JOURNAL OF VIROLOGY, Mar. 1977, p. 1105-1112 Copyright © 1977 American Society for Microbiology
Vol. 21, No. 3 Printed in U.S.A.
Characterization of Vesicular Stomatitis Virus mRNA Species Synthesized In Vitro D. P. RHODES, G. ABRAHAM, R. J. COLONNO, W. JELINEK, AND A. K. BANERJEE* Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110,* and Rockefeller University, New York, New York 10021
Received for publication 5 October 1976
The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed. Virions of vesicular stomatitis virus (VSV) ues and those expected based on the previously contain a single-stranded RNA genome with a reported molecular weights for the NS protein molecular weight of 3.6 x 106 to 4 x 106 and five (40,000 to 45,000) and M protein (29,000) is disstructural proteins designated L, G, N, NS, and cussed. M (24). Virus-specific mRNA isolated from infected cells (19) or synthesized in vitro by the MATERIALS AND METHODS virion-associated RNA polymerase (18) can be resolved by velocity sedimentation into four Virus and RNA. VSV (Indiana serotype) was size classes, which sediment at 31S, 17S, 14.5S, grown in baby hamster kidney cells (BHK-21, clone and 12S. In vitro translation of these purified 13), adapted to suspension culture, and purified as mRNA's has demonstrated that the 17S mRNA described previously (3). VSV genome RNA was codes for the G protein, the 14.5S mRNA codes extracted from purified virus with phenol-sodium dodecyl sulfate (SDS) and purified by velocity sedifor the N protein, the 12S mRNA codes for both mentation through sucrose gradients containing the NS and M proteins (8, 9, 15), and the 31S SDS. mRNA is believed to code for the L protein (17). 32P-labeled VSV mRNA was synthesized in vitro Past attempts to resolve the 12S mRNA into using one a-32P-labeled nucleoside triphosphate and two species have not succeeded, although se- three unlabeled nucleoside triphosphates as dequence complexity (23) and translation studies scribed previously (10). Methylated VSV mRNA was suggest that it may consist of two distinct synthesized in vitro using S-[methyl-3H]adenosylmethionine as the only labeled substrate (21). The mRNA species. In the present work, we show that in the in mRNA's were purified by SDS-phenol extraction, Sephadex G-50 chromatography, velocity sedimenvitro synthesized 12S mRNA can be separated tation through SDS-sucrose gradients, and chrointo two species by polyacrylamide gel electro- matography on oligo(dT)-cellulose columns, accordphoresis after first annealing the 12S mRNA to ing to previously published procedures (5, 11). genome RNA and then digesting away the sinRNA annealing. Two to five micrograms of unlagle-stranded regions of the resulting RNA du- beled genome RNA (0.05 to 0.125 optical density plexes with RNase T2. We have identified the units at 260 nm) was added to 0.1 to 0.25 ,±g of smaller mRNA component as the message for purified, labeled in vitro mRNA; The solution was the M protein and the larger mRNA as the adjusted to a final volume of 0.1 ml containing 0.3 M 0.03 M sodium citrate, pH 6.5, and incubated message for the NS protein. Comparison of the NaCl, at 72 to 74°C for 4 h, cooled to room temperature, and electrophoretic mobilities of VSV mRNA du- diluted with 0.1 ml of sterile water, the RNA was plexes with the double-stranded RNAs of the precipated with ethanol. reovirus genome gave molecular weight estiDigestion and analysis of annealed RNA. Anmates of 190,000 and 148,000 for the messages nealed RNA was collected by centrifugation and coding for the VSV proteins NS and M, respec- dissolved in 40 1.l of sterile water. After adding 5 Al tively. The apparent conflict between these val- of 100 mM sodium acetate, pH 4.5, 1 ,l of 2 M NaCl, 1105
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and 1 ,ul of RNase T2 (500 U/mi), the samples were incubated at 370C for 30 min. Digestion was stopped by adding 5 1.l of 10% SDS, and the samples were prepared for electrophoresis by adding 1 Al of 1 M Tris-hydrochloride, pH 8.0, 2 pl of bromophenol blue, and 25 pul of glycerol. Electrophoresis was on 5% polyacrylamide gels (0.6 by 10 cm) as described previously (6) for 16 to 19 h at 4 mA/gel, and gels were fractionated into 1-mm slices with a Gilson automatic gel crusher. Fractions from gels containing 3H radioactivity were incubated overnight at 50'C in 0.6 ml of 15% H202, and radioactivity was assayed in 10 ml of ACS (Amersham/Searle) scintillation cocktail. When :12P-labeled RNA was to be recovered from the gel slices, each fraction was suspended in 0.8 ml of 0.5 M NaCl, 0.1% SDS, 20 mM Tris-hydrochloride, pH 7.5, and 10 mM EDTA, and radioactivity was determined by Cherenkov counting. The fractions containing the peak of 32P radioactivity were then incubated for 24 h at 37TC with gentle shaking, gel particles were removed by centrifugation, and the supernatants were concentrated by vacuum dialysis against 0.1% SDS; the RNA was recovered by precipitation with ethanol. Nearest-neighbor analysis. a- [32P]AMP-labeled RNA was hydrolyzed for 18 h at 37°C with 0.3 N KOH, and each digest was neutralized with Dowex 50 (H +) and analyzed by high-voltage paper electrophoresis as described previously (4). Two-dimensional fingerprinting. a-[32P]GMP-labeled duplex RNAs were eluted from gels as described above, denatured by incubation for 30 min at 37°C in 90% Me2SO, and reprecipitated with ethanol. These samples, and the corresponding single-stranded poly(A)-containing mRNA's, were digested with RNase T1, and two-dimensional fingerprints were prepared as described by Barrell (7). Chemicals and enzymes. a_-32P-labeled ribonucleoside triphosphates and S-[methyl-3H]adenosylmethionine were purchased from New England Nuclear Corp. (Boston, Mass.). RNases T2 and T1 (produced by Sankyo Co., Japan) were purchased from Calbiochem (San Diego, Calif.). ACS scintillation cocktail is a product of Amersham/Searle (Arlington Heights, Ill.).
RESULTS Separation of 12S mRNA into two components. VSV mRNA was synthesized in vitro using 4-[:12P]ATP as the labeled substrate, and the poly(A)-containing 12S and 14.5S mRNA's were purified by sedimentation through sucrose gradients and chromatography on oligo(dT)-cellulose. Each of the purified mRNA's was then annealed to VSV genome RNA, digested with RNase T2, and analyzed by electrophoresis on 5% polyacrylamide gels (Fig. 1). The duplexes derived from the 12S mRNA were resolved into two major components, whereas the duplex from the 14.5S mRNA migrates as a single major band with a lower electrophoretic mobility. Similar experiments with purified 17S and 31S in vitro mRNA's each showed the presence
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of a single band of duplex RNA with respectively lower electrophoretic mobilities (see Fig. 2 and 3). These higher-molecular-weight duplexes usually showed a small amount of radioactivity migrating at the same rates as the duplexes derived from the 14.5S and 12S mRNA's, indicating that they are contaminated with varying amounts of the smaller messages, presumably aggregates, which are not removed during the sucrose gradient purifi-
cations (data not shown). Absence of poly(A) after T2 digestion of annealed mRNA's. A possible reason for the presence of two duplexes derived from the 12S mRNA detected in polyacrylamide gels could be due to failure of RNase T2 to remove the poly(A) from all of the annealed mRNA molecules. Thus, the faster-migrating band could correspond to duplexes from which the poly(A) had been removed, whereas the slow-moving band could correspond to duplexes that had retained a single-stranded poly(A) "tail." The absence of slower-moving duplexes in digests of the annealed 14.5S, 17S, and 31S mRNA's argues against this possibility. More direct evidence for two species in the 12S size range was provided by determining the nucleotide compositions of alkaline digests of each of the a[32P]AMP mRNA duplexes and comparing these with the compositions of alkaline digests of the same messages either lacking or containI
3
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0 v
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20
60 40 MIGRATION (mm)
80
100
FIG. 1. Separation of two duplex RNAs derived from VSV in vitro 12S mRNA by polyacrylamide gel electrophoresis. VSV mRNA was synthesized in vitro in the presence of a-[32P]ATP, and the 12S and 14.5S mRNAs were purified by sedimentation twice through SDS-sucrose gradients and by chromatography on oligo(dT)-cellulose. Portions of the purified 12S and 14.5S were annealed to VSV genome RNA, digested with RNAse T2, and analyzed by electrophoresis on 5% polyacrylamide gels for 16 h at 4 mA! gel. The brackets indicate gel fractions that were pooled for nearest-neighbor analysis (see Table 1). Symbols: 0, duplexes prepared from 12S mRNA; 0, duplexes prepared from 14.5S mRNA.
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VSV mRNA SPECIES
ing poly(A). The results of these nearest-neighbor transfer analyses are shown in Table 1. Digests of the poly(A)-containing mRNA's (Table 1, lines 2 and 5) contained 52 to 54% 3'AMP, reflecting the contribution ofthe poly(A). However, analysis of the corresponding RNA duplexes after RNase digestion to remove single-stranded regions, including poly(A), showed that the relative amount of ApA sequences decreased to 29 to 32% (Table 1, lines 3, 6, and 7). These values are similar to the 29% value (Table 1, lines 1 and 4) found for the poly(A)-lacking mRNA's that had not bound to oligo(dT)-cellulose during the purification of
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the mRNA's. The results indicate that the two RNA duplexes derived from the 12S mRNA do not differ substantially in their poly(A) content.
Identification of the mRNA species coding for the M and NS proteins. The presence of two different mRNA molecules in the 12S mRNA fraction is in agreement with the previous evidence that the VSV 12S mRNA codes for two viral proteins, M and NS (8, 9). To identify which of the two 12S mRNA's codes for each of these proteins, we have taken advantage of the fact that each VSV gene has a different sensitivity to UV irradiation (1, 2). Using a coupled transcription-translation system, Ball and White (2) have shown that expression of the NS gene was more resistant to UV irradiation than was expression of the M gene. We have con-
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I
I R
xL
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MIGRATION (mm)
FIG. 2. Molecular weight determination of RNA duplexes derived from five VSV in vitro mRNA's. VSV mRNA was synthesized in vitro using [32P]CTP as the labeled precursor, and the product was purified by oligo(dT)-cellulose column chromatography and SDS-sucrose gradient sedimentation to isolate four size classes of poly(A)-containing mRNA. Approximately 104 cpm each of the 31S, 1 7S, and 14.5S and 2 x 104 cpm of the 12S mRNA species were mixed together and annealed to VSV genome RNA. After adding 10O cpm of [3H]uridine-labeled reovirus double-stranded RNA, the mixture was digested with RNAse T2 and analyzed by gel electrophoresis as described in Materials and Methods. Symbols: *, 32P-labeled VSV RNA duplexes; 0, 3Hlabeled reovirus double-stranded RNA. a-
MIGRATION (mm)
FIG. 3. Retention of methyl-3H-labeled 5'-termini of VSV in vitro mRNA's during preparation of duplexes. Methylated VSV mRNA was synthesized in vitro in the presence of S-[methyl-3H]adenosylmethionine and purified by phenol extraction and chromatography on Sephadex G-100 and oligo(dT)cellulose columns. The poly(A)-containing 3Hlabeled RNA was used to prepare RNA duplexes as described in Materials and Methods and analyzed by electrophoresis for 19 h on a 5% polyacrylamide gel.
TABLE 1. Nearest-neighbor analysis of a-[32P]AMP-labeled mRNA species Total cpm Sample prepn CpA (%) ApA (%) GpA (%) UpA %) a 14.5S 23.9 28.6 30.2 17.4 38,817 b 52.4 14.5S 16.4 19.2 61,814 11.9 c Duplex 3 26.6 31.8 26.2 15.4 7,204 a 12S 23.0 28.8 30.5 17.7 34,459 b 12S 16.4 53.6 18.7 11.5 47,550 c 34.4 Duplex 4 19.4 29.1 17.1 6,690 24.4 30.9 26.4 c Duplex 5 18.3 4,664 a 14.5S and 12S mRNA's labeled with a-V32P]AMP were fractionated on oligo(dT)-cellulose into poly(A)lacking (preparation a) and poly(A)-containing (preparation b) components. RNA duplexes (preparation c) prepared from poly(A)-containing mRNA's were eluted from the gel fractions indicated by the brackets in Fig. 1. Each RNA was hydrolyzed with KOH, and the resulting 3'-nucleoside monophosphates were analyzed by paper electrophoresis (4). RNA species
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firmed this result by translation of purified 12S logarithms of their molecular weights (20). We mRNA's synthesized by UV-irradiated VSV have used this relationship to estimate the mo(data not shown). To identify which of the two lecular weights of the transcribed portion [not 12S mRNA's codes for each of these proteins, including the poly(A)] of each of the five VSV mRNA's after first converting the messages we have determined the relative resistances of duplex 4 and duplex 5 (and, therefore, their into the corresponding double-stranded RNAs, as described above. VSV mRNA was synthecorresponding mRNA's) to UV irradiation. Identical samples of purified VSV were ex- sized in vitro using a-[32P]CTP as the labeled posed to UV irradiation for various times and precursor, and the individual poly(A)-containthen used to synthesize mRNA in vitro with a- ing mRNA's were purified as before. Portions of [32P]UTP as the labeled substrate. Each mRNA the purified mRNA's were then recombined to product was purified by oligo(dT)-cellulose provide a mixture containing equal amounts of chromatography and sucrose gradient sedimen- 32p radioactivity in each of the five mRNA's. tation to isolate the poly(A)-containing 12S This mixture was annealed with an excess of mRNA molecules. As expected, the higher genome RNA and, after adding [3H]uridinedoses of UV irradiation resulted in a decrease labeled reovirus genome RNA as internal moin the amount oftotal RNA synthesized, as well lecular weight markers, the mixture was dias a decrease in the relative amount of mRNA gested with RNase T2 and analyzed by polysedimenting at 12S (data not shown). Each acrylamide gel electrophoresis (Fig. 2). In addipoly(A)-containing 12S mRNA was annealed tion, four separate gels of the individual 31S, with an excess of unlabeled genome RNA, di- 17S, 14.5S, and 12S mRNA species were run in gested with RNase T2, and analyzed by electro- parallel (data not shown). The results showed phoresis on a 5% polyacrylamide gel, with a that RNA duplexes 1, 2, and 3 are derived from result similar to that shown in Fig. 1. Table 2 the 31S, 17S, and 14.5S mRNA's, respectively, shows the ratios of the duplex 4 RNA to the and duplexes 4 and 5 are both derived from the duplex 5 RNA with increasing doses of UV 12S mRNA. irradiation. The results clearly indicate that When the logarithm of the molecular weight the synthesis of duplex 4 RNA was more re- of each of the 10 reovirus double-stranded RNA sistant to UV irradiation, since there was more segments is plotted as a function of the migrathan a 50% increase in the ratio after 21 s of UV tion of that segment in the gel shown in Fig. 2, exposure compared with the unirradiated control. From these results, coupled with the obTABLE 3. Molecular weights of VSV mRNA and servation made by Ball and White (2), we conprotein species clude that the mRNA of duplex 4 codes for the Mol wt (x 10-6) Coding Proteins viral NS protein and the mRNA in duplex 5 Duplex potencodes for the viral M protein. RNA tialb Molecular weights of VSV mRNA's. The species dsRNAa ssRNA (mol wt Species (Mlw1tc Xl (l ) 103) relative mobilities of double-stranded RNAs during electrophoresis in polyacrylamide gels is 1 3.776 1.89 L 210.0 190 2 1.141 0.571 63.4 G 69 approximately inversely proportional to the TABLE 2. Effect of UV irradiation on transcription of the 12S mRNA species Duration of UV dose
32P-labeled double-stranded RNA
(RNA duplex 4/RNA
duplex 5)
0 7 14 21
1.39 1.64 1.94 2.17
a Samples of purified VSV were irradiated with UV light for 0, 7, 14, or 21 s (2) and then used to synthesize mRNA in vitro in the presence of a[32P]UTP; the poly(A)-containing 12S mRNA was purified from each product. Each sample was used to prepare duplexes, and the relative amounts of 32P in duplex 4 and duplex 5 were determined after electrophoresis through a 5% polyacrylamide gel as in Fig.
1.
3 4 5
0.739 0.385 0.298
0.369 0.192 0.149
N 41.0 50 21.4 NS 40-45 16.6 M 29 a The molecular weights (x 10-6) of doublestranded (ds) reovirus genome RNA segments used for the determination of the molecular weights of VSV duplex RNAs are as follows: L3 = 2.79; L2 = 2.71; L, = 2.55; M3 =1.62; M2 = 1.55; Ml = 1.46; S4 = 0.88; S3 = 0.75; S2 = 0.65; S, = 0.61. (The L, M, and S denote the large, medium, and small genome RNA segments of reovirus.) The molecular weights of the VSV duplex RNA species were calculated from the straight line giving the least-squares best fit for the log molecular weights of the 10 reovirus dsRNA's (see Fig. 1). bCoding potential of each mRNA species was calculated as one-ninth the corresponding singlestranded (ss) RNA molecular weight. c The molecular weights of VSV proteins are from Wagner et al. (25).
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the 10 points fall very close to a straight line (data not shown). This line was used to calculate the molecular weight of double-stranded RNA corresponding to the migration in the gel of each of the five VSV duplex RNAs. The results are summarized in Table 3, along with the apparent molecular weights of the corresponding VSV proteins. The calculated molecular weights for duplexes 1, 2, and 3 are in reasonable agreement with the apparent molecular weights of the corresponding VSV proteins L, G, and N, respectively. However, the molecular weights calculated for the VSV duplex 4 and 5 RNAs are about one-half of the sizes needed to code for the VSV NS and M proteins, respectively. Several possible reasons for this apparent discrepancy are detailed in the Discussion. The most obvious possibility is that the annealed mRNA's have been overdigested during the RNase T2 treatment so that the doublestranded RNAs in duplexes 4 and 5 are shorter than the actual lengths of the mRNA's from which they were prepared [other than removal of the poly(A) from the 3'-ends of the mRNA's]. Protection of the 5'-termini of VSV mRNA's from RNase T2 after annealing to genome RNA. One way to test for the possibility of overdigestion was to follow the fate of the 5'-termini of the mRNA's. For this purpose, methylated VSV mRNA was synthesized in vitro using S-[methyl-3H]adenosylmethionine as the only labeled substrate so that only the 5'termini would be labeled (21). After the poly(A)-containing mRNA's were purified, they were annealed with excess unlabeled genome RNA, digested with RNase T2, and analyzed by polyacrylamide gel electrophoresis. The 3H radioactivity migrated through the gel in four bands (Fig. 3) corresponding to RNA duplexes 2, 3, 4, and 5 in Fig. 3. 3H radioactivity was not detected in the position of duplex 1 because the 31S mRNA is always synthesized in the smallest amount (18), and, furthermore, only the 5'termini of the mRNA's are labeled in this experiment. Nearly 80% of the 1H radioactivity used for hybridization was recovered in the gel without making any allowance for the lower counting efficiency when counting sliced gels. Thus, virtually all of the 5'-termini of the messages must be retained under the RNase T2 digestion conditions used in these experiments. Two-dimensional fingerprints of mRNA duplexes eluted from gels. A second test for possible overdigestion of the RNA duplexes by RNase T2 was carried out by comparing twodimensional fingerprints of the T2-digested duplexes with the fingerprints of the purified RNA used to prepare the duplexes. mRNA was
VSV mRNA SPECIES
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synthesized in vitro using a-[32P]GTP as the labeled precursor, and the poly(A)-containing 12S mRNA species were purified. A portion of the mRNA was annealed to genome RNA, digested with RNase T2, and analyzed by electrophoresis on a 5% acrylamide gel as in Fig. 1. RNA duplexes 4 and 5 were eluted from the gels, denatured with Me2SO, and then digested with RNase T1. A portion of the purified 12S a[32P]GMP-labeled mRNA from the same in vitro RNA product was also digested with RNase T1 under identical conditions. A two-dimensional fingerprint was prepared frm each T1 digest using the procedure of Barrell (7). All of the large oligonucleotides (designated as spots 1 to 8) present in the T1 digest of the purified 12S mRNA (Fig. 4A) are present in either the fingerprint of RNA duplex 4 (spots 2, 4, 6 to 8) or the fingerprint of RNA duplex 5 (spots 1, 3, and 5) (Fig. 4C). Thus, with this sensitive fingerprinting technique, we could not detect the loss of any sequences from the mRNA molecules as a consequence of digesting the annealed mRNA's with RNase T2. However, it should be noted that extra spots appear in the digests of the RNA duplexes eluted from gels, especially in the smaller oligonucleotide region, apparently due to enzymatic contamination of these samples during the handling involved in elution from gels and denaturation. DISCUSSION The virion-associated RNA polymerase of VSV synthesizes multiple species of mRNA in vitro that code for the five virus structural proteins. Although the 31S, 17S, and 14.5S mRNA species appear to be monocistronic and code for the viral L, G, and N proteins, respectively, the 12S mRNA size class presumably includes two mRNA species of similar molecular weights and codes for both the M and NS proteins. In an attempt to separate these two mRNA species, we have taken advantage of the fact that double-stranded RNA molecules are better resolved by polyacrylamide gel electrophoresis than their single-stranded counterparts (13). Accordingly, 12S mRNA was hybridized with genome RNA and the resulting RNA duplexes obtained after treatment with RNase T2 were resolved in a 5% polyacrylamide gel. By this procedure, two distinct mRNA species were obtained (Fig. 1). From the relative resistance of their synthesis to UV irradiation (Table 2), we have demonstrated that the slower-moving RNA (duplex 4) codes for the viral NS protein and the faster-moving RNA (duplex 5) codes for the M protein. A similar technique for separating the multiple mRNA species of Newcastle disease virus has been reported (14).
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FIG. 4. Two-dimensional fingerprints of RNase Ti digests of 12S mRNA's. VSV mRNA was synthesized in vitro in the presence of a-[32P]GTP, and the poly(A)-containing 12S mRNA was purified as detailed in Materials and Methods. A portion of this mRNA was annealed to genome RNA and digested with RNase T2, and the products were resolved by electrophoresis to separate the component duplexes 4 and 5 as described in the legend to Fig. 1. The fractions containing RNA duplexes 4 and 5 were located by Cherenkov counting, pooled separately, and denatured with Me2SO. Each RNA sample was digested with RNase T1, and a twodimensional fingerprint was prepared as described by Barrell (7); the 32P-labeled oligonucleotides were located by autoradiography. First dimension, electrophoresis on cellulose-acetate at pH 3.5 (right to left); second dimension, homochromatography on DEAE thin-layer plates (from bottom to top). (A) Fingerprint of poly(A)-containing 12S mRNA before annealing to genome RNA. (B) Fingerprint of mRNA strand of RNA duplex 4. (C) Fingerprint of mRNA strand of RNA duplex 5. The numbers indicate which large oligonucleotides in (A) are derived from the mRNA strands of RNA duplex 4 or 5.
The molecular weights of the individual VSV mRNA duplexes were determined using the above method by comparing their electrophoretic mobilities to those of the 10 doublestranded genome RNAs of reovirus, whose molecular weights are known (16). The results clearly show that the molecular weights of the
single-stranded 31S, 17S, and 14.5S mRNA species (calculated as one-half of the molecular weights of the corresponding RNA duplexes) are compatible with their coding potentials (Table. 3). On the other hand, the molecular weights ofthe mRNA's coding for the NS and M proteins (RNA duplexes 4 and 5, respectively)
VSV mRNA SPECIES
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.
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.
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inconsistent with their coding assignments. These two mRNA molecules appear to be approximately one-half the size needed to code for proteins with the estimated molecular weights of the VSV NS (40,000 to 45,000) and M (29,000) proteins. We have considered several possibilities for this apparent discrepancy in molecular weights. (i) The most serious possibility could be that the RNA duplexes were overdigested during RNase T2 treatment. However, this seems unlikely since (a) the 3H-labeled reovirus double-stranded RNA used as molecular weight standards was present during the RNase T2 digestion and was not affected by the RNase treatment, and (b) the mRNA's coding for the proteins L, G, and N after duplex formation are are
1111
apparently not degraded since their molecular weights as duplexes are reasonably in agreement with the corresponding single-stranded molecular weights (Table 3). Moreover, the recovery of virtually all of the 5'-termini (labeled with methyl-3H groups) in the duplex RNAs (Fig. 3) shows that there is no reduction in length of the duplexes from this end of the molecules. Furthermore, the presence of all of the characteristic large oligonucleotides (Fig. 4) in two-dimensional T1 fingerprints of RNA duplexes 4 and 5 after elution from gels argues against this possibility. Similarly, no overdigestion was detected by comparing T1 fingerprints of RNA duplex 3 and the corresponding single-stranded 14.5S N protein mRNA (data not shown). (ii) We have used the linear relationship between the electrophoretic mobilities and the logarithms of the molecular weights of the reovirus genome double-stranded RNA segments to calculate the molecular weights of the VSV mRNA duplexes. A possibility exists that this relationship does not remain linear at the lowmolecular-weight region, and the extrapolation may not be valid. However, it is worthwhile to point out that if one calculates the molecular weights of the corresponding mRNA's predicted from the estimated molecular weights ofthe NS and M proteins, one finds that RNA duplex 5 should migrate no more than 6 mm farther than the smallest reovirus double-stranded RNA segment and, thus, would require very little extrapolation. Similarly, RNA duplex 4 would require no extrapolation since it would have an electrophoretic mobility within the range of the reovirus double-stranded RNA standards. (iii) A third possibility is that the estimated molecular weights of the mRNA's are correct and that the molecular weights of the proteins are erroneous. The phosphoprotein NS is known to migrate anomalously in SDS-polyacrylamide gels depending on the pH condition. Thus, its true molecular weight is still unknown. On the other hand, the M protein with an estimated molecular weight of 29,000 may actually represent a dimer. A number of proteins, particularly membrane proteins, have been shown to bind less than the normal amount of SDS, and some proteins behave primarily as dimers during SDS-polyacrylamide gel electrophoresis despite the use of totally denaturing conditions (12, 22). Although it is still unclear which of the possibilities is the cause for the discrepancy, we believe that the second and the third possibilities stated above need careful investigation. In addition, the mo-
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lecular weights of the mRNA's should also be determined by an independent (preferably chemical) technique. Finally, no mRNA species other than the five shown in Fig. 2 have been detected in any of these experiments. Therefore, it seems likely that these five mRNA species, in addition to the small "leader" RNA previously described (10), constitute the only transcription products made in vitro by the virion-associated RNA polymerase of VSV and that these six transcripts may account for virtually the entire coding capacity of the VSV genome RNA. ACKNOWLEDGMENT We thank Alba LaFiandra for kindly supplying us with 3H-labeled reovirus genome RNA.
11.
12. 13.
14.
15. 16.
LITERATURE CITED 1. Abraham, G., and A. K. Banerjee. 1976. Sequential transcription of the genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:1504-1508. 2. Ball, L. A., and C. N. White. 1976. Order of transcription of genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:442-446. 3. Banerjee, A. K., S. A. Moyer, and D. P. Rhodes. 1974 Studies on the in vitro adenylation of RNA by vesicular stomatitis virus. Virology 61:547-558. 4. Banerjee, A. K., U. Rensing, and J. T. August. 1969. Replication of RNA viruses. X. Replication of a natural 6S RNA by the Q13 RNA polymerase. J. Mol. Biol. 45:181-193. 5. Banerjee, A. K., and D. P. Rhodes. 1973. In vitro synthesis of RNA that contains polyadenylate by virionassociated RNA polymerase of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 70:3566-3570. 6. Banerjee, A. K., and A. J. Shatkin. 1970. Transcription in vitro by reovirus-associated ribonucleic acid-dependent polymerase. J. Virol. 6:1-11. 7. Barrell, B. G. 1971. Fractionation and sequence analysis of radioactive nucleotides, p. 751-779. In G. L. Cantoni and D. R. Davis (ed.), Procedures in nucleic acid research, vol. 2. Harper and Row, New York. 8. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975. Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 72:274-278. 9. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975. Translation and identification of the viral mRNA
17.
species isolated from subcellular fractions of vesicular stomatitis virus-infected cells. J. Virol. 15:1012-
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1019. 10. Colonno, R. J., and A. K. Banerjee. 1976. A unique
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23.
24.
RNA species involved in initiation ofvesicular stomatitis virus RNA transcription in vitro. Cell 8:197-204. Colonno, R. J., and H. 0. Stone. 1975. Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme. Proc. Natl. Acad. Sci. U.S.A. 72:2611-2615. Furthmayr, H., and V. T. Marchesi. 1975. Subunit structure of human erythrocyte glycophorin A. Biochemistry 15:1137-1144. Ito, Y., and W. K. Joklik. 1972. Temperature-sensitive mutants of reovirus. I. Patterns of gene expression by mutants of groups C, D, and E. Virology 50:189-201. Kaverin, N. V., 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. Knipe, D., J. K. Rose, and H. F. Lodish. 1975. Translation of individual species of vesicular stomatitis viral mRNA. J. Virol. 15:1004-1011. Martin, S. A., and H. J. Zweerink. 1972. Isolation and characterization of two types of blue tongue virus particles. Virology 50:495-506. Morrison, T., M. Stampfer, D. Baltimore, and H. F. Lodish. 1974. Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells. J. Virol. 13:72-72. Moyer, S. A., and A. K. Banerjee. 1975. Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus. Cell 4:37-43. Moyer, S. A., M. J. Grubman, E. Ehrenfeld, and A. K. Banerjee. 1975. Studies on the in vivo and in vitro messenger RNA species of vesicular stomatitis virus. Virology 67:463-473. Peacock, A. C., and C. W. Dingman. 1968. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 1:668-674. Rhodes, D. P., S. A. Moyer, and A. K. Banerjee. 1974. In vitro synthesis of methylated messenger RNA by the virion-associated RNA polymerase of vesicular stomatitis virus. Cell 3:327-333. Robinson, N. C., and C. Tanford. 1975. The binding of deoxycholate, Triton X-100, sodium dodecyl sulfate, and phosphatidylcholine vesicles to cytochrome b5. Biochemistry 14:369-378. Rose, J. K., and D. Knipe. 1975. Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species. J. Virol. 15:994-1003. Wagner, R. R. 1975. Reproduction of rhabdoviruses, p. 1-80. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R. MacLeod. 1972. Classification of rhabdovirus proteins: a proposal. J. Virol. 10:12281230.
Vol. 21, No. 3 Printed in U.S.A.
Characterization of Vesicular Stomatitis Virus mRNA Species Synthesized In Vitro D. P. RHODES, G. ABRAHAM, R. J. COLONNO, W. JELINEK, AND A. K. BANERJEE* Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110,* and Rockefeller University, New York, New York 10021
Received for publication 5 October 1976
The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed. Virions of vesicular stomatitis virus (VSV) ues and those expected based on the previously contain a single-stranded RNA genome with a reported molecular weights for the NS protein molecular weight of 3.6 x 106 to 4 x 106 and five (40,000 to 45,000) and M protein (29,000) is disstructural proteins designated L, G, N, NS, and cussed. M (24). Virus-specific mRNA isolated from infected cells (19) or synthesized in vitro by the MATERIALS AND METHODS virion-associated RNA polymerase (18) can be resolved by velocity sedimentation into four Virus and RNA. VSV (Indiana serotype) was size classes, which sediment at 31S, 17S, 14.5S, grown in baby hamster kidney cells (BHK-21, clone and 12S. In vitro translation of these purified 13), adapted to suspension culture, and purified as mRNA's has demonstrated that the 17S mRNA described previously (3). VSV genome RNA was codes for the G protein, the 14.5S mRNA codes extracted from purified virus with phenol-sodium dodecyl sulfate (SDS) and purified by velocity sedifor the N protein, the 12S mRNA codes for both mentation through sucrose gradients containing the NS and M proteins (8, 9, 15), and the 31S SDS. mRNA is believed to code for the L protein (17). 32P-labeled VSV mRNA was synthesized in vitro Past attempts to resolve the 12S mRNA into using one a-32P-labeled nucleoside triphosphate and two species have not succeeded, although se- three unlabeled nucleoside triphosphates as dequence complexity (23) and translation studies scribed previously (10). Methylated VSV mRNA was suggest that it may consist of two distinct synthesized in vitro using S-[methyl-3H]adenosylmethionine as the only labeled substrate (21). The mRNA species. In the present work, we show that in the in mRNA's were purified by SDS-phenol extraction, Sephadex G-50 chromatography, velocity sedimenvitro synthesized 12S mRNA can be separated tation through SDS-sucrose gradients, and chrointo two species by polyacrylamide gel electro- matography on oligo(dT)-cellulose columns, accordphoresis after first annealing the 12S mRNA to ing to previously published procedures (5, 11). genome RNA and then digesting away the sinRNA annealing. Two to five micrograms of unlagle-stranded regions of the resulting RNA du- beled genome RNA (0.05 to 0.125 optical density plexes with RNase T2. We have identified the units at 260 nm) was added to 0.1 to 0.25 ,±g of smaller mRNA component as the message for purified, labeled in vitro mRNA; The solution was the M protein and the larger mRNA as the adjusted to a final volume of 0.1 ml containing 0.3 M 0.03 M sodium citrate, pH 6.5, and incubated message for the NS protein. Comparison of the NaCl, at 72 to 74°C for 4 h, cooled to room temperature, and electrophoretic mobilities of VSV mRNA du- diluted with 0.1 ml of sterile water, the RNA was plexes with the double-stranded RNAs of the precipated with ethanol. reovirus genome gave molecular weight estiDigestion and analysis of annealed RNA. Anmates of 190,000 and 148,000 for the messages nealed RNA was collected by centrifugation and coding for the VSV proteins NS and M, respec- dissolved in 40 1.l of sterile water. After adding 5 Al tively. The apparent conflict between these val- of 100 mM sodium acetate, pH 4.5, 1 ,l of 2 M NaCl, 1105
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and 1 ,ul of RNase T2 (500 U/mi), the samples were incubated at 370C for 30 min. Digestion was stopped by adding 5 1.l of 10% SDS, and the samples were prepared for electrophoresis by adding 1 Al of 1 M Tris-hydrochloride, pH 8.0, 2 pl of bromophenol blue, and 25 pul of glycerol. Electrophoresis was on 5% polyacrylamide gels (0.6 by 10 cm) as described previously (6) for 16 to 19 h at 4 mA/gel, and gels were fractionated into 1-mm slices with a Gilson automatic gel crusher. Fractions from gels containing 3H radioactivity were incubated overnight at 50'C in 0.6 ml of 15% H202, and radioactivity was assayed in 10 ml of ACS (Amersham/Searle) scintillation cocktail. When :12P-labeled RNA was to be recovered from the gel slices, each fraction was suspended in 0.8 ml of 0.5 M NaCl, 0.1% SDS, 20 mM Tris-hydrochloride, pH 7.5, and 10 mM EDTA, and radioactivity was determined by Cherenkov counting. The fractions containing the peak of 32P radioactivity were then incubated for 24 h at 37TC with gentle shaking, gel particles were removed by centrifugation, and the supernatants were concentrated by vacuum dialysis against 0.1% SDS; the RNA was recovered by precipitation with ethanol. Nearest-neighbor analysis. a- [32P]AMP-labeled RNA was hydrolyzed for 18 h at 37°C with 0.3 N KOH, and each digest was neutralized with Dowex 50 (H +) and analyzed by high-voltage paper electrophoresis as described previously (4). Two-dimensional fingerprinting. a-[32P]GMP-labeled duplex RNAs were eluted from gels as described above, denatured by incubation for 30 min at 37°C in 90% Me2SO, and reprecipitated with ethanol. These samples, and the corresponding single-stranded poly(A)-containing mRNA's, were digested with RNase T1, and two-dimensional fingerprints were prepared as described by Barrell (7). Chemicals and enzymes. a_-32P-labeled ribonucleoside triphosphates and S-[methyl-3H]adenosylmethionine were purchased from New England Nuclear Corp. (Boston, Mass.). RNases T2 and T1 (produced by Sankyo Co., Japan) were purchased from Calbiochem (San Diego, Calif.). ACS scintillation cocktail is a product of Amersham/Searle (Arlington Heights, Ill.).
RESULTS Separation of 12S mRNA into two components. VSV mRNA was synthesized in vitro using 4-[:12P]ATP as the labeled substrate, and the poly(A)-containing 12S and 14.5S mRNA's were purified by sedimentation through sucrose gradients and chromatography on oligo(dT)-cellulose. Each of the purified mRNA's was then annealed to VSV genome RNA, digested with RNase T2, and analyzed by electrophoresis on 5% polyacrylamide gels (Fig. 1). The duplexes derived from the 12S mRNA were resolved into two major components, whereas the duplex from the 14.5S mRNA migrates as a single major band with a lower electrophoretic mobility. Similar experiments with purified 17S and 31S in vitro mRNA's each showed the presence
J. VIROL.
of a single band of duplex RNA with respectively lower electrophoretic mobilities (see Fig. 2 and 3). These higher-molecular-weight duplexes usually showed a small amount of radioactivity migrating at the same rates as the duplexes derived from the 14.5S and 12S mRNA's, indicating that they are contaminated with varying amounts of the smaller messages, presumably aggregates, which are not removed during the sucrose gradient purifi-
cations (data not shown). Absence of poly(A) after T2 digestion of annealed mRNA's. A possible reason for the presence of two duplexes derived from the 12S mRNA detected in polyacrylamide gels could be due to failure of RNase T2 to remove the poly(A) from all of the annealed mRNA molecules. Thus, the faster-migrating band could correspond to duplexes from which the poly(A) had been removed, whereas the slow-moving band could correspond to duplexes that had retained a single-stranded poly(A) "tail." The absence of slower-moving duplexes in digests of the annealed 14.5S, 17S, and 31S mRNA's argues against this possibility. More direct evidence for two species in the 12S size range was provided by determining the nucleotide compositions of alkaline digests of each of the a[32P]AMP mRNA duplexes and comparing these with the compositions of alkaline digests of the same messages either lacking or containI
3
S
0 v
I 0
_
20
60 40 MIGRATION (mm)
80
100
FIG. 1. Separation of two duplex RNAs derived from VSV in vitro 12S mRNA by polyacrylamide gel electrophoresis. VSV mRNA was synthesized in vitro in the presence of a-[32P]ATP, and the 12S and 14.5S mRNAs were purified by sedimentation twice through SDS-sucrose gradients and by chromatography on oligo(dT)-cellulose. Portions of the purified 12S and 14.5S were annealed to VSV genome RNA, digested with RNAse T2, and analyzed by electrophoresis on 5% polyacrylamide gels for 16 h at 4 mA! gel. The brackets indicate gel fractions that were pooled for nearest-neighbor analysis (see Table 1). Symbols: 0, duplexes prepared from 12S mRNA; 0, duplexes prepared from 14.5S mRNA.
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VSV mRNA SPECIES
ing poly(A). The results of these nearest-neighbor transfer analyses are shown in Table 1. Digests of the poly(A)-containing mRNA's (Table 1, lines 2 and 5) contained 52 to 54% 3'AMP, reflecting the contribution ofthe poly(A). However, analysis of the corresponding RNA duplexes after RNase digestion to remove single-stranded regions, including poly(A), showed that the relative amount of ApA sequences decreased to 29 to 32% (Table 1, lines 3, 6, and 7). These values are similar to the 29% value (Table 1, lines 1 and 4) found for the poly(A)-lacking mRNA's that had not bound to oligo(dT)-cellulose during the purification of
1107
the mRNA's. The results indicate that the two RNA duplexes derived from the 12S mRNA do not differ substantially in their poly(A) content.
Identification of the mRNA species coding for the M and NS proteins. The presence of two different mRNA molecules in the 12S mRNA fraction is in agreement with the previous evidence that the VSV 12S mRNA codes for two viral proteins, M and NS (8, 9). To identify which of the two 12S mRNA's codes for each of these proteins, we have taken advantage of the fact that each VSV gene has a different sensitivity to UV irradiation (1, 2). Using a coupled transcription-translation system, Ball and White (2) have shown that expression of the NS gene was more resistant to UV irradiation than was expression of the M gene. We have con-
W
x
u
I
I R
xL
U
MIGRATION (mm)
FIG. 2. Molecular weight determination of RNA duplexes derived from five VSV in vitro mRNA's. VSV mRNA was synthesized in vitro using [32P]CTP as the labeled precursor, and the product was purified by oligo(dT)-cellulose column chromatography and SDS-sucrose gradient sedimentation to isolate four size classes of poly(A)-containing mRNA. Approximately 104 cpm each of the 31S, 1 7S, and 14.5S and 2 x 104 cpm of the 12S mRNA species were mixed together and annealed to VSV genome RNA. After adding 10O cpm of [3H]uridine-labeled reovirus double-stranded RNA, the mixture was digested with RNAse T2 and analyzed by gel electrophoresis as described in Materials and Methods. Symbols: *, 32P-labeled VSV RNA duplexes; 0, 3Hlabeled reovirus double-stranded RNA. a-
MIGRATION (mm)
FIG. 3. Retention of methyl-3H-labeled 5'-termini of VSV in vitro mRNA's during preparation of duplexes. Methylated VSV mRNA was synthesized in vitro in the presence of S-[methyl-3H]adenosylmethionine and purified by phenol extraction and chromatography on Sephadex G-100 and oligo(dT)cellulose columns. The poly(A)-containing 3Hlabeled RNA was used to prepare RNA duplexes as described in Materials and Methods and analyzed by electrophoresis for 19 h on a 5% polyacrylamide gel.
TABLE 1. Nearest-neighbor analysis of a-[32P]AMP-labeled mRNA species Total cpm Sample prepn CpA (%) ApA (%) GpA (%) UpA %) a 14.5S 23.9 28.6 30.2 17.4 38,817 b 52.4 14.5S 16.4 19.2 61,814 11.9 c Duplex 3 26.6 31.8 26.2 15.4 7,204 a 12S 23.0 28.8 30.5 17.7 34,459 b 12S 16.4 53.6 18.7 11.5 47,550 c 34.4 Duplex 4 19.4 29.1 17.1 6,690 24.4 30.9 26.4 c Duplex 5 18.3 4,664 a 14.5S and 12S mRNA's labeled with a-V32P]AMP were fractionated on oligo(dT)-cellulose into poly(A)lacking (preparation a) and poly(A)-containing (preparation b) components. RNA duplexes (preparation c) prepared from poly(A)-containing mRNA's were eluted from the gel fractions indicated by the brackets in Fig. 1. Each RNA was hydrolyzed with KOH, and the resulting 3'-nucleoside monophosphates were analyzed by paper electrophoresis (4). RNA species
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firmed this result by translation of purified 12S logarithms of their molecular weights (20). We mRNA's synthesized by UV-irradiated VSV have used this relationship to estimate the mo(data not shown). To identify which of the two lecular weights of the transcribed portion [not 12S mRNA's codes for each of these proteins, including the poly(A)] of each of the five VSV mRNA's after first converting the messages we have determined the relative resistances of duplex 4 and duplex 5 (and, therefore, their into the corresponding double-stranded RNAs, as described above. VSV mRNA was synthecorresponding mRNA's) to UV irradiation. Identical samples of purified VSV were ex- sized in vitro using a-[32P]CTP as the labeled posed to UV irradiation for various times and precursor, and the individual poly(A)-containthen used to synthesize mRNA in vitro with a- ing mRNA's were purified as before. Portions of [32P]UTP as the labeled substrate. Each mRNA the purified mRNA's were then recombined to product was purified by oligo(dT)-cellulose provide a mixture containing equal amounts of chromatography and sucrose gradient sedimen- 32p radioactivity in each of the five mRNA's. tation to isolate the poly(A)-containing 12S This mixture was annealed with an excess of mRNA molecules. As expected, the higher genome RNA and, after adding [3H]uridinedoses of UV irradiation resulted in a decrease labeled reovirus genome RNA as internal moin the amount oftotal RNA synthesized, as well lecular weight markers, the mixture was dias a decrease in the relative amount of mRNA gested with RNase T2 and analyzed by polysedimenting at 12S (data not shown). Each acrylamide gel electrophoresis (Fig. 2). In addipoly(A)-containing 12S mRNA was annealed tion, four separate gels of the individual 31S, with an excess of unlabeled genome RNA, di- 17S, 14.5S, and 12S mRNA species were run in gested with RNase T2, and analyzed by electro- parallel (data not shown). The results showed phoresis on a 5% polyacrylamide gel, with a that RNA duplexes 1, 2, and 3 are derived from result similar to that shown in Fig. 1. Table 2 the 31S, 17S, and 14.5S mRNA's, respectively, shows the ratios of the duplex 4 RNA to the and duplexes 4 and 5 are both derived from the duplex 5 RNA with increasing doses of UV 12S mRNA. irradiation. The results clearly indicate that When the logarithm of the molecular weight the synthesis of duplex 4 RNA was more re- of each of the 10 reovirus double-stranded RNA sistant to UV irradiation, since there was more segments is plotted as a function of the migrathan a 50% increase in the ratio after 21 s of UV tion of that segment in the gel shown in Fig. 2, exposure compared with the unirradiated control. From these results, coupled with the obTABLE 3. Molecular weights of VSV mRNA and servation made by Ball and White (2), we conprotein species clude that the mRNA of duplex 4 codes for the Mol wt (x 10-6) Coding Proteins viral NS protein and the mRNA in duplex 5 Duplex potencodes for the viral M protein. RNA tialb Molecular weights of VSV mRNA's. The species dsRNAa ssRNA (mol wt Species (Mlw1tc Xl (l ) 103) relative mobilities of double-stranded RNAs during electrophoresis in polyacrylamide gels is 1 3.776 1.89 L 210.0 190 2 1.141 0.571 63.4 G 69 approximately inversely proportional to the TABLE 2. Effect of UV irradiation on transcription of the 12S mRNA species Duration of UV dose
32P-labeled double-stranded RNA
(RNA duplex 4/RNA
duplex 5)
0 7 14 21
1.39 1.64 1.94 2.17
a Samples of purified VSV were irradiated with UV light for 0, 7, 14, or 21 s (2) and then used to synthesize mRNA in vitro in the presence of a[32P]UTP; the poly(A)-containing 12S mRNA was purified from each product. Each sample was used to prepare duplexes, and the relative amounts of 32P in duplex 4 and duplex 5 were determined after electrophoresis through a 5% polyacrylamide gel as in Fig.
1.
3 4 5
0.739 0.385 0.298
0.369 0.192 0.149
N 41.0 50 21.4 NS 40-45 16.6 M 29 a The molecular weights (x 10-6) of doublestranded (ds) reovirus genome RNA segments used for the determination of the molecular weights of VSV duplex RNAs are as follows: L3 = 2.79; L2 = 2.71; L, = 2.55; M3 =1.62; M2 = 1.55; Ml = 1.46; S4 = 0.88; S3 = 0.75; S2 = 0.65; S, = 0.61. (The L, M, and S denote the large, medium, and small genome RNA segments of reovirus.) The molecular weights of the VSV duplex RNA species were calculated from the straight line giving the least-squares best fit for the log molecular weights of the 10 reovirus dsRNA's (see Fig. 1). bCoding potential of each mRNA species was calculated as one-ninth the corresponding singlestranded (ss) RNA molecular weight. c The molecular weights of VSV proteins are from Wagner et al. (25).
VOL. 21, 1977
the 10 points fall very close to a straight line (data not shown). This line was used to calculate the molecular weight of double-stranded RNA corresponding to the migration in the gel of each of the five VSV duplex RNAs. The results are summarized in Table 3, along with the apparent molecular weights of the corresponding VSV proteins. The calculated molecular weights for duplexes 1, 2, and 3 are in reasonable agreement with the apparent molecular weights of the corresponding VSV proteins L, G, and N, respectively. However, the molecular weights calculated for the VSV duplex 4 and 5 RNAs are about one-half of the sizes needed to code for the VSV NS and M proteins, respectively. Several possible reasons for this apparent discrepancy are detailed in the Discussion. The most obvious possibility is that the annealed mRNA's have been overdigested during the RNase T2 treatment so that the doublestranded RNAs in duplexes 4 and 5 are shorter than the actual lengths of the mRNA's from which they were prepared [other than removal of the poly(A) from the 3'-ends of the mRNA's]. Protection of the 5'-termini of VSV mRNA's from RNase T2 after annealing to genome RNA. One way to test for the possibility of overdigestion was to follow the fate of the 5'-termini of the mRNA's. For this purpose, methylated VSV mRNA was synthesized in vitro using S-[methyl-3H]adenosylmethionine as the only labeled substrate so that only the 5'termini would be labeled (21). After the poly(A)-containing mRNA's were purified, they were annealed with excess unlabeled genome RNA, digested with RNase T2, and analyzed by polyacrylamide gel electrophoresis. The 3H radioactivity migrated through the gel in four bands (Fig. 3) corresponding to RNA duplexes 2, 3, 4, and 5 in Fig. 3. 3H radioactivity was not detected in the position of duplex 1 because the 31S mRNA is always synthesized in the smallest amount (18), and, furthermore, only the 5'termini of the mRNA's are labeled in this experiment. Nearly 80% of the 1H radioactivity used for hybridization was recovered in the gel without making any allowance for the lower counting efficiency when counting sliced gels. Thus, virtually all of the 5'-termini of the messages must be retained under the RNase T2 digestion conditions used in these experiments. Two-dimensional fingerprints of mRNA duplexes eluted from gels. A second test for possible overdigestion of the RNA duplexes by RNase T2 was carried out by comparing twodimensional fingerprints of the T2-digested duplexes with the fingerprints of the purified RNA used to prepare the duplexes. mRNA was
VSV mRNA SPECIES
1109
synthesized in vitro using a-[32P]GTP as the labeled precursor, and the poly(A)-containing 12S mRNA species were purified. A portion of the mRNA was annealed to genome RNA, digested with RNase T2, and analyzed by electrophoresis on a 5% acrylamide gel as in Fig. 1. RNA duplexes 4 and 5 were eluted from the gels, denatured with Me2SO, and then digested with RNase T1. A portion of the purified 12S a[32P]GMP-labeled mRNA from the same in vitro RNA product was also digested with RNase T1 under identical conditions. A two-dimensional fingerprint was prepared frm each T1 digest using the procedure of Barrell (7). All of the large oligonucleotides (designated as spots 1 to 8) present in the T1 digest of the purified 12S mRNA (Fig. 4A) are present in either the fingerprint of RNA duplex 4 (spots 2, 4, 6 to 8) or the fingerprint of RNA duplex 5 (spots 1, 3, and 5) (Fig. 4C). Thus, with this sensitive fingerprinting technique, we could not detect the loss of any sequences from the mRNA molecules as a consequence of digesting the annealed mRNA's with RNase T2. However, it should be noted that extra spots appear in the digests of the RNA duplexes eluted from gels, especially in the smaller oligonucleotide region, apparently due to enzymatic contamination of these samples during the handling involved in elution from gels and denaturation. DISCUSSION The virion-associated RNA polymerase of VSV synthesizes multiple species of mRNA in vitro that code for the five virus structural proteins. Although the 31S, 17S, and 14.5S mRNA species appear to be monocistronic and code for the viral L, G, and N proteins, respectively, the 12S mRNA size class presumably includes two mRNA species of similar molecular weights and codes for both the M and NS proteins. In an attempt to separate these two mRNA species, we have taken advantage of the fact that double-stranded RNA molecules are better resolved by polyacrylamide gel electrophoresis than their single-stranded counterparts (13). Accordingly, 12S mRNA was hybridized with genome RNA and the resulting RNA duplexes obtained after treatment with RNase T2 were resolved in a 5% polyacrylamide gel. By this procedure, two distinct mRNA species were obtained (Fig. 1). From the relative resistance of their synthesis to UV irradiation (Table 2), we have demonstrated that the slower-moving RNA (duplex 4) codes for the viral NS protein and the faster-moving RNA (duplex 5) codes for the M protein. A similar technique for separating the multiple mRNA species of Newcastle disease virus has been reported (14).
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RHODES ET AL.
A a*
a_4 I~
4
I*
.4,
B
0
,-
o
ed
O
V
pv ft,
76
7 6
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8Q 1
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04
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FIG. 4. Two-dimensional fingerprints of RNase Ti digests of 12S mRNA's. VSV mRNA was synthesized in vitro in the presence of a-[32P]GTP, and the poly(A)-containing 12S mRNA was purified as detailed in Materials and Methods. A portion of this mRNA was annealed to genome RNA and digested with RNase T2, and the products were resolved by electrophoresis to separate the component duplexes 4 and 5 as described in the legend to Fig. 1. The fractions containing RNA duplexes 4 and 5 were located by Cherenkov counting, pooled separately, and denatured with Me2SO. Each RNA sample was digested with RNase T1, and a twodimensional fingerprint was prepared as described by Barrell (7); the 32P-labeled oligonucleotides were located by autoradiography. First dimension, electrophoresis on cellulose-acetate at pH 3.5 (right to left); second dimension, homochromatography on DEAE thin-layer plates (from bottom to top). (A) Fingerprint of poly(A)-containing 12S mRNA before annealing to genome RNA. (B) Fingerprint of mRNA strand of RNA duplex 4. (C) Fingerprint of mRNA strand of RNA duplex 5. The numbers indicate which large oligonucleotides in (A) are derived from the mRNA strands of RNA duplex 4 or 5.
The molecular weights of the individual VSV mRNA duplexes were determined using the above method by comparing their electrophoretic mobilities to those of the 10 doublestranded genome RNAs of reovirus, whose molecular weights are known (16). The results clearly show that the molecular weights of the
single-stranded 31S, 17S, and 14.5S mRNA species (calculated as one-half of the molecular weights of the corresponding RNA duplexes) are compatible with their coding potentials (Table. 3). On the other hand, the molecular weights ofthe mRNA's coding for the NS and M proteins (RNA duplexes 4 and 5, respectively)
VSV mRNA SPECIES
VOL. 21, 1977 f
0
0 .
.
03
.
FIG. 4C
inconsistent with their coding assignments. These two mRNA molecules appear to be approximately one-half the size needed to code for proteins with the estimated molecular weights of the VSV NS (40,000 to 45,000) and M (29,000) proteins. We have considered several possibilities for this apparent discrepancy in molecular weights. (i) The most serious possibility could be that the RNA duplexes were overdigested during RNase T2 treatment. However, this seems unlikely since (a) the 3H-labeled reovirus double-stranded RNA used as molecular weight standards was present during the RNase T2 digestion and was not affected by the RNase treatment, and (b) the mRNA's coding for the proteins L, G, and N after duplex formation are are
1111
apparently not degraded since their molecular weights as duplexes are reasonably in agreement with the corresponding single-stranded molecular weights (Table 3). Moreover, the recovery of virtually all of the 5'-termini (labeled with methyl-3H groups) in the duplex RNAs (Fig. 3) shows that there is no reduction in length of the duplexes from this end of the molecules. Furthermore, the presence of all of the characteristic large oligonucleotides (Fig. 4) in two-dimensional T1 fingerprints of RNA duplexes 4 and 5 after elution from gels argues against this possibility. Similarly, no overdigestion was detected by comparing T1 fingerprints of RNA duplex 3 and the corresponding single-stranded 14.5S N protein mRNA (data not shown). (ii) We have used the linear relationship between the electrophoretic mobilities and the logarithms of the molecular weights of the reovirus genome double-stranded RNA segments to calculate the molecular weights of the VSV mRNA duplexes. A possibility exists that this relationship does not remain linear at the lowmolecular-weight region, and the extrapolation may not be valid. However, it is worthwhile to point out that if one calculates the molecular weights of the corresponding mRNA's predicted from the estimated molecular weights ofthe NS and M proteins, one finds that RNA duplex 5 should migrate no more than 6 mm farther than the smallest reovirus double-stranded RNA segment and, thus, would require very little extrapolation. Similarly, RNA duplex 4 would require no extrapolation since it would have an electrophoretic mobility within the range of the reovirus double-stranded RNA standards. (iii) A third possibility is that the estimated molecular weights of the mRNA's are correct and that the molecular weights of the proteins are erroneous. The phosphoprotein NS is known to migrate anomalously in SDS-polyacrylamide gels depending on the pH condition. Thus, its true molecular weight is still unknown. On the other hand, the M protein with an estimated molecular weight of 29,000 may actually represent a dimer. A number of proteins, particularly membrane proteins, have been shown to bind less than the normal amount of SDS, and some proteins behave primarily as dimers during SDS-polyacrylamide gel electrophoresis despite the use of totally denaturing conditions (12, 22). Although it is still unclear which of the possibilities is the cause for the discrepancy, we believe that the second and the third possibilities stated above need careful investigation. In addition, the mo-
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lecular weights of the mRNA's should also be determined by an independent (preferably chemical) technique. Finally, no mRNA species other than the five shown in Fig. 2 have been detected in any of these experiments. Therefore, it seems likely that these five mRNA species, in addition to the small "leader" RNA previously described (10), constitute the only transcription products made in vitro by the virion-associated RNA polymerase of VSV and that these six transcripts may account for virtually the entire coding capacity of the VSV genome RNA. ACKNOWLEDGMENT We thank Alba LaFiandra for kindly supplying us with 3H-labeled reovirus genome RNA.
11.
12. 13.
14.
15. 16.
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