JOURNAL OF VIROLOGY, Mar. 1977, p. 1094-1104 Copyright ©) 1977 American Society for Microbiology

Vol. 21, No. 3 Printed in U.S.A.

RNA Synthesis of Vesicular Stomatitis Virus VII. Complete Separation of the mRNA's of Vesicular Stomatitis Virus by Duplex Formation GORDON J. FREEMAN,* JOHN K. ROSE, GAIL M. CLINTON, AND ALICE S. HUANG Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,* and Department ofBiology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138 Received for publication 28 September 1976

Full-length virion RNA and complementary mRNA's of vesicular stomatitis virus can be annealed to each other, digested with RNases, and then separated as five unique duplex RNA molecules on polyacrylamide slab gels. Similar RNA duplexes were detected whether mRNA or virion RNA was the radioactive component and whether the mRNA was synthesized in vitro or in vivo. The sharp banding pattern of these RNA molecules was dependent on treatment with RNase T2, suggesting that removal of poly(A) is necessary. Identification of the coding region contained in each RNA duplex was based on their previous identification as single-stranded mRNA on formamide-containing, polyacrylamide gels. Because the two smallest mRNA's had not been previously separated, their identification was based on their in vitro transcriptional gene order. In the order of increasing mobilities on the slab gels, the RNA duplexes are identified as the hybrid of the region of the genome RNA hybridized to the complementary mRNA coding for the large protein, the glycoprotein, the nucleocapsid protein, the core-associated NS protein, and the matrix protein (L, G, N, NS, and M). Several lines of evidence support the presence of undegraded complete mRNA, excluding poly(A), in these RNA duplexes. Also, the two smallest mRNA's, separated by duplex formation, were denatured, and their individual oligonucleotide fingerprints were determined. From chemical length determinations, the molecular weights of the mRNA, minus poly(A), are 2.78 x 105 and 2.5 x 105, respectively, for the mRNA's of the NS and M proteins. Vesicular stomatitis virus (VSV) is an enveloped, single-stranded RNA virus (see 28). The genome is a covalent linear RNA molecule of 3.82 x 106 daltons (11, 19). During viral multiplication there is synthesis of multiple, smaller mRNA's with the opposite polarity of virion RNA (10, 18). These virus-specific mRNA's account for almost all of the genetic information found in the virion RNA. Detailed analyses have shown that the mRNA's contain poly(A) at the 3'-end (8, 23) and methylated, capped nucleotides at the 5'-end (1, 20, 21). The mRNA's can be separated into four fractions by either sucrose density gradient centrifugation or formamide polyacrylamide gel electrophoresis (5, 16, 22). The 28S mRNA, isolated from sucrose gradients, was translated in vitro into the large (L) protein (16, 24). The smaller mRNA's were separated on formamide polyacrylamide gels into bands 2, 3, and 4 (22). RNA from band 2 codes for the glycoprotein (G); RNA from band 3 codes for the nucleocapsid (N) protein; and RNA from band 4 codes for both

the nucleocapsid-associated (NS) and matrix (M) proteins (15). Utilizing the fact that VSV synthesizes mRNA complementary to the genome RNA, hybrid RNA molecules can be formed between virion RNA and each of the virus-specific mRNA's. If these duplex molecules can be isolated after RNase digestion of the unhybridized single strands, then the hybrids would be expected to be separated readily on polyacrylamide gels designed to detect small differences in double-stranded RNA (13). Using this approach, we have been able to separate and identify all of the VSV mRNA's. (This work is based on studies to be submitted by G.J.F. to the Faculty of Arts and Sciences of Harvard University in partial fulfillment of the requirements for the Ph.D. degree.) MATERIALS AND METHODS Cells and virus growth. Standard B particles of the Indiana serotype of VSV were used throughout. Their history and growth in Chinese hamster ovary

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cells have been described (25). Baby hamster kidney cells obtained from Amiya Banerjee were occasionally used for virus growth. There was no detectable difference in the hybridized RNA obtained from the two different cell lines. Reovirus (Dearing strain, type 3) was obtained from Bernard Fields. The virus was grown in L cells as described by Cross and Fields (7). Preparation of VSV mRNA. When unlabeled virus-specific RNA was needed, cells were infected with VSV at a multiplicity of 3 and suspended at a concentration of 2 x 106 cells/ml in Joklik minimal essential medium, buffered at pH 7.4 with the organic buffer HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid; 20 mM), plus nonessential amino acids and 2% fetal calf serum. For 32P-labeled virus-specific RNAs, the procedure outlined by Rose and Knipe (22) was used. Both labeled and unlabeled mRNA were purified as described previously (22). In summary, RNA in the cytoplasm was extracted with chloroformphenol. The poly(A)-containing RNA was purified by binding to and elution from oligo(dT)-cellulose. Preparation of virion RNA. Sucrose gradientpurified virions (25) were mixed with sodium dodecyl sulfate to a final concentration of 2% sodium dodecyl sulfate and warmed at 370C for 1 min. The solubilized material was layered on a 15 to 30% (wt/ wt) sucrose gradient containing 0.04% sodium dodecyl sulfate in buffer (0.1 M NaCl; 0.01 M Tris, pH 7.4; and 0.01 M EDTA) and centrifuged at 76,000 x g for 10 h at 220C. RNA sedimenting at 40S was pooled and ethanol precipitated. To detect the 40S RNA in the gradient fractions, either the optical density at 260 nm was continuously monitored on a Gilford recording spectrophotometer or a Geiger counter was used to detect those fractions containing 32p. RNA-RNA hybridization. For RNA-RNA hybridization, the method of Ito and Joklik (12) was followed. In short, RNA samples stored in ethanol were precipitated by centrifugation at 100,000 x g for 1 h at 40C; the precipitates were dried in vacuo and dissolved in 0.001 M EDTA, pH 7.4, and the RNA was denatured by adding 9 volumes of dimethyl sulfoxide (Me2SO), with incubation at 450C for 30 min. The solution was then adjusted to 63% Me2SO in 0.03 M NaCl, 0.01 M Tris (pH 7.4), 0.002 M EDTA; for annealing, the final total RNA concentration was between 25 and 100 ,g/ml, and the samples were incubated at 370C for 24 h. After hybridization, the RNA was ethanol precipitated and dissolved in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0). RNases were added to a final concentration of 1 ,ug/ml for RNase A, 12.5 U/ ml for RNase T1, and 10 U/ml for RNase T2, and the samples were incubated at 3700 for 30 min. All three RNases were used except in the experiment shown in Fig. 3. RNases were then removed by extraction with chloroform-phenol as described above for the purification of mRNA. Yeast RNA (10 ,tg/ml) was added as carrier, and the RNA was ethanol precipitated. Electrophoresis of double-stranded RNA in polyacrylamide slab gels. RNA was precipitated out of ethanol by centrifugation at 8,000 x g for 20 min at

VSV RNA DUPLEXES

1095

40C in a Brinkmann microcentrifuge, dried in vacuo, and dissolved in 8 ,ul of electrophoresis buffer (0.25 M Tris, 0.01 M EDTA, to pH 8.4 with acetic acid). Four microliters of a solution of 60% sucrose-0.5% bromophenol blue in electrophoresis buffer was added to each sample. Acrylamide gels were formed in an E-C (St. Petersburg, Fla.) slab gel apparatus with 5% acrylamide, 0.13% bisacrylamide, 0.04% N,N,N',N'-tetramethylethylenediamine, and 0.6% ammonium persulfate in electrophoresis buffer (7). Electrophoresis was at 50 V for 40 to 48 h with constant buffer circulation. The slab gels were transferred onto filter paper, dried in a Hoefer gel dryer, and exposed to Cronex 4 X-ray film. When necessary, the wet gel was covered with Saran Wrap, exposed to X-ray film, and autoradiographed. The regions of the gel containing the labeled RNA were excised, using the film as a template. The gel band was cut into small pieces and placed in the conical portion of a 10-ml disposable plastic pipette that had been cut off to give a volume of about 2 ml. The tip of the pipette was plugged with glass wool. A dialysis bag was slipped over the tip of the pipette, and the RNA was electrophoresed out of the gel into the dialysis bag at 1.5 mA/ tube for 12 h. At the end of electrophoresis the polarity of the current was reversed for 2 min to prevent trapping of the RNA in the pores of the dialysis membrane. The solution was removed from the dialysis bag, and the RNA was ethanol precipitated. RNA fingerprinting. The homochromatography method of fingerprinting was exactly as described by Rose and Knipe (22) and essentially as that described by Barrell (4). In summary, RNA samples in low salt were digested with RNase T1, and the oligonucleotides were separated in the first dimension by electrophoresis at pH 3.5 on cellulose acetate strips and in the second dimension by homochromatography on polyethyleneimine-cellulose plates. Radioisotopes and chemicals. ATP, GTP, CTP, and UTP were purchased from Schwarz/Mann. Carrier-free [32p]- and a-[32P]UTP (136 Ci/mmol) were purchased from New England Nuclear Corp. Triton X-100 and yeast RNA were purchased from Sigma. Acrylamide (electrophoresis grade), N,N'-methylene-bisacrylamide, and N,N,N',N'-tetramethylethylenediamine were all purchased from Eastman Kodak Co. Ammonium persulfate and Me2SO were purchased from Mallinckrodt. Sodium dodecyl sulfate was purchased from Matheson, Coleman, and Bell. Oligo(dT)-cellulose was purchased from Collaborative Research. RNase A was purchased from Worthington. RNases T1 and T2 were purchased from Calbiochem. Cellulose acetate strips were purchased from Schleicher and Schuell. Polyethyleneimine plates were purchased from Brinkmann.

RESULTS Hybridization of mRNA's with virion RNA and separaion of the duplex molecules. To avoid degraded RNA in the hybridizations, great care was taken during the RNA purification to prevent degradation by RNases. Du-

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plexes were made by melting the RNA at 450C in 90% Me2SO and annealing at 370C in 63% Me2SO. This gentle procedure avoids the thermal degradation of RNA seen in hybridizations done at higher temperatures. To determine whether the genome RNA of VSV and its complementary mRNA species would anneal to form complete duplexes, RNA from purified virions was hybridized with polyadenylated mRNA from infected cells. After annealing, the RNA was ethanol precipitated, dissolved in buffer containing 0.3 M NaCl, and digested with RNases A, T1, and T2. The RNase-resistant duplexes were separated on polyacrylamide slab gels designed to separate reovirus double-stranded RNA (7). The RNA was visualized by staining with ethidium bromide. Figure 1 (slot a) shows the separation achieved and the banding pattern of the duplex RNAs formed between the mRNA's and virion RNA. Double-stranded RNA from reovirions is shown in slot b for comparison. The major RNase-resistant duplexes of VSV RNA are seen as five bands, which have been designated 1, 2, 3, 4a, and 4b. Band 0, the largest of the duplexes with an estimated single-stranded molecular weight of 2.8 x 106, using reovirus RNA markers, appeared to consist of double-stranded RNA formed between two complementary 40S molecules. This conclusion was based on the following observations despite the low estimate for its molecular weight. Full-sized 40S RNA complementary to virion RNA is made during viral replication and is polyadenylated (17); therefore, it would have been isolated along with the mRNA fraction. Also, when highly purified virion RNA was self-annealed, 3.3% became RNase resistant, and the unique duplex formed migrated identically to band 0 (data not shown). Molecular weight determination of the single-stranded mRNA found in the duplex molecules. The molecular weights of reovirus double-stranded RNA determined by gel mobility (14) and electron microscopy (26) were used to estimate the molecular weights of the VSV duplexes. One-half the molecular weight of the duplex was taken as the molecular weight of the single-stranded mRNA encoded by the viral genome. VSV mRNA, estimated this way, had molecular weights in the range 0.2 x 106 to 1.76 x 106 (Table 1). If, as will be demonstrated, bands 1, 2, 3, 4a, and 4b contained mRNA's for the L, G, N, NS, and M proteins, respectively, the molecular weights were 7% higher for the L message and 5 to 24% lower for the other mRNA's than pub-

.I.j L

s.~~~~~~

1'

40

4b

W

FIG. 1. Ethidium bromide-stained polyacrylamide slab gel of duplexes formed between VSV mRNA and virion RNA. The hybridization mixture contained 4 pg of unlabeled virion RNA and 50 pg of unlabeled, oligo(dT)-selected VSV mRNA. After hybridization and digestion with RNases, the duplexes were subjected to electrophoresis in a slab gel with reovirus RNA in an adjacent slot. The wet gel was then stained with 0.5 pg of ethidium bromide per ml of buffer (40 mM Tris, pH 7.9; 5 mM sodium acetate; 1 mM EDTA) for 0.5 h at 22°C. The gel was photographed with light at 260 nm. (a) VSV mRNA hybridized to virion RNA; (b) reovirus double-stranded RNA.

lished values obtained by complexity analysis and gel mobility (15, 22). These discrepancies were probably a result of using reovirus RNA as molecular weight markers. Alternatively, the RNAs may have become considerably smaller after hybridization and nuclease diges-

VOL. 21, 1977

VSV RNA DUPLEXES

TABLE 1. Estimated molecular weights of VSV mRNA calculated from the mobilities of their duplex RNAs Bands 1 2 3

4a 4b

10-6) Mol wt (X Expectedc Gene assign- Estimatedb menta L G

N NS M

1.76 0.58 0.41 0.23 0.20

31.65 0.73 0.53

(0.28) (0.26)

aGene assignments were based on the results shown in Fig. 4, 5, and 6. bThese were the average molecular weights of single-stranded RNAs from three different electrophoretic determinations, using reovirus RNA as markers. The molecular weights of reovirus RNA are based on two different methods: (i) from their mobilities on acrylamide gels (14) and (ii) from their contour length when measured by electron microscopy (26). c The expected molecular weight minus poly(A) was obtained from Knipe et al. (15). Molecular weights given in parentheses were calculated from the chemical length determinations shown in Table 2, using 321 as the average molecular weight of a nucleotide.

tion. Evidence in support of the fact that the duplexes contained the complete length of the mRNA, excluding the poly(A) at the 3'-end, is summarized in the Discussion. Hybridization of 32P-labeled mRNA and virion RNA and separation of the duplex molecules. To insure that radioactively labeled RNA gave results equivalent to those with unlabeled mRNA, both 32P-labeled mRNA's and virion RNA were separately hybridized to an excess of cold RNA of the opposite polarity (Fig. 2). When the virion RNA was labeled, 90.1% of the virion RNA was RNase resistant after hybridization. In contrast, when the mRNA was labeled, 98.6% of the mRNA was RNase resistant after hybridization. Incomplete annealing with labeled virion RNA and unlabeled mRNA might be expected from the finding that the genome RNA contains sequences not found in mRNA (6), but it might also be explained by an incomplete saturation with certain mRNA species. The major bands 2, 3, 4a, and 4b were present under both conditions of annealing; however, band 0 was observed only when the virion RNA was labeled and band 1 was more readily seen when the virion RNA was the radioactively labeled component in the hybridization (Fig. 2, slot b). When labeled genome RNA was hybridized to an excess of mRNA, then detection of less abundant species of oligo(dT)-selected RNA

1097

(band 0 and 1) should have been increased relative to the more abundant RNAs in bands 2, 3, 4a, and 4b. Presumably, this explains the detection of bands 0 and 1 in Fig. 2b, but not in Fig. 2a where the unlabeled genome RNA was in excess. The amount of material in band 0 and 1, even when detectable, is small; this is presumably due to the increased susceptibility of these large molecules to nicking by 32p decay or by other nonspecific factors. Although it is not clearly shown in Fig. 2, an examination of a less highly exposed film indicates that the relative intensities of the bands shown in slot a revealed an order of decreasing intensity for bands 3, 4a, 4b, 2, and 1 (cf. Fig. 5 and 7, slot a). Such an order is consistent with the suggestion that the relative molar amounts of the VSV mRNA species transcribed in the cell is a reflection of the gene order (27), thus permitting a tentative assignment of the _-

-3-b2 .4a-

a

b

FIG. 2. Autoradiograph of a polyacrylamide slab gel containing the RNase-resistant hybrids formed between virion RNA and mRNA of VSV. Two hybridization mixtures containing 250 Mg of unlabeled 40S virion RNA and 4 x 107 cpm of 32P-labeled mRNA, or 107 cpm of 32P-labeled virion RNA and 400 Mg of unlabeled mRNA, were treated as described in Materials and Methods. A sample of approximately 90,000 cpm of RNase-resistant duplexes from each sample was separated on gels. (a) Unlabeled virion RNA hybridized to labeled mRNA; (b) labeled virion RNA hybridized to unlabeled mRNA.

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mRNA's for the N, NS, M, G, and L proteins, in that order, in bands 3, 4a, 4b, 2, and 1. Effect of RNases in the generation of unique RNA duplexes formed between mRNA and virion RNA. Because the duplexes shown in Fig. 1 and 2 were generated after hybridization by treatment with all three RNases (A, T1, and T2), it was of interest to determine whether unique hybrids were obtained by digestion with only one or two of the RNases. To test this, samples of hybridization mixtures containing 32P-labeled VSV mRNA and excess unlabeled virion RNA were treated with RNases A, T1, and T2 (Fig. 3, slot a); A and T1 (slot b); A and T2 (slot c); T1 and T2 (slot d); A alone (slot e); T1 alone (slot f); and T2 alone (slot g). Only those hybrids exposed to RNase T2 gave sharp band-

ing patterns, suggesting that heterogeneity was caused by poly(A) (Fig. 3, slots a, c, d, and

g).

Identification of the mRNA's. One method for determining the correct assignments between the duplex molecules and the mRNA species was first to separate the single-stranded mRNA species on formamide-containing polyacrylamide gels and then to hybridize each of the separated single-stranded RNA species to virion RNA. Previously, three of the mRNAcontaining fractions separated on formamide gels have been identified by translating them in in vitro systems (15). Using the nomenclature of Knipe et al. (15), band 2 contained mRNA for the G protein, band 3 contained mRNA for the N protein, and band 4 contained

.A.

*o.

._

a

b

C

d

e

f

40 4b

q

FIG. 3. Presence of RNase-resistant duplexes formed between VSV mRNA and virion RNA when treated with different RNases and their combinations. After hybridization of4.1 mg of virion RNA with 2.5 x 106 cpm of 32P-labeled, oligo(dT)-selected mRNA, the RNA was divided into seven samples and digested with different RNases prior to their separation on polyacrylamide slab gels. The concentrations ofeach RNase used were as indicated in Materials and Methods. (a) RNases A, T1, and T2; (b) RNases A and T1; (c) RNases A and T2; (d) RNases T1 and T2; (e) RNase A; (f) RNase T1; (g) RNase T2.

VOL. 21, 1977

VSV RNA DUPLEXES

mRNA's for the NS and M proteins. Band 1 was heterogeneous and contained too little mRNA for in vitro translation. After separation on formamide gels, the 32p_ labeled single-stranded VSV mRNA's were annealed with unlabeled virion RNA in excess, and the resultant duplexes are shown in Fig. 4. Band 1 from formamide gels gave poor results. Most of the RNA eluted from this region did not rebind to oligo(dT)-cellulose. When the unbound RNA was hybridized to virion RNA, a fuzzy band was seen in the region expected for duplexes containing the RNA for the L protein (Fig. 4, slot a). In addition, there was readily detectable radioactivity in bands 2, 3, 4a, and

-.1

.-2-~ :I..

*--3.4

. _.

...A

..

-4a-4b-

a

b cde

FIG. 4. Autoradiograph of the RNA duplexes formed between virion RNA and the individual VSV mRNA species, which had first been separated by banding on formamide polyacrylamide gels. Individual VSV 32P-labeled mRNA species were purified on a 3.75% polyacrylamide gel containing 98% formamide as described previously (22). After autoradiography, the individual bands 1 through 4 were excised, and the RNA was freed ofgel contaminants by again binding and eluting from oligo(dT)-cellulose. The isolated mRNA species were individually hybridized with 40S RNA purified from virions, and the RNA duplexes were separated on polyacrylamide slab gels as described in Materials and Methods. (a) Band 1 mRNA, which failed to rebind to oligo(dT)-cellulose; (b) band 2 mRNA, oligo(dT)-selected; (c) band 3 mRNA, oligo(dT)-selected; (d) band 4 mRNA, oligo(dT)-selected; (e) total mRNA isolated as described for Fig. 2, slot a.

1099

4b. Band 2 from formamide gels yielded duplex molecules at band 2 on the slab gel; mRNA was also detected in duplexes at band 3 and in trace amounts in bands 4a and 4b (Fig. 4, slot b). Band 3 from the formamide gel contained mainly mRNA forming duplexes migrating at the corresponding band 3 position on the slab gel; there was trace contamination by mRNA forming duplexes that migrated at bands 4a and 4b. Band 4 from the formamide gel contained only RNA that formed duplexes migrating at the 4a and 4b positions. Therefore, the numbering system designated for the RNA duplex-containing bands in the slab gels corresponded to the numbering system used for the bands in the single-stranded, RNA-containing formamide gels. In summary, for the gel containing duplex RNA the conclusions were that band 1 consisted of mRNA for L protein, band 2 consisted of mRNA for G protein, band 3 consisted of mRNA for N protein, and bands 4a and 4b consisted of mRNA for the NS and M proteins. Identification of the mRNA's for the NS and M proteins. To determine which of the bands, 4a or 4b, coded for the NS and M proteins specifically, advantage was taken of the fact that Ball and White (2, 3) determined the transcriptional gene order of VSV by a coupled in vitro transcription-translation system using VSV irradiated with UV light. Because the synthesis of the mRNA's for N, NS, M, G, and L proteins increases, in this order, in sensitivity to UV irradiation, the labeled, in vitro synthesized mRNA species made by UV-irradiated VSV, when annealed to virion RNA, should confirm the assignments made in the previous experiment, as well as determine which of the duplexes contained mRNA coding for the NS or M protein. Figure 5 shows the duplex RNAs formed by hybridizing virion RNA to 32P-labeled mRNA's made in vitro by VSV that had not been irradiated (slot b) or had been UV irradiated at two different dosages (slots c and d). Slot a in Fig. 5 contained mRNA from an in vivo preparation. Comparison of mRNA made in vitro to those made in vivo indicate that duplexes with identical mobilities were formed by RNA from both sources. In our hands, in vitro RNA banding at position 0 or 1 was not detected, suggesting that 40S antigenome RNA and the L-protein mRNA were not synthesized in this in vitro system. Examination of slots b through d (Fig. 5) shows that band 4b appeared to decrease in intensity faster than band 4a with increasing amounts of UV irradiation. These autoradiograms and those of other additional slots were scanned. The amount of each RNA found in

1100

J. VIROL.

FREEMAN ET AL.

bands 2, 4a, and 4b synthesized by UV-irradiated virus was expressed as the remaining percentage of the same RNA species synthesized by unirradiated virus (Fig. 6). RNA in band 2 appeared to be the most sensitive to UV irradiation, with band 4b being the next most sensitive and 4a being the least sensitive. Since band 2 is known to contain mRNA for the G protein, it follows that band 4b contained mRNA for the M protein, thus identifying band 4a as the one containing mRNA for the NS protein. Oligonucleotide fingerprints of mRNA obtained from bands 4a and 4b. To demonstrate that bands 4a and 4b contained different nucleotide sequences, each being a subset of the sequences previously identified for the combined mRNA's of the NS and M proteins (22), an oligonucleotide fingerprint analysis was performed on the separated RNA. To do so, 32P_ labeled RNA from bands 4a and 4b were obtained from a preparative slab gel. A sample

i

4a 4b

*

*

a£0

100 .3 80

Z.

60

0

*

A

4a

-

01-

-

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.- -1-.

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4b

114T

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0

40k

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2

20 _

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FIG. 5. Radioautograph of slab gel containing RNA duplexes formed between in vitro synthesized 32P-labeled mRNA's and virion RNA. In vitro polymerase assay mixtures in different volumes contained the following, in a 300-pl sample: 30 pg of UVirradiated or unirradiated, purified standard VSV; 15 ,mol of Tris buffer, pH 7.5; 1.6 ,umol of MgCl, 1.6 ,umol of dithiothreitol; 0.08% Triton X-100; 30 ,mol of NaCl; 0.2 ,umol of S-adenosylmethionine; 17% of a cytoplasmic extract prepared by Dounce homogenization of 2 x 106 Chinese hamster ovary cells in 1 ml of Tris buffer; and 5 jjCi of a-[32P]UTP. They were incubated for 4.5 h at 31 °C. Incorporation of a-[32P]UTP into acid-precipitable material was linear during this period. The RNA was extracted with phenol-chloroform, oligo(dT)-selected, hybridized to an excess of unlabeled virion RNA, and digested with RNases as described in Materials and Methods. Equivalent counts of 32P-containing duplex RNA were placed in each slot. A sample of duplex RNA formed between in vivo synthesized 32P-labeled mRNA and virion RNA was co-electrophoresed on

in 2

4

6

8

10

UV DOSE SECONDS FIG. 6. Identification of the mRNA's in the duplex molecules banding at 4a and 4b. In vitro synthesized 32P-labeled mRNA by unirradiated and UV-irradiated VSV were hybridized to virion RNA as described for Fig. 5. The resultant duplexes were separated on acrylamide slab gels. Radioautograms of individual slots were scanned on a Gilford recording spectrophotometer, and the individual peaks were integrated. After normalizing the values for peaks representing bands 2, 4a, and 4b to that obtained for band 3, the results were plotted as the remaining percentage of each mRNA in bands 2, 4a, and 4b synthesized by UV-irradiated VSV compared with that synthesized by unirradiated VSV. The UV dose was 5.45 ergslmm2 per s.

the same gel. (a) RNA synthesized in vivo; (b) RNA synthesized in vitro by unirradiated VSV; (c) RNA synthesized in vitro by VSV irradiated at 49.05 ergs/ mm2; (d) RNA synthesized in vitro by VSV irradiated at 59.95 ergs/mm2.

VOL. 21, 1977

was rerun on a slab gel to demonstrate the negligible contamination of one band by the other (Fig. 7). After the duplexes were denatured and the RNA was treated with RNase T1, the oligonucleotides were subjected to electrophoresis and homochromatography (22). As expected, the oligonucleotide fingerprint patterns indicated that the two mRNA's were different from each other (Fig. 8). In comparing these patterns with the published pattern for the combined mRNA's for the NS and M proteins, it is apparent that Rose and Knipe (22) calculated the total sequence complexity from nucleotides all obtained from the mRNA for the M protein. We have been unable to obtain sequence complexities from these fingerprints (Fig. 8) because of losses during preparation of the RNA for oligonucleotide analysis. However, the oligonucleotide patterns obtained show all of the large unique sequences found in the fingerprint of the combined mRNA's for the NS and M proteins, excluding poly(A). These results and those described below give an indication that the RNAs in the duplexes had not lost sequences other than poly(A) during nuclease digestion. Bands 4a and 4b retain their 5'-termini and are intact. To examine further whether the mRNA's had lost sequences other than poly(A) during the preparation of double strands, RNAs from bands 4a and 4b were analyzed to see if the 5'-termini were intact. Denatured 32P_ labeled RNA from each duplex molecule was digested with RNases T1, T2, and A, and the mononucleotides were separated from the 5'termini by pH 3.5 electrophoresis on DEAE paper (21). Autoradiograms showed the presence of 5'-termini for each of the mRNA's derived from duplex structures (data not shown). These termini were identical to those previously described by Rose (21), which indicates that the mRNA's from bands 4a and 4b were intact. Table 2 gives the quantitation of the radioactivity in these termini relative to the total radioactivity in the RNA, thus yielding estimated lengths for the mRNA found in bands 4a and 4b. The average value of the two mRNA's (see Table 1) is only 8% greater than the average found previously for the combined mRNA's (15, 22), indicating that, at most, only a small fraction of the sequences, presumably poly(A), was lost during duplex formation and nuclease digestion.

VSV RNA DUPLEXES

1101

2 '4A

3

_4a -4b

a b c FIG. 7. Preparative separation of duplex RNA in bands 4a and 4b by slab gel electrophoresis. 32p_ labeled mRNA was prepared and annealed to virion RNA in excess, as described for Fig. 2, slot a. After separation of the duplexes on a slab gel, bands 4a and 4b were cut out and eluted by electrophoresis as described in Materials and Methods. The RNAs were passed through a CF-11 column (9) to further reduce the contamination by acrylamide fragments. Samples of each RNA duplex were rebanded on slab gels; the remaining duplex RNA was fingerprinted (see Fig. 8). (a) 32P-labeled mRNA and virion RNA duplexes; (b) separated band 4a; (c) separated band 4b.

digested with RNases, and then separated as five major bands on polyacrylamide gels. Each of these bands contains a duplex RNA, which consists of the region of the genome RNA and the complementary mRNA coding for one of the five structural proteins of VSV. Molecular weight determinations of the duplex molecules, using reovirus double-stranded RNA as markers, result in values that can be as much as 24% lower than expected molecular weights obtained by complexity analysis (15, 22). However, several lines of evidence suggest that the single-stranded RNA in the duplex DISCUSSION molecules is very close to full-sized VSV Full-length virion RNA and complementary mRNA without its poly(A). First, the duplex mRNA's of VSV can be annealed to each other, molecules are of unique sizes, indicating the

1102

FREEMAN ET AL.

CG

*.i4!_.

.NW.UG.-.4

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J. VIROL.

AG 1t

4a

:l

;

A_ ..4.

.m _.;s

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..;.

...,.

....

..

...... ;. A.

..wi:.

OFF

an.

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4b

UG

2?

**CG

AG

4>

L~ cellulose acetate,

ct -4i

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FIG. 8. Fingerprints of the NS and M protein mRNA's separated by duplex formation with virion RNA. 32P-labeled mRNA separated in bands 4a and 4b were obtained exactly as described for Fig. 7 and fingerprinted after RNase Ti digestion of the denatured duplex RNAs (22). The oligonucleotides numbered 1 through 6 correspond to those described by Rose and Knipe (22). (4a) NS protein mRNA; (4b) M protein mRNA.

absence of any random breaks in the RNA chains. Also, when the mobility of the duplex RNAs is graphed versus the logarithm of the molecular weights obtained by complexity analysis (15), a linear relationship is obtained. Second, if certain regions, like the ends of the RNAs, are more susceptible to nuclease digestion, then more extensive nuclease treatment

should decrease the size of the duplexes. This is not the case within the range of fivefold-higher concentrations of RNases (data not shown). Third, when the duplex RNAs are denatured and then separated on formamide-polyacrylamide gels, the single-stranded mRNA's migrate with only a slightly higher mobility than native VSV mRNA molecules and the bands

VOL. 21, 1977

VSV RNA DUPLEXES

TABLE 2. Quantitation of 5'-terminia 32P CpM Total radio- Calculated Band

activity inleghnu

C and D the RNAs

cleotides)

A

B

4a

183

52

47

48,802

865

4b

156

40

39

37,621

800

The isolation and characterization of the multiple 5'-termini derived from VSV mRNA's have been described previously (21). Termini A, B, C, and D are m7G5'ppp5' (m)AmpAp, m7G5'ppp5' (m)AmpmAmpCp, pppAp, and pppGp, respectively. Radioactivity in the region of termini C and D was summed because C and D were not resolved in this electrophoretic separation. The radioactivity in the 5'-termini was used to calculate the length in nucleotides of each RNA by assuming that the heterogeneous termini were derived from each RNA species. Thus, the total radioactivity in the termini divided by the average number, five, of phosphates in the termini gave the radioactivity per phosphate. This number divided into the total radioactivity yielded the calculated number of phosphates (nucleotides) in each mRNA species. a

1103

seen with in vitro products has an estimated molecular weight of 106 for single-stranded RNA and banded between bands 1 and 2. The characterization of this large RNA is in progress. The analysis of single-stranded RNA from formamide acrylamide gels indicates that in formamide gels there is contamination of all of the mRNA bands by the mRNA's for the NS and M proteins. This may explain the presence of NS and M polypeptides resulting from in vitro translation of the mRNA for the G protein (15). The significant contamination ofthe L and G protein mRNA's with the N protein mRNA is probably due to cross contamination of relatively closely migrating RNA species in the formamide gels, but the possibility exists that nonspecific trapping of RNA intermediates and smaller mRNA's occurs in the formamide polyacrylamide gels. This contamination by transcriptive complexes may be especially true for band 1, where the actual amount of mRNA for the L protein is so small that further analysis of this particular mRNA was not possible (Fig. 4, slot a). When the electrophoretic patterns of the duplexes are examined, minor bands are present. Although these have not been further analyzed, the smaller duplexes may consist of unique breakdown products or, more interestingly, defective transcription resulting from internal initiations or initiations on RNA from defective interfering particles. Large RNA found in duplexes may be incomplete digestion products. The possibility exists, however, that read through or lack of processing occurs occasionally during transcription and that the information in adjacent cistrons becomes linked in large RNA molecules. Only further linkage analysis of these RNAs will determine whether such molecules exist. Despite such unanswered questions, it is clear that the methodology of duplex formation for isolating both mRNA sequences and coding regions on the genome of VSV is highly reproducible and quantitative. Using such an analysis, partial RNase digestions may yield a gene order through linkage analysis. Also, based on the findings of Ito and Joklik (13) with heterologous duplex RNA from reovirus and temperature-sensitive mutants, lesions in VSV temperature-sensitive mutants may similarly be localized and subsequently mapped.

are much sharper (data not shown). Both the faster migration rate and the sharpness of the bands can be accounted for by the loss of poly(A). Lastly, a chemical length determination of the mRNA in the duplex bands 4a and 4b not only demonstrates that the molecules are intact at their 5'-ends, but also that they give chain lengths (Table 2) that correspond very closely to the molecular weight values previously determined for the combined mRNA's of the NS and M proteins (15, 22). Given all of this direct and indirect evidence, our data, nevertheless, cannot exclude the loss of less than 50 bases from the 3'-end. RNA duplex formation allows the separation of the NS and M mRNA's of VSV for the first time. Taking the coding capacity of these mRNA's (Table 2), it can be calculated that the NS and M proteins should have maximum molecular weights of 31,700 and 29,300, respectively. This type of calculation, especially for the phosphorylated NS protein, is probably a closer reflection of the real molecular weight of the protein. Similar separations by RNA duplex formation are obtained with in vitro synthesized mRNA (Fig. 5). The in vitro products, when annealed to virion RNA and then digested with RNases, migrate identically to duplex moleACKNOWLEDGMENTS cules formed between mRNA synthesized intraWe thank Norma Hewlett and Donald Rao for expert cellularly and virion RNA. In vitro products, in technical support, Bernie Fields for advice on reovirus, and our hands, do not form hybrids migrating to the David Baltimore for suggesting the experiment shown in positions for band 0 or 1. The largest duplex Fig. 5.

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This work was supported by Public Health Service research grant AI 10100 from the National Institute of Allergy and Infectious Diseases. G. M. C. is an American Cancer Society Postdoctoral Fellow. A. S. H. is a Research Career Development awardee of the Public Health Service, Institute of Allergy and Infectious Disease. G. J. F. is a trainee supported by Public Health Service grant T32 CA 09031 from the National Cancer Institute.

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