Purification of plant viruses and virus coat proteins by high performance liquid chromatography

Journal of Virological Methods, 28 (1990) 245-256

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Elsevier

VIRMET 01020

Purification of plant viruses and virus coat proteins by high performance liquid chromatography Merete Albrechtsen

and Morten Heide

Plant Protection Centre, Danish Research Service for Plant and Soil Science, Lyngby, Denmark

(Accepted 13 February 1990)

Summary High performance liquid chromatography (HPLC) gel filtration has been successfully applied in the purification of elongated and isometric plant viruses. Two different approaches have been tested. In one approach, semi-purified virus particles were dissociated with lithium chloride and the released coat proteins purified by HPLC gel filtration. The purified coat protein was highly immunogenic and gave rise to very specific antisera reacting with intact virus particles as well as with SDS-denatured coat protein monomers. This method is generally applicable only for elongated viruses since many isometric viruses are not dissociated by the lithium chloride treatment. The second approach consisted in gel filtration-of native, undissociated virus particles and could be used both with elongated and isometric viruses. Both methods were fast and simple to perform and removed all or most of the contaminating plant proteins as judged by sodium dodecylsulphate gel electrophoresis followed by silver staining or by immunoblotting with antiserum against healthy plant extracts. With both methods the recovery of virus coat protein was about 30% on average. High performance

liquid chromatography;

HPLC; Virus purification

Correspondence to: M. Albrechtsen, Plant Protection Centre, Danish Research Service for Plant and Soil Science, Lottenborgvej 2, DK-2800 Lyngby, Denmark.

0166~0934/90/$03.50Q 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Introduction The diagnosis of plant virus diseases today relies to a large extent on serological techniques such as enzyme-linked immunosorbent assay (ELISA), dot immunobinding (DIB) and immunosorbent electron microscopy (ISEM) (see, e.g., Clark and Adams, 1977; Miller and Martin, 1988; Mime and Lesemann, 1984; Towbin and Gordon, 1984). The success of these methods is dependent on the quality of the antiserum. In the case of the fast and sensitive methods suitable for routine use, such as ELISA and DIB, the absence of background reaction with healthy plant components is essential. Although cross-absorption of existing antisera with healthy plant extracts is possible (e.g., Lange and Heide, 1986), it is of course preferable to avoid eliciting antibodies to healthy plant components in the first place. This means immunizing with very pure virus preparations, since even a very slight contamination of the immunogen with plant (glyco-)proteins may give rise to a quite substantial immune response. Most virus purification schemes comprise repeated cycles of differential centrifugation and/or polyethylene glycol (PEG) precipitation, procedures which may cause virus aggregation with consequent loss of virus. The last step in the purification is usually some form of gradient ultracentrifugation, the most common being rate zonal centrifugation in sucrose gradients. This method tends to give problems with elongated viruses which are prone to an aggregation that causes them to sediment through the gradient at different rates with consequent loss of resolution and poor recovery. Another commonly used procedure is equilibrium density centrifugation in cesium chloride, which is applicable even to aggregated viruses. However, this method is quite expensive although the cost may be reduced somewhat by recovery and reuse of the cesium salt. Furthermore, not all viruses are stable in high cesium chloride concentrations; this problem may be solved by the use of cesium sulphate which, however, is even more expensive. Finally, equilibrium density centrifugation is time consuming, tying up the ultracentrifuge for around 20 h at a time unless preformed gradients are employed. Chromatographic methods, which avoid high osmotic pressures and centrifugal fields, have also been used for virus pu~fication, in particular gel filtration on various matrices such as granulated agar or agarose (e.g., van Regenmortel, 1962; Huttinga, 1975), controlled-pore glass beads (Barton, 1977), or granulated dextran or polyacrylamide gels (e.g., Lee et al., 1988). These methods do not seem to have gained wide popularity, however, presumably because they have appeared too cumbersome relative to other established methods. But recent technological advances in column chromatography have drastically reduced the time and effort needed for both development and routine use of chromatographic methods. In particular, the development of high performance liquid chromatography (HPLC) columns which can be used for protein purification has provided new possibilities for virus purification. We here present examples of the use of HPLC gel filtration in the purification of both elongated and isometric plant viruses for use as immunogens. To our knowledge, HPLC chromatography has so far only been used to separate peptides

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derived from virus coat proteins (Shukla et al., 1988) but not previously to isolate intact coat proteins or whole virus particles. We believe HPLC chromatography to be a valuable addition to the range of methods available for virus purification.

Materials and Methods Viruses and antisera

Isolates of lettuce mosaic virus (LMV), tomato ringspot virus (TRSV), and dipladenia mosaic virus (a presumed potyvirus (Paludan et al., 1988); herein called DiMV) were supplied by Dr Paludan, Department of Virology, the Danish Research Service for Plant and Soil Science, Denmark. The bean yellow mosaic virus (BYMV) isolate was kindly supplied by Dr Bos, Research Institute for Plant Protection, Wageningen, The Netherlands. The viruses were cultivated in Nicotiana benthamiana grown in a glasshouse. Antiserum against LMV was the generous gift of Dr Vetten, Biologisches Bundesanstalt, Braunschweig, Federal Republic of Germany. Antiserum against extracts of healthy Nicotiana leaves was prepared in the following way: 50 g frozen leaves of Nicotiana benthamiana, N. clevelandii, and N. tabacum cv. Xanthi in a 1:l: 1 mixture were homogenized in 100 ml cold 0.05 M Tris-HCl, 0.01 M potassium chloride, 0.005 M ethylene diamine tetraacetate (EDTA), pH 7.8, containing 0.1% (w/v) each of ascorbic acid and cysteine. The homogenate was passed through a double layer of gauze and centrifuged at 15 000 x g/30 min/2”C. The supernatant was dialyzed overnight at 2°C against two changes of 1 1 distilled water and subsequently lyophilized. The residue was redissolved in 0.05 M potassium phosphate, 0.1 M sodium chloride, pH 7.8, and stored in aliquots at -20°C. The protein content was determined by the method of Bradford (1976) using bovine serum albumin as standard. For each immunization, an aliquot containing 150-200 l.r.8 protein was mixed with an equal volume of Freund’s incomplete adjuvant (DIFCO Laboratories). Two New Zealand White rabbits were immunized intramuscularly ten times each over a period’of twelve months. Antiserum against DiMV coat protein was prepared as follows: coat protein was isolated from lithium chloride-treated DiMV (see below) and stored at a concentration of 0.5 mg/ml. For immunization, aliquots were mixed with an equal volume of Freund’s incomplete adjuvant. Two New Zealand White rabbits were immunized intra-muscularly four times each over three months, each rabbit receiving a total of 400 pg protein. Basic virus purification

LMV and DiMV were purified by a modification of the method of Alconero et al. (1986) for purification of pea seed-borne mosaic virus. Briefly, 100 g of leaves were homogenized in 200 ml 0.5 M sodium borate buffer, pH 7.5, and the extract clarified by the addition of 50 ml each of chloroform and carbon tetrachloride.

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After two low speed centrifugations virus was precipitated from the supernatant with 4% PEG (6000 kDa) and resolubilized in 0.05 M borate buffer. The final cesium chloride gradient centrifugation used by Alconero et al. was replaced by two cycles of differential centrifugation. BYMV was purified similarly to LMV and DiMV except that a 0.5 M potassium phosphate buffer was used instead of 0.5 M sodium borate and the PEG precipitation step was omitted. TRSV was purified essentially as described by State-Smith (1966) except that the buffer used was 0.05 M potassium phosphate, 0.005 M EDTA, 0.01 M potassium chloride, pH 7.0, and the final sucrose density gradient centrifugation was replaced by centrifugation through a 20% sucrose cushion. Purified viruses were stored in buffer at -80°C. HPLC

of intactviruses

Gel filtration was performed on an 0.8 X 30 cm Protein Pak Glass 2OOSWcolumn (Waters) using a Waters 650 HPLC apparatus equipped with a Waters LambdaMax 481 in-line spectrophotometer set at 280 nm. Between 0.25 and 2 mg virus, purified as described above, was used for each experiment. The virus preparation was thawed, diluted in column buffer (0.05 M potassium phosphate, 0.05 M sodium chloride, 0.005 M EDTA, pH 7.2), and centrifuged at 1000 rpm for 2 min in an Eppendorf microcentrifuge to remove aggregated virus. The supernatant was loaded on the column using a 100 ~1 loop. Virus was eluted with Column Buffer at 0.8 mllmin at room temperature and lo-drop fractions (approx. 0.5 ml) were collected. The virus was reconcentrated from the void volume fractions by centrifugation at 86500 x gavfor 2 h. HPLC of lithium chloride-treated virus Virus purified -by the basic procedures described above was diluted to 5-20 mg/ml with distilled water, mixed with an equal volume of 4 M lithium chloride and kept at -20°C for 48-72 h. This procedure has previously been found to precipitate virus RNA, releasing soluble coat protein (Moghal and Francki, 1976). The sample was then thawed and centrifuged in 400 ~1 microcentrifuge tubes at 50000 x g,, for 2 h to pellet RNA and other insoluble components. The supernatant containing soluble virus coat protein was fractionated by HPLC gel ~ltration exactly as described above for chromatography of intact viruses. Coat protein peak fractions were identified by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and were pooled, dialyzed against distilled water, and concentrated by lyophilization. Quantification of virus by ELISA Virus recovery after HPLC chromatography of intact viruses was determined by an indirect ELISA method as follows. A 2-fold dilution series of the semi-purified

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virus preparation (before HPLC chromatography) was prepared in 0.2 M sodium carbonate buffer pH 9.5, and the virus recovered after HPLC chromatography was diluted in the same buffer; 100 ~1 of each dilution were used for coating flatbottomed 96-well microtitreplates (Nunc Immunoplate I, 4-39454), 2 h at room temperature. The plates were washed twice with phosphate buffered saline (PBS; 0.05 M sodium phosphate, 0.14 M sodium chloride, pH 7.4) and residual protein binding capacity was blocked with 10% (v/v) normal horse serum in PBS, 200 pi/well for 1 h at room temperature. The plates were incubated overnight at 4°C with 100 tJ/well anti-virus antiserum diluted 1:2000 in PBS containing 5% (v/v) normal horse serum. After three washes each 5 min with PBS containing 0.05% (v/v) Tween 20 the plates were incubated for 3 h at room temperature with 100 PYwell alkaline phosphatase-coupled swine anti-rabbit IgG antibodies diluted 1:2000 in PBS containing 5% (v/v) normal horse serum. After three washes as above the enzyme reaction was developed with p-nitrophenylphosphate substrate (Boehringer Mannheim Cat. No. 415294) in 1 M diethanolamine buffer, pH 9.8. SDS-PAGE

and immunoblotting

Electrophoresis was performed on 12.5% polyacrylamide slab gels according to Laemmli (1970). Gels were stained for protein with Coomassie Brilliant Blue or by the silver staining method of Morrissey (1981). Alternatively, gels were electroblotted as described by Towbin et al. (1979) except that polyvinylidene difluoride membranes (Immobilon TM, Millipore) were used instead of nitrocellulose and the transfer buffer contained only 15% (v/v) methanol. Excess protein binding capacity of the membranes was blocked with 10% (v/v) horse serum in TST buffer (0.05 M Tris, 0.5 M NaCl, 0.5% (v/v) Tween 20, pH 10.2) for 1 h. Immunolabelling was performed by incubating overnight at 4°C with the primary antiserum diluted in TST buffer, washing in three changes of TST buffer 10 min each, incubating for 3-4 h at room temperature with alkaline phosphatase-conjugated secondary antibody in TST buffer and washing as before. The enzyme reaction was developed with 5-bromo-4-chloroindoxyl phosphate as described by Blake (1984). Molecular weight markers for SDS-PAGE were from Pharmacia: phosphorylase b (94000 kDa), bovine serum albumin (67000 kDa), ovalbumin (43000 kDa), carbonic anhydrase (30000 kDa), trypsin inhibitor (20100 kDa), alpha-lactalbumin (14400 kDa). Dot-blots were performed by spotting 3 ~1 aliquots on nitrocellulose membranes, allowing the membranes to dry at room temperature for 30 min and then blocking and immunolabelling as described above.

Results Viruses purified by the basic purification methods described in Materials and Methods were often found to contain contaminating host plant proteins. In some cases this was evident on SDS-polyacrylamide gels stained for protein (as in Figs. 1

2.50

B

c

Fig, 1. A: Purification of coat protein from LMV by HPLC gel filtration analysed by SDS-PAGE and silver staining. Lane 1, LMV purified by the basic procedure (see Materials and Methods); lane 2, LhIV coat protein after lithium chloride treatment and HPLC gel filtration. B and C: HPLC purification of intact LMV particles analysed by SDS-PAGE and (B) Coornassie staining, or (C) immunoblotting with antiserum against healthy Nicotiana leaf extract. Lane 1, LMV purified by the basic procedure; lane 2, LMV after HPLC gel filtration. The bars indicate the positions of molecular weight markers (from top to bottom, 94000,67000,43000,30000,20100, and 14400 kDa). A280

mex . 0.037

O.OlO-

A

mm z 0.014

B

0.008-

0.006-

0.004-

0.002 -

i oJFig. 2. Traces showing the absorbance at 280 nm of the column eluate during HPLC gel filtration of (A) intact LhW particles, and (B) lithium chloride-dissociated LMV. Arrows indicate sample injection points, Bars beneath the traces show which fractions were pooled and reconcentrated.

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Fig. 3. Purification of tomato ringspot virus by HPLC gel filtration of intact virus particles. (A) SDSPAGE and silver staining. (B) Immunoblot labelled with antiserum against tomato ringspot virus. (C) Immunoblot labelled with antiserum against healthy Nicotiana leaf extract. Lane 0, molecular weight markers (94000, 67000, 43000, 30000, 20100, and 14400 kDa); lane 1, tomato ringspot virus before HPLC gel filtration (approx. 10 ug total protein); lane 2, as lane 1 but only approx. 2 pg protein; lane 3, after gel filtration. The coat protein bands are marked by arrows.

and 3). In other cases, the virus preparation looked pure on silver stained SDS gels (as in Fig. 4A) but reacted strongly with an antiserum against healthy Nicotiana leaf extract (see Fig. 4C). With the aim of obtaining purer material for use as immunogens, the virus preparations were treated with lithium chloride and the solubilized material was submitted to HPLC gel filtration. When the peak coat protein fractions were pooled and reconcentrated and analyzed by SDS-PAGE a considerable enrichment of coat protein over contaminants was observed. As a representative example, the purification of LMV coat protein is shown in Fig. lA, lanes 1 and 2. The Am profile of the column eluate is shown in Fig. 2B. Similar results were obtained with several other rod-shaped viruses including pea early browning virus (a tobravirus), potato virus X (a potexvirus), and a presumed potyvirus, DiMV (results not shown). The recovery of virus coat protein was estimated by running SDS polyacrylamide gels of the purified coat protein and of the original virus preparation in various dilutions and comparing the staining intensities of the coat protein bands after protein staining. The recovery ranged from 15 to 50% in different experiments.

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Coat protein purified from DiMV by the lithium chloride method was used as immunogen. The resulting rabbit antisera reacted very strongly with DiMV coat protein in immunoblotting experiments, but only weakly or not at all with coat protein from a number of other potyviruses. The antisera also recognized DiMV in dot-blots of DiMV-infected Nicotiana clevelundii leaf extracts, with only a barely discernible background reaction against healthy Nicotiana leaves being observed in some experiments. Finally, the antisera were found to decorate DiMV particles in ISEM experiments, demonstrating that the antibodies recognize DiMV coat protein in intact virus particles (results not shown). Several attempts at dissociating the isometric viruses tomato bushy stunt virus, tomato ringspot virus and arabis mosaic virus with lithium chloride were unsuccessful, even using pre-swelling of the virus with EDTA as done by Erickson and Rossmann (1982) for the dissociation of southern bean mosaic virus. Treatment with 1 M calcium chloride (Yamazaki and Kaesberg, 1963) or with 6 M urea also failed to release appreciable amounts of coat protein. However, we observed that gel filtration of native, undissociated virus particles often produced virus preparations substantially free of contaminating plant proteins. As an example, Fig. 3 shows a tomato ringspot virus preparation obtained by the method described in Materials and Methods (lanes 1 and 2) and the same virus preparation after a single cycle of HPLC gel filtration (lanes 3).

Fig. 4. Purification of DiMV by HPLC gel filtration of intact virus particles. (A) SDS-PAGE and silver staining. (B) Dot-blot labelled with antiserum against DiMV. Each lane shows a 2-fold dilution series. (C) Dot-blot prepared identically to B but labelled with antiserum against healthy Nicotiuna leaf extract. Lane 1, DiMV before HPLC gel filtration; lane 2, after HPLC gel filtration. The bars indicate the positions of molecular weight markers (94000, 67000, 43000,30000,20100, and 14400 kDa).

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Next, gel filtration of intact (undissociated) rod-shaped viruses was attempted. The recovery of virus coat protein was estimated by SDS-PAGE or by indirect ELISA and ranged from 8 to 50%. In some cases, all detectable plant contaminants were removed in a single chromatography step as shown for LMV in Fig. 1B and C, and for DiMV in Fig. 4. The Azm elution profile for LMV is shown in Fig. 2A. However, with some heavily contaminated BYMV preparations some contaminants co-eluted with the virus in the void volume fractions (not shown). With a view to obtaining a better separation of virus from contaminants, the effect of urea on the integrity of several rod-shaped plant viruses was tested. For most rodshaped viruses including BYMV, pre-incubation of virus with 1 M urea in neutral buffer made no difference to the elution profile on subsequent gel filtration, but 6 M urea caused considerable disintegration of the viruses (results not shown). Consequently, the BYMV preparation was preincubated with 1 M urea for 15 min at room temperature before gel filtration in buffer without urea. This procedure resulted in a slightly better separation of virus from plant proteins, but also in a somewhat lower recovery of virus coat protein as judged by subsequent SDSPAGE.

Discussion The feasibility of using gel filtration (also called exclusion chromatography or permeation chromatography) in the purification of plant viruses has been demonstrated repeatedly (e.g., Cech, 1962; van Regenmortel et al., 1962; Huttinga, 1975; Barton, 1977). Barton (1977) for instance showed that gel filtration on controlled pore glass could separate intact viruses from contaminating plant material as efficiently as sucrose density gradient centrifugation and with less damage to labile viruses. Various other gel filtration matrices have been employed, but none seem to have gained wide popularity. As an illustration, of the hundreds of suggested purification methods described in the Commonwealth Mycological Institute and Association of Applied Biologists Descriptions of Plant Viruses, less than 10% mention gel filtration techniques (of these, most use controlled pore glass or some form of granulated agarose gel as matrix). A possible explanation for the general lack of enthusiasm among plant virologists for these methods is that they have appeared too awkward for routine use. With traditional matrix materials, quite long columns are needed to bring about a satisfactory separation of virus from contaminants, and these long columns are rather difficult to prepare and - particularly in the case of agarose gel matrices - are slow and delicate to operate. However, the gel filtration columns used in HPLC chromatography permit very good separations to be obtained in much shorter times than previously and with much less effort. At the same time, the gentleness of the traditional gel filtration methods should be preserved in the HPLC version since only moderate pressures are employed (max. 120 psi). The other strategy described in this paper, dissociation of virus particles and use of the purified coat protein as immunogen, has not been much used, possibly due

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to earlier reports of reduced or altered immunogenicity of solubilized coat proteins (e.g., Loor, 1967; Moghal and Francki, 1976). Some of these authors used harsher procedures for dissociation of the virus, such as treatment with pyrrolidine and formaldehyde or with SDS, which would be expected to denature the protein. However, Moghal and Francki (1976) used lithium chloride to dissociate various potyviruses but failed to obtain useful antisera in mice. In our hands, coat protein released from DiMV (a presumed potyvirus) by treatment with lithium chloride was highly immunogenic in rabbits and the antisera reacted well both with denatured coat protein and with intact virus particles. This is in agreement with Goodman et al. (1976) who found that potato virus X coat protein released with lithium chloride could reassemble into virus particles, suggesting only minimal denaturation of the protein. The lithium chloride dissociation method, with or without pretreatment with EDTA, was found not to work for the isometric viruses tomato ringspot virus, arabis mosaic virus, and tomato bushy stunt virus, which belong to the nepovirus and tombusvirus groups. However, other isometric viruses may be dissociable by this method, e.g., Erickson and Rossmann (1982) showed that southern bean mosaic virus could be dissociated by lithium chloride after EDTA pretreatment. The column used for the experiments reported here is designed for optimal separation of proteins in the 5000 to 60000 dalton range. This is appropriate for the purification of released virus coat protein, but in the case of intact virus particles an even better separation might be obtained with a column with a larger exclusion limit. Only proteins smaller than approximately 100000 dalton are retarded on the column used here, while larger proteins elute together with the virus particles in the void volume. A larger exclusion limit would allow the separation of larger proteins from the virus particles. No pre-column or other pretreatment of the sample other than a short microcentrifuge spin was used, but no problems with blockage of the column have been encountered to date. The recovery of virus coat protein was about 30% on the average in the experiments recorded here. Considering the degree of purification obtained this compares quite favourably with other alternative purification procedures. Barton, for instance, (1977) calculated the recovery of different viruses after sucrose gradient centrifugation to between 0 and 70%) with an average of 43%. The loss of virus in the HPLC experiments happened to a large extent during sample loading, which in our particular HPLC apparatus occurs via a valve system with a considerable dead space. With practice the sample loss in this device may be greatly reduced. The recovery might also be increased by pooling a larger number of fractions after gel filtration. In these experiments, only the peak fractions were recovered to ensure optimal purification. In conclusion, HPLC gel filtration of either intact or dissociated plant viruses can be used as a fast and simple last step in a virus purification scheme to provide very pure material for immunization or other purposes. In particular for labile or aggregation-prone viruses this would seem to be the method of choice. As more and more laboratories acquire HPLC or FPLC equipment, we believe that gel

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filtration chromatography will become an increasingly popular, fast and inexpensive alternative or supplement to existing ultracentrifugation techniques.

Acknowledgements

The authors wish to thank MS Pemille Petersen for expert technical assistance. This work was supported in part by the Centre for Plant Biotechnology, Denmark.

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