- Email: [email protected]
Properties of mouse leukemia viruses
VIROLOGY
49, 345-358
(1972)
of Mouse
Properties III. Electron
Microscopic
Appearance
Techniques MILAN
as well
V. NERMUT,’
Max-Planck-Znstitut
fiir
Leukemia
as Revealed
after
as Freeze-Drying
HERMANN
Virusforschung,
FRANK,
April
Conventional
Preparation
and Freeze-Etching WERNER
AND
Biologisch-Medizinische Accepted
Viruses
Abteilung,
SCHAFER Tcbingen,
Germany
17, 1972
Rauscher, Friend, and Gross strains of murine leukemia viruses (MuLV) were studied with negative staining, freeze-drying, and freeze-etching techniques. Negative staining of the virus particles with uranyl acetate (UA) and the combination of phosphotungstic acid (PTA) negative staining with freeze-drying showed that the virus particles are roughly spherical and are covered with “knobs” about 80 A in diameter which are weakly bound to the viral membrane. Freeze-etching of the virus particles confirmed the existence of the “knobs.” A diameter of about 1060 A was determined for the virus particles in freeze-dried preparations. Freeze-drying and shadowing of the viral cores liberated by Tween 80 and ether, ether alone or Triton X-100, revealed that their surfaces have a regular pattern of hexagonally arranged subunits with a diameter of about 60 8 and a center-to-center distance of about 75 A. The existence of pentons as well as the configuration of the shadows of freeze-dried cores suggested that the viral core possesses icosahedral symmetry. Negative staining of fixed virus particles resolved the core shell into two tracks, a fact which indicates the existence of two components. A smooth membranous layer underlying the layer of regularly arranged subunits wss observed in liberated cores. The diameter of the viral cores was about 800 h; as determined after freezedrying. The internal component of the core, the nucleoid, is a filamentous structure, which may possess helical symmetry. Considering the above described substractures, we have proposed a model of MULV.
leukemia viruses (MuLV) by the presence of at least seven different antigenic components (Schafer et al., 1972a,b). In order to obtain more information on the fine structures of MuLV’s, we used conventional procedures as well as such recently developed techniques as freeze-etching and negative staining followed by freeze-drying (Nermut and Frank, 1971). Several previously unknown substructures were thereby revealed.
INTRODUCTION
In the last decade several papers dealing with the fine structure of RNA-containing oncogenic viruses of the C type have appeared (see reviews of Bader, 1969; Vigier, 1970; Gross, 1970). The principal techniques employed were ultrathin sectioning and negative staining of intact and degraded virus particles. However, there remains considerable uncertainty as to the true shape and fine architecture of the surface and interior of the particles, the complexity of which is attested to, in the case of murine 1 Present cal Research,
address: National Institute Mill Hill, London NW
MATERIALS
@ 1972 by Academic Press, Inc. of reproduction in my form reserved.
METHODS
Viruses. Friend, Rauscher, and occasionally Gross murine leukemia virus were
for Medi7, England. 345
Copyright All rights
AND
346
NERMUT,
FRANK,
used. Friend virus was produced in STU, Rauscher virus in JLSV5, and Gross virus in rat thymus cell cultures. All were purified by ultracentrifugation followed by banding in a potassium citrate density gradient. The corresponding techniques have been described in previous communications of this series (Schafer et al., 1972a,b). Treatment of virus particles with Tween 80 and ether was performed as described by de Thb and O’Connor (1966). Samples for electron microscopy were removed after every step, i.e., after Tween, after addition of ether, and after density gradient centrifugation. Treatment with ether alone was performed as follows: 1 part of cold peroxide-free ether (Merck, Darmstadt, Germany) was added to 2 parts of concentrated virus suspension and shaken or stirred for 45 min at 4’. The ether and the water phases were separated by lowspeed centrifugation and the remaining ether was removed from the water phase by bubbling with nitrogen. The sample was then layered on a potassium citrate density gradient containing Ca2+ and Mg”+ ions (Lacour et al., 1970) and centrifuged in a Spinco SW 39 rotor at 30,000 rpm to equilibrium. The material banding at a density of about 1.26 g/ml was separated, diluted with Tris. Mg buffer and centrifuged at 22,000 rpm for 80 min in a Spinco rotor 40. The pellet was washed once again with the same buffer and then used for freeze-drying. Treatment with T&m X-100 (Roehm and Haas Co., Philadelphia, Pennsylvania) was done with a 0.02 % solution at 36” for 20 min. Samples for negative staining were removed immediately (washing was performed on the grids), and the remaining material was submitted to a rate zonal density gradient centrifugation on potassium citrate to remove debris and membranous structures. Washing was then performed as described for the ether treatment. Dimethyl sdfoxde (DMSO) (Merck, Darmstadt, Germany) was added to the virus suspension at a concentration of 20 % 30 min before freeze-drying. Trypsin (2 X tryst. 180 EU/m.l, Serva, Heidelberg, Germany) was dissolved in 1M/ 15 phosphate buffer, pH 7.4, as a 0.5 % solu-
AND
SCIldFER
tion and was diluted with distilled water 5-10 times before use. Pronase (lyophilized, Serva, Heidelberg, Germany) was used as a 0.2 % solution in phosphate-buffered saline pH 7.2 (PBS). Purified phospholipase C from Clostrklium welchii (Sigma Chemical Co., St. Louis, Missouri) was used in 0.25 mg/ml concentration in PBS. Snake venom from Ancistrockm piscivorus (Ross Allen’s Institute Inc., Florida) was used in a concentration of 1 mg/ml in PBS. Treatment with the enzymes as above was performed for a given time (see results) at 37” in a water bath. Bu.ers. Phosphate-buffered saline (PBS), pH 7.2, contained 0.7% NaCI, 0.31% Na2HP0, and 0.034% KH2P0,. Tris. Mg buffer contained 0.01 M Tris(hydroxymethyl)aminomethane with 0.001 M MgSO, (final pH was 6.7). Negative staining was done with 2 % phosphotungstic acid (PTA) pH 5.0, 6.0, or 7.2, or with 1% uranyl acetate (UA) pH 4.4, or with 3 % ammonium molybdate (AM), pH 5.3 or 6.7. Staining was done by first placing a drop of the material on the grid for adsorption and then applying the negative stain. Carbon-coated Formvar films were used throughout. Fixation. The material was fixed either with 5 % glutaraldehyde (GA) or 0.2 or 1.0 % osmium tetroxide. After the material had been adsorbed onto the grid, this was placed on a drop of the fixative for 10 min and washed with distilled water before negative staining. Freeze-drying. The grids containing the adsorbed material were carefully rinsed with volatile buffer (e.g., ammonium acetate) or double-distilled water, and, after the liquid had been drained off with filter paper, the grids were immediately frozen in liquid nitrogen. The frozen grids were then quickly transferred to a four-specimen stage of the Balzers freeze-etching apparatus BA 500 M (Balzers AG, Balzers, Liechtenstein) which was previously cooled to - 150” and dabbed with liquid Freon 22 in order to ensure an immediate good thermal contact between grid and stage. The grids were then gently nudged into the identations of the stage and
ELECTRON
MICROSCOPY
OF
secured by a cover plate. This last step was especially important because otherwise the light-weight grids frequently slid out of the stage during the drying process. The chamber was then evacuated and the temperature of thr specimen stage was raised to -8.5”. The microtome arm was simultaneously cooled to - 150’ and then moved over the specimen stage. After at least 20 min drying, the knife arm was removed and the grids were shadowed with platinum-carbon (Pt-C) at an angle of 3045”. In some experiments the shadow-casting was done from two directions. In practice, the right holder of the freeze-etching device (normally destinrd for carbon evaporation) was used as the second source of Pt-C and adjusted at approximately the same height and distance from the stage as the left one. The resulting angle between the two directions was about 80”. Freeze-drying of negatively stained specimens. The grids with adsorbed virus particles dvere washed as described above and placed on a drop of either PTA or AM. After 15 set the grids were removed, drained of excess stain, and immediately dipped in liquid nitrogen. Drying of the film usually required 3 or more seconds, so that the grid could conveniently be frozen before drying. Further procedures were the same as described for freeze-drying, except that the shadowing was omitted, and, before the chamber was aired, the temperature of the stage was raised to about +40” in order to avoid water condensation on t’hr preparation due to moisture in the air. A white coating composed of dried lamellalike PTA formed on the grids and must be carefully blown away in order to uncover the thin PTA layer on the supporting film. The adsorbed virus material is found predominantly in this layer. Freeze-etching was performed as described previously without the addition of any protective substances to the material before freezing (Nermut and Frank, 1971). dgar filtration was done according to Kellenberger and Arber (1957). Specimens were shadowed with Pt-C at the same angle employed when the preparations were freezedried.
MOUSE
LEUKEMIA
VIRUS
347
Electron microscopy. Specimens were observed with a Siemens Elmiskop I A electron microscope at 80 kV and a nominal magnification of 40,000 or 80,000 X. The real magnification was evaluated as described by Frank and Day (1970). RESULTS
Shape, Surface, and Size of MuLIP
Particles
Negative staining of the viruses with phosphotungstic acid (PTA) or ammonium molybdate (AM) revealed particles of various shapes (Fig. la). Some of the particles were empty, and many of them possessed characteristic tails. The surface of most of the particles was smooth, and only rarely were knoblike protrusions (henceforth to be referred to as knobs) visible on their periphery (Fig. lb). These knobs were 5&70 8 long. Negative staining with uranyl acetate (UA) produced rather surprising results. The virus particles now appeared as spheres of uniform size. Their surface was covered with knobs of about 80 A in diameter (Fig. 2). The knobs did not appear to be solid globules, but, instead resembled rings or clusters of smaller subunits. Their arrangement on the surface was not very regular, although hexons could occasionally be seen. Some particles were partially or entirely devoid of knobs. When the particles were subjected to negative staining with PTA combined with freeze-drying, they were predominantly round (Fig. 3a). On many of the particles one could detect knobs having a structure similar to those recognizable after conventional negative staining with UA (Fig. 3b). Specimensprepared by agar filtration were subjected to shadow-casting with Pt-C. This preparation produced flat particles displaying a prominent center, causing them to resemble a fried egg (Fig. 4). With this technique, however, knobs were not detectable. Freeze-dried and shadowrd virus particles cast long and, more important, often angular shadows, indicating that they were not ideal spheres (Fig. 5). In order to analyze this in 2 The term MuLV (murine leukemia virus) was used throughout, since the mouse leukemia virus strains Friend, Rauscher, and Gross delivered analogous elerbon microsropic results.
348
NERMUT,
FRANK,
ANI)
SCHAFER
The abbreviations PTA, UA, or AM indicate negative staining with, respectively, phosphotungstate, pH 6.0; many1 acetate, pH 4.4; ammonium molybdate, pH 6.7. FIG. 1. (a) Native virus particles (Rauscher) stained with PTA show a remarkable pleomorphism with tailed and broken particles. (b) One of the rare particles (Friend) showing knobs on the periphery after PTA staining. X 132,000. FIG. 2. The same suspension as in Fig. la (Rauscher), but stained with UA. The particles are uniformlyroundand covered with knobs. One particle (arrow) has no knobs. Scale line = 1000 1. X 132,000. FIG. 3. Virus particles (Friend) after PTA staining followed by freeze-drying. Note the absence of tails (compare with Fig. 1) and a somewhat smaller diameter (compare with Fig. 2). Knobs are seen on the surface (b). X132,000.
more detail, we shadowed freeze-dried particles from two directions. At least someof the particles revealed angular shadowssimilar to those cast by an icosahedron model (Fig. 6). It should be mentioned, however, that the angles were not as distinct as those observed on influenza virus (Nermut and Frank,
1971). No surface structures were detected on the freeze-dried particles because they were rather high and, therefore, electron dense. When specimens were freeze-dried and shadowed after treatment with Tween 80, one could observe more or less flattened
ELECTRON
MICROSCOPY
OF
membranous structures with knobs (loo-120 A in diameter) on their surface (Fig. 7). The possibility that these structures were empty viral envelopes is supported by the finding of liberated viral cores in the same Tween SOtreated preparations. Freeze-etching revealed round particles with knobs only on their periphery, the others apparently having been removed by the fracturing (Fig. 8). In these preparations, like those described in the preceding paragraph, the knobs had a diameter of about 120 A. The knobs were removed by the action of trypsin (3 hr), pronase (3 hr), and DMSO (Fig. 9) ; such particles had a similar morphology regardless of treatment. Removal of such protrusions from Rous sarcoma virus by a proteolytic enzyme has been described recently by Rifkin and Compans (1971). It is evident from the results described that negative staining with UA, freezedrying plus shadowing, and negative staining combined with freeze-drying produced the best preserved particles. Thus, these techniques were employed in investigation of the diameters of virus par$cles. The lowest average diameter, 1080 A, was found after freeze-drying and shadowing. Allowing for the thickness of the carbon metal film, one can estimate that the virts particles have a diameter of about 1060 A. This is in good %greement with the average diameter of 1060 A found by Bader et al. (1970) in thin sections. Much larger particles containing two cores were observed occasionally (Fig. lob). Particles treated by either negative staining with PTA followed by freeze-drying or negative staining with UA, were found to haye somewhat larger diameters (1300 and 1440 A, respect,ively) . Internal
Structure
of Virus
Particles
When the virus particles were fixed with GA or 0~0~ before PTA negative staining, the stain penetrated into the particles thus revealing someof their internal components. It is obvious, that the particles contained an internal body, “the core” (Fig. lOa); the more rarely observed larger particles have two cores (Fig. lob). In well preserved particles (Fig. 1Oa)the core is limited by a distinct
MOUSE
LEUKEMIA
VIRUS
349
shell, about 80 8 thick. This shell is often resolved into two separate tracks, 2030 A thick (Fig. lOc), which are not always parallel to each other (Fig. lOd, e). Occasionally, only one track was observed (Fig. lob, f). The type of fixation did not influence the appearance of the tracks. Definite substructures were not revealed inside the core shell by this preparation procedure. However, unfixed negatively stained (UA) particles which had been slightly damaged by various treatment’s (Tween-ether, Triton X-100, phospholipase C) showed not only a better preserved core shell with regular striations (Fig. ll), but also a filamentous structure in the interior which appeared somewhat thicker than the core shell and was often faintly striated (Fig. llc, d). This filamentous structure, which we designate as the nucleoid, appeared mostly as a ring (Fig. llb) and only sometimes as a spiral (Fig. llf). In few cases, if t,he nucleoid was not completely penetrated by stain, some parallel running strands became visible (Fig. 1lg small arrows), giving t’he impression t’hat it is a hollow sphere formed by a wound filament. Freeze-etching was a technique which we considered highly promising for studying the internal organization of the virus particles and, in particular, for revealing either the core surface or its internal filamentous component. The results obtained, however, were somewhat disappointing. In the rare cases where a superficial layer was removed from the particles, the exposed part was smooth (Fig. 12a, particles labeled B). We observed no particles where the core was broken, only particles where the core had been completely removed (Fig. 12, particles IEbeled C). In such cases elements SO-100 A in diameter occasionally remained on interior of the viral envelope. Some virus particles were completely removed, leaving only single knobs behind (Fig. 12b, particles labeled D). In subsequent experiments we tried to release the cores by treatment with Tweenether (de The and O’Connor, 1966), ether alone or various detergents (sodium dodecyl sulfate, sodium deoxycholate, Brij 35, Brij 58, Nonidet P-40, Triton X-100). Both conventional negative staining and freeze-dry-
Fms. 4-9.
X132,000.
ELECTRON
MICROSCOPY
OF
ing with shadowing were used to prepare the samples for electron microscopy. When Tween-ether treated viruses were freezedried, particles smaller in size than intact virus particles, often displaying a regular hexagonal pattern were found (Fig. 13a). We assume that they represent the viral cores. After citrate density gradient centrifugation of Tween-ether treated virus, they were found in the lower band (1.26 g/ml), but only in low concentration together with much debris. If the virus particles were treated with ether alone, the yield of cores in the lower band was significantly higher. Of the above-mentioned detergents, only Triton X-100 liberated intact cores in substantial numbers. A hexagonal arrangement of subunits was visible on the core surface if the cores were somewhat’ collapsed. These subunits, 60 A in diameter, had $ center-to-center distance of about 75-80 A. In a few cases pentons were detected, but we failed to find two pentons on the same core and thus could not establish the triangulation number of the core. In Fig. 13b such a subunit with 5-fold symmetry, indicated by its five nearest neighbors, is encircled, whereas in Fig. 13c one of the five neighboring six-coordinated subunits, i.e., a hexon, is marked. These two figures also show that the subunits are not solid globules but are composed of radially arranged “structure units.” The uncollapsed cores showed a long shadow which, in some cases, was more distinctly angular than that of the complete virus particles, however, the surface substructure was not revealed (Fig. 14). Apparently, these cores were too high to permit resolution of the substructure. The usually FIG. 4. Virus
MOUSE
LEUKEMIA
351
VIRUS
angular form of their shadow, even after shadowing from two directions, was considered by us to be more similar to the shadow cast by an icosahedron model (Fig. 15) than the shadows cast by the whole particle, thus suggesting a regular symmetry. Negative staining of liberated cores revealed either a striation of their shell (Fig. 16a) or subunits on the periphery of the cores (Figs. 16b and c). On some cores the subunits were seen to be situated on a smooth membranous structure (Fig. 16c and 4. The core diameter was determined for fixed intact virus particles negatively stained with PTA and for liberated cores which had been freeze-dried and shadowed. The mean diameters ranged between 740 and 800 h. DISCUSSION
The shape of MuLV particles has been determined to be spherical both in ultrathin sections and after negative staining of fixed particles (Bernhard, 1958; Dalton, 1962; de Harven and Friend, 1964; de The and O’Connor, 1966). However, if the native virus particles are stained negatively with PTA, many of them display a taillike protrusion (Dalton et al., 1962). We found that no tails were formed when PTA negative staining is followed by freeze-drying or when UA was used as negative stain. This is presumably due more to the stabilizing effect of UA on membrane lipids than to its low pH, as “tails” were seen after very acid PTA (pH 4.4), too. In addition, UA clearly revealed projections (“knobs”) at the surface of the particles. After PTA negative staining these knobs were seen only rarely, and then exclu-
particles (Gross) prepared by agar filtration and shadowed with 45”. Scale line = 1000 A. FIG. 5. Freeze-dried particles (Friend) shadowed under the same conditions as in FIG. 6. (a and c) Virus particles (Friend) after freeze-drying and two-directional icosahedron model correspondingly illuminated from two directions showing shadows particle ones. FIG. 7. Virus particles (Friend) treated with Tween 80 for 30 min, freeze-dried particle (A) is full and knobs cannot be seen. The others are more or less emptied spondingly improved visibility of the knobs. FIG. 8. Freeze-etched particles (Friend) showing knobs on the periphery. FIG. 9 Particles (Friend) treated with 20% DMSO for 30 min and freeze-etched face. The knobs were completely removed.
Pt/C
at an angle
of
about
Fig. 4. shadowing. (b) An similar to the virus and shadowed. (B-D) with
show
a smooth
One corre-
sur-
Fro. 10. Virus particles (a, c, d, f Rauscher; b, e, Friend) fixed with lo/o 0~04 (a, d, e) or 5vi GA (k, c, f) and stained negatively with PTA (a-e) or AM (f). T@ particle in b contains two cores. The core shell (arrows) is seen either as a compact structure about 80 A thick (a), or as a thin track (b and f), or as two parallel tracks (c) or nonparallel tracks (d and e). Scale line in Fig. 10a = 1000 A. X220,000. FIG. 11. Partially opened virus particles (a, b, d, f, g, Friend; c, e, Rauscher) revealing internal organization after negative staining with UA. A, viral envelope; B, core shell; C, internal filament (nucleoid). Core shell is usually striated. Particles were treated with snake venom (a), Triton X-100 (b), phospholipase C and neuraminidase followed by Tween and ether (c, e), Tween and ether (d, g) and nontreated (f). X220,000.
sively on the periphery of the particles. Still better preservation of the knobs is achieved by PTA negative staining combined with freeze-drying. This indicates that the normal
drying process either removes the knobs or deforms them in such a way as to render them unrecognizable. That, the knobs are indeed real struct,ures, not artifacts, could be
ELECTRON
MICROSCOPY
OF MOUSE
clearly demonstrated by freeze-drying and shadowing as well as by freeze-etching of the virus particles. The lability of the knobs may explain why they were not always detected by other authors using conventional preparation techniques. Nowinski et al., (1970) for example, concluded that MuLV does not possess “surface projections.” Parsons (1963)) however, described slender “outer spikes” on Gross leukemia virus particles, and Zeigel and Rauscher (1964) at times found peripheral “knobs” or spikes. Freeze-drying followed by shadowing suggested that the particles are not true spheres. Their shadows are angular; however, their corners are not very distinct, i.e., they are “soft.” In contrast, the isolated cores often displayed shadows that were distinct and more angular than the previous ones and which were similar to those cast by an icosahedron model. Some evidence indicative of a polygonal shape for the cores or the virus particles have been published previously (Dalton et al., 1962; Zeigel and Rauscher, 1964; de The and O’Connor, 1966). Our studies support the possibility that the virus cores have cubical symmetry: the cores are covered by small globular subunits in a closeand very regular hexagonal arrangement. These globules often show radially arranged “structure units.” The occasional finding of a penton (Fig. 13b) suggeststhat the core surface has icosahedral symmetry. The icosahedral shape of the core may determine the shape of the whole particle. However, the corners of t.he particles are indistinct, probably due to the plasticity of the viral envelope. The demonstration of the hexagonal pattern by techniques other than freezedrying and shadowing seemsto be very difficult. Some indications for the existencz of hexagonally arranged subunits 50-60 A in size were obtained earlier by Zeigel and Rauscher (1964), but at this time it was not yet known “whether these patterns represent viral coat plus knobs (surface) or are internal subsurface structures.” Only very rarely did we find this structure with negative staining, probably because most of the cores were penetrated by stain and only among the unpenrtrated ones were a few found that were
LEUKEMIA
VIRUS
353
stained only from one side, thus revealing the hexagonal pattern. A further element is apparently present in the core shell below the layer of regularly arranged subunits. We supposethat the two small tracks, seen sometimes instead of the striated layer after negative staining with PTA, originate from the core shell (striated layer). The tracks show branching, thus indicating the existence of two components. Our observations on isolated cores suggest that the outer track represents the subunit layer, whereas the inner track seemsto be a smooth membrane. Both components can be distinguished in Figs. 16b-d, where the subunit layer was apparently partially removed. Some information on the material situated inside the core has been presented in earlier publications. In ultrathin sections several authors described a distinct ring underneath the core shell which could represent a helical nucleocapsid (de The and O’Connor, 1966; Zeigel et al., 1966; de Tkaczevski et al., 1968). Zeigel and Rauscher (1964) liberated strands with helical pattern from particles of the Rauscher virus by treatment with distilled water. In this work both the core shell and a filamentous nucleoid structure could be revealed, but only by the use of UA for negative staining. The striation of these filaments may suggest a helical symmetry. However, the real arrangement of the filamentous nucleoid cannot yet be determined. The possibility exists, that the various appearance of the internal structures (rings, spirals, possibly hollow spheres) depends upon the degree of penetration of the strain. Alternatively, the rings or spirals could represent degradation products of a hollow sphere or all three forms could represent different stagesof viral maturation. Nowinski et al. (1970) and Sarkar et al. (1971) proposed that the nucleoid exists as a hollow sphere formed by supercoiling of helical strands. This is probably correct, but the structure considered by those authors (at least in the case of Rauscher leukemia virus) is apparently identical with the core shell described by us. After negative staining with PTA, which these authors used cxclusively, the core shell, but never the inner filamentous element, was recognizable in our experiments.
ELECTRON
MICROSCOPY
OF MOUSE
cl dl FIG. 17. Schematic representation of the probable fracture planes found after freeze-etching of particles. (a) Removal of surface knobs. (b) Splitting of the viral membrane. (c) Removal of the core except for some subunits of the core shell lying on the internal face of the viral membrane. (d) Removal of the whole particle except for some knobs (see discussion).
Recently, Luftig and Kilham (1971) reported differential responses of Rauscher leukemia virus particles to staining with UA and PTA which agreed reasonably well with our findings. However, their different conclusions regarding structural configuration of the virus particle, e.g., failure to observe surface knobs, different size measurements and a whorled internal substructure, appear relat.ed partly to the condition of the starting material and partly to different interpretation of the structure observed. The virus examined by these workers was twice frozen and thawed, a procedure we have found detrimental to virus structure and consequently avoided in our studies. Indeed, freezing may not only remove knobs, but
LEUKEMIA
VIRUS
355
also could so disrupt the internal structure that one could not readily differentiate between the core shell and the inner filamentous component. It seems likely that such a situation prompted these authors to term as “nucleoid” this entire, complex core structure which we have here described. We could clearly demonstrate the core shell only after UA staining of slightly damaged but unfixed virus particles. The morphology of the freeze-etched particIes deserves special consideration. The observed fracture patterns are summarized in Fig. 17 and documented in Fig. 12. The origin of the A-labeled particles is well understood. The “knobs” seen on the periphery of the particles were uncovered by the etching process. However, the failure of the fracturing process to expose the hexagonal globular surface of the cores or their interior cannot be explained easily. The exposed surface of the B-labeled particles looks smooth, an observation that cannot be accounted for by the lack of resolut\on. The hexagonal globules are about 60 A in diameter and are seen clearly after freeze-drying. In freezeetched preparations where a fine carbon replica (without any organic material) is examined, the resolution is even better. Thus we assume that the outer membrane was split in the middle, so that the core surface was still covered by the lower part of it (Fig. 17b). Such an explanation is in agreement with the data published by Branton (1966) and Pinto da Silva and Branton
FIG. 12. Virus particles (Friend) prepared by freeze-etching. In particles marked A only the knobs were removed from the surface. In B particles apart of the outer membrane has been split off in addition, in C particles only a part of the outer membrane remains, and in D the whole particle wm removed. A schematic re resentation of the different fracture planes is given in Fig. 17. X 132,000. Scale line in Fig. 12a = 1000 % . FIG. 13. Virus cores (Friend) released by Tween-ether treatment. Freeze-drying and Pt/C shadowing. (a) Cores which are somewhat collapsed reveal a regular pattern of hexagonally arranged subunits. (b) A penton (encircled) found on the corner of a core. (c) A neighboring hexon. (a) X 132,000; (b, c) x350,000. FIG. 14. Uncollapsed Tween-ether cores (Friend) do not reveal any substructure but csst an angular shadow after freeze-drying. X132,000. FIG. 15. (a and c) Uncollapsed cores (Friend) (Triton X-100) after freeze-drying with two-directional shadowing. The shadows are compared with those of an icosahedron model (b). X 132,000. FIG. 16. Isolated cores (Friend), stained negatively with PTA (a, b), UA (c) and AM (d), sometimes showed a striation of the core shell (a), ~EJshown in Fig. 11. The shells were sometimes dissolved (b-d), revealing a smooth membrane (black arrows) covered with globules in certain spots (white arrows). The cores were liberated by the action of snake venom (a), Triton X-100 (b, c) and et’her (d). X220,000. Scale line = 1000 ii.
NERMUT,
356
FRANK,
AND SCHAFER
membrane
Y
viral
envelope
nucleoid ~nucleocopsid?)
1060;1.-----FIG.
18.
Schematic representation
(1970). A similar phenomenon has also, been observed in influenza virus by BLchi et al. (1969) and Nermunt and Frank (1971). When the virus particle was fractured horizontally, the whole core was apparently removed, so that only “shallow dishes” were found (C in Fig. 12). In such casesthe internal face of the outer membrane was revealed. Small elements seen upon it could then represent some of the core subunits which were left behind. The structures labeled D in Fig. 12b are very similar in appearance to those labeled C, but they do not cast any shadow. In this casethe whole virus particle has been removed, and the few tiny granules left behind are probably outer “knobs” that remained embedded in the ice and were then freed by etching. Our ideas about the fine structural organization of MuLV are represented in Fig. 18, which shows a transverse section of a virion. The main part of the virion is the “virus core,” presumably possessingcubical symmetry. It contains a strand, called “nucleoid,” as its chemical nature has not yet been sufficiently analyzed to use the term nucleocapsid. This structure is covered by the “core shell,” which consists of two structurgl elements: (1) a “core membrane” about 30 A thick; (2) a layer of globular or ringlike
1 of a MuL virion.
“morphological subunits” of about 60 A in diameter forming a close hexagonal pattern. The “viral core” is covered by the “viral envelope” gonsisting of a “viral membrane” (about 80 A thick) and the “knobs” (about 80 8 in diameter). Our findings in certain regards are similar to the observations made by de ThB and O’Connor (1966)) but our interpretation differs in several points. Their “outer shell” corresponds to our “core shell” and their ‘(inner shell” to our “nucleoid.” Padgett and Levine (1966) draw a scheme of Rauscher virus which also is not greatly different from our concept. However, this was done on the basisof experiments with snake venom preparations, which were later shown to be contaminated with herpeslike particles (Monroe et al., 1968). Similar data about the fine structure of avian myeloblastosis virus have been published recently by Gelderblom et al. (1972) suggesting that these structural properties are common to many, if not all, C-type particles. At the present stage of the investigations, it cannot be decided with which of the viral structural elements the various MuLV antigens are correlated. Our current efforts to isolate constituents of viral envelopes and
ELECTRON
MICROSCOPY
OF MOUSE
cores and to determine their antigenic composition should help to solve this problem. ACKNOWLEDGMENTS The authors wish to thank Mrs. U. Michel and Miss A. Kleih for skillful technical assistance, Mrs. I. Thiering, Miss R. Szabo, and Mr. G. Berger for preparation of micrographs and Dr. R. L. Witter for kind revision of the manuscript.
J. P. (1969). RNA tumor viruses. In “The Biochemistry of Viruses” (H. B. Levy, ed.), pp. 293-328. Dekker, New York. B~DER, J. P., BROWN, N. R., and BADER, A. V. (1970). Characteristics of cores of avian leukosarcoma viruses. Virology 41,718-728. BXCHI, T., GERHARD, W., LINDENMANN, J., and M~;.HLETHALER, K. (1969). Morphogenesis of influenza A virus in Ehrlich ascites tumor cells as revealed by thin-sectioning and freeze-etching. J. Viral. 4,769-776. BERNHARD, W. (1958). Electron microscopy of tumor cells and tumor viruses. Cancer Res. 18, 491-509. BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. Nat. A cad. Sci. U.S.A. 55, 1048-1056. DALTON, A. J. (1962). Micromorphology of murine tumor viruses and of affected cells. Fed. PTOC. Fed. Amer. Sot. Exp. Biol. 21,936-941. DALTON, A. J., HAGUENAU, F., and MOLONEY, J. B. (1962). Morphology of particles associated with murine leukemia as revealed by negative staining: Preliminary report. J. Nat. Cancer Inst. 29, 1177-1179. DE HARVEN, E., and FRIEND, C. (1964). Structure of virus particles partially purified from the blood of leukemia mice. Virology 23,119-124. DE TI&, G., and O’CONNOR, T. E. (1966). Structure of a murine leukemia virus after disruption with Tween-ether and comparison with two myxoviruses. Virology 28,713-728. DE TKACZEVSKI, L., DE HARVEN, E., and FRIEND, C. (1968). Structure and leukemogenic activity of a murine leukemia virus. J. Viral. 2,365-375. FRANK, H., and DAY, L. A. (1970). Electron microscopic observations on fd bacteriophage, its alkali denaturation products and its DNA. Virology 42, 144-154. GELDERBLOM, H., BAUER, H., BOLOGNESI, D. P., and FRANK, H. (1972). Morphogenese und Aufbau von RNS-Tumorviren: Elektronenoptische Untersuchungen an Viruspartikeln vom C-Typ. BADER,
Zentralbl. Hyg. Abt.
Bakteriol. Parasitenk. 1 Orig. 220, 79-90.
Infektionskr.
VIRUS
357
L. (1970). “Oncogenic Viruses,” 2nd. Pergamon, Oxford. KELLENBERGER, E., and ARBER, W. (1957). Electron microscopical studies of phage multiplications. I. A method for quantitative analysis of particle suspensions. Virology 3,245-255. LACOUR, F., FOURCADE, A., UERGER, C., and DELAIN, E. (1970). Coiled structure of the nucleocapsid of avian myeloblasosis virus. J. Gen.
GROSS,
Viral. LUFTIG,
REFERENCES
LEUKEMIA
9, 89-92.
R. B., and KILHAM, S. S. (1971). An electron microscope study of Rauscher leukemia virus. Virology 46, 277-297. MONROE, J. H., SHIBLEY, G. P., SCHIDLOVSKY, G., NAKAI, T., HOWATSON, A. F., WIVEL, N. W., and O’CONNOR, T. E. (1968). Action of snake venom on Rauscher virus. J. Nat. Cancer Inst. 40, 135;145. NERMUT, M. V., and FRANK, H. (1971). Fine structure of influenza AP (Singapore) as revealed by negative staining, freeze-drying and freezeetching. J. Gen. Viral. 10,37-51. NOWINSKI, R. E., OLD, L. J., SARKAR, N. II., and MOORE, D. H. (1970). Common properties of the oncogenic RNA viruses (Oncorna viruses). Virology 42, 1152-1157. PADGETT, F., and LEVINE, A. S. (1966). Fine structure of the Rauscher leukemia virus as revealed by incubation in snake venom. Virology 30, 623430. PARSONS, D. F. (1963). Structure of the Gross leukemia virus. J. Nat. Cancer Inst. 30,569-583. PINTO D.* SILVA, P., and BRANTON, D. (1970). Membrane splitting in freeze-et.ching. Covalently bound ferritin as a membrane marker. J. Cell Biol. 45,598-605. RIFKIN, D. B., and COMPANS,
R. W. (1971). Identification of the spike proteins of Rous sarcoma virus. Virology 46,485-489. SARXAR, N. H., NOWINSKI, R. C., and MOORE, D. H. (1971). Helical nucleocapsid structure of the oncogenic ribonucleic acid viruses (Oncorna viruses). J. Viral. 8,564-572. SCHSFER, W., FISCHINGER, P. J., LANGE, J., and PISTER, L. (1972a). Properties of mouse leukemia virus. I. Characterization of various antisera and serological identification of viral components. Virology47,197-209. SCHAFER, W., LANGE, J., FISCHINGER, P. J., FRANK H., BOLOGNESI, D. P., and PISTER, L. (1972b). Properties of mouse leukemia viruses. II. Isolation of viral components. Virology 47, 210-228. VIGIER, P. (1970). RNA oncogenic viruses: Structure, replication, and oncogenicity. Progr. Med. Viral. 12, 240-283. ZEIGEL, R. F., and RAUSCHER, F. J. (1964). Elec-
358
NERMUT,
FRANK,
tron microscopic and bioassay studies on a murine leukemia virus (Itauscher). I. Effect,s of physicochemical treatments on the morphology and biological activity of the virus. J. 1Vat. Cancer Imt. 32, 1277-1307.
AND
SCHAFER
ZEIGEL, 11. F., TYSILYLL, R. L., O’CONNOR, T. P:. TEETER, E., and ALLEN, B. V. (196G). Observations on the morphology of a murine leukemia virus (Kauscher) propagated in tissue cultures. Nat. Cancer Inst. Monogr. 22, 237-263.
49, 345-358
(1972)
of Mouse
Properties III. Electron
Microscopic
Appearance
Techniques MILAN
as well
V. NERMUT,’
Max-Planck-Znstitut
fiir
Leukemia
as Revealed
after
as Freeze-Drying
HERMANN
Virusforschung,
FRANK,
April
Conventional
Preparation
and Freeze-Etching WERNER
AND
Biologisch-Medizinische Accepted
Viruses
Abteilung,
SCHAFER Tcbingen,
Germany
17, 1972
Rauscher, Friend, and Gross strains of murine leukemia viruses (MuLV) were studied with negative staining, freeze-drying, and freeze-etching techniques. Negative staining of the virus particles with uranyl acetate (UA) and the combination of phosphotungstic acid (PTA) negative staining with freeze-drying showed that the virus particles are roughly spherical and are covered with “knobs” about 80 A in diameter which are weakly bound to the viral membrane. Freeze-etching of the virus particles confirmed the existence of the “knobs.” A diameter of about 1060 A was determined for the virus particles in freeze-dried preparations. Freeze-drying and shadowing of the viral cores liberated by Tween 80 and ether, ether alone or Triton X-100, revealed that their surfaces have a regular pattern of hexagonally arranged subunits with a diameter of about 60 8 and a center-to-center distance of about 75 A. The existence of pentons as well as the configuration of the shadows of freeze-dried cores suggested that the viral core possesses icosahedral symmetry. Negative staining of fixed virus particles resolved the core shell into two tracks, a fact which indicates the existence of two components. A smooth membranous layer underlying the layer of regularly arranged subunits wss observed in liberated cores. The diameter of the viral cores was about 800 h; as determined after freezedrying. The internal component of the core, the nucleoid, is a filamentous structure, which may possess helical symmetry. Considering the above described substractures, we have proposed a model of MULV.
leukemia viruses (MuLV) by the presence of at least seven different antigenic components (Schafer et al., 1972a,b). In order to obtain more information on the fine structures of MuLV’s, we used conventional procedures as well as such recently developed techniques as freeze-etching and negative staining followed by freeze-drying (Nermut and Frank, 1971). Several previously unknown substructures were thereby revealed.
INTRODUCTION
In the last decade several papers dealing with the fine structure of RNA-containing oncogenic viruses of the C type have appeared (see reviews of Bader, 1969; Vigier, 1970; Gross, 1970). The principal techniques employed were ultrathin sectioning and negative staining of intact and degraded virus particles. However, there remains considerable uncertainty as to the true shape and fine architecture of the surface and interior of the particles, the complexity of which is attested to, in the case of murine 1 Present cal Research,
address: National Institute Mill Hill, London NW
MATERIALS
@ 1972 by Academic Press, Inc. of reproduction in my form reserved.
METHODS
Viruses. Friend, Rauscher, and occasionally Gross murine leukemia virus were
for Medi7, England. 345
Copyright All rights
AND
346
NERMUT,
FRANK,
used. Friend virus was produced in STU, Rauscher virus in JLSV5, and Gross virus in rat thymus cell cultures. All were purified by ultracentrifugation followed by banding in a potassium citrate density gradient. The corresponding techniques have been described in previous communications of this series (Schafer et al., 1972a,b). Treatment of virus particles with Tween 80 and ether was performed as described by de Thb and O’Connor (1966). Samples for electron microscopy were removed after every step, i.e., after Tween, after addition of ether, and after density gradient centrifugation. Treatment with ether alone was performed as follows: 1 part of cold peroxide-free ether (Merck, Darmstadt, Germany) was added to 2 parts of concentrated virus suspension and shaken or stirred for 45 min at 4’. The ether and the water phases were separated by lowspeed centrifugation and the remaining ether was removed from the water phase by bubbling with nitrogen. The sample was then layered on a potassium citrate density gradient containing Ca2+ and Mg”+ ions (Lacour et al., 1970) and centrifuged in a Spinco SW 39 rotor at 30,000 rpm to equilibrium. The material banding at a density of about 1.26 g/ml was separated, diluted with Tris. Mg buffer and centrifuged at 22,000 rpm for 80 min in a Spinco rotor 40. The pellet was washed once again with the same buffer and then used for freeze-drying. Treatment with T&m X-100 (Roehm and Haas Co., Philadelphia, Pennsylvania) was done with a 0.02 % solution at 36” for 20 min. Samples for negative staining were removed immediately (washing was performed on the grids), and the remaining material was submitted to a rate zonal density gradient centrifugation on potassium citrate to remove debris and membranous structures. Washing was then performed as described for the ether treatment. Dimethyl sdfoxde (DMSO) (Merck, Darmstadt, Germany) was added to the virus suspension at a concentration of 20 % 30 min before freeze-drying. Trypsin (2 X tryst. 180 EU/m.l, Serva, Heidelberg, Germany) was dissolved in 1M/ 15 phosphate buffer, pH 7.4, as a 0.5 % solu-
AND
SCIldFER
tion and was diluted with distilled water 5-10 times before use. Pronase (lyophilized, Serva, Heidelberg, Germany) was used as a 0.2 % solution in phosphate-buffered saline pH 7.2 (PBS). Purified phospholipase C from Clostrklium welchii (Sigma Chemical Co., St. Louis, Missouri) was used in 0.25 mg/ml concentration in PBS. Snake venom from Ancistrockm piscivorus (Ross Allen’s Institute Inc., Florida) was used in a concentration of 1 mg/ml in PBS. Treatment with the enzymes as above was performed for a given time (see results) at 37” in a water bath. Bu.ers. Phosphate-buffered saline (PBS), pH 7.2, contained 0.7% NaCI, 0.31% Na2HP0, and 0.034% KH2P0,. Tris. Mg buffer contained 0.01 M Tris(hydroxymethyl)aminomethane with 0.001 M MgSO, (final pH was 6.7). Negative staining was done with 2 % phosphotungstic acid (PTA) pH 5.0, 6.0, or 7.2, or with 1% uranyl acetate (UA) pH 4.4, or with 3 % ammonium molybdate (AM), pH 5.3 or 6.7. Staining was done by first placing a drop of the material on the grid for adsorption and then applying the negative stain. Carbon-coated Formvar films were used throughout. Fixation. The material was fixed either with 5 % glutaraldehyde (GA) or 0.2 or 1.0 % osmium tetroxide. After the material had been adsorbed onto the grid, this was placed on a drop of the fixative for 10 min and washed with distilled water before negative staining. Freeze-drying. The grids containing the adsorbed material were carefully rinsed with volatile buffer (e.g., ammonium acetate) or double-distilled water, and, after the liquid had been drained off with filter paper, the grids were immediately frozen in liquid nitrogen. The frozen grids were then quickly transferred to a four-specimen stage of the Balzers freeze-etching apparatus BA 500 M (Balzers AG, Balzers, Liechtenstein) which was previously cooled to - 150” and dabbed with liquid Freon 22 in order to ensure an immediate good thermal contact between grid and stage. The grids were then gently nudged into the identations of the stage and
ELECTRON
MICROSCOPY
OF
secured by a cover plate. This last step was especially important because otherwise the light-weight grids frequently slid out of the stage during the drying process. The chamber was then evacuated and the temperature of thr specimen stage was raised to -8.5”. The microtome arm was simultaneously cooled to - 150’ and then moved over the specimen stage. After at least 20 min drying, the knife arm was removed and the grids were shadowed with platinum-carbon (Pt-C) at an angle of 3045”. In some experiments the shadow-casting was done from two directions. In practice, the right holder of the freeze-etching device (normally destinrd for carbon evaporation) was used as the second source of Pt-C and adjusted at approximately the same height and distance from the stage as the left one. The resulting angle between the two directions was about 80”. Freeze-drying of negatively stained specimens. The grids with adsorbed virus particles dvere washed as described above and placed on a drop of either PTA or AM. After 15 set the grids were removed, drained of excess stain, and immediately dipped in liquid nitrogen. Drying of the film usually required 3 or more seconds, so that the grid could conveniently be frozen before drying. Further procedures were the same as described for freeze-drying, except that the shadowing was omitted, and, before the chamber was aired, the temperature of the stage was raised to about +40” in order to avoid water condensation on t’hr preparation due to moisture in the air. A white coating composed of dried lamellalike PTA formed on the grids and must be carefully blown away in order to uncover the thin PTA layer on the supporting film. The adsorbed virus material is found predominantly in this layer. Freeze-etching was performed as described previously without the addition of any protective substances to the material before freezing (Nermut and Frank, 1971). dgar filtration was done according to Kellenberger and Arber (1957). Specimens were shadowed with Pt-C at the same angle employed when the preparations were freezedried.
MOUSE
LEUKEMIA
VIRUS
347
Electron microscopy. Specimens were observed with a Siemens Elmiskop I A electron microscope at 80 kV and a nominal magnification of 40,000 or 80,000 X. The real magnification was evaluated as described by Frank and Day (1970). RESULTS
Shape, Surface, and Size of MuLIP
Particles
Negative staining of the viruses with phosphotungstic acid (PTA) or ammonium molybdate (AM) revealed particles of various shapes (Fig. la). Some of the particles were empty, and many of them possessed characteristic tails. The surface of most of the particles was smooth, and only rarely were knoblike protrusions (henceforth to be referred to as knobs) visible on their periphery (Fig. lb). These knobs were 5&70 8 long. Negative staining with uranyl acetate (UA) produced rather surprising results. The virus particles now appeared as spheres of uniform size. Their surface was covered with knobs of about 80 A in diameter (Fig. 2). The knobs did not appear to be solid globules, but, instead resembled rings or clusters of smaller subunits. Their arrangement on the surface was not very regular, although hexons could occasionally be seen. Some particles were partially or entirely devoid of knobs. When the particles were subjected to negative staining with PTA combined with freeze-drying, they were predominantly round (Fig. 3a). On many of the particles one could detect knobs having a structure similar to those recognizable after conventional negative staining with UA (Fig. 3b). Specimensprepared by agar filtration were subjected to shadow-casting with Pt-C. This preparation produced flat particles displaying a prominent center, causing them to resemble a fried egg (Fig. 4). With this technique, however, knobs were not detectable. Freeze-dried and shadowrd virus particles cast long and, more important, often angular shadows, indicating that they were not ideal spheres (Fig. 5). In order to analyze this in 2 The term MuLV (murine leukemia virus) was used throughout, since the mouse leukemia virus strains Friend, Rauscher, and Gross delivered analogous elerbon microsropic results.
348
NERMUT,
FRANK,
ANI)
SCHAFER
The abbreviations PTA, UA, or AM indicate negative staining with, respectively, phosphotungstate, pH 6.0; many1 acetate, pH 4.4; ammonium molybdate, pH 6.7. FIG. 1. (a) Native virus particles (Rauscher) stained with PTA show a remarkable pleomorphism with tailed and broken particles. (b) One of the rare particles (Friend) showing knobs on the periphery after PTA staining. X 132,000. FIG. 2. The same suspension as in Fig. la (Rauscher), but stained with UA. The particles are uniformlyroundand covered with knobs. One particle (arrow) has no knobs. Scale line = 1000 1. X 132,000. FIG. 3. Virus particles (Friend) after PTA staining followed by freeze-drying. Note the absence of tails (compare with Fig. 1) and a somewhat smaller diameter (compare with Fig. 2). Knobs are seen on the surface (b). X132,000.
more detail, we shadowed freeze-dried particles from two directions. At least someof the particles revealed angular shadowssimilar to those cast by an icosahedron model (Fig. 6). It should be mentioned, however, that the angles were not as distinct as those observed on influenza virus (Nermut and Frank,
1971). No surface structures were detected on the freeze-dried particles because they were rather high and, therefore, electron dense. When specimens were freeze-dried and shadowed after treatment with Tween 80, one could observe more or less flattened
ELECTRON
MICROSCOPY
OF
membranous structures with knobs (loo-120 A in diameter) on their surface (Fig. 7). The possibility that these structures were empty viral envelopes is supported by the finding of liberated viral cores in the same Tween SOtreated preparations. Freeze-etching revealed round particles with knobs only on their periphery, the others apparently having been removed by the fracturing (Fig. 8). In these preparations, like those described in the preceding paragraph, the knobs had a diameter of about 120 A. The knobs were removed by the action of trypsin (3 hr), pronase (3 hr), and DMSO (Fig. 9) ; such particles had a similar morphology regardless of treatment. Removal of such protrusions from Rous sarcoma virus by a proteolytic enzyme has been described recently by Rifkin and Compans (1971). It is evident from the results described that negative staining with UA, freezedrying plus shadowing, and negative staining combined with freeze-drying produced the best preserved particles. Thus, these techniques were employed in investigation of the diameters of virus par$cles. The lowest average diameter, 1080 A, was found after freeze-drying and shadowing. Allowing for the thickness of the carbon metal film, one can estimate that the virts particles have a diameter of about 1060 A. This is in good %greement with the average diameter of 1060 A found by Bader et al. (1970) in thin sections. Much larger particles containing two cores were observed occasionally (Fig. lob). Particles treated by either negative staining with PTA followed by freeze-drying or negative staining with UA, were found to haye somewhat larger diameters (1300 and 1440 A, respect,ively) . Internal
Structure
of Virus
Particles
When the virus particles were fixed with GA or 0~0~ before PTA negative staining, the stain penetrated into the particles thus revealing someof their internal components. It is obvious, that the particles contained an internal body, “the core” (Fig. lOa); the more rarely observed larger particles have two cores (Fig. lob). In well preserved particles (Fig. 1Oa)the core is limited by a distinct
MOUSE
LEUKEMIA
VIRUS
349
shell, about 80 8 thick. This shell is often resolved into two separate tracks, 2030 A thick (Fig. lOc), which are not always parallel to each other (Fig. lOd, e). Occasionally, only one track was observed (Fig. lob, f). The type of fixation did not influence the appearance of the tracks. Definite substructures were not revealed inside the core shell by this preparation procedure. However, unfixed negatively stained (UA) particles which had been slightly damaged by various treatment’s (Tween-ether, Triton X-100, phospholipase C) showed not only a better preserved core shell with regular striations (Fig. ll), but also a filamentous structure in the interior which appeared somewhat thicker than the core shell and was often faintly striated (Fig. llc, d). This filamentous structure, which we designate as the nucleoid, appeared mostly as a ring (Fig. llb) and only sometimes as a spiral (Fig. llf). In few cases, if t,he nucleoid was not completely penetrated by stain, some parallel running strands became visible (Fig. 1lg small arrows), giving t’he impression t’hat it is a hollow sphere formed by a wound filament. Freeze-etching was a technique which we considered highly promising for studying the internal organization of the virus particles and, in particular, for revealing either the core surface or its internal filamentous component. The results obtained, however, were somewhat disappointing. In the rare cases where a superficial layer was removed from the particles, the exposed part was smooth (Fig. 12a, particles labeled B). We observed no particles where the core was broken, only particles where the core had been completely removed (Fig. 12, particles IEbeled C). In such cases elements SO-100 A in diameter occasionally remained on interior of the viral envelope. Some virus particles were completely removed, leaving only single knobs behind (Fig. 12b, particles labeled D). In subsequent experiments we tried to release the cores by treatment with Tweenether (de The and O’Connor, 1966), ether alone or various detergents (sodium dodecyl sulfate, sodium deoxycholate, Brij 35, Brij 58, Nonidet P-40, Triton X-100). Both conventional negative staining and freeze-dry-
Fms. 4-9.
X132,000.
ELECTRON
MICROSCOPY
OF
ing with shadowing were used to prepare the samples for electron microscopy. When Tween-ether treated viruses were freezedried, particles smaller in size than intact virus particles, often displaying a regular hexagonal pattern were found (Fig. 13a). We assume that they represent the viral cores. After citrate density gradient centrifugation of Tween-ether treated virus, they were found in the lower band (1.26 g/ml), but only in low concentration together with much debris. If the virus particles were treated with ether alone, the yield of cores in the lower band was significantly higher. Of the above-mentioned detergents, only Triton X-100 liberated intact cores in substantial numbers. A hexagonal arrangement of subunits was visible on the core surface if the cores were somewhat’ collapsed. These subunits, 60 A in diameter, had $ center-to-center distance of about 75-80 A. In a few cases pentons were detected, but we failed to find two pentons on the same core and thus could not establish the triangulation number of the core. In Fig. 13b such a subunit with 5-fold symmetry, indicated by its five nearest neighbors, is encircled, whereas in Fig. 13c one of the five neighboring six-coordinated subunits, i.e., a hexon, is marked. These two figures also show that the subunits are not solid globules but are composed of radially arranged “structure units.” The uncollapsed cores showed a long shadow which, in some cases, was more distinctly angular than that of the complete virus particles, however, the surface substructure was not revealed (Fig. 14). Apparently, these cores were too high to permit resolution of the substructure. The usually FIG. 4. Virus
MOUSE
LEUKEMIA
351
VIRUS
angular form of their shadow, even after shadowing from two directions, was considered by us to be more similar to the shadow cast by an icosahedron model (Fig. 15) than the shadows cast by the whole particle, thus suggesting a regular symmetry. Negative staining of liberated cores revealed either a striation of their shell (Fig. 16a) or subunits on the periphery of the cores (Figs. 16b and c). On some cores the subunits were seen to be situated on a smooth membranous structure (Fig. 16c and 4. The core diameter was determined for fixed intact virus particles negatively stained with PTA and for liberated cores which had been freeze-dried and shadowed. The mean diameters ranged between 740 and 800 h. DISCUSSION
The shape of MuLV particles has been determined to be spherical both in ultrathin sections and after negative staining of fixed particles (Bernhard, 1958; Dalton, 1962; de Harven and Friend, 1964; de The and O’Connor, 1966). However, if the native virus particles are stained negatively with PTA, many of them display a taillike protrusion (Dalton et al., 1962). We found that no tails were formed when PTA negative staining is followed by freeze-drying or when UA was used as negative stain. This is presumably due more to the stabilizing effect of UA on membrane lipids than to its low pH, as “tails” were seen after very acid PTA (pH 4.4), too. In addition, UA clearly revealed projections (“knobs”) at the surface of the particles. After PTA negative staining these knobs were seen only rarely, and then exclu-
particles (Gross) prepared by agar filtration and shadowed with 45”. Scale line = 1000 A. FIG. 5. Freeze-dried particles (Friend) shadowed under the same conditions as in FIG. 6. (a and c) Virus particles (Friend) after freeze-drying and two-directional icosahedron model correspondingly illuminated from two directions showing shadows particle ones. FIG. 7. Virus particles (Friend) treated with Tween 80 for 30 min, freeze-dried particle (A) is full and knobs cannot be seen. The others are more or less emptied spondingly improved visibility of the knobs. FIG. 8. Freeze-etched particles (Friend) showing knobs on the periphery. FIG. 9 Particles (Friend) treated with 20% DMSO for 30 min and freeze-etched face. The knobs were completely removed.
Pt/C
at an angle
of
about
Fig. 4. shadowing. (b) An similar to the virus and shadowed. (B-D) with
show
a smooth
One corre-
sur-
Fro. 10. Virus particles (a, c, d, f Rauscher; b, e, Friend) fixed with lo/o 0~04 (a, d, e) or 5vi GA (k, c, f) and stained negatively with PTA (a-e) or AM (f). T@ particle in b contains two cores. The core shell (arrows) is seen either as a compact structure about 80 A thick (a), or as a thin track (b and f), or as two parallel tracks (c) or nonparallel tracks (d and e). Scale line in Fig. 10a = 1000 A. X220,000. FIG. 11. Partially opened virus particles (a, b, d, f, g, Friend; c, e, Rauscher) revealing internal organization after negative staining with UA. A, viral envelope; B, core shell; C, internal filament (nucleoid). Core shell is usually striated. Particles were treated with snake venom (a), Triton X-100 (b), phospholipase C and neuraminidase followed by Tween and ether (c, e), Tween and ether (d, g) and nontreated (f). X220,000.
sively on the periphery of the particles. Still better preservation of the knobs is achieved by PTA negative staining combined with freeze-drying. This indicates that the normal
drying process either removes the knobs or deforms them in such a way as to render them unrecognizable. That, the knobs are indeed real struct,ures, not artifacts, could be
ELECTRON
MICROSCOPY
OF MOUSE
clearly demonstrated by freeze-drying and shadowing as well as by freeze-etching of the virus particles. The lability of the knobs may explain why they were not always detected by other authors using conventional preparation techniques. Nowinski et al., (1970) for example, concluded that MuLV does not possess “surface projections.” Parsons (1963)) however, described slender “outer spikes” on Gross leukemia virus particles, and Zeigel and Rauscher (1964) at times found peripheral “knobs” or spikes. Freeze-drying followed by shadowing suggested that the particles are not true spheres. Their shadows are angular; however, their corners are not very distinct, i.e., they are “soft.” In contrast, the isolated cores often displayed shadows that were distinct and more angular than the previous ones and which were similar to those cast by an icosahedron model. Some evidence indicative of a polygonal shape for the cores or the virus particles have been published previously (Dalton et al., 1962; Zeigel and Rauscher, 1964; de The and O’Connor, 1966). Our studies support the possibility that the virus cores have cubical symmetry: the cores are covered by small globular subunits in a closeand very regular hexagonal arrangement. These globules often show radially arranged “structure units.” The occasional finding of a penton (Fig. 13b) suggeststhat the core surface has icosahedral symmetry. The icosahedral shape of the core may determine the shape of the whole particle. However, the corners of t.he particles are indistinct, probably due to the plasticity of the viral envelope. The demonstration of the hexagonal pattern by techniques other than freezedrying and shadowing seemsto be very difficult. Some indications for the existencz of hexagonally arranged subunits 50-60 A in size were obtained earlier by Zeigel and Rauscher (1964), but at this time it was not yet known “whether these patterns represent viral coat plus knobs (surface) or are internal subsurface structures.” Only very rarely did we find this structure with negative staining, probably because most of the cores were penetrated by stain and only among the unpenrtrated ones were a few found that were
LEUKEMIA
VIRUS
353
stained only from one side, thus revealing the hexagonal pattern. A further element is apparently present in the core shell below the layer of regularly arranged subunits. We supposethat the two small tracks, seen sometimes instead of the striated layer after negative staining with PTA, originate from the core shell (striated layer). The tracks show branching, thus indicating the existence of two components. Our observations on isolated cores suggest that the outer track represents the subunit layer, whereas the inner track seemsto be a smooth membrane. Both components can be distinguished in Figs. 16b-d, where the subunit layer was apparently partially removed. Some information on the material situated inside the core has been presented in earlier publications. In ultrathin sections several authors described a distinct ring underneath the core shell which could represent a helical nucleocapsid (de The and O’Connor, 1966; Zeigel et al., 1966; de Tkaczevski et al., 1968). Zeigel and Rauscher (1964) liberated strands with helical pattern from particles of the Rauscher virus by treatment with distilled water. In this work both the core shell and a filamentous nucleoid structure could be revealed, but only by the use of UA for negative staining. The striation of these filaments may suggest a helical symmetry. However, the real arrangement of the filamentous nucleoid cannot yet be determined. The possibility exists, that the various appearance of the internal structures (rings, spirals, possibly hollow spheres) depends upon the degree of penetration of the strain. Alternatively, the rings or spirals could represent degradation products of a hollow sphere or all three forms could represent different stagesof viral maturation. Nowinski et al. (1970) and Sarkar et al. (1971) proposed that the nucleoid exists as a hollow sphere formed by supercoiling of helical strands. This is probably correct, but the structure considered by those authors (at least in the case of Rauscher leukemia virus) is apparently identical with the core shell described by us. After negative staining with PTA, which these authors used cxclusively, the core shell, but never the inner filamentous element, was recognizable in our experiments.
ELECTRON
MICROSCOPY
OF MOUSE
cl dl FIG. 17. Schematic representation of the probable fracture planes found after freeze-etching of particles. (a) Removal of surface knobs. (b) Splitting of the viral membrane. (c) Removal of the core except for some subunits of the core shell lying on the internal face of the viral membrane. (d) Removal of the whole particle except for some knobs (see discussion).
Recently, Luftig and Kilham (1971) reported differential responses of Rauscher leukemia virus particles to staining with UA and PTA which agreed reasonably well with our findings. However, their different conclusions regarding structural configuration of the virus particle, e.g., failure to observe surface knobs, different size measurements and a whorled internal substructure, appear relat.ed partly to the condition of the starting material and partly to different interpretation of the structure observed. The virus examined by these workers was twice frozen and thawed, a procedure we have found detrimental to virus structure and consequently avoided in our studies. Indeed, freezing may not only remove knobs, but
LEUKEMIA
VIRUS
355
also could so disrupt the internal structure that one could not readily differentiate between the core shell and the inner filamentous component. It seems likely that such a situation prompted these authors to term as “nucleoid” this entire, complex core structure which we have here described. We could clearly demonstrate the core shell only after UA staining of slightly damaged but unfixed virus particles. The morphology of the freeze-etched particIes deserves special consideration. The observed fracture patterns are summarized in Fig. 17 and documented in Fig. 12. The origin of the A-labeled particles is well understood. The “knobs” seen on the periphery of the particles were uncovered by the etching process. However, the failure of the fracturing process to expose the hexagonal globular surface of the cores or their interior cannot be explained easily. The exposed surface of the B-labeled particles looks smooth, an observation that cannot be accounted for by the lack of resolut\on. The hexagonal globules are about 60 A in diameter and are seen clearly after freeze-drying. In freezeetched preparations where a fine carbon replica (without any organic material) is examined, the resolution is even better. Thus we assume that the outer membrane was split in the middle, so that the core surface was still covered by the lower part of it (Fig. 17b). Such an explanation is in agreement with the data published by Branton (1966) and Pinto da Silva and Branton
FIG. 12. Virus particles (Friend) prepared by freeze-etching. In particles marked A only the knobs were removed from the surface. In B particles apart of the outer membrane has been split off in addition, in C particles only a part of the outer membrane remains, and in D the whole particle wm removed. A schematic re resentation of the different fracture planes is given in Fig. 17. X 132,000. Scale line in Fig. 12a = 1000 % . FIG. 13. Virus cores (Friend) released by Tween-ether treatment. Freeze-drying and Pt/C shadowing. (a) Cores which are somewhat collapsed reveal a regular pattern of hexagonally arranged subunits. (b) A penton (encircled) found on the corner of a core. (c) A neighboring hexon. (a) X 132,000; (b, c) x350,000. FIG. 14. Uncollapsed Tween-ether cores (Friend) do not reveal any substructure but csst an angular shadow after freeze-drying. X132,000. FIG. 15. (a and c) Uncollapsed cores (Friend) (Triton X-100) after freeze-drying with two-directional shadowing. The shadows are compared with those of an icosahedron model (b). X 132,000. FIG. 16. Isolated cores (Friend), stained negatively with PTA (a, b), UA (c) and AM (d), sometimes showed a striation of the core shell (a), ~EJshown in Fig. 11. The shells were sometimes dissolved (b-d), revealing a smooth membrane (black arrows) covered with globules in certain spots (white arrows). The cores were liberated by the action of snake venom (a), Triton X-100 (b, c) and et’her (d). X220,000. Scale line = 1000 ii.
NERMUT,
356
FRANK,
AND SCHAFER
membrane
Y
viral
envelope
nucleoid ~nucleocopsid?)
1060;1.-----FIG.
18.
Schematic representation
(1970). A similar phenomenon has also, been observed in influenza virus by BLchi et al. (1969) and Nermunt and Frank (1971). When the virus particle was fractured horizontally, the whole core was apparently removed, so that only “shallow dishes” were found (C in Fig. 12). In such casesthe internal face of the outer membrane was revealed. Small elements seen upon it could then represent some of the core subunits which were left behind. The structures labeled D in Fig. 12b are very similar in appearance to those labeled C, but they do not cast any shadow. In this casethe whole virus particle has been removed, and the few tiny granules left behind are probably outer “knobs” that remained embedded in the ice and were then freed by etching. Our ideas about the fine structural organization of MuLV are represented in Fig. 18, which shows a transverse section of a virion. The main part of the virion is the “virus core,” presumably possessingcubical symmetry. It contains a strand, called “nucleoid,” as its chemical nature has not yet been sufficiently analyzed to use the term nucleocapsid. This structure is covered by the “core shell,” which consists of two structurgl elements: (1) a “core membrane” about 30 A thick; (2) a layer of globular or ringlike
1 of a MuL virion.
“morphological subunits” of about 60 A in diameter forming a close hexagonal pattern. The “viral core” is covered by the “viral envelope” gonsisting of a “viral membrane” (about 80 A thick) and the “knobs” (about 80 8 in diameter). Our findings in certain regards are similar to the observations made by de ThB and O’Connor (1966)) but our interpretation differs in several points. Their “outer shell” corresponds to our “core shell” and their ‘(inner shell” to our “nucleoid.” Padgett and Levine (1966) draw a scheme of Rauscher virus which also is not greatly different from our concept. However, this was done on the basisof experiments with snake venom preparations, which were later shown to be contaminated with herpeslike particles (Monroe et al., 1968). Similar data about the fine structure of avian myeloblastosis virus have been published recently by Gelderblom et al. (1972) suggesting that these structural properties are common to many, if not all, C-type particles. At the present stage of the investigations, it cannot be decided with which of the viral structural elements the various MuLV antigens are correlated. Our current efforts to isolate constituents of viral envelopes and
ELECTRON
MICROSCOPY
OF MOUSE
cores and to determine their antigenic composition should help to solve this problem. ACKNOWLEDGMENTS The authors wish to thank Mrs. U. Michel and Miss A. Kleih for skillful technical assistance, Mrs. I. Thiering, Miss R. Szabo, and Mr. G. Berger for preparation of micrographs and Dr. R. L. Witter for kind revision of the manuscript.
J. P. (1969). RNA tumor viruses. In “The Biochemistry of Viruses” (H. B. Levy, ed.), pp. 293-328. Dekker, New York. B~DER, J. P., BROWN, N. R., and BADER, A. V. (1970). Characteristics of cores of avian leukosarcoma viruses. Virology 41,718-728. BXCHI, T., GERHARD, W., LINDENMANN, J., and M~;.HLETHALER, K. (1969). Morphogenesis of influenza A virus in Ehrlich ascites tumor cells as revealed by thin-sectioning and freeze-etching. J. Viral. 4,769-776. BERNHARD, W. (1958). Electron microscopy of tumor cells and tumor viruses. Cancer Res. 18, 491-509. BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. Nat. A cad. Sci. U.S.A. 55, 1048-1056. DALTON, A. J. (1962). Micromorphology of murine tumor viruses and of affected cells. Fed. PTOC. Fed. Amer. Sot. Exp. Biol. 21,936-941. DALTON, A. J., HAGUENAU, F., and MOLONEY, J. B. (1962). Morphology of particles associated with murine leukemia as revealed by negative staining: Preliminary report. J. Nat. Cancer Inst. 29, 1177-1179. DE HARVEN, E., and FRIEND, C. (1964). Structure of virus particles partially purified from the blood of leukemia mice. Virology 23,119-124. DE TI&, G., and O’CONNOR, T. E. (1966). Structure of a murine leukemia virus after disruption with Tween-ether and comparison with two myxoviruses. Virology 28,713-728. DE TKACZEVSKI, L., DE HARVEN, E., and FRIEND, C. (1968). Structure and leukemogenic activity of a murine leukemia virus. J. Viral. 2,365-375. FRANK, H., and DAY, L. A. (1970). Electron microscopic observations on fd bacteriophage, its alkali denaturation products and its DNA. Virology 42, 144-154. GELDERBLOM, H., BAUER, H., BOLOGNESI, D. P., and FRANK, H. (1972). Morphogenese und Aufbau von RNS-Tumorviren: Elektronenoptische Untersuchungen an Viruspartikeln vom C-Typ. BADER,
Zentralbl. Hyg. Abt.
Bakteriol. Parasitenk. 1 Orig. 220, 79-90.
Infektionskr.
VIRUS
357
L. (1970). “Oncogenic Viruses,” 2nd. Pergamon, Oxford. KELLENBERGER, E., and ARBER, W. (1957). Electron microscopical studies of phage multiplications. I. A method for quantitative analysis of particle suspensions. Virology 3,245-255. LACOUR, F., FOURCADE, A., UERGER, C., and DELAIN, E. (1970). Coiled structure of the nucleocapsid of avian myeloblasosis virus. J. Gen.
GROSS,
Viral. LUFTIG,
REFERENCES
LEUKEMIA
9, 89-92.
R. B., and KILHAM, S. S. (1971). An electron microscope study of Rauscher leukemia virus. Virology 46, 277-297. MONROE, J. H., SHIBLEY, G. P., SCHIDLOVSKY, G., NAKAI, T., HOWATSON, A. F., WIVEL, N. W., and O’CONNOR, T. E. (1968). Action of snake venom on Rauscher virus. J. Nat. Cancer Inst. 40, 135;145. NERMUT, M. V., and FRANK, H. (1971). Fine structure of influenza AP (Singapore) as revealed by negative staining, freeze-drying and freezeetching. J. Gen. Viral. 10,37-51. NOWINSKI, R. E., OLD, L. J., SARKAR, N. II., and MOORE, D. H. (1970). Common properties of the oncogenic RNA viruses (Oncorna viruses). Virology 42, 1152-1157. PADGETT, F., and LEVINE, A. S. (1966). Fine structure of the Rauscher leukemia virus as revealed by incubation in snake venom. Virology 30, 623430. PARSONS, D. F. (1963). Structure of the Gross leukemia virus. J. Nat. Cancer Inst. 30,569-583. PINTO D.* SILVA, P., and BRANTON, D. (1970). Membrane splitting in freeze-et.ching. Covalently bound ferritin as a membrane marker. J. Cell Biol. 45,598-605. RIFKIN, D. B., and COMPANS,
R. W. (1971). Identification of the spike proteins of Rous sarcoma virus. Virology 46,485-489. SARXAR, N. H., NOWINSKI, R. C., and MOORE, D. H. (1971). Helical nucleocapsid structure of the oncogenic ribonucleic acid viruses (Oncorna viruses). J. Viral. 8,564-572. SCHSFER, W., FISCHINGER, P. J., LANGE, J., and PISTER, L. (1972a). Properties of mouse leukemia virus. I. Characterization of various antisera and serological identification of viral components. Virology47,197-209. SCHAFER, W., LANGE, J., FISCHINGER, P. J., FRANK H., BOLOGNESI, D. P., and PISTER, L. (1972b). Properties of mouse leukemia viruses. II. Isolation of viral components. Virology 47, 210-228. VIGIER, P. (1970). RNA oncogenic viruses: Structure, replication, and oncogenicity. Progr. Med. Viral. 12, 240-283. ZEIGEL, R. F., and RAUSCHER, F. J. (1964). Elec-
358
NERMUT,
FRANK,
tron microscopic and bioassay studies on a murine leukemia virus (Itauscher). I. Effect,s of physicochemical treatments on the morphology and biological activity of the virus. J. 1Vat. Cancer Imt. 32, 1277-1307.
AND
SCHAFER
ZEIGEL, 11. F., TYSILYLL, R. L., O’CONNOR, T. P:. TEETER, E., and ALLEN, B. V. (196G). Observations on the morphology of a murine leukemia virus (Kauscher) propagated in tissue cultures. Nat. Cancer Inst. Monogr. 22, 237-263.