95, 24-35

VIROLOGY

Isolation

MICHAEL

(1979)

and Characterization of a Large “Hairpin” Avian Retrovirus RNA1 L. PERDUE,

Department of Microbiology

WERNER

and Immunology,

WUNDERLI,3 Duke

Accepted

University December

Medical

Segment

AND WOLFGANG Center,

Durham,

North

from

K. JOKLIK4 Carolina

27710

22, 1978

Avian retrovirus RNA (both ‘70 S and 35 S) possesses several attributes of doublestranded (ds) RNAs. Among them are an affinity for hydroxyapatite equal to that of authentic ds RNAs and exceeding that of any other single-stranded RNA tested except hnRNA, and an unexpectedly high melting temperature of some of its sequences, indicating the presence of intramolecular base-paired regions. Limited digestion with pancreatic RNase A of Prague C strain of Rous sarcoma virus as well as B77 and RAV-2 35 S RNA yielded a product that accounted for about 7% of the total viral RNA, behaved like reovirus ds RNA when chromatographed on hydroxyapatite, possessed a T, that was similar to that of reovirus ds RNA, was almost as susceptible to RNase III as reovirus ds RNA under conditions when reovirus messenger RNA was completely resistant, and could be isolated as a relatively homogeneous component following centrifugation in sucrose density gradients or electrophoresis in formamide-containing polyacrylamide gels. Its properties were consistent with the interpretation that it is a highly (but not perfectly) base-paired hairpin about 350 base pairs long. It was mapped by determining which of various size classes of poly(A)-containing fragments of viral RNA contained it and found to be located in the region between 5000 and 6000 nucleotides from the 3’-terminus of nondefective viral RNA; this region is at, or close to, the junction of the pol and env genes. The fact that the RNA of the helper virus free Bryan high-titer strain of RSV, which lacks most of the env gene, did not yield such a hairpin fragment agrees with this conclusion. INTRODUCTION

namely the 35 S subunits, are intimately associated in a head-to-head arrangement via numerous specific interactions (Darlix et al., 1978), the most important of which is mediated by a highly base-paired nucleotide sequence about 50 base pairs (b~)~ long centered at about 100 residues from their 5’termini (Haseltine et al., 1977) [see also Bender and Davidson (1976) for the RNAs

The 70 S RNA of avian retroviruses is a complex of two large and several small nucleic acid molecules, the precise structure of which is dictated by base-pairing interactions. Three classes of such interactions have been described. First, analysis of the effect of enzymes such as RNase III, Tl ribonuclease and Sl nuclease has shown that the two major components of the complex,

5 Abbreviations used: ds, double-stranded; ss, single-stranded; HAP, hydroxyapatite; hnRNA, heterogeneous nuclear RNA; RSV-PrC, the Prague C strain of Rous sarcoma virus; RSV-SRB, the Schmidt-Ruppin B strain of Rous sarcoma virus; RSV-BH(-), the Bryan high-titer strain of RSV genome in the absence of helper virus; ASV, avian sarcoma virus; ffu, focus forming unit; DEF, duck embryo fibroblasts; CEF, chick embryo fibroblasts; Me,SO, dimethyl sulfoxide; SSC, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2; Kb, kilobase; bp, base pair; TCA, trichloroacetic acid; NTE buffer, 10 m&f NaCl, 10 nu’l4 Tris.HCl, 1 mM EDTA, pH 7.4.

1 This work was supported by Research Grant AI08909 from the National Institutes of Health. * USPHS Postdoctoral Fellow (5-F32-CA05280). Present Address: Division of Experimental Pathology, Department of Pathology, University of Kentucky, Lexington, Ky. 40506. 3 Fellow of the Swiss Stiftung fiir medizinisch-biologische Stipendien during the initial phases of this work. Present Address: NESTECIForschungsabteilung, Postfach 88, 1814 La Tour-de-Peilz, Switzerland. 4 To whom reprint requests should be addressed. 0042-6822/79/070024-12$02.00/O Copyright All rights

0 19’79 by Academic Press, Inc. of reproduction in any form reserved.

24

“HAIRPIN”

SEGMENT

FROM

of mammalian RNA tumor viruses]. Second, tRNAtrP molecules are hydrogen-bonded to these 35 S subunits via nucleotide sequences about 100 residues from their 5’-termini (Harada et al., 1975; Shine et al., 1977), and a 135 nucleotide-long DNA molecule is hydrogen-bonded to their middle region (Darlix et al., 1977). Third, like other ss RNAs, the 35 S RNA subunits themselves possess a great deal of secondary structure (Bader and Ray, 1976). Among indications in this direction are their susceptibility to ds RNA-specific nucleases RNase III and IV and their resistance in the presence of high salt to ribonuclease Tl and Sl nuclease (Travnicek and Riman, 1973; Leis et al., 1978; Darlix et al., 1978), their ability to intercalate ethidium bromide (Cavalier-i, 1974), and the fact that they exhibit a hyperchromic shift when denatured (see Riggin et al., 1975, for the RNA of Moloney murine leukemia virus). Further, electron microscopic observation of the RNA of RD-114, the endogeneous baboon C-type virus BKD, and woolly monkey sarcoma virus under partially denaturing conditions has revealed the presence of a large loop near the center of each 35 S subunit, as well as two hairpin structures about 250 and 1000 bp long respectively (Kung et al., 1975, 1976). Avian retrovirus RNAs do not show such structures, but this may be because they are less stable, rather than because they do not exist. Hairpin regions of the length observed by Kung et aZ. (1976) are not present in RNAs except in hnRNA (Jelinek and Darnell, 1972; Jelinek et al., 1974), where they have been postulated to play a role in its processing to mRNA. There is good evidence that 35 S retrovirus RNA is also processed since the env and src genes cannot be translated from it (von der Helm and Duesberg, 1975; Pawson et al., 1977; McGinnis et al., 1978), but must be translated from 28 S and 21 S mRNA species that are derived from it (Hayward, 1977; Stacey et al., 1977; Pawson et al., 1977; Beeman and Hunter, 1977; Purchioet al., 1977; Weisset al., 1977). The studies reported in this paper were undertaken to determine the level of intramolecular base-pairing in avian retrovirus RNA, to determine whether any of its regions are unusually rich in hydrogen

AVIAN

RETROVIRUS

RNA

25

bonds, and if so, to isolate, characterize and map such regions. MATERIALS

AND

METHODS

Virus and cells. The Prague C and Schmidt-Ruppin B strains of RSV, the B77 strain of ASV, and two td mutants of RSVPrC and B77 were propagated in Peking duck embryo flbroblasts by infecting subconfluent secondary monolayers at multiplicities of 0.1 to 1.0 ffu per cell and transferring them to roller culture bottles 3 to 4 days later. The cells were maintained and passaged as previously described (Stone et al., 1974). Virus was harvested either manually or by employing a Smith-Kozoman Autoharvester (Smith and Quade, 1976) set to collect at 2- or 4-hr intervals. Following removal of cellular debris, virus was concentrated by centrifuging at 23,000 rpm for 45 min and resuspended in 10 n&f Tris * HCl, pH 8, containing 1 m&? EDTA. All virus stocks were the progeny of DEF infected with the supernatant fluid from a single soft agar clone of transformed DEF. The RNA of all RSVPrC and B77 virus preparations comprised 80-90% a type subunits (Duesberg and Vogt, 1973a; Stone et al., 1974); the RNA of the two td mutants comprised only b type subunits. RAV-2 and RSV-BH(-) (kindly provided by Dr. H. Hanafusa) was propagated in chfgs- CEF. Preparation

and radiolabeling

of RNA.

Cellular and viral RNAs labeled with 32P were isolated from cells exposed to 500 &i/ml ortho-[32P]phosphate (New England Nuclear, carrier-free) in phosphate-free medium for 12-16 hr following a 6 to 8 hr period of phosphate starvation. To label RNA with tritium, [3H]uridine (New England Nuclear) was added to the medium at the rate of 200 Z.&i/ml and incubation was continued for 12-16 hr before harvest. Viral RNA was purified as described by Smith and Quade (1976). Briefly, RNA was extracted from crude virus pellets by digestion with proteinase K (Beckman Inst., Palo Alto, Calif.) followed by extraction with phenol. 70 S RNA was isolated in 1530% (w/v) sucrose density gradients and concentrated by centrifugation from 70%

26

PERDUE,

WUNDERLI,

ethanol. 35 S RNA was isolated either by centrifuging 70 S RNA in 5-20% (w/v) sucrose/80% Me,SO [in 10 n-&f Tris *HCl, 10 mM LiCl, 1 mM EDTA (pH S)] density gradients or by heating 70 S RNA to 70” in NTE buffer for 3 min and centrifuging in 15-30% (w/v> sucrose (in NTE buffer) density gradients. Ribosomal RNA, hnRNA, and reovirus messenger and genome RNAs were prepared by published procedures (Hizi et al., 19’77; Pagoulatos and Darnell, 1970; McCrae and Joklik, 1978; Ito and Joklik, 1972). [3H]Uridine-labeled VSV 42 S RNA was prepared from virions purified from infected mouse L fibroblasts kindly provided by Dr. Nancy Davis and Dr. Gail Wertz of the Department of Bacteriology, University of North Carolina, Chapel Hill, North Carolina. Thermal denaturation assays. Thermal denaturation and renaturation assays were performed in a Beckman Acta V Recording Spectrophotometer. RNA samples in various dilutions of SSC were placed into jacketed cuvettes in which the temperature was controlled by a circulating water bath. Heating and cooling rates were maintained at approximately l”/min. Temperatures were measured by a thermistor immersed in a cuvette containing only 1xSSC and were recorded manually on the absorbance protiles. Preparation

of poly(A)-containing

frag-

ments of 35 S RNA. Fragments of 35 S RNA were generated by sonic oscillation of 70 S or 35 S RNA using a Branson sonifier and heating to 80” for 5 min before adsorption to and elution from oligo(dT)cellulose. The fragments were separated into size classes by centrifugation in 15-30% sucrose density gradients, using 35 S viral, 28 S and 18 S ribosomal, and 4 S RNA as markers. Enzymatic reactions. Digestion with pancreatic ribonuclease A (Worthington Biochemicals, Freehold, N. J.) was carried out as follows unless stated otherwise: between 1 and 5 pg of RNA, dissolved in 0.51.0 ml of 1 M NaCl, 0.01 M Tris.HCl, pH 7.4, were treated with 0.01-0.05 pg enzyme/ml (lo-20 RNase molecules per RNA molecule) at 0” for 30 min. Ribonuclease III, purified from Esche-

AND

JOKLIK

coli, was a generous gift of Dr. Jonathan Leis, Duke University Medical Center. The reaction mixture for it was: 20 mM Tris.HCl, 100 nul4 KCl, 10 m&f MgCl,, 0.1 nub! dithiothreitol, pH 7.9. Chromatographic techniques. Hydroxyapatite columns (HTP, BioRad Laboratories, Richmond, Calif.) were packed in 5-ml disposable syringes using Millipore prefllters as bed supports. Column volumes of 0.5 to 1.0 ml were ample to adsorb up to 50 Fg of RNA which was the most used in any experiment. The syringes were housed in a Plexiglas water bath kept at 60” and up to 40 columns could be run simultaneously under conditions of batch-wise elution. Flow rates of 0.5 to 1.0 ml/min could be used without noticeable loss of resolution. For gradient elution an external mixing device was attached to the syringes and a peristaltic pump was used to control flow rate and column pressure. Oligo(dT)-cellulose columns (Collaborative Research, Waltham, MASS) were prepared and poly(A)-containing RNA was isolated as described by Wang et al. (1976). Gel electrophoresis. Electrophoresis was performed in gels containing 99% formamide, 4.25% acrylamide, 0.75% bis-acrylamide, and 0.02 M phosphate buffer, pH 7.0 (Duesberg and Vogt, 1973b). Slab gels were poured between two glass plates separated by 0.2-cm spacers. Samples of nuclease-digested 32P-labeled viral RNA were precipitated with ethanol and resuspended in 90% formamide buffered with 0.02 M phosphate, pH 7.0. After loading the samples, the gels were run at 100-200 V and 25 mA for 2-3 hr. The top plates were then removed and the gels were covered and sealed with Saran Wrap. Autoradiography was for 16-32 hr employing preflashed Kodak Royal X-Omat RP film. richia

RESULTS

Thermal

Denaturation

Assays

Optical melting curves have been utilized extensively for the approximate quantitation of the secondary structure of ss RNAs. Although many structural and physicochemical features are actually measured when nucleic acids are melted, the largest

“HAIRPIN”

SEGMENT

FROM AVIAN

30 t g 20 8 1 10 I3o a 20 c s 10 B 0 1020 30 40 50 60 700 1020 3040 30 60 70 FIG. 1. Thermal denaturationkenaturation profiles. Thermal denaturation and renaturation was performed on several RNA species dissolved in 0.1 x SSC (0.018 M Na+) and the optical density was measured at 254 nm. Hyperchromic shift profiles are shown for: (A) RSV-PrC RNA; (B) td mutant RSV-PrC RNA; (C) reovirus mRNA synthesized in vitro; and (D) 28 S HeLa cell ribosomal RNA.

observable effects are caused by disruption of hydrogen bonds and consequent relaxation of secondary structure (Davidson, 1972). Most ss RNAs exhibit increases in absorbance at 260 nm greater than 20% when melted, and comparative measurements on different RNAs are thought to provide some insight into the relative extent of intramolecular base-pairing. When 70 S or 35 S RNA from RSV-PrC or B77 virus was denatured and renatured in 0.1 x SSC, the bulk of the intramolecular base pairs displayed a melting curve and T, roughly comparable to that of ribosomal RNA or messenger RNA such as reovirus mRNA, but a small proportion of them (component II) did not denature until the temperature reached 60-30” (Fig. 1, Table 1). The results suggested that 6-9% of the sequences in viral RNA were held in a much more stable base-paired configuration than the remainder. The shape of the denaturation and renaturation .curves indicated that the same secondary configuration was not reached upon cooling. This appeared to be caused primarily by thermal scissions resulting in bimolecular rather than unimolecular renaturation events; this was suggested by the fact that no intact 35 S RNA could be recovered following melting, and the average size of the RNA after heating to the upper limits of the melt-

RETROVIRUS

27

RNA

ing curves for extended periods of time was 18 S (data not shown). The second hyperchromic shift was not due to thermal scissions because when 40% Me&SO was included in the 0.1 x SSC, the T,s were lowered by 19”, component II was still observable, and 35 S RNA could be recovered intact upon cooling. We did not observe any differences between the profiles obtained for 70 S and 35 S RNA. However the work of others (for example, Darlix et al., 1978) suggests that subtle differences in base-pairing interactions do exist between them. Chromatography

on Hydroxyapatite

Chromatography on HAP has been employed to a limited extent for characterizing the secondary structure of RNAs. In general, all ss RNAs elute from HAP at between 0.14 and 0.18 M sodium phosphate at 60” (Bernardi, 1969). The only RNAs which have so far been found to elute at a higher

0

2

4

6

6

10 12 FRACTION

14

16

I6

20

22

FIG. 2. Elution of RNAs from HAP. Gradient elution from HAP columns of RSV-PrC RNA was performed at 60”, employing gradients of 0.10 to 0.50 M phosphate (pH 6.8). In (A) 23 S L cell ribosomal RNA labeled with 3H was cochromatographed with 32P-labeled ‘70 S RNA; in (B) 3H-Labeled 42 S VSV RNA was cochromatographed with 32P-labeled 35 S RSV-PrC RNA purified in a MeJlO/sucrose density gradient. Onemilliliter fractions were collected; phosphate molarities were determined by measurement of refractive index.

28

PERDUE,

WUNDERLI, TABLE

AND JOKLIK 1

COMPARISON OF T, VALUES, PERCENTAGE HYPERCHROMICITIES, AND RELATIVE HYPERCHROMICITIES FOR SEVERAL RNAs

RNA 35 s B77 RNA RSV-PrC RNA 70 s B77 RNA

Concentration of ssc 0.1x 0.1x

0.1x 1.0x 40% DMSO”

RSV-PrC RNA

0.1x

td RSV-PrC RNA

0.1x

T, ascending (“)

T, descending (“I

Percentage hyperchromicity”

Relative hyperchromicity”

I’ II I II

40 71 38 70

35 33 -

24 4 25 4

69 11 71 11

I II I II I II I II I II

40 72 58 22 52 38 72 38 71

34 54 33 33 34 -

23 5 23

66 14 66

25 4 25 4

71 11 71 11

Reovirus mRNA

0.1x 1.0x

29 46

33 53

20 21

57 60

HeLa cell Ribosomal RNA

0.1x 1.0x

39 57

40 59

26 24

74 69

L cell ribosomal RNA 28 s 18 s

0.1x 0.1x

40 38

43 41

27 26

77 74

n Percentage increase of optical density to lower plateau, or from lower to higher plateau, for components I and II, respectively. b Determined by dividing the percentage hyperchromicity by 35% [the value for fully double-stranded reovirus RNA (Warrington et al., 1973)]. e I and II refer to the lower and upper components of the melting curves as observed in Fig. 1. ‘l In 1.0 X SSC (see text).

molarity (0.20-0.30 M) are ds RNAs such as reovirus RNA and the replicative intermediates of viral genomes such as that of turnip yellow mosaic virus RNA (Bokstahler, 1967) and alfalfa mosaic virus RNA (Pinck et al., 1968>, and hnRNA (Jelinek and Darnell, 1972). When 70 S or 35 S RNA from either RSV-PrC or RAV-2 was cochromatographed with a variety of RNAs including reovirus ds RNA, hnRNA, messenger RNAs, ribosomal RNAs and negativestranded RNAs, the retrovirus RNAs required higher phosphate concentrations for

elution than all ss RNAs except hnRNA (Fig. 2); in fact, they eluted at the same phosphate concentration as reovirus ds RNA (Table 2). In order to ascertain at what temperature the structure or structures that were responsible for the increased affinity for HAP were disrupted (melted), thermal chromatography on HAP was performed. RNAs were applied to columns of HAP in buffer containing Me,SO (to avoid thermal scission); they were then eluted by gradually raising the temperature while constantly perfusing the columns with the same

“HAIRPIN” TABLE

SEGMENT

FROM AVIAN

2

ELUTION CHARACTERISTICS OF VARIOUS RNAs FROM HAP

RNA preparation”

Range of phosphate concentrations required for elution” Of)

70 S RSV-PrC RNA 70 S td RSV-PrC RNA 35 S RSV-PrC RNA 70 S B77 RNA 70 S td B77 RNA 35 S RSV-SRB RNA DEF hnRNA HeLa cell hnRNA Reovirus ds RNA DEF 45 S RNA Reovirus mRNA DEF ribosomal RNA HeLa cell ribosomal RNA L cell ribosomal RNA VSV (42 S) genome RNA

0.22-0.26 0.22-0.26 0.24-0.28 0.22-0.28 0.22-0.28 0.24-0.28 0.20-0.30 0.20-0.30 0.22-0.26 0.20-0.24 0.14-0.18 0.17-0.20 0.17-0.21 0.16-0.20 0.16-0.20

n RNA samples were prepared as described under Materials and Methods and dissolved in 0.10 M phosphate buffer, pH 6.8, at 60”. They were applied to columns containing 0.5 ml packed HAP at a rate of 0.5 ml/min and eluted with 0.1-0.5 M phosphate gradients. * The ranges of elution were determined by the limits at which approximately 50% of the RNA was eluted about each peak fraction. This included from three to seven fractions, depending on the RNA.

buffer. The MezSO concentration chosen (40%) was expected to lower the T,s by about 18” [Me&SO reduces the T, of reovirus ds RNA by 0.45’1% in 0.18 M sodium phosphate (Henderson and Joklik, unpublished observations)]. In order to counteract this effect and make the results comparable to those obtained in the absence of Me&SO, such as those in the experiments described in Fig. 1 and Table 1, the sodium ion concentration was increased lo-fold to 0.18 M, which increases the T, by about 18” (see Table 1). The results are shown in Fig. 3. The thermal chromatogram of 35 S RNA indicated a T, of 69”. This compared with values of 71-72” for component II in 0.018 M NaCl in the absence of Me2S0, using the technique described in Fig. 1 and Table

RETROVIRUS

RNA

29

1. When DEF hnRNA was chromatographed in the same manner, about one-half of it bound to HAP in 40% Me$O-0.18 M sodium phosphate at 40” and exhibited a thermal chromatography profile that was very similar to that of RSV-PrC 35 S RNA; its T, was 67”. These data confirmed the view that the feature causing high affinity for HAP was an unusually high degree of intramolecular base-pairing. Isolation

of the Base-Paired

Sequences

It proved possible to isolate the sequences causing increased afflnity for HAP and responsible for component II of the hyperchromicity curves by digesting RSV-PrC 35 S RNA with pancreatic RNase A. The following conditions were used: 1.0 M NaCI, O”, 30 min, molar enzyme/RNA ratio 2O:l. When digested in this manner a fraction was obtained that amounted to about 7% of the RNA, bound to HAP at 60” and required phosphate concentrations exceeding 0.20 M for elution. By contrast, VSV 42 S RNA yielded no such fraction. This “ds” RSV-PrC RNA fraction exhibited the following properties: (a) when denatured in 90% Me&SO and chromatographed on HAP it eluted at a phosphate

60

42

46

50 54

58 62 66

70 74 76

62

86

FIG. 3. Thermal chromatography of 35 S RSV-PrC RNA and DEF hnRNA on HAP. RNA samples were applied to HAP columns at 40” in 40% Me$O/O.18 M phosphate buffer, pH 6.8. While washing the columns constantly with this buffer, the temperature of the system was raised at a rate of 0.5”/min. Fractions were collected at 2” intervals; RNA in them was precipitated with TCA, and radioactivity in it was measured. The following amounts of RNA were used: RSV-PrC 35 S RNA, 200 ng; DEF hnRNA, 80 ng.

30

PERDUE,

WUNDERLI.

concentration of 0.26 M (Fig. 4); (b) when subjected to thermal chromatography on HAP as described in Fig. 3, it exhibited an elution profile very similar to those of undigested viral RNA and hnRNA, with a T, of 67”; (c) it was susceptible to digestion by 50 @g/ml pancreatic RNase A when in 0.01 il4 NaCl, but was resistant when in 1.0 M NaCl; (d) it was almost, but not quite, as susceptible to digestion by RNase III as reovirus ds RNA (72 and 92% respectively) under conditions when ribosomal RNA and reovirus messenger RNA were completely resistant; and (e) it was resistant to digestion by DNase I and sensitive to alkali (0.3 N, 37”, 3 hr), demonstrating that it contained no detectable DNA. These data indicated that the 7% “ds” fraction possessed a high content of basepaired sequences; further, the fact that no 9

t S-

“: 8 E T k

. .* .

T-

AND JOKLIK TABLE BASE COMPOSITION BASE-PAIRED

3

OF THE ISOLATED SEQUENCES”

35 S RSV-PrC RNA

Cytosine Adenine Guanine Uracil Purine (%) (G + ‘2) (“ro) A/U G/C

Isolated basepaired sequencesb

Counts per minute

%

Counts per minute

%

29,113 23,804 31,004 19,946

28.03 22.92 29.85 19.20

10,613 10,374 13,075 11,753

23.19 22.66 28.48 25.69

52.77 57.88 1.19 1.06

51.14 51.67 0.88 1.23

“ The RNA was obtained from virus grown in the presence of otiho-[32P]phosphate for 15 hr. Its specific activity was 8 x lo5 cpm/gg (2.4 x lo3 cpmlpmol). Its base composition was determined by the procedure of Sebring and Salzman (1964). * Isolated by limited digestion with RNase A followed by chromatography on HAP (see above).

65432-

‘I 0

2

4

6

8

10 12 14 16 IS FRACTION

20 22

24

FIG. 4. Chromatography of the RNaae-resistant fraction of RSV-PrC 35 S RNA, melted in Me$O, on HAP. IjgP]-labelled RSV-PrC 35 S RNA in 1.0 M NaCl was digested at 0” for 30 min with pancreatic RNase A (20 enzyme molecules per 35 S RNA molecule). Dextrsn sulfate (500 pg/ml) was then added and the sample made 0.10 M with respect to sodium phosphate, pH 6.8. It was then chromatographed on HAP as described in the legend to Fig. 2. Ninety-two percent of the sample eluted at phosphate concentrations below 0.20 M, while 8% required higher concentrations for elution. This latter fraction was collected and precipitated with 5 mM cetyl trimethylammonium bromide after the addition of 10 pg tRNA as carrier. The precipitate was suspended in 1 M NaCl and reprecipitated with 2.5 vol 95% EtOH, resuspended in 200 ~1 90% Me&O, 10 mM Tris.HCl, 1 m&f EDTA, pH ‘7, and heated at 3’7” for 5 min. The sample was then diluted go-fold into 0.1 M phosphate, pH 6.8, and rechromatographed on HAP. The highest CaT value which could be reached by the “da” fraction under these conditions was about 1 X 10e5 mol.sec/liter.

special annealing following melting in Me&SO was necessary for refolding into the ds configuration suggested that the molecules comprising this fraction were hairpin in nature. Nucleotide Base Composition Fraction

of the “de”

Although conditions for digesting RSVPrC 35 S RNA had been devised to isolate segments rich in base-paired secondary structure, it remained possible that sequences rich in purines, and thus resistant to pancreatic RNase A, had in fact been selected. In order to rule out this possibility the isolated base-paired “ds” sequences were hydrolyzed with alkali and the products subjected to electrophoresis on DEAEcellulose in order to determine their nucleotide base composition. As shown in Table 3, the purinelpyrimidine ratio of the “ds” fraction was close to unity. Its (G + C) content was 51.7%, which was lower than the value for unhydrolized RNA, namely 57.9% [which was comparable to values in the literature (for example, Bishop et al.,

“HAIRPIN”

SEGMENT

FROM AVIAN

REOVIRIJS S3 RNA

7s

5s 4s

III

7

I

;

1”

FRACTION

FIG. 5. Electrophoresis of the “double-stranded” fraction in formamide-polyacrylamide gels. 3ZP-labeled “double-stranded” RNA was isolated by elution from HAP with 0.30 M phosphate (see Fig. 2). It was precipitated with 5 mM cetyl trimethylammonium bromide after the addition of 10 pg/ml tRNA as carrier. The pellet following centrifugation at 10,000 g for 10 min was collected, washed with cold 70% ethanol, and dissolved in formamide as described under Materials and Methods. 3H-Labeled 4 S, 5 S, and ‘7 S RNAs from L cells and reovirus genome RNA segment S3 were included in the sample. The mixture was heated at 37” for 5 min and loaded onto a 5% polyacrylamide gel containing 99% formamide (lo-cm disc gel). Electrophoresis was for 4 hr at 5 mA (100 VI. The gel was sliced into l-mm disks, and assayed for “H and 32P.

19’70)]. These data indicated that the reason why the “ds” fraction was resistant to RNase A in 1.0 M NaCl was not an abnormally high purine content. Size of the “ds” Fraction The “ds” fraction of RSV-PrC 35 S RNA sedimented in sucrose and MepSO-containing sucrose density gradients at rates corresponding to 7-8 S and 10 S respectively (data not shown). Its size was determined more definitively by electrophoresing it in denaturing -- polvacrvlamide gels. As shown 1 ”

RETROVIRUS

RNA

31

in Fig. 5, when electrophoresed in 99% formamide-5% polyacrylamide gels the “ds” fraction behaved as a reasonably homogeneous species. Its rate of migration, between that of reovirus S3 RNA (about 1000 bp) and L cell 7 S RNA (about 200 nucleotides), indicated that it comprised a population of molecules 600 to ‘700 nucleotides long. Since it accounted for about 7% of RSV-PrC 35 S (about 10 Kb), these results indicated that the “ds” fraction represented a single region of the viral genome which was in all likelihood hairpin in nature. Further evidence concerning the size and homogeneity of the “ds” fraction was obtained by autoradiography of gels in which limited pancreatic RNase A digests of RSVPrC and RAV-2 RNA had been electrophoresed (Fig. 6). A species of molecules was formed from both viral RNAs which migrated faster than denatured reovirus S4 RNA (also about 1000 bp) but slower than the poly(A) sequences contained in viral RNA [loo-200 nucleotides (Quade et al., 1974)]. Its migration rate was consistent with a length of 600-700 nucleotides. This

ABC - Reovirus

S4 RNA

-rrds”

segment

- Poly

(A)

FIG. 6. Autoradiograms of limited RNase A digests of the RNAs of RSV-PrC, RAV-2 and RSV-BH(-) electrophoresed in 99% formamide-polyacrylamide gels. 32P-labeled RSV-PrC RNA (A), RAV-2 RNA (B), and RSV-BH(-) RNA (C) was digested with RNase A as described in the legend to Fig. 4. The digests were precipitated with ethanol and resuspended in 30% formamide, 0.02 M phosphate, pH 6.8. After addition of 3H-labeled reovirus genome RNA species S4, the samples were loaded onto 5% polyacrylamide/ 99% formamide slab gels, electrophoresed and autoradiographed. The position of the reovirus RNA was determined by slicing the gel after autoradiography.

32

PERDUE,

WUNDERLI,

AND JOKLIK

species was not formed when the RNA of RSV-BH( -) was digested in similar manner. Location of the “ds” Fraction Within Viral 35 S RNA

The location of the sequences comprising the “ds” fraction in the RSV-PrC 35 S RNA molecule was determined as follows. Viral 35 S RNA was fragmented as described under Materials and Methods and the resulting poly(A>containing molecules were ordered with respect to size by centrifugation in sucrose density gradients. They were then treated with pancreatic RNase A under the limiting digestion conditions described above, keeping the molar enzyme/RNA ratio constant, and the digests were chromatographed on HAP to determine whether they contained material capable of binding in 0.18 M, but eluting in 0.40 M phosphate buffer. The results are shown in Fig. ‘7. With increasing length of poly(A)-containing molecules, an increasing proportion of their 4

15

SIZE

OF POLY (A)-CONTAINING

FRAGMENT

(kb)

FIG. 8. Mapping the “double-stranded” RNA segment by measuring the affinity of sized poly(A)-contaming fragments of RSV-PrC 35 S RNA for HAP. The poly(A)-containing fragments were prepared and sized as described in the legend to Fig. 7. Molecules in each size class were then applied to HAP at 60” in 0.18 M phosphate. Columns were washed first with the same buffer and then eluted with 0.40 M phosphate, both at 60”. The amount of RNA that was eluted by 0.40 M phosphate buffer was measured and plotted against the average size of the molecules in each fraction.

digestion products exhibited HAP binding properties characteristic of ds RNA, until a peak was reached at about 5 Kb, when the proportion reached 12%. Thereafter it declined to a value of 7% for full length mol5 E ecules, in agreement with the results deBo ” ” ” ” ” scribed above. The peak value of 12% of I 2 3 4 5 6 7 8 9 IO SIZE OF POLY (A)-CONTAINING FRAGMENTS IKb) 5 Kb indicated that molecules in the “ds” fraction were about 600 nucleotides long, in FIG. 7. Mapping the “double-stranded” RNA segagreement with the results described above. ment by measuring the amount of HAP-binding maThese data place the sequence in the “ds” terial in RNase A digests of poly(A)-containing RSVPrC 35 S RNA fragments of various sizes. 32P-labeled fraction near the center of the RSV-PrC 35 poly(A)-containing fragments were prepared as de- S RNA molecule. In further experiments, the ability of the scribed under Materials and Methods and fractionated by centrifugation in 5-30s (w/v) sucrose density gra- undigested poly(A)-containing fragments dients, using 4, 18, 28, and 35 S RNAs in separate themselves to bind to HAP like ds RNA density gradients as size markers. The various frac- was determined. The results presented in tions were then digested with pancreatic RNase A Fig. 8 show that fragments longer than 5 Kb (molar enzyme/RNA ratio 20:1, 1.0 M NaCl, 0”, 30 bound to HAP in 0.18 M phosphate and min) and chromatographed on HAP at 60” using two were eluted in 0.40 M phosphate, while successive batch elutions with 0.18 and 0.40 M phosfragments shorter than 5 Kb bound prophate. The amount of material eluting with 0.40 M gressively less and less. This result confirms phosphate was then plotted as a percentage of the total amount of RNA in the sample against the size the conclusion that the 600-700 nucleotide sequence responsible for ds RNA behavior of the poly(A)-containing viral RNA fragments from which it was generated. is located close to the middle of the viral ii-//y

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35 S RNA molecule which is further supported by the fact that this sequence is absent from the RNA of RSV-BH(-) (see above). This RNA lacks most of the env gene (Duesberg et al., 1975) and there is evidence that the deletion encompasses its 5’-end (Vogt, personal communication), which would be located near the middle of the molecule. DISCUSSION

The results presented in this paper demonstrate that the RNA of avian RNA tumor viruses possesses a segment that confers upon it an affinity for HAP equal to that of authentic ds RNAs and greatly exceeding that of all other ss RNAs tested with the exception of hnRNA. This segment, which can be isolated by treating 35 S viral RNA with RNase A under conditions of limited digestion, seems to be a single large hairpin about 300 bp long that is highly base-paired and located near the center of the 35 S RNA molecule. This position is of great potential interest. Avian RNA tumor viruses express themselves via three species of mRNA: the complete 35 S RNA molecule which codes for the gag and pol genes, a 28 S mRNA species that codes for the env gene, and a 21 S mRNA species that probably codes for the arc gene (von der Helm and Duesberg, 1975; Pawson et al., 1977; McGinnis et a!. , 1978; Hayward, 1977; Stacey et al., 1977; Beemon and Hunter, 1977; Purchio et al., 1977; Weiss et al., 1977). Since the gag plus pol genes are approximately the same size as the env plus src genes, the region where the 300 bp hairpin is located appears to be at, or very close to, the location where translation of the 35 S RNA molecule is terminated. It is conceivable that the hairpin region specifically blocks the translation of 35 S RNA beyond the pol gene, and that it functions during the splicing out of the pol and gag genes during the generation of env and src gene mRNA which are known to possess a leader sequence derived from the 5’-terminus of the 35 S RNA molecule at their own 5’-termini (Mellon and Duesberg, 1977; Krzyzek et al., 1978; see also Rothenberg et al., 1978). It may well prove to be a useful

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map position marker, either by conferring on the RNA fragment under investigation the ability to bind to HAP in the presence of 0.2 M phosphate, or by yielding material after limited digestion with RNA that is capable of binding to HAP. It is tempting to speculate that the hairpin structure described here corresponds to the 250 bp hairpin demonstrated in BKD and woolly monkey sarcoma virus RNA by means of electron microscopy under partially denaturing conditions by Kung et al. (1976) and which they showed to be located near the center of the molecule. While such a structure has not yet been demonstrated in ASV RNA, the results presented in this paper suggest that an analogous structure is in fact present in it, although it may well possess a somewhat lower thermal stability. HnRNA of eukaryotic cells contains double-stranded sequences similar in nature to that described here. Jelinek (1977) has recently demonstrated that these regions are transcribed directly from repeated DNA sequences. It will be interesting to determine whether there is any sequence homology between the base-paired sequences of retrovirus RNA and cellular hnRNA. REFERENCES

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