Properties of the product synthesized by vesicular stomatitis virus particles

J. Mol. Biol. (1971) 58, 799-814

Properties of the Product Synthesized by Vesicular Stomatitis Virus Particles

D. H. L. BISHOP AND POLLY ROY

The Institute of Cancer Research, Columbia University College of Physicians and Surgeons

99 Fort Washington Avenue, New York City, N. Y. 10032, U.S.A.

(Received 9 November 1970, and in revised form 18 January 1971)

The product RNA synthesized by vesicular stomatitis virus particles has been characterized. One-half to one-quarter of the viral genome is transcribed in reactions run at 37°C. Transcription is repetitive. The product is heterogeneous and probably consists of several distinct polynucleotides. Product and template complexes are present in multistranded structures. At least 50% of the viral particles (VSV-It), 30% of the VSV-II defective particle and probably none of the VSV-III defective particles, have enzyme. The defective particles do not have complementary sequences to each other or to the VSV-I viral RNA. The product transcribed at 37°C by VSV particles is complementary to VSV-I and VSV-II, but not to VSV-III RNA. The RNA from all three viral particles are linear, covalent, polynucleotides. Product is synthesized by base-pairing to the template RNA and not by end addition.

1. Introduction

Vesicular stomatitis virus possesses a transcriptase enzyme in the viral particles (Baltimore, Huang & Stampfer, 1970). The enzyme activity can be demonstrated in the presence of a non-ionic detergent, Triton NlOl, or Nonidet NP40, and in the viral particle it will only copy the endogenous viral template RNA (Bishop & Roy, 1971). The kinetics of product synthesis, together with a pulse-chase product analysis, has shown that product RNA is initially synthesized in association with the template RNA and subsequently released as a heterogeneous mixture of free species having molecular weights in the range of 2 to 10 x 105-compatible with being messenger RNA. The template RNA is undegraded during the synthesis (Bishop & Roy, 1971).

The experiments documented in this paper were performed to answer the following questions concerning the product synthesis.

(1) How many of the viral particles have functional enzyme ? (2) Is the transcription repetitive ? (3) Is the whole template transcribed 1 (4) What structural form (double-stranded or multistranded) do the replicating template-product complexes possess ? (5) What identity or complementarity does VSV-It RNA have with the RNA from the defective particles, VSV-II and VSV-III ?

t Abbreviations used: VSV, vesicular stometitis virus; dodecyl SOP, sodium dodecyl sulfate. 799

800 D. H. L. BISHOP AND P. ROY

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FIG. 1. Limited ribonuclease digestion of Qfl multistrrtnded end double-stranded RNA. Qfl multistranded or double-stranded RNA was isolated from a lo-min in vitro Qfl replioase

reaction primed with 3H-labeled Qfi RNA, using [w~~P]UTP to label the product species. The products of the reaction were purified from triphosphates by Sephadex G60 column chromato- gr8phy (see Biehop & Roy, 1071), alcohol-precipitated from the void volume snd resolved by gel electrophoresis using B preswollen %a-acrylamide cross-linked 2.4% polyaarylamide gel (Bishop, Claybrook & Spiegelman, 1067). Electrophoresis wee at 6Ov, 1Omnand 18Omin (see Pace, Bishop & Spiegelman, 1067). The gel was sliced and pairs of O.&mm gel slices &ted at 4°C for 8 hr with @6 ml. of O*lSm-N&l, 0.1% dodecyl SO1, 0.01 ra-Tris*HCl buffer, 0.006 M-EDTA (pH 7) in capped scintillation counting vials with gentle reciprocal shaking. The eluants oontaining multistrsnded Q/3 RNA (Qfl-FS) were pooled and concentrated by alcohol precipitetion. Double-stranded QB

VESICULAR STOMATITIS VIRUS PRODUCTS

2. Materials and Methods (a) Cell line : virus preparation

801

The preparation of vesicular stomatitis virus (infectious particle: VSV-I) and two defective particles (VSV-II, VSV-III) f rom BHK21 cells infected with VSV is described elsewhere (Roy & Bishop, 1971). Reovirus double-stranded RNA was generously given by Dr Acs, Institute for Muscle Disease, Inc., New York City.

(b) Enzyme assays; preparation of product and tewvplate RNA from reaction mixtures; gel electrophoresis

Reaction assays and techniques to purify RNA from reaction mixtures have been described (Bishop & Roy, 1971). Gel electrophoresis was conducted in his-acrylamide cross-linked, 2.2% polyacrylamide gels in E or 3E buffer, containing 0.1% dodecyl SO, (Bishop, Claybrook & Spiegelman, 1967). When RNA samples were pretreated with ribonuclease, or annealed in 0.4 M-NaCl, the gel and electrophoresis buffer contained 3E buffer (approximately 0.45 M-sodium acetate), and the electrophoresis was for 3 hr at 50 V and 20 mA per gel. Otherwise, E buffer (approximately 0.15 M-sodium acetate) was used and the electrophoresis conducted for 2 hr at 50 v and 10 mA per gel.

(c) Ribonuclease treatment of template and product RNA

Limited ribonuclease treatment of RNA samples was used to digest single-stranded RNA but leave double-stranded regions intact. The conditions chosen were developed using Q/l multistranded (Qfl-FS) and double-stranded (&6-HS) RNA (Pace, Bishop & Spiegelman, unpublished observations), and are illustrated in Fig. 1. RNA samples (50 pl.), suspended in O-1 M-NaCI, 0.01 M-TrisHCl buffer, 0.005 M-EDTA (pH 7), were treated with ribonuclease T, (1 pg/ml. final concentration), for 10 min at room temperature. The digestion was stopped by addition of dodecyl SO, (1 ye final concentration), then glycerol added (20 ~1.) and the mixture loaded directly on a polyacrylamide gel. The profiles obtained, after electrophoresis, of multistranded Qfi RNA (before or after ribonuclease treatment), and double-stranded Q/3 RNA (also before or after ribonuclease treatment), are shown in Fig. 1. Evidently the conditions used for limited ribonuclease digestion removed the single-stranded tails from Q/3-FS RNA but left intact the double-stranded regions. Multistranded Qfl-FS RNA was converted into a form electrophoretically in- distinguishable from double-stranded Q/3-HS RNA. Similar conditions were chosen for the limited ribonuclease digestion of VSV reaction products described here.

(d) Annealing of template and product RNA

Annealing of RNA is dependent on the temperature, salt concentration, length of incubation and mole concentrations of the RNA samples (see Roy & Bishop, 1971). At concentrations greater than 1 pg/ml., polio plus and minus strarlds anneal quantitatively in 0.4 M-NaCl, 0.01 M-Tris*HCl buffer, pH 7.4 at 60°C in 20 min (Roy & Bishop, 1970). Consequently, similar conditions were chosen for annealing VSV template and product species. The annealing volume was 50 ~1. and always contained more than 1 pg VSV RNA/ml. of 0.4 M-NaCl, 0.01 M-Tris.HCl buffer, pH 7.5. The specific activity of the 3H-labeled viral RNA (counted in Aquasol, New England Nuclear, Boston, Mass.), was 2 x lo5 cts/min/pg, whilst that of the 32P product (calculated from the ape&i cactivity of the [a-32P]UTP, assuming 25 moles O/e UMP in the product), was 15 x 105 cts/mm/pg. The amount of product species in the annealing mixture varied with the mdividual

RNA (Qp-HS) was similarly isolated from relevant gel slices. The RNA samples, suspended in 50 ~1. of 0.1 M-NaCl, 0.01 M-Tris.HCl buffer, 0.005 M-EDTA (pH 7.0), were treated with ribonu- cleese T, (1 pg/ml. final concentration) for 10 min at room temperature. The digestion was stopped by addition of dodecyl SOI (1% final concentration), glycerol added (20 ~1.) and the mixture loaded directly on a preswollen 2.4% polyacrylamide gel. After electrophoresis at 60 v and 10 mA per gel for 180 min the gels were sliced, dissolved in H,O, and counted in Kinard’s scintillation fluid (Pace el al., 1967). The profiles of Q,%FS RNA before (a) or after (b) ribonuclease treatment and Q,&HS RNA also before (c) or after (d) digestion are given. SMW indicates small molecular weight species.


sample. The counting efficiency in the presence of a dissolved l-mm gel slice, relative to that obtained by counting directly in Aquasol, was 68% for 3H and 77% for 3aP. The cross-over from 3aP counts into the 3H channel was 2.4% and 5.6% in the absence or presence of a dissolved gel slice.

(e) Melting vesicular stomtitis virus template and product RNA species

RNA samples at a concentration of 5’pg/ml. of 0.01 a6-sodium phosphate buffer (pH 7.0), 0.005 M-EDTA, were melted by heating 20- to 50-~1. volumes at 100°C for 30 set and cooling quickly. In the absence of the phosphate buffer, the recovery of intact VSV RNA was only 20% under otherwise identical conditions.

3. Results

In order to determine the number of viral particles which possess enzyme, reaction mixtures were extracted and examined for the involvement of template viral RNA in complexes with product RNA species (see Discussion). The degree of completeness of the transcription process was examined by determining the relative mass of product to template RNA in these native complexes, as well as in synthetic hybrids obtained by annealing all the product and template species in the extracted RNA. This latter technique also showed that the transcription process is repetitive. These experiments were performed with viral preparations which contained both the infectious VSV-I and the two defective particles, so that information could be obtained on the partici- pation of all three viral particles in the transcription process.

(a) Properties of the product synthesized by vesicular stonaatitis virus particles

A crude preparation of [3H]uridine-labeled VSV particles (containing VSV-I, -11 and -111) was incubated under standard reaction conditions, utilizing[a?-3aP]UTP to label the product species. RNA was extracted at the indicated time intervals. The template and product RNA species were analyzed by gel electrophoresis (see Materials and Methods). The following observations can be made by a critical examination of the unannealed proilles obtained (Fig. 2) :

(1) As demonstrated previously (Bishop t Roy, 1971), product is initially synthesized in association with the template species and subsequently accumulates in the small molecular weight region (molecular weights 10 to 2 x 105). (2) The free product is heterogeneous. But the size distribution is quite specific. There is very little free product in the size range lower than 2 x IO5 daltons (fifth to sixth cm of the gel) (see Bishop & Roy, 1971). It was shown previously (Bishop&Roy, 1971) that there is little intra- or extra-viral ribonuclease activity. Consequently, we are led to the conclusion that there are several RNA species liberated. (3) As the time-course of the experiment progressed, the recovery of free 3H- labeled VSV-I RNA diminished (see Fig. 3 for a definition of “free VSV-I RNA”). There was a concomitant involvement of the 3H label in slower moving complexes. At 60 minutes, half of the 3H-labeled VSV-I RNA was involved in these complexes (see Fig. 3). This suggests, therefore, that 50°h of the viral particles have active enzyme (see Discussion). In the lo-, 20- and 30-minute samples, about 33% of the 3H-labeled VSV-I RNA was involved in these complexes. (4) Throughout the time-course of the experiment, the recovery of free VSV-III RNA hardly altered (see Fig. 4), suggesting that the VSV-III particles do not

VESICULAR STOMATITIS VIRUS PRODUCTS 803

have an active enzyme-so that they are not involved in the slower moving template-product complexes. (5) Also, during the time-course of the experiment, the recovery of free VSV-II RNA dropped 30%. This suggests that the VSV-II particles have enzyme but that the enzyme is either less active than that of VSV-I, or that fewer particles possess enzyme. It will be shown in a subsequent communication that VSV-II preparations contain no more than 30% of the particle transcriptase activity that VSV-I possesses, whilst VSV-III has less than 5% of the VSV-I particle transcriptase. (6) Bearing in mind that the specific activity of the 32P product label was about eight times that of the 3H viral label, an estimation can be made of the mass of product relative to the mass of template in the mpkxes. The term “mass” is used rather than size because it will be shown that at five minutes the complexes are multistranded with respect to product species. The relative mass of product to template is about 1: 20 for the 5-minute sample, 1: 16 for the lo-minute and 20-minute samples, 1: 8 and 1: 16 for the complexes in the first or second three mm of gel for both the 30-minute and 60minute samples. Obviously these esti- mations are rough and do not give the upper or lower limits of the product- template masses. However, by way of comparison, Qfl multistranded RNA (Q/3- FS) has about 1.5 : 1 product to template RNA ratio in the in vitro reaction product (Bishop, unpublished observations).

What these VSV product-template figures suggest therefore is that free product RNA is liberated from complexes which have not formed a complete complement of product on the template RNA.

(b) Properties of the annealed reaction products

The reaction products of the time-course experiment described above were annealed (see Materials and Methods) and subjected to electrophoresis (Fig. 2). The following observations can be made from evaluation of the profiles obtained.

(1) Annealing resulted in the formation of slower moving 32P product-3H template hybrids. (2) The free product of the unannealed 5-, lo- and 20-minute samples was complementary to the viral RNA, and was recovered, after annealing, in the hybrids. (3) For the 5-minute and lo-minute samples, free 3H-labeled VSV-I viral RNA remained after annealing. No free product RNA species were recovered, indicating that, both before and after annealing, there was an excess of template RNA over product RNA molecules. (4) For the 20-minute sample after annealing there was almost no residual free 3H-labeled VSV-I RNA and no free 32P-labeled product RNA, indicative of an apparent parity between template and product molecules. (5) For the 30-minute and 60-minute annealed samples, all of the VSV-I 3H- labeled viral RNA was recovered in the hybrids. Unannealed 32P-labeled product was present in the small molecular weight region of the gels. These observations point to the conclusion that at these times, there is excess product to template molecules and that even though, at best, 5074 of the viral particles have active enzyme, there are sufficient product molecules to hybridize to all the virion 3H-labeled RNA present in the extract. Consequently the transcription process is repetitive,

62

go4 D. H. L. BISHOP AND P. ROY

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(a) Nat annealed (a) Annealed

(b) Nat annealed

Distance moved ( cm) Distance moved (cm)

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(b) Annealed

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Fm. 2. Gel electrophoresis of vesicular stomatitia virus reaction products with or without an annealing pretreatment.

A lOO-fold standard reaction (12.5-ml. vol.), containing 1 mg of [3H]uridine-labeled VSV particles (2 x 10s cts/min/pg RNA), and [w~~P]UTP to label the product RNA, was incubated at 37% (see Bishop & Roy, 1971 for details). Samples (1.26 ml.) were withdrawn at intervels, extracted by phenol in the presence of dodecyl SO1, the RNA separated from triphosphetes by Sephadex GSO column chromatography and precipitated from the void volume (for details see Bishop & Roy, 1971). The RNA was suspended in 0.4 M-N&I, 0.01 M-TrisHCl buffer (pH 7.4), to give a concentration of greater than 1 pg of 3H-labeled RNA/ml. A portion of each sample was subjected to gel electrophoresis on his-acrylamide cross-linked, 2.2% polyacrylamide gels containing 3E buffer with or without an annealing pretreatment (see Materials and Methods). After electrophoresis (see Materials and Methods), the gels were sliced, dissolved in HaOa and counted in Aquasol (see Bishop & Roy, 1971). (a) 5-min sample; (b) lo-min sample; (o) 20-min sample. (d) 30-min sample: (e) 60-min sample.

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(d) Not onneoled

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In relation to the question of whether the free unannealed product is complementary or identical to the template RNA, purified viral RNA (1 pg) was added to a portion of the 60minute reaction product and the mixture annealed and subjected to gel electrophoresis. All of the product was recovered in the hybrids; there WES no free product remaining. Consequently it can be concluded that all of the product RNA species synthesized in the ir, vitro reaction are complementary to the virion RNA. The product species do not replicate autonomously. (6) The recovery of VSV-I RNA, in its free or hybrid state after annealing (and compared to their recoveries before annealing), is shown in Figure 3. The

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FICA 3. The recovery of free or complexed VSV-I RNA. The amount of 3H label in the gel profiles of Fig. 2 (see Fig. 6 for the zero-time sample) wss

computed and the percentage of label in the VSV-I, VSV-II and VSV-III regions divided by their respective molecular weights (Bishop & Roy, 1971: 4.4 x 106 for VSV-I, 1.9 x lo6 for VSV-II and 1.1 X IO8 for VSV-III). The figures obtained (“relative moles of RNA”) were plotted against the time interval that the sample was withdrawn. For VSV-I RNA the reoovery of label in the “free” VSV region (8) (about 7 to 12 mm of the gel) and “ complexed” region (b) (from 0 to 0 mm) are given. The relative moles of RNA for the annealed profiles are also shown.

recoveries, expressed as relative moles of RNA, summarize the involvement of the template as described in the preceding paragraphs. The “relative moles of RNA” were calculated from the amount of VSV-I RNA recovered in the gel as free (or complexed or hybridized) 3H species, expressed as percentage of the total computed sH label recovered-divided by its molecular weight. The value of expressing the results in this manner is that one is thereby comparing in molar tern what happens simultaneously to the three viral RNA species (VSV-I, VSV-II and VSV-III-present in the same viral preparation). (7) The recovery of free VSV-III RNA throughout the time-course experiment, either before or after annealing, is shown in Figure 4 in terms of its relative moles of RNA. Apparently, VSV-III RNA does not anneal to the product RNA sp- thesized during the reaction. (8) The recovery of free VSV-II RNA (relative moles of RNA) throughout the time-course experiment (again before or after annealing) is shown in Figure 5. (As with VSV-I RNA, VSV-II RNA anneals to product species with comparable molar eficie?acy. It was pointed out previously that free 3H-labeled VSV-II RNA in the unannealed samples decreases 30% during the reaction, indicating enzymic activity of the VSV-II particles. Therefore it is probable that free


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FIQ. 4. The recovery of free VSV-III RN A. The recovery of free 3H-labeled VSV-III RNA before or after annealing (rel&ive moles of

RNA), is plotted against the time interval the sample was withdrawn (see Fig. 3 for explanation).

I I I 20 40 60

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FIQ. 6. The recovery of free VSV-II RNA. The recovery of free “H-labeled VSV-II RNA, before or after annealing (relative moles of

RNA), is plotted against the time interval the sample was withdrawn (see Fig. 3 for details).

product synthesized on VSV-II RNA templates will be present in the reaction mixture.) It is also possible that the VSV-I product species hybridize to VSV-II RNA, and this could account for the annealing of the VSV-II RNA. (9) The relative mass of product : template (calculated from the 32P and 3H labels) in the hybrids for the B-minute annealed sample was 1:2Q; for the lo-minute and 20-minute samples it was 1:16; whilst for both the 30- and 60.minute samples it was 1: 8. This also suggests that the hybridized product did not cover the entire template-however, these figures do not tell us what were the ranges of transcribed templates in the hybrid or native complexes.

(c) Homology between VSV-I, VSV-II and VSV-III In order to determine if VSV-I, VSV-II and VSV-III have cmpleme~tary sequences

to each other, RNA extracted from the viral particles was subjected to gel electrophoresis with or without an annealing pretreatment. As shown in E’igure 6, the distribution of label was essentially identical with or without annealing, indicating that the three RNA species are not complementary to each other. Purthermore, none of the particle types contain plus and minus complementary species of RNA, as demonstrated by Robinson (1970) for the viral RNA of two myxoviruses.


Distance moved (cm)

C-4

FIG. 6. Homology of VSV-I, VSV-II and VSV-III RNA. RNA was isolated by phenol extraction of a [sH]uridine-labeled VSV particle preparation.

The RNA (at a concentration of 6 rg/ml. of 0.4 n-NaCl, 0.01 Y-TrisHCl buffer, pH 7.5) was subjected to annealing before electrophoresis in 2.2% polyaorylamide gels containing 3E buffer (Materials and Methods). After electrophoresis the gels were sliced, dissolved and counted in Aquasol (Bishop & Roy, 1971). The proliles of an unannealed (a) as well as an annealed (b) sample are given.

(d) Melting the vesicular stomatitis virus reaction product

The reaction product from the lo-minute sample (Fig. 2) was melted by heat treatment (see Materials and Methods) and subjected to gel electrophoresis (Fig. 7). No breakdown of 3H-labeled RNA to small molecular weight species was observed. No dissociation of the VSV-I RNA into VSV-II or VSV-III species was evident. The product RNA was completely dissociated from the template species and was recovered with the same distribution pattern as found previously for the unannealed free 3aP.labeled RNA species. Product RNA is therefore not covalently linked to template molecules.

(e) Pro&t analysis by ribonuclease treatment

We have demonstrated that both in the unannealed native complexes and annealed hybrids, the mass of product is smaller than the mass of template present-even at times when free product RNA molecules are being released from the complexes. Although from the relative mass determinations an average of one-eighth of the template was associated with product RNA in the native complexes, this figure does not tell us what is the longest length of hydrogen-bonded template-product regions present in the complexes.

In order to determine the range of size of the hydrogen-bonded template-product species, two prerequisites were necessary.

(1) To isolate the double-stranded RNA regions of the complexes by limited ribonuclease digestion. (2) To size them by gel electrophoresis.


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Fro. 7. Melting the RNA of a vesicular stomatitis virus reaction product. A portion of the RNA isolated from a IO-min incubation of sH.labeled VSV particles in a

reaction mixture containing [a-saP]UTP to label product RNA (Fig. 2) was melted as described in Materials and Methods. The gel electrophoresis profile obtained after melting is given. Electro- phoresis in E buffer in a 2.2% polyacrylamide gel was at 50 v and 10 mA for 120 min.

The conditions for limited digestion of RNA-whereby single-stranded regions would be removed but double-stranded regions left intact--were realized using Q/3 multistranded RNA (Materials and Methods). Limited digestion of the 3H-labeled viral VSV RNA totally solubilized the 3H label. Reovirus double-stranded RNA was used to obtain the necessary calibration of the gels for sizing double-stranded RNA (Fig. 8).

The results of limited digestion of the native reaction products from a kinetic experiment are shown in Figure 9. (Note that the speciJic activity of the proo7u.d in this experiment was 4 x lo5 ctslmin/pg-see figure legend.) The following conclusions were drawn.

(1) The double-stranded RNA species isolated from the complexes were heterogeneous in size. The digestion products of the IO-minute and B-minute samples had a smaller size distribution than those of later samples.

(2) None of the double-stranded RNA’s approached 8x lo6 or even 4 x lo6 molecular weight, indicating that there was no hybrid equivalent to a totally transcribed template. The percentage of total 32P label in the 2 to 4 x lo6 molecular weight size range was 0, 2, 3, 3 and 2% for the 5-, lo-, 20-, 30- and GO- minute samples, respectively. The majority of the double-stranded RNA was


Molecular weight ( x IO61

Distance moved (cm)

FIQ. 8. Gel electrophoresis of reovirus double-stranded RNA. Reovirua [3H]uridine-labeled double-stranded RNA was subjected to electrophoresis in a 2.2%

polyacrylsmide gel containing 3E buffer at 50 v and 20 mu for 180 min, The gel was sliced, dissolved in H,Oa and counted in Aquasol (Bishop & Roy, 1971). Three main bands of RNA (2.5 x 106, 1.4 x 10e and 0.8 x lo8 molecuIar weight-Shatkin, Sipe & Loh, 1968) were evident. The mobility of double-stranded RNA was used to calibrate the gels recorded in Figs 9 and 10, assuming a linear relationship between the logarithm of the molecular weight and distance moved (Shatkin eb al., 1963).

recovered in the O-5 to 2 x lo* molecular weight region and constituted 46, 30, 15, 6 and 3% of the total 32P for the 5, lo-, 20-, 30- and 60-minute samples, respectively. (3) ,Although all of the product RNA in the undigested 5-minute sample was associated with template (see Fig. 2 for example), 50% of the 3aP label was solu- bilked by the limited ribonuclease treatment (computed as the label present in the 6- to $-cm region of the gels). This indicates that half of the product in the complexes of the &minute reaction is in a single-stranded state and that the native complex is multistranded, containing single-stranded product tails. The percentage of the total label solubilized by the limited digestions of the other samples was 65, 80, 90 and 94% for the lo-, 20-, 30- and 60-minute samples, respectively. (4) The over-all 32P-labeled product to 3H-labeled template naass ratio of the ribonuclease-resistant species present in the 0- to 6-cm of the gels approached parity. Finally, a similar profile of ribonuclease-resistant RNA species was obtained

when the 6Ominute reaction product was pre-annealedprior to ribonuclease treatment (Fig. 10). However, the amount of 3H label recovered in the double-stranded species was greater than that found with the unannealed sample (see Fig. 9). Assuming that all of the 3aP label that could anneal had annealed to the template (see Results,

Molecular weigh,

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FIG. 9. Limited ribonuclease digestion of the 1 RNA was isolated from a time-course experiment (similar to that described in Fig. 2) in which [~x-~~P]UTP was

lated at different t,ime intervals wore subjected t,o limited ribonucleaw treatment (see Materials and Methods and rwunted in Aquasol. (a) 5.min sample; (b) IO-min sample; (c) 20.min sample: (tl) 30-min sample; (e) 60.min aa oated. The theoretical positions for double-stranded RNA (size-range 1 to 0.1 x 10s molecular weight) (see Fig.

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%NA isolated from VSV-primed reactions. used to label the RNA products of a [3H]uridine-lebeled VSV-primed reaction. Samples of the RNA iso- Fig. I), and subsequently resolved by gel electrophoresis. The whole gel was sliced, dissolved in H,Oz and

mple. The positions where single-stranded VW-I, VSV-II and VSV-IIr RNA would be recovered are indi- J) are &o indicated. The specific activity of the product RNA in this experiment was 4x lo5 cts/min/pg.

VESICULAR STOMATITIS VIRUS PRODUCTS

Molecular weight (x IO6 1

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Fra. 10. Limited ribonuclease digestion of an annealed RNA sample isolated from a VSV- primed reaction.

The RNA from a 60-min reaction (Fig. 9) was pm-annealed (Materials and Methods) before limited ribonuolease digestion (Materials and Methods and Fig. 1) and gel electrophoresis (Fig. 9). The positions where single-stranded VSV-I, VSV-II and VSV-III RNA would be recovered are indicated. The theoretical positions for double-stranded RNA (size-range 4 to 0.1 x lo* molecular weight) (see Fig. 8) are also indicated.

section (b)), then, since 70% of the total 3H label was solubilized, this indicates that 70% of the viral RNA had no cornplementury 32P RNA to anneal to. This also indicates that a minimum of one-third of the template had been transcribed in vitro. It should be noted (Fig. 10) that 65% of the 32P label of the annealed 60-minute sample was solubilized, agreeing with the earlier postulate that for the later samples there are excess free product molecules after the annealing treatment.

When a similar experiment was performed with the 20-minute reaction product, only 10% of the 32P label was solubilized when the sample was pre-annealed prior t#o

digestion (in comparison to 80% solubilization for the unannealed samples).

4. Discussion The results obtained in this investigation concern the nature of the product synthe-

sized by vesicular stomatitis virus particles. The following conclusions are drawn. (1) At least 50% of the VSV-I particles possess transcriptase enzyme. The number of enzyme molecules per particle is unknown. (2) Fewer of the VSV-II particles contain active transcriptase. Probably VSV-III particles contain no active enzyme at all. (3) All the product synthesized in the reaction was complementary to VSV-I and VSV-II RNA but not to VSV-III RNA,


(4) None of the RNA species isolated from the three viral particles is complementary to any other species. The viral ribonucleic acids are linear covalent polynucleotides. (5) Product species are synthesized in multistranded complexes in which they are, after extraction, partially hydrogen-bonded to template molecules. (6) Transcription is repetitive but not all the template is transcribed in reactions run at 37°C. RNA species are heterogeneous and have molecular weights of between 2 and 10 x lo5 daltons.

(a) The nature of the complexes i&nti$ed by gel electrop~re& of rea&m products

Much of the work described in this paper is based on the assertion that the slow- moving complexes isolated from a reaction mixture represent product and template associations. The belief is that in the reaction these ‘complexes ’ are generated by the transcriptaee and represent, in part, hydrogen-bonded product-template association. It has been assumed that involvement of the 3H label in complexes is indicative of active viral enzyme. These assertions are based on the following evidence.

(1) The “ complexes ” are not present in an unincubated reaction mixture (see Bishop & Roy, 1971). (2) The initial product is mostly (see Fig. 2(a)) or totally (Bishop $ Roy, 1971) present in the first 1 cm of the gels. There is template label in the same gel region which was not present in the unincubated control (Bishop & Roy, 1971). (3) An [c+~~P]UTP pulse of a IO-minute-old reaction (initially incubated with cold UTP) labels only the complexes, indicating that these are the actively grow- ing sites of product synthesis (Bishop & Roy, 1971). The “pulsed” material can be chased into free, fast-moving product species. (4) All the complexes disappear on melting; almost all the 3H template label is then recovered as free undegraded viral RNA species-equivalent to the initial gel profile of the 3H-labeled viral preparation; all the 3aP product label is recovered as free single-stranded fast-moving species (Pig. 7). Ribonuclease treatment of the melted RNA gives a ribonuclease resistance of 0.2% for the 3H- labeled RNA and 15o/o for the 32P-labeled RNA (compare Pig. 9(b) in which the ribonuclease resistance of the 32P and 3H RNA labels is about 30% and 5%, respectively, for the unmelted complete IO-min reaction products). (5) Annealing reaction products generates slow-moving hybrids of template and product molecules (Fig. 2) and increases the total amount of 32P- and 3H- labelled ribonuclease-resistant species (see Fig. 10 and Results section).

The native complexes observed are not artifacts of the electrophoresis process (see Fig. 7). When a melted IO-minute reaction product was extracted by phenol and precipitated by alcohol, prior to electrophoresis, a profile of 3H- and 32P-labeled RNA species was obtained which was identical in distribution to that given in Figure 7. However, annealing the same melted reaction product gave a profile similar to that shown in Figure 9(b). These observations, together with the evidence presented above, indicate that the native complexes are not derived during the course of an extraction process from free product and free template molecules.

The fact that almost all the 32P-labeled product of the &minute reaction (Fig. 2(a)) was present in the native complexes isolated from the reaction mixture, but that only 50% of the 32P-label was ribonuclease resistant (Fig. 9(a)), suggests that the


complexes are multistranded, viz. contain more than one homologous product strand per template strand.

It should be stressed, however, that these complexes represent the RNA extracted from enzyme-RNA species and consequently do not necessarily indicate the state of the RNA present in the unextracted reaction mixture.

(b) Transcription is repetitive

The observations which lead to the conclusion that transcription is repetitive are the following : in the B-minute sample, the product is in a multistranded form (see Results section). During the enzyme reaction (involving only 50% of the VSV-I particles and 30% of the VSV-II particles), product RNA is made in sufllcient quantity to anneal to all the VSV-I (and VSV-II) RNA present. The ratio of product-template mass in the complexes is 1: 8 (at best) even when free RNA species are present in the reaction mixture. The free product species of later samples can be totally annealed only on the addition of viral RNA, so that it can be concluded that the free species are extra identical copies of the product RNA already present in the hybrids.

(c) Not all the template is transcribed

Evidently from the results obtained it is suggested that complete transcription is not a prerequisite to the release of product species, as is the case in the QR system.

Several pieces of evidence lead us to also suggest that the template was incom- pletely transcribed even by 60 minutes (or very infrequently completely transcribed under the conditions of the experiments performed here).

The natural complexes in the unannealed samples have a Iow product-template mass ratio even at 60 minutes.

No evidence of a mass parity was obtained for the hybrids generated by annealing the total reaction products. Moreover, ribonuclease digestion of the native or annealed reaction product left residual double-stranded RNA species with a 1 x lo6 molecular weight average-the largest double-stranded RNA obtained after digestion had a 3 x lo6 molecular weight. However, it could be argued that the length determinations of the double-stranded species after ribonuclease digestion are not conclusive, especially if product synthesis is multiply initiated along the t’emplate with intervening single- stranded template regions.

It will be shown in a subsequent communication that complete transcription of the viral genome can be obtained in reactions run at 28°C (Bishop, manuscript submitted for publication). The kinetics of transcription at 28°C are linear t,hrough 6 hours of incubation, whereas transcription at 37°C cuts off between 60 and 90 minutes (Aaslestad, Clark, Bishop & Koprowski, manuscript in preparation). Moreover, the initial rate of transcription at 28°C is three- to fourfold greater than that at 37°C (Aaslestad et al., manuscript in preparation). Complete transcription in reactions conducted at 28°C takes about 60 to 70 minutes (Bishop, manuscript submitted for publication). The reasons for these differences in transcription rate and comple- tion between reactions run at 37°C and 28°C are not known.

(d) Are more than one messenger species produced?

Evidence that several polynucleotides are liberated after transcription of the viral RNA is suggested by the heterogeneity of the free product and double-stranded RNA in the complexes. It was shown previously that there is little nuclease activity in


the viral preparation (Bishop & Roy, 1971), and also that there is little product in the 2 x IO5 or smaller molecular weight range. How many different product species are present is impossible to estimate, especially with the contribution from VW-II particles in the reaction mixture. Also, there is always the possibility that premature abortion of transcription contributes to the heterogeneity obtained (although one might expect therefore to get more RNA in the lower molecular weight regions).

The results discussed in this paper suggest that when a virus initially infects a cell, several identical copies of messenger RNA are synthesized-presumably to condition the cell for subsequent processes of the infection. The value for the virus of possessing a transcriptase is manifold. Many messenger RNA species are produced from one template, their production is sequestered and chance, irrevocable nicking of the one viral genetic tape the virus possesses is minimized.

We appreciate the advice and encouragement of Dr Sol Spiegelman and the expert technical assistance of Andrew Czernik.

This investigation was supported by U.S. Public Health Service research grant CA-02332 from the National Cancer Institute.

REFERENCES

Baltimore, D., Huang, A. S. & Stampfer, M. (1970). Proc. Nat. Acad. Sci., Wash. 66, 563. Bishop, D. H. L., Claybrook, J. R. t Spiegelman, S. (1967). J. Mol. Biol. 26, 290. Bishop, D. H. L. & Roy, P. (1971). J. MOE. Biol. 57, 513. Pace, N. R., Bishop, D. H. L. & Spiegelman, S. (1967). J. ViiroZogy, 1, 771. Robinson, W. S. (1970). Nature, 225, 944. Roy, P. & Bishop, D. H. L. (1970). J. WoZogy, 6, 604. Roy, P. & Bishop, D. H. L. (1971). Biochka. biophys. Acta. 235, 191. Shatkm, A. J., Sipe, J. D. & Loh, P. (1968). J. Virology, 2, 986.

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Properties of the product synthesized by vesicular stomatitis virus particles

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