The distribution and rate of accumulation of TSWV in N. rustica
was compared at two different times of the year, February and September (Table 3.1 and 3.2). In February the overall rate of accumulation was slower than in September. From this observation it was concluded that the optimal time of harvest for purification is subject to seasonal variation.
A modified procedure for purification of TSWV is described consisting of differential centrifugation, treatment with antiserum against sap of healthy N. rustica
and zone- and density-gradient centrifugation in sucrose gradients and finally a concentration by high speed centrifugation (Scheme 3.1). When large amounts of infected leaves were to be processed the virus was purified by means of centrifugation in a zone-rotor (Scheme 3.2). This procedure gave very good results.
Antisera were raised in rabbits against purified TSWV. These sera reacted with healthy N. rustica
sap in agargel double diffusion studies. This reaction could be abolished by absorption with either healthy N. rustica
sap or polysaccharides purified from N. rustica
(Fig. 4.5 and 4.6). Unabsorbed sera reacted with the major structural proteins, following their electrophoretic separation in SDS-gels (Fig. 4.1), however, absorption greatly diminished the intensity of the reactions. The positions of the precipitin lines corresponded with those of the structural proteins visualized by staining. From these observations it was concluded that the reaction of TSWV-antiserum with the structural proteins must be due mainly to host derived polysaccharides covalently bound to the structural proteins.
An unabsorbed antiserum gave three precipitin lines with TSWV in agar gel double diffusion studies (Fig. 4.2). Absorption of this antiserum with increasing concentrations of intact virus resulted in the disappearance of all but one of the precipitin lines (Fig. 4.10).
Polyacrylamide gel electrophoresis of purified virus following disruption with SDS, ME and iodoacetamide revealed 7 proteins (referred to as protein 1-7, in order of increasing molecular weight) (Fig. 5.1). Protein 1-4 were major proteins,
proteins 5, 6 and 7 occurred in very small amounts and were designated minor proteins.
Proteins 2, 3, 4 and 5 gave a clear reaction with PAS-reagent, indicating their glycoprotein nature (Fig. 5.3). Protein 1 gave a very weak reaction with PAS-reagent. It was not possible to demonstrate the presence of carbohydrate in proteins 6 and 7 due to their small amounts. A PAS positive zone migrating in front of the bromophenol blue tracker dye was detected. The material contained in this zone could be extracted from purified virus with chloroform-methanol (Fig. 5.5). From the properties of this zone viz. the presence of carbohydrate, solubility in chloroform-methanol, reaction with TSWV-antiserum (Fig. 4.1), and the absence of staining with coomassie brilliant blue, it was concluded that this zone contained glycolipids.
Determination of molecular weights in SDS-gels was carried out according to Segrest and Jackson (1972). The asymptotic minimum molecular weights derived for proteins 1-5 were 27,000 d; 52,000 d; 58,000 d; 78,000 d and 90,000 d respectively (Fig. 5.6).
The technique of lactoperoxidase catalysed iodination of intact and Nonidet P 40 disrupted virus was used to locate the individual proteins. From the differential labeling of proteins 1-4 it was concluded that protein 1 was localized inside the virus envelop and proteins 2, 3 and 4 were external to the envelop (Fig. 5.10 and 5.11, and Table 5.5). Furthermore the radioactivity profiles strongly suggested that protein 5 was also localized outside the envelop.
The action of an unknown proteolytic enzyme present as a contaminant in a preparation of galactose oxidase suggested a similar distribution of the proteins. Incubation of intact virus with the enzyme preparation led to the degradation of all structural proteins except protein 1.
TSWV nucleic acid, extracted from purified virus with phenol-SDS, was non-infectious under the test conditions used. Following electrophoresis of the extracted nucleic acid in 2% polyacrylamide gels containing 0,5% agarose, 5 bands were observed (Fig. 6.1). The amount of material contained in the different bands varied with the season. Bands 1-4 were sensitive to RNases and resistant to DNase (Fig. 6.2 and 6.3). This was a strong indication for the presence of single stranded RNA in these bands. Precipitation in 2 M LiCl (Fig. 6.4), resistance to heat (Fig. 6.5) and mobility in gels of increasing percentage acrylamide (Fig. 6.6) (Harley et al. 1973) confirmed the single stranded character.
Band 5 appeared very resistant to the action of RNases and stained with coomassie brilliant blue. This band therefore probably represents a complex between protein and nucleic acid.
The molecular weights of RNAs 1, 2, 3 and 4 (corresponding to bands 1-4) were estimated under both non-denatured (Loening and Ingle, 1967) and denatured conditions (Reijnders et al., 1973). The values obtained for RNAs 1, 2, 3 and 4. by the two methods agreed well. From this the molecular weights 1.3 x 10 6
d, 1.7 x 10 6
d, 1.9 x 10 6
d and 2.5-2.8 x 10 6
d for RNAs 1, 2, 3 and 4 were derived.
Following dissociation of the envelop of TSWV with Nonidet P 40, and centrifugation on a glycerin gradient, both protein I and infectivity showed the same distribution along the gradient (Fig. 7.1 and 7.2). From this it was concluded that protein 1 was associated with the nucleic acid.
The results obtained are discussed in relation to known data about the arrangement of proteins in particles of other enveloped viruses. It is concluded that protein I is localized inside the lipid bilayer and associated with the RNA. The proteins 2, 3 and 4, in view of their properties, have to be localized on the outside of the lipid bilayer and probably one or more of them are associated with the projections.
On the basis of the results obtained the cryptogram of TSWV may be written as: R/1 : Χ/Σ7.4 : S/S : S/Th. The classification of TSWV in a monotypic group (Harrison et al., 1971 b) is in agreement with the data presented here concerning the RNA and protein composition of TSWV.