Vesicular stomatitis virus—A new interfering particle, intracellular structures, and virus-specific RNA

41, 615-630

VIROLOGY

Vesicular

(1970)

Stomatitis

VirusStructures,

A New and

;\I. I’ETRIC’ Department

of Biolog~y,

Interfering

Virus-Specific .~ND

Particle,

Intracellular

RNA

I,. PREVEC

McMu.ster

Cniversiby,

Accepied

March.

Hamilton,

Ontario,

Cnnadn.

28, 197%

A st,rain of the Indiana serotypc of vesicular stomatitis virtts (VSV) designated IIlL-LT produces a defective particle which difiers from the T particle described h) others. This “long T” particle is approximately one-half the length of the infectiotts B particle and cont,ains a single-stranded IiNA having a sediment at ion coefficient of 30 S. Infection of L cells with the HII-I,T strain of \-SV resttlts in the intracellular accumulation of viral nttcleoprolein strtxtttres similar to nttcleoprotein St ruct,ures derived from mature B and “long T” particles. In the infected cell, virrts-specific RN.4 species with sedimental ion coefficients of 30 9 and 15 S appear to be associated with polyribosomes. INTROI)IJCTION

Infection of cells in culture with vesicular stomatitis virus (VSV) results, under the proper conditions, in the production not only of more infectious, bullet-shaped B particles, but also of shorter, noninfectious T particles (Hackett et al., 1967; Huang et al., 1966). The latter are capable of specific interference with the production of infectious particles (Hackett et al., 1967; Huang and Wagner, 1966b). Using the Indiana serotype of VSV, other workers have shown that the T particle, while similar to the B particle in general morphology, antigenicity (Huang et al., 1966) and protein constituents (Wagner et al., 1969a), is one-third its length and contains a RNA genomc of molecular w-eight 1.1 X lo6 daltons which is also approximately one-third the length of the B particle genome (Huang and Wagner, 1966a; Nakai and Howatson, 1968). In this paper we will report that the T particle produced in our system, while similar antigenically and in protein constituents to the B particle (Rang and Prevec, 1969), differs 1 Research stttdent, Institttte of Canada.

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from the T particle described above, both in overall length and size of genome. Infection of cells with VSV has been shown to lead to the production of various classes of virus-specific RKA within the infected cells (Schaffer et al., 196s; Stnmpfer et al., 1969). We have investigated virus-specific RNA present in association with cytoplasmic structures in cells infected with the “long T” producing strain of VSV, and from these studies suggest some possible functional assignments for the single-stranded RSA species. MATERIALS

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Cells and viruses. The growth and maintenance of a subline of Earle’s L cell in suspension culture and its use for growth and plaque titration of VSV have been prcviously described (Rang and I’revec, 1969). Two strains of the Indiana serotypc of VSV used in these studies were obtained from Dr. ,411an Howatson, Department of Medical Biophysics, University of Toronto. One, designated IND-ST, appears to be identical to the Indiana serotype viruses used in other laboratories. The second strain, designated HR-LT, was derived from the InTD-ST strain by selecting for virus which

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was resistant to inactivation by repeated heat’ing at 43°C for 3 hours. Chemicals and isotopes. Actinomycin D was a gift from Dr. Dorian, of the Merck, Sharp and Dohme Co., Montreal. Radioisotopes, uridine-3H (29CQ’mmole) and 14C-amino acid mixtures (1.5 mCi/mg) were purchased from the Kew England Nuclear Corporation. Radioactivity was monitored on a Beckman liquid scintillation counter as previously described (Mang and Prevec, 1969). Infection and Labelling. Approximately lo8 cells were collected by centrifugation from growing cultures and resuspended in 10 ml of MEM (Grand Island Biological Company, catalog No. F13) growth medium containing appropriately diluted virus. After an adsorption period of 30 min at 37” the cells were resuspended to a concentration of 6 X lo5 cells/ml with ME;\1 supplemented with 2% fetal calf serum. The cultures were incubated at 37°C. Actinomycin D at a final concentration of 2 pg/ml was added 30 min after resuspension of the cultures. For uridine-3H labeling, the radioisotope was added to a final concentration of 2 ,.&i/ml 3 hours after addition of actinomycin D, and the entire culture was harvested after a further 2 hours. To label with 14C-amino acids, the infected cells were collected by centrifugation 2.5 hours after addition of actinomycin D and resuspended in prewarmed MEM containing one-twentieth the normal amino acid concentration and 2% fetal calf serum. After 30 min of incubation at 37” in this amino acid-deficient medium, the 14C-labeled amino acid mixture was added to a final concentration of 0.2 pCi/ml. After 1.5 hours of further incubation, a sufficient volume of 50 X MEM amino acid mixture, neutralized to pH 7.0, was added to bring the amino acid concentration in the culture to 5 times that of normal MEM. After a further incubation period of 10 min the culture was harvested. Preparation and analysis of cytoplasmic extracts. The labeled, infected cells were collected by centrifugation at 1000 0 for 5 minutes at 4” and the cells were washed three times by resuspension and recentrifugation in ice-cold phosphate-buffered saline (PBS). The final washed cell pellet was

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resuspended in 1 ml of sodium RSB (0.01 M NaCl, 0.0015 111 &IgCl,, and 0.01 AT TrisHCl, pH 7.5) and the cells were allo\ved to swell for 10 min. The swollen cells were broken with 20 strokes in a tight-fitting glass Dounce homogenizer. The nuclei were removed by centrifugation at 7000 g for 10 min, and the supernatant fraction was layered on sucrose gradients for analysis. Linear sucrose gradients, l&30 % sucrose dissolved in sodium RSB+ (0.01 ;I( NaCl, 0.015 Ji MgCl?, and 0.01 iI4 Tris-HCl pH 7.5) were prepared in 38.ml centrifuge tubes. The gradients were centrifuged at 81,000 g and 4” for 4 hours in a Beckman L2-65B ultracentrifuge. The gradients were analyzed, and successive l-ml fractions were collected on an ISCO ultraviolet analyzer (254 nm) and fraction collector. Half of each fraction was precipitated with cold 5 % TCA, and the precipitate was collected on nitrocellulose filters and assayed for radioa,ctivity. RNA extraction and analysis. Appropriate fractions from the sucrose gradient analysis of cytoplasmic extracts were pooled, and sodium dodecyl sulphate (SDS) was added to a final concentration of 0.5%. The RNA was precipitated by adding two volumes of cold ethanol and storing at -20” for 12 hours. If required, yeast RNA carrier was added. The precipitate was collected by centrifugation and redissolved in 0.1 ml of STE-SDS buffer (0.1 M NaCl, 0.01 U Tris-HCl, pH 7.4, 0.001 M EDTA and 0.5 % SDS), and the solution was layered on 5 ml, 5-20% linear sucrose gradients made in STE-SDS buffer. The gradients were centrifuged at 180,000 y for 2 hours at 15” in a Spine0 SW 50 rotor. In some cases a small amount of radioactive ribosomal RNA from L cells was premixed with the sample to serve as an internal standard. These gradients were analyzed either as described above or by collecting successive 0.16-ml fractions directly into Gelman glass fibre filters in scintillation vials. The filters were then dried, and radioactivity was determined. Electron microscopy. All samples were examined on carbon-coated formvar grids in a Philips 300 or a Zeiss 1311 9 electron microscope. Samples taken from sucrose gradients were washed a number of times

with distilled water on the grid. The virus particles were negatively stained with 2 % phosphotungstic acid (pH 6.S) while ribonucleoprotein particles (RNT-‘) were nega. tively stained with 2 o/o unbuffered uranyl acetate.

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Icigure 1 reveals the differences in the types of pa,rticles present in lysates of L cells infected with either IND-ST (wild type) VSV or the HR-LT strain of VSV. The lysate from IND-ST infected cells contained two major light-scattering and UV-absorbing bands and a minor band located between these tT\-o. This result is identical to the published result of Huang et al. (1966b) for wild-type Indiana VSV. The most rapidly sedimenting band is the region of infectious or B particles while the most slowly sedimenting (fraction 10) is the region of “short T” particles. Between these two regions is a less prominent band whose physical ma.keup will become apparent after considering the particles produced by HRLT infection. From this figure it is evident that infection of cells with HR-LT strain of VSV yields only two types of particles. One of these, the most rapidly sedimenting, is the infectious B particle whereas the other band contains a noninfectious, complement-fixing particle (Kang and Prevec, 1969) which sediments faster than the “short T” particle of IND-ST. The lower panel of Fig. 1 confirms the dist’inct identity in sucrose gradients of the noninfectious particle from HR-LT infection and the “short T” particle of IND-ST infection. In this gradient equal quantities of virus shown in upper panels were consedimented. Electron microscopic studies of both the B and ‘(short T” particles resulting from INDST infection have been described by Nakai and Howatson (196s). B particles of the HR-LT strain are morphologically indisdistinguishable from those of the IXD-ST strain (Fig. 2A, B). In contrast, however, the defective particle produced in HR-LT infection, while structurally similar to the R

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FIG. 1. Sucrose gradient, fractionation of virus particles produced by t,he IIR-LT and INISST strains of VRV. Two cell cultures, each 108 cells, were separately infected with the RR-LT strain and the INIX-ST strain of VSV at an m.o.i. of 10. Actinomycin I> (2 pg/ml) was added to both cul. tnres at 30 min post-infection, and 1.5 horns later uridine-1% (0.02 &i/ml) was added to t,he IIR-LT infected culture and uridine-“FT (2 #Z/ml) to the IND-ST infected cnlture. The cultures were harvested after 15 hours, the cells were removed by centrifugntion at 600 y for 10 min, and virus particles were collected from the cell-free snpernatant by centrifngation at -IO,000 q for 2 hours and then resuspended in 1.5 ml of PM. One milliliter of virns yield from each strain and a mixture of 0.5 ml of virns from each strain were separately layered on 5 to 3Oc/;, sllcrose gradients made in PBS. The gradients were centrifnged for I hor~r at 48,000 g and 5” in a Spinco SW 25.1 rotor and then collected; optical density (252 nm) was monitored using an IX0 gradient, collector. ltadioactivity in the mixed sample was determined by precipitating the sample with 576 cold TCA and collecting the precipitate on nitrocellrdosc filters; these were dried, and radioactivity was determined in a Beckman scintillation colmlcr. “II cormts (O---O), *4C counts (O----O) and optical density (- - -) are plotted. The bot,torn of the ccntrifugc t rlbcl is to the right.

618

FIG. 2. Electron photomicrographs and of the IND-ST strain (B) WC?re partially purified and negatively

(A,)

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of B and T p Iaarticles. Infectious defective “ ‘1 ong T” particles strained wit1 h 2% phosphotungstate.

PCarticle (Fig. 2C) is slightly more than OIEhr Jf the length of the B particle (Fig. 3). Fj .gure 2D shows, for comparison, a preparaticIn of “short T” particles which are one-

B particles of the HR-LT strain (C) and “short T” particles (D) Approximately X 85,000.

third the length of a B particle (Fig. 3, Huang et al., 1966; Nakai and Howatson, 1968). For convenience we shall refer to the larger defective particle produced by HR-LT

VESICULAIt

r

40 -

LONG

STOMATITIS

Brown et al., 1967), values of 42-45 S for RNA from B particles (Fig. 4a) and 20 S for RNA extracted from “short T” particles (Fig. 4~) vvere obtained. The RSA of the “long T” particle, however, sediments as a distinct peak at 30-32 S (Fig. 4b). Assuming a relationship between molecular w-eight and sedimcntntion coefficient of the form Jl = aS” and taking molecular weight values for B particle RNA of 3.5 X 10fi and for “short T” of 1.1 X 10” daltons (Nakai and Howatson, 1968), an estimate of 1.7-2.0 X lo6 daltons is obtained for the molecular weight of the “long T” genome. Thus the ratios of the RSA molecular weights of the three particles are in excellent agreement with the ratios of the overall lengths of the intact particles.

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Since VSV replication is insensitive to the action of actinomycin D (Huang and Wagner, 1966), this antibiotic can be used to suppress host cell RNA synthesis and thus allow the selective study of viral RNAcontaining structures in the infected cell. In Fig. 5a is plotted the optical density and radioactivity present in the cytoplasm of uninfected cells, which were treated in all other respects identically to infected cultures. From the radioactivity profile it can be seen that, with the exception of the top of the gradient, very little radioactivity is associated with any particular cellular structures. This result ensures that 2 pg/ml of actinomycin D under the conditions used was sufficient. to inhibit the synthesis of most Rn‘A molecules in uninfected cells. Figure 5b shows the optical density and radioactive profile obtained for cytoplasm extracted from infected cells. This has been centrifuged for a longer time than the uninfected control for better resolution of smaller particles. The optical density profile is very similar to that for uninfected cells. Beginning at the top of the tube there is a peak of radioactivity associated with the 40 S ribosomal subunit and two additional peaks at approximately 100 and 140 S. As well as in these peaks, there exists a generally high level of radioactivity throughout the polyribosome region of the gradient.

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FIG. 3. Histogram infectious B particles Particle lengths were graphs similar to those

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of length distribution and defective T particles. obtained from photomicroof Fig. 2.

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infection as a “long T” particle. To justify the designation “T” derived from “transmissible interfering component” (Cooper and Bellett, 1959) we have demonstrated in our laboratory that the “long T” particle is capable of homologous interference. The results of the interference studies will be presented in detail elsewhere. RNA Extracted from “Lmg

T” Particles

Since the overall length of the “long T” particle is intermediate between that of the “short T” and B particles it would be expected that the RNA genome of this particle would also be intermediate in size compared to the other two particles. In agreement with other workers (Huang and Wagner, 1966a;

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FIG. 4. Sucrose gradient analysis of RNA from B (a), “long T” (b), and “short T” (c) particles. 3H-labeled virions were purified from gradients as described in Fig. 1. SDS (0.5%) was added to appropriate gradient fractions, and the RNA was precipitated by the addition of two volumes of cold ethanol and some yeast RNA as carrier. The RNA precipitates along with a small amount of 14C-labeled L cell RNA as marker were centrifuged in 5-20y0 sucrose gradients in STE-SDS buffer. After centrifugation at 130,000 g for 2 hours at 15” in a Spinco SW 50 rotor, successive fractions were collected onto filter Dads by dripping from the bottom of the gradient tube. The 3H counts in viral RNA (O---O) and the “C counts in riboeomal and transfer RNA (0- - -0) are plotted. Centrifugation was from right to left.

The presence of radioactive peaks at 140 S and 100 S was significant, since, as reported (Kang and Prevec, 1969) the RNP particles extracted from purified B and “long T” virus particles also have sedimentation coefficients of 140 and 100 S, respectively. It was therefore possible that the radioactivity peaks observed in infected cell cytoplasm were due to viral RNP structures. While the 100 S radioactive peak is always present in large amount, not all experiments show a large 140 S peak of radioactivity. One such experiment which nevertheless demonstrates an interesting feature of 140 S material is shown in Figs. 5c and d. Figure 5c shows a gradient of cytoplasmic material prepared identically to that of Fig. 5b. The gradient fractions, after collection, were split into two equal portions; one portion was precipitated directly with TCA and the radioactivity was determined while the other portion was treated with 5 pg/ml of RNase for 30 min at 37” and then precipitated with acid. It can be seen that the RNA sedimenting at 40 S is entirely RNase sensitive, as is most of the RNA in the polyribosome region

of the gradient. In contrast, the 100 S peak is almost completely resistant to RNase. The RnTase resistant material extends from 100 S to a small peak at 140 S. In contrast to this result, when a duplicate portion of cytoplasmic extract was treated with 5 pg/ml of RNase for 10 min at 0” prior to centrifugation, the result shown in Fig. 5d was obtained. As can be seen from the optical density profile, the polyribosome structures have been destroyed and the number of free ribosomes greatly increased. The only radio. activity remaining in the gradient is completely RNase resistant and sediments at 100 S. No trace of RNase-resistant material at 140 S remains, suggesting that the nuclease treatment has converted the 140 S material to fragments with a new sedimentation coefficient. Virus-SpeciJic RNA in Cytoplasmic Extracts The fractions selected for RNA extraction were those containing 40 S ribosomal subunits, single ribosomes, 100 S region and 140 S region. The results of sucrose gradient analysis of RNA extracted from each of

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5. Cytoplasmic extracts of infected and uninfected cells. Cells were infected or mock-infected with HR-LT VW at 50 m.o.i. Actinomycin 1) (2 @g/ml) was added at 30 min post-infection, and uridine3H (2 PC/ml) 3 hours later. The cultures were harvested at 5 hours post-infection, and cytoplasmic extra&s were prepared and analyzed on linear 15-30 7; sucrose gradients in RSB+ at 58,000 g and 4°C for 3 hours (a) or 6 hours (b-d). The optical density at 254 nm (- - -) was continuously monitored as l-ml gradient fractions were collected. Each fraction was precipitated onto filters with 57. TCB, and the radioactivity was determined. (a) A mock-infected culture. (b) Cytoplasmic extract of an infected culture. (c) Cytoplasmic extract of a second infected culture in which the fractions of the gradient were split into two portions; one portion was acid precipitated directly and radioactivity was determined (O---O), while the other portion was treated for 30 min at 37” with 5 pg/ml of RNase and then acid precipitated and counted (0 --0). (d) A duplicate of the sample shown in (c) except that the extract was treated for 10 min at 0” with 5 pg/ml RNase prior to sucrose gradient analysis. After collection of the gradient fractions, each fraction was again split in two and acid was either precipitated directly for radioactive determination (0 - - - 0) or treated with 5 pg/ml RNase for 30 min at 37” prior to precipitjation and radioactivity determination (O- - -0). Centrifugation was from left to right’. FIG.

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FIG. 8. Analysis of virus-specific RNA found in selected fractions of cytoplasmic extract from infected cells. Uridine-3H-cont,aining fractions from the 40 S region (a), 80 S region (b), 100 S region (c), and 140 S region (d) of a cytoplasmic extract identical to that seen in Fig. 3b were separately pooled. SDS was added to a final concentration of 0.5y0, and the RNA was precipitated with two volumes of cold ethanol. The RNA precipitate was redissolved in 0.1 ml of STE-SDS buffer and layered on linear 5 to 20$& sucrose gradients in STE-SDS buffer. After centrifugation at 130,000 9 and 15” for 2 hours in a Spinco SW 50.1 rotor, successive fractions were collected by dripping from the bottom of the gradient tube. The optical density (260 nm) (- - -) of each fraction was determined; then the material in each fraction was precipitated onto nitrocellulose filters with 5% cold TCA, and the radioactivity (O---O) was determined. Centrifugation was from right to left.

these regions is presented in Fig. 6. RKA extracted from material of the 40 S region of a cytoplasmic extract consists solely of 15 S ltNA species. This result suggests that, the 15 S virus specific RNA in the 40 S

cytoplasmic fraction is either bound to cellular components or attains a higher sedimentation coefficient in the presence of cellular cytoplasmic extract (Girard and Raltimore, 1966). We have not distinguished

between these possibilities, but because of the high sensitivity of this RSA fraction to RKase it is probably not protected by associated protein. The amount of labeled RNA associated with single ribosomes is generally small and variable, suggesting that this label results from breakdown of some polyribosomes during the extraction procedure. In I’ig. 6 we see that the major species of RNA in this fraction are 15 S and 30 S. Both t’he 100 S and 140 S regions of the cytoplasmic gradient contain virus-specified 30 S and 15 S ItNA species. In addition, the 140 S region contains virtually all the 43 S viral RNA present in the cell cytoplasm whereas the 100 S region contains a large amount of the 30 S species of RNA. This result is consistent with the notion that 140 S and 100 S RN!? structures contain 43 S and 30 S RNA, respectively, while some additional 30 S and 15 S virus-specific messenger RNA is associated with the polyribosomes present in these regions of the cyboplasmic gradient. Virus-Specific hxts

Protein

in

Cytoplamic

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If, as me suggest, the RNA containing fractions which sediment at 100 S and 140 S are RSI’ particles of “long T” and B virions, then it should be possible to label these fractions with radioactive amino acids. When infected cells were labeled with 14Camino acids and the cytoplasmic fraction was examined, radioactivity was found associated with both the 100 S and 140 S fraction. Some radioactivity was also associated with protein which remained at the top of the gradient (I+Yg.7). It has previously been demonstrated that purified VSV virions contain three major and one minor structural proteins (Iiang and l’revec, 1969); Wagner et al., 1960a, b), one of which (VP,) arises from the viral nucleoprotein. The proteins of the 1Otl S, 140 S, and low molecular weight region of infected cell cytoplasm were analyzed and compared to the viral structural proteins on polyacrylamide gels. Figure Qa presents a control run of purified viral protein and is similar to the results already published. The remaining diagrams

1 FRACTION

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FIG. i. 1GAmino acid labeling of cytoplasmic fractions from infcctcd cells. Some lo8 cells were infected with the III’I-LT strain of WV and treated with act,inomycin 1) as described in Fig. 1. At 3 hours posbinfection the infected cells were collected by centrifuySation at 600 g for 5 min and resllspended in MEM cant aining one-twcntietjh the normal amino acid concentration. A mistllrc of 14C-amino acids (0.2 &i!ml) was added, and after a flIrther 2 hollrs of incrtbntjion the cells \vvrre harvested. The cytoplasn~ic fraction was obtained and analyzed c,n sIwxse gradients as described in Fig. 5. Continrwrls optical density (254 nm) along the gradient (- - -) and the radioactivity in SIICcessive l-nil fractions (O---a) after precipitat,ion with cold 5(‘; TCA are shown. Single rihosomes (80 S) arc located in fraction 15, and centrifrlgation was from left IO right.

in this figure show the radioactive profile on polyacrylamide of 14C-labeled protein from each of the regions of the cytoplasmic gradient either alone or in admixt’ure with 14Clabeled viral proteins identical to that shown in Fig. Sa. While the mixtures do not show exact additivity of counts, perhaps as a result of varying counting efficiency due to gel slicing, it is nevertheless evident that the 100 S, 140 S, and low molecular weight cytoplasmic extract fractions contain principally V1’3. This result supports the hypothesis that the 140 S and 100 S structures are viral RNP purticlcs.

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The apparent absence of the viral coat proteins VP2 and VP4 from the cytoplasmic extract suggested that these proteins may be associated with structures which are separat*ed from the cytoplasmic fraction during removal of nuclei. That this was indeed the cast is demonstrated in Fig. 9. Extracts of whole infected cells show all four viral protein fractions whereas the cytoplasmic fraction contains principally VP3. It is interesting to note that no significant protein fraction other than those already described as virus structural proteins were present in the infected cell extract. Particles Together with the biochemical evidence presented above, the most definitive evidence that the 140 S and 100 S regions contain viral 1iNP structures comes from electron microscopic examination. Figure 10 shows the structures present in material from the 140 S and 100 S region of sucrose gradients as seen by electron microscopy. As expected, ribosomes and aggregates of ribosomes can be observed in the photomicrographs. The long, wavy structures are morphologically identical to the nucleoprotein of VSV obtained by disruption of whole virus as described by Nakai and Homatson (1968). In some of the photomicrographs the nucleoprotein structures were sufficiently extended to be measured with a map measuring device after projection. Five measurements on material from the 140 S region showed lengths of 3.4 to 3.9 pm while six measurements of the 100 S particles gave lengths of 1.3 to 1.9 pm. These lengths are consistent with the expected length of nncleoprot,ein from I3 and “long T” particles.

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FIG. 9. Comparison of viral prot,ein in t,ot,al cell extract and in cytoplasmic fraction extract. Cells, which had been incubated with 0.5 pg/ml of actinomycin 11 for 12 hours previously, were infected and labeled with r4C-amino acids as described in Fig. 7. The labeled cell culture was split into two port,ions, and the cells were collected by centrifugation at 000 g for 10 min. One cell pellet was resuspended and, after homogenization and centrifugation as described in Methods, t,he cytoplasmic extract was treated with SDS, urea, and mercaptoethanol. The second cell pellet, was treated directly with these reagents and briefly with ultrasonics to reduce the viscosity. Roth extracts were dialyzed overnight against SDS, urea, and mercaptoethanol as described previously (Kang and Prevec, 1969) and, after the addition of 3H-amino acid labeled viral proteins as markers, the proteins were examined on polyacrylamide gel. (A) Cyt,oplasmic extract. (B) Total cell extract. 3H-marker proteins (0- - -0); %-extract proteins (O---O).

FIG. 8. Polyacrylamide gel analysis of ‘“C-amino acid-labeled fractions from infect,ed cell cytoplasm. ltadioactive fractions from the top of the gradient (free protein), t,he 100 S region, and the 140 S region of a gradient identical to that in Fig. 7 were separately pooled. The sucrose was removed by dialysis, and the material of the pooled fractions was concentrated by flash evaporation. After the fractions were treated wit.h urea, acet.ic acid, SDS, and mercaptoethanol, samples containing 20,000 cpm were put, on acrylamide gels either alone or along with 40,000 r4C-counts of marker virus proteins. The gels were run for 17 hours at 3 V/cm and then sliced; the radioactivity in each slice was determined and plotted. The figures show: virus marker alone (a), free protein alone (b) and with virus marker (b’), protein from 100 S region alone (c) andwith virus marker (c’), and protein from 140 S region alone (d) and with virus marker (d’).

FIG. 10. Electron photoxnicrographs cytoplasmic extract sucrose gradient. a 250,000 X magnification of a portion of the bar represents 0.1 ~.r.

of material present, in the 100 S (A) and 140 S (U) region (I a Both of these are at a magnification of 103,000 X. In (C) is shown of the ribonucleoprotein structllres shown in A and R. The length 626

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Some shorter extended fragments were seen in both preparations, but we assume these resulted from breakage during preparation. Origin and Function of RNP Particles It is possible that the RNP particles are not normal cytoplasmic constituents, but are produced from mature virions during homogenization of cells. In order to check this possibility unlabeled cells and uridine-3H-labeled purified B and T particles were mixed and carried through the normal extraction procedure used to obtain cytoplasmic fractions. In this case no radioactive particles of 140 S or 100 S were observed on sucrose gradients, and under the conditions of centrifugation described in Fig. 1 the virus particles were pelleted to the bottom of the gradient tube. If deoxycholate (0.5 % final concentration) was added to the cytoplasmic extract prior to centrifugation, then 100 S and 140 S RNP particles were indeed produced. This experiment suggests that the RNP particles in the cytoplasmic extract did not result from chemical or mechanical disruption of completed virions during the extraction procedure. If intracytoplasmic RNl’ particles are precursors to mature virus it should be possible to demonstrate the loss of these particles from the cytoplasm during infection. Infected cell cultures were pulsed for 10 min with radioactive amino acids at 4 hours post-infection. Excess unlabeled amino acids were added, and a portion of the culture was removed at various times thereafter. Cytoplasmic extracts were prepared, and the radioactivity remaining in 140 S, 100 S, and low molecular weight protein fractions was determined and plotted as shown in Fig. 11. The total radioactivity in the cytoplasmic extract decreased during the chase period, losing 25 % of the initial radioactivity during the 45.min period of observation. In contrast, the radioactivity in the RNl’ particles was relatively constant, perhaps even increasing slightly during this period. The low molecular weight protein fraction accounted for all the radioactivity lost from the cytoplasmic extract. The results suggest that, under our condit,ions of infection, the matjuration of 140 S

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FIG. 11. Kinetics of labeled proteins in infected cell cytoplasm during a chase with unlabeled amino acids. Some 3 X 10R cells were infected and treated with actinomycin 1) and low amino acid medium as described for Fig. 7. Thirty minutes after resuspension of the cells in amino acid-deficient medium, ‘Y-amino acid mixture (0.2 &i/ ml) was added to the culture. ilfter 10 minllt,es an eqllal volume of MEM containing sufficient unlabeled amino acid concentrate t,o yield a final amino acid concentration of 5 times that in MEM was added. At the indicated times after the addition of chase amino acids, equal samples were withdrawn from the cldtllre, and the cytoplasmic fractions hvere ext,racted and analyzed on sucrose gradients. The radioactivity in free protein, 100 S region, and 140 S region of the gradients for each time point was computed and normalized to optical density in the extract. The normalized counts for each region and the total counts in each gradient are plotted.

and 100 S RNl’ particles to complete virions proceeds extremely slowly, if it occurs at all.

The results presented in this paper point out three distinct facts regarding t’he replication of VSV in L cells. 1. Infection of I, cells with the IIYD-ST strain of VSV results in the production of infectious B particles, defective “short T” particles and, to a limited extent, an intermediate-sized particle.

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Infection of L cells with the HR-LT strain of VSV, leads to the production of infectious B particles and only one type of defective particle. This particle we have called “long T.” 2. Subviral nucleoprotein particles with sedimentation coefficients of 100 S and 140 S appear to accumulate in the cytoplasm of cells infected with mixed B and “long T” particles of the HR-LT strain. These subviral particles are equivalent to the nucleoprotein structures released by detergent treatment of “long T” and B particles. 3. Virus-specific RNA fractions with sedimentation coefficients of 15 S and 30 S appear to be associated with infected-cell polyribosomes. These species of RNA may therefore serve as messenger RNA species directing the synthesis of virus-specified proteins. The presence of large, infectious, bulletlike particles together with smaller, defective particles has been reported for both the Indiana and New Jersey serotypes of VSV (Huang et al., 1966; Huang and Wagner, 1966b; Hackett et al., 1967). In all cases, including this paper, the size and morphology of the infectious B particles appears to be approximately the same. The defective particles of the Indiana serotype observed by other workers appear to be identical to those we term “short T” particles derived from the IND-ST strain. The overall length (Huang et al., 1966; Nakai and Howatson, 1968), length of nucleoprotein core (Nakai and Howatson, 1968) and sedimentation coefficient of constituent RNA (Brown et al., 1967; Huang and Wagner, 1966a) of the “short T” particle are all consistent with a particle whose length is about one-third that of a B particle. In contrast, L cells infected with the HR-LT strain of the Indiana serotype produce defective particles whose overall length and sedimentation coefficient of constituent RNA are consistent with a particle which is approximately one-half the length of a B particle. The presence of RNP precursors to mature virions has been demonstrated in cells infected with WEE virus (Sreevalsan and Allen, 1968) and Semliki Forest virus (Friedman and Brezesky, 1967). While the evidence presented in this paper is consistent

PREVEC

with the hypothesis that the 100 S and 140 S structures are identical to the internal RNP structures of “long T” and B virions, no precursor function has been demonstrable for these structures. We would suggest that, under conditions of interference (i.e., when T particles are present in the infecting inoculum) the R9P structures accumulate within the infected cell. Experiments to be reported elsewhere show that when pure B particles are used for infection, neither 100 S nor 140 S structures normally accumulate in the cytoplasm. While observations on the RNase sensitivity of 140 S RNP particles are inconclusive, they nevertheless suggest that this structure may have one or more points of nuclease susceptibility. Wagner et al. (196913) have reported that the RNP from B virions is stable to large concentrations of RNase. Their equilibrium gradient procedure would be incapable, however, of detecting isolated breaks in the nucleoprotein strand. The final observation to be considered is the finding that 15 S, and perhaps 30 S, RNA is associated with polyribosomes and may therefore serve some function as messenger RNA. As has already been described (Kang and Prevec, 1969; Wagner et al., 1969a), the protein constituents of the VSV virion can be separated into three major and one minor protein fractions. Using the molecular weight values for the major proteins as determined by Wagner et al. (1969a), we can estimate that RNA molecules of molecular weight 3.4 X 105, 5.9 X 105, and 8.1 X lo5 could be required to serve as monocistronic messengers for each protein. RNA molecules of this size should have sedimentation coeflicients in the range of 11 to 1s S. In fact, when the 15 S RNA from polyribosomes of VSV infected cells is examined by more lengthy centrifugation on sucrose gradients, as seen in Fig. 12, it appears to be composed of three distinct classes of RlSA with sedimentation coefficients estimated at IS S, 15 S, and 13 S. This result would then suggest that each virion protein is indeed coded for by a distinct mRNA in contrast to the situation observed in picornaviruses (Summers and hIaBel, 196s). The possible function of 30 S RXA, which may be acting as messenger, and its relation-

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Childs Memorial Furld for Medical Itesearch. WC acknowledge the advice of Dr. Allen Howatson, University of Toronto, regarding the techniques of electron microscopy. The excellent technical assistance of Mr. I). Takayesu is gratefully acknowledged. REFERENCE8

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FIG. 12. Higher resolution analysis of 15 S polyribosomal RN4. A cytoplasmic extract was prepared from lO* cells infected with HR-LT VSV after labeling with uridine-“II in the presence of actinomycin 1). The polyribosomal fraction was obtained from sucrose gradients as shown in Fig. 3. RNA was extracted from the polyribosomes by treating with Sl>S and precipitating with ethanol. This RNA was then put on a 5-20% sucrose gradient in STE-Sl)S together with 14C-labeled ribosomal RNA as marker and centrifuged at 250,000 g and 15°C for 4 hours. Successive fractions were collected by dripping from the bottom of the gradient t.ubc, and the 14C (O---O) and 3H (O- - -0) radioactivit,y was determined. Centrifugationwas from left to right.

ship to the 30 S RNA present in the “long T” particle remains to be determined. The finding by Stampfer et al. (1969) that a species of single-stranded RKA sedimenting at 28 S (probably equivalent to 30 S in this paper) was present in cells infected with a virus strain resembling the IND-ST strain is of interest in this regard. Since the normal Indiana, strain makes little if any “long T” particles (Huang et cd., 1966), the above observation may also suggest that 30 S RKA acts as messenger in VSV-infected cells. ACKiXOWLEIXMENTS This work was supported by grants from the Nat ions1 Cancer Institute of Canada, the Nat ional Itesearch Coluncil of Canada, and the Jane Coffin

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