Molecular organization and stabilizing forces of simple RNA viruses

VIROLOGY

70, 1-16 (1976)

Molecular

Organization

IV. Selective

and Stabilizing

Forces of Simple

Interference with Protein-RNA Interactions Sodium Dodecyl Sulfate

S. BOATMAN’

AND

RNA Viruses

by Use of

J. M. KAPER

Plant Virology Laboratory, Plant Protection Institute, ARS, U. S. Department of Agriculture, Maryland 20705 Accepted October 2,1975

Beltsville,

The effect of the anionic detergent sodium dodecyl sulfate (SDS) on a number of simple isometric RNA viruses and empty capsids was tested. Some viruses showed extreme sensitivity, e.g., cucumber mosaic (CMV), brome mosaic (BMV) and alfalfa mosaic (AMV) virus, some extreme resistance, e.g., turnip yellow mosaic (TYMV) and tomato bushy stunt (TBSV) virus, and some intermediate resistance, e.g., southern bean mosaic virus (SBMV) and bacteriophage t2, to dissociation into components by this detergent. In the viruses most sensitive to SDS, virion dissociation is apparently caused by disruption of the electrostatic protein-RNA interactions which are responsible for stabilizing the virions. It is proposed that dodecyl sulfate CDS-) ions bind by means of the hydrocarbon chain to specific binding sites on the virion so that the sulfate groups are near lysine- or arginine-phosphate interaction points; these interactions are neutralized and the phosphates are repulsed, resulting in virion dissociation. Based on this hypothesis several predictions were made, tested experimentally and found valid: (1) Virions become more resistant to SDS as the contribution of the protein-protein interactions to virus stability increases. This was confirmed by testing the sensitivities of a number of viruses, including those mentioned above, to SDS. (2) Capsids devoid of nucleic acid are less sensitive to SDS than the respective intact virions. The behavior of TYMV, BMV and bacteriophage f2 virions and capsids with SDS confirmed this prediction. (3) Positive and neutral detergents do not cause dissociation of the SDS-sensitive viruses. (4) Reassembly of viruses stabilized by protein-RNA interactions is inhibited by SDS but not by positive detergents. Predictions (3) and (4) were confirmed by appropriate experiments with CMV or BMV and SDS, dodecyltrimethylammonium chloride (DTAC) and TritonX-100. Measurements of amounts of detergent bound at low SDS concentrations showed that CMV and BMV have much greater affrnitives than TYMV for DS- ions. It is suggested that SDS is useful as a probe for protein-RNA interactions and that relative sensitivity to SDS could be used in categorizing viruses according to stabilizing interactions. INTRODUCTION

The effect of the anionic detergent sodium dodecyl sulfate (SDS) on several viruses suggests that SDS can interfere effectively with protein-nucleic acid interactions. Sreenivasaya and Pirie (1938) first utilized SDS to obtain ribonucleic acid (RNA) from tobacco mosaic virus (TMV); infectious TMV-RNA was later isolated by ’ Present address: Hollins College, Hollins College, Va. 24020. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

Fraenkel-Conrat et al. (1957) using this detergent. More recently, Brakke (1971) was able to obtain good yields of RNA from brome mosaic virus (BMW and TMV by using SDS in the presence of bentonite and salt. SDS is often used in conjunction with phenol to extract viral RNA. SDS has also been widely utilized to disrupt viruses in order to determine protein constituents and molecular weights of subunits by polyacrylamide-gel electrophoresis (Maizel, 1971). Similarly, Lane and

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BOATMAN

Kaesberg (1971) examined BMV-RNA by gel electrophoresis after treating BMV with SDS. It has been thought that, in these instances, SDS acted mainly as a protein denaturant and solubilizer. Proteins to which large amounts of SDS are bound assume a common rod-like shape, apparently with considerable secondary structure (Reynolds and Tanford, 1970al which causes their electrophoretic migration in polyacrylamide gels to be dependent only (or mainly) on molecular weight. The binding of SDS has been studied in detail for only a few proteins, and much of this work has been done with serum albumin (Tanford, 1968; Steinhardt and Reynolds, 1969). These and other studies indicate that detergents in general are effective in denaturing proteins at very low concentrations, in many instances causing large and cooperative conformational changes. For example, native bovine serum albumin (BSA) has binding sites for a few (about 121dodecyl sulfate (DS1 ions at low SDS concentrations. With only slightly greater amounts of SDS the protein denatures, adopting a conformation that can bind large amounts of DS (Steinhardt and Reynolds, 1969). Several proteins have been found to bind the same (large) amount of DS per gram at SDS concentrations above 5 x lo-” M Reynolds and Tanford, 1970a; Nelson, 1971). The binding of SDS to the coat protein of bacteriophage dX174 has been studied (Carusi and Sinsheimer, 19631, but, in general, few investigations of the interaction of SDS with viruses have been made (Hersh and Schachman, 1956; Carusi and Sinsheimer, 1963; Kruseman et al., 1971). Viruses are very useful models for the study of biological systems in which macromolecules self-assemble into active and complex structures. Previous papers in this series have dealt with stabilizing interactions in turnip yellow mosaic virus (TYMV) (Kaper, 1971) and in cucumber mosaic virus (CMV) (Kaper and Geelen, 1971). In these studies, TYMV was shown to be stabilized mainly by protein-protein interactions with a contribution from pHdependent protein-RNA linkages. CMV represents the opposite extreme of the sta-

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KAPER

bility spectrum and is stabilized by protein-RNA interactions, protein-protein interactions being very weak or nonexistent. There is a constant search for probes which will allow direct interference with the noncovalent interactions responsible for stabilizing aggregates of macromolecules which become biologically active structures. A number of such probes is available. They usually entail physical or chemical modification of the biopolymers in conjunction with spectrophotometric or other methods to monitor the interference. In this paper, evidence is presented that SDS can serve as such a probe and that it specifically disrupts protein-RNA interactions in a number of simple isometric viruses. A mechanism for the action of SDS on these viruses is proposed, and possible uses for SDS as a specific probe for the stabilizing interactions of viruses are suggested. A preliminary version of this paper was presented at the First John Innes Symposium, Norwich, England (Boatman and Kaper, 1973). MATERIALS

AND

METHODS

Apparatus. Instruments for analytical ultracentrifugation, ultraviolet spectra, and optical density determinations were essentially the same as those utilized previously (Kaper, 1971). Rate-zonal centrifugations in sucrose density gradients were carried out in a Spinco Model L3-50 or L2-65 ultracentrifuge. Sucrose gradients were scanned with an ISCO fractionator and an ultraviolet flow cell at 254 nm. Polyacrylamide-gel electrophoresis was performed on the Savant Instruments, Inc., Model DEC-12 cell with Plexiglas tubes (15.2 x 0.45 cm) and the Vokam power supply from Shandon Scientific Co., Inc. Ultraviolet absorption scans of the polyacrylamide gels were obtained with a Gilford Model 2400 spectrophotometer and a gel scanning attachment. Electron microscopy was carried out with the JEOL Model lOOB electron microscope. A Nippon Kogaku Shadograph was used to measure virions 2 Mention of any specific equipment, trade products or commercial companies does not constitute its endorsement by the U. S. Government over similar products or companies not mentioned.

SDS AND

PROTEIN-RNA

in electron photomicrographs. Counting of 35S-labeled SDS in binding studies was done with a Beckman LS-250 liquid scintillation counter with Aquascint liquid scintillation cocktail, obtained from NuclearChicago. Reagents,

viruses, capsids, and RNAs.

SDS was obtained from Matheson, Coleman, and Bell, and was recrystallized from 95% ethanol. The recrystallized sample was shown to be pure by thin-layer chromatography. SDS was also obtained from Schwarz/Mann and used without further purification. 35S-labeled SDS was obtained from AmershamSearle. Dodecyltrimethylamine hydrochloride (DTAC) and dodecylamine hydrochloride (DAH) were obtained from Eastman Organic Chemicals. Triton X-100 was obtained from Rohm and Haas. All salts were obtained from Fisher Scientific Company and were analytical grade. Cucumber mosaic virus strain S (CMVS) was purified from infected squash plants (Cucurbita pep0 L. cv. Caserta Bush) according to Van Regenmortel (1964). Brome mosaic virus (BMV) was isolated from barley (Hordeurn vulgare L. cv. Moore) (the infected tissue was a gift of H. E. Waterworth, U. S. Department of Agriculture, Glen Dale, Md.) or wheat (Triticum aestivum L. cv. Thorne), which was a gift of S. A. Tolin (Virginia Polytechnic Institute and State University, Blacksburg). A slight modification of the method of Bockstahler and Kaesberg (1962) was utilized. High speed centrifugation was carried out at 30,000 rpm in a Spinco 30 rotor for 3.5 hr, at 39,000 rpm in a Spinco 40 rotor for 2 hr, or at 50,000 rpm in a Spinco 65 rotor for 1.5 hr. Low speed centrifugation was carried out at 10,000g for 10 min, and pellets were resuspended in 0.02 M sodium acetate, pH 5.0. Turnip yellow mosaic virus (TYMV) was isolated from infected Chinese cabbage plants (Brassica pekinensis Rupr. cv. Wong Bok) according to the method of Dunn and Hitchborn (1965). Peanut stunt virus (PSV) was isolated from cowpea (Vigna unguiculata (L.) Walp cv. Early Ramshorn) either by dialysis followed by differential centrifugation according to Scott

INTERACTIONS

3

(1963) or from sucrose gradients (Tolin, 1967). Alfalfa mosaic virus (AMV) was purified by the method of Hull et al. (1969) from tobacco (Nicotiana tabacum L. cv. Xanthi NN); the infected tissue was a gift of S. A. Tolin, as were broad bean mottle virus (BBMV), cowpea chlorotic mottle virus (CCMV), and the soybean (Glycine max (L.) Merr. cv. Bansei) tissue infected with bean pod mottle virus (BPMV) from which BPMV was isolated according to the method of Bancroft (1962). Bacteriophage f2 was obtained from Miles Laboratories; carnation mottle virus (CarMV) and southern bean mosaic virus (SBMV) were gifts of H. E. Water-worth; tobacco ringspot virus (TRSV) was obtained from I. R. Schneider (U. S. Department of Agriculture, Beltsville, Md.); tomato bushy stunt virus (TBSV) from R. L. Steere (U. S. Department of Agriculture, Beltsville, Md.); and turnip crinkle virus (TCV) was a gift of A. C. H. Durham (Cambridge). AMV components were a gift of L. van Vloten-Doting (Leiden). BMV capsids were prepared essentially by the method of Bancroft et al. (1968). They were also prepared by dialyzing BMV protein against 0.02 M sodium acetate, pH 5.2, with 0.2 M sodium chloride. TYMV capsids were prepared by two methods; the artificial top component by treatment of TYMV with urea according to the method of Jonard and Hirth (1966), and the naturally occurring top component from purified TYMV by rate-zonal centrifugation in sucrose density gradients in a Spinco Ti 14 zonal rotor. CMV-RNA was obtained by phenol extraction of CMV (Kaper and West, 1972), and BMV-RNA essentially by the method of Bockstahler and Kaesberg (1965). The quality of the RNA was analyzed by polyelectrophoresis as deacrylamide-gel scribed below. Treatment of viruses and capsids with detergents (and salt). In a standard reac-

tion of a detergent with a virus, virus was added to a mixture of buffer, Cleland’s reagent (1,4-dithiothreitol), detergent and salt (if any) so that the final concentrations of virus, buffer and Cleland’s reagent were 0.2 mg/ml, 0.02 M, and 0.001 M, re-

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BOATMAN

spectively. The mixing was done either at 20 or at o”, depending on the experiment. One-percent SDS solutions were prepared fresh daily. Buffers used were sodium phosphate, pH 6.0 and 7.0, and sodium acetate, pH 5.25. Tris-HCI was sometimes used at pH 7.2, but Tris-acetate at pH 5.8 caused precipitation of BMV when SDS was present. The samples were examined by analytical ultracentrifugation at 20” in a Spinco TiAN-F rotor. Observations of three samples per run were made at 4-min intervals after the rotor had reached a speed of 29,500 rpm. The shortest possible time between sample preparation and its first examination at the desired speed in the ultracentrifuge was 20 min. To observe the sedimentation of RNAs, the centrifuge was accelerated to 59,780 rpm. Intact virus or phenol-extracted RNA in the same buffer (and salt) were routinely included in the same rotor as controls in each experiment. In similar experiments CMV and BMV concentrations were varied from 1 to 6 mg/ml and SDS concentrations from 3.5 to 17 mJ4 (O.l-0.5%). The reaction mixtures were diluted to 0.2 mg/ml with the corresponding buffer prior to examination in the analytical ultracentrifuge. To test for possible effects of dilution, mixtures of BMV and SDS at pH 6.0 were submitted without dilution to rate-zonal centrifugation. Binding studies with sodium dodecyl 135Slsulfate. The amount of SDS bound to CMV, BMV, and TYMV was determined by equilibrium dialysis using 35S-labeled SDS (specific radioactivity, 9.4 mCi/mol) in 0.02 M sodium phosphate, pH 6.4 or 6.0. SDS solutions were prepared for each binding experiment by dissolving together amounts of labeled and unlabeled SDS to give the desired radioactivity and were stored in the cold to minimize hydrolysis of the SDS. Virus samples were prepared for dialysis in solutions containing the same concentration of SDS with which they were to be dialyzed in order to minimize the time required to reach equilibrium (Cassel et al., 1969). Dialysis tubing was heated at 75” in 1 M KC1 for 10 min, followed by 10

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min in 0.1 M EDTA and several rinses with distilled water. All SDS concentrations employed in binding studies (0.021.5 ti) were below the critical micelle concentration, and virus concentrations were 0.2 mg/ml (less than 0.02% protein). Dialysis was carried out at 20” for 72 hr against frequent changes of the appropriate SDS-buffer mixture. The amount of SDS bound was determined by subtracting the radioactivity of undialyzed SDS solutions from that of an equal volume of the contents of the dialysis bags containing known amounts of the viruses. Controls consisting of undialyzed virus-SDSbuffer mixtures and the final buffer-SDS dialysates were included in all experiments. Appropriate corrections were made for quenching. A few experiments were carried out at 4”, but the limited solubility of SDS in the cold prevented the use of a wide range of SDS concentrations at low temperatures. Polyacrylamide-gel electrophoresis. Electrophoresis of RNA was carried out at pH 7.2 as described by Kaper and West (1972). Electrophoresis of viruses, with or without pretreatment with SDS, was done at pH 7.2 or 7.0 in Tris-acetate buffer with 0.25-0.3 mg of virus per gel. Isolation and examination of BMV intermediate. The swollen intermediate, formed when BMV is treated at pH 6.0 with SDS, was isolated from sucrose density gradients as follows: BMV at 1 mg/ml in 0.02 M sodium phosphate, pH 6.0, was combined with 0.048% SDS and 1 ml was layered on each of six gradients for ratezonal centrifugation. The peaks corresponding to the intermediate as detected by the fractionator were collected. The pooled fractions, diluted with 0.02 M sodium phosphate, pH 6.0, were centrifuged at 40,000 r-pm in a Spinco 40 rotor for 2.5 hr, and the resulting pellets were resuspended in the same buffer. A portion of the isolated intermediate was extracted with phenol and the RNA obtained was examined by polyacrylamide-gel electrophoresis as described above. Drops of liquid from the gradient zones were placed on collodion-coated, carbonbacked copper grids for examination in the

SDS AND

PROTEIN-RNA

electron microscope. After 1 min, each drop was blotted and the grid was rinsed several times with triple-distilled water and negatively stained with 1% uranyl acetate. Intact virus and the original mixture of BMV and SDS were prepared for electron microscopy in the same manner. Dissociation -reassociation and reassembly . Dissociation-reassociation and reas-

sembly of CMV were carried out according to Kaper and Geelen (1971). In a typical reassembly experiment 0.1 ml of a protein solution containing 0.16 mg of CMV protein in 2 M LiCl, 0.001 M Cleland’s reagent, 0.02 M Tris-HCl, pH 7.2, was added to 0.04 mg of RNA in 0.1 ml of 0.02 M sodium phosphate, pH 7.0. After 10 min in an ice bath, 0.8 ml of 0.02 J4 Tris-HCl, pH 7.2, was added. In a typical dissociation-reassociation experiment, CMV or BMV at 2 mg/ml was combined with 2 M LiCI, 0.001 M Cleland’s reagent, and 0.02 M Tris-HCl, pH 7.2. Reassociation was accomplished by diluting 0.1 ml of the above mixture to 1.0 ml with 0.02 M Tris-HCl, pH 7.2. In the experiments in which the effect of SDS or DTAC on reassociation was tested, the detergent was added to the buffer used to make the final dilution. RESULTS

AND

DISCUSSION

Preliminary Results and Working Hypothesis

In preliminary experiments, it was observed that the isometric peanut stunt virus (PSV), a legume-infecting strain of CMV, dissociated in the presence of very low, and in a narrow range of, concentrations of SDS (Boatman et al., 1973). In addition, the degree of dissociation was dependent upon both virus and SDS concentrations. These observations were tested and verified with CMV-S, which, like PSV, was very sensitive to dissociation by SDS. On the other hand, TYMV is quite resistant to dissociation in the presence of SDS (Stols and Veldstra, 1965; this paper). The fact that two viruses which are very similar architecturally (Finch and Klug, 1966; Finch et al., 1967) behave so differently with the same reagent seems

INTERACTIONS

5

puzzling until one considers differences in the bonds which stabilize these virions. Kaper (1971) has pointed out that TYMV seems to be stabilized mainly by proteinprotein interactions of a hydrophobic nature, with only weak, pH-dependent protein-RNA linkages. In contrast, CMV appears to be stabilized mainly by proteinRNA interactions, with insignificant protein-protein interactions contributing to the overall stability of the virion (Kaper and Geelen, 1971). SDS is thought to exert its denaturing effect on proteins by hydrophobic binding of the hydrocarbon chain to binding sites on the protein with some contribution from electrostatic binding of the sulfate group near positively charged residues (Steinhardt and Reynolds, 1969). The most obvious proposal for a mechanism of SDS action on viruses is that the DS- ions bind to the viral protein and interfere sufficiently with its tertiary and quaternary structure that the mutual interaction of the subunits and their interaction with the RNA becomes thermodynamically less favorable than interaction with the solvent and/or SDS. While this mechanism is not incompatible with much of the experimental data, it suffers from the fact that it cannot explain two important observations which will be discussed below: first, the high dissociation efficiency of SDS seems confined to viruses which are thought to derive a great deal of their stability from protein-RNA interactions, and, second, nucleic acid-containing virions are much more sensitive to SDS than the respective empty capsids. Thus, RNA clearly seems to be involved in the action of SDS on viruses. To account for this, an alternative mechanism of SDS action could be the binding of DS ions specifically to interfere with protein-RNA interactions stabilizing SDS-sensitive viruses. This interference could come about through (a) disruption of ionic protein-RNA interactions by the ionic part of the detergent or perhaps through (b) direct interference of the hydrocarbon chain of the DS ions with specific nonpolar protein-RNA interactions. Although most of the data do not rule out (b), they offer more direct support for (a). Therefore, we proposed the following hy-

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pothesis for the mechanism of action of SDS on viruses stabilized by protein-RNA interactions: Dodecyl sulfate ions (DS1 bind to protein subunits via the DS-hydrocarbon chains; if this binding brings the DS- sulfate groups close to regions where electrostatic interactions occur between positively charged residues of the protein subunits and the negatively charged phosphate groups of the RNA, they can neutralize these interactions and repel the phosphates thereby disrupting the ionic protein-RNA interactions. Thus, CMV, stabilized by its protein-RNA interactions, is degraded to protein and RNA, while TYMV, stabilized by protein-protein interactions, is not degraded. This hypothesis leads directly to several predictions which can be tested experimentally: (1) The more important the proteinprotein interactions, relative to proteinRNA interactions, the more resistant the virus will be to degradation by SDS; (2) capsids of viruses, when they exist, should be less sensitive than virions to SDS; (3) positive and neutral detergents with structures similar to that of SDS should not cause virus dissociation at the low concentrations at which SDS is effective. The following sections describe results of experiments which were carried out to test these and other predictions and to secure information concerning the nature of the degradation products. Effect of SDS on Different

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temperature effect on the SDS degradation of CMV and BMV between 5 and 25”. A slightly increased degradation observed at 40” may be due to the beginnings of thermal degradation, aided by the SDS. Effect of SDS on Capsids Prediction (2) was tested with virions and empty capsids from several viruses. Naturally occurring TYMV capsids were unaffected by 350 mM (10%) SDS at 20”, while the intact virus was 30% degraded at that SDS concentration. In addition, when the naturally occuring capsids were heated with 35 m&f, (1%) SDS at 60” for 2 min, these were unaffected. The virus was completely degraded under the same conditions. Alkali-prepared capsids of bacteriophage f2 have also been found to be much less sensitive than the virions to SDS (R. W. Henkens, personal communication). BMV capsids were prepared by self-assembly from BMV protein subunits (Bancroft, 1970). When capsids in 0.2 M sodium

Virions

As stated earlier (Boatman and Kaper, 1973), prediction (1) was confirmed by examining the effect of SDS on a number of simple RNA viruses, Thus, as shown in Fig. 1 and Table 1, PSV, CMV and AMV, stabilized mainly by protein-RNA interactions, were found to be very sensitive to SDS; TYMV and bacteriophage fz, stabilized mainly by protein-protein interactions, were resistant to SDS; and BMV at pH 6.0, stabilized by a mixture of proteinRNA and protein-protein interactions, was of intermediate sensitivity to SDS. BMV with only protein-RNA interactions present (pH 7.0) exhibits a sensitivity to SDS much like that of CMV etc. Tests showed that there is little or no

mM

SDS

FIG. 1. Effect of SDS on BMV, pH 7.0 (0-O); PSV, pH 7.0 (x-x); CMV, pH 7.0 (n-tb); CMV, pH 6.0, (+-+); BMV, pH 6.0 (0--O) at 0.2 mg/ml in 0.02 M NaPO, at 20”. Reaction mixtures were prepared from stock solutions of viruses at 1 mgiml and examined as soon as possible after preparation (about 20 min) by analytical ultracentrifugation. Measurements of residual intact virus were made after 20 min at 29,500 rpm, and an intact virus control was included with each experiment.

SDS AND PROTEIN-RNA

7

INTERACTIONS

TABLE 1 SDS CONCENTRATIONS AT WHICH DIFFERENT VIRIONS DISSOCIATE COMPARED WITH THEIR LYSINE AND AMINO ACID CONTENT

Virus” AMV BMV, pH 7.0 PSV CMV, pH 6.0 and pH 7.0 BBMV, pH 6.0 CCMV, pH 6.0 BMV, pH 6.0 TCV CarMV n SBMV TYMV TBSV TRSV BPMV

Dissociation begins knkf) 0.035 0.069 0.17 0.17 0.31 0.45 0.62 0.69 8.7 17.0 35.0 31.0 N N N

50% dissociation hQ4

Lysineh (%)

Basic amino acids* (%ir)

0.17 0.14 0.28 0.42

7.1 6.9 5.3 6.3

12.5 13.8 13.3 14.6

0.69 0.90 0.87 0.89 69 35-170 87 690 -

7.9 6.6 6.9 7.3 6.5 4.9 2.6 3.7 3.2 4.1 4.5

14.2 11.5 13.8 12.8 11.5 7.3 10.0 5.3 8.2 7.8 7.5

” Viruses at 0.2 mglml in 0.02 M NaPO,, pH 6.0 or 7.0, were mixed with several concentrations of SDS and examined immediately in the analytical ultracentrifuge. N, the virus was not affected by 700 mM (20%) SDS. * All lysine and basic amino acid percentages were calculated from data reviewed by Matthews (19701, except for PSV (Boatman, Tolin and Kaper, unpublished results) and bacteriophage f2 (Weber, et al., 1966).

chloride, 0.01 M sodium acetate, were combined with pH 5.2, 1 r&4 (0.03%) SDS, onethird of the capsids degraded (see Fig. 4). BMV virions, under the same conditions, were completely degraded by 0.5 mM (0.015%) SDS! (See below for effects of salts.) In 35 mM (1%) SDS 25% of the capsids still remained intact. BMV capsids were in all instances considerably more resistant tc SDS than were the virions. Since capsids lack the negative phosphates of RNA, there would be no repulsion between these and the DS sulfates. However, in the above experiments, approximately half of each capsid preparation degraded at a rather low SDS concentration, though one at which virus was completely degraded. Pfeiffer and Hirth (1974) have determined the ranges of pH and ionic strength in which various aggregates of BMV protein exist. Their work suggests that the pH and ionic strength we used to prepare and test BMV capsids are at the very limit of the range in which capsids form. Under these conditions, part of the capsids could be quite fragile because of imperfect assem-

bly and would be more subject to degradation at low SDS concentrations. Effect of Other Detergents

As stated earlier (Boatman and Kaper, 1973), prediction (3) was confirmed by examining the effect of the cationic detergents dodecyltrimethylammonium chloride (DTAC) and dodecylamine hydrochloride (DAH) and the nonionic detergent Triton X-100 on CMV and PSV. These viruses were unaffected by DTAC, DAH and Triton X-100. In another experiment, CMV was incubated with 0.18 rniW DTAC prior to being made 0.7 mM (0.02%) in SDS. In 0.7 rni&f SDS alone CMV was completely degraded. The CMV in the mixture of DTAC and SDS was essentially undegraded. The DTAC may have formed a 1:l complex with the SDS as a consequence of which there would be a reduction of the free SDS concentration to 0.52 mM. However, CMV in 0.52 mM SDS is still 75% degraded (cf. Fig. 1). Thus, some DTAC probably was

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bound to the CMV, protecting it from the action of SDS. These experiments with positively charged detergents (DTAC and DAH) offer compelling evidence that virus dissociation by SDS results from repulsion between negative sulfate of the DS ions and the RNA phosphates. If the hydrocarbon chain directs the binding of the detergent, then the SDS and DTAC probably bind at the same sites, but DTAC, having a positive instead of a negative charge, should not cause dissociation of the virus. No dissociation was observed. However, these results should be interpreted with caution, since DTAC probably does not bind to the virions as strongly as DS ions (Nozaki et al., 19741 and, if it does bind, may not bind to the same sites as DS. On the other hand, the blocking of the SDS effect by DTAC suggests that DTAC competes with SDS for the same binding sites. Effects of SDS on Reassembly and on Reversible Dissociation of CMV and BMV A fourth set of predictions arises from the working hypothesis, although with some reservations. These predictions follow automatically from the previous ones: (1) Inhibition of reassembly of virions from the protein subunits and RNA should occur at equally low or lower concentrations of SDS than those causing dissociation; (2) with those viruses for which capsids can be reassembled from the protein subunits, SDS should be less inhibitive for capsid self-assembly than for virion reassembly; (3) positively charged detergents should not inhibit reassembly of virions. The above predictions were first tested with the reversible dissociation of BMV and the self-assembly of BMV capsids. Reassociation of BMV from its components was prevented by 0.09 mM (0.0025%) but not by 0.035 n1J4 (0.001%) SDS. Capsid formation was successful when the dialysis was carried out in the presence of 0.035 mM (0.001%) SDS. Use of higher SDS concentrations was not feasible, because the low temperature and ionic strength at which capsid self-assembly must be performed prevents the use of SDS solutions

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of sufficient concentration. Attempts to prepare capsids by the more rapid dilution method failed, and therefore the effect of SDS on capsid self-assembly could not be rigorously tested. As expected, reassociation of BMV from its components in the presence of a much higher DTAC concentration (0.35 n&f or 0.01%) was not inhibited. CMV reassociation from its components was less sensitive to inhibition by SDS. It was prevented by 0.17 r&f (0.005%) SDS, but at half this concentration (0.09 mM or 0.0025%) a somewhat heterogeneous mixture of nucleoproteins sedimenting slightly more slowly than virions was formed. DTAC also did not affect the reassociation at 0.35 mM (0.01%). Self-assembly of CMV capsids has thus far proved impossible (Kaper and Geelen, 19711, and therefore the influence of SDS on this process could not be tested. Free protein subunits, with which reassembly of capsids or virions can occur, in all likelihood have the same tertiary structure as those in the intact virions. However, the subunits may bind SDS with greater ease since in their free state the exposed intersubunit contact areas probably possess additional DS binding sites. This could explain why SDS inhibits virion or capsid reassembly more effectively than it promotes their dissociation. Such a consideration enables us to predict that, due to repulsion of the RNA, virion reassembly should be more effectively inhibited by SDS than capsid reassembly, even though this could not be tested experimentally. Effects of Salts on Degradation

by SDS

To test the sensitivity of BMV to SDS under the same conditions that were used with the capsids, BMV was combined at pH 5.2 and 0.2 M sodium chloride with SDS at 0.52 mM (0.015%). The virus was completely degraded, and about one-third of the uv-absorbing material sedimented at a rate similar to that of the slowly sedimenting intermediate observed when BMV was combined with SDS at pH 6.0. At pH 5.2 and 6.0, the BMV virions were not degraded by 0.52 mM SDS or 0.2 M sodium chloride alone.

SDS AND

PROTEIN-RNA

To determine the generality of this apparent synergism between SDS and salts in the degradation of these viruses, CMV at pH 7.0 was combined with 0.17 mM (0.005%) SDS and lithium chloride in concentrations of O-O.4 M. CMV was not degraded by 0.17 mM SDS or by 0.4 M lithium chloride, but the combination of this low concentration of SDS and increasing amounts of lithium chloride resulted in increasing degradation of the CMV (see Fig. 2). Similarly, CMV was unaffected by 0.1 M sodium sulfate and only about 50% was degraded by 0.42 mM (0.012%) SDS; however, when CMV was combined with a mixture of 0.42 n&f SDS and 0.1 M sodium sulfate the virus was completely degraded. In this experiment the sodium sulfate was combined with CMV before addition of the SDS. Thus, in addition to demonstrating further the generality of the salt enhancement of SDS sensitivity, this result shows that sulfate ions do not compete with SDS. The facts that sodium sulfate alone does not degrade CMV (Boatman and Kaper, unpublished results) and that chloride ion (cf. Fig. 2) also enhances the SDS effect suggest that the sulfate group of SDS is

‘oo-i 0 20

0‘

'0 \

1 0 0.1

, 0.2

I 0.3

1

‘0 1 0.4

M LiCl

FIG. 2. Effect of LiCl on the degradation of CMV at 0.2 mg/ml in 0.02 M NaPO,, pH 7.0, with 0.17 mM (0.005%) SDS at 20”. Reaction mixtures were prepared from 1-mg/ml stock solutions of CMV and examined immediately after preparation in the analytical ultracentrifuge. Measurements of residual intact virus were made after 20 min at 29,500 rpm, and an intact CMV control was included with each experiment.

INTERACTIONS

9

not solely responsible for the specificity of the action of SDS. The synergistic action of salts and SDS could be due to increased binding of SDS by virions at higher ionic strengths. Such increased binding was observed by Nelson (1971), but several investigators have reported decreased SDS binding at higher ionic strength, presumably due to lower effective concentration of free SDS monomer (Pitt-Rivers and Impiombato, 1968; Reynolds and Tanford, 1970a,b; Fish et al., 1970). It is also possible that the binding of SDS to the virions weakens the protein-RNA interactions so that they are more sensitive to salt and the virions dissociate at a lower concentration of salt and SDS than in the presence of either alone. Preliminary binding studies with CMV and SDS suggest that the latter is the case. SDS and Other Viruses

In order to determine whether the working hypothesis of SDS interference with the stabilizing interactions of viruses could be generalized, other viruses were tested for stability in the presence of SDS. The results of these tests are shown in Table 1. Alfalfa mosaic virus (AMV), which is thought to be stabilized mainly by protein-RNA interactions (Hull, 1969), proved to be the most sensitive to SDS of any of the viruses examined; degradation was complete at 0.17 n&f (0.005%) SDS. This behavior is entirely consistent with the working hypothesis. The individual AMV components seemed to have essentially the same sensitivities to SDS. At the other extreme of the stability spectrum, tomato bushy stunt virus (TBSV) and tobacco ringspot virus (TRSV) were apparently unaffected even by 700 n-&f (20%;) SDS at 20”. Broad bean mottle virus (BBMV) at pH 6.0 exhibited a sensitivity intermediate between CMV and BMV, dissociating between 0.3 and 1.0 m&f (0.009 and 0.03%) SDS. After incubation at pH 7.0, BBMV was 86% degraded by 0.34 mM (0.01%) SDS and thus was somewhat more sensitive than CMV at this pH. The sensitivity differential for BBMV between the lower and higher pH was not as great as with BMV

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virus chlorotic mottle and cowpea (CCMV). CCMV, which, like BMV, is stabilized by a combination of protein-RNA and pH-dependent protein-protein interactions (Bancroft, 19701, behaved in every way like BMV with SDS. CCMV dissociated at pH 5.5 over a range of 0.34-1.36 mM (O.Ol-0.04%) SDS with the formation of a more slowly sedimenting intermediate, like that of BMV. After incubation at pH 7.0, CCMV was completely degraded by 0.34 mM (0.01%) SDS. Turnip crinkle virus (TCV) was degraded over a range of 0.1-35 mM SDS and was 50% degraded at a very low concentration of SDS, 0.85 mM (0.025%). Carnation mottle virus (CarMV) was about 10% degraded at 9 mit4 (0.27) SDS, about 50% at 35 mM (1%) and 75% degraded at 70 mM (2%) SDS. Southern bean mosaic virus (SBMV) exhibited no degradation at 35 mM (1%) but was degraded partially with the formation of a more slowly sedimenting intermediate and a product which sedimented like RNA at 90 m&f (2.5%) SDS (Fig. 3a). The intermediate sediments at about one-third the rate of the virus and appears to be similar to the “subviral entity” found by Sehgal and Sinha (1974). Hull (personal communication) has found that when SBMV is pretreated with EDTA, it degrades between

C

b

FIG. 3. Effect of SDS on SBMV (a) and bacteriophage f2 (b) at 0.2 mg/ml in 0.02 M NaPO,, pH 7.0, and 20”. The reactants were combined and examined in the analytical ultracentrifuge immediately. The photoelectric scanning patterns (265 nm) represent sedimentation patterns of (a) SBMV combined with 90 m&f (2.5%) SDS and (b) bacteriophage fz combined with 175 mM (5%,) SDS, 20 min after the centrifuge reached a speed of 29,500 rpm.

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0.001 and 0.01% SDS. Apparently the SBMV is stabilized by divalent cations, and, upon their removal by chelation with EDTA, the resulting virions become sensitive to SDS because their primary means of stabilization are now the protein-RNA interactions. Further degradation was observed at 170 and 350 mM (5 and 10%) SDS, but complete degradation of the virus was not accomplished. TYMV proved to be very resistant, showing only slight dissociation at 680 mM (19%) SDS. Bacteriophage f2 exhibited intermediate resistance to SDS, degrading slightly at 17 mM (0.5%) and about 50% at 170 n&f (5%) SDS (Fig. 3b). Table 1 lists the viruses tested in the order of increasing degree of resistance to SDS. The viruses near the top of Table 1 are most sensitive and have a strong contribution from protein-RNA interactions to the overall stability of the virions, while those near the bottom are stabilized mainly by protein-protein interactions (cf. Kaper, 1973). Binding

of SDS to Viruses

It was important to determine how much SDS was bound by viruses sensitive and resistant to SDS. Figure 4 shows that both BMV and CMV bound SDS at low detergent concentrations and that at all concentrations tested CMV bound more SDS than BMV. On the other hand, TYMV bound no detergent under these conditions. Ultracentrifugal examination of the equilibrated samples showed that CMV was completely degraded at 0.17 mM (O.O05%c)SDS and pH 6.4 and that BMV was 85% degraded. This contrasts with a somewhat greater stability of these viruses under normal (nondialysis) SDS dissociation conditions (cf. Fig. 1). This apparent disagreement in the results from the two types of experiments can be explained by examining a number of factors peculiar to the dialysis conditions employed. When dialyzed with 0.17 n&f SDS, CMV bound 50 DS-/subunit and was degraded. Under nondialysis conditions of dissociation, the binding of DS ions would deplete the reaction mixture of free SDS and would still fail to attain a

SDS AND

-5

-4

-3 LOG

-2 M

PROTEIN-RNA

-1

SDS

FIG. 4. Binding of SDS to BMV (O-0); CMV (0-k and TYMV (a---a). Viruses at 0.2 mg/ ml in 0.02 M NaPO,, pH 6.4, and 0.017 m&f (0.0005%), 0.17 mkf (0.005%), 0.34 IlLW (O.Ol%), 0.7 n&f (0.02%), and 1.4 mM (0.04%) SDS containing the appropriate amount of “?S-labeled SDS were dialyzed against several changes of the same solution for 72 hr at room temperature. Aliquots of the virus samples, undialyzed virus samples, dialysate and undialyzed solution were counted, and the amount of SDS bound was calculated as follows: [(Radioactivity (cpm) of 1.0 ml of dialyzed virus cpm of 1.0 ml of dialysate)/cpm of 1.0 ml of a l.Ommol/ml solution of SDS1 + [(A,,,, of dialyzed virus/ E!?, of virus) x (percentage of protein in virus/ molecular weight of protein)].

level of 50 DS-/subunit. For instance, when CMV is incubated with 0.17 n&f SDS under the conditions of Fig. 1, binding of 10 DS ions/subunit would decrease the SDS concentration to 0.08 mM, and this would be insuffIcient for CMV degradation even under dialysis conditions. A second factor, which could explain the decreased stability of BMV, is the fact that, at pH 6.4, BMV virions have begun to swell (Incardona et al., 1973) and thus presumably have weakened protein-protein interactions. These binding experiments expose a possible weakness in the working hypothesis. SDS binding data demonstrate that TYMV’s resistance to SDS was caused directly by its failure to bind DS- ions at low SDS concentrations and was not necessarily due to the strength with which protein-protein interactions stabilized the capsid as assumed by the working hypothe-

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11

sis. If other SDS-resistant viruses also fail to bind DS ions, such a correlation would suggest a predisposition of protein subunits in viruses stabilized by proteinRNA interactions to bind DS ions and thus to be more susceptible to the dissociative effect of SDS. Nelson (1971) has indeed demonstrated that, although most proteins bind DS ions easily, a few do so very poorly and are therefore resistant to denaturation by SDS. If a predisposition to bind DS- ions (but not structurally similar positively charged detergents) can be demonstrated for certain viral proteins, it would also be important to learn whether this property exists only when the subunits are integrated with the RNA into virions or whether capsids bind DS ions equally well. It is not yet known what structural characteristics predispose proteins to bind detergent anions such as those of SDS. This property may rely upon the proper combination and location of apolar and basic residues as determined by the primary structures of the protein. There is a significant correlation between the sensitivity to SDS of the viruses tested and the lysine content of their protein subunits (Table 1). This suggests that the lysyl side chains may at least partially determine the specific locations of the DS-binding sites by electrostatic interaction between the ammonium (amino) group of lysine and the sulfate of DS. Nozaki et al. (1974) have suggested that the polar groups of ionic detergents do play a role in binding of the detergent with proteins and that, while hydrophobic interactions of the detergent with protein are important in directing the binding, the polar end would be attracted to side chains of the opposite charge. A mechanism in which SDS behaves as a bifunctional ligand was invoked to explain the stabilization by SDS of human serum albumin against denaturation by urea (Markus et al., 1964).Under certain conditions, SDS could even reverse the effects of urea. Also, Habeeb (1966) proposed that SDS binds to bovine serum albumin so that the sulfates are near lysyl e-amino groups. In this report, our working hypothesis proposes a similar bifunctionality for

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SDS in which the hydrocarbon part directs binding and the sulfates, which may aid in directing the binding, exert repulsive rather than attractive forces. Examination of Virus Degradation Products A knowledge of the nature of the degradation products formed when viruses are combined with SDS should be helpful in elucidating the mode of action of SDS on these viruses. CMV (pH 7.0), BMV (pH 7.0), and BBMV (pH 6.0) were combined with concentrations of SDS at which small amounts of intact virus remained. Analytical sedimentation patterns showed small amounts of rapidly sedimenting material closely resembled the respective phenolisolated multicomponent RNA’s. Thus at the pH’s indicated, these viruses are probably directly degraded into free RNA and protein. As reported earlier (Boatman and Kaper, 19731, at pH 7 sedimentation patterns for completely degraded BMV and CMV were very much like those for phenol-extracted RNA, while those of PSV showed material sedimenting more rapidly than phenol-extracted RNA. This was in general agreement with results from polyacrylamide electrophoresis and indicates that PSV, although initially more sensitive to SDS, apparently yields fragments which have residual protein bound to the RNA components. As mentioned earlier (Boatman and Kaper, 19731, BMV forms a more slowly sedimenting intermediate when combined with SDS at and below pH 6.0. The swelling may be due to repulsion between the sulfates of bound DS- ions and other sulfates or phosphates. At low SDS concentrations the virions do not dissociate because the pH-dependent protein-protein interactions remain intact, but at higher SDS concentrations more DS ions bind and the linkages cannot maintain their integrity. The isolated intermediate appeared in the electron microscope as slightly irregular particles with diameters which were 1220% (3-5 nm) greater than those of the virus at the same pH (see Fig. 5). This was determined by measuring 20 particles each

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of intact BMV and the swollen intermediate and averaging the values obtained for each. When a sample of the virus was combined with SDS at a concentration at which virus, intermediate, and RNA all were present and this mixture was applied immediately to a grid, normal virus particles, “swollen” particles and partially degraded particles were seen in the preparation (see Fig. 5). The differences in diameter between virion and intermediate were confirmed by comparison of the two on these grids. The intermediate appeared to be quite stable and could be kept for weeks without appreciable change. RNA from the BMV intermediate was also extracted with phenol and examined by gel electrophoresis. It resembled the RNA pattern obtained from intact virus except that it appeared somewhat degraded, probably due to ribonuclease sensitivity of the swollen particles. Although the A 26,jA280ratio of the intermediate was indicative of loss of a small amount of RNA (1.56 vs 1.63 for intact BMW, there was no evidence that there was preferential loss of any one RNA component. A similar BMV intermediate, which appeared and disappeared as pH increased from 5.9 to 7.0 at high ionic strength, has been observed by Pfeiffer and Hirth (1974b). Thus, it seems that SDS degrades most of the viruses tested to RNA and protein. Little is known, however, about the state of aggregation of the released protein at the low SDS concentrations employed. In the case of CMV, ultracentrifugation using schlieren optics indicates that the released protein sediments very slowly and might be either monomers or dimers of the protein subunits. The Use of SDS to Probe Stabilizing Interactions The variation in structural sensitivity of small RNA viruses to SDS (Table 1) suggests that, in principle, this compound could be used as a probe to measure the relative importance of protein-protein and protein-RNA interactions in the stabilization of small RNA viruses and would allow a crude categorization of viruses according

SDS AND

PROTEIN-RNA

INTERACTIONS

F IG. 5. BMV, pH 6.0 (top); a 0.2-mglml suspension of BMV in 0.2 M NaPO,, pH 6.0, with 1.0 mkf (0 .03%) SDZj (middle); BMV swollen intermediate, pH 6.0 (bottom). All samples were stained with 1% u ranyl ace1Late. Bar represents 100 nm. The swollen intermediate was isolated from 0.5-300/c sucrose density of a l-mg/ml mixture of BMV in the same buffer vvith 1 ml clients in 0.02 M NaPO,, pH 6.0. One milliliter mM (0.03%) SDS was layered on the gradient.

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BOATMAN

to their stabilizing interactions on this basis alone (Kaper, 1972, 1973). If such a categorization is attempted, there seem to be among these viruses representatives of five kinds of behavior with SDS (exposure to SDS is brief, periods of up to 1 hr): (i) Dissociation begins at a low concentration and is complete within a narrow range (AMV, CMV, PSV, TAV (Habili and Francki, 1974); BMV, BBMV, and CCMV at pH 7.0; SBMV after EDTA treatment); (ii> dissociation begins at a moderately low concentration and is complete within a narrow range (BMV, BBMV, and CCMV, all at pH 6.0 or lower); (iii) dissociation begins at a relatively low concentration and is complete within a wide range (no identifiable intermediate formed) (TCV); (iv) dissociation begins at a moderate concentration and continues over a wide range with no definite point at which complete degradation is reached (seemingly specific intermediates sedimenting more slowly than virions may be formed) (CarMV, f2, SBMV, TYMV); (v) no dissociation is observed at any SDS concentration (TBSV, TRSV, BPMV). The structures of the viruses in these classes are maintained by different combinations of various kinds of stabilizing interactions: Class (i) is stabilized mainly by protein-RNA interactions. Members of class (i) bind DS ions strongly and rapidly, while members of class (VI probably do not bind DS- ions at all under the conditions of the experiment. This would suggest that the surfaces of class (i) viruses have a certain degree of hydrophobic character and differ greatly from the surfaces of class (v) viruses. Class (ii) viruses probably bind DS- more slowly and less strongly than class (i), but the initial unfolding of the virus protein gives forms that bind DS more strongly resulting in complete dissociation. The behavior of viruses in classes (iii) and (iv) is more difflcult to rationalize. It is probable that these viruses owe their stability in large part to protein-protein interactions. Once the SDS concentration exceeds the critical micelle concentration (about lo-” M at ionic strength 0.11, the limiting concentration of the monomer has been attained, and it is possible that viruses interact with the mi-

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celles. The interaction of SDS with icosahedral viruses is complex, and perhaps the relatively simple working hypothesis presented here ultimately will prove to be inadequate in explaining these phenomena. One thing seems certain: The nucleic acid has an important role in the ability of SDS to degrade icosahedral viruses. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Carolyn K. West, Rita Tickel, and W. R. Povish and thank Dr. S. A. Tolin for assistance with electron microscopy. Sandra Boatman wishes to thank Dr. Russell L. Steere for his hospitality in allowing her to use the facilities of the Plant Virology Laboratory and thanks Hollins College for research grants. REFERENCES J. B. (1962). Purification and properties of bean pod mottle virus and associated centrifugal and electrophoretic components. Virology 16, 419-427. BANCROFT, J. B. (19701. The self-assembly of spherical plant viruses. A&m. Virus Res. 16,99-134. BANCROFT, J. B., WAGNER, G. W., and BRACKER, C. E. (19681. The self-assembly of a nucleic acid-free pseudo-top component for a small spherical virus. Virology 36, 146-149. BOATMAN, S., and KAPER, J. M. (1973). Forces responsible for the generation of virus structures: The use of SDS to probe protein-RNA interactions. In “Generation of Subcellular Structures” (R. Markham, J. B. Bancroft, D. R. Davies, D. E. Hopwood, and R. W. Horne, eds.), pp. 123-134. North-Holland/American Elsevier, Amsterdam. BOATMAN, S., TOLIN, S. A., and KAPER, J. M. (1973). A comparison of properties of peanut stunt virus and cucumber mosaic virus. Phytopathology 63, 801. BOCKSTAHLER, L. E., and KAESBERG, P. (19621. The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2, l-9. BOCKSTAHLER, L. E., and KAESBERG, P. (1965). Isolation and properties of RNA from bromegrass mosaic virus. J. Mol. Biol. 13, 127-137. BRAKKE, M. K. (19711. Degradation of brome mosaic and tobacco mosaic viruses in bentonite. Virology 46, 575-585. CARUSI, E. A., and SINSHEIMER, R. L. (1963). The physical properties of a protein isolated from bacteriophage &X174. J. Mol. Biol. 7, 388-400. CASSEL, J. G., GALLAGHER, J., REYNOLDS, J. A., and STEINHARDT, J. (1969). The role of transport phenomena in ion binding studies of serum albumin. Biochemistry 8, 170661713. DUNN, D. B., and HITCHBORN, J. H. (19651. The use BANCROFT,

SDS AND

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of bentonite in the purification of plant viruses. Virology 35, 171-192. EMERSON, M. F., and HOLTZER, A. (1967). On the ionic strength dependence of micelle number. II. J. Phys. Chem. 71, 1898-1907. FINCH, J. T., and KLUG, A. (1966). Arrangement of protein subunits and the distribution of nucleic acid in turnip yellow mosaic virus. II. Electron microscope studies. J. Mol. Biol. 15, 344-364. FINCH, J. T., KLUG, A., and VAN REGENMORTEL, H. M. V. (1967). The structure of cucumber mosaic virus. J. Mol. Biol. 24, 303-305. FISH, W. W., REYNOLDS, J. A., and TANFORD, C. (19701. Gel chromatography of proteins in denaturing solvents. Comparison between sodium dodecyl sulfate and guanidine hydrochloride denaturants. J. Biol. Chem. 245, 5166-5168. FRAENKEL-CONRAT, H., SINGER, B., and WILLIAMS, R. C. (1957). Infectivity of viral nucleic acid. Biochim. Biophys. Acta 25, 87-91. HABEEB, A. F. S. A. (19661. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal. B&hem. 14, 328-336. HABILI, N., and FRANCKI, R. I. B. (1974). Comparative studies on tomato aspermy and cucumber mosaic viruses. II. Virus stability. Virology 60, 29-36. HERSH, R. T., and SCHACHMAN, H. K. (1958). On the size of the protein subunits in bushy stunt virus. Virology 6, 234-243. HULL, R. (1969). Alfalfa mosaic virus. Advan. Virus Res. 15. 365-433. HULL, R., REES, M. W., and SHORT, M. N. (1969). Studies on alfalfa mosaic virus. I. The protein and nucleic acid. Virology 37, 404-415. INCARDONA, N. L., MCKEE, S., and FLANEGAN, J. B. (1973). Noncovalent interactions in viruses: Characterization of their role in the pH and thermally induced conformational changes in bromegrass mosaic virus. Virology 53, 204-214. JONARD, G., and HIRTH, L. (1966). Action de l’uree sur le virus de la mosaique jaune du navet: Formation de capsides artificielles. C. R. Acad. Sci. Paris 263D, 1909-1912. KAPER, J. M. (1971). Studies on the stabilizing forces of simple RNA viruses. I. Selective interference with protein-RNA interactions in turnip yellow mosaic virus. J. Mol. Biol. 56, 259-276. KAPER, J. M. (1972). Experimental analysis of the stabilizing interactions of simple RNA viruses, In “RNA Viruses: Replication and Structure,” (E. M. J. Jaspars and A. Van Kammen, eds.1, F.E.B.S. Symposium 27, pp. 19-41. North-Holland, Amsterdam. KAPER, J. M. (1973). Arrangement and identification of simple isometric viruses according to their dominating stabilizing interactions. Virology 55, 299-304.

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KAPER, J. M., and GEELEN, J. L. M. C. (1971). Studies on the stabilizing forces of simple RNA viruses. II. Stability, dissociation, and reassembly of cucumber mosaic virus. J. Mol. Biol. 56, 277294. KAPER, J. M., and WEST, C. K. (1972). Polyacrylamide gel separation and molecular weight determination of the components of cucumber mosaic virus RNA. Prep. Biochem. 2, 251-263. KRUSEMAN, J., KRAAL, B., JASPARS, E. M. J., BOL, J. F., BREDERODE, F. TH., and VELDBTRA, H. (1971). Molecular weight of the coat protein of alfalfa mosaic virus. Biochemistry 10, 447-455. LANE, L. C., and KAESBERG, P. (1971). Multiple genetic components in bromegrass mosaic virus. Nature New Biol. 232, 40-43. MAIZEL, J. V., JR. (1971). Polyacrylamideelectrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5. pp. 179-246. Academic Press, New York. MARKUS, G., LOVE, R. L., and WISSLER, F. C. (1364). Mechanism of protection by anionic detergents against denaturation of serum albumin. J. Biol. Chem. 239, 3687-3693. MATTHEWS, R. E. F. (1970). “Plant Virology.” Academic Press, New York. NELSON, C. A. (1971). The binding of detergents to proteins. I. The maximum amount of dodecyl sulfate bound to proteins and the resistance to binding of several proteins. J. Biol. Chem. 246, 38953901. NOZAKI, Y., REYNOLDS, J. A., and TANFORD, C. (1974). The interaction of a cationic detergent with bovine serum albumin and other proteins. J. Biol. Chem. 249, 4452-4459. PFEIFFER, P., and HIRTH, L. (1974). Aggregation states of brome mosaic virus protein. Virology 61, 160-167. PITT-RIVERS, R., and IMPIOMBATO, F. S. A. (1968). The binding of sodium dodecyl sulphate to various proteins. Biochem. J. 109, 825-830. REYNOLDS, J. A., ~~~TANFORD, C. (1970a). The gross conformation of protein-sodium dodecyl sulfate complexes. J. Biol. Chem. 245, 5161-5165. REYNOLDS, J. A., and TANFORD, C. (1970b). Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. Proc. Nat. Acad. Sci., USA 66, 1002-1007. SCOTT, H. A. (1963). Purification of cucumber mosaic virus. Virology 30, 103-106. SEHGAL, 0. P., and SINHA, R. C. (1974). Characteristics of a nucleoproteinaceous subviral entity resulting from partial degradation of southern bean mosaic virus. Virology 59, 499-508. SREENIVASAYA, M., and PIRIE, N. W. (1938). The disintegration of tobacco mosaic virus preparations with sodium dodecyl sulphate. Biochem. J.

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32,1707-1710. STEINHARDT, J., and REYNOLDS, J. A. (19691.1n “Multiple Equilibria in Proteins.” Academic Press, New York. STOLS, A. L. H., and VELDSTRA, H. (1965). Interaction of turnip yellow mosaic virus with quaternary ammonium salts. Virology 25, 508-515. TANFORD, C. (19681. Protein denaturation. Part A. Characterization of the denatured state. A&m.

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Protein Chen. 23, 121-282. TOLIN, S. A. (19671. Properties ofpeanut stunt virus. Phytopathology 57, 834. VAN REGENMORTEL, H. M. V. (19641. Purification of plant viruses by zone electrophoresis. Virology 23, 495-502. WEBER, K., NOTANI, G., WIKLER, M., and KONIC~ BERG, W. (19661. Amino acid sequence of the f2 coat protein. J. Mol. Biol. 20, 423-425.