5 IO 15 20 25 30 35 3’-termini are protected from S, nuclease FRACTION NUMBER by base-pairing. FIG. 1. Sedimentation of 32P-labeled reovirus ge- digestion, presumably Denatured RNA. To ensure that the S, nome RNA in a sucrose gradient containing formamide. Ds RNA purified by gel filtration was treated with nuclease is active against single-stranded 50% formamide as described in the Methods section reovirus RNA under the conditions of diand layered onto a 15%30% sucrose gradient in 50%’ gestion employed, 32P-labeled genome formamide, 1 x SSC, 1 mM EDTA, and 20 mM RNA was denatured with dimethyl sulfoxTris buffer, pH 7.9. Centrifugation was for 8 hr at ide and then digested with S,. More than 60K (SW-65) at 20”. Fractions were collected from the 50% of the radioactivity was acid soluble bottom and radioactivity in each fraction was deterafter 1 hr, and all the RNA was converted mined by Cerenkov counting. The samples indicated to low-molecular-weight material that sedby the bracket were combined, precipitated in ethaimented near the top of a sucrose gradient nol, and analyzed by electrophoresis in a 5% poly(Fig. 5). acrylamide gel (Shatkin et al., 1968) (inset).
chromatography on DEAE-cellulose in 7 M urea (Banerjee and Shatkin, 1971). As seen in Fig. 3, most of the radioactivity elutes at 0.08 M NaCl in the position of mononucleotides. The alkaline digests of both untreated RNA (Fig. 3A) and S,treated RNA (Fig. 3B) contain in the elution position of the 5’-terminal nucleo-
FRACTION
NUMBER
FIG. 2. Sedimentation of SzP-labeled ds RNA after S, nuclease digestion. Ds RNA was analyzed on a 5’%-25% sucrose gradient after incubation for 2 hr at 45°C in 0.3 M NaCl, 1 mM EDTA, 30 mM NaAc buffer, pH 4.5, with (0- -0) and without (O---O) S, nuclease. After centrifugation for 7 hr at 60,000 rpm (SW-45) at O”, 0.2.ml fractions were collected. Fractions l-10 were counted in Aquasol and 11-24 by Cerenkov radiation.
100
MUTHUKRISHNAN
n
I DO-
AND SHATKIN I
A
I
I
M NoCl - 0.3 50 -
FRACTION NUMBER FIG. 3. DEAE-cellulose chromatography of KOH digests of S,-treated and untreated ds RNA. The RNA in fractions lo-23 of Fig. 2 was pooled and precipitated with ethanol. The pellets of untreated (A) and S,-treated RNA (B) were dissolved in 0.3 N KOH, incubated at 37” for 18 hr, neutralized (Banerjee and Shatkin, 1971), and loaded onto 1 x 20.cm columns of DEAE-cellulose. The oligonucleotides were eluted with a gradient of 0.0-0.3 M NaCl containing 7 M urea and 50 mMTris, pH 8.0. Fractions of 1.8 ml were collected and counted in 9.5 ml 2-methyoxyethanol (methyl cellosolve) and 7.5 ml spectrofluor-toluene. The arrow indicates the elution position of ppGp (Banerjee and Shatkin, 1971).
0
2
4
6
s IO FRACTION
I2 14 NUMBER
16
IS
20
22
substrate. tRNA containing 3’-terminal . ..CpC - sH was prepared as described in the Methods section. It was treated with S, nuclease under the conditions described in the Methods section. As shown in Table 1, more than 50% of the 3’-terminal SHlabeled residues were hydrolyzed within 30 min. Similar findings were obtained with tRNA containing 3’[3H]CCA. Thus, S, nuclease can recognize the 3’-terminal unpaired bases in tRNA when the singlestranded tail is only two residues in length.
FIG. 4. Sedimentation of 3’-3H-labeled ds RNA after S, nuclease digestion. 3’-JH-labeled ds RNA (15 pg, 60,000 cpm) was incubated with and without S, nuclease and analyzed by sedimentation in 5-25% sucrose as in Fig. 2. The fractions collected from the gradient were counted in Aquasol. Incubation with (0- -0) and without (04) S, nuclease.
Action of Pancreatic RNA
3’-Termini of tRNA. As a test for the ability of S, nuclease to hydrolyze a short single-stranded tail in double-stranded RNA, tRNA which contains an unpaired 3’-terminal . ..CpCpA extending from a double-stranded stem was used as a model
The results indicate that both the 5’ and 3’ ends of reovirus genome RNA are resistant to the single-strand specific nuclease, S1, and suggest the absence of singlestranded tails in the RNA segments. The resistance of formamide-sucrose purified ds RNA to pancreatic and T, RNases is less
and T, RNases on ds
REOVIRUS
GENOME
101
FRAGMENTS
tively. Thus, even under conditions of relatively high ionic strength, the 3’ termini (and possibly 5’ termini as well) of ds RNA are sensitive to T, and pancreatic RNase digestion. Inactivity of ds RNA as FrimerlTemplate for AMV Reverse Transcriptuse
0
2
4
6
8
10 12 14 16 FRACTION NUMBER
18
20
22
24
FIG. 5. Analysis of denatured 3T-labeled reovirus genome RNA after digestion with S, nuclease. Ds RNA dissolved in 10 mM Tris buffer, pH 7.4, 1 mM EDTA was denatured by adding 9 vol of dimethyl sulfoxide and incubating at 37’ for 30 min. The denatured RNA, recovered by ethanol precipitation, was digested with S, nuclease and sedimented in sucrose as in Fig. 2 TABLE S, NUCLEASE
HYDROLYSIS
I OF 3’.TERMINAL
C FROM
tRNA” Time (min)
wm Acid-soluble
cpmb in pC
0
0
0
15 30 60 120
5,287 8,048 10,376 11,780
3,996 8,274 9,798 13,038
“The tRNA (14,000 cpm) was digested with S, nuclease as described in the Methods section. The reactions were stopped by chilling and addition of an equal volume of 10% trichloroacetic acid. The acidsoluble material was collected after filtration through a Millipore filter, extracted with ether to remove the acid, evaporated to dryness, and analyzed by highvoltage paper electrophoresis at pH 3.5. b A portion (-25%) of the released radioactivity migrated with authentic C, and the values reported are the sum of the radioactivity in pC plus C.
complete, however. Treatment of 32Plabeled ds RNA with either RNase in 0.3 A4 salt yielded 0.25% of the total radioactivity as low-molecular-weight material (Fig. 6A, fractions l-7). At least a portion of this material can be accounted for as 3’-terminal nucleotides from the results shown in Fig. 6B. Incubation of 3’-SH-labeled ds RNA with T, or pancreatic RNase released 35% and 100% of the radioactivity, respec-
Duplex structures containing 5’ singlestranded regions function as primer/ternplate for avian sarcoma virus reverse transcriptase (Baltimore and Smoler, 1971); the virion-associated enzyme adds deoxyribonucleotides to the 3’-end of a 4 S RNA primer that is base-paired with 35 S RNA genome subunits (Faras et al., 1973). Purified ds RNA was tested for primer/template activity with AMV reverse transcriptase as described in the Methods section Incorporation of 3H-labeled deoxynucleotides into acid-insoluble products was 0.03, 0.04, and 2.3 pmoles for reaction mixtures containing only endogenous primer/template, 20 pg of reovirus ds RNA,
I &-
i -
b 40 I x
A
f
” 20 : 0
4
8 FRACTION
12
16
20
24
NUMBER
FIG. 6. Sedimentation of pancreatic and T, RNase digestion products of ds RNA. Ds RNA labeled uniformly with [32P]phosphate (A) or at the 3’-termini with [SH]borohydride (B) was incubated with pancreatic RNase or T, RNase or no enzyme as described in the Methods section. The incubation mixtures were analyzed in 5%-25% sucrose gradients as in Fig. 2. Control (O--O), pancreatic RNase (.A), T, RNase (A-A).
102
MUTHUKRISHNAN
AND SHATKIN
and 10 pg of activated salmon sperm DNA (Fry and Weissbach, 1973), respectively. Complementarity of Terminal quences in ds RNA
I
I
I
KOH
----G’
I
I
U’
C’
---T---l---T
A’ ;
Base Se-
Since it has been reported that the 5’ termini contain G (Banerjee and Shatkin, 1971; Miura et al., 1974) and the 3’ ends are C (Banerjee and Shatkin, 1971) in reovirus RNA, it was of interest to determine if the penultimate bases are also complementary. To characterize the 3’-penultimate residues, ds RNA labeled at the 3’-termini by [SH]borohydride reduction was denatured and digested with T, or pancreatic RNase. T, RNase digestion will yield the 3H-labeled trialcohol derivative of cytidine (C’) from RNA containing G as the 3’penultimate residue. Similarly, pancreatic RNase will yield C’ from RNA containing a 3’-penultimate pyrimidine. The digestion products were analyzed by paper chromatography (Fig. 7). The total amount of ‘H-labeled 3’ termini was determined by analysis of KOH digests of the RNA. After T, RNase treatment, 45% of the total amount of 3’ ends based on the KOH value were recovered as C’, consistent with the 3’ sequence . ..GCOH in one strand. Pancreatic RNase treatment released the remaining 3’ termini as C’, and all the 3’ ends were released with a mixture of the two RNases. The complementary strand thus has a pyrimidine next to the 3’ cytidine. The same results were obtained when individual ds RNA segments were analyzed, consistent with . ..GCOH and . ..PyrCou at the 3’ ends of the two strands in each duplex. The 3’-penultimate residues were further identified by the stepwise procedures described by Hunt (1970). They included: (1) periodate oxidation of the denatured reovirus genome RNA; (2) P-elimination with aniline; (3) alkaline phosphatase digestion; with periodate; (5) (4) reoxidation [SH]borohydride reduction; (6) KOH hydrolysis of the 3H-labeled RNA; and (7) paper chromatography of the digest. As shown in Fig. 8, the 3’-penultimate nucleotides are G and U. If the 3’ and 5’-termini of reovirus ds RNA are base-paired, the 5’ sequences for
t
$ "
T, + Pam. RNose
1
20 IO 5 0 20 IO 5
-0
4
a
12
I6
io
24
ie
i2
DISTANCE MIGRATED (cm)
FIG. 7. Paper chromatography of the digestion products of 3’-3H-labeled reovirus RNA. Denatured 3’-8H-labeled RNA (30,000 cpm each) was digested with 0.3 N KOH for 18 hr at 37”; T, RNase (500 units/ml), and pancreatic RNase (20 rg/ml) for 18 hr at 37” in 10 mA4 Tris buffer, pH 7.4, and 1 mM EDTA; pancreatic RNase (100 pg/ml, 30 min, 37”) or T, RNase (500 units/ml, 18 hr, 37’). At the end of the incubations, the KOH hydrolysate was neutralized by passage through Dowex 50-H+ (Banerjee and Shatkin, 1971). A mixture of the four authentic trialcohol derivatives of cytidine (C’), uridine (U’), adenosine (A’), and guanosine (G’) (12 ~(g each) was added to the four digested RNA samples which were then analyzed by descending chromatography (De Wachter and Fiers, 1967) on Whatman No. 1 paper in a solvent system consisting of n-butanol: isobutyric acid: H,O:NH,OH (30:15:10:1). the two complementary strands would be GC... and GA... . Reovirus ds RNA is synthesized via single-stranded genome transcripts that are converted subsequently to genome RNA (Schonberg et al., 1971). Consequently, the single-stranded RNA corresponds to one of the strands of the duplex segments. We, therefore, determined the 5’-penultimate base of the single-stranded RNA by phosphate transfer experiments. Three single-stranded RNA preparations were synthesized in vitro by
REOVIRUS 401
1
’
GENOME
I
103
FRAGMENTS DISCUSSION
Model I for reovirus genome assembly requires that the ds RNA segments contain single-stranded tails. The single-strand L$P\d ii i[ specific nuclease, S1, would be expected to degrade these regions. No S,-sensitive ma0 4 i Ii I6 0 24 2s 32 36 terial was detected in ds RNA that had DISTANCE MIGRATED (4 been purified by sedimentation in forFIG. 8. Paper chromatography of the KOH digesmamide-sucrose under conditions detion products of reovirus RNA ‘H-labeled in the signed to remove noncovalently-associated 3’-penultimate positions. Denatured RNA was labeled with [3H]borohydride after removing the 3’ nucleic acids. S, nuclease degrades uncytidine as described in the Methods section. The paired nucleotide sequences without base RNA was digested with 0.3 N KOH and analyzed as preference by both exo- and endonudescribed in the legend of Fig. 7. cleolytic cleavage (Ando, 1966; Sutton, 1971; Shishido and Ando, 1972). In addiincubating the virion-associated RNA po- tion, the enzyme hydrolyzed the unpaired, lymerase (Borsa and Graham, 1968; Shat- 3’-terminal dinucleotide in tRNA, indicatkin and Sipe, 1968) with [LY-~~P]UTP, ing that it can recognize short single-CTP, or -ATP as the labeled precursor. stranded tails in RNA under the conditions The product RNAs were purified by gel used in these experiments. We thus should filtration and shown to consist of the three have detected single-stranded tails of two size classes of mRNA (1, m and s, Fig. 10 or more residues in reovirus genome RNA, inset) as reported previously (Banerjee and including purine-rich sequences (Millward Shatkin, 1970; Skehel and Joklik, 1969; and Nonoyama, 1970). The amount of ds Levin et al., 1970; Bellamy and Joklik, [32P]RNA used for S, digestion in our 1967). The RNAs were hydrolyzed with experiments was sufficient to detect the KOH which results in the transfer of the phosphate from the penultimate nucleotide to the 5’ nucleotide. Chromatography on DEAE-cellulose was used to separate the 5’-terminal residues (-5 charge) from the mononucleotides (-2 charge) (Banerjee and Shatkin, 1971). When [(Y-~~P]ATP or [N-~~P]UTP were the radioactive precursors, no radioactivity eluted in the position of -5 charge at 0.18 M NaCl (Fig. 9). The minor peaks eluting at 0.11 M NaCl in the position of -3 net negative charge are probably resistant dinucleotides (Lane and Butler, 1959). When [cx-~‘P]CTP was used, 0.2% of the radioactivity in the KOH digest eluted at 0.18 M NaCl in the position of FRACTION NUMBER 5’-termini (Fig. 10). Thus, the in vitro FIG. 9. DEAE-cellulose chromatography of KOH single-stranded RNA corresponding to the digests of reovirus mRNA synthesized in uitro with + strand of the duplex (Hay and Joklik, [(Y-~~P]ATP and [~x-~~P]UTP as the radioactive pre1971) contains the 5’ sequence GC... which cursors. Single-stranded RNA was synthesized under is complementary to one of the 3’ se- standard conditions as described in the Methods quences, . ..GCOH. The other 3’ sequence, section with (A) [w~~P]UTP (0.2 mM, sp act = 0.5 . ..UCOH. is complementary to the minus Ci/mmole) or (B) [u-~*P]ATP (0.2 mM, sp act _ 1.2 strand 5’ sequence, GAU... (Mirua et al., Ci/mmole) as the radioactive precursors. The purified 1974; Chow, N. L., and Shatkin, A. J., RNA was digested with KOH, and the digests were unpublished results). analyzed as described in the legend to Fig. 3.
