JOURNAL OF VIROLOGY, Oct. 1978, P. 324-336

Vol. 28, No. 1

0022-538X/78/0028-0324$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Transcription and Translation of Newcastle Disease Virus mRNA's In Vitro PETER L. COLLINS,* LAWRENCE E. HIGHTOWER, AND L. ANDREW BALL Microbiology Section, Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268

Received for publication 6 April 1978

Transcription directed in vitro by Triton-activated Newcastle disease virus (NDV) was stimulated and prolonged by the presence of cytoplasmic extracts of animal cells. The RNA products closely resembled those of NDV transcription in vivo by several criteria: binding to oligodeoxythymidylic acid-cellulose, the mobility and relative abundance of each major band resolved by polyacrylamide gel electrophoresis, and the ability to direct the accurate cell-free synthesis of polypeptides corresponding to the NDV proteins HN, Fo/Fl, NP, and M. Synthesis of a novel polypeptide related to NP but of higher apparent molecular weight was also detected. These results indicated that cell-free transcription under these conditions was a close facsimile of NDV transcription in vivo. In addition, both in vitro and in vivo, NDV polypeptides were synthesized in nonequimolar amounts which reflected the order of the genes in the transcriptional map: NP, Fo, M, (47K, HN), L. Strains AV and HP, virulent strains which have differences in biological activities, exhibited differences in the polypeptides synthesized in infected cells and in cell-free systems.

Newcastle disease virus (NDV) is an avian paramyxovirus (11). Its genome consists of a single negative strand (3) of RNA with a molecular weight of approximately 5.4 x 106 (25). Six unique NDV-specific proteins have been described (23). Three are associated with the viral envelope (41): the larger glycoprotein, HN, has hemagglutinin and neuraminidase activities. The smaller glycoprotein, F, has cell-fusion activity and consists of two disulfide-linked polypeptide chains, F1 and F2 (40), generated by proteolytic processing of a larger precursor, Fo (23, 34). A nonglycosylated protein, M, is a structural component of the inner surface of the envelope. Two unique polypeptides are associated with the ribonucleoprotein core: the nucleocapsid protein NP and the large protein L (16). The other known unique protein, 47K, is a minor structural protein and has not been well characterized. The combined polypeptide molecular weights of these proteins account for about 90% of the coding capacity of the NDV genome. The products of NDV transcription in vivo are polyadenylated RNAs that are complementary to the genome and have sedimentation coefficients of 35S, 22S, and 18S (8). Under denaturing conditions, most of the 35S and 22S RNA sediments at 18S, suggesting that aggregates or conformational variants of 18S species cosediment with these larger classes (43, 47). A small amount of 22S RNA is resistant to denaturation 324

(47), but it has not been shown to contain any unique species. The 35S RNA has been translated in vitro to yield products that included the L protein (33), indicating that this class contains a unique, large mRNA species. The 18S RNA has been resolved into five distinct bands by polyacrylamide gel electrophoresis (6, 14, 47) and has been translated in vitro to yield products corresponding to HN, Fo, NP, 47K, and M (13, 33). The 6 distinct species of RNA contained in the 35S and 18S classes agree in number and coding capacities with the known unique NDV polypeptides. The 18 to 22S RNAs anneal to about 60% of the NDV genome (8, 37), and on the basis of its size the 35S mRNA could account for the remaining 40% of the genome. Similarly, the transcription products of Sendai virus, a closely related paramyxovirus, sediment as 33S, 22S, and 18S classes. The 22S RNA is sensitive to denaturation, and the 33S and 18S RNAs together anneal to the entire Sendai genome (38). From these results, it is apparent that most of the unique mRNA and protein species of NDV have been identified. In vitro, a virion-associated RNA nucleotidyl transferase (EC 2.7.7.6) can be activated by nonionic detergent (24) or detergent and high-salt treatment (16) to synthesize RNA that is 22S or less in size and which resembles intracellular NDV mRNA in being complementary to the genome, polyadenylated, capped, and methylated (6, 14-17, 24, 46). Although the NDV RNA

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325

made in vitro hybridized to as much as 49% of min, preincubation at 37°C for 45 min, and Sephadex the genome, its translation in cell-free systems G-25 gel filtration (2, 19). Preparation of NDV RNA in vitro. Reaction yielded only NP and NP fragments (B. SpanierCollins, Ph.D. thesis, Harvard University, Cam- mixtures for transcription of NDV RNA contained: 30 Tris-hydrochloride (pH 7.6), 33 mM NH4Cl, 7 bridge, Mass., 1975; S. R. Weiss, Ph.D. thesis, mM mM KCI, 50 mM NaCl, 4 mM magnesium acetate, 1 Harvard University, Cambridge, Mass., 1975), mM dithiothreitol, 0.2 mM spermidine, 1 mM ATP, and there was no evidence that additional com- 0.5 mM GTP and CTP, 0.1 mM UTP, 10 mM creatine plete transcripts were synthesized by the reac- phosphate, 80 ug of creatine kinase per ml, 0.4% Triton tion in vitro. Similarly, characterization of the N-101, 100 to 200 ,kg of viral protein per ml, and 30% RNAs transcribed in vitro by parainfluenza virus (vol/vol) cell extracts (to give a final concentration of type 5 (simian virus 5), another paramyxovirus, about 1.5 mg of protein per ml). The RNA used in this suggested that a region of the genome equivalent work was rountinely synthesized in the presence of to about one gene was expressed during a 4-h mouse L-cell extracts. Incubation was at 30°C for 8 h. The RNA products were labeled by including reaction (9). [5,6-3H]UTP (Amersham Corp.) in the reaction mixHere we report that transcription by deter- ture to a final specific activity of 0.98 Ci/mmol. Isogent-activated NDV was stimulated and pro- topic incorporation was monitored as described before longed when cell extracts were included in the (1). reaction mixture. Analyses of the products sugExtraction of RNA made in vitro. To isolate the gested that the reaction closely resembles NDV total RNA product for polyacrylamide gel analysis, transcription in infected cells and that the mech- the transcription reaction mixture was adjusted to 1 anism that controls NDV RNA synthesis in vivo mg/ml in proteinase K (EM Laboratories) and to 0.1 M NaCl, 0.01 M Tris-hydrochloride (pH 8.8), 2 mM also regulates the process in vitro. EDTA, 0.5% sodium dodecyl sulfate (SDS), and 1% 2(Some of these results were presented in pre- mercaptoethanol buffer [46]). After inliminary form at the Third Cambridge Confer- cubation at room (solubilizing temperature for 45 min, the RNA ence on Virology held in Cambridge, England, was extracted with phenol-chloroform-isoamyl alcohol in August 1977.) (13) and concentrated by precipitation in 70% ethanol and 0.1 M sodium acetate at -20°C. To obtain a MATERALS AND METHODS preparation of polyadenylic acid [poly(A)]-containing Virus preparation and assay. NDV strains AV RNA for translation in vitro, the reaction mixture was (Australia-Victoria, 1932) and HP (Israel-HP, 1935) adjusted to 0.4 M NaCl and 0.1% SDS and passed were grown in embryonated eggs as previously de- through a column of oligodeoxythymidylic acid scribed (21) except that the virus was concentrated by [oligo(dT)]cellulose (Collaborative Research, type 3, centrifugation of allantoic fluids at 18,000 rpm in a 0.2 g per 1-ml sample). The RNA that remained bound Spinco type 19 rotor for 2 h at 4°C. Standard prepa- during washes of binding buffer (0.4 M NaCl, 10 mM rations contained 1 x 1010 to 3 x 1010 PFU/ml and 1 Tris-hydrochloride [pH 7.6], 0.02% SDS) and eluted in mg of protein per ml. Virus used for transcription in a low-salt buffer (10 mM Tris-hydrochloride [pH 7.6], 0.1 vitro was further purified: the virus suspension was mM EDTA, 0.02% SDS) was enriched for transcripts dialyzed against 300 volumes of 100 mM NaCl, 10 mM Tris-hydrochloride (pH 7.6), and 1 mM EDTA (NTE) for 2 h at 4°C and subjected to centrifugation in linear gradients of 20 to 65% sucrose in NTE in a Spinco SW27.1 rotor at 24,000 rpm for 17 h at 4°C. The virus band was harvested, diluted fivefold with NTE, pelleted, resuspended in a small volume of NTE, and stored at -70°C. The transcriptase activity was stable for several months. Plaque assays were performed on secondary cultures of chick embryo cells incubated at 40°C with an overlay of 0.8% agarose in NCI medium (GIBCO) containing 2% calf serum. Cell culture. Secondary cultures of chick embryo cells were made from 2-day-old primary cultures that had been prepared as previously described (10). Cultures were seeded at 1.0 x 106 cells per 60-mm plate in NCI medium containing 6% calf serum. Cells were confluent after incubation for 2 days at 37°C and were promptly used. Cell extracts. Cytoplasmic extracts for use in both cell-free transcription and translation reactions were prepared from mouse L-929 cells grown in suspension culture or from aged primary cultures of chick embryo cells. The major steps in the preparation were: Dounce homogenization, centrifugation at 10,000 x g for 10

that had been completed and polyadenylated (4, 31). The RNA preparation was concentrated by ethanol precipitation. Preparation of NDV RNA from infected cells. Secondary cultures of chick embryo cells were infected at a multiplicity of 5 PFU/cell. Beginning with an adsorption period of 45 min, the cells were incubated at 40°C in Eagle minimal essential medium (MEM). At 3.25 h postinfection, 5 ug of actinomycin D/ml (Sigma Chemical Co.) was added; at 4 h, 100 MCi of [3H]uridine/ml (Amersham Corp.; specific activity, 39 Ci/mmol) was added; and at 9 h, the cells were lysed in cold solubilizing buffer. The lysate was then incubated with proteinase K, and the RNA was extracted with phenol-chloroform-isoamyl alcohol and concentrated by ethanol precipitation. Alternatively, to obtain an mRNA preparation for translation in vitro, cells were lysed in cold solubilizing buffer lacking SDS but containing 1% Triton N-101 and 0.5% sodium deoxycholate. This procedure minimized solubilization of nuclei from the plate. Once removed from the plate, the lysate was adjusted to 0.4 M NaCl and 0.5% SDS, and the RNA was isolated by oligo(dT) cellulose chromatography and concentrated by ethanol precipitation. Eighty-five percent of the radioactive RNA ex-

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tracted from NDV AV-infected cells bound to the oligo(dT) cellulose column and could be recovered in low-salt buffer. Cell-free translation of NDV mRNA. Reaction mixtures for translation of NDV mRNA contained: 26 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-hydrochloride (pH 7.6); 33 mM NH4Cl; 27 mM KCI; 3 mM magnesium acetate; 1 mM dithiothreitol; 0.2 mM spermidine; 1 mM ATP; 0.5 mM GTP, CTP, and UTP; 10 mM creatine phosphate; 80 Ag of creatine kinase per ml; 50 ,iM amino acids except methionine; 10 jiM S-adenosyl methionine; and 30% (vol/vol) cell extracts. NDV mRNA translation was optimal under these conditions. mRNA made in vitro was added to a concentration of about 2 to 3 jig/mi. At these concentrations, amino acid incorporation by the system was proportional to input NDV mRNA. Incubation was at 30°C for 2 h. Proteins were labeled by including [35S]methionine (Amersham Corp.; specific activity, about 400 Ci/mmol) at a concentration of 1 jiM in the reaction mixture. Isotopic incorporation was monitored as described elsewhere (2). The reaction was stopped by the addition of 1 volume of two times concentrated SDS-polyacrylamide gel electrophoresis sample buffer (0.125 M Tris-hydrochloride [pH 6.8], 20% glycerol, 10% 2-mercaptoethanol, 4.6% SDS

[261).

Preparation of NDV polypeptides from infected cells. Secondary cultures of chick embryo cells were infected at a multiplicity of 5 PFU/cell as described above. At 6 h postinfection, the cells were washed well with MEM lacking methionine and incubated for 30 min at 40°C in MEM containing 1% (0.15 mg/liter) of the normal methionine content. [35S]methionine was included at a concentration of 30 to 50 jiCi/ml. The cells were then solubilized directly in polyacrylamide gel electrophoresis sample buffer. Polyacrylamide gel electrophoresis: RNA. RNA was analyzed by electrophoresis in 2% polyacrylamide-0.6% agarose slab gels (14 by 17 by 0.15 cm) containing 6 M urea according to the procedure of Floyd et al. (18). This gel was polymerized on top of a 2-cm support of 9% polyacrylamide gel. RNA samples for electrophoresis were dissolved in 36 mM Tris-phosphate (pH 7.8)-i mM EDTA, containing 10% glycerol and 70% formamide. Immediately prior to loading, the samples were heated to 70°C for 2 min and cooled quickly to 0°C. Electrophoresis was at 130 V for 8 h at 4'C or at 100 V for 7 h at room temperature, as indicated. Gels were fixed with 10% acetic acid, stained with toluidine blue, destained, prepared for fluorography, and exposed to Kodak XR-5 X-ray film that had been presensitized to linearize response (5, 29). The resulting fluorograms were scanned with a Joyce Loebl Chromoscan densitometer, and the relative amount of radioactivity in each peak was determined by measuring the area under the peak. Polyacrylamide gel electrophoresis: proteins. Proteins were analyzed by electrophoresis in 11.5% polyacrylamide slab gels by the procedure of Laemmli (26). Electrophoresis was for 17 h at a constant current of 17.5 mA per gel. The fixed dried gels were analyzed by autoradiography using Kodak XR-5 X-ray fim and were quantitated by densitometer scanning. Tryptic peptide analysis. The procedure of two-

J. VIROL.

dimensional analysis of tryptic digests of polypeptides labeled with [35S]methionine has been described before (2). The digests were applied to 18 by 18 cm sheets of glass-fiber paper impregnated with silica gel (Gelman ITLC sheets, type SA). Separation in the first (horizontal) dimension was by electrophoresis at pH 6.5 at 300 V for 3 h. Separation in the second (vertical) dimension was by ascending chromatography in butanol-acetic acid-water (3:1:1) for 2.5 h. The dried sheets were analyzed by autoradiography using Kodak XR-5 X-ray fllm.

RESULTS NDV AVtranscription in vitro. Detergentactivated NDV synthesized about 0.2 Mg of RNA/100 jig of viral protein in a 1-ml reaction mixture lacking cell extracts. Under these conditions, transcription continued for 4 to 6 h. When cell-free extracts of mouse L-cells or primary chick embryo cells, which had no detectable endogenous RNA synthetic activity, were included in the reaction mixture, the viral transcriptase was stimulated 30- to 50-fold. The reaction continued for more than 8 h, resulting in the synthesis of 7.5 Mg of RNA/100 Mg of viral protein in a 1-ml reaction mixture (Fig. 1). The concentration of genome RNA in the reaction mixture was about 1.3 ,ig/ml, assuming a virion RNA content of 0.92% (wt/wt) (35), so the RNA products were synthesized in at least fivefold excess over the template. The ability of cell extracts to stimulate and prolong vesicular stomatitis virus transcription in vitro and to protect the transcriptase products has been described elsewhere (2). Although the basis of these effects remains unclear, they were

8

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FIG. 1. RNA transcription in vitro executed by detergent-activated NDV strain AV in the presence (0) or absence (-) of a mouse L-cell extract in the reaction mixture. Synthesis of RNA was monitored by the incorporation of label from [3H]UTP, and based on its specific activity, this was expressed in micrograms of RNA per 1 ml of reaction mixture.

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not related to the protein synthetic activity of these extracts since the same effects were observed in the presence of cycloheximide (50 ,ug/ ml) and postribosomal supematant fluids of cytoplasmic extracts. In the presence of cell extracts, both NDV transcription and polyadenylation of transcripts were largely insensitive to changes in the concentrations of magnesium acetate (3.0 to 5.0 mM), Triton N-101 (0.05 to 0.50%), or monovalent cations (40 to 120 mM).

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Analysis of NDV AV RNA by polyacryl6 * amide gel electrophoresis. The following samples were subjected to electrophoresis at 4°C in polyacrylamide-agarose gels containing 6 M 5 urea: NDV RNA extracted from infected cells 4 (Fig. 2, channels a and d), total NDV RNA made -18s in vitro (Fig. 2, channel b), and the fraction of 23!ii3 NDV RNA made in vitro that bound to oligo(dT)-cellulose (Fig. 2, channel c). The major bands have been designated 1 to 8 in order of decreasing electrophoretic mobility. Bands 1 to 5 represent the five distinct species of 18S NDV mRNA. This is the most abundant class of viral RNA extracted from infected cells and was the major product of the reaction in vitro. On the basis of their electrophoretic mobilities and relative abundances, bands 1 to 5 of the RNA transcribed in vitro were indistinguishable from those made in vivo. Bands 6 and 7, representing the 22S RNA, were present in larger amounts in the product made in vitro than in NDV RNA FIG. 2. Comparison by polyacrylamide gel electroextracted from infected cells. In addition, a small phoresis of NDV RNA made in vitro or extracted and variable amount of the NDV RNA made in from infected cells. The following samples, prepared vitro comigrated with authentic 35S RNA (band as described in Materials and Methods, were sub8). Occasionally, the gel profile of the RNA made jected to electrophoresis at 4°C in a 2% polyacrylagarose slab gel containing 6 M urea: (a in vitro contained minor bands smaller than amide-O.6% and d) RNA extracted from actinomycin D-treated band 1. These smaller species often did not bind NDV-infected cells (b) total NDV RNA transcribed to oligo(dT)-cellulose columns. The fraction of in vitro, (c)poly(A)-containingNDVRNA transcribed NDV RNA made in vitro that bound to in vitro, (e) RNA extracted from actinomycin Doligo(dT)-cellulose varied from 55 to 85%, some- treated uninfected chick cells. The major NDV bands what less than the 80 to 90% binding that has have been numbered to 8 in order of increasing been reported for the 18 to 22S NDV RNAs molecular weight, and the positions of 28S and 18S extracted from infected cells (47). All eight dis- L-cell rRNA markers run in adjacent channels are tinct species made in vitro were represented in shown. Viral RNA bands 3 and 4 were not resolved the same relative proportions in the poly(A)- under these conditions. A fluorogram of the dried gel is shown. containing fraction. NDV RNA extracted from infected cells, and the total product synthesized in vitro, were also this band of NDV RNA extracted from infected subjected to electrophoresis in aqueous poly- cells contains two distinct species (L. A. Ball, P. acrylamide-agarose-urea gels at room tempera- L. Collins, and L. E. Hightower, in B. W. J. ture (Fig. 3a and b, respectively). Under these Mahy and R. D. Barry, ed., Negative Strand -L

1

conditions, NDV RNA bands 3 and 4 could be clearly resolved, showing that the mobilities and relative abundances of RNAs 3 and 4 were the same whether made in vitro or extracted from infected cells. Furthermore, RNA band 2 was partially resolved into a doublet, and this is in agreement with our previous observation that

Viruses and the Host Cell, in press). However, distinct bands corresponding to the expected electrophoretic mobilities of RNAs 6 to 8 were detected under these conditions. The products of NDV transcription in vitro were also subjected to electrophoresis in polyacrylamide gels containing 6 M urea in 98% no

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Under standard conditions, chick cell-free systems directed by NDV mRNA that had been transcribed in vitro incorporated 0.025 nmol of methionine per ,ug of mRNA. This corresponds to about 0.05 to 0.10 ,ug of protein synthesized per ug of mRNA. This latter calculation was based on the assumption that the methionine content of the NP protein is representative of the total translation product and is based on published amino acid analyses of the NP protein of NDV strains Ulster, Texas, and Beaudette

(32). t

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FIG. 3. Comparison by polyacrylamide gel electrophoresis of NDV-AVRNA made in vitro or extracted from infected cells. (a) NDV RNA extracted from infected cells and (b) the total product of NDV transcription in vitro were subjected to electrophoresis at room temperature in a 2% polyacrylamide-0.6% agarose slab gel containing 6 M urea. NDV RNA bands I to 5 are labeled, and the positions of internal 28S and 18S L-cell rRNA markers are shown. Densitometer tracings of fluorograms of the dried gels are shown.

formamide (45). The resulting gel profile (not shown) contained eight bands that resembled in electrophoretic mobilities and relative amounts the eight RNA bands resolved by electrophoresis under nondenaturing conditions. Furthermore, the pattem was strikingly similar to published profiles of NDV RNA extracted from infected cells and analyzed by polyacrylamide gel electrophoresis in fornamide (47). Cell-free translation of NDV AV mRNA made in vitro. The addition of NDV mRNA synthesized in vitro to a chick cell-free system under standard conditions stimulated the incorporation of [35S]methionine 20- to 50-fold above endogenous levels during a 2-h reaction.

NDV polypeptides extracted from infected cells were analyzed by discontinuous SDS-polyacrylamide slab gel electrophoresis in conjunction with autoradiography (Fig. 4, channel c), resulting in a higher degree of resolution than had been previously obtained (21-23, 30, 39). The most prominent bands of the polypeptides extracted from chick embryo cells 6 h after infection with NDV are the viral proteins: L, HN, Fo, NP, M, and F1, the larger subunit of the glycoprotein F (40). An additional virus-specific polypeptide having an apparent molecular weight in our gel system of 53,000 (P53) was sometimes found. Peptide analysis of P53 showed that it was not related to any of the other major NDV proteins (not shown). Therefore, it may correspond to the unique viral protein that had been previously designated 47K, rather than to the 53,000-dalton NP fragment that had been designated NP1 (23). The major products of chick cell-free systems directed by NDV RNA made in vitro (Fig. 4, channel b) included polypeptides that corresponded in mobility to authentic NP, F1, and M. Although no major polypeptides synthesized in vitro comigrated with L, HN, or Fo, there were additional products having apparent molecular weights of 59,000 (P59) and 67,000 (P67). In addition, the gel pattem contained a continuum of minor virus-specific species having molecular weights of less than 54,000, some of which were identified by tryptic peptide analysis as being F1 and NP fragments (not shown). In the absence of added messenger, chick cellfree systems synthesized small amounts of endogenous polypeptides (Fig. 4, channel a). Messenger preparations isolated from transcription reaction mixtures that lacked NDV slightly stimulated amino acid incorporation. We attribute this effect to mRNA species contributed by the cytoplasmic extracts present during transcription in vitro. However, no new discrete polypeptides were detectable by gel electrophoresis under standard conditions of analysis (not shown). Cell-free translation of NDV AV mRNA

NDV TRANSCRIPTION AND TRANSLATION IN VITRO

VOL. 28, 1978

b

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67

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(Fig. 5, channel b). The resulting gel profile was indistinguishable from the pattern of products made in response to NDV mRNA that had been synthesized in vitro. Tryptic peptide analysis. The relationships between NDV polypeptides synthesized in chick cell-free systems and those extracted from infected chick embryo cells were investigated by tryptic peptide analysis. The cell-free system products analyzed were those synthesized in response to NDV mRNA that had been made in vitro. Tryptic digests were subjected to two-dimensional mapping, and the methionine-containing peptides were visualized by autoradiography (Fig. 6). The maps of P67 and HN were indistinguishable, establishing that these species were related. The cell-free systemi product that comigrated

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HN FIG. 4. SDS-polyacrylamide gel electrophoresis of the translation products directed by ND V-AVmRNA made in vitro. The polypeptides synthesized by a chick cell-free system (a) in the absence of added messenger and (b) directed by NDV mRNA made in vitro were subjected to electrophoresis in parallel with extracts from (c) NDV A V-infected and (d) uninfected chick cells. Polypeptides were labeled with [3S]methionine as described in Materials and Methods, and an autoradiogram of the dried gel is shown. The major NDVpolypeptides are marked.

extracted from infected cells. As shown in Fig. 4, comparison of the viral polypeptides synthesized in vitro and those extracted from infected cells demonstrated clear differences. Therefore, despite the close similarity between the gel profiles of NDV RNA made in vitro and in vivo (Fig. 2 and 3), it was possible that these polypeptide dissimilarities reflected functional defects in the mRNA's synthesized by the cellfree transcription reaction. To test this possibility, authentic NDV mRNA was extracted from infected cells and used to direct protein synthesis in chick cell-free systems. The products were analyzed by polyacrylamide gel electrophoresis

P67

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FIG. 5. Analysis bypolyacrylamide gel electrophoresis of the translation products directed by NDVAV mRNA extracted from infected cells. The polypeptides synthesized by chick cell-free systems in response to mRNA extracted from (a) uninfected and (b) NDVinfected chick embryo cells were subjected to electrophoresis in parallel with extracts from (c) NDV-infected and (d) uninfected chick embryo cells. Polypeptides were labeled with [3S]methionine, and an autoradiogram of the dried gel is shown. The major NDVpolypeptides are marked.

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FIG. 6. Two-dimensional tryptic peptide analysis of NDV polypeptides synthesized by a chick cell-free system directed by NDVAVmRNA made in vitro and of NDVAVpolypeptides extracted from infected cells. [35S]methionine-labeled tryptic digests were spotted at bottom center (0). Resolution in the first dimension (horizontal) was by electrophoresis at pH 6.5 (the anode was to the right) and in the second dimension

(vertical) by ascending chromatography. All maps dried sheets are shown.

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with NP also corresponded by peptide mapping, although the maps contained differences. Mapping of the M-sized band of the polypeptides

directly comparable in scale. Autoradiograms of the

made in vitro showed that this gel band contained a species corresponding to M, but also contained an NP fragment.

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; Surprisingly, the maps of Fo and F, from infected cells did not correspond closely. This was also observed when peptide maps were generated by limited proteolysis in SDS by Staphylococcus aureus protease in conjunction with gel electrophoresis, by the procedure of Cleveland et al. (12) (not shown). A possible explanation is presented in the Discussion. When mapped by both procedures, the cell-free system product that comigrated with F1 corresponded more closely with F1 than with Fo or any other viral polypeptide. The map of P59 resembled that of NP, but also contained similarities with the map of the Frelated cell-free system product. However, mapping by the procedure of Cleveland et al. established that P59 was related to NP and not to any of the other viral polypeptides (not shown). Cell-free translation of mRNA made in vitro by NDV strain HP. NDV strains A V and HP are virulent strains that differ in some biological properties, particularly in cell-fusion activity (7). The SDS-polyacrylamide gel patterns of labeled polypeptides extracted from chick em-

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bryo cells infected with strains A V and HP (Fig. 7, channels h and g, respectively) were found to have clear differences. In particular, the F1 proteins of strains AV and HP did not comigrate. Under the conditions of electrophoresis shown in Fig. 7, the F1 protein of strain HP was not resolved from the NP protein. However, when subjected to electrophoresis in 8.5% SDS-polyacrylamide gels, F1 migrated slightly slower than NP (not shown). This difference was repeated in the gel pattern of polypeptides synthesized in chick cell-free systems in response to NDV HP and AVmRNA made in vitro (Fig. 7, channels e and f, respectively). Under the conditions shown in Fig. 7, the gel pattern of HP polypeptides synthesized in vitro also contained a single band in the NP/ F1 region. However, NP and F1 proteins of strain HP synthesized in vitro, like their counterparts extracted from infected cells, could be resolved by electrophoresis in 8.5% gels (not shown). There was an additional difference between the strains: the products of cell-free systems directed by NDV HP mRNA did not contain detectable

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COLLINS, HIGHTOWER, AND BALL

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FIG. 7. Comparison bypolyacrylamide gel electrophoresis of NDVAV and HPpolypeptides synthesized in vitro with those extracted from infected cells. The gel channels contain: the products synthesized by an L-cell cell-free system (a) without added messenger, (b and c) in response to mRNA that had been transcribed in vitro by NDV HP and NDV AV, and the products synthesized by a chick cell-free system (d) without added messenger, and (e and t) in response to mRNA that had been transcribed in vitro by NDVHP and NDVAV. Extracts from chick embryo cells that had been infected with (g) NDV HP and (h) NDVAV, or that were (i) uninfected, were subjected to electrophoresis in parallel. Polypeptides were labeled with [35S]methionine, and an autoradiogram is shown. amounts of a polypeptide comigrating with the A V P59 polypeptide. Cell-free systems were also prepared from mouse L-cells and were programmed with NDV HP and A V mRNA preparations that had been made in vitro (Fig. 7, channels b and c, respectively). The NDV polypeptides synthesized by the two systems corresponded in electrophoretic mobility and relative amount. In particular, the P59 polypeptide of strain AV was synthesized in both systems. The similarity in the responses of the two cell-free systems is in accordance with the observation that, although chick cells support productive NDV infection and L-cells do not, the block in NDV reproduction in L-cells occurs late in maturation (44). Relative molar amounts of NDVAV poly-

peptides. The distribution of radioactivity among the NDV polypeptides from infected cells was determined by quantitation of polyacrylamide gel patterns similar to those shown in Fig. 4, 5, and 7. The tryptic peptide analyses suggest that none of the NDV polypeptides contains disproportionate amounts of methionine, and this is further supported by the observation that the distribution of radioactivity among the methionine-labeled polypeptides agrees closely with figures previously reported for NDV polypeptides labeled under similar conditions with a 14C-amino acid mixture (22). Therefore, the distribution of [35S]methionine can be used to calculate approximate molar ratios among the NDV polypeptides (Table 1). In the same way, the relative molar amounts

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TABLE 1. Distribution of radioactivity among ND V-A Vpolypeptides extracted from infected cells or synthesized in vitro Extracts from infected cells Polypeptides

% Total major viral

Cell-free systems

Polypeptides Molar ratiob

% Total major viral

Molar ratio' bandsa bands' NP 43.5 100 NP-related (NP + P59) 62.5 100 F-related (Fo + F1) 27 65c F-related (F,-sized species) 18.5 31 M M 13 41 26 12d HN 11.5 22e HN-related (P67) 7 9 L 5 3 a Determined by weight of areas under peaks of densitometer tracings of gels such as shown in Fig. 4, 5, and 7. The values for polypeptides from infected cells are based on quantitation of 10 gels and are in close agreement with published results (21, 22). The values for cell-free system products are based on quantitation of seven gels. 'Molar ratios were determined by relating amount of a polypeptide to its molecular weight and expressing the result in terms of 100 equivalents of NP. The molecular weights of NDV polypeptides have been published (21, 22). c For this calculation the molecular weights of the polypeptide portions of Fo and F1 were assumed to be 54,000. This is the apparent size of F, and the F-related polypeptide made in vitro. d This peak also contained an NP fragment (Fig. 6). e The molecular weight of P67, the 67,000-dalton HN-related cell-free system product, was used in this calculation. P67 may represent the nonglycosylated form of HN (13).

of the major NDV polypeptides synthesized in cell-free systems directed by NDV mRNA's were estimated. The gel patterns of polypeptides synthesized in response to NDV mRNA's made in vitro and in vivo were quantitatively, as well as qualitatively, indistinguishable. In addition, the gel pattern of products made in cell-free systems prepared from mouse L-cells and chick cells were quantitatively very similar. Accordingly, these different cases have been considered together in Table 1 as a single category. The order of the relative molar amounts of NDV polypeptides extracted from infected cells was, in decreasing amount: NP, (Fo + F1), M, HN, L. For NDV polypeptides made in cell-free systems, the order was: (NP + P59), Fo/Fl, M, P67. DISCUSSION In the presence of cytoplasmic extracts, NDV directed the synthesis of RNA in fivefold excess of input genome RNA, indicating that at least some regions of the genome were repeatedly transcribed in vitro. The effects of the cytoplasmic extracts on viral transcription remain unexplained. In this paper we present characterizations of only the RNAs made when cytoplasmic extracts were included in the reaction mixture. However, preliminary comparison of viral RNAs made in the presence and absence of the extracts indicated that there were qualitative as well as

quantitative differences. When analyzed by polyacrylamide gel electrophoresis, the products of transcription in vitro resolved into eight bands that comigrated with distinct bands of viral mRNA's extracted from

infected cells. Bands 1 to 5, which are the major products of NDV transcription, were synthesized in the same abundances relative to each other in vitro as in vivo. Band 8, representing the remaining known NDV mRNA, was present in reduced amounts among the products of transcription in vitro. This may reflect sensitivity of this large RNA to degradation in the reaction mixture during the 8-h incubation. Band 2 of the polyacrylamide gel pattern of NDV RNA occasionally was resolved into a doublet (Fig. 3 and Ball et al., in press). We have previously presented evidence that band 2 of NDV RNA extracted from infected cells was composed of two species transcribed from genes with different UV target sizes (Ball et al., in press). Similar results were obtained when transcriptional mapping was conducted in vitro (our unpublished observations). Both species of band 2 of NDV RNA made in vitro bind to oligo(dT)cellulose (not shown). The relationships between the bands of NDV RNAs resolved by gel electrophoresis and the individual NDV polypeptides are not known, except for band 8, which contains the L-protein mRNA. However, the relative amounts of the NDV polypeptides synthesized by cell-free systems in response to NDV mRNA were the same whether the messenger preparation had been transcribed in vitro or extracted from infected cells. Therefore, the mRNA's for the HN, NP, Fo/Fl, and M proteins accumulated in about the same relative amounts in vitro and in vivo. Considered together with the similarity between the gel patterns of NDV RNA made in vitro and in vivo, these results suggest that regulation of

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NDV transcription operated with fidelity during detergent-activated transcription. This process is probably analogous to primary transcription by NDV in vivo, since no evidence of NDV genome replication in vitro was found in this work, or in a previous study (42). These results are therefore in accordance with the observation that the control of transcription of NDV genes appears to be largely independent of genome replication (6; B. Spanier-Collins, Ph.D. thesis, Harvard University, Cambridge, Mass., 1975). Transcription and translation in cell-free systems have the advantage of a very low background of endogenous synthetic activities. Synthesis of polypeptides corresponding to HN, NP, Fo/Fl, and M by cell-free systems in response to NDV mRNA that had been made in vitro is proof that these proteins are specified by viral genes. The finding of an additional species of polyadenylated RNA in band 2 underscores the possibility that unidentified unique NDV polypeptides exist, perhaps analogous to the small nonstructural proteins described for Sendai virus and simian virus 5 (28, 36). Detection of additional proteins and RNAs synthesized in NDV-directed cell-free systems would constitute rigorous proof that they are NDV gene products. NDV AV mRNA made in vitro or extracted from infected cells directed the synthesis of P59, an NP-related polypeptide having a larger apparent molecular weight than NP. The possibility that NP is generated by the proteolytic processing of a larger primnary translation product has been discussed before (39; L. E. Hightower and M. D. Smith, in B. W. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cell, in press). P59 might represent such a precursor, or an intermediate in the sequential processing of an even larger primary gene product. Interestingly, the substitution of the arginine analog canavanine for arginine in vivo (Hightower and Smith, in press) and in vitro (our unpublished data) resulted in a reduction in the amount of radioactivity in the 56,000dalton NP band and the appearance of heterogeneous, NP-related bands having apparent molecular weights of up to 62,000 to 64,000. Such a pattern could arise from an inhibition of proteolytic cleavage by incorporation of canavanine residues into a precursor. Alternatively, P59 might result from aberrant initiation or termination of transcription or translation of the NP protein mRNA. Or P59 might represent a phosphorylated class of NP, analogous to the two phosphorylated classes of Sendai virus NP that have higher apparent molecular weights than the more abundant nonphosphorylated class

(27).

J. VIROL.

The tryptic peptide maps of the cell-free system product P67 and the HN glycoprotein were very similar, showing that the two polypeptides were clearly related. It seems probable that P67 represents the nonglycosylated form of HN, since cell-free systems are generally deficient in glycosylating activity (13). It has been clearly demonstrated that the virion structural glycoprotein F is generated by proteolytic processing of a larger precursor glycoprotein, Fo (34; Hightower and Smith, in press). Furthermore, the methionine-containing tryptic peptides of Fo and F1 from infected cells largely correspond when mapped by high-voltage paper ionophoresis at pH 3.5 (23) and by electrophoresis on thin-layer plates at pH 6.5 (our unpublished data). However, Fo and F1 did not correspond closely by peptide analysis when electrophoresis of tryptic peptides was followed by thin-layer chromatography in a second dimension (this paper), or when mapping was performed by gel electrophoresis of S. aureus protease limited digests. These results suggest that differences in polypeptide and carbohydrate content resulting from proteolytic processing can strongly influence peptide analysis under some conditions. The cell-free system product that comigrated with authentic F1 corresponded more closely with F1 than Fo, both on the basis of two-dimentional tryptic peptide mapping (Fig. 6) and polyacrylamide gel electrophoresis of S. aureus protease limited digests (not shown). Our identification of the F-related cell-free system product was further substantiated by the observation that the differences in apparent sizes of the authentic F1 proteins of strains AV and HP coincided with a similar difference between the F-related proteins synthesized in cell-free systems. For strain AV, canavanine treatment of infected cells blocks the processing of Fo (Hightower and Smith, in press), and similarly the substitution of canavanine for arginine in cellfree systems directed by mRNA of strain AV blocked the accumulation of the 54,000-dalton F-related species (our unpublished data). Therefore, this polypeptide, rather than being the nonglycosylated form of Fo, may have resulted from cleavage of a larger primary translation product by proteases in the cell-free system. The comparison of strains A V and HP in this work is a clear demonstration of polypeptide differences between virulent strains of NDV. In this specific case, the major polypeptide difference detected, the size of the F, protein, correlates with the major known biological difference, cell-fusion activity. Since the F-related cell-free system products are probably nonglycosylated, the different mobilities of the F1 glycoproteins

VOL. 28, 1978

NDV TRANSCRIPTION AND TRANSLATION IN VITRO

of strains HP and A Vare due to the polypeptide portions, rather than the carbohydrate moieties. There was an additional difference between the strains: no polypeptides which comigrated with AV P59 were detected among the products of strain HP. It has recently been shown that, for both NDV and Sendai virus, transcription is initiated at a single promoter site followed by sequential readthrough of a single transcriptional unit containing all of the genes (20; Ball et al., in press). The transcriptional maps ofthe two paramyxoviruses contained clear similarities. For NDV, the order of transcription is: NP, Fo, M, (47K, HN), L (Ball et al., in press). The possibility was raised (20; Ball et al., in press) that the regulation of paramyxovirus gene expression is similar to that of the rhabdovirus vesicular stomatitis virus in that there is a polar effect on the frequency of transcription of the genes contained within the transcriptional unit (1) with no detectable subsequent control at the level of mRNA translation

(45).

For NDV, no significant temporal control of mRNA and protein synthesis has been detected (6, 22, 43), and the abundances of viral polypeptides in infected cells approximately reflects the composition of the virus particle (22). The relative amounts of radioactivity in the gel bands of NDV mRNA's (Fig. 2, 3, and reference 43) suggest that viral mRNA's are synthesized in nonequimolar amounts both in vitro and in vivo, although it remains to be proved that each gel band contains one mRNA species. Translation of this mRNA in cell-free systems yielded nonequimolar amounts of NDV polypeptides. The relative molar amounts of polypeptides synthesized in cell-free systems and extracted from infected cells decrease in the same order: NP, Fo, M, HN, L. This order is the same as the transcriptional map and suggests that the regulation of NDV gene expression is largely a consequence of the mechanism of transcription. ACKNOWLEDGMENTS We thank Carol White for kind assistance throughout this work, Glenn Smith for assistance in presensitizing film and preparing prints, and Liz Jean for help in preparing the manuscript. This work was supported by Public Health Service grants HL 19490 from the National Heart and Lung Institute and CA 14733 from the National Cancer Institute. We benefited greatly from the use of a cell culture facility supported by the latter grant. L.E.H. received additional support from the University of Connecticut Research Foundation. P.L.C. was a National Science Foundation graduate fellow. LITERATURE CITED 1. Ball, L. A., and C. N. White. 1976. Order of transcription of genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:422-446. 2. Ball, L. A. and C. N. White. 1978. Coupled transcription

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