The 50S and 35S RNAs from Newcastle Disease Virus-Infected Cells

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1 J. gen. ViroL 0977), 35, Printed in Great Britain 439 The 50S and 35S RNAs from Newcastle Disease Virus-nfected Cells By BONNE B. SPANER* AND MCHAEL A. BRATT University of Massachusetts Medical School, Worcester, Massachusetts 01605, U.S.A. (Accepted 4 January 977) SUMMARY Electrophoretic analyses of the 5oS and 35S Newcastle disease virus-specific RNAs from infected cells before and after heat denaturation make it possible to demonstrate that these regions each contain single-stranded RNAs with corresponding S values as well as partially base-paired structures. The partially basepaired structures which sediment at 5oS (4 to 6oS) have a distribution in gels similar to that of the in vitro transcriptive intermediates, and they remain when 5oS RNA synthesis (replication) is blocked by cycloheximide. The partially basepaired 35S RNA is more homogeneous and is neither labelled in the in vitro transcription reaction nor when infected cells are treated with cycloheximide. These base-paired structures may, therefore, be involved in transcription and replication, respectively. NTRODUCTON Newcastle disease virus (NDV) is a paramyxovirus with a single-stranded 5oS RNA genome of molecular weight 5"4 x lo 6 (Kolakofsky, Boy de la Tour & Delius, 1974). Cells infected with NDV contain virus-specific RNA which, after selective labelling with ah-uridine in the presence of actinomycin D, can be separated by velocity sedimentation into three major size classes: 5oS, 35S and 18 to 22S (Bratt & Robinson, 967). The 18 to 22S is at least 95 % single-stranded, is complementary to virion RNA (Bratt & Robinson, 967), is associated with polysomes and released from them by EDTA (Bratt & Robinson, 1967) and contains polyadenylic acid [poly (A)] sequences (Weiss & Bratt, 1974). The 18 to 22S RNAs separate into 6 to 8 species when fractionated by electrophoresis on SDS aqueous gels (Collins & Bratt, 1973) but the denaturing conditions of formamide gel electrophoresis reveal only five apparently discrete species (Weiss & Bratt, 1976). Messenger RNA activity has been ascribed to the 18 to 228 RNAs although only two (or perhaps three) of NDV's six polypeptides (Hightower, Morrison & Bratt, 1975) could be unequivocally identified among the cell-flee translation products (Morrison et al. 1975). Among the missing polypeptides was the largest, 'L', whose estimated molecular weight of 2.2 xo 5 (Hightower et al. 1975) considerably exceeds the o'85 O 5 estimated coding capacity of the largest 18S RNA species. Considerably less is known about the RNAs in the 35S and 5oS regions. They contain variable amounts of base-pairing as indicated by ribonuclease resistance (Bratt & Robinson, 967; Zhdanov & Kingsbury, 1969; Bratt & Robinson, 971). Preferential labelling in short ah-uridine pulses has suggested that the base-pairing is associated with intermediates in RNA synthesis. Some 35S RNA is also found associated with polysomes (Bratt & Robinson, 1967) but whether it represents single-stranded mrna or partially base-paired * Present address: Department of Biology, Wheaton College, Norton, Massachusetts o2766.

2 440 a. B. SPANER AND M. A. BRATT intermediates is unclear. Bratt & Robinson (1967) also showed that when labelled for a long period (2 h), 90 ~ of the RNA in the 35S region is complementary to virion RNA. RNA, presumably including single-stranded 5oS virion RNA, in the 5oS region was shown to be 3o ~ complementary to virion RNA. We now report the characterization of the RNAs from the 35S and 5oS regions using polyacrylamide gel electrophoresis. The 35S and 5oS regions both contain partially basepaired structures as well as single-stranded molecules with these S values. Additional studies, conducted under conditions where replication does not occur, suggest possible roles for these base-paired structures. Elsewhere, we report that the single-stranded RNA in the 35S region has the characteristics of mrna and directs the synthesis of the L polypeptide (Morrison et al. 975; B. B. Spanier, C. W. Clinkscales, M. A. Bratt & T. G. Morrison, unpublished observations). METHODS Preparation of virus. Virus was grown in eggs and purified as previously described (Weiss & Bratt, 974). The strains of virus used were NDV-AV (Australia-Victoria, 932) and NDV-HP (srael HP, 1935). NDV-AV was used to analyse the 5oS and 35S regions of intracellular virus-specific RNA. NDV-HP was used in the preparation of 32P-labelled virion RNA (because it grows to high titres in eggs) and in the in vitro polymerase reaction. Different NDV strains synthesize the same three major size classes of virus-specific RNA, although in differing proportions- NDV-AV synthesizes more 18 to 22S RNA relative to 5oS RNA than NDV-HP (Bratt, 969). The 8 to 2zS RNA of differing strains of NDV contain RNA species with similar electrophoretic mobilities (Collins & Bratt, ~973; Spanier-Collins, 975). n addition, virion RNA from one strain of NDV anneals 9o to OO ~ of the intracellular complementary 18 to 22S RNA from different strains (Kingsbury, 1966; M. A. Bratt, unpublished observations). Therefore, in many types of of experiments on the nature of NDV-specific RNAs, the strain of virus is unimportant. However, the conclusions about relative proportions of single-stranded and partially base-paired RNAs may be valid only for NDV-AV. a2p-labelled RNA. Eight-day-old embryonated eggs were injected with mci per egg of a2p-phosphate (New England Nuclear) in o- m standard medium (Eagle's minimal essential medium with 2"5 ~ calf serum, 2"5 ~ (w/v) tryptose phosphate broth and o'7 NaHCO~). One day later, each egg was injected with lo 4 p.f.u, of NDV-HP. After two additional days incubation at 39 C, virus was harvested and purified (Weiss & Bratt, 974), and the RNA extracted with buffer-saturated phenol and precipitated twice in ethanol (Bratt & Robinson, 1967). Preparation of cell cultures. Secondary cultures of chick embryo cells were prepared and grown in a 5 ~ CO2 atmosphere as previously described (Bratt & Gallaher, 1969), but in standard medium. Cultures were plated at 5 x io G cell per OO mm tissue culture plate and used after 48 h incubation at 4o C. ntracellular RNA. Virus-specific RNA was labelled in the following ways: () cultures were infected with 5 p.f.u./cell of virus, and at 3"z5 h after infection, were treated with actinomycin D 0o #g/ml; generously supplied by Merck, Sharp, and Dohme, Rahway, New Jersey) in labelling medium (Eagle's minimal essential medium supplemented with 2 ~ dialysed calf serum and o'o7 ~ NaHCO3). At 4 h after infection, oo #Ci/ml of 3Huridine (New England Nuclear, 28 Ci/mmol) was added. At 9 h p.i., the cells were solubilized in solubilizing buffer [o- M-NaC1, o.oi M-tris ph 8"5, o'ooz M-EDTA, ~ SDS and

3 NDV: 5oS and 35S RNAs 441 (v]v) mercaptoethanol], extracted with phenol (saturated with solubilizing buffer lacking SDS) and precipitated twice in ethanol (Bratt & Robinson, 967). (2) When cycloheximide (Actidione; Calbiochem) was present from the start of infection, zo #g/ml actinomycin D and 50/~g[ml cycloheximide were added 30 min before infection at an m.o.i, of 50 p.f.u./cell. 3H-uridine (~5o #Ci/ml) was added at the time of infection in the presence of the two drugs. Cells were solubilized at 6 h p.i. (3) When cycloheximide was used late in infection, the m.o.i, was 5 p.f.u.]cell, 5o/xg/ml of cycloheximide was added at 4"5 h p.i., 2o/~g/ml of actinomycin D was added at 6"5 h p.i., and ~oo #Ci/ml of ah-uridine was added from 7 to 8 h. (4) Host cell RNA (to be used as markers) was labelled with 2 #Ci/ml of 14C-uridine (New England Nuclear, 52"7 mci/mm) for 3o h. n vitro RNA. 'n vitro' RNA is the product of the virion-associated polymerase reaction (Huang, Baltimore & Bratt, 97~), in which virion RNA is the template for the transcription of complementary 18 to 228 RNA. n vitro RNA was labelled with 3H-GTP (New England Nuclear, 5"66 Ci/mmol) and prepared as described by Weiss & Bratt 0974). Heat treatment of RNA. RNA was heated in a boiling water bath for 2 min in either SDS-standard buffer or E buffer (see below) plus 0"3 ~o SDS, and then quenched in ice water. This treatment rendered 96 to loo ~ of the RNA sensitive to ribonuclease. Sucrose gradient sedimentation. RNA extracts dissolved in 0"3 ml SDS-standard buffer (o. M-NaC1, o-o u-tris ph 7"4, o.oo2 M-EDTA and o'5 ~o SDS) were layered on to 2 ml linear 5 to 3o % sucrose gradients containing the same buffer (Clavell & Bratt, 97 ). The gradients were centrifuged for 4 h at 18o ooo g (39 ooo rev/min) in a Spinco SW 41 rotor at 22 C, and fractions were collected as described previously (Weiss & Bratt, ~974). n all sedimentation analysis figures, the top of the gradient is on the right. The 5oS, 35S and i8 to 22S RNAs pooled from sucrose gradients and electrophoresed individually were selected from regions corresponding to the following in Fig. : fraction numbers 3 to ~3 for 5oS RNA, 14 to 2 for 35S RNA and 22 to 3 for ~8 to 22S RNA. Gel electrophoresis. Labelled RNA was subjected to electrophoresis in o'5 ~o agarose-1.2 % acrylamide, ethylene diacrylate cross-linked gels (o x 0"6 cm inside diameter) prepared in the following way. Agarose (o'25 g' special grade') was added to 20 ml of water, dissolved by heating to boiling, and then cooled to 50 C in a waterbath. Electrophoresis (E) buffer was 0"04 m-tris, O'O2 M-sodium acetate and o.oo~ M-EDTA, ph 7'2. A mixture containing.o ml of a 30 ~ stock solution of acrylamide, o.c6o ml ethylene diacrylate, 5 ml of 5 E buffer and 8"72 ml of water was de-gassed. TEMED (N,N,N',N'-tetramethylethylenediamine, 0-02 ml) was added and the mixture was incubated at 50 C. Ammonium persulphate (0"4 ml of a o ~ solution, final concentration 0-8 mg/ml) was added to the agarose solution and mixed. Ten ml of the agarose solution was added to the acrylamide mixture at 50 C and the mixture was quickly used to make the gels, since the agarose hardens rapidly when cooled below 50 C. [The agarose is added to stiffen the gels because of the low percent acrylamide. At the concentration used, it probably does not contribute to the molecular sieving effect (Peacock & Dingman, 968).] The gels were pre-electrophoresed for about 20 rain before samples were applied. Electrophoresis was done at room temperature (23 C) with a constant current of 5 ma per gel for 4 h using E buffer plus 0" 3 ~ SDS (Bishop, Claybrook & Spiegelman, 967). The 0"5 ~ agarose-l.8 ~ acrylamide gels were prepared and electrophoresed as above except that 1"5 ml of the 3o ~ stock solution of acrylamide was used and the gels were electrophoresed for 2 h. Gels were then frozen and sliced into mm fractions with a Joyce Loebl gel slicer. Slices were dissolved in o. M-piperidine and counted in 5 ml of modified

4 442 B. B. SPANER AND M. A. BRATT 50S 35S 18~2S [ 1 if 2O Fraction number Fig.. Sedimentation analysis of intracellular NDV-AV RNA. 3H-abelied intracelular RNA (O) was subjected to sucrose gradient velocity sedimentation as described in Methods. A portion of each fraction was treated with ribonuclease (V). Bray's solution (Hightower & Bratt, 1974). The direction of migration of all gels in the figures is from left to right. Ribonuelease treatment. Ribonuclease digestion was done by incubating RNA at 37 C for 3 min in a mixture containing 5o t~g of bovine pancreatic RNase (Schwarz Bioresearch) per ml and 25 units of T RNase (Calbiochem) per ml in 2 x SSC (o'3o M-NaC1 and o.o 3 M-sodium citrate). RESULTS Base-paired and single-stranded 35S and 5oS RNAs The three size classes of NDV-specific intracellular RNA labelled with ~H-uridine fiom 4 to 9 h p.i. are shown in Fig. t : 8 to 22S (5 ~ resistant to ribonuclease), 35S (O ribonuclease-resistant) and 5oS (35 ~ ribonuclease-resistant). To distinguish between partially base-paired and single-stranded species which sediment together, 18 to 2zS, 35S and 5oS RNAs were separately pooled from a sucrose gradient, and heated to OO C for 2 min to render the RNA single-stranded. Fig. 2 shows velocity sedimentation analyses of these RNAs before and after heating. Sedimentation of the i8 to ~2S RNA is not significantly affected by heating (Fig. 2a, b). n contrast, heating the RNA from the 35S region reduces the amount of RNA sedimenting at 35S, while increasing the amount of RNA in the 8S region (Fig. 2e, d). Heating RNA from the 5oS region dramatically reduces the amount of RNA sedimenting at 5oS, while increasing the amount sedimenting in the 18 to 35S regions (Fig. 2e, f).

5 ND V: 5oS and 35S RNAs (a) 28S 18 (c) 28S 18S 3.0 (e) 28S 18S 20 / O X.=_ 30,... --~---,... a t - (b) 28S 8S 8-0 (d) ' 18S ' (f) i 28s 18s 20 _==;~;==~ 1 e \ J Fraction number Fig. 2. Sedimentation analysis of i8 to 22S, 35S and 5oS RNA (NDV-AV) before and after heating. all-labelled 18 to 2zS, 35S and 5oS RNA (0)were separately pooled from a sucrose gradient and sedimented again before and after heating. Samples'were counted directly in Aquasol. (a) 18 to 22S RNA; (b) 8 to 2zS RNA after heating; (c) 35S RNA; (d) 35S RNA after heating; (e) 5oS RNA; (f) 5oS RNA after heating. Arrows mark 8S and z8s RNA from extinciton determinations. The effect of heating is seen in greater detail after electrophoresis of the 18 to 2zS, 35S and 5 o S RNAs in o'5 ~ agaro se- 1.2 % polyacrylamide gels (Fig. 3). The pattern of unheated 18 to 22 S RNA (Fig. 3 a) is similar to that seen in higher percent gels (Collins & Bratt, J973), although the resolution is considerably lower. Heating causes no significant shift in the migration of this RNA (Fig. 3b). lit has recently been shown (Roux & Kolakofsky 975; Weiss & Bratt, 1976 ) that treatment of the ~zs RNAs of NDV and Sendai virus with heat, dimethyl sulphoxide, or formamide converts most of this RNA to species which sediment and/or electrophorese with the 8S RNAs. A comparison of Fig. 3 a and b suggests that the zzs RNA is diminished to some extent after heating. Quantification of this is difficult because of the small proportion of 2zS RNA present in this preparation.] Fig. 3(e) shows that 35S RNA can be resolved into a major peak which migrates more slowly than 28S RNA and an even more slowly migrating minor peak (slices 41 to 45). Heating this RNA (Fig. 3d) reduces the minor peak, while increasing the amount of more rapidly migrating species in the 18 to 2zS regions; most of the RNA continues to migrate as the major peak. Fig. 3 (e) shows the heterogeneous pattern of RNA from the 5oS region before heating. After heating (Fig. 3f), the slowest migrating heterogeneous RNA disappears, leaving a small peak in the 5oS region, which, as shown below, corresponds to virion RNA. Concomitantly, there are increases in the faster migrating species - 35S and 18 to 22S. Therefore, the 5oS and 35S regions both contain single-stranded species with the

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7 NDV: 5oS and 35S RNAs 445 (a)! "0 20 L, i (b) 1.0 ] ? X E m (c) ,5 2O Slice number Fig. 4. Electrophoretic analysis of virion 0~DV-HP) and intracellular 5oS RNA (NDV-AV) before and after heating. (a) 32P-labelled virion RNA ( ) was co-electrophoresed with all-labelled intracellular 5oS RNA (0). (b) Heated 82P-labelled virion RNA ( ) was co-electrophoresed with all-total intracellular RNA (0). (c) Heated 3~P-labelled virion RNA (G) was co-electrophoresed with heated 8H-intracellular 5oS RNA (0).

8 4 446 B. B. SPANER AND M. A. BRATT [ i 28s 18s? 6 X Fraction number Fig. 5. Reconstruction experiment. 3H-labelled ~8 to 22S virus-specific RNA (NDV-HP) was extracted with five oo mm plates of secondary chick embryo cells either infected ([]) with NDV- HP or mock-infected (Q), and subjected to velocity sedimentation analysis. Each fraction was counted directly in Aquasol. Arrows mark 8S and 28S cell RNA from extinction determinations. corresponding S values as well as partially base-paired structures which release smaller single-stranded species when heated. Possible artifactual origins of the 35S and 5oS RNAs Under certain denaturing conditions (in the presence of formamide or dimethyl sulphoxide), 5oS virion RNA sediments at about 35S (Kolakofsky et al 974; B.B. Spanier, unpublished observations). We therefore felt it necessary to eliminate the possibility that the heat-resistant 35S RNA seen in the gels is simply an artifact of heating 5oS virion RNA. 32P-labelled 5oS RNA isolated from virions was heated and co-electrophoresed with intracellular 5oS RNA. Fig. 4a shows untreated 32p-labelled virion RNA co-electrophoresed with 3H-intracellular 5oS RNA. The virion RNA is a single sharp peak which coincides with one of the intracellular peaks. Fig. 4b shows heated 3zP-labelled virion RNA coelectrophoresed with untreated total intracellular RNA. The total RNA is separated in the gel into the heterogeneous 5oS region, the minor and major 35S species, and the 22S and 8S RNAs. Although some 32p-labelled RNA has broken down, most remains as a single peak coinciding with the same peak in the 5oS region. Clearly, heating virion RNA does not convert it to a 35S peak. This is also shown in Fig. 4(c), where both 3zP-labelled virion 5oS RNA and 3H-intracellular 5oS RNA were heated before electrophoresis. Again, the heated virion RNA coincides with a 5oS peak and not with the 35S peak. Therefore, the single-stranded 35S species is not an artifact of heating single-stranded 5oS RNA. We were also concerned that the RNase-resistant structures might be artifacts produced by the annealing of completely single-stranded RNA species during the preparation of the RNA as suggested by the study of Kolakofsky, Boy de la Tour & Bruschi (974). This was particularly important since the infected cells contain a large excess of 18 to 22S RNA complementary to virion RNA, and the products of heating the RNA from the 35S and 5oS

9 NDV: 5oS and 35S RNAs 447 regions both migrate and sediment mainly in the 18 to 2zS regions. To investigate this possibility, a reconstruction experiment was done. Unlabelled, uninfected and infected cells were dissolved in solubilizing buffer containing 3H-labelled ~8 to 2zS RNA (from the same number of infected cells) pooled from a sucrose gradient. The RNA was then extracted with phenol, and the distribution of the labelled x8 to 2zS RNA in the sucrose gradients was examined. As shown in Fig. 5, the vast majority of radioactivity again sediments in the 8 to zzs region. Less than 5 //o of the radioactivity sediments faster than 228 in the presence of RNA from infected cells, and less than 8 ~ with RNA from uninfected cells. This is considerably less than z5 ~ (half the usual value of at least 5o ~ of the total NDV-specific RNA sedimenting faster than zzs) which one would expect if the ZH-labelled ~ 8 to zzs RNA formed hybrids, since it is competing with an equal amount of unlabelled 18 to ~zs RNA from the infected cells. Since the values are so low, and because there is no difference between the infected and uninfected cell samples, we conclude that the partially base-paired RNA structures normally seen in the 5oS and 35S regions are not produced by artifactual annealing of single-stranded x 8 to 22S RNA and the 5cS RNA during the preparation of the RNA for gradient or gel analyses. Further analysis of base-paired RNAs The ability of acrylamide gel electrophoresis to separate single-stranded and partially base-paired RNAs within the 35S and 5oS regions makes it possible to characterize these RNAs further. Portner & Kingsbury 0972) ascribed distinct functions to the base-paired RNAs extracted from cells infected by Sendai virus, another paramyxovirus. A relatively homogeneous species sedimenting at z4s was implicated in replication, and heterogeneous base-paired structures sedimenting between 28S and 6oS were implicated in transcription. One approach they used was to compare the RNAs synthesized in the presence and absence of cycloheximide. nhibitors of protein synthesis have no effect on the distribution of 18 to 22S RNAs (Collins & Bratt, 973) but are known to decrease incorporation of ahuridine into the RNA sedimenting at 5oS relative to the more slowly sedimenting species (Clavell & Bratt, 97; Robinson, 97 ; Portner & Kingsbury, i972; Weiss & Bratt, ~975). The inhibition of incorporation into the 5oS RNA is assumed to represent an inhibition of replication. Another striking effect of cycloheximide is to decrease the RNase-resistance of the 35S region from 2o ~ to 5 ~ (Spanier-CoUins, 975). Fig. 6 contains an electrophoretic analysis of the 35S RNAs which accumulate in the presence and absence of cycloheximide. Higher percentage gels (o'5 ~ agarose and 1.8 acrylamide) and longer electrophoresis times (only the 28S cellular ribosomal RNA marker remains on the gel) allowed for considerably better separation than that obtained in Fig. 3. The pattern for the RNA from untreated cultures (Fig. 6a) shows that the major singlestranded species (fractions 36 to 5) has been fully resolved from the minor species (fractions 26 to 3 l) which in Fig. 3 was shown to be heat labile. n contrast, the minor species is missing from the 35S RNA synthesized in the presence of cycloheximide (Fig. 6). Fig. 7 contains a similar electrophoretic analysis (using the conditions described in Fig. 3) of the 5oS RNA which accumulates in the presence and absence of cycloheximide. The distribution of the 5oS RNA from untreated cells (Fig. 6a) is identical to that already seen in Fig. 3 e and 4 a. The partial base-pairing (indicated by ribonuclease resistance) of the most slowly migrating species is consistent with the disappearance of RNA from this region on heating (Fig. 3 e, f). The small amount of 5oS RNA which accumulates in cycloheximidetreated cells (Fig. 6b) shows the same broad distribution as that in untreated cells. The elimination of the relatively homogeneous heat-labile minor component of the 35S

10 448 B. B. SPANER AND M. A. BRATT 20 8 (a)! 1 (a) i X.=_ E 7 &, ~ 15 (b) i 15 j (b) i i J Slice number Fig Slice number Fig. 7 Fig. 6. Electrophoretic analysis of the 35S intracellular RNA (NDV-AV) synthesized in the presence and absence of cycloheximide. (a) 3H-labelled 35S RNA (O) synthesized late in infection was electrophoresed in a o'5 ~ agarose-1.8 ~ acrylamide gel. (b) 8-t-labelled 35S RNA (O) synthesized late in infection in the presence of cycloheximide was electrophoresed in a parallel gel. 14C-labelled 28S ribosomal RNA (O) was co-electrophoresed in each gel. Fig. 7. Electrophoretic analysis of the 5oS intracellular RNA (NDV-AV) synthesized in the presence and absence of cycloheximide. (a) 3H-labelled 5oS intracellular RNA (O) was subjected to electrophoresis. The RNA was eluted from the gel by incubating each slice overnight in o5 ml z x SSC on a shaker at 4 C and the RNase resistance (V) of each slice was determined. (b) ZHlabelled 5oS RNA (O) synthesized from o to 6 h after infection in the presence of cycloheximide was subjected to electrophoresis in a paralled gel. RNA by cycloheximide (Fig. 6), and the lack of effect of this drug on the distribution of the heterogeneous heat-labile, partially base-paired structures which sediment around 5oS (Fig. 7), tentatively implicate the former as an intermediate in replication and the latter as an intermediate in transcription. Base-paired RNA has also been implicated as an intermediate in in vitro transcription by the polymerase of the virion (Clavell & Bratt, 971 ). Fig. 8 shows a velocity sedimentation analysis of the products of incubating ~2P-labelled virus in an in vitro polymerase reaction with 3H-GTP for o, ]5 and 2O rain. With no incubation (Fig. 8a) the a2p-labelled 5oS template RNA is seen as a sharp peak and no

11 NDV: 5oS and 35S RNAs 449 [(a) ] [(b) l S 115~- 50S 28S 18S x= (c) / 5os 2gs lgs~ \ x 5 5 l i Fraction number Fig. 8. Sedimentation analysis of in vitro polymerase reaction products. n vitro polymerase reactions containing 32P-labelled virions (NDV-HP) and 8H-GTP were incubated for (a) o rain, (b) 15 rain, and (c) 12o rain, the RNA extracted and subjected to velocity sedimentation analysis. Samples were TCA-precipitated. 32P-labelled virion RNA ( ) and 8H-GMP incorporated during the reaction (0) are shown. n (c) the RNase resistance of the incorporated SH-GMP (,) is shown. la) i (b) 6-0 3"0 7._= 4.0 x x._= +~ 2"0 0.5! & Slice number Fig. 9- Electrophoretic analysis of the 42S intermediate from an in vitro polymerase reaction and 5oS intracellular RNA. (a) 3H-RNA (41) sedimenting faster than 28S, from an in vitro polymerase reaction (NDV-HP), was electrophoresed. 8S and 28S ribosomal RNA are indicated by arrows. (b) asp-labelled 5oS intracellular RNA (NDV-AV) [C)] was electrophoresed in a parallel gel with 3H-labelled 18S and 28S ribosomal RNA (0). incorporation of ZH-GMP has occurred. After 5 rain incubation (Fig. 8b), 3H-GMP has been incorporatedin to the 42S region (fractions 8 to 6) and also into the 18 to 22S region. After 12o rain incubation (Fig. 8c), a distinct peak of the 3H-GMP still sediments in the 42S region and exhibits an RNase resistance of4o ~ compared to 2 ~ for the 8 to 22S RNA. The z2p-labelled 5oS template now has a more slowly sedimenting shoulder which coincides with the 8H-labelled 42S product RNA. The 42S structure represents an intermediate in RNA synthesis based on its preferential labelling in short pulses, and its RNase resistance (Clavell & Bratt, 1970, and because, as shown here, the sedimentation constant of some of the 3zP-labelled 5oS template changes to 42S in the in vitro polymerase reaction. The partial conversion of template RNA from 5oS 30 VlR 35

12 450 B. B. SPANER AND M. A. BRATT to 42S suggests that only a portion of the templates become involved in the polymerase reaction. This intermediate must serve a transcriptive function since no replication occurs in vitro, and the single-stranded product of the in vitro polymerase reaction consists of 18 to 22S RNA which is complementary to virion RNA (Huang et al 971), possesses poly (A) (Weiss & Bratt, i974) and can function as a message for the cell-free synthesis of at least one virus polypeptide (T. G. Morrison, S. R. Weiss, B. Spanier, L. E. Hightower & M. A. Bratt, unpublished observations). Electrophoretic analysis of the 'transcriptive intermediate' (actually all of the RNA sedimenting more rapidly than 28S) from an in vitro polymerase reaction (Fig. 9a) shows a migration pattern which is similar to that ofintracellular 5oS RNA (Fig. 9b). No 35S RNA has ever been detected in the in vitro reaction, and neither the major nor minor components of 35S RNA are seen here. The similar electrophoretic behaviour of the in vitro transcriptive intermediate and the intracellular RNA which sediments in the 5oS region, and the absence, in vitro, of 35S RNA of any type provides additional suggestive evidence for a transcriptive role for the base-paired RNAs in the 5cS region and a replicative role for the heat-labile minor 35S component. DSCUSSON We have characterized the 5oS and 35S RNAs of NDV using electrophoretic separation in agarose-acrylamide gels. By comparing heated and unheated 5oS and 35S RNA, we have demonstrated the presence of both ribonuclease-resistant structures and completely singlestranded 5oS and 35S species. The single-stranded 5oS RNA which remains after heating co-migrates with 5oS virion RNA and represents less than 3 o % of the total RNA in the 5oS region. The single-stranded 35S species is the messenger RNA for the largest virion protein, L (Morrison et al. 975; B. B. Spanier, C. W. Clinkscales, M. A. Bratt & T. G. Morrison, unpublished observations). Based on their kinetics of labelling (Bratt & Robinson, 97~ ) and the reconstruction experiment in Fig. 5, the partially base-paired RNAs presumably represent intermediates that exist in the infected cell. The amount of base-pairing that we measure after extraction and preparation of the RNA may be an exaggeration of what is actually present in the native structures in the infected cell (Clavell & Bratt, 197i; Oberg & Philipson, 971), but does not seem to represent artifactual annealing of completely single-stranded RNA. The partially base-paired structures in the 5oS region (representing about 75 % of the NDV-specific radioactivity of that region) migrate in gels heterogeneously and more slowly than singe-stranded 5oS RNA. This is consistent with a theoretical conformation ofpartialy base-paired intermediates with single-stranded tails which impede migration. t is possible that a variable number and random conformation of the tails causes the broad, heterogeneous spread of a single structure in the gels; alternatively, the heterogeneity could represent several different structures. The partially base-paired structure in the 35S region migrates more slowly than the singlestranded 35S species. t appears as a minor peak (about 25 ~o of total NDV-specific radioactivity in the 35S region) next to the major species, but can be clearly separated from the single-stranded 35S species in '8 % acrylamide gels (Fig. 6a). Kaverin & Varich (1974) also observed a pattern of minor and major peaks of 35S RNA in gels, but did not characterize them further. The partial separation by gel electrophoresis of the single-stranded and partially basepaired RNAs in the 5oS and 35S regions provides a method for the analysis of the relative proportions of these RNAs with different strains and under different conditions (length of

13 NDV: 5oS and 35S RNAs 451 labelling time, time after infection, etc.), as well as the analysis of RNA synthesis in the absence of replication as described here. The similarities among the in vitro transcriptive intermediate, the 5oS region from cycloheximide-treated infected cells, and intracellular 5oS RNA suggest that the 4o to 6oS region contains transcriptive intermediates which migrate in a heterogeneous pattern in gels. [t should be pointed out that although the gel pattern of the in vitro intermediate is very similar to that of 5oS intracellular RNA, complete transcription has not been detected in the in vitro polymerase reaction. Complete molecules of 35S RNA are not detected and some of the 18 to z2s RNAs may be missing (Weiss, 1976), although, like the 18 to 2zS RNA from infected cells, this RNA corresponds to 60 ~ of the genome (Spanier-Collins, 975).] The effect of cycloheximide (a) to decrease the ribonuclease resistance in the 35S region from zo ~ to 5 ~o (Spanier-Collins, 975) and (b) to eliminate the more slowly migrating minor component in the 35S region, plus the lack of a 35S intermediate in the in vitro polymerase reaction, suggests that the partially base-paired minor component of the 35S region might play a role in replication. However, one would expect the generation of complete 5oS strands (albeit a small amount) after denaturing a replicative intermediate. We have observed this only once in our heating experiments, when a very large quantity of 35S RNA was heated (B. B. Spanier, unpublished observations). A similar problem, perhaps due to the small number of complete 4oS strands and their fragmentation during heating, was reported in the vesicular stomatitis virus (VSV) system (Schincariol & Howatson, 1972 ). Therefore, it is unclear whether the 35S intermediate is involved in transcription or replication. Annealing experiments after purification of the 35S structure may resolve that question. Another inconsistency which is unexplained is that the decrease in labelling of the 5oS region caused by cycloheximide is greater than the proportion of single-stranded 5oS RNA in the total 5oS region. Thus, the inhibition of virion 5oS RNA synthesis may not, alone, account for the effect of cycloheximide on the 5oS region. Analysis of this is impeded because cycloheximide stimulates incorporation of 3H-uridine into single-stranded 35S and 18 to zzs RNA (Weiss & Bratt, 1975 ; Spanier-Collins, 1975). Studies of the effect of cycloheximide on absolute incorporation of 3H-uridine into single-stranded and base-paired RNA would give a clearer indication of the quantitative effect of cycloheximide. Earlier annealing studies on NDV reported that when RNA is labelled for a long period, 9 ~ of the 35S RNA is complementary to virion RNA (Bratt & Robinson, 967). Conversely, 35S RNA annealed to 70 ~ ofvirion RNA, while 18 to 22S RNA annealed to 5o % ofvirion RNA. Together, the 35S and 8 to 22S RNA annealed to 7 ~o of the virion RNA, suggesting some overlap of sequences. n the light of our results, the ambiguity in the former experiment can probably be attributed to: (a) cross-contamination of 35S and 18 to 22S RNA in sucrose gradients, and (b) the presence of both single-stranded 35S RNA and a 35S intermediate containing strands of both polarities. Sendal virus also synthesizes 5oS, 35S and 8S RNA in infected cells (Blair & Robinson, 1968). ts ~8S RNA can function as messenger RNA in a cell-free translation system (Kingsbury, 1973). Roux & Kolakofsky (1975) have also recently demonstrated a singlestranded 33S RNA in Sendal virus-infected cells. They have also provided evidence suggesting that some Sendal virus 8S RNA also sediments as an aggregate at 33S. The presence of similar aggregates of 8S RNA in NDV-infected cells might account for the generation of more 8S RNA than expected from the heating of total 35S RNA (see Fig. 3c, d); the decrease in the minor 35S peak after heating does not appear to be sufficient to generate all the RNA smaller than 35S. 3o-2

14 452 B.B. SPANER AND M. A. BRATT One significant difference between the two viruses is found in the partially base-paired RNAs. Portner & Kingsbury 0972) demonstrated that Sendai virus RNA intermediates sedimenting from 28S to 6oS appear to be involved in transcription, while a homogeneous 24S RNA complex appears to be involved in replication. We do not find a 24S partially base-paired structure in the NDV system, although the 35S structure we detect may prove to be analogous. f so, it will be of interest to determine the basis for the difference in sedimentation of these structures. We gratefully acknowledge the technical assistance of Eivor Houri and Reed Kotler; helpful discussions with Dr Trudy Morrison, Dr C. Worth Clinkscales, Dr Susan Weiss, and Charles Madansky; the help of Denise Brouillette and Patricia Theriault in the preparation of the manuscript; the National Science Foundation for research grant GB-3o595X and the National nstitute of Allergy and nfectious Disease for research grant A ~2467, under which this study was conducted and the National nstitute of Allergy and nfectious Disease for the training grant A-oo387 under which B.B.S. was a predoctoral trainee. REFERENCES BSHOP, D. H. L., CLAYBROOK, J. a. & SPEGELMAN, S. (967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. Journal of Molecular Biology, 26, BLAR, C. D. & ROBNSON, W. S. (1968). Replication of Sendai virus.. Comparison of the viral RNA and virusspecific RNA synthesis with Newcastle disease virus. Virology 35, BRATT, U. A. (969). RNA synthesis in chick embryo ceils infected with different strains of NDV. Virology 38, BRATT, M. A. & 6ALLAHER, W. R. (1969). Preliminary analysis of the requirements for fusion from within and fusion from without by Newcastle disease virus. Proceedings of the National Academy of Sciences of the United States of America 64, BRATX, M. A. & ROBNSON, W. S. (967). Ribonucleic acid synthesis in ceils infected with Newcastle disease virus. Journal of Molecular Biology 23, -2. BRATT, M. n. & ROBNSON, W. S. (197). Evidence for an RNA intermediate in cells infected with Newcastle disease virus. Journal of General Virology io, CLAVELL, L.A. & BRATT, M.A. 097). Relationship between the ribonucleic acid-synthesizing capacity of ultraviolet-irradiated Newcastle disease virus and its ability to induce interferon. Journal of Virology 8, COLLNS, B. S. & BRATT, M. A. (973). Separation of the messenger RNAs of Newcastle disease virus by gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America 70, m~htower, L.n. & BRATT, M.A. (974). Protein synthesis in Newcastle disease virus-infected chicken embryo cells. Journal of Virology x3, 788-8oo. H~HTOWER, L. E., MORRSON, T. ~. & BRATT, M. a. 0975). Relationships among the polypeptides of Newcastle disease virus. Journal of Virology x6, 599 6O7. ~JtrAN~, A. S., BALTMORE, D. & BRATT, M.g. (197). Ribonucleic acid polymerase in virions of Newcastle disease virus: comparison with the vesicular stomatitis virus polymerase. Journal of Virology 7, ~AVERN, N. V. & VARCH, N. L. (974). Newcastle disease virus-specific RNA: polyacrylamide gel analysis of single-stranded RNA and hybrid duplexes. Journal of Virology r3, o. ~NCSBURV, O. W. (1966). Newcastle disease virus RNA.. Preferential synthesis of RNA complementary to parental viral RNA by chick embryo cells. Journal of Molecular Biology x8, 2o KNGSBURV, D. W. (973). Cell-free translation of paramyxovirus messenger RNA. Journal of Virology xz, lo2o-o27. J~OLAKOFSKV, D., BOY DE LA TOUR, E. & BRUSCH, a. (974). Self-annealing of Sendai virus RNA. Journal of Virology x4, KOLA~OFSKV, D., BOY DE Lh XOUR, E. & DELUS, n. (974). Molecular weight determination of Sendai and Newcastle disease virus RNA. Journal of Virology r3, MORRSON, T. G., WESS, S., HGHTOWER, L. E., SPANER-COLLNS, B. & BRATT, M. A. (975). Newcastle disease virus protein synthesis. nstitut National de la Sant~ et de la Recherche Mddicale 47, o. 6BERG, B. & ~'HLH'SON, L. 097)- Replicative structures of poliovirus RNA in vivo. Journal of Molecular Biology 58,

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