Identification of Polysomal RNA in BHK Cells Infected by Sindbis Virus

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1973, p Copyright 1973 American Society for Microbiology Vol. 11, No. 4 Plrintd in U.S.A. Identification of Polysomal RNA in BHK Cells Infected by Sindbis Virus DEBORAH MOWSHOWITZ Department of Biological Sciences, Columbia University, New York, New York Received for publication 13 November 1972 Polysomes were prepared from Sindbis virus-infected BHK cells. The major species of RNA in these polysomes was identified as 26S RNA (interjacent RNA) by (i) disrupting the polysomes with EDTA; (ii) treating the infected cells with puromycin; and (iii) isolating polysomes from cells infected with a temperaturesensitive mutant that does not form nucleocapsids. Small amounts of 42S RNA and 33S RNA were also found in polysomes. Cells infected by group A arboviruses contain two major species of virus-specific, singlestranded RNA: 42S RNA, which is identical to that found in the virion, and 26S RNA, which has been called interjacent RNA (13). Infected cells synthesize large amounts of 26S RNA. This synthesis appears to be an indispensable part of the infection process, since one temperaturesensitive mutant of Sindbis virus (ts24), which has a lesion in the synthesis of 26S RNA, is blocked in virus production (10). Two different roles have been proposed for 26S RNA: (i) as precursor to 42S RNA (6) and (ii) as a mrna species (15). The first possibility is no longer reasonable because it has been found that 42S RNA can be made in the absence of concomitant 26S RNA synthesis in cells infected by ts24 (10), and that 42S RNA and 26S RNA are synthesized on separate double-stranded RNA forms in cells infected by wild-type Sindbis (D. T. Simmons and J. H. Strauss, J. Mol. Biol., in press). In addition, it has been established by nucleic acid hydridization (D. T. Simmons and J. H. Strauss, J. Mol. Biol., in press) and other methods (1) that 26S RNA corresponds to a unique third of the 42S RNA. Thus, 42S RNA cannot be an aggregate of several 26S RNA molecules. Since 26S RNA cannot be a precursor to 42S RNA, we undertook a series of experiments to test the second possibility, that 26S RNA acts as mrna during infection. MATERIALS AND METHODS Tissue culture and viruses. BHK-21 cells (originally obtained from P. Marcus) were grown in monolayers in plastic bottles in Eagle medium containing 10%o fetal calf serum, 0.01 M N -2 hydroxyethyl - piperazine-n' -2' -ethanesulfonic acid (HEPES) and 50 pliters of neomycin per ml. Continuous monolayer cultures were main- 535 tained by periodic trypsinization and seeding. The cells were infected in Eagle medium containing 5% fetal calf serum and the same concentration of HEPES and neomycin. Wild-type Sindbis virus and the temperature--sensitive mutant ts2 were obtained from B. Burge. The isolation and characterization of the mutant have been described (2, 3, 17). To prepare virus stocks, confluent monolayers were infected with virus at very low multiplicities ( PFU per cell) at either 37 C (wild-type virus) or 27 to 30 C (ts2). The virus was adsorbed to the monolayers for 1 h, culture medium was added, and the infected cultures were incubated for 24 to 72 h. The culture fluid was removed and used as a stock preparation of virus. Infection prior to labeling. Virus was absorbed to confluent or nearly confluent monolayers at a concentration of approximately 10 PFU per cell. After 1 h, medium containing 1 pliter of actinomycin D per ml was added, and the cultures were incubated until 4 to 5 h after infection, the time at which the rate of virus-specific RNA synthesis reaches a maximum (unpublished data). The absorption and incubation were carried out at 37 C (wild-type virus) or at 27 to 30 C (ts2). Cultures infected by ts2 at a low temperature were shifted to 37 C at 15 min before labeling with either amino acids or nucleosides. Amino acid labeling. At 4 to 5 h after infection, the medium was removed from infected monolayers, the cells were washed with lowamino acid medium (Eagle medium containing neomycin, HEPES, 3% dialyzed calf serum, 1 pg of actinomycin per ml and 1/10 the normal concentration of amino acids) and incubated for 1 h in low-amino acid medium. The medium was then removed and replaced with the same medium containing 10 to 20 uci of "4C-labeled "reconstituted protein hydrolysate" algae profile (Schwartz Mann, Orangeburg, N.Y.) per ml. The labeling period was ended, and the cells were fractionated as described below. At 5 h after infection, cell-specific protein synthesis is inhibited at

2 5365IOWSHOWITZ J. VIROL. least 85%;j,, and viral-specific proteini synthesis reaches a maximum, so that at least 60 to 70% of the total protein synthesis is viral-specific (14). Nucleoside labeling. At 4 to 6 h after infection, the medium was removed and replaced with the the same medium containing: [2-'4C]uridine, 50 mci/mmol; [5-3H]uridine, 30 Ci/mmol; or [8-3HJadenosine, 17 Ci/mmol (all three isotopes from Schwartz/Mann, Orangeburg, N.Y.). The high-molecular-weight radioactive RINA synithesized under these conditions (after treatmenit with actiniomycii 1)) is virtuially all viralspecific, as described previouisly (8) and showii in Fig. 1. At the appropriate time, the labeling period was ended, and the cells were fractionated as described below. Preparation of cytoplasmnic extracts. At the end of the labeling period the culture bottles were plunged into ice water. The cultures were rinsed twice with cold phosphate-btuffered saline anid once with cold isotonic buffer (0.14 MI NaCl; M MgCl2; 0.01 Ml Tris, ph 8.0). These solutions and all others used in the fractionation (except the Nonidet P-40 [NP40]) were sterilized by heat or treatment with diethyl pyrocarbonate ("Baycovin" from Naftone, Inc., Chicago, Ill.) to destroy ribonuclease. All manipulations were carried out at 4 C until sodium dodecyl sulfate (SDS) was added. The rinsed monolayers were dissolved in isotonic buffer containing 0.5% NP40 and centrifuged at 800 X g for 2 min to pellet the nuclei. The supernatant fluid constituted the cytoplasmic extract. Gradien-t analysis of polysomes. Cytoplasmic extracts were anialyzed for their polysome A 15-hA 42S 26S - B 26S 0.i in A Ii lo ~- Gel Slice Number FIG. 1. Polyacrylambide gel electrophoresis of RNA from sucrose gradient fractions. Fractions 2 to 7 (cores and polysomes) and fractions 8 to 10 (66S particles) of the gradietnts shown in Fig. 3 were pooled, and the RNA was analyzed on polyacrylamide gels as described in Materials and Methods. The two samples shown in each panel were antalyzed separately and the patterns were aligned by the position of the marker in each gel. Panel A, RNA from the core-polysome region of the gradients: control (@); treated with EDTA; (0). Panel B, RNA from 65S particles: control (-); treated with EDYTA (0). content by centrifugation through 7 to 47% (wt/wt) sucrose in isotonic buffer at 4 C. The centrifugation was for 100 min at 39,000 rpm in a Spinco SW40 rotor or 16 h at 13,000 rpm in a Spinco SW27 rotor. The shorter time was used whenever RNA was to be recovered from the extracts. The gradients were collected by pumping the material through a Gilford recording spectrophotometer into tubes in ice (if the RNA was to be rerun) or into tubes containing 1 to 2 drops of 20% SDS. One of the following procedures was then used: (i) each fractioni was precipitated with cold 10% trichloroacetic acid in the presence of 0.05 mg/ml bovine serum albumin and collected onto membrane filters (Millipore Corp.); or (ii) a small part of each fraction was placed onto a piece of filter paper and precipitated on the paper with 10% trichloroacetic acid. The papers or filters were washed with 5% trichloroacetic acid, the papers were washed with ethanol, and both were dried and counted in toluene-based scintillation fluid. Gradient analysis of ribonucleoprotein derived from polysomes. The appropriate fractions of a 7 to 47% sucrose gradients were collected in ice, pooled; and mixed with 1.5 times their volume of EDTA-containing buffer (0.01 M EDTA; 0.2 M NaCl; 0.01 M Tris, ph 7.2). All or part of the mixture was then analyzed by centrifugation through 20 to 40% sucrose in EDTA-containing buffer for 14.5 h at 22,000 rpm in a Spinco SW25.3 rotor at 4 C. The gradients were collected, and the radioactivity was counted as described above. Preparation of RNA for gel electrophoresis. Cytoplasmic extracts containing labeled RNA were adjusted to 0.5 to 2%7o SDS, 0.05 M sodium acetate, 0.01 M EDTA, and were phenol-extracted at 50 C by the method of Warner et al. (15). The extract was made 0.1 M in NaCl, and the RNA was precipitated by the addition of 2 vol of ethanol and stored at -20 C until needed. Fractions of sucrose gradients containing labeled RNA were collected into tubes containing 1 to 2 drops of 20% SDS, and yeast RNA was added to an approximate concentration of 0.1 mg/ml. In some experiments, the RNA was precipitated by the addition of 2 vol of ethanol and stored at -20 C until needed. In other experiments, the fractioils were made 0.01 M in EDTA and 0.05 M in sodiurmi acetate. One vol of chloroform anid 1 vol of phenol were added, and the fractions were stored at -20 C prior to phenol extraction and precipitation with ethanol as described above. Polyacrylamide gel electrophoresis of RNA. RNA precipitates were collected by centrifugation and dissolved in a mixture containing 20% glycerol, 0.5% SDS, and bromophenol blue. Samples (10- to 50-Aliter) were mixed with 5- to 10-,liter samples of marker ([14C]uridine labeled RNA from infected cell extracts) and analyzed on 2.4% polyacrylamide gels containing 0.5% agarose and SDS essentially as described by Levin and Friedman (8), except that the gels and the buffers contained 10% glycerol. Only single-stranded RNA

3 VOL. 11, 1973 molecules migrate significant distances into the gels under the conditions used. The gels were frozen in a hexane dry-ice bath and sliced (16). The slices were hydrolyzed at 60 C for 90 miii in a Protosol (New England Nuclear Corp.) -based mixture (Protosol-water-toluene, 9: 1: 10), cooled to 0 C anid counited in toluene-based scintillation fluid. Polyacrylamide gel electrophoresis of protein. Fractions of sucrose gradients containing protein were pooled and made 10% in trichloroacetic acid. The resulting precipitates were collected by centrifugation, washed with 5% trichloroscetic acid, then 1% trichloroacetic acid. and then acetone. The dried precipitates were then suspended in M sodium phosphate buffer, ph 7.0; SDS was added to 1%, and mercaptoethanol was added to 0.1%. The samples were heated at 37 to 60 C for approximately 1 h until dissolved. Then they were analyzed on 7.5% acrylamide gels containing glycerol and overlayered with glutathione as described by Strauss et al. (14). RESULTS Preparation of polysomes from infected cells. It is notoriously difficult to prepare undegraded polysomes from monolayer cells infected with arboviruses (5). Therefore the first experiments were designed to demonstrate that relatively intact polysomes could be prepared from BHK cells infected with Sindbis virus by using our cell fractionation procedure. Infected monolayers were exposed briefly to 14C-amino acids to label nascent polypeptide chains; the monolayers were washed and dissolved in isotonic buffer (ph 8) containing 0.5% NP40. The nuclei were removed by low-speed cenitrifugation, and half the cytoplasmic extract was layered on a 7 to 47% sucrose gradient. The remaining half was treated with EDTA to disrupt the polysomes (4) and then layered on a gradient. The gradients were then centrifuged under conditions designed to separate monosomes, subribosomal particles, and polysomes from each other, so that any degradation of polysomes to monosomes would be clearly evident. As shown in Fig. 2, a large part of the radioactivity incorporated in a 90-s pulse of amino acids was associated with very large structures that were disrupted by EDTA. Very little radioactivity was associated with single ribosomes, indicating that the extracts contained undegraded polysomes. To confirm that the very large structures were indeed polysomes, two infected monolayers were exposed to [UIC]amino acids as above, and one monolayer was exposed to puromycin for 5 min to release the nascent peptide chains from polysomes (4). Cytoplasmic extracts were prepared from the monolayers and analyzed on gradients RNA IN BHK CELLS 0~ a. I-) H L._ a z *YL 300 _ 200 _ 100 _ I() 20 r FrGoctioil Number 537O P FIG. 2. Sucrose gradien2t analysis of cytoplasmic extracts from infected cells pulse-labeled with [14CJamino acids. A BHK monolayer infected with Sindbis virus was labeled for 90 s at 6 h after intfectiont with 20 JACi of a [1'C] aminzo acid mixture per ml. An extract was prepared from the mionolayer, onie-half was treated with EDTA (final contcn, M), and both halves of the extract were an alyzed onl 7 to 47% sucrose gradienlts for 16 h as described int Materials and Methods. Sedimentation was from right to left. Control extract (0); extract treated with EDTA (0). as in the previous experiment. As shown in Fig. 3, most of the radioactivity associated with large structures was released by puromycin, indicating that the large structures were polysomes. They were assumed to be mostly viral polysomes, because most of the protein synthesized under these labeling conditions is viral-specific (14). In the infected cells treated with puromycin and the extracts treated with EDTA, some radioactivity remained associated with particles of about 1408 (Fig. 2 and 3). These particles are viral nucleocapsids or cores containing 42S RNA and the inner protein of the virion. Cores are assumed not to be disrupted by EDTA because they have been prepared previously in large amounts from cytoplasmic extracts treated with EDTA (3, 17) and because the amount of core protein associated with the particles in cytoplasmic extracts is not affected by EDTA treatment (unpublished data). Identification of RNA in polysomes, cores, and 65S particles. To identify the species of RNA associated with viral polysomes, a cytoplasmic extract was prepared from infected cells

4 538 MOWSHOWITZ J. VIROL. X6, ,000_ E Fraction Number FIG. 3. Sucrose gradient analysis of cytoplasmic extracts from infected cells pulse-labeled with [14C]_ amino acids in the presence or absence of puromycin. BHK monolayers infected with Sindbis virus were labeled for 90S at 6 h after infection with 10 IACi of a ["4Clamino acid mixture per ml. One monolayer was fractionated immediately. The other was treated with 200s g of puromycin per mlfor 5 min (still in the presence of the label). Extracts were prepared from both monolayers and analyzed on 7 to 47% sucrose gradients for 16 h as described in Materials and Methods. Sedimentation was from right to left. Control (@); puromycin treated (0). (treated with actinomycin) anid labeled with [3H]uridine for 1 h late in infection. The extract was divided in half. One half was treated with EDTA to disrupt the polysomes and both halves were layered on 7 to 47% sucrose gradients and centrifuged as before. The results are shown in Fig. 4. The control extract contained a broad peak of radioacitivity in the region of the gradient that contained the polysomes. About half of the radioactive RNA in this peak was released by EDTA into particles sedimenting at about 658; this RNA was assumed to be polysomal. The remaining RNA sedimented as 140S particles after EDTA treatment; it was assumed to be in cores. The RNA from the 65S particles, the 140S particles, and the broad peak present before EDTA treatment (presumably a mixture of cores and polysomes) were analyzed on polyacrylamide gels. The broad peak of polysomes and cores contained 42S RNA, 26S RNA, and a small amount of a third species, 33S RNA (Fig. 1). This minor species has been seen before in arbovirus-infected cells (7, 8); its significance is discussed in detail below. Most of the 26S and 33S RNA associated with the polysomes and cores was released by EDTA (1A) so that both 26S and 33S RNA appeared to be mrna species. Virtually 0. I U re) la CK c H c4, D 2000 r S I A 5 10 Fraction Number FIG. 4. Sucrose gradient analysis of cytoplasmic extracts from infected cells labeled with ['H]uridine. A monolayer infected with Sindbis virus was labeled from 4 to 5 h after infection with 50 pci of [(Hluridine per ml. An extract was prepared from the monolayer and half was treated with EDTA (final concn, M). Both halves were then analyzed on 7 to 47% sucrose gradients for 100 min as described in Materials antd Methods. Sedimentation was from right to left. A 0O-pliter portion of each fraction was precipitated on filter paper as described in Materials and Methods. Control extract (0); extract treated with EDTA (0). all of the 42S RNA plus a small amount of 26S RNA remained associated with cores in this and all other experiments. The significance of the core-associated 26S RNA is not known, but this 26S RNA cannot be a breakdown product of 42S RNA, because the recovery of 42S and 26S RNA is not affected by EDTA treatment (Table 1). As shown in Fig. 1B, the 65S particles generated by EDTA treatment contained primarily 2'6S and 33S RNA, so that the particles appeared to be generated from polysomes. Similar particles containing heterodisperse RNA with an average sedimentation coefficient of 26S have been observed before in extracts treated with EDTA (5, 17). These particles were probably generated from polysomes also, but contained degraded RNA. The 65s particles contained a trace amount of 42S RNA. This 42S RNA was assumed to be a contaminant, since (i) the amount of 42S RNA associated with 65S particles was so small, (ii) I I

5 VOL. VINA 11, 1973 IN BHK CELLS TABLE 1. Polyacrylamide gel analy8is of RNA species from polysomes, cores, and 65S particlesg Fraction of cytoplasmic extract analyzed RNA speciesb (counts/min) 42s 26s Polysomes + cores. 98, , particles... 7,185 80,053 Cores... 93,100 23,200 A mixture of cores and polysomes was prepared, treated with EDTA, and separated into cores and 65S particles as described in the legend to Fig. 5. The RNA from portions of the original mixture (cores + polysomes), the cores (+ pellet), and the 65S particles was analyzed on polyacrylamide gels as described in Material and Methods. b The total counts/min present in each fraction of the entire gradient as calculated from the counts/min recovered from each gel. the amount was unaffected by EDTA (or other treatments), and (iii) the method of preparation used did not completely separate cores from 658 particles (Fig. 4). Origin of 65S particles. To show directly that 658 particles were generated from polysomes, the EDTA treatment described above was repeated in a slightly different way. Infected cells were labeled with ['H]uridine for 1 h late in infection, and the extract was centrifuged on a 7 to 47% sucrose gradient (Fig. 5). The middle portion of the gradient corresponding to the peak of cores and polysomes was pooled, treated with EDTA, and centrifuged on a gradient containing EDTA. A portion of the RNA in the pooled peak of cores and polysomes and in the two peaks and pellet from the EDTA gradient was analyzed on polyacrylamide gels. The results (Table 1) showed the following. (i) The amount of 26S RNA recovered from 65S particles corresponds to the amount lost from the polysome region, indicating that the 65S particles and the 268 RNA they contain are generated from structures the size of polysomes. (ii) No 42S is lost during the treatment with EDTA, thus ruling out the possibility that 42S RNA is converted to 26S RNA during the treatment with EDTA. (iii) No significant amounts of 42S or 26S RNA are generated during the treatment with EDTA, indicating that singlestranded RNA species are not being generated from double-stranded replicative structures (which do not enter the gels). To confirm that 65S particles were generated from polysomes and not other large structures, two monolayers were labeled with ['H]adenosine a 200 V ro 0 j 539n FRACrION NUMBER FIG. 5. EDTA-sucrose gradient analysis of the polysome region of a cytoplasmic extract. A cytoplasmic extract was prepared from an infected monolayer labeled with 50 pci of ['Hluridine per ml from 4 to 5 h after infection. The extract was analyzed on a 7 to 47% sucrose gradient for 100 min as described in Materials and Methods. The top of this gradient was discarded and the remainder collected in three equal fractions. The fraction containing most of the cores and polysomes (the portion of the gradient corresponding to fractions 4.5 to 7 of the control extract in Fig. 4) was diluted with EDTAcontaining buffer, and 0.9 of the total was analyzed on 20 to 40o sucrose gradients containing EDTA as described in Materials and Methods. for 2 h late in infection. One monolayer was treated with puromycin during the last 10 min of labeling. Cytoplasmic extracts were prepared from both monolayers and analyzed on sucrose gradients as before. As shown in Fig. 6, puromycin generated 65S particles. The RNA in the particles was analyzed on polyacrylamide gels; as in previous experiments, it was mostly 26S RNA plus a small amount of 33S RNA. 42S RNA amociated with polysomes. The next experiment was done to determine if 42S RNA was associated with polysomes late in infection. In all the experiments described above, the RNA in the cores and structures heavier than cores was combined prior to gel electrophoresis. When this was done, the 42S RNA did not appear to be mrna, because the amount recovered as cores and heavier structures was unaffected by EDTA or puromycin. A different picture emerged when the RNA in the cores and heavier structure was analyzed separately (Fig. 7). In this experiment, an infected monolayer was labeled with

6 540 MOWSHOWITZ J. VIRWOL. F 65S 0- o0 42S ou 2 extracts of infected cells labeled with ['H]adeno8ine in the presence and absence of puromycin. Two infected monolayers, A and B, were labeledfrorn 4 to 6 h after infection with [1H]adenosine. (A, 300 ;&Ci/ml; B, 60,uCi/ml). Puromycin (200 ;&g/ml) was added to B during the last 10 min of the labeling period. Cytoplasmic extracts were prepared from both monolayers. One-tenth of the extract from A and all of the extract from B were analyzed on 7 to 47% sucrose gradients for 100amin as described in Materials and Methods. A 10-,uliter portion of each fraction was precipitated on membrane filters (Millipore Corp.) and counted as described in Materials and Methods. A, Control extract (a); B, extract of cells treated with puromycin (O). [Iffluridine for 1 h late in infectioni. A cytoplasmic extract was prepared, half was treated with EDTA, and both halves were layered on 7 to 47% sucrose gradients and centrifuged as described. The results were similar to those shown in Fig. 4. The RNA from the cores and that from structures heavier. than cores was pooled and analyzed on polyacrylamide gels. Both 42S RNA and 26S were released from structures heavier than cores by EDTA, so that both species appeared to be in polysomes (Fig. 7). However, the 42S RNA which was released was recovered from the core peak after EDTA treatment, suggesting that the released 42S RNA plus its associated protein sedimnented at about 140S a.5 l, SLICE NUMBER FIG. 7. Polyacrylamide gel electrophoresis of RNA from sucrose gradient fractions. An infected monolayer was labeled from 4.5 to 6.5 h after infection with 200 jci of [3H]uridine per ml. A cytoplasmic extract was prepared, half was treated with EDTA (final conen, M), and both halves were analyzed on 7 to 47% sucrose gradient8 as described in Materials and Methods. The gradients closely resembled those of Fig. $ and 5. The fractions containing the cores and heavier structures were pooled, and the RNA was analyzed on polyacrylamide gels as described in Materials and Methods. The two samples shown in each panel were analyzed separately, and the patterns were aligned by the position of the 42s marker in each gel. Panel A, RNA from structures heavier than cores: control *; treated with EDTA 0. Panel B, RNA from cores: treated with EDTA 0. and was indistinguishable from cores in these gradients. An identical result was obtained by using puromycin instead of EDTA. to disrupt the polysomes. It was concluded that a fraction of the 42S RNA in infected cells at the time of maximal RNA synthesis might be mrna; the rest was definitely in cores. RNA synthesis in cells infected with ts2. In the experiments described above, polysomal RNA was prepared by selectively disrupting the polysomes in a polysome-core mixture. The next series of experiments was designed to do the opposite (disrupt the cores and leave the polysomes intact). Since there was no simple way to do this by physical means, we examined RNA synthesis in cells infected by the temperaturesensitive mutant, ts2. Ts2 was chosen because it has been shown (11) that cells infected by this mutant continue to synthesize proteins and RNA at the nonpermissive temperature, but do not

7 VOL. 11, 1973 RNA IN BHK CELLS 541 assemble the components into cores or virions because of a defect in the core protein or in its processing (3, 17). To show that the infected cells contained polysomes at the nonpermissive temperature, cells were infected at 29 C and labeled briefly at 37 C with [14Clamino acids. Extracts of the cells were prepared and centrifuged on 7 to 47% sucrose gradients in the presence and absence of EDTA. The gradients looked exactly like those shown in Fig. 2; most of the radioactivity appeared to be incorporated into nascent chains associated with polysomes or into soluble protein. No radioactivity appeared in cores, but this could have been because of the short labeling time (Fig. 2). To confirm that the cells infected by ts2 contained no cores at the nonpermissive temperature, two monolayers were infected with ts2 at 29 C. One monolayer was shifted to 37 C late in infection and both were labeled for about 2 h with [3Hjuridine. Then cytoplasmic extracts were prepared, treated with EDTA, layered on 20 to 40% sucrose gradients containing EDTA, and centrifuged. These conditions were chosen to give a maximum separation of cores from 65s particles. No RNA was associated with cores in cells labeled at 37 C (Fig. 8). Since the cells infected by ts2 contained no cores at 37 C, they were labeled for a short time to analyze the polysomal RNA. The cells were infected with ts2 at 29 C, shifted to 37 C late in infection, and labeled for 0.5 h with ['Hiuridine. A cytoplasmic extract was prepared; half was layered on a 7 to 47% sucrose gradient, and half was treated with EDTA before layering. Both gradients were centrifuged as above (Fig. 9). As expected from the previous experiments, all of the RNA in the polysome region of the gradient appeared to be in polysomes and not in cores; i.e., it sedimented in the same region of the gradient as the polysomes before treatment with EDTA, and it sedimented as 65S particles after treatment with EDTA. The RNA in the whole cytoplasmic extract and in appropriate fractions of the gradients (pellet, core-polysome region, and 65S particles) was pooled, phenol extracted, and examined on polyacrylamide gels. As shown in Fig. 10, 26S RNA was the primary RNA species recovered from 65S particles and from intact polysomes untreated with EDTA or puromycin. The gels of the pellets are not shown because they contained very little 26S or 42S RNA. A small amount of 42S RNA was found in the polysome-core region of these gradients before and after treatment with EDTA. This viral RNA could have been in residual cores or in polysomes since both cores and disrupted polysomes containing 42S RNA appear to sediment at 140S. At the 1200r 900q tl,, k FRACTION NUMBER FIG. 8. Sucrose gradient analysis of cytoplasmic extracts from cells infected with tsf and labeled with ['H]uridine. Two infected monolayers were infected with tsf at 29 C. At 6.5 h after infection, one was 8hifted to 37 C, and both were labeled from 6.75 to 9 h after infection with 10 pci of ['H]uridine per ml. Cytoplasmic extracts were prepared from both monolayers, treated with EDTA (final concn, M) and centrifuged on 20 to 40%01 sucrose gradients containing EDTA for 14.5 h at 22,000 rpm in a Spinco SM25.8 rotor at 4 C. Sedimentation was from right to left. A 20-pliter portion of each fraction was precipitated on filter paper and counted as described in Materials and Methods. Labeled at 29 C (@); labeled at 37 C (0). time these experiments were started, there were conflicting reports (3, 17) regarding the amount of 42S RNA synthesized in ts2-infected cells. We had hoped that the cells would contain normal amounts of 42S RNA which was not in cores; it would then have been possible to establish the S value and intracellular location of "free" 42S RNA. However, the ts2-infected cytoplasmic extracts that we examined contained neither cores nor significant amounts of "free" 42S RNA. DISCUSSION The experiments described in this paper demonstrate that 26S RNA is the major species of polysomal RNA found late in infection in BHK cells infected with either wild-type Sindbis virus or a coreless mutant. Kennedy (7) recently showed that 26S RNA is the major species of RNA associated with membrane-bound polysomes in chick cells infected by a coreless mutant

8 542 MOWSHOWITZ J. VIROL S 2S a- Il 500 ~~~~~~~~42S 42S C.)~ \v< S Fraction Number FIG. 9. Su-crose gradient analysis of cytoplasmic extracts of cells infected with ts and labeled with [1H]uridine at the nonpermissive temperature. A monolayer was infected with ts2 at 29 C and shifted to 37 C at 4.75 h after infection. The monolayer was then labeled with 100,uCi of ['H]uridine per ml from a to 6.5 h after infection. A cytoplasmic extract was prepared fromn the cells, half was treated with EDTA (final concn 0.25 M), and both halves of the extract were analyzed on 7 to 47%0 sucrose gradients for 100 min as de-scribed in Materials and Methods. A 20- pliter portion of each fraction Wfl8 precipitated on mekmbrane fflters (Millipore Corp.) and counted as described. Control extract (a); extract treated with EDTA (O). of Semliki Forest virus (a type A arbovirus). We have found that 26S RNA is also the major species of polysomal RNA in Chinese hamster ovary (CHO) cells infected by wild-type Sindbis vii (unpublished data). Since 26S RNA is associated with polysomes in all three cases and since approximately 80% of the 26S RNA in the infected BHK and CHO cells is in polysomes, a major function of interjacent RNA must be to act as mrna in arbovirus-infected cells. Since 26S RNA is the major mrna species present during the period when virion protein synthesis is maximal, it seems likely that 26S RNA is the mrna for the structural proteins of Gel Slice Number FIG. 10. Polyacrylamide gel electrophoresis of sucrose gradient fractions. Fractionts I to 8 (cores and polysomes) and 9 to 15 (65s particles) of the gradients shown in Fig. 9 were pooled. The RNA was p4enol extracted and analyzed on polyacrylamide gels as described in Materials and Methods. The two samples shown in each panel were analyzed separately, and the patterns were aligned by the position of the 26SRNA marker in each gel. Panel A, RNA from cores and polysomes: control (0); treated with EDTA (0). Panel B, RNA from 65s particles: control (0); treated with EDTA (0). the virion. The 268 RNA is large enough (1.8 X 106 daltons) to code for all three known virion proteins (combined molecular weight = 128,000; reference 12), or for the polypeptide which is suspected to be precursor to all three (estimated molecular weight = 130,000; reference 9). Any function which is assigned to 26S RNA must be consistent with what is already known about the Sindbis mutant ts24, which is temperature sensitive for initiation of all RNA synthesis and for continuation of 26S RNA synthesis. It is not temperature-sensitive for continuation of 42S RNA synthesis. The simplest interpretation of this result is that initiation of 42S RNA synthesis is dependent on prior synthesis of a protein encoded by 26S RNA and that synthesis of 26S RNA is in turn dependent on a protein which is defective in ts24. The 26S RNA is certainly the major RNA species associated with arbovirus-specific polysomes late in infection, but it is probably not the only polysomal RNA in infected cells. Two other RNA species, 42S RNA and 33S RNA, also seem to be associated with polysomes. Viral RNA (42S) must act as mrna at the start of infection because Sindbis virus RNA is infectious. Viral RNA appears to be polysomal late in infection because it is found in structures larger than cores which are disrupted by puromycin and EDTA. However, the RNA released from polysomes by these reagents cannot be

9 VOL. 1 l, 1973 RNA IN BHK CELLS 543 recovered after treatment in a structure distinct from cores. The 33S RNA is a min;or species which is founid in very small amounts in infected cells. It appears to be polysomal because it is released from large structures by both EDTA and puromycin. However, the amount of 33S RNA recovered in the cytoplasmic extracts and its distribution throughout the various fractions of the gradients varied from experiment to experiment. In some cases the 33S RNA was found in the polysomecore fraction, in others in the pellet of the gradient (all in the absence of EDTA). One possible explanation is that the 33S RNA is associated with polysomes that are incompletely released from membranes by NP40, so that variable amounts are discarded with the nuclei and the remainder is partitioned between the pellet and polysomes. This seems a reasonable explaniation, because Kennedy has found that small amounts of 33S RNA are associated with membrane bound-polysomes in Semliki-infected chick cells. ACKNOWLEDGMENTS I thank James E. Darnell for many valuable suggestions and discussions and Lois Williams for technical assistance. This work was supported by Public Health Service grant CA from the National Cancer Institute and grants GB27691X and VC1O1B from the National Science Foundation and from the American Cancer Society, respectively. LITERATURE CITED 1. Arif, B. M., and P. Faulkner Genome of Sindbis virus. J. Virol. 9: Burge, B. W., and E. R. Pfefferkorn Isolation and characterization of conditional-lethal mutants of Sindbis virus. Virology 30: Burge, B. W., and E. R. Pfefferkorn Functional defects of temperature-sensitive mutants of Sindbis virus. J. Mol. Biol. 35: Darnell, J. E Ribonucleic acids from animal cells. Bacteriol. Rev. 32: Eaton, B. T., T. P. Donaghue, and P. Faulkner Presence of poly(a) in the polyribosomeassociated RNA of Sindbis-infected BHK cells. Nature N. Biol. 238: Kaariainen, L., and P. J. Gomatos A kinetic analysis of the synthesis in BHK 21 cells of RNAs specific for Semliki forest virus. J. Gen. Virol. 5: Kennedy, S. I. T Isolation and identification of the virus-specified RNA species found on membrane-bound polyribosomes of chick embryo cells infected with Semliki forest virus. Biochem. Biophys. Res. Commun. 48: Levin, J. G., and R. M. Friedman Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J. Virol. 7: Pfefferkorn, E. R., and M. K. Boyle Selective inhibition of the synthesis of Sindbis virion proteins by an inhibitor of chymotrypsin. J. Virol. 9: Scheele, C. M., and E. R. Pfefferkorn Inhibition of interjacent ribonucleic acid (26S) synthesis in cells infected by Sindbis virus. J. Virol. 4: Scheele, C. M., and E. R. Pfefferkorn Virus-specific proteins synthesized in cells infected with RNA+ temperature-sensitive mutants of Sindbis virus. J. Virol. 5: Schlesinger, M. J., S. Schlesinger, and B. W. Burge Identification of a second glycoprotein in Sindbis virus. Virology 47: Sonnabend, J. A., E. M. Martin, and E. Mecs Viral-specific RNA in infected cells. Nature (London) 213: Strauss, J. H., B. W. Burge, and J. E. Darnell Sindbis virus infection of chick and h1amster cells: synthesis of virus-specific proteins. Virology 37: Warner, J. R., R. Soeiro, H. C. Birnboim, M. Girard, and J. E. Darnell Identification by zone sedimentation of a heterogeneous fraction separate from ribosomal precursor RNA. J. Mol. Biol. 19: Weinberg, R. A., U. E. Loening, M. Willems, and S. Penman Acrylamide gel electrophoresis of HeLa cell nucleolar RNA. Proc. Nat. Acad. Sci. U.S.A. 58: Yin, F. H., and R. Z. Lockart, Jr Maturation defects in temperature-sensitive mutants of Sindbis virus. J. Virol. 2: