Purification and fine structure of Kilham's rat virus

EXPERIMENTAL AND MOLECULAR PATHOLOGY 4, 130-140 (1965)

Purification and Fine Structure of Kilham’s Rat Virus

C&AR VASQUEZ AND CARLOS BRAILOVSKY

Institut de Recherches SW le Cancer, Villejuif (Seine), France

Received October 12, 1964

The Kilham rat virus (K-rat or RV virus), isolated by Kilham and Olivier (1959) from metastasizing sarcomas of the rat liver associated with Cysticercus fascioZaris,

was described as producing no apparent illness in its natural host. Later (1961a, b) Kilham reported that the virus, widely spread as a latent infection in normal rats, produced an acute illness and death within 3-6 days of the intracerebral inoculation of suckling hamsters. The animals which survived the acute phase became mongoloid dwarfs with deformities of the teeth (Baer and Kilham, 1962a, b). In spite of the fact that the K-rat virus had a number of properties in common with the polyoma virus, it has been shown experimentally to be distinctly different. No neoplasms were induced in animals inoculated with this agent, and salivary glands, cultivated in vitro,

reacted quite differently to both viruses (Dawe et al., 1961). Attempts to provoke neoplastic transformation in newborn Mastomys inoculated

with the same virus were unsuccessful (Rabson et al., 1961). However, the animals deveIoped an acute fatal disease histopathologically characterized by Feulgen-positive intranuclear inclusions in many organs and tissues. These inclusions suggested the DNA nature of the virus.

Electron microscopic studies were carried out simultaneously by Dalton et al.

(1963) and Bernhard et al. ( 1963). Dalton et al. showed the presence of small electron-dense particles in thin sections of lymphnodes, spleen, liver, and kidney from hamsters infected with the virus. Bernhard et al. studied cultures of rat (AT-strain) and hamster (BHK-strain) fibroblasts and were able to show not only characteristic morphologic changes in the nuclei but also the presence of naked virus particles (Fig. I). These particles, 150-180 A in diameter, were either dense or revealed circular profiles (Fig. lb). Both groups of authors concluded that it was impossible to dis- tinguish the K-rat virus from the closely related H-virus group (‘Chandra and Toolan, 1961; Portella, 1963; Toolan et al., 1964) since they were identical in size and shape. A more recent work by Payne et al. (1964) deals with a similar very small DNA rat virus called X14, which was found to have the same serotype as Kilham’s rat virus and Toolan’s HS-virus. On negatively stained preparations, this agent had a mean diameter of 22 mu.

Further electron microscopic studies with purified K-rat virus particles were made by Breese et al. (1964) in an attempt to elucidate more completely their fine structure. Using the negative stain technique, they found particles morphologically similar to those seen in thin sections, but the number of capsomeres and the symmetry of the virus were not determined and reconstruction of the capsid was not made.

In the present work we have attempted a new approach to solve these problems.

130

KILHAM’S RAT VIRUS

F ‘IG. 1. (a) Segment of the nucleus and cytoplasm of a virus-infected hamster fibroblast Vii-1 us particles; Chr., chromatin; M, nuclear membrane; r, ribosomes. It appears from this (

tror 1 micrograph that the virus particles have approximatively the size of ribosomes. X 60,000.

Ma ny “empty” virus particles in the nucleus. X 90,000.

L Gus culture. The stock of K-rat virus was obtained from kidneys of newb han nsters which had been inoculated at the age of 24 hours and killed on the 5th d

MATERIALS AND METHODS

v, elec-

(b)

lay.

132 &AR VASQUEZ AND CARLOS BRAILOVSKY

The tissue was ground in a mortar and diluted to 10% in Stoker’s medium. The mixture was frozen and thawed 3 times and centrifuged at 3000 rpm for 15 minutes; the supernatant was stored at -20°C. The virus was inoculated at 1.2 X IO* plaque- forming units (PFU) per Roux bottle of confluent rat embryo cells. When the first signs of CPE were detectable, the growth was stopped by freezing at -20°C. The thermally disrupted cell suspension was centrifuged at 2500 rpm for 15 minutes, and the pellet was retained for purification.

V~YUS purification. A preliminary isolation of the nuclei of the infected cells was carried out using the technique described by Zalta et al. (1962) and later modified by Hubert et al. (1962). In the last step, the nuclear pellet was retained and the preparation was observed in the optical microscope for possible contamination. The nuclei were disrupted in distilled water by means of ultrasonication (1 megacycle, 200 W for 1 minute). The material was digested for 1 hour at 37°C by adding 0.2 ml of DNAse (0.1% ) per 5 cc of nuclear suspension in order to remove the DNA. The solution was then centrifuged in a Spinco preparative centrifuge (swinging bucket rotor SW39) for 16 hours at 35,000 rpm and 5°C in a preformed density gradient of potassium tartrate (4). The samples of droplets were collected by puncture of the bottom of the tubes.

The infectivity of the fractions was tested on secundary cultures of rat embryo cells (1.5 X 10F cells per Falcon dish) grown in Stoker’s medium with 10% calf serum and 1% penicillin-streptomycin. When the cultures were confluent after about 24 hours, they received 0.5 ml of the fraction to be tested. After 2 hours of incubation an agar layer was added. The cultures remained 3 days at 37°C in the presence of 5% COa. After addition of a 0.1% solution of neutral red, the plaques became clearly visible the 4th culture day.

It could thus be shown that the various fractions we used for our electron microscopic study contained between 1 X lo5 and 1.2 X 10s PFU per milliliter.

Electron microscopy. Droplets of samples containing the virus were placed on grids covered with carbon and Formvar films. The grids were washed in distilled water and stained with 2% potassium phosphotungstate adjusted to pH 7.3 with NaOH. They were examined immediately afterward with a Siemens Elmiskope I microscope, equipped with double condenser illumination, at magnifications of 40,000-80,000.

In order to find out the best method of visualizing the capsomeres, the usual con- centration of phosphotungstate was decreased or increased (l--4$&) and used at different pH’s (3, 6, and 9). Since the images of the virus particles were not improved, a pretreatment with ultrasonication was tried, but without success. Negative staining was also carried out with uranyl acetate at pH 4, but no advantage was found com- pared with phosphotungstate. Attempts to disrupt the capsomeres in urea of high molarity were made, but in no case was it possible to break the capsid. Treatment in a IO M urea solution for as long as 24 hours had no visible action on the capsid.

RESULTS

Virus purification. The method employed for virus purification was remarkably successful. During the purification of the nuclear fraction, the virus remains enclosed in the nuclear envelope. Thus the cytoplasm can be removed without adsorption of

mi xny virus particles which could easily be lost. Neither the weak ultrasonication US to disrupt the nuclear membrane nor the DNAse employed afterward to digest t ch romatin damaged the virus.

KILHAM’S RAT VIRUS 133

;e d :he

Samples of the virus were studied both with shadow-casting and negative staini .ng

FIG. 2. Low magnification of the Kilham rat virus after purification. Platinum shadowing x 30,000.


(F Vi1

of

‘igs. 2 and 3). The preparation was so pure that on many grids a great number us particles were observed with only occasional scattered cellular debris. The virus fine structure. Because of its very small size, the symmetry and structt the virus were difficult to analyze. In typical preparations two classes of vir

of

n-e ‘US

FIG. 3. Low magnification of the virus stained with phosphotungstic acid (PTA). Note the presence of intact and hollow particles. x 120,000.


particles were present: intact and hollow ones (Figs. 3 and 4). In both classes, two distinct forms were observed: one with visible capsomeres along the peripheral profile of the particle and another without this particular feature. All the particles had the same diameter, which was estimated to be 180 k 15 A. In the hollow particles, the diameter of the core was about 120 A.

FIG. 4. Groups of hollow and intact virus particles. Capsomeres are difficult to see. X 240,000.

The shape of the capsids appeared angular in profile. As can be seen in Fig. 5, some of these profiles can be interpreted as hexagons, but the general impression is that they are not perfect polyhedrons but rather that they are deformed by concavities and protuberances. Some of the reversed contrast pictures suggest that the edges of


the hexagons are actually concave (Fig. 6). Capsomeres protruded along the border of the polyhedral periphery and were specially marked in the vertexes (see arrows, Fig. 6). The capsomeres could be counted on the periphery of selected particles. In some cases, evidence for 9 or 10 peripheral capsomers was found (Figs. 5 and 6))

FIG. 5. (a) Reverse contrast. Some profiles of viruses appear angular. It is possible to count

9 or 10 peripherical capsomeres. (b) Two reverse prints of one virus particle (right) showing the presence of one capsomere apparently surrounded by six other. X 440,000.

while in others it was possible to see clearly only a few. In addition, it appeared that the axes of the capsomeres corresponded to the radii of the virion and that they were thus tilted in relation to each other (Fig. 6a).

The symmetry of the capsomeres was difficult to establish. In reversed prints the


capsomeres were more clearly seen than on normal prints, and it seemed possible to observe both 5-fold and 3-fold symmetry. If selected particles were studied it was possible to see triangular patterns over the surface of the capsid (Figs. 5 and 6).

The approximate dimensions of the capsomeres are 30 A in length and 20 A in width, with an apparent groove between them of about 10 A. No evidence of hollow capsomeres was obtained.

FIG. 6. (a) Negative print on which it seems possible to show polyhedral shapes deformed by knobs and concavities. Sometimes triangulation of the capsomeres may be found (arrows). In one

particle (high magnification in the right corner) the tilting of the peripherical capsomeres is visible.

(b) Sixfold position of a model where it is not possible to count the peripherical capsomeres since there is a superposition effect. (c) Fivefold position of the same model where the peripheral

capsomeres are more distinctly visible. In both cases the tilting of the capsomeres, lying along the

radii of the virion, make it difficult to observe the points of symmetry.


DISCUSSION

From these studies on the fine structure of the capsid of Kilham’s rat virus, some facts suggest the type of symmetrical arrangement of the capsomeres. The rat-K virus is a naked virus of a uniform, small size (180 ii in diameter) and is composed of a capsid and a core. The capsid is roughly hexagonal in shape and has concavities and protuberances on its periphery. The more or less hexagonal shape of the virus indi- cates that it might have a deltahedron structure. The pattern of a typical icosahedron with P = 1 according to Caspar and Klug (1962) and a triangulation number of Tl or T4 does not correspond to the profiles we were able to see. The only deltahedron which could fit was the lowest number of the second class, P = 3 with a triangulation number of T = 3 or T = 12. This deltahedron with icosahedral symmetry is a pentagonal dodecahedron with pentagonal pyramids placed on each face. We can omit, as the least favorable possibility, the deltahedron with T = 12, since the number of capsomeres must be in the order of 122, which is too high to fit with our observations. The T = 3 solution seems the best. This deltahedron has 60 identical triangular faces, 12 vertexes on the S-fold axes, and 20 vertexes in the 6-fold axes.

According to our findings it is possible to detect on the surface of the virus a symmetrical arrangement of groups of capsomeres surrounded by six or one surrounded by five. In some perspectives it is possible to see that adjoining groups have a common edge. As we have previously concluded, the capsomeres can be arranged in symmetrical triangular facets. It is possible to construct a model if the base is the pentagonal dodecahedron T = 3 and columnar units, which represent the capsomeres, are used; if one of such capsomeres is put in each of the 32 vertexes of the deltahedron, we obtain a model which appears as an approximation to the virus studied (Fig. 6).

In both the virus and the model the symmetrical array of capsomeres is the same, and approximately the same hexagonal profile can be observed.

If we look at the model in the S-fold position (Fig. 6c) it is possible to count up to ten capsomeres on the periphery, as we can do in some of the virus particles. But on the model in the 6-fold position we are not able to count any capsomeres (Fig. 6b). This may explain why we cannot count the capsomeres on all of the virus particles and why their exact number is difficult to evaluate. Since the axes of the capsomeres lie along the radii of the virion, they are tilted in respect to each other, and the number of visible capsomeres is dependent upon the orientation of the virus particle.

The most likely hypothesis is that the K-rat virus is a pentagonal dodecahedron with 3 2 capsomeres.

We can discard the 12 capsomeres solution for two main reasons: first, we observe in the K-rat virus a 3-fold symmetrical arrangement of the capsomeres. This arrangement cannot be achieved with 12 capsomeres in icosahedral symmetry. Second, the size of the capsomeres (20 A) is so small that 12 of them would not cover the surface of the capsid. The phage +X174 (Hall et al., 1959; Melnick et al., 1963; Tromans and Horne, 1961) is of the same size as the K-rat virus and has 12 capsomeres, but each capsomere is 70 ii in diameter.

What about the possibility of 42 capsomeres? The K-rat virus is considerably smaller than the viruses of the papova group (see review of Horne and Wildy, 1963). In this group, 12 and more capsomeres can be easily counted in the periphery and as


many as 26 can be seen on one side of the particle. We can count a maximum of 9-10 capsomeres at the periphery of our agent, and less than 16 capsomeres are visible on anyone side of the particle. These reasons lead us to exclude the presence of 42 capsomeres.

Up to now, three groups of investigators have indicated the existence of viruses (all are RNA-viruses) with 32 capsomeres arranged in icosahedral symmetry. Huxley and Zubay (1960) and Nixon and Gibbs (1960) simultaneously interpreted the fine structure of the turnip yellow mosaic virus. Huxley and Zubay thought the capsomeres to be arranged on or near the vertexes of either a pentagonal dodecahedron or a rhombic triocontrahedron, and found that both possibilities agree with the studied particles. Nixon and Gibbs suggested a model with a regular icosahedral symmetry; both investigators reconstructed the virus; ping-pong or tennis balls were used to represent the hollow capsomeres of 70 J% diameter. Later, Mayor (1964), in a study of the picornavirus group (Melnick et al., 1963), showed rhombic-shaped sets of capsomeres, which obviously indicated a rhombic triocontrahedron form. Our interpretation of the fine structure of the K-rat virus agrees in part with the studies of Huxley and Zubay. Our model differs significantly in that we used columnar and not round capsomeres and thus introduced the phenomenon of tilting which is not shown spherical capsomeres.

The roughly hexagonal profile with concavities and protuberances, the constant number of peripherical capsomeres, and the evidence of an icosahedral symmetry of 5:3 : 2 with hexagonal sets of capsomeres having common edges are facts which are in agreement with a pentagonal dodecahedron interpretation. In this polyhedron, each of the vertexes is occupied by elongated capsomeres, but it is impossible to determine if they are pentameres and hexameres to meet the mathematical requirement of 60T structural units.

The existence of a capsid with a triangulation number of T = 3 and 32 capsomeres probably forms a protective coat with virtually no gaps, and makes an especially strong protective shell for the viral nucleic acid.

The K-rat virus is the first DNA virus for which such a shape and symmetry has been tentatively proposed. We can presume that the capsid of the closely related H-virus group may present a similar fine structure.

SUMMARY

Kilham’s rat virus has been purified and studied with the negative stain method. Its ultrastruc-

tural analysis is discussed and an interpretation of its shape and symmetry is tentatively proposed. The virus was found to have a mean diameter of 180 A. It has an angular profile and seems to

be roughly hexagonal, with concavities and protuberances. The number of peripherical capsomeres

does not exceed 10. The findings can be explained with a model representing a pentagonal dodeca-

hedron. The capsomeres measure approximately 30 A in length and 20A in width. Their number is likely to be 32, thus forming a protective coat with very small gaps.

ACKNOWLEDGMENTS

We wish to thank M. Alain Niveleau and Danielle Boullier for their technical assistance, Dr. Emma Shelton for the correction of the manuscript, and Professor P. Tournier and Dr. W. Bern-

hard for their stimulating discussion and critical remarks. We also express our gratitude to Funda-

leu, Buenos Aires, and the Mutuelle de 1’Education Nationale, Paris, for their generous financial aid. One of us (C. Brailovsky) is a research fellow of the University of Buenos-Aires.


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Purification and fine structure of Kilham's rat virus