Electron microscopy of RNA strands released from individual reovirus particles*1

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J. Mol. Biol. (1968) 34, 137-147

Electron Microscopy of RNA Strands released from Individual Reovirus Particles C. VASQUEZ AND A. K. KLEINSOHMIDT

Department of Biochemistry New York University X&o01 of Medicine New York, New York, U.S.A.

(Received 30 December 1967) A gradual release of double-stranded RNA occurred after the spreading of reovirus particles on urea in the cold. Electron microscopy of virus-protein monolayers showed that each virion expelled about 11 fragments of S-3 p maximum total length. This gives an estimated RNA molecular weight between 17 and 22 million daltons per virion. The length distribution of the fragments was t&modal, with peaks at O-38,0.66 and l-13 p, but there was large variation of filament lengths in each peak.

1. Introduction The capsid of the reovirus shows an organization typical of isometric virus particles (Caspar, 1965). It consists of two layers. The outer layer is composed of 540 structural units and has icosahedral symmetry (Vasquez & Tournier, 1964). The inner layer (Vasquez & Tournier, 1962) envelops the core containing double-stranded RNA (Gomatos & Tamm, 1963). Different extraction procedures yield fragments of the viral genome rather than a single filamentous molecule (Gomatos & Stoeckenius, 1964; Kleinschmidt, Dunnebacke, Spendlove, Schaffer & Whitcomb, 1964; Bellamy, Shapiro, August 85 Joklik, 1967; Dunnebacke & Kleinschmidt, 1967; Iglewski & Franklin, 1967; Vasquez & Kleinschmidt, 1967 (Proc. 25th Meeting Electron Microscopy Xoc. Amer., p. 92); Watanabe & Graham, 1967). The extracted RNA is not infectious in susceptible cell cultures (Gomatos & Stoeckenius, 1964). Electron microscopy using the monolayer technique showed a t&modal length distribution of the reovirus RNA (Kleinschmidt et al., 1964; Dunnebacke & Kleinschmidt, 1967; Vasquez & Kleinschmidt, 1967 (see above). A corresponding trimodal distribution of doublestranded RNA was found by sucrose density-gradient centrifugation and polyacrylamide gel electrophoresis whether the RNA was derived from purified virus or infected tissue (Bellamy et al., 1967; Watanabe & Graham, 1967). Three classes of messenger RNA of reovirus-infected cells were found which, under proper conditions, hybridize with the correspondent trimodal heat-denatured viral RNA (Bellamy & Joklik, 1967a; Watanabe & Graham, 1967; Watanabe, Prevec & Graham, 1967). In addition to the double-stranded fragments, a large number of small polynucleotide strands, rich in adenylic acid, were found in both virions and infected tissue culture preparations (Bellamy & Joklik, 196’73). One-step release of the RNA for electron microscopy has been used by various investigators to minimize its breakdown. Filaments as long as 7 p could be obtained 137

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by phenol extract,ion (Kleinschmidt et al., 1964), osmotic shock (Gomatos & Stoeckenius, 1964), or brief exposure to sodium perchlorate (Dunnebacke & Kleinschmidt, 1967); however, most of the filaments were shorter than 7 p and showed a great variation in length. Pretreatment with urea in the cold has also been used in experiments for electron microscope studies (Granboulan & Niveleau, 1967). Filaments of various lengths were observed. About half of the filaments had a mean length of 5-l CL.If the reovirus genome consisted of a single nucleic acid filament it would appear that, upon extraction, it is broken into fragments. In order to throw further light on the nature of the reovirus genome, we carried out studies designed to observe the RNA during its release from the virion. By spreading the virus on a urea subphase, it was found that an average of 11 fragments were released over a period of ten minutes. The total composite fragment length per virion was 8.3 p. This gives an estimated molecular weight of 17 to 22 million, which is higher than the previously reported values (Gomatos & Tamm, 1963; Granboulan & Niveleau, 1967).

2. Materials and Methods Purified reovirus type 3 was a gift of Drs Bellamy and Joklik, Albert Einstein College of Medicine, New York. Drs Tournier and Granboulan, Institut de Recherches sup le Cancer, Villejuif, France, also kindly supplied us with samples of the same type. The virus was grown in HeLa cells, BHK cells, and mouse L cells. It was purified according to previously published procedures. Purified viral RNA was kindly supplied by Drs Bellamy and Joklik as fractions from a sucrose density-gradient centrifugation (Bellamy et al., 1967). The RNA was in solution in SSC (0.15 N-NaCl-O-015 M-sodium citrate, pH 7). These preparations were used without further purification. DFPt-chymotrypsin was purchased from Worthington, Inc. (a) Two-step release The procedure of Granboulan & Niveleau (1967) was used. The intact virus was incubated in 4 M-wea (neutralized and filtered) at 4°C for 5 min. Ammonium acetate (1 M) and DFP-chymotrypsin (0.01 ye) were added. This mixture was immediately spread in the cold room onto a subphase of 0.015 M-ammonium acetate, adjusted to pH 8 with sodium hydroxide or 0.3 M-ammonium acetate, pH 7. We call this method a two-step extraction; the first step is the treatment with urea, the second is the spreading. In some was modified as follows: in the first step, 3 to 8 M-urea was used cases, the extraction for 5 min; in the second step, the spreading with DFP-chymotrypsin in 1 M-ammOnkII acetate was performed on 2, 3, or 4 M-urea instead of ammonium acetate as a subphase. (b) One-step release This was carried out as follows. The intact virus, appropriately diluted in a solution of 1 M-ammonium acetate, and O.Olo/o DFP-chymotrypsin was spread at 4°C onto a subphase of urea, the concentration of which varied from 2 to 8 M. In some experiments, a mixture of sodium perchlorate (mostly 4 M) and urea (mostly 4 M) was used as a subphase. (c) Spreading The spreading procedure was routinely carried out using a Teflon-coated aluminium trough (200 ml. capacity) and a wetted glass ramp. Details of the technique are described eksewhere (Dunnebacke & Kleinschmidt, 1967). Following the spreading, the fihn was transferred to grids after varying periods of time (0 to 20 min). (d) Electron Carbon (platinum

supports, specimen

t Abbreviation

microscopy

reinforced with Formvar or collodion, mounts, Siemens type). A mixed monolayer

used: di-isopropyl

phosphofluoridate.

were mounted was transferred

on grids to grids

PLATE I. Reovirus particles negatively (a) Intact virus particles; (b) virus particles The bar represents 0.5 p.

stained by potassium phosphotungstate, after 5 min in vitro treatment with 4 M-urea

pH 7. at 4’ ‘2

[ facinq p. 138

PLATE II. Electron micrographs of reovirus particles at different stages of disruption; uraniumshadowed. A virus-protein monolayer was transferred from 4 M-urea subphase to grids (a) after 4 min (incomplete spiders); (b) after ‘7 min (complete spiders); (c) after 10 min (detachment of fragments from a spider; and (d) after 12 min (splash). The bar indicates 0.5 p.

PLATE III. filaments.

A field showing spider The bar indicates 1 p.

and splash

forms

of reovirus

particles

as well

as freed

RNA

PLATE IV. Double-stranded RNA showing strand separation (uranyl acetate stained). The RNA corresponds to the M fraction of the sucrose density gradient. The protein film yielding these denatured strands was prepared in 4 m-urea and 4 M-sodium perchlorate at room temperature. The fragments are locally denatured showing strand separation internally (a) and at the free ends (b). The bar indicates 0.25 p.

RNA

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by touching the film surface. The grid was then rinsed with water for 30 sec. Next, the adherent water droplet was replaced by ethanol; the grid surface was dried and metalshadowed as previously described (Dunnebacke & Kleinschmidt, 1967). In some eases, after rinsing with water, the grid was positively stained with many1 acetate (O*OOOlM) in acetone for 30 see, and rinsed briefly with ethanol (Gordon & Kleinsohmidt, 1968). The staining was carried out at room temperature. Intact virus particles and virus particles treated with 4 M-urea were negatively stained using 3% phosphotungstate, pH 7 (Vasquez & Tournier, 1964). Electron micrographs were taken with an Elmiskop 1A at a magnification of 7200 or higher. The magnification was calibrated with a grating replica (Fullam, Inc.). Filaments

were measured by projecting the negative on paper, tracing the filaments with pencil, and measuring the lengths with a map ruler. Some measurements were made with a Grafacon 1OlOA board, connected with a PDP-8 digital computer, by measuring the

projected negatives (Basilica, Kleinschmidt & Murphy, manuscript in preparation).

3. Results (a) Two-step release

Virus particles were incubated with 4 M-urea for five minutes at 4°C and spread on 0.015 M-ammonium acetate, pH 8, according to Granboulan & Niveleau (1967). Electron micrographs showed scattered fragments mostly shorter than 1.5 p and a smaller number of capsid remnants. Similar results were obtained when O-3 Mammonium acetate, 2,3, or 4 M-urea (all at pH 7) were used as subphases. Performing the experiments at room temperature had no influence on the length of the fragments. Contour length measurements from two preparations are shown in Figure l(a) and (b). The longest filaments found in these preparations were 4.8 p; length distribution maxima of the histogram were at 0.36, 0.67 and 1.15 TV. Five minutes incubation with urea prior to spreading did not degrade the virus completely. This was shown in control experiments where the urea-treated particles were negatively stained (Plate I). We assume that the spherical appearance of the particles in Plate I(b) is due to the maintenance of the inner layer (Vasquez & Tournier, 1962) after the outer layer has been stripped off by the action of urea. Liberation of RNA does not occur until the partially degraded virus is spread on a subphase. (b) One-step release The above procedure does not allow one to estimate the number of RNA fragments released per virion. For this purpose, the intact virus was spread onto a subphase of 4 M-Urea at 4°C. After various periods of time, the protein film was sampled for electron microscopy. The results are presented diagrammatically in Figure 2. There was a gradual decomposition of the virus particles while bound to the protein film. With 5 M-urea, for example, there was disintegration of the capsid within three minutes. This was followed by the appearance of aggregated filaments attached to the altered virus particles. These forms are referred to as incomplete “spiders”. During the period from four to seven minutes, a form called spider predominated. At the end of this period, the spider form disintegrated, and the RNA was found as separated fragments, localized to the area previously occupied by the spider. This form is called “splash”. After 12 minutes or more, the RNA fragments began to be widely dispersed throughout the protein monolayer. The forms shown in Figure 2

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represent the predominant pattern for a given time. Other forms were present but in minor amounts; for example, in one experiment at ten minutes, 75 spiders and 32 splashes were counted. In addition, dispersed filaments were seen in fields containing splashes and spiders. The number of free filaments increased as the number of splash forms became larger.

3o

(6)

1000 Filaments

Length (p)

FIG. 1. Length distribution of reovirus RNA fragments up to 1.8 p. (a) RNA filaments extracted in 4 ~-we& and spreed on 0.3 M-ammonium acetate, (b) extracted in 4 ~-urea and spread on 4 rd-urea; and (c) released by spreading the intact virus on 4 M-ure& (one-step release). The ordinate is the percentage number of filaments, per 0.1 p interval. The arrows indicate peaks.

Spider and splash forms are seen in the electron micrographs of Plates 11 and III, showing the progress of dissolution of the virus particles. Within three minutes after spreading on 4 M-Urea, no release was observed. In the fourth minute, increasing numbers of aggregated filaments were found emerging from the virus particles (Plate II(a)). The disintegration proceeded showing the appearance of spiders (Plate II(b)) and detached fragments (Plate II(c)). Plate II(d) shows a typical splash form. The spider forms were analyzed with regard to their total filament length. The histogram in Figure 3 shows the complete total length of filaments per spider at various sampling times, Of 58 spiders measured at four minutes, 14 (24%) had total

RNA

STRANDS

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141

lengths between 4 and 4.5 p. The period from four to ten minutes shows a progressive increase in the number of spiders having a total length from 5 to 8-5 p. None of them had a length greater than 8-5 pG

M

s

,

Intact

7

Destruction

,

lncofnplete

Is;,

v TA? r/l’

virus particle

Complete

y I,

of copsid

spider

spider

Splash

Dispersed

filaments



FIG 2. Disintegration of reovirus by urea. The scheme shows the gradual disruption of the capsid, the extrusion of the RNA fragments, and the dispersal of fragments (vertical rows) by nrea at 4°C. The right-hand side column gives the time of exposure to urea, i.e. the time elapsed between spreading and mounting of a given sample.

Spiders were also analyzed with regard to the number of fragments per spider. Figure 4 represents the same experiments as Figure 3, but plotted to show the relative number of fragments per spider at different sampling times. The number of fragments per spider was estimated by assuming that eaoh fragment displayed two free ends. This assumption rests on the observation that the average number of fragments per splash (see below) was about half the average number of free ends per spider. The results show that the number of fragments per spider increased with time. In another series, 39 splashes were measured. As in Figure 5, the maximum total length observed was about 8.3 p. When these fragment lengths were plotted in a histogram (Fig. 5, inset), the peaks (O-35, O-70 and 1.15 p) corresponded closely to the length distribution maxima of Figure l(a), (b) and (c). Figure 5 shows from bottom to top a decrease both in the number of filaments and in the total length per

-i

-

RNA

0

STRANDS

FROM

INDIVIDUAL

REOVIRIONS

/

I

1

I

I

,

1

1

I

1

2

3

4

5

6

7

8

9

143

Length (4 FIG. 5. Composite length and fragment lengths of RNA of 39 splashes. Each line (in p) represents one splash. Inset: Length distribution histogram of 340 fragments of 39 splashes. The peaks are indicated in /z lengths (L).

splash. These results can be explained by dispersal of filaments from the region of the splash. As stated above, the number of dispersed filaments increased with time. Both Figures 3 and 5 indicate a maximum total length of about 8.3 CL.Shorter lengths may be due to the omission of filaments that remained within the capsid, or that of aggregated filaments, or of filaments dispersed from the region of the spider or splash. To verify the latter assumption, the following experiment was done. A grid having spiders and splashes after 12 minutes of exposure to urea, was photographed completely. A typical field is illustrated in Plate III. All of the filament,s visible in non-overlapping fields were measured, counted (806 filaments), and divided by the number (70) of spiders and splashes. This should give the average length and number of filaments per particle. The results showed a mean filament length of 8.3 p, in agreement with the maximum value of Figures 3 and 5. The average number of filaments per particle was 11.5. When purified virus was spread, not on urea, but on O-3 M-ammonium acetate, no disruption of virus occurred. Furthermore, RNA fragments were only rarely seen. This shows that the large number of fragments observed when the virus was spread on urea originated from the virus particles, and were not present in the purified preparations as contaminants. The electron microscopic length distributions can be correlated to the molecular weight distribution observed upon centrifugation of reovirus RNA in sucrose gradients. In order to compare the electron microscopic RNA distribution with the triple-peaked sedimentation profiles of Bellamy et al. (1967), we used in Figure 6

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KLEINSCHMIDT

the weight distribution of lengths (Hall & Doty, 1958) of all free filaments measured. The longest filament found was 7.5 f~, in agreement with earlier observations (Kleinschmidt et al., 1964; Dunnebacke & Kleinschmidt, 1967; Granboulan C% Niveleau, 1967). In the histogram of Figure 6, the trimodal distribution of fragments is evident and is essentially the same as the sedimentation profile (cf. Fig. 2, of Bellamy et al., 1967).

Length

L $1

FIQ. 6. Percentage of total length of 8021 RNA filaments as a function of increasing length. Ordinate gives percentage of number of filaments in each 0.1 p interval x L. This distribution is equivalent to a weight-distribution of lengths.

(c) Electron rnicroscop~ of isolated RNA Reovirus RNA, centrifuged through a sucrose density gradient, was kindly provided by Drs Bellamy and Joklik. Fractions 8, M and L (Bellamy et al., 1967) were mixed with ammonium acetate, pH 7. The results of a series of length measurements are shown in Figure 7. The mean lengths of each of the three peaks were: 0.38 & 0.09 p (S), 0.64 & 0.26 p (M), and 1.10 & 0.27 p (L). The calculated molecular weights, S = 0.8 x 10”; M = 1.3 x 106; and L = 2.3 x lo6 (assuming 2.1 x 106/p) are identical to those of Bellamy et al. (1967), based on sedimentation coefficients of 10.5 s (S), 12 s (M), and 14 s (L). To study the denaturation of double-stranded reovirus RNA, mixtures of urea and sodium perchlorate, pH 7, at room temperature, were used for a one-step denaturation of RNA in the spread film. Denaturation without strand separation occurred at 4 M-urea and sodium perchlorate, frequently showing (Plate IV) pairs of intertangled RNA strands.

4. Discussion The sequential release of double-stranded RNA from individual reovdrus particles has been visualized by electron microscopy. We have also measured the contour lengths of the liberated RNA fragments and attempted to assess the number of

RNA

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40 T=0.38 kO.09 -I z=oY54 f 0.26 :: II I 1 Z=l-IO

-L 0.27

Length L (~1 FIG. 7. Length distribution histogram of three peaks of RNA from sucrosedensity gradients. Each peak is shown independently. Solid line, S peak (small length); dashed line, M peak (medium

length); dotted line, L peak (long length). Total number of filaments = 741.

fragments per particle. These fragments were uniform in width but variable in length; all were linear and unbranched. The shortest fragments were 0.05 p and the longest 7.5 p. Partial denaturation of the double-stranded RNA was observed after spreading at room temperature on a mixture of urea and sodium perchlorate. Our measurements showed the presence of three major peaks of double-stranded RNA similar to those observed in previous electron microscopic investigations using different extraction procedures. The molecular weight of the RNA within each peak, derived from mean Glament lengths, was also similar to that calculated from sedimentation coefficients (Bellamy et al., 1967). Length variations of each of the peaks were constantly found (see Figs 1, 6 and 7). It is unlikely that the variations observed are due to instrumental errors. These errors are estimated to be less than 4% (Lang, Bujard., Wolff & Russell, 1967). Distortions of length due to spreading on high concentrations of urea are a possible source of error. However, the same variation occurs when reovirus RNA is spread on ammonium acetate or on aqueous solutions of 0.5% formaldehyde (Dunnebacke & Kleinschmidt, 1967). The fact that these results are obtained under a variety of carefully controlled conditions would seem to rule out the possibility that the observed fragmentation is due to shear. Bellamy et al. (1967) have reported that reovirus contains about 20% singlestranded RNA, rich in adenylate, having a sedimentation coefficient less than 4 s. We have not observed such molecules, but it is possible that visualization of this RNA is beyond the resolution limit of our present technique. Our observations show that (a) from the fourth minute on, a majority of the spiders have six to nine fragments (Fig. 4), and (b) the average number of fragments per splash is 8.7 (Fig. 5). However, if one counts all visible fragments in a large number 10

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KLEINSCHMIDT

of non-overlapping fields (from protein-film samples after 12 minutes) and divides by the number of spiders plus splashes, one obtains an average of 11.5 fragments per particle. Since, as mentioned earlier, filaments were often seen dispersed from the region of the spiders and splashes, (a) and (b) are likely to be underestimates. As regards the total length of RNA, the maximum length both of spiders (Fig. 3) and splashes (Fig. 5) reached a limiting value slightly greater than 8 p (longer composite lengths were never found) and the same measurements which resulted in an average &lament number of 11.5 gave a maximum total length of 8.3 p. Moreover, the mean fragment length of all 8021 filaments measured (see Fig. 6) was 0.71 p. If we assume an average of 11.5 fragments per particle, we obtain a total length of 8.2 p. Thus, the total length of the RNA of reovirus appears to be slightly more than 8 f~. This figure is close to our value for the longest measured intact filaments (7.4 p), and those reported by others. If we accept the total RNA length to lie between 8 and 8.3 p, then a molecular weight corresponding to these lengths can be estimated. The calculated molecular weights for double-stranded RNA per unit length vary between 2-l million per micron (Bellamy et aE., 1967), 2-3 million per micron (Langridge & Gomatos, 1963), and 2.6 million per micron (Arnott, Wilkins, Fuller & Langridge, 1967). Hence, calculated molecular weights of reovirus RNA fall between 17 and 22 x 106. These values are higher than those previously reported (Gomatos & Tamm, 1963; Granboulan & Niveleau, 1967). If the reovirus RNA consists of a single filament, its peculiar fragmentation into smaller filaments upon release from the virion, giving a trimodal length distribution, and the large variation of filament length within each peak, remain to be explained. Watanabe & Graham (1967) suggest that fragmentation occurs at weak regions of the chain. Whether such regions contain the adenine-rich strands described by Bellamy & Joklik (19673) is a matter for conjecture. We are indebted to Mr H. Annus, Mr C. N. Gordon, Miss W. Hellmann, and Miss M. Pendergast for their assistance. We also wish to thank Dr S. Ochoa for helpful suggestions in the preparation of the manuscript. This work was supported by a grant from the John A. Hartford Foundation, Inc., New York, and by grant FR 05399-04 from the National Institutes of Health, United States Public Health Service. One of us (C.V.) is a recipient of an Eleanor Roosevelt Fellowship of the American Cancer Society, awarded by the International Union Against Cancer, and is on leave of absence from the Carrera de1 Investigator (Consejo Naci’onal de Investigaciones Cientificas y Tecnicas), Argentina.

REFERENCES Arnott, S., Wilkins, M. H. F., Fuller, W. & Langridge, R. (1967). J. Mol. Bid. 27, 525. Bellamy, A. R. & Joklik, W. K. (1967a). J. Mol. Biol. 29, 19. Bellamy, A. R. & Joklik, W. K. (19673). Proc. Nat. Acad. Soi., Wash. 58, 1389. Bellamy, A. R., Shapiro, L., August, J. T. & Joklik, W. K. (1967). J. Mol. Biol. 29, 1. Caspar, D. L. D. (1965). In Viral and Rickettsial Infections of Man, ed. by F. L. Horsfall, Jr. & I. Tamm, p. 86. Philadelphia: Lippmcot Comp. Dunnebacke, T. H. & Kleinschmidt, A. K. (1967). 2. Nutur. 22b, 159. Gomatos, P. J. & Stoeckenius, W. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1449. Gomatos, P. J. & Tamm, I. (1963). PTOC. Nat. dead. Sci., Wash. 49, 707. Gordon, C. N. & Kleinschmidt, A. K. (1968). Biochim. biophyys. Acta, 155, 305. Granboulan, N. & Niveleau, A. (1967). J. Microscopic, 6, 23. Hall, C. E. & Doty, P. (1958). J. Amer. Chem. SOS. 80, 1269.

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Iglewski, W. J. & Franklin, R. M. (1967). J. v’irology, 1, 302. Kleinschmidt, A. K., Dunnebacke, T. H., Spendlove, R. S., Schaffer, F. L. & Whitcomb, R. F. (1964). J. Mol. Biol. 10, 282. Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. BioZ. 23, 163. Langridge, R. & Gomatos, P. J. (1963). Science, 141, 694. Vasquez, C. & Tournier, P. (1962). Virology, 17, 503. Vasquez, C. & Tournier, P. (1964). Virology, 24, 128. Watanabe, Y. & Graham, A. F. (1967). J. ViroZogy, 1, 665. Watanabe, Y., Prevec, L. & Graham, A. F. (1967). Proc. Nat. Acad. Sci., Wash. 58, 1040.

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