JOURNAL OF VioLoGY, Dec. 1975, p. 1426-1434 Copyright ©) 1975 American Society for Microbiology

Vol. 16, No. 6 Printed in U.SA.

Isolation of RNA Transcripts from the Entire Sendai Viral Genome LAURENT ROUX AND DANIEL KOLAKOFSKYl* Departement de Biologie Moleculaire, Universite de Geneve, 30, Quai Ernest Ansernet, 1211 Geneve 4, Switzerland Received for publication date

Three classes of viral transcripts (18S, 248, and 33S) were isolated from viral ribonucleoproteins in Sendai virus-infected cells. Hybridization studies with virion minus strand genome RNA demonstrated that the 188 RNA contained transcripts from 60% of the viral genome while the 33S RNA contained transcripts from the entire viral genome. Brief heat or ME2SO treatment of the 33S RNA demonstrated that this RNA was composed of two classes: RNA which continued to sediment at 33S (33S* RNA) and 18. RNA aggregates (18S9* RNA). The 33S* RNA was determined to be a transcript from the 40% of the viral genome not protected by the 18. RNA. The aggregated 18S RNA does not appear to be an artifact of isolation.

Cells infected with negative strand viruses genome. They found that the 18S, 22S, and 35S such as Sendai virus, Newcastle disease virus RNA hybridized to 50, 40, and 70% of the viral (NDV), and vesicular stomatitis virus (VSV), genome, respectively, but that a mixture of the contain virus-specific RNAs which are smaller three RNAs hybridized to no more than 70% of than, and complementary to, the viral genome the viral genome. Similarly, Kaverin and Var(7, 15, 17). In our present understanding of the ich (6) found that 18S RNA from NDV-infected events which occur during infection, viral nu- cells hybridized to only 65% of the viral gecleocapsids containing the virion polymerase nome, but they did not test the other faster enter the cell cytoplasm and are transcribed sedimenting virus-specific RNAs. Portner and into multiple, monocistronic mRNAs. These Kingsbury (14) isolated all RNA sedimenting virus-specific mRNAs are then used to synthe- more slowly than 5GS RNA from Sendai virussize viral proteins, some of which are required infected cells and found that this RNA could to replicate the viral genome via a full-length hybridize to 80% of the viral genome. It was plus strand or antigenome intermediate. A sec- therefore of interest to determine more exactly ond round of transcription from progeny minus what fraction of the viral minus strand genome strand genomes then occurs, followed by the was transcribed into mRNAs in Sendai virusincreased synthesis of viral proteins, and fi- infected cells, since the alternatives to complete nally assembly and budding off of virus at the transcription of the viral genome [(i) that either not all the information contained in the viral host cell plasma membrane (7, 15, 17). The above replication scheme seems to be an genome was used to code for viral proteins, or adequate description of the events occurring (ii) that some of the information contained in during rhabdovirus infection, since virus-spe- the viral genome was directly translated from cific mRNAs isolated from VSV-infected cells 50S plus strands (antigenomes)] would reprehave been shown to represent the transcripts of sent a fundamental difference from the current the entire viral genome (11, 18). Analogous in- replication scheme outlined above. The recent formation about the viral mRNAs found in para- finding (9a) that Sendai virus-infected cells influenza virus-infected cells, however, has in- contain seemingly larger amounts of antigedicated that the above replication scheme may nomes than would be required simply as innot be equally applicable to parainfluenza virus termediates in genome replication is consistent replication. For example, Bratt and Robinson with the latter alternative. In this paper we described the isolation of (2) isolated three sedimentation classes of RNA (18S, 22S, and 35S) from NDV-infected cells virus-specific mRNAs from Sendai virus-inwhich were each complementary to the viral fected cells under conditions which minimize the contamination of these RNAs with deI Present address: Department of Microbiology, Univergraded genome RNA and/or incomplete genome RNA caused by disruption of the viral replicasity of Utah Medical Center, Salt Lake City, Utah 84112. 1426

VOL. 16, 1975

RNA TRANSCRIPTS FROM SENDAI VIRAL GENOME

tive intermediates. In agreement with previous studies (1, 2), we have found three sedimenta-

tion classes of complementary virus-specific RNA (18S, 24S, and 33S). However, brief exposure to heat or ME2SO denaturation of the virus-specific RNAs has demonstrated that all of the 24S RNA and approximately 65% of the 33S RNA are composed of aggregated 18S RNA. Hybridization studies with 32P-labeled minus strand genome RNA have shown that the 188 RNA is composed of transcripts from 60% of the viral genome, and that the 33S RNA which continues to sediment at 33S after disaggregation treatment represents a transcript from the other 40%o of the viral genome. MATERIALS AND METHODS Isolation of 3H-labeled viral mRNAs. Confluent secondary cultures of chicken embryo fibroblasts in 10-cm tissue culture dishes were infected with 20 mean egg infectious dose (EID50) of the Harris strain of Sendai virus per cell. After 60 min at 31 C, the infecting medium was removed and replaced with 4 ml of Dulbecco modified Eagle medium containing 0.5% lactalbumin hydrolysate per dish, 1% calf serum, and 1 ,ug of actinomycin D per ml, and the incubation continued at 37 C. [3H]uridine (25 Ci/ mmol) was added to the culture medium (20 i&Ci/ml) 45 min later and the cultures were harvested at 18 h postinfection. The infected cells were allowed to swell for 15 min at 0 C in 10 mM Tris-chloride, pH 7.5, 10 mM NaCl, 20 mM EDTA (NEB buffer [18]) at a concentration of 5 x 107 cells/ml and disrupted by Dounce homogenization. The homogenate was centrifuged for 10 min at 4,000 x g and the resulting supernatant was directly layered onto gradients of 10 to 30% sucrose in NEB buffer containing a 4-ml cushion of 70% sucrose and centrifuged for 5 h at 25,000 rpm (7 C) in the Spinco SW27 rotor. Fractions containing the viral ribonucleoproteins (RNPs, see Fig. 1A) were precipitated by the addition of 2 volumes of ethanol, collected by centrifugation, suspended in TNE (25 mM Tris-chloride, pH 7.4, 50 mM NaCl, 1 mM EDTA containing 0.5% sodium dodecyl sulfate [SDS]), and further purified by sedimentation velocity (cf. Fig. 1B, 1C). Preparation of 32P-labeled Sendai 50S minus strands (32P-labeled genome RNA). Four confluent cultures of MDBK cells in 75-cm2 Falcon tissue culture flasks were infected with 20 mean egg infectious dose of the Harris strain of Sendai virus per cell, and incubated at 31 C with 6 ml of Dulbecco modified Eagle medium containing 0.1 the normal amount of inorganic phosphate per flask, 0.5% lactalbumin hydrolysate, and 50 ACi of 32pO4 per ml. At 18 h postinfection, the medium was removed (and discarded) and replaced with the above medium containing 10 uCi of 32PO, per ml. At 42 h postinfection, the medium was harvested and replaced with medium without 32PO4. At 64 h postinfection, the medium was again harvested and combined with the 18- to 42-h harvest. The combined medium was centrifuged for 10 min at 5,000 x g to remove a cellular

1427

debris, and virus was then sedimented through a 25ml cushion of 25% glycerol in TNE by centrifuging for 90 min at 37,000 rpm in the International A 170 rotor. The virus pellet was dissolved in 0.6 ml of TNE containing 1% SDS, layered onto three 5 to 23% sucrose gradients containing 100 mM LiCl, 20 mM Tris-chloride, pH 7.4, 1 mM EDTA, and 0.1% SDS, and centrifuged for 105 min at 50,000 rpm (7 C) in the SW56 rotor. The gradients were then fractionated by puncturing the bottom of the tube and monitored for radioactivity by Cerenkov counting, and those fractions containing the 5S RNk were precipitated with ethanol. The virion 50S RNA was then collected by centrifugation, dissolved in 0.2 ml of 0.4 M NaCl, 20 mM Tris-chloride, pH 7.4, 2 mM EDTA, and 0.1% SDS, and annealed for 30 min at 75 C. The annealed solution was made up to 1.0 ml with STES (0.1 M NaCl, 0.05 M Tris-chloride, pH 7.4, 1 mM EDTA, and 0.1% SDS), 0.18 ml of ethanol was added, and the solution was passed through a 2-ml column of cellulose powder (Whatman CF11) in 85% STES and 15% ethanol to remove double-stranded (ds) and multistranded RNA, which represented 60% of the preparation. 32P-labeled genome RNA, prepared in this manner, contained approximately 700,000 counts/ min per gg and was found to be less than 3% resistant to RNase after further self-annealing. Determination of the percentage of the 32P-labeled viral genome hybridized. Samples of 32p_ labeled genome RNA (1,000 to 2,000 counts/min) and varying amounts of 3H-labeled virus-specific mRNAs as indicated in Fig. 4 and 5 were combined in a total volume of 10 ul containing 0.3 M NaCl, 0.4 M Tris-chloride, pH 7.5, and 0.002 M EDTA, and annealed for 2 h at 75 C in sealed capillaries. After annealing, the contents of each capillary were transferred into 1.6 ml of the above annealing buffer. To one-half of each sample, 30 ;Lg of ribosomal RNA was added, the RNA was precipitated by the addition of 80 ;l of 100% trichloroacetic acid, and the precipitate was collected on membrane filters (Millipore Corp.) to determine the total trichloroacetic acid-precipitable radioactivity present. The other half of each sample was incubated with 32 ,ug of RNase A for 30 min at 25 C and then precipitated with trichloroacetic acid as described above to determine the percentage of radioactivity present as RNA-RNA hybrids. Sendai virions grown in MDBK cells have been shown to contain small quantities of cellular RNAs (9a). Since these contaminating RNAs are single stranded, they cochromatograph with free minus strand viral genomes on cellulose columns. Minus strand or genome RNA prepared as described above is therefore always enriched in these contaminating nonviral RNAs. To determine the extent of this contamination, samples of 32P-labeled genome RNA preparations were annealed with a large excess of heat-denatured 3H-labeled genome-ds RNA of relatively low specific activity (6,000 counts/min per jtg). This genome dsRNA has been shown to contain all sequences present in the viral genome (8). Since the 3H-labeled genome-dsRNA was found to reassociate completely after annealing and this dsRNA repre-

1428

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ROUX AND KOLAKOFSKY

sented a 100-fold excess over the 32P-labeled genome RNA, the fraction of 32P-labeled RNA sensitive to RNase after annealing was judged to represent the nonviral RNA contamination. In the experiments reported in this paper, the 32P-labeled genome RNA used was found to be from 80 to 92% pure by this test, and the percentage of 32P-labeled genome RNA present in hybrids was therefore determined as: (32p counts/min resistant to RNAse after annealing with 3H-labeled mRNA/32P counts/min resistant to RNAse after annealing with 3H-labeled genome dsRNA) x 100. When calculated in this way, saturating amounts of 3H-labeled 18S mRNA were found to hybridize with 60 + 2% of the 32P-labeled viral genome in six different experiments.

RESULTS Isolation of virus-specific 18S, 24S, and 33S RNAs. Late in infection when synthesis of virus-specific mRNA is maximal, Sendai virusinfected cells contain large amounts of viral genomes and antigenomes packaged as viral nucleocapsids. At any time during infection, some of the viral nucleocapsids will be in the process of synthesizing both 50S plus and minus strands in replicative intermediates. Since the methods commonly used to isolate mRNA from infected cells, e.g., phenol extraction or SDS treatment of cytoplasmic extracts, are known to either anneal or release incomplete RNA chains from replicative intermediates, mRNA isolated by these methods is contaminated with either partially dsRNAs or incomplete RNA chains. To minimize such contamination, cytoplasmic extracts of Sendai virus-infected cells labeled with [3H]uridine in the presence of actinomycin D were treated with EDTA to dissociate polysomes and sedimented in 10 to 30% sucrose gradients which contained a cushion of 70% sucrose. Under these conditions (Fig. 1A), cytoplasmic extracts of Sendai-infected cells contained three sedimentation classes of virus-specific RNA: a broad band of presumptive viral RNPs which sedimented from 40 to 60S, an even broader band of presumptive viral RNPs which sedimented from 60 to 90S, and the major band which sedimented faster than 150S and had sedimented into the 70% sucrose cush-

18

2j1

I\

60S 40S

I I

115

15 9 10o x

CI-

L0.5

= 0.5

6

6

4

4

2

2 1

10

20

30

FRACTION NUMBER

ml cushion of 70% sucrose and centrifuged for 5 h at 25,000 rpm (7 C) in the Spinco SW27 rotor. After centrifugation, the optical density profile (260 nm) of the gradients was monitored to determine the position of the 40S and 60S ribosomal subunits, and fractions ofapproximately 1 5 ml were collected. Samples of each fraction (50 pi) were spotted on filter paper disks which were washed with 6% trichloroacetic acid followed by 95% ethanol, and counted by liquid scintillation to determine radioactivity (panel A). Gradient fractions were pooled as indicated in panel A and precipitated by the addition of2 volumes ion. of ethanol, and the precipitate was collected by centrifugation. The precipitates were dissolved in TNE containing 0.5% SDS, and a sample of fractions I (panel C) and H (panel B) were mixed with 14C_laFIG. 1. Isolation of virus-specific mRNAs from beled mouse kidney cell RNA and sedimented on 5 to Sendai virus-infected cells. Chick embryo fibro- 23% sucrose gradients in TNE containing 0.1% SDS blasts were infected with Sendai virus, virus-specific for 105 min at 50,0(O rpm (8 C) in the Spinco SW56 RNA was labeled with [3H]uridine in the presence of rotor. Gradient fractions were collected by puncturactinomycin D, and cytoplasmic extracts of the in- ing the bottom of the tube, mixed with 3 ml of Triton fected cells were prepared as described. Samples of cocktail (600 ml of toluene, 300 ml of Triton X-100, cytoplasmic extract (2.5 ml) were layered onto 10 to 100 ml of water, and 5.5 g of Permablend IIIlliter) 30% sucrose gradients in NEB buffer contianing a 3- and counted by liquid scintillation.

RNA TRANSCRIPTS FROM SENDAI VIRAL GENOME

VOL. 16, 1975

Fractions I, II, and III were pooled as indicated in Fig. 1A, precipitated with ethanol, and collected by centrifugation. The precipitates were dissolved in TNE containing 0.5% SDS, and a sample of each fraction was mixed with 14C-labeled mouse kidney cell RNA and analyzed on SDS sucrose gradients. As shown in Fig. 1C, fraction I consisted mostly of 188 RNA and a small amount of 24S RNA. Fraction II contained three sedimentation classes, 18S, 24S, and 33S RNA, present in approximately equal amounts (Fig. 1B). Fraction III was found to consist mostly of genome 50S RNA and a small amount of more slowly sedimenting RNA (data not shown). To isolate the 188, 24S, and 338 RNAs, fractions I and II RNA were sedimented on preparative 5 to 23% sucrose gradients (5 h, 38,000 rpm, 8 C in the Spinco SW40 rotor), 188 RNA was pooled from the fraction I RNA gradient, and 24S and 33S RNA were pooled from the fraction II RNA gradients as indicated in Fig. 1C and 1B, respectively. The purified 24S, and 33S RNAs were precipitated with ethanol, collected by centrifugation, and dissolved in TNE containing 0.5% SDS. Virus-specific 188, 24S, and 33S RNAs so isolated were found not to self-anneal and to be completely complementary to virion genome RNA (Table 1). Sucrose gradient analysis of 24S RNA. 24S RNA, isolated as described above, sedimented as a sharp 24S band when recentri-

1429

fuged in SDS sucrose gradients (Fig. 2A). To determine whether the 24S RNA contained other aggregated RNAs, the 24S RNA was heated for 4 min at 60 C in low-salt buffer (TNE) and resedimented in a sucrose gradient. Heat-treated 24S RNA sedimented mostly at 188, with a prominent shoulder at 248 (Fig. 2B). This suggested that the 24S RNA was composed mostly, if not entirely, of aggregated 3

3

2

-F

S

2 C-.) x cL-

C-)

TABLE 1. Annealing properties of 3H-labeled virus-

specific RNAsa Additions

1 Lg of Sendai 50S 3 Ag of Sendai

Min at

70 C

2

3H counts/min resistant to RNase

18S

245

33S

0 60 60

18 25 1125

2 5 475

15 24 495

60

1050

475

560

973

460

506

50S

Total 3H counts/min added

a [3Hluridine labeled virus-specific 18S, 24S, and 33S RNA, isolated as described in Fig. 1, were annealed with and without egg-grown Sendai 50S RNA in 10 1zl of 0.3 M NaCl, 0.4 M Tris, pH 7.4, and 0.002 M EDTA in sealed capillaries. The contents of each capillary were then transferred to 0.8 ml of the above annealing buffer and digested with 33 Ag of RNase A for 30 min at 25 C. The remaining RNA was co-precipitated with 30 Ag of carrier rRNA, collected on membrane filters (Millipore Corp.) and counted by liquid scintillation.

0

FRACTION NUMBER FIG. 2. Sucrose gradient analysis of 24S virusspecific RNA. Fraction H RNA, isolated as described in Fig. IA, was separated into 18S, 24S and 33S fractions as shown in Fig. IB by sedimentation on 5 to 23% sucrose gradients (5 h at 39,000 rpm in the Spinco SW40 rotor). The RNA in each fraction was recovered by ethanol precipitation and samples ofthe 3H-labeled 24S RNA were mixed with 14C-labeled mouse kidney cell RNA and (A) sedimented directly on5 to23% sucrose gradients as described in Fig. IB, (B) heated for4 min atWf0 C in TNE before sedimentation, or (C) dried in vacuum, suspended in 5 id of water with 45 1 of99% ME2SO added, heated for 10 min at37 C, and sedimented on 0 to 10% sucrose gradients in 99% ME2SO 49) for 4.5 h at 50/)AW rpm (265 C) in the Spinco SW56 rotor.

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ROUX AND KOLAKOFSKY

18S RNA. 24S RNA was therefore heated for 10 min at 37 C in 90% ME2SO, and then sedimented in a 99% ME2SO-sucrose gradient (9), conditions which are known to denature the fully ds form of the viral genome (Daniel Kolakofsky, unpublished data). ME2SO-treated 24S RNA sedimented as a band only slightly slower than mouse cell 18S RNA in a 99% ME2SOsucrose gradient, with little or no evidence of any 24S RNA being present (Fig. 2C). Virusspecific 24S RNA, therefore, appears to consist of 18S RNA aggregates which sediment at 24S. Sucrose gradient analysis of 33S RNA. Virus-specific 33S RNA was found to sediment mostly as 33S RNA with a broad 24 to 28S shoulder when recentrifuged in SDS sucrose gradients (Fig. 3A). When the 33S RNA was heated for 4 min at 60 C in TNE buffer and resedimented on a SDS sucrose gradient, this RNA was now found to sediment as two bands of 188 and 33S with a broad shoulder of RNA sedimenting between the two bands (Fig. 3B). Furthermore, the 33S RNA again sedimented as 2 bands, with sedimentation values of 23S and 17S relative to mouse cell rRNA (whose sedimentation values are arbitrarily set at 28S and 18S in ME2SO), when ME2SO was treated and centrifuged in a 99% ME2SO-sucrose gradient. Virus-specific 33S RNA therefore appears to be composed of two RNA classes, namely 188 RNA which has aggregated to sediment at 338 (18S* RNA) and 33S RNA which continues to sediment at 33S in aqueous sucrose gradients and sediments at 23S ME2SO in ME2SO-sucrose gradients (33S* RNA). To demonstrate that the 33S RNA which sediments at 33S after heat treatment in aqueous sucrose gradients is the same RNA species which sediments at 23S in ME2SO-sucrose gradients, 33S* RNA was isolated from a sucrose gradient as shown in Fig. 3B and resedimented on a 99% ME2SO-sucrose gradient. The results (Fig. 3D) demonstrate that under these conditions, 338* RNA sediments mostly at 23S ME2SO, with only a small peak of RNA sedimenting at 18S ME2SO. We conclude that 33S RNA isolated from fraction II RNA contains approximately 35% 33S* RNA and 65% 18S RNA which has aggregated to sediment at 33S (188* RNA). 18S RNA isolated from fraction I RNA, on the other hand, continued to sediment as a single 18S band after heat or ME2SO treatment in both aqueous and ME2SO-sucrose gradients (data not shown). Virus-specific RNA isolated from viral RNPs as described in Fig. 1 is therefore composed of approximately 66% 188, 23% 24S, and 11% 33S RNAs. However, since most, if not all, of the 24S RNA, and 65% of the 33S RNA, are composed of aggregated 188 RNA

J. VIROL.

2

2

0

1.1

CD x

x

OL-

2

M: C3L-

L-.

=-

--r

nvI

20 10 FRACTION NUMBER

30

FIG. 3. Sucrose gradient analysis of 33S virus-spe-

cific RNA. Samples of 3H-labeled 33S RNA, isolated

as described in the legend to Fig. 2, were mixed with "4C-labeled mouse kidney RNA and (A) sedimented directly on 5 to 23 % sucrose gradients as described in Fig. IB, (B) heated for 4 min at 60 C in TNE before sedimentation, or (C) ME2SO treated and sedimented in a sucrose-ME2SO gradient as described in Fig. 2C. 3H-labeled 33S RNA which continued to sediment at 33S after heating (33S*) was isolated as described in panel B, treated with 90% ME2SO and sedimented in a sucrose-ME2SO gradient (panel D).

which sediments at 24 and 33S (cf. Fig. 2 and 3), virus-specific RNA in Sendai virus-infected cells, after disaggregation, is composed of approximately 96% 188 RNA and only 4% 33S* RNA. In three other experiments (data not shown), 33S* RNA was found to represent from 4 to 8% of the total virus-specific mRNAs. Hybridization of 18S and 33S viral mRNAs to the viral genome. Tritium-labeled virus-specific 18S, 24S, and 33S RNAs isolated

VOL. 16, 1975

RNA TRANSCRIPTS FROM SENDAI VIRAL GENOME

from the viral RNPs did not self-anneal and were completely complementary to the viral 50S RNA genome (Table 1). It was therefore of interest to determine what fraction of the viral genome could be hybridized to the virus-specific 18S and 33S RNAs. Constant amounts of 32p_ labeled virion 50S minus strand RNA of highspecific activity (32P-labeled genome RNA, see Materials and Methods) were therefore annealed with increasing amounts of either 3Hlabeled 188 or 3H-labeled 33S RNA and the percentage of the 32P-labeled genome RNA in dsRNA hybrids was determined. As previously demonstrated (2, 6), virus-specific 18S RNA cannot hybridize to the entire genome, but converted only 60% of the viral genome to hybrids. However, in contrast to previous reports (2), virus-specific 33S RNA can hybridize to the entire genome (Fig. 4). Since virus-specific 33S RNA is composed both of 33S RNA which continues to sediment at 33S after disaggregation (33S*) and aggregated 188 RNA (18S* RNA), it seemed likely that the 33S* RNA was responsible for protecting only that part (40%) of the viral genome not protected by the 188 RNA. Accordingly, mixtures of a constant amount of 32P-labeled genome RNA and 3H-labeled 188 RNA (420 counts/min, cf. Fig. 4) were annealed with increasing amounts of 3H-labeled 18S, 33S, 188*, and 33S* RNAs, and the percentage of the 32p_ labeled genome RNA in hybrids was determined. Saturating amounts of 3H-labeled 18S RNA again hybridized to only 60% of the viral genome, but both the 33S and the 33S* RNA hybridized to 100% of the 32P-labeled genome RNA (Fig. 5). 188* RNA, on the other hand, could not protect 100% of the viral genome when similar amounts of radioactive RNA were added. Since the relative specific activities of all the 3H-labeled virus-specific RNAs varied by less than 20% (see Discussion), the slower rate at which 3H-labeled 18S* RNA protectd the viral genome suggests that the 188* RNA contains considerably less of those viral sequences not protected by the 188 RNA. The ability of the 18S* RNA to protect more than 60% of the viral genome is most likely due to the presence of small amounts of degraded 33S* RNA formed during the disaggregation of the 338 RNA. We conclude that the virus-specific 18S RNA is composed of transcripts from 60% of the viral genome and that the virus-specific 33S* RNA represents transcripts from the remaining 40% of the viral genome. Nature of the unstable virus-specific 24S and 33S RNAs. Since 18S and 33S* virusspecific RNAs represent transcripts from the entire viral genome and virus-specific 18S, 24S,

1431

100

80 m

CL.

60

40 20

n

0

3

6 9 12 3H-CPM x 10-2 ADDED FIG. 4. Hybridization of 3H-labeled 18S and 33S RNA with 32P-labeled genome RNA. Various amounts of 3H-labeled 18S RNA (prepared by sucrose gradient sedimentation of fraction I RNA, cf. Fig. 1C) and 3H-labeled 33S RNA (prepared by sucrose gradient sedimentation of fraction II RNA, cf. Fig. IB) were mixed with 2,)00 counts/min of 3P-labeled genome RNA and annealed for 2 h at 75 C as described. The percentage of 3P counts/min in hybrids was determined as described.

100 90 0=

80

C=

70

60 50

0

3

6

9

12

3H-CPM x 10-2 ADDED OVER 420 CPM lBS FIG. 5. Hybridization of 3H-labeled 18S, 3H-labeled 33S, 3H-labeled 18S*, and 3H-labeled 33S* RNAs with 3P-labeled genome RNA. Various amounts of 3H-labeled 18S RNA, 3H-labeled 33S RNA (see legend to Fig. 4), and 3H-labeled 18S* RNA and WH-labeled 33S* RNA (prepared by sucrose gradient centrifuigation from heated WH-labeled 33S RNA, cf. Fig. 3B) were combined with a mixture of 420 counts/min of 3H-labekd 18S RNA and 2,000 counts/min ofPP-labeled genome RNA and annealed for2 h at 75 C. The percentage of 3P-labeled RNA in hybrids was determined as described.

and 33S RNAs are all complementary to genome RNA (Table 1), the 18S RNA which sediments as aggregates at 24S and 33S cannot contain any viral sequences not present in the 188 and 33S*. Nevertheless, the apparent ho-

1432

ROUX AND KOLAKOFSKY

J. VIROL.

mogeneity of the aggregated 18S RNA on su- RNPs as described in Fig. 1 and the amount of gradients (cf. Fig. 2A and 3A) suggests radioactivity in the 18S, 24S, and 33S virusthat the aggregation may be specific and there- specific RNAs was estimated by sedimentation fore of possible biological significance. We have analysis in sucrose gradients. The proportion of therefore examined two possible explanations radioactivity in 24S and 33S RNA did not diminof its origin. (i) Is the aggregated 188 RNA ish in the hour following the 1-h labeling period which sediments at 24S and 33S an artifact of even though no additional [3H]uridine was inthe isolation procedure? Since aggregation is corporated into virus-specific RNA (Table 2, ii). most likely to occur during ethanol precipita- There is, therefore, no evidence to suggest that tion of the RNPs, a sample containing all viral 33S and 24S virus-specific RNAs are processed RNPs (both fractions I and II) was isolated as to the virus-specific 18S RNA. described in Fig. 1A, diluted twofold with TNE DISCUSSION containing 1% SDS, and directly sedimented in 15 to 30% SDS sucrose gradients. Such virusThis work demonstrates that (i) Sendai virusspecific RNA was found to contain the same infected cells contain, in addition to genome relative proportions of 18S, 24S, and 338 RNA length RNA, only two sedimentation classes of as a companion sample of the same viral RNPs virus-specific RNA (18S and 33S) stable to brief which were ethanol precipitated and then sedi- heating in low-salt buffer or to ME2SO denaturmented in identical sucrose gradients (data not ation; (ii) the 188 RNA contains sequences shown). Aggregated 18S RNA which sediments complementary to 60% of the viral genome and at 24S and 33S therefore does not appear to be the 33S* RNA contains sequences which are an artifact of the ethanol precipitation of the complementary to the other 40% of the viral viral RNPs. (ii) Does aggregated 18S RNA genome; (iii) the entire viral minus strand gewhich sediments at 24S and 33S arise from the nome is therefore transcribed during Sendai processing of 188 RNA from larger RNA precur- virus infection. sors? To investigate whether 33S and 24S RNA Since the 188 RNA represents transcripts contained RNAs which were precursors to the from 60% of the 5 x 106 dalton viral genome (8, 18S RNA, chicken embryo fibroblasts were lavirus-specific 18S RNA must consist of sevbeled for 0 to 18 h, 15 to 18 h, and from 17 to 18 h !9), eral RNAs whose total molecular weight is appostinfection, with [3H]uridine in the presence proximately 3 x 106. Assuming an average moof actinomycin D. Virus-specific RNA isolated lecular weight of 600,000 for 18S RNA, this from the viral RNPs as described in Fig. 1 was would suggest that the virus-specific 18S RNA found to contain the same relative proportion of 18S, 24S, and 33S RNA whether the viral RNA TABLE 2. Stability of virus-specific 24S and 33S was labeled for 1, 3, or 18 h (data not shown, see RNAa Fig. 1). Thus, if processing of the 24S and 338 3H RNA did occur, the half-life of the putative % 3H counts,/ precursor RNAs must be considerably less than Experimental counts/min in: min conditions the shortest time of labeling used (i.e., 1 h). We in viral therefore examined the fate of RNA labeled for RNPs 18S 24S 33S 1 h over shorter periods. Two sets of four cul(i) 1-h [3H]uridine 25,410 54 27 19 tures each of Sendai virus-infected chicken embryo fibroblasts were treated with 1 ,ug of actino- (ii) 1-hpulse[3H]uridine 29 26,750 47 24 mycin D per ml and 10 mM glucosamine (to pulse + 1-h deplete the uridine 5'-triphosphate pool [161) at chase in pres17 h and 15 min postinfection. (i) [3H]uridine (20 ence of glucosawas added to h each set of at cultures 18 ACi/ml) mine and cold postinfection and one set of cultures was haruridine vested at 19 h postinfection. At 19 h postinfeca Cytoplasmic extracts were prepared from Sention, medium was removed from the other set of cultures, the cultures were washed once with dai virus-infected chicken embryo fibroblasts which phosphate-buffered saline, and fresh culture were pulse labeled with [3H]uridine for 1 h with and a 1-h chase in the presence of glucosamine medium containing 1 pLg of actinomycin D per without non-radioactive uridine (for details see text). ml, 10 mM glucosamine, and 100 Ag of nonradio- and Virus-specific RNPs were isolated from the cytoactive uridine per,ul was added. These cultures plasmic extracts and the percentage of radioactivity were harvested at 20 h postinfection. (ii) Virusin 18S, 24S, and 33S RNA was estimated as despecific RNA was then isolated from the viral scribed in Fig. 1. crose

VOL. 16, 1975

RNA TRANSCRIPTS FROM SENDAI VIRAL GENOME

is composed of five different RNA species. Although there is as yet no information on the further separation of Sendai virus-specific 18S RNA, 188 RNA from NDV-infected cells has been analyzed by polyacrylamide gel electrophoresis. Our data are consistent with those of Kaverin and Varich (6) and Collins and Bratt (3), who found that NDV 188 RNA could be separated into six and seven components, respectively. Kaverin and Varich (6) also determined that the NDV 18S RNA represented transcripts from 65% of the viral genome as opposed to the 60% we have found for Sendai 18S RNA. Further experiments will be required to determine whether this small difference is reproducible. The virus-specific 33S* RNA, on the other hand, contains all the information present in the viral genome which is not contained in the 18S RNA. Since 40% of the viral genome represents 2 x 106 daltons of RNA, and a single RNA chain of this size can be expected to sediment at approximately 33S, the 33S* RNA most probably represents a single transcript from the viral genome. The similarities between Sendai and VSV genome transcription, like other aspects of their replicative cycle, are now quite striking. In both VSV and Sendai virus-infected cells there are only two sedimentation classes of plus strand RNA smaller than genome length: a major class of smaller RNAs which is heterogeneous, and a minor class of larger RNA which is homogeneous. From the data presented in this paper, we estimate that 96% of the radioactivity incorporated into virus-specific RNA other than genome length RNA is found in 188 RNA and only 4% in 33S* RNA. In other experiments (not described), the amount of label in 33S* RNA varied from 4 to 8%. To determine the relative abundance of the 18S and 338* RNAs, we have estimated the relative specific activities of these RNAs from the annealing experiments described in Fig. 5 by calculating the amount of 3H radioactivity relative to 32P radioactivity resistant to RNase digestion when saturating amounts of the 3H-labeled virus-specific RNAs were added. Since the relative specific activities of the virus-specific 18S, 33S, and 33S* RNAs where found to vary by less than 20% (data not shown), the relative abundance of the 18S and 33S* RNA can be calculated directly from the isotopic labeling of the virusspecific RNA. Assuming a molecular weight of 2.0 x 106 for the 338* RNA and a composite molecular weight of 3.0 x 106 for the 188 RNA, we estimate that there are 8 to 16 times more of

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each 18S RNA than 33S* RNA in the cytoplasmic extract of Sendai virus-infected cells prepared as described. Although the possibility that the 338* RNA is specifically lost, e.g., bound to membranes, during the isolation procedure cannot be excluded, we feel that this explanation is unlikely since reextraction of the nuclear and membrane pellet which remains after Dounce homogenization with 0.5% Triton X-100 and 0.1% deoxycholate did not release a greater proportion of 338 RNA relative to 18S RNA than normally found in the first cytoplasmic extract (data not shown). It therefore appears that the transcription of individual mRNAs from the Sendai viral genome is quantitatively controlled. Although the function of the virus-specific 338* RNA is not known, it seems probable that this RNA, like the VSV 288 RNA (13, 19), is a large monocistronic mRNA. The supporting evidence for this is (i) the 338* RNA does not self-anneal and is completely complementary to the viral genome RNA, and (ii) the 338* RNA is not found in nucleocapsids but sediments in sucrose gradients as a broad band slightly faster than the 18S RNA RNPs. Recently, the presence of a very large polypeptide (150,000 to 200,000 daltons [4]) has been detected in purified preparations of parainfluenza virions (4, 5, 10, 12, 20). Should this protein, like the VSV L protein, be viral coded (13, 19), it seems probable that the Sendai 33S* RNA is the mRNA which codes for this large polypeptide. However, molecular weight estimations of RNA by sucrose gradient sedimentation and large polypeptides by electrophoresis in high percentage polyacrylamide gels are not very accurate. More precise molecular weight estimates will be required to determine whether the 33S* RNA is a single, monocristronic mRNA. The reason(s) why approximately 25% of the virus-specific 188 RNA isolated from cytoplasmic extracts sediments at 24S and 33S is not clear. The experiments that we have so far described appear to rule out the most likely explanations, namely, that the aggregated 188 RNA is an artifact of the isolation procedure or that the unstable 248 and 338 RNAs are precursors to the 18S RNA. Although the conspiratorial view that these unstable RNAs are simply the result of nonspecific aggregation seems probable, the apparent homogeneity of the 24S RNA on sucrose gradients suggests that the aggregation may be specific. Furthermore, since the aggregation does not seem to be an artifact of isolation, it is possible that some of

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ROUX AND KOLAKOFSKY

the 18S virus-specific RNAs exist as aggregates in infected cells (note the heterogeneity of the larger RNPs in Fig. 1A). Experiments to further examine the nature of the aggregated 18S RNA are now in progress. ACKNOWLEDGMENTS We thank Pierre-Francois Spahr for generous advice, encouragement and support, Andree Bruschi for excellent technical assistance, and Monique Visini for her patience in preparing the manuscript. This work was supported by research grant 3.331.074 from the Fonds National Suisse de la Recherche Scientifique.

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LITERATURE CITED Blair, C. D., and W. S. Robinson. 1968. Replication of Sendai virus, comparison of viral RNA and virus specific RNA synthesis with Newcastle disease virus. Virology 35:537-549. Bratt, M. A., and W. S. Robinson. 1967. Ribonucleic acid synthesis in cells infected with Newcastle disease virus. J. Mol. Biol. 23:1-27. Collins, B. S., and M. A. Bratt. 1973. Separation of messenger RNAs of Newcastle disease virus by gel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. 70:2544-2548. Hightower, L. E., and M. A. Bratt. 1974. Protein synthesis in Newcastle disease virus-infected chicken embryo cells. J. Virol. 13:788-800. Hosaka, Y., and Y. K. Shimizu. 1972. Artificial assembly of envelope particles of HVJ (Sendai virus). Virology 49:627-639. Kaverin, N. I., and N. L. Varich. 1974. Newcastle disease virus-specific RNA: polyacrylamide gel analysis of single-stranded RNA and hybrid duplexes. J. Virol. 13:253-260. Kingsbury, D. W. 1974. The molecular biology of paramyxoviruses. Med. Microbiol. Immunol. 160:73-83. Kolakofsky, D., E. Boy de la Tour, and A. Bruschi.

J. VIROL. 1974. Self-annealing of Sendai virus RNA. J. Virol. 14:33-39. 9. Kolakofsky, D., E. Boy de la Tour, and H. Delius. 1974. Molecular weight determination of Sendai and Newcastle disease virus RNA. J. Virol. 13:261-269. 9a. Kolakofsky, D., and A. Bruschi. 1975. Antigenomes in Sendai virions and Sendai virus infected cells. Virology 66:185-199. 10. Lamb, L. A. 1975. The phosphorylation of Sendai virus proteins by a virus particle associated protein kinase. J. Gen. Virol. 26:249-263. 11. Leamnson, R. N., and M. E. Reichmann. 1974. The RNA of defective vesicular stomatitis virus particles in relation to viral cistrons. J. Mol. Biol. 85:551-568. 12. Marx, P. A., A. Portner, and D. W. Kingsbury. 1974. Sendai virion transcriptase complex: polypeptide composition and inhibition by virion envelope proteins. J. Virol. 13:107-112. 13. Morrison, T., M. Stampfer, D. Baltimore, and H. Lodish. 1974. Translation of vesicular stomatitis mRNA by extracts from mammalian and plant cells. J. Virol. 13:62-72. 14. Portner, A., and D. W. Kingsbury. 1970. Complementary RNAs in paramyxovirions and paramyxovirusinfected cells. Nature (London) 228:1196-1197. 15. Prevec, L. 1974. Physiological properties of vesicular stomatitis virus and some related rhabdoviruses. In E. Kurstak and K. Maramorosch (ed.), Viruses, evolution and cancer. Academic Press Inc., New York. 16. Scholtissek, C. 1971. Detection of an unstable RNA in chick fibroblasts after reduction of the GTP pool by glucosamine. Eur. J. Biochem. 24:358-365. 17. Shatkin, A. J. 1974. Animal RNA viruses: genome structure and function. Annu. Rev. Biochem. 43:643-665. 18. Stamminger, G., and R. A. Lazzarini. 1974. Analysis of the RNA of defective VSV particles. Cell 3:85-93. 19. Stampfer, M., and D. Baltimore. 1973. Identification of the vesicular stomatitis virus large protein as a unique viral protein. J. Virol. 11:520-526. 20. Zaides, V. M., L. M. Selimova, 0. P. Zhirnov, and A. G. Bukrinskaya. 1975. Protein synthesis in Sendai virus infected cells. J. Gen. Virol. 27:319-327.