Ribonucleic acid synthesis of vesicular stomatitis virus

VIROLOGY

42, 946-957 (1970)

Ribonucleic

ALICE Department

Acid

Synthesis

III. Multiple

Complementary

S. HUANG,

DAVID

of Biology,

Massachusetts

of Vesicular

Virus

Messenger RNA Molecules’

BALTIMORE2 Institute Accepted

Stomatitis

AND

of Technology, August

MARTHA

STAMPFER3

Cambridge, Massachusetts

01139

4, 1970

Infection of Chinese hamster ovary cells by vesicular stomatitis virus (VSV), in the absence of any detectable interference by defective particles, leads to a replacement of host polyribosomes by a distinct class of smaller polyribosomes. These small polyribosomes synthesize only virus-specific polypeptides and are as active in protein synthesis as polyribosomes from uninfected cells. The 28 S and 13 S single-stranded RNA species made during viral replication are associated with the viral polyribosomes after separation of the polyribosomes by rate zonal and isopycnic centrifugations. Release of the 28 S and 13 S RNA species from polyribosomes by EDTA and hybridization of these RNA species to virion RNA shows that they have nucleotide sequences which are complementary to those of the virion RNA. The finding of multiple pieces of messenger RNA, all smaller than 40 S virion RNA, suggests that VSV polypeptides are synthesized from individual molecules of RNA, rather than from one large messenger RNA. These studies also demonstrate the presence of newly synthesized nucleocapsid-like structures which contain heterogeneous 23-40 S RNA. INTRODUCTION

sedimentation constant as RNA extracted from B particles (Huang and Wagner, 1966; During the replication of vesicular stomaBrown et aI., 1967) and is similar to virion titis virus (VSV) in Chinese hamster ovary RNA by base ratio analysis (Newman and cells, more than nine speciesof viral specific Brown, 1969). RNA are synthesized (Stampfer et al., 1969). In order to find which of the singleThey include duplex RNA molecule, at least stranded species functions as messenger three partially ribonuclease-resistant RNA RNA, polyribosomes of VSV-infected Chinese species, and a minimum of five singlehamster ovary cells were first characterized stranded RNA species. When cells are inby their ability to synthesize virus-specific fected with purified B particles of VSV and proteins and then examined for attached interference by defective T particles (Huang viral RNA. Although it can be argued that et al., 1966) is completely absent, the pattern not all polyribosome-associated viral RNA of RNA synthesis becomesless complicated. is necessarily messenger RNA, no better Under these conditions, single-stranded criterion is presently available. In this study RNA detected by sucrose gradient centriwe therefore have defined as messengerRNA fugation falls into three categories: 40 S, any viral RNA which is associatedwith poly28 S, and 13 S. The 40 S RNA has the same ribosomes during both rate zonal and isopycnic sedimentation, and which is dis1 This investigation was supported by American sociated from polyribosomes by EDTA Cancer Society Grant E-512 and U.S. Public treatment. Health Service Grant AI-08388. 2 Faculty Research Awardee of the American Cancer Society. 3 Predoctoral fellow of the National Science Foundation.

MATERIALS

AND METHODS

Virus and cells. VSV and its growth in Chinese hamster ovary cells have been de946

mRNA OF VESICULAR scribed previously (Stampfer et al., 1969). The preparation of standard B particles of VSV, completely uncontaminated by defective interfering T particles, is described elsewhere (manuscript in preparation). The medium used for growth of cells and virus and for all the experimental infections was Joklik-Modified Eagle’s minimum essential medium for Spinner cultures (Grand Island Biochemical Co., Grand Island, New York) which was supplemented with nonessential amino acids and 7 % fetal calf serum. For amino acid incorporation, cells were resuspended in leucine-free minimum essential medium, supplemented with nonessential amino acids and 5 % dialyzed fetal calf serum. Bu.er solutions. Three different buffer solutions were used to separate polyribosomes and ribosomal subunits in sucrose gradients. They are reticulocyte standard buffer (RSB: 0.01 M Tris, pH 7.4; 0.01 M NaCl; 0.0015 M MgCl,) ; high salt buffer (HSB: 0.01 M Tris, pH 7.4; 0.5 M NaCl; 0.05 MgCl,); and reticulocyte standard buffer containing EDTA (NEB: 0.01 M Tris, pH 7.4; 0.01 M NaCl; 0.02 M EDTA). For separating deproteinized RNA molecules, sucrose gradients were made with 0.5% sodium dodecyl sulfate buffer (0.01 M Tris, pH 7.4; 0.1 M NaCl; 0.001 M EDTA; 0.5% sodium dodecyl sulfate). Infections. Exponentially growing Chinese hamster ovary cells at 4 X lo5 cells/ml were concentrated lo-fold from suspension cultures and either not infected or infected with standard B particles of VSV at a multiplicity of 10. Actinomycin (10 pg/ml) was added and viral attachment was allowed to proceed at 37” for 30 min, after which the cells were diluted 2-fold with medium. All further incubations were at 37” prior to harvesting the cells. At this stage, 2 ml of the suspended, infected cells were removed to a 13 X 100 mm test tube containing 0.3 clCi of uridine-14C. At hourly intervals up to 6 hours after infection, 0.1 ml was removed and assayed for acid-precipitable radioactivity in a planchet counter as previously described (Stampfer et al., 1969). This provided a monitor for virus growth during each experiment.

STOMATITIS

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947

For amino acid incorporation, cells were harvested by centrifugation at the indicated times and resuspended at one half the volume in leucine-free medium. After 5 min of incubation, radioactive leucine was added. For uridine incorporation, radioactive uridine was added directly to the suspended cell cultures. Cells were harvested into a mixture of liquid and frozen Earle’s saline, washed and homogenized by standard techniques (Penman et al., 1964) except that Chinese hamster ovary cells were allowed to swell in reticulocyte standard buffer for 10 min at 4” prior to homogenization. When applicable, marker nucleocapsid in the form of uridinelabeled B particles was added to cytoplasmic extracts, which were then brought to 1% sodium deoxycholate and 1% BRIJ-58 just prior to sucrose gradient centrifugation. Centvifugations. All sucrose gradient centrifugations were performed in the L2-65B Beckman ultracentrifuge using the SW 27 rotor with large buckets. Sucrose gradients of 15-30% were used and collected through a Gilford recording spectrophotometer into fractions containing about 1.4 ml. Glutaraldehyde fixation and isopycnic centrifugation in preformed CsCl gradients have been described in detail (Baltimore and Huang, 1968). Acid-precipitable radioactivity from all gradients was assayed as described (Huang and Baltimore, 1970). Polyacrylamide gel electrophoresis. Cytoplasmic extracts or purified virions were solubilized in 1% sodium dodecyl sulfate and 3 % urea at 37” for 1 hour. They were then dialyzed against buffer containing mercaptoethanol, concentrated and electrophoresed on 7 % acrylamide gels in the presence of sodium dodecyl sulfate as previously described (Jacobson and Baltimore, 1968a) except that just prior to electrophoresis the polypeptides were further reduced and acetylated by the following procedure. A one-tenth volume of freshly prepared dithiothreitol (10 mg/ml) was added to a sample which was then incubated at 37” for 30 min. A one-tenth volume of iodoacetamide (35 mg/ml) was then added. 14C-amino acid-labeled VSV, used for marker

948

HUANG,

BALTIMORE,

STAMPFER

proteins, has been described in detail (Burge and Huang, 1970). Materials. n-Leucine-14C at 240 mCi/ mmole, leucine-4, 5-3H at 5 Ci/mmole, mCi/mmole and uridine-2-14C at -50 uridine-5-3H at 3 17 Ci/mmole were ob-

a. CONTROL 6 l/4 HR

b. 2

tained from New England Nuclear Corp., Boston, Massachusetts. Ribonuclease A was purchased from Worthington Biochemical Co., Freehold, New Jersey. Ribonuclease Tl and dithiothreitol (Cleland’s reagent) were obtained from Calbiochem,

c. 3 l/4

HR

HR

0.3

I1.-!

4260

0

.I-4.5

i d. 4 l/4

HR

_ie. 5 l/4

!O FRACTION

HR

20

30 NUMBER

IO

20

30

TOP

FIG. 1. Breakdown of host polyribosomes after infection by VSV. Aliquots of 4 X 10’ infected cells were harvested at (b) 2 hours, (c) 3.25 hours, (d) 4.25 hours, (e) 5.25 hours, and (f) 6.25 hours after infection, and the cytoplasmic extracts were analyzed on sucrose gradients containing reticulocyte standard buffer by centrifugation at 95,000 g for 2 hours at 4”. (a) Uninfected cells, after incubation with actinomycin for 6.25 hours, were similarly harvested and analyzed. The left scale on the ordinate indicates the absorbance to the left of the break in the experimental curve and the right scale indicates the absorbance to the right of the break in the experimental curve in each frame.

mRNA

OF

VESICULAR

Los Angeles, California. Iodoacetamide was purchased from Sigma Chemical Co., St. Louis, Missouri. VSV-RNA, used in the hybridization studies, was obtained from B particles by sodium dodecyl sulfate disruption and further purified on sucrose gradients as previously described (Stampfer et al., 1969). The various sources of other reagents have been previously acknowledged (Huang and Baltimore, 1970). RESULTS

Breakdown

of Host Cell Polyribosomes

by VSV

The normal polyribosome pattern of Chinese hamster ovary cells was identical to the pattern shown in Fig. lb and was, therefore, not depicted here. Actinomycintreated, uninfected cells showed a gradual reduction in the percentage of ribosomes in polyribosomes, the greatest loss occurring in the largest polyribosomes (Fig. la). In the actinomycin-treated, infected cultures no change was evident up to 2 hours (Fig. lb). During the next hour there was a striking reduction in the larger polyribosomes and an increase in the smaller ones (Fig. lc). The small polyribosomes became more prominent by 4.25 hours after infection (Fig. Id), but by 6.25 hours there were few polyribosomes left in the infected cell (Fig. lf). Because the growth of VSV under these conditions was essentially complete by 6 hours, the small polyribosomes were the most likely sites of viral protein synthesis. The greatest number of these smaller polyribosomes appeared between 3 and 4 hours after infection, and, therefore, all subsequent experiments were done during that period after infection. In order to display the small polyribosomes, sucrose gradients were centrifuged for longer times, and the buffer solution in the sucrose gradients was changed to a high salt buffer which causes better separation of polyribosomes and dissociates free monoribosomes into their 60 S and 40 S subunits (Huang and Baltimore, 1970). The ODs,o pattern for the small polyribosomes from VSV-infected cells was highly reproducible, showing at least four major peaks at 110 S, 130 S, 155 S, and 175 S

STOMATITIS

949

VIRUS

(Fig. Zd-f). The peak at 110 S, just ahead of the 60 S subunit, always appeared to be split. Synthesis and Release of Nascent Polypeptides by Polyribosomes from Uninfected and V/W-Infected Cells To test whether the small polyribosomes were active in protein synthesis, infected cells were compared to uninfected cells for their ability to incorporate radioactive amino acids into polyribosome-associated, nascent polypeptides and to release completed chains. Figure 2 shows such an experiment where the cells were exposed to leucine-14C for 3, 5, and 10 min. In unin-

0.3

0.2

0

IO

20

30

0 IO FRACTION

20 NUMBER

0

10

20

3,”

FIG. 2. Synthesis of nascent polypeptides by uninfected and VSV-infected cells. (a, b, and c) uninfected and (d, e, and f) infected cells were exposed to leucine-W at a final concentration of 0.3 rCi/ml at 3.17 hours after infection. Aliquots of 4 X 10’ cells were harvested at 3, 5, and 10 min after addition of leucine-‘4C. Cytoplasmic extracts were analyzed on sucrose gradients containing high salt buffer by centrifugation at 95,000 g for 2.45 hours at 4”. --, absorbance at 260 nm; O-----O, W. Pellets in (a) and (d) contained 1602 cpm and 1632 cpm, respectively.

950

HUANG,

BALTTMORE.

fected Chinese hamster ovary cells, as in other eukaryotic cells (Kuff and Roberts, 1967; Baltimore, unpublished observations), there was an increasing ratio of radioactivity to ODZ60 as the size of the polyribosomes increased (Fig. 2a, b, and c). However, the pattern in VSV-infected cells was unusual: there was a general distribution of radioactivity over the polyribosomes and also a sharp peak at about 120 S which rapidly increased in size as the time of exposure to radioactive leucine was lengthened (Fig. 2d, e, and f). In order to understand this pattern of incorporation, the nature of the 120 S peak was investigated. The possibility existed that the 120 S peak represented viral proteins associated with viral RNA in nucleocapsids. Preliminary experiments showed that successive treatment of B particles with the detergents, sodium deoxycholate (0.1%) and BRIJ-5S (O.l%), at 4°C resulted in a greater than 90% yield of nucleocapsids, which had a sedimentation constant in sucrose of 120 S and a buoyant density in CsCl of 1.31 g/ml. The nucleocapsids were also completely resistant to ribonuclease. These results are in close agreement with those reported previously (Kang and Prevec, 1969; Wagner et al., 1969). To examine the relationship of the 120 S, amino acid-labeled peak to nucleocapsids, a culture of infected cells was exposed to leucine-14Cfor 3.5 min and half of the culture was harvested. The other half received a large excess of unlabeled leucine, and was then incubated for another 12 min and harvested. Cytoplasmic extracts were prepared and, just prior to detergent treatment, B particles, labeled with uridine-3H, were added to serve as a marker for nucleocapsid in the sucrose gradients. Figure 3a shows that during the 3.5-min labeling period most of the radioactivity was incorporated into either polyribosomeassociated, nascent polypeptides or completed chains which sedimented near the top of the sucrose gradient. Little, if any, radioactivity appeared to sediment with nucleocapsids (BNC), although an obvious peak of radioactivity was present which sedimented slightly more slowly than the marker nucleocapsids.

STAMPFER 30 20

- a.

b.

IO N h x I 0”

6 4 2 C FRACTION

NUMBER

FIG. 3. Synthesis and release of nascent polypeptides by VSV-infected cells. At 3.25 hours after infection, 8 X 107 cells were exposed to leucine-W at 0.3 &i/ml. After 3.5 min, half of the cells were harvested. The other half received nonradioactive leucine at a final concentration of 52.4 &ml and was incubated for another 12 min before harvesting. Sucrose gradient centrifugation was exactly as described for Fig. 2. O-----O, 14C; o-0, uridine-3H-labeled viral nucleocapsid marker (BNC).

By 12 min after the addition of nonradioactive leucine, all the incorporated radioactivity either cosedimented with marker nucleocapsids or sedimented near the top of the gradient (Fig. 3b). These results indicated that the region ahead of the 60 S ribosomal subunit contained two structures which were labeled with radioactive leutine: (1) nascent polypeptides which were removed from polyribosomes by a chase with excess leucine and (2) nucleocapsids which increased in radioactivity during a chase. The data in Fig. 2 allow an estimation of the rate of synthesis and release of polypeptides in uninfected and VSV-infected cells. For this calculation, the radioactivity incorporated into completed polypeptide chains (the top fractions of the sucrose gradients) was expressed as a percent of the total amount of incorporated leucine (including radioactivity in the pellets). After a 3 min exposure of uninfected Chinese hamster ovary cells to radioactive leucine (Fig. 2a), 44% of the radioactivity was found in completed polypeptide chains. In VSV-infected cells (Fig. ad), 50% was

mRNA

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951

found in completed chains. After longer periods of incorporation such calculations for the infected cells became difficult because of the increasing amount of radioactivity in nucleocapsid-like material and because of the movement of newly synthesized polypeptides to other parts of the cell and to the extracellular medium which was not included in the cytoplasmic extracts (see following section). Nevertheless, the results of the 3-min pulse indicate that the small polyribosomes were approximately as active in protein synthesis as normal host polyribosomes. VSV-Specific Protein Synthesis by Viral Polyribosomes In order to demonstrate that the small polyribosomes were indeed VSV-specific and not residual host polyribosomes, the proteins from the cytoplasm labeled under similar conditions as the previous experiment were examined by polyacrylamide gel electrophoresis. Uninfected Chinese hamster ovary cells produced a wide spectrum of polypeptides (Fig. 4a); infected cells synthesized a small number of polypeptides which all comigrated with VSV structural proteins (Fig. 4b). These results, although clearly showing only viral specific protein synthesis in infected cells, differ from other published patterns for VSV protein synthesis by the absenceof nonstructural viral polypeptides (Wagner et al., 1970) and by the reduced amount of polypeptide 2, which includes a glycoprotein (Burge and Huang, 1970). To see whether these proteins were found in the nuclear fraction which was usually separated from the cytoplasm and discarded, nonnuclear material was extracted from the nuclear fraction with a mixture of Tween40 and sodium deoxycholate as described by Penman (1966). Electrophoresis of this extract showed that the two other nonstructural viral proteins as well as polypeptide 2 were contained in the material extracted by the mixed detergents. Although newly synthesized VSVspecific polypeptides were distributed in different parts of the infected cell, polyribosomes obtained from the detergentwashed nuclear fraction of infected cells

I

0

-

IO

20 30 DISTANCE

40 mm

50

+

FIG. 4. SDS-polyacrylamide gels of polypeptides synthesized by uninfected and VSV-infected cells. Two aliquots of 4 X lo7 cells, (a) uninfected, and (b) infected, were exposed at 3.25 hours after infection to 1eucineJH at a final concentration of 5 #i/ml. Nonradioactive leucine at 52.4 pg/ml was added 5 min later, and the cells were incuba.ted for another 20 min before harvesting. Onefifth of each of the cytoplasmic extracts was prepared for acrylamide gel electrophoresis. O-----O, Marker ‘4C-amino acid-labeled VSV; @---a, 3H. had the same ODsso pattern as polyribosomes obtained from the cytoplasmic extract. We can conclude, then, that the small polyribosomes were indeed VSVspecific and made only identifiable viral proteins. Separation of RNA in Nucleocapsiclsfrom RNA Attached to Polyribosomes When infected cells were exposed to radioactive uridine for 30 min or longer, only single-stranded viral RNA specieswere labeled. To determine which of them were polyribosome-associated and, therefore, viral messenger RNA, extracts of infected cells were labeled for 2 hours and examined on sucrose gradients for the association of viral RNA with cytoplasmic structures.

952

HUANG,

BALTIMORE,

Figure 5a shows three major areas of radioactivity: one roughly coincident with the polyribosomes and nucleocapsid, another just ahead of the 60 S ribosomal subunit and the third overlapping the 405 ribosomal subunit. If the uridine-3H found in the polyribosome region were in messenger RNA, then EDTA treatment, which dissociates polyribosomes, would also release the radioactivity from polyribosomes. When half of the cytoplasmic extract shown in Fig. 5a was first treated with EDTA and then layered on a sucrose gradient containing EDTA (NEB), the viral polyribosomes dissociated to 50 S and 30 S subunits and the only radioactivity left in the polyribosome region cosedimented at 120 S with marker nucleocapsids (BNC). This experiment substantiates the leucine incorporation studies where nucleocapsid-like material was synthesized and found to sediment in the 120 S region (see Figs. 2 and 3). As another means of distinguishing nucleocapsids from polyribosomes, the RNAcontaining structures sedimenting in the polyribosome region in sucrose gradients were further characterized by isopycnic centrifugation in CsCl. Almost all the incorporated uridine from the 120 S region of the sucrose gradient containing high salt buffer (taken from fraction c, Fig. 5a) was found in structures with buoyant densities of 1.57 g/ml and 1.31 g/ml (Fig. 6~). Similar results were obtained when other fractions from the pol;yribosome region of the same sucrose gradient were analyzed in CsCl. It was previously shown that polyribosomes exposed to high salt buffer have a density of 1.57 g/ml (Huang and Baltimore, 1970). VSV nucleocapsids are known to have a density of 1.31 g/ml. Therefore, the only viral RNA sedimenting in the polyribosome region of sucrose gradients was either associated with polyribosomes or nucleocapsids. This conclusion is further supported by the data in Fig. 6d where it is shown that the remaining material from the 120 S region in sucrose gradients containing EDTA (taken from fraction d, Fig. 5b) has the buoyant density of nucleocapsids. Nonspecific association of RNA with cytoplasmic proteins would

STAMPFER

BNC +

60s 4

I 5

15 FRACTION

25 NUMBER

FIG. 5. Sucrose gradients of viral RNA in cytoplasmic extracts before and after treatment with EDTA. Infected cells were exposed to 2 &i/ml of uridine-3H at 2 hours and harvested at 4 hours after infection. Half of the cytoplasmic extract was layered on a sucrose gradient containing high salt buffer. The other half was treated with EDTA (0.02 M) and layered on a sucrose gradient containing EDTA. The gradients were centrifuged at 95,000 9 for 3 hr at 4°C. The small letters in the figure relate to data in Figs. 6 and 7. 0 -0, 3H from 0.1 ml of each fraction; -, total absorbance at 260 nm.

result in a density of 1.40 g/ml (Baltimore and Huang, 1970). This experiment demonstrates that the only structures dissociated by EDTA are polyribosomes. Messenger RNAs

of VXV

To identify the viral RNA species that serve as messenger RNA, selected fractions (w, x, y, and z) from the two sucrose gradients shown in Fig. 5 were examined for

mRNA I

c. -*\

OF VESICULAR

I

STOMATITIS

VIRUS

I-

HSB

I w.

3 - 1.6

x\

--Y. .X., --x

6 POLYRIBOSOMES

28s 4

I

18s 4

- 1.5 2 - 1.4 1

x\xxx\x

953

-- 1.3 5 - 1.2

, ENC-

1

8 6

Y

0-

IO FRACTION

20 NUMBER

Y.

28s 4

18S 4

30

FIG. 6. Buoyant densities in CsCl of RNAcontaining structures sedimenting in sucrose in the polyribosome region. Samples of 1.2 ml each of fraction c shown in Fig. 5a (HSB gradient) and of fraction d shown in Fig. 5b (NEB gradient) were fixed with glutaraldehyde. Half of each of the samples was layered onto two separate 2&400/, preformed CsCI gradients and the other halves on two other 35-52y0 preformed CsCl gradients. (c) CsCl gradients containing material from the HSB gradient are graphed together and (d) similarly for mat,erial from the NEB gradient. X-X, Buoyant density; e---O, 3H.

their content of different viral RNA species on sucrose gradients containing sodium dodecyl sulfate (Fig. 7). The results of matched fractions from the original two gradients were plotted together in order to demonstrate the loss and gain of RNA species at different levels of the gradients caused by the EDTA treatment. Figure 7 shows that at the positions marked w and x in Fig. 5, some of the 28 S and all of the 13 S RNA specieswere removed when the cytoplasmic extracts were treated with EDTA. Furthermore, Fig. 7 shows that at the positions marked y and z in Fig. 5,

FRACTION

NUMBER

FIG. 7. Viral RNA species associated with polyribosomes. Samples of 0.4 ml from the fractions w, x, y, and z of both sucrose gradients shown in Fig. 5 were exposed to 0.25% sodium dodecyi sulfate and layered on individual sucrose gradients containing 0.5% sodium dodecyl sulfate. The gradients were centrifuged at 52,000 g for 16 hours at 22’. Each of the two gradients of the matched fractions from the two original sucrose gradients are graphed together. O-----O, 3H from the NEB gradient; e-0, 3H from the HSB gradient.

I

954

WANG,

BALTIMORE,

STAMPFER

the content of, respectively, 28 S and 13 S RNA species was increased by EDTA treatment. Thus, both 28 S and 13 S RNA molecules were attached to polyribosomes and, by definition, must be messengerRNA for the synthesis of viral proteins. These experiments also indicate that only 20 % of each of the 28 S and 13 S RNA species in the cytoplasmic extract were localized on polyribosomes. The remainder was found at the top of the gradient containing high salt buffer, even after 2 hours of exposure to radioactive uridine. Figure 7 further shows that the nucleocapsids contain heterogeneous 2840 S RNA species (Fig. 7w and x).

the RNA extracted from virions (Schaffer et al., 1968; Stampfer, unpublished observations). However, because not al! the 13 S or 28 S RNA specieswere found to be polyribosome-associated, we directly tested for complementarity of the labeled viral messenger RNAs. To isolate the messenger RNAs, virus-specific polyribosomes from uridine-labeled infected cells were first separated on a sucrosegradient. The sucrose fractions containing VSV-specific polyribosomes (equivalent to fractions Nos. 3-17 of Fig. 5a), were pooled and the polyribosomes concentrated by centrifugation. Viral messengerRNA was dissociated from polyribosomes by resuspending the pellet in 0.02 M EDTA (NEB) followed by reHybridization of Messenger RNA to VVSV centrifugation in a sucrose gradient conRNA taining EDTA (Fig. 8). Three well defined, Molecular hybridization has shown that radioactive peaks were evident at the same all of the 13 S and much of the 28 S RNA positions as those shown in Fig. 5b. The species made during VSV replication are fastest sedimenting peak at 120 S reprecomplementary in nucleotide sequence to sented nucleocapsids, the next fastest sedimenting peak contained 28 S messenger TABLE ANNEALING OF VESICULAR BNC n b

4

0.4

1

t

AZ,,

Messenger

13 s 28 s

5

IO 15 FRACTION

20 NUMBER-

FIG. 8. Isolation of mRNA from VSV-specific polyribosomes. Cells (8 X 107) were exposed to 20 &i/ml of uridineJH from 2 to 3.75 hours after infection and harvested. The cytoplasmic extract was first separated on a sucrose-RSB gradient which was centrifuged for 2.5 hours at 95,000 g at 4”. The gradient fractions containing VSV polyribosomes were pooled, and the polyribosomes were pelleted at 54,000 g for 1.25 hours at 4”. The pellet was resuspended in 1 ml of 0.02 M EDTA (NEB). Half of this was layered over a sucroseNEB gradient and centrifuged at 45,000 g for 12 hours at 4’. -, Total absorbance at 260 nm; m-0, 3H in 0.15 ml of each fraction.

RNA RNA=

$$ Ribonuclease-resistance annealed to No added

0.3

0

RNA

1

VIRAL MESSENGER STOMATITIS VIRUS

0.0 0.7

RNA

WITH

when

VSV RNA 99 101

a Labeled RNA from fractions 21 (28 S) and 26 (13 S) of the gradient shown in Fig. 8 was treated with O.O5oj, SDS and precipitated with 2 volumes of ethanol in the presence of carrier yeast tRNA The precipitate was resuspended in at -20”. 0.30 M sodium chloride and 0.03 M trisodium citrate (2X - SSC). Mixtures were made containing 1500 cpm 3H-labeled messenger RNA and either 8 pg of virion RNA or no added RNA and brought to 0.1% SDS in 0.4 ml of 2X - SSC. The mixtures were boiled for 5 min and then allowed to anneal for 120 min at 70”. The samples were chilled to 4” and divided into two equal portions, one of which was treated with 50 pg/ml RNase A and 25 units of RNase Tl for 30 min at 22”. All samples were then acid precipitated and assayed as described for the gradient fractions. The results are presented as percentage of ribonucleaseresistant RNAJH after annealing.

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955

messenger RNA in relation to control mechanisms for VSV protein synthesis, the 13 S group of messengerRNA must be better separated and a correlation made between each messenger RNA and the polypeptide coded by the messengerRNA. Although these studies ident.ified only viral messenger RNA attached to polyribosomes during the height of viral protein synthesis, the RNA undoubtedly represents all the viral messenger RNA used during the infection because the same viral polypeptides are synthesized throughout the infection (Wagner et al., 1970; Mudd and Summers, 1970; Huang, unpublished observations) . DISCUSSION The lack of associationof virion RNA with Only 28 S and 13 S RNA speciescomple- polyribosomes and the noninfectious nature mentary in base sequence to virion RNA of purified RNA from virions (Huang and appear to be associated with VSV-specific Wagner, 1966) led us to predict and find an polyribosomes. Preliminary acrylamide gel RNA polymerase in the virion of VSV analysis of the 13 S messengerRNA indi(Baltimore et al., 1970). Because the product cates that there are at least two different made by this polymerase also has only RNA species in this group. VSV messenger complementary nucleotide sequences to RNA is, therefore, found in multiple species, VSV-RNA, the enzyme is probably a suggesting that VSV polypeptides are syn- transcriptase. These studies on VSV mesthesized from separate RNA strands. This senger RNA and the VSV polymerase contrasts to poliovirus replication, where the show two phenomena which have not messengerRNA is a single species identical been previously demonstrated. First is to virion RNA (Penman et al., 1963; Sum- the synthesis of smaller pieces of complemers and Levintow, 1965) and where in- mentary RNA, presumably from a large dividual polypeptides are formed from single-stranded RNA template, and second cleavage of large precursor polypeptides is the function of these smaller pieces as (Summers and Maize& 1968; Jacobson and messenger RNA. Studies on Newcastle Baltimore, 1968b; Kiehn and Holland, 1970). diseasevirus suggestthat similar phenomena It has not been possible to demonstrate may be operative during viral replication cleavage of polypeptides during VSV repli- (Kingsbury, 1966; Bratt and Robinson, cation (Wagner et al., 1970; Mudd and 1967). Whether any cellular messengerRNA Summers, 1970). is similarly synthesized from a larger singleIndividual pieces of messengerRNA may stranded RNA template remains to be seen. form the basis of control for the disproIn the process of investigating the viral portionate synthesis during VSV replica- polyribosomes, another intracellular, urition of structural proteins over nonstruc- dine-labeled structure has been identified. tural proteins (Mudd and Summers, 1970; It sediments with polyribosomes but has a Wagner et al., 1970). Such control could be buoyant density of 1.31 g/ml in CsCl exerted by selective transcription of certain indicating a high protein content. Studies messenger RNA pieces or by alteration of using radioactive leucine also showed struccertain messengerRNAs so that initiation tures sedimenting with polyribosomes, which of translation is prevented. In view of the continued to be labeled either with inlatter hypothesis, there are 28 S and 13 S RNA species which are not associated creasing periods of exposure to radioactive with polyribosomes and their function is leucine (Fig. ad-f) or after a chase by the not known. To understand the function of addition of nonradioactive leucine (Fig. RNA released from polyribosomes and the slowest sedimenting peak contained 13 S messenger RNA from polyribosomes. The released 28 S and 13 S messenger RNAs found in the peak fractions of Fig. 8 were hybridized to virion RNA (Table 1). Both 13 S and 28 S messenger RNA hybridized completely to virion RNA. Under these conditions, there is no detectable selfcomplementarity of virion RNA (Stampfer, unpublished results) and therefore virion RNA and messenger RNA contain completely separate and complementary base sequences.

956

HUANG,

BALTIMORE,

3b). The similarities in sedimentation of both the leucine- and uridine-labeled materials suggest that the two different precursors are labeling the same structure, which has properties similar to viral nucleocapsids. These intracellular nueleocapsids can be formed by the association of newly made polypeptides with RNA as soon as 3 min after the addition of labeled amino acids. The reaction appears to be very specific, because the complex is formed mainly with RNA similar in base sequences to virion RNA (unpublished observations). No 13 S RNA is found in nucleocapsids. Specific associations have been found for Sendai virus RNA where nucleocapsids are formed very rapidly from newly made 57 S RNA (Blair and Robinson, 1970). However, in the case of VSV the slower sedimentation of some of the newly formed intracellular nucleocapsids compared to marker nucleocapsids (Fig. 3a) and their content of heterogeneous 2840 S RNA (Fig. 7w and x) indicate that they are not identical to nucleocapsids from virions. These smaller nucleocapsids are not precursors of smaller defective VSV particles, because only standard B particles are produced under these conditions (manuscript in preparation). It is not known whether these intracellular nucleocapsids are joined to form nucleocapsids of B particles or serve only as a mechanism to sequester certain RNA molecules from the pool of intracellular RNA. The breakdown of host polyribosomes by VSV is clearly demonstrated here. The rapidity of this breakdown, compared to uninfected cells treated with actinomycin (Fig. la), suggests that VSV prevents host protein synthesis direct.ly rather than by inhibition of cellular RNA synthesis (Huang and Wagner, 1965). ACKNOWLEDGMENTS We thank Mrs. Donna Smoler, Miss Esther Bromfeld, and Mr. David Lynn for excellent technical assistance. We are grateful to Dr. John A. Mudd for making available data prior to publication. REFERENCES BALTIMORE, D . , and pycnic separation

HUANG, A. S. (1968). Isoof subcellular components

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