Partial purification and some properties of tobacco etch virus induced intranuclear inclusions

61, 200-209

VIROLOGY

Partial

(1974)

Purification

and Some Induced

HJALMAR Plant

Pathology

KNUHTSEN, Department,

Plant

Properties

lntranuclear ERNEST Virus

May

Etch Virus

Inclusions’

HIEBERT,

Laboratory,

Accepted

of Tobacco

University

AND of Florida,

D. E. PURCIFULL Gainesville,

Florida

32611

15, 1974

Tobacco etch virus (TEV) induced intranuclear inclusions (NI) were isolated from tissue of Datura stramonium L. by homogenization in buffer followed by Triton X-100 detergent treatment and by a combination of successive low speed and sucrose density-gradient centrifugations. The isolated NI, which appeared similar to those seen in situ, were rectangular with a striated surface. The NI were readily dissociated in sodium dodecyl sulfate (SDS), 6 M urea and 67% formic or acetic acids. Spectral analysis of dissociated NI revealed a typical protein spectrum with a maximum at 277 nm and a minimum at 250 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of NI revealed two major zones and two minor zones all of which migrated at different rates than viral or cytoplasmic inclusion protein. Molecular weights were estimated to be 49,800 and 54,500 for protein in the major zones and 95,600 and 101,400 for protein in the minor zones. An antiserum produced to purified NI reacted specifically with NI antigens, but not with TEV or extracts from uninoculated D. coat protein, TEV-induced cytoplasmic inclusions, stramonium L. No reaction was detected with purified NI tested against antisera to virus or to cytoplasmic inclusions. INTRODUCTION

Tobacco etch virus (TEV) induces two distinct types of inclusions in infected host cells. Kassanis (1939) reported the occurrence of thin, rectangular, intranuclear crystals (NI) in TEV-infected plants. The NI since have been described as truncate, four-sided pyramids (Matsui and Yamaguchi, 1964) and although they are typically found in the karyoplasm of nuclei, they are occasionally observed in cytoplasm (Sheffield, 1941). The other inclusion type induced by TEV, which occurs only in the cytoplasm, consists of the lamellate “pinwheels” and laminated aggregates (Edwardson, 1966; Edwardson et al., 1968). The latter inclusions have been isolated and shown to consist of protein that is distinct from viral coat protein and host protein (Hiebert et al., 1971; Purcifull et al., 1973; Hiebert and McDonald, 1973). Reports regarding the nature of the NI 1 Journal Experiment

paper Station.

No.

5049.

Florida

Agricultural

are limited to in situ studies. Rubio-Huertos and Garcia-Hildago (1964), who studied the ultrastructure of the NI, suggested that they consisted of virus crystals. Cytochemical studies by Takahashi (1962) and Hooker and Summanwar (1964) suggested that the NI were composed of protein without accompanying nucleic acid. The autoradiographic studies by Hayashi and Matsui (1967) and the proteolytic enzymatic treatment of ultrathin sections of TEV infected tissue by Shepard (1968) have supported the suggestion that NI consist primarily of protein. Shepard et al. (1974) using immunoferritin techniques, showed that antibody prepared against TEV, dissociated capsid protein and cytoplasmic inclusions did not react with NI in fractured plant cells. We have investigated the nature of TEVNI by isolating and studying them in uitro. This report presents a purification procedure and a partial characterization of these inclusions.

TEV MATERIALS

Plant

Material

AND

INTRANUCLEAR

INCLUSION

METHODS

and TEV Source

Used

Tobacco etch virus (American Type Culture Collection No. PV-69) was cultured in Nicotiana tabacum var. Havana 425 for purification of virus (Purcifull, 1966) and cytoplasmic inclusions (Hiebert et al., 1971). TEV-NI were isolated from D. stramonium L. Plants were held at 20- 30” and harvested between 25 and 30 days after inoculation. Intranuclear

Inclusion

Purification

The TEV-NI were extracted by homogenization of infected leaves and isolated by Triton X-100 treatment of the homogenate followed by a combination of successive low speed and sucrose density-gradient centrifugations. Purification Method II was developed in the later stages of this study and the significance of the method will be presented below. Purification

Method

I

Tobacco etch virus infected tissue was blended for 1 min at low speed in a Waring Blendor, using 3 ml of an ice cold solution of grinding buffer (GB) which consisted of 0.005 M, pH 7.0 sodium phosphate buffer with 0.005 M 2-mercaptoethanol, 0.01 M magnesium chloride, and 0.25 M sucrose, per gram of tissue. The tissue homogenate was filtered through two layers of cheesecloth and 1 layer of Miracloth. It was then fractionated by centrifugation at 1000 g (all values given are max g) for 10 min in a Sorvall RC2-B refrigerated centrifuge. The pellets were washed with GB (2 ml of buffer per gram of original tissue) and centrifuged at 1000 g for 10 min. The resulting pellet was resuspended in cold buffer (RB) which consisted of 0.005 M, pH 7.0, phosphate buffer with 0.005 M 2-mercaptoethanol and 0.01 M sodium chloride. Triton X-100 (Rohm and Haas) was added to a final concentration of 5% and the resulting mixture was stirred for 1 hr at 4”. The preparation was washed twice by subjecting it to centrifugation at 1000 g for 10 min, discarding the supernatants and resuspending the pellets in RB. The final pellet was

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201

resuspended in 40% sucrose prepared with RB. The suspension was subjected to homogenization in the Sorvall Omnimixer at 9600 rpm for 4 min and layered on sucrose gradients prepared just prior to use. Five milliliters of tissue extract (equivalent of 25 g of original tissue) was added to each gradient. The gradients consisted of 5.0 ml layers of 80, 70, 60, and 50% w/v sucrose in RB. The gradients were centrifuged at 15,000 rpm for 20 min in the SW 25.1 rotor in the Spinco model L preparative ultracentrifuge. The NI, which either layered on top of or slightly penetrated the 80% zone, were collected in droplets after puncturing the bottom of the tube with a needle. The NI rich fraction was diluted 1: 1 with resuspending buffer and centrifuged at 1000 g for 10 min. The supernatant was discarded and the pellet was resuspended in 40% sucrose, homogenized and layered on gradients as described above. This procedure was repeated through a total of three successive gradients to increase the purity of the NI. The final pellet was resuspended in a small volume of RB. Purification Method II consisted of the same procedure as Method I but with 0.5% sodium sulfite added to all solutions used in purification. Microscopy The NI were examined in situ in epiderma1 strips of TEV infected D. stramonium leaves after clearing the cells by immersing tissue in 5% Triton X-100 for 3-5 min and staining with bromophenol blue (Christie, 1971). The NI were monitored during purification by light microscopic examination of unstained preparations or preparations stained with bromophenol blue. Purified NI were mounted on Formvar coated grids and stained directly with 0.2% uranyl acetate (pH 4.0) for electron microscopy. Purified NI, which were examined by ultrathin section microscopy, were prepared in a similar manner as that described by Hiebert et al. (1971) for purified cytoplasmic inclusions, except for an addition of 2% uranyl acetate in 95% ethanol during the dehydration series. The ultrathin sections

202

KNUHTSEN.

HIEBERT

were mounted on a Formvar coated grid, post stained with uranyl acetate-lead citrate and examined with a Philips 200 electron microscope. Ultraviolet

Absorption

Spectrophotometry

It was necessary to dissociate NI since the purified NI solutions were too turbid for spectrophotometry. The NI preparations were held for 60 min either at room temperature in 1.0% sodium dodecyl sulfate (SDS), or at 4’ in 67% acetic or 67% formic acid. The solutions were centrifuged 10 min at 1100 g prior to spectrophotometry in order to remove insoluble material. Spectrophotometric analyses were performed in the Beckman ACTA C II recording spectrophotometer. Molecular Weights of Constitutent NI Proteins by Polyacrylamide Gel Electrophoresis

Molecular weights of NI proteins were estimated by using the SDS polyacrylamide gel electrophoresis technique described previously (Hiebert and McDonald, 1973). Preparation of the Antiserum and Serology

Antiserum to TEV-NI was prepared by four injections of NI purified by Method I. The NI preparations, which were unreactive with antiviral, anticytoplasmic inclusion or anti-tobacco sera, were emulsified (1: 1) in Freund’s complete adjuvant prior to injection. Two injections with NI protein 7 days (A 280 = 26-28) were administered apart. Booster injections, consisting of lo-12 A,,, units, were given 60-67 days after the initial injection. The rabbit was bled intermittently over a period of several months beginning 2 wk after the first injection. Serological comparisons were performed by immunodiffusion tests utilizing the detergent method of Gooding and Bing (1970) to dissociate the antigens into small components which diffuse rapidly in the agar plates. The gels consisted of 0.85% Noble agar, 0.5% SDS, and 1% sodium azide in water. Virus concentration was adjusted to 1.0 mg/ml and the NI and cytoplasmic preparations to 1.0 A,,, units/ml. Wells

AND

PURCIFULL

were cut in agar gel plates with an adjustable well cutter (Grafar Corp., Detroit, MI) with a distance of 0.5 cm between the well edges. Reactants were added at room temperature and the plates incubated several days at 25”. Preparation of Protein from Uninoculated Plants

Host proteins were extracted from uninoculated plants by low and high speed centrifugations and by ammonium sulfate precipitation. Four fractions were obtained after D. stramonium tissue was homogenized in grinding buffer under the same conditions described above for NI purification (Method I). The extract was centrifuged at 9150 g for 10 min to yield a low speed pellet. The supernatant was centrifuged at 89,000 g for 16 hr to yield a high speed pellet. The resultant supernatant was subjected to ammonium sulfate precipitation at 40 and 80% saturation. RESULTS

Purification

The characteristic shape, size and density of the TEV-NI were important features in developing the purification procedure. The NI can be readily seen and differentiated from cellular components by light microscopy (Fig. 1) and therefore this was used to monitor the progress of purification. The TEV-NI in Datura are so large that they will readily sediment in a few minutes at 1000 g or move through dense and viscous sucrose in a gradient at relatively low centrifugal forces. This property greatly simplified the purification procedure. The purity of the isolated NI was evaluated by microscopy and serology. Electron microscopy of purified NI revealed little contamination by viral particles, cytoplasmic inclusions, bacteria and host material. Purified NI did not react with antisera either to TEV induced cytoplasmic inclusions or to TEV in immunodiffusion tests. Yields, determined by direct count with a light microscope and a hemacytometer, ranged from 2 x lo8 to 8 x lOa inclusions per kg of tissue. Absorbance produced by

TEV

FIG. 1. Photomicrographs (b). N, nucleus; n, nucleolus;

INTRANUCLEAR

INCLUSION

of TEV-NI stained with bromophenol CI, cytoplasmic inclusion; NI, nuclear

purified NI degraded in SDS ranged from 50 to 145 A,,, units/kg of tissue. Yields obtained with purification Method II were usually about 60-70s of those obtained with Method I.

Microscopy

of NI

A light microscopic comparison of NI purified by Method I with those seen in situ indicates that the purified NI have retained their distinctive structure (Fig. 1). The purified NI consist of thin, hyaline, rectangular plates which are frequently offset at 45” when stacked on top of each other (Fig. lb). Electron microscopy revealed an apparent difference between NI purified by Method I vs Method II. NI purified by Method I have sharp, distinct edges with relatively little fragmentation (Fig. 2a) whereas NI purified by Method II show considerable fragmentation and rounded edges (Fig. 2b). Electron microscopy (at magnifications greater than 50,000) revealed a periodic substructure on the surface of the NI stained in either uranyl acetate or ammo-

PURIFICATION

blue showing NI in situ (a) and purified inclusion; bar, 5 Km.

203

NI

nium molybdate, but not in phosphotungstate, at the early stages in purification Method I and at all stages in Method II. The multidirectional striations of the NI consist of a main set of axes (arbitrarily designated as primary) (Fig. 3) which are intersected by other main axes at 90”. A secondary set of axes that are also intersected by other secondary axes at 90” intersect the primary axes at 45’ (best seen by viewing Fig. 3 at a low angle). The primary axes meet the edges of the rectangular NI at 45” while the secondary axes run parallel to these edges. The distance between the centers of the striations of the primary axis is 102 * 2 A and 73 * 2 A for striations of the secondary axis. These values have been estimated from electron micrographs of purified NI using catalase crystals as an internal standard as described by Luftig (1967). The TEV-NI striations with periodicities similar to those shown in purified preparations have been observed in thin sectioned tissue (J. R. Edwardson, personal communication) and by freeze etch microscopy in situ and

FIG. 2. Electron I (a) and by Method

micrographs of TEV-NI II (b). Bar, 3.3 pm.

stained

with

0.2% uranyl

acetate

comparing

NI purified

by Method

FIG. 3. Electron micrograph showing surface of TEV-NI purified in the presence of 0.5% sodium sulfite (Method II) and stained with 0.2% uranyl acetate. Two distinct sets of axes are best seen (outlined by the inset) when micrograph is viewed from a low angle. The primary axes (designated arbitrarily) are outlined by solid lines and the secondary axes by broken lines in the inset. 204

TEV

INTRANUCLEAR

INCLUSION

with purified NI (McDonald and Hiebert, 1974). The morphology of purified NI in ultrathin sections (Fig. 4a, c) is similar to that of NI in situ (Fig. 4b). Figure 4a shows

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205

that purified NI are thin, multilayered, truncated pyramids as observed by Matsui and Yamaguchi (1964). The curvature in the purified NI is not commonly seen in situ and it may be due to distortion of the

FIG. 4. Electron micrographs of thin sections through a compacted curved and truncated pyramid structures (a) and at high magnification (b) shows an electron micrograph of a thin section of TEV-NI in situ (b), and 320 for (cl.

mass of purified TEV-NI showing typical showing constituent layers (c) (arrow). for comparison. Bar, 1600 nm for (a) and

206

KNUHTSEN.

HIEBERT

inclusions upon removal from the cellular environment. The purified NI (Fig. 4a), as well as those in situ (4b), show separations between laminations. Figure 4c, a high magnification cross-sectional view of purified NI, reveals the fragile nature of the laminations and suggests that these laminations are not held together strongly. Spectrophotometry

The NI were found to dissociate in the protein denaturants, such as 1.0% SDS, 6 M urea, 67% acetic acid, and 67% formic acid. The spectrum of SDS disrupted inclusions was typical of proteins, with a maximum absorbance at 277, a minimum at 250, and with a tryptophan shoulder at 290 nm (Fig. 5). Polyacrylamide

Gel Electrophoresis

Analysis of NI (purified by Method I) by SDS 6% polyacrylamide gel electrophoresis revealed two major proteins with estimated

AND

PURCIFULL

molecular weights of 49,800 and 54,400, and two minor proteins with estimated molecular weights of 95,600 and 101,400 for components designated NIPl, NIP2, NIP3, and NIP4, respectively (Fig. 6B). There is a noticeable amount of high molecular weight protein near the top of the gel from the NI purified by Method I (Fig. 6B) which suggests that a portion of these NI are resistant to SDS dissociation. Analysis of NI purified by Method II revealed similar results except that only a trace of component NIP4 and high molecular weight protein was present (Fig. 6A). A comparison of the NI protein electropherograms with those obtained with TEV capsid (Fig. 6C) and cytoplasmic inclusions (Fig. 6D) proteins clearly show that NI proteins are distinct from either of them. The possibility that NI in TEV-infected tissue represent an aggregation of host proteins was examined. Various fractions DEPTH

(CM)

I

.6-

6 6 v

*

7 6

ABCDEFGH

240

260

260 WAVELENGTH

300

320

(nm)

FIG. 5. Ultraviolet absorption spectrum of purified TEV-NI degraded by 1.0% SDS and centrifuged prior to spectroscopy.

FIG. 6. Polyacrylamide gel electrophoresis of SDS dissociated proteins of TEV-NI, TEV induced cytoplasmic inclusions, viral coat protein (TEV), protein from uninoculated plants, and various protein markers. TEV-NI purified in the presence (A) and in the absence (B) of 0.5% sodium sulfite. TEV-NI proteins are designated NIPl-4 and correspond to proteins of MW 49,800, 54,400, 95,600, and 101,400, respectively. (C) TEV coat protein (V); (D) TEV cytoplasmic inclusions (CD, (E) protein from uninfected datura precipitated by ammonium sulfate at 40% saturation, (F) carbonic anhydrase (29,006). (G) glutamate dehydrogenase (53,000), (H) bovine serum albumin (67,000). Electrophoresis was from top to bottom in a 6% gel slab for 3 hr at 160 v with pulsed power supply at 300 cps.

TEV

INTRANUCLEAR

INCLUSION

from healthy tissue (described in Materials and Methods) were analyzed by polyacrylamide gel electrophoresis. Only the fraction obtained by 40% ammonium sulfate precipitation contained significant amounts of protein with a size similar to NI protein (NIPB) (Fig. 6E). However, this fraction did not react with NI antiserum. The electrophoretic behavior of the SDS dissociated NI proteins was further studied by varying the gel concentration. Gels of 3% acrylamide revealed only two zones (Fig. 7A) with estimated molecular weights of 50,700 and 99,500. Gels of 10% (Fig. 7B) revealed similar results to those seen with 6% gels except that the separation was improved.

207

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94,000 53,000

Serology Purified preparations of NI were immunogenic and serologically distinct from either the virus or cytoplasmic inclusions as determined by reciprocal testing in immunodiffusion tests (Fig. SA). The TEVNI prepared by Methods I and II gave reactions of identity when tested against the TEV-NI antiserum. The TEV-NI antiserum titer was one-half against TEV-NI and it also reacted with sap from TEVinfected Datura (Fig. 8B, well 3). Occasionally a faint reaction was observed against crude sap from healthy tissue (Fig. 8B, well 4), but this was believed to be nonspecific since normal serum occasionally precipitated spontaneously with D. stramonium sap. None of the concentrated fractions from uninoculated Datura reacted with the NI-antiserum. To test the serological specificity of NI induced in different plant species, NI were concentrated from extracts of TEVinfected Nicotiana tabacum L., Datura stramonium L., and Cassia tora L. by a modification of Method II in which 0.5% SDS was added to the extract at the stage normally intended for sucrose densitygradient centrifugation. When tested against the NI antiserum, the NI isolated from these plants showed reactions of identity in double diffusion plates (Fig. SC). Extracts from healthy tissue prepared in a similar manner did not react.

A FIG. 7. Polyacrylamide dissociated proteins of II) in 3% (a) and 10% (b) of the protein markers glutamate dehydrogenase gel. Electrophoresis was gel slab (a) for 2 hr and at 160 V with a pulsed

B gel electrophoresis of SDS TEV-NI (purified by Method polyacrylamide. The position phosphorylase A (94,000) and (53,000) are shown for each from top to bottom in the 3% in the 10% gel slab (b) for 6 hr power supply at 300 cps.

DISCUSSION

Our results indicate that the NI induced by TEV are quite stable and can be readily purified in high yields. The physical appearance of the purified NI as revealed by microscopy is the only criterion to date that we have to evaluate whether or not the purified NI have been denatured. We believe the presence of well-defined striations on the surface of the isolated NI is an indication of their integrity because striations are seen on NI in ultrathin sections of TEV infected tissue (J. R. Edwardson, personal communication) or on NI examined in situ by freeze-etch electron microscopy (McDonald and Hiebert, 1974) and because the striations are lost gradually over a period of several hours in the

FIG. 8. Serological analyses of TEV-NI. Central wells contain (A) TEV-NI. (B, C) TEV-NI antiserum. Peripheral wells in (A) contain: (1) TEV, (2) TEV antiserum, (3) TEV cytoplasmic inclusions, (4) TEV cytoplasmic inclusion antiserum, (5) antiserum to NI, (6) proteins fractionated from uninoculated Datum strumonium by centrifugation for 16 hr at 89,000 g. Peripheral wells in (B) contain: (1) TEV cytoplasmic inclusions, (2) TEV-NI, (3) crude extracts from TEV infected Daturu in 1% SDS, (4) crude extracts from uninoculated Datura in 1% SDS, (5) extracts from uninoculated D. strumonium concentrated by centrifugation at 9000 g, and (6) TEV. Peripheral wells in (C) contain: well 1, TEV-NI extracted from Nicotiunu tubucum var Havana 425; well 2, TEV-NI from Datum; and well 3, TEV-NI from Cussia toru. Wells 4, 5, and 6 in (C) contain extracts from uninoculated N. tubucum, Duturu, and C. toru. Antisera were undiluted. TEV was used at a concentration of 1 mg/ml and the purified inclusion preparations were used at a concentration of 1 ODU/ml at 280 nm.

absence of sodium sulfite (purification Method I). The differences in yield between Method I vs Method II may be attributed to the apparent oxidation of NI in the absence of sulfite resulting in stabilization of the protein. This would reduce erosion and fragmentation of the NI and subsequent loss during purification. The fact that a portion of NI purified by Method I is resistant to dissociation by SDS in the presence of mercaptoethanol or by 6 M urea (Hiebert, unpublished) supports the oxidation concept. The multiplicity of NI protein composition as revealed by SDS polyacrylamide electrophoresis is not clear. The electrophoretic response of NIP1 and NIP2 to varying gel concentrations suggests that the mobility difference between NIP1 and NIP2 may be due to shape or size and not to charge. The position of NIP3 and NIP4 on the polyacrylamide gels suggests that they may be dimers of NIP1 and NIPB, respectively. Although the SDS polyacrylamide electrophoresis results do not clearly define the NI protein composition, it is clear that the NI protein is distinct from that of the viral capsid protein, cytoplasmic inclusion protein, and healthy host protein.

The primary constituent of the NI appears to be protein which is serologically distinct from viral capsid, cytoplasmic inclusions and host proteins. Protein yields, based on A,,, of the purified preparations, suggest that massive quantities of serologitally unique protein are produced and assembled into NI in TEV infected tissue. The origin, function, and physiological significance of the TEV-NI are unknown. The structural and immunochemical distinctness of the NI demonstrated herein, and the constancy of their association with TEV infection in different plant species (Kassanis, 1939; Purcifull and Edwardson, 1968), suggest that the NI proteins are coded from the viral genome. ACKNOWLEDGMENTS The authors thank Dr. John Edwardson and Mr. R. G. Christie for assistance concerning cytological aspects of the study and Mrs. R. C. Wase for technical assistance. This investigation was supported by the National Science Foundation Research Grant (GB-32093). REFERENCES R. G. (1971). A rapid diagnostic technique for plant viruses. Proceedings of the Pest Control Conference, University of Florida Vol. 5, pp. 65-68. EDWARDSON, J. R. (1966). Electron microscopy of cytoplasmic inclusions in cells infected with rodCHRISTIE,

TEV

INTRANUCLEAR

INCLUSION

shaped viruses. Amer. J. Bat. 53, 359-364. EDWARDSON, J. R. PURCIFULL, D. E., and CHRISTIE, R. G. (1968). Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses. Virology 34, 250-263. GOODING, G. V., JR. and BING, W. W. (1970). Serological identification of potato virus Y and tobacco etch virus using immunodiffusion plates containing sodium dodecyl sulfate. Phytopathology 60, 1293. HAYASHI, T., and MATSUI, C. (1967). Electron microscopy of host cells infected with tobacco etch virus. IV. Tritiated uridine and leucine uptake in intracellular virus and intranuclear crystalline inclusions. Virology 33, 47-54. HIEBERT, E. and MCDONALD, J. G. (1973). Characterization of some proteins associated with viruses in the potato Y group. Virology 56,349-361. HIEBERT, E., PURCIFULL, D. E., CHRISTIE, R. G., and CHRISTIE, S. R. (1971). Partial purification of inclusions induced by tobacco etch virus and potato virus Y. Virology 43, 638-646. HOOKER, W. and SUMMANWAR, A. S. (1964). Intracellular acridine orange fluorescence in plant virus infections. Exp. Cell Res. 33, 609612. KASSANIS, B. (1939). Intranuclear inclusions in virus infected plants. Ann. Appl. Biol. 26, 705-709. LUFTIG, R. (1967). An accurate determination of the catalase crystal period and its use as an internal marker for electron microscopy. J. Ultrastract. Res. 20, 91-102. MATSUI, C. and YAMAGUCHI, A. (1964). Electron microscopy of host cells infected with tobacco etch virus. I. Fine structures of leaf cells at later stages of

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infection. Virology 22, 40-47. MCDONALD, J. G. and HIEBERT, E. (1974). Ultrastructure of the crystalline inclusion induced by tobacco etch virus visualized by freeze-etching. J. Ultrastrut. Res., in press. PURCIFULL, D. E. (1966). Some properties of tobacco etch virus and its alkaline degradation products. Virology 29, 8-14. PURCIFULL, D. E. and EDWARDSON,, J. R. (1968). Nuclear crystals in Zinma and other non-solanceous plants infected with tobacco etch virus. Phytopathology 58, 532-533. PURCIFULL, D. E., HIEBERT, E., and MCDONALD J. G. (1973). Immunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group. Virology 55, 275-279. RUBIO-HUERTOS, M. and GARCIA-HILDAGO, F. (1964). Ultrathin sections of intranutilear and intracytoplasmic inclusions induced by severe etch virus. Virology 24, 84-90. SHEFFIELD, F. M. L. (1941). II. The cytoplasmic and nuclear inclusions associated with severe etch virus. J. Roy. Microsc. Sot. 61, 30-45. SHEPARD, J. F. (1968). Electron microscopy of subtilisin treated tobacco etch virus nuclear and cytoplasmic inclusions. Virology 36, 20-29. SHEPARD, J. F., GAARD, G. and PURCIFULL, D. E. (1974). A study of tobacco etch virus induced inclusions using indirect immunoferritin procedures. Phytopathology 64, 418-425. TAKAHASHI, W. N. (1962). Effect of viral infection on the nuclei of the host. Phytopathology 52, 20.