Molecular organization and stabilizing forces of simple RNA viruses

VIROLOGY

71, 185-198 (1976)

Molecular Organization and Stabilizing Forces of Simple RNA Viruses V . The Role of Lysyl Residues in the Stabilization of Cucumber Mosaic Virus Strain S

J . M . KAPER Plant Virology Laboratory, Plant Protection Institute, Agricultural Research Service, U .S . Department of Agriculture, Beltsville, Maryland 20705 Accepted December 29, 1975

Experiments have been carried out to identify the chemical groups responsible for the stabilization of CMV (strain S) . At pH 10.3 C MV-protein and CMV-RNA reassociated into nucleoproteins that possess no specific viruslike structure . In rate zonal centrifugation the nucleoproteins sedimented as three zones trailing the virus marker, The nucleoproteins were isolated and analyzed for RNA component contents with polyacrylamide electrophoresis . In order of increasing sedimentation rates, they contained RNA components 4, 3, and 1 + 2 . Separate treatment of CMV-protein and CMV-RNA at pH 10 .3, followed by neutralization, had no effect on their capability to reassemble normally at pH 7 . The nucleoproteins formed at pH 10 .3 could be dissociated by 2 .0 M LiCI . The protein reisolated in this manner could also be reassembled at pH 7 with CMV-RNA to form normal virions . These experiments suggest that the aborted reassembly at alkaline pH is caused mainly by a deprotonation of protein amino-acid residues and RNA bases with a pK around 10, and that reprotonation can reverse any pH-induced conformational perturbation of CMVprotein . Reaction of CMV with the lysyl-specific reagent trinitrobenzene-sulfonic acid (TNBS) led to structural transformation of the virions into slower sedimenting trinitrophenylated (TNP)-nucleoproteins with undefined structures . These nucleoproteins also contained CMV-RNA components 4, 3, and 1 + 2 in order of increasing sedimentation rates . The time-course of the TNBS reaction and the structural transformation suggested that approximately 3 of 15 lysyl residues in each subunit are essential to the stability of the virion . Protein could easily be prepared from 3TNP-CMV by means of 2 .0 M LiCI dissociation . The yellow color and the spectral properties provided direct identification of CMV-protein as the site of trinitrophenylation . CMV with nine TNP substituents per subunit failed to dissociate under the influence of 2 .0 M LiCI . Reassembly at pH 7 .2 failed when 3TNP-CMV-protein was used as protein constituent . Reaction of the D-strain of CMV with TNBS was much slower . The structural transformation occurred at a higher TNP substitution level, and was more gradual than with CMV-S . This observation is in agreement with the generally greater stability of CMV-D . Of several bromoviruses tested, the reactivity with TNBS decreased in the following order : BBMV > CCMV > BMV . All but BMV degraded in their entirety under the conditions investigated .

most exclusively stabilized by proteinRNA interactions . Several lines of evidence suggest that these interactions consist mainly of ionic linkages between positively charged amino-acid residues on CMV-protein and the negative phosphate groups of CMV-RNA . These linkages are

INTRODUCTION

In previous publications from this laboratory (Kaper and Geelen, 1971 ; Kaper, 1972 ; Kaper, 1973) we argued that cucumber mosaic virus (CMV)' is probably al' Unless mentioned specifically, the S strain of CMV is referred to . 185 Copyright Academic Press, All rights of reproduction in any form reserved . C©

1976

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broken at high concentrations of certain neutral salts (cf. Kaper, 1975), and can be remade by lowering the salt concentration, giving rise to reassembled virions . Boatman and Kaper (1976) have shown that CMV and other viruses, stabilized by protein-RNA interactions, are degraded at very low concentrations of the anionic detergent sodium dodecyl sulfate (SDS) . They proposed that DS - ions bind to CMV protein in proximity to positive amino-acid residues . The latter would be neutralized, and simultaneously the bound DS - ions would also repel the RNA, causing disintegration of the virion structure . Circumstantial evidence for some type of involvement of protonated lysyl residues in the stabilization of the structure of CMV virions was produced by Kaper and Geelen (1971), who showed that the virus was sensitive to alkaline pH . Virions degraded into slower sedimenting nucleoproteins at pH 10 . In addition, the normal reassembly of CMV-S could be aborted at pH 10 or higher, and resulted in the formation of nucleoproteins sedimenting at 36, 60, and 75 S . The coincidence of the pH's of degradation and anomalous component reassembly of CMV with the pK that would be expected of lysyl residues in proteins (Tanford, 1962) led Kaper and Geelen (1971) to propose that deprotonation of the lysyl residues in CMV would perturb the normal minimum free energy conformation of the virion . This could lead to a structural transition of CMV and/or to anomalous reassociation of its components. In this publication it will be shown that the nucleoproteins resulting from abnormal (pH 10 .3) reassociation of CMV components consist of nonspecific protein-RNA reassociation products, the size of which is determined by the size of the RNA components of CMV . Furthermore, the reaction of CMV with the lysyl-specific reagent trinitrobenzenesulfonic acid (TNBS) (Means and Feeney, 1971), and its consequences for the virion structure and its reassembly properties will constitute direct evidence for a pivotal role of lysyl residues in the stabilization of CMV and structurally related viruses .

Some of this work was reported at the Third International Congress of Virology, Madrid, 1975 . MATERIALS AND METHODS

Apparatus . The instruments used in this study were described in earlier publications of this series (Kaper, 1971 ; Kaper and Geelen, 1971 ; Re and Kaper, 1975; Boatman and Kaper, 1976) . Materials . All chemicals used were of analytical or best available grade, and were purchased from commercial supply houses . Commercial TNBS was recrystallized from 5 N HCl . CMV-S and CMV-D were cultivated, isolated, and purified as previously described (Lot and Kaper, 1973) . CMV-Q was cultivated, isolated, and purified like CMV-S ; CMV-R and peanut stunt virus (PSV) like CMV-D (Lot and Kaper, manuscript in preparation) . Brome mosaic virus (BMV) was obtained with the method of Bockstahler and Kaesberg (1962) . Cowpea chlorotic mottle virus (CCMV) and broad bean mottle virus (BBMV) were gifts of S. A. Tolin, Virginia Polytechnic Institute and State University. Methods . Preparation of protein from CMV . In the procedure described in preceding publications (Kaper, 1969 ; Kaper and Geelen, 1971), CMV was added to a mixture of LiCI, Tris-HC1 (pH 7.2), and Cleland's reagent, such that the final strengths of these reagents were 2 .0, 0.02, and 0.001 M, respectively . For the work described in this publication, the Tris-HC1 buffer was omitted from the dissociation mixture . This omission did not affect the protein preparation in terms of yield or reassembly capability . In view of the scaling-up of the reassembly required in many experiments, several tests were performed in which the virus concentration in the dissociation mixture was as high as 20 mg/ ml. This also had no effect on the final protein preparation . Virus was added to the reagent mixture at room temperature (ca . 22°) . The dissociation mixture was left at room temperature for about 2 min, and then transferred to an icebath for 30 min, during which an RNA precipitate devel-



ROLE OF LYSYLS IN CMV STABILIZATION

oped . This was removed by low-speed centrifugation . The supernatant contained the protein, which had to be kept in the cold . In the absence of a reliable extinction coefficient, the concentration of CMV-protein was estimated on the assumption that the dissociation of the virus by this method is quantitative . Ultraviolet spectra of CMV-protein were measured upon dilution in 2 .0 M LiCI . Reassembly methods . Reassembly or reassociation of CMV-protein with CMVRNA, either by the dilution or the dialysis methods, was basically performed as described previously (Kaper and Geelen, 1971) . However, in view of the different pH manipulations in the anomalous reassociation experiments, some modifications were introduced. Furthermore, the methods were scaled-up in terms of initial protein and RNA concentrations, such that the theoretically expected concentration level of reassembled CMV could reach 2 mg/ml . In practice, CMV-protein in 2 .0 M LiCI (at 1O X its final reassembly concentration) was mixed with 0 .08 its volume of 1 .0 M buffer at the preselected reassembly pH . To this protein solution a stoichiometric amount of RNA in distilled water was added such that a 1 :4 dilution of the protein was accomplished . The mixture was left in an ice bath for 10 min, and was subsequently further diluted to lOx the original protein solution volume (reassembly by dilution) . Alternatively, the mixture was dialyzed against the final reassembly solvent (reassembly by dialysis) . TNBS reactions. Reactions with TNBS were carried out at constant temperature in the spectrophotometer . CMV at approximately 0.3 mg/ml in 0 .1 M Na phosphate buffer (pH 8) or 0 .1 M Na borate buffer (pH 9) was allowed to react with the reagent at final strength of 0 .05% (=10x excess over total lysyl amino groups in CMV) . Optical density readings at 345 nm were compared with those of a 0 .05% TNBS solution in the same buffer . The degree of trinitrophenylation per subunit was calculated, and corrected for TNBS disappearance according to methods described by Goldfarb (1966), and Scheele and Lauffer (1969) . Because the light scattering of CMV solutions at

187

this concentration was negligible at 345 run, it was not considered in the calculation of the results . To examine the degree of structural transformation caused by the reaction with TNBS, samples were withdrawn for ultracentrifugal examination . Several methods for inhibition of the reaction and the resulting transformation were tested . Cooling to 0°, addition of excess lysine, dialysis, or a combination of these methods failed to be completely effective . A combination of cooling to 0° and ultracentrifugation as soon as feasible appeared the most satisfactory way to trap the structurally transformed virus at the time of inhibition . However, analytical ultracentrifugation could not be used to examine these samples because of the high ultraviolet absorbance of the samples due to the TNBS excess . Rate zonal centrifugation on sucrose gradients was the most suitable method because the TNBS remained at the top of the gradient . This had the additional advantages of allowing separation of the TNBS from the virus or the reaction products and preventing further reaction . During the reaction a virus control was maintained under the same conditions of concentration, temperature, and pH, for a length of time corresponding to the sample that was withdrawn last . It was ultracentrifuged simultaneously with the trinitrophenylated samples . In the scaled-up version of the reaction, virus and TNBS concentrations were increased 10-fold . The final pH of the reaction mixture in the pH 8 buffer was 7 .2 under these conditions ; in the pH 9 buffer there was virtually no pH change . Because of the increased virus and reagent concentrations, direct monitoring of the reaction was impossible . Consequently, the reaction was carried out in a water bath, and at set time intervals aliquots of the reaction mixture and TNBS control were diluted 1:50 in the corresponding buffers for optical density comparison at 345 ran . The time-course and the structural transformations of the scaled-up reaction under these conditions closely resembled the pH 8 reaction at lower concentrations . This result was fortuitous, and was probably due to the fact that the increased reaction



J . M . KAPER

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rate due to the higher reactant concentrations was approximately compensated for by a concomittant decrease in reaction rate due to the lower pH . Standard methods . Rate zonal centrifugation was performed on 0 .2-0 .8 M sucrose gradients in various buffers and/or salt solutions, depending on the purpose of the experiment . All runs were performed with the Beckman S W 41 rotor .- Nucleoprotein mixtures were ultracentrifuged at 40,000 rpm for either 90 min (CMV approximately in the middle of the tube), or 150 min (CMV close to bottom of the tube) . CMV-RNA samples were ultracentrifuged at 32,000 rpm for 16 hr . Polyacrylamide-gel electrophoresis was carried out as described previously (Kaper and West, 1972) . If nucleoproteins were examined for their RNA components, 1% SDS was included in the application solvent . CMV concentrations were estimated using an extinction coefficient of E 1, 2 1, of 50 (Kaper et al ., 1965) . Negative staining for electron microscopy was done with a 1% uranyl acetate solution in distilled water . RESULTS

Anomalous Reassociation of CMV Components at Moderately Alkaline pH Isolation and analysis of nucleoprotein products for RNA content . For isolation of the nucleoprotein products resulting from abnormal (pH 10 .3) reassociation after rate zonal centrifugation on sucrose gradients, the scaled-up method of reassembly was used . Under these conditions, the final concentration of total nucleoprotein was equivalent to a CMV concentration of 1 .22 .0 mg/ml . Samples of pH 10 .3 reassociation mixture were centrifuged in sucrose gradients made up with reassembly solvent buffered at pH 10 .3 . The sedimentation pattern of Fig . 1A shows three major zones at 0 .40, 0 .61, 0 .73 of the depth of a ' Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U .S . Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable .

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Rate zonal centrifugation patterns of different CMV materials on sucrose gradients . (A) Nucleoproteins NP 1 .4 obtained in pH 10 .3 reassociation of CMV-RNA and CMV-protein . Arrow indicates position of CMV marker . Centrifugation conditions : 165 min at 40,000 rpm at 5' . (B) CMV reassembled at pH 7 .2 . Arrow indicates CMV marker sedimentation; a and b are zones collected from the gradient for RNA component polyacrylamide-gel electrophoresis (ef. Fig . 2B) . Centrifugation conditions: 90 min at 40,000 rpm, and 5' . (C) CMV-RNA isolated by means of phenol-SDS extraction (Kaper and West, 1972) . Centrifugation conditions : 960 min at 32,000 rpm and 5' . Sedimentation is from left to right . FIG. 1 .

CMV marker at pH 7,2 . In addition there is a faster leading shoulder, a slow minor zone, and some slow sedimenting material at the meniscus . For comparison, a normal (pH 7 .2) reassembly sedimentation pattern of CMV is shown (Fig . 1B), and also a sedimentation pattern of CMV-RNA (Fig . 1C) . The sedimentation patterns shown in Figs . 1A and C are strikingly similar except for the different centrifugation conditions . The three major zones NP4, NP3, and NP1+2 (cf. Fig . IA) were collected, pooled with corresponding zones of tubes run simultaneously, and concentrated by ultracentrifugation . The pellets thus obtained exhibited poor solubility in distilled water, in low ionic strength pH 7 buffers, and also in the pH 10 .3 reassembly solvent . They were dissolved in solvents containing SDS . Their ultraviolet spectra were characteristic of nucleoproteins with maxima and minima at about 260 and 240 nm, respectively . Figure 2A shows the polyacrylamide-gel electrophoresis patterns of the nucleoprotein zones . The three major centrifugal zones with increasing sedimentation rates contained the CMVRNA components 4, 3, and 1 + 2, respectively . Apparently, under alkaline reassembly conditions, CMV-RNA and protein

ROLE OF LYSYLS IN CMV STABILIZATION

recombine into structures containing the individual CMV-RNA segments, and the latter also determine the sedimentation rates of the nucleoproteins formed . For comparison, Fig . 2B shows polyacrylamide-gel electrophoresis patterns of the RNA of pH 7 .2 reassembled CMV, isolated and concentrated from sucrose gradient centrifugation fractions (cf. Fig . 1B) . All CMV-RNA components were recovered from the band sedimenting with the rate of CMV, and from the next faster sedimenting zone, suggesting that the latter was a virus aggregate . Figure 2C gives the electrophoretic pattern of isolated CMV-RNA . Further evidence that the RNA components of CMV determine the size of the nucleoproteins formed in the pH 10 .3 reassociation, was obtained from experiments in which the RNAs of CMV strains with characteristically different RNA component composition (Lot and Kaper, manuscript in preparation) were used to reassociate with CMV-S protein . The sedimentation patterns of the reassociated nucleoproteins reflected faithfully the RNA composition of the strains used (Fig . 3) . Attempts were made to visualize the different nucleoprotein particles obtained in pH 10 .3 reassociation by means of electron microscopy . To this end, either unfractionated pH 10 .3 reassociation mixtures or the zones fractionated by sucrose gradient centrifugation (but before concenEration) were examined directly, or after dialysis to neutrality in 0.02 M Tris-HC1-0 .2 M LiCl (pH 7.2) in both the presence and the absence of 2% formaldehyde . In neither of the samples examined were discreet particles observed, in contrast to CMV reassembled at pH 7 .2 . However, there usually was an undefined, amorphous, stain-excluding mass visible in these electron micrographs, probably representing nonspecific aggregates of the nucleoproteins . Failure of pH 10 .3 exposure to affect reassembly capacity of CMV components . CMV-protein preparations were prepared for pH 10 .3 abnormal reassociation, but then reneutralized by the addition of a sufficient amount of 1 .0 M Tris-HC1 (pH 7.2) (0 .16 the volume of the initial protein solution), prior to mixing with RNA and

189

FIG . 2 . Polyacrylamide-gel electrophoresis patterns of RNA components in fractions of CMV reas . sembly mixtures centrifuged on sucrose gradients . (A) RNA components found in the major nucleoprotein fractions NP 1-4 from pH 10 .3 reassociation of CMV components (Fig . IA) . (B) RNA components found in fractions a and b (Fig . 1B) . (C) Phenol-SDS extracted CMV-RNA .

final dilution with 0 .02 M Tris-HCl (pH 7 .2) . The pH of the final reassembly mixture was 7 .4 . Controls for this experiment consisted of the standard pH 7 .2 reassembly, an abnormal reassociation at pH 10 .3 (cf. Fig . 1), and a pH 7 .2 reassembly in which there was a second addition of 1.0 M Tris-HCI to make the ionic strength conditions comparable with the reneutralized reassembly mixture . The temporary exposure of CMV-protein to pH 10 .3 had not affected its reassembly capacity, because the sedimentation pattern of its reassembly product, and those of both pH 7 .2 controls were identical, and comparable to the one shown in Fig . 113. The pH 10 .3 reassembly control was analogous to that shown in Fig . IA . A short pH 10 .3 exposure of CMV-RNA was tested in a similar manner . As with the protein, there was no effect on its reassembly capacity after neutralization . Dissociation of nucleoproteins resulting from pH 10 .3 reassociation of CMV components . Earlier in this subsection it was mentioned that upon dialysis of the pH 10 .3 dissociation mixture to pH 7 .2 in 0 .02 M Tris-HCl-0 .2 M LiCl, no discreet viruslike structures could be detected . Similar failure to convert the pH 10 .3 nucleoprotein particles into virions by neutralization was also reported by Kaper and Geelen (1971) . However, since pH 10.3 treatment of the individual CMV components had not affected their reassembly capacity, it was important to know whether their



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FIG . 3 . Rate zonal centrifugation patterns . Top row : pH 10 .3 reassociation mixtures of CMV-S protein with the RNAs of (A) CMV-Q, (B) CMV-D, (C) CMV-R, and (D) PSV . Centrifugation conditions are 165 min at 40,000 rpm and 5° . Bottom row: RNA preparations used in reassociation experiments of top row . Centrifugation conditions are 960 min at 32,000 rpm and 5° .

actual recombination into anomalous nucleoprotein structures had affected their reassembly capacity, particularly that of the protein . To test this, the pH 10 .3 nucleoproteins had to be redissociated first . Reassociation at pH 10 .3 was carried out under scaled-up conditions, so that the concentration of the resulting nucleoprotein mixture was 2 mg/ml . This mixture was redissociated by addition of concentrated LiCl and Cleland's reagent to final concentrations of 2 .0 and 0 .001 M, respectively . The mixture was left in an icebath for 30 min, after which the nucleic acid precipitate was removed by low speed centrifugation . The supernatant contained the protein, which was neutralized by the addition of 0 .16 its volume of 1 .0 M TrisHCI (pH 7.2) . The ultraviolet spectrum showed that the protein was somewhat contaminated with RNA . Redissociation could also be done at pH 7 .2, if the pH 10 .3 reassociation mixture was neutralized before addition of LiCI and Cleland's reagent . This resulted in a protein that was virtually uncontaminated with RNA . Reassembly with protein obtained from pH 10 .3 reassociated nucleoproteins . The

protein preparations obtained in both the pH 10 .3 and pH 7 .2 dissociations of the pH 10 .3 reassociation products, were mixed with 3 vol of a stoichiometric amount of RNA in water, and dialyzed overnight at 4° against 0 .2 M LiCl-0 .02 M Tris-HCl (pH 7.2) . Upon ultracentrifugation, both samples showed that reassembly had taken

place normally . On the other hand, when the two (pH 10 .3 and pH 7 .2) dissociation mixtures of the pH 10 .3 reassociation products were directly diluted (1 :4) with 0.02 M Tris-HCl (pH 7.2), and dialyzed against 0.2 M LiCl-0 .02 M Tris-HCl (pH 7 .2) [reversible dissociation procedure, cf . Kaper and Geelen (1971)], the reassembly was imperfect due to aggregation of a large proportion of the nucleoprotein . Reaction of TNBS with the Lysyl Residues of CMV-S

The purpose of the TNBS reactions was to determine whether chemical alteration of the lysyl residues would interfere with the stabilization and/or reassembly of CMV . Reaction with CMV-protein, CMV-protein prepared at a concentration of 4 mg/ ml in 2.0 M LiCI was mixed with either concentrated pH 7 .2 or pH 10 .3 buffers and a 20-fold excess of TNBS adjusted to the respective pH's . Final conditions were: protein, 3 mg/ml ; buffer, 0.06 M; LiCl, 1 .5 M ; incubation 1 hr at about 25° . The pH 10 .3 sample was reneutralized by addition of concentrated pH 7.2 buffer . To both protein suspensions RNA was added for reassembly, either by dilution or by dialysis . Controls consisted of protein solutions treated similarly but omitting the TNBS . Ultracentrifugal analysis showed that all the controls resulted in normal reassembly. All TNBS-treated samples failed to give reassembly . However, these results



ROLE OF LYSYLS IN CMV STABILIZATION could not be taken as definitive due to the uncertainties created by the formation of precipitates in the TNBS-treated protein samples . Reaction with virions : Kinetics and structural consequences . TNBS reacts with CMV-S in a characteristic manner . The reaction rate is influenced by reactant concentrations, temperature and pH . For the purpose of this work, reactions were performed under conditions of approximately 10X excess TNBS over total eamino groups of lysyl residues present . Temperature of the reactions was kept constant at values close to room temperature (21-24°), and the pH was 8 or 9, unless stated otherwise . Figure 4 shows the timecourse of the reaction of TNBS with CMVS at pH 8 and pH 9 . Its most characteristic feature is the presence of two inflection points caused by an initial rapid reaction which slows down, a subsequent resurgence of the reaction rate, which then slows down again, and, after a long time (>24 hr) reaches a plateau when approximately the expected number of e-amino groups per subunit (cf. Van Regenmortel et al ., 1972 ; Habili and Francki, 1974) have reacted .

19 1

Samples were withdrawn from the reaction mixtures at the times and levels of trinitrophenylation indicated by the arrows (Fig . 4) . They were cooled to 0°, and examined by rate zonal centrifugation on sucrose gradients as soon as feasible . The results of these ultracentrifuge analyses are shown in the insets of Fig . 4 . Here the depth of sedimentation of untreated CMV, or of the controls kept under the same conditions with omission of TNBS, was identical to that in the sedimentation patterns of samples withdrawn at very low reaction levels, e .g ., diagrams a8 and a9 . The large amount of absorbance at the top of the gradients represents the unreacted excess TNBS . In both reactions a beginning of structural change in the virion is discernible at about the first inflection point, which is at a reaction level of approx 2-3 amino groups per subunit (cf. diagrams cS and b9) . The structural change is practically completed at a reaction level of 4-5 amino groups (sedimentation diagrams d8 and c9) . This sequence of structural events is illustrated in Fig . 5 for the pH 9 reaction . Here electron micrographs of samples withdrawn at trinitrophenyla-

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13 21 17 HOURS FIG. 4 . Time-course of the reaction of TNBS with lysyl residues of CMV-S at pH 8 and pH 9, at approximately 23` . The insets are rate zonal centrifugation patterns of samples withdrawn at the times and trinitrophenylation levels indicated by the arrows . They reflect the approximate degree of structural transformation undergone by the virions .



d . M . KAPER

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Fin . 5 . Electron micrographs (magnification 225,000 x) and rate zonal centrifugation patterns of samples withdrawn from a pH 9 reaction mixture of TNBS with CMV-S at the following levels of trinitrophenylation : (A) 0 .8, (B) 1 .9, (C) 3 .5, and (D) 7 .6 amino groups per subunit . The sedimentation patterns probably reflect a somewhat larger degree of structural transformation due to incomplete inhibition .

tion levels of about 0 .8, 1 .9, 3 .5, and 7 .6 amino groups per subunit, are displayed. The insets of the electron micrographs are the sucrose gradient sedimentation pat-

terns of the samples withdrawn for electron microscopy . It should be kept in mind, however, that these patterns probably represent a somewhat higher level of trinitro-

ROLE OF LYSYLS IN CMV STABILIZATION

phenylation, and thus a larger degree of structural change, because the inhibition technique of cooling to 0° is not completely effective. Failure of reaction with CMV-RNA . CMV-RNA at a concentration equivalent to that present in the virus in the reaction of virions with TNBS described above, failed to give any increase in optical density at 345 nm relative to the TNBS control . This was taken as a demonstration of the inability of the amino groups of the RNA bases to react with TNBS . Direct evidence that in reactions with virions the site of trinitrophenylation is the protein component will be given at the end of this subsection . Further characterization of the trinitrophenylation products . Sedimentation diagrams of the pH 9 reactions showed a good definition of the reaction products, which sedimented to approx 0 .45, 0 .63, and 0 .76 of the depth of an appropriate CMV control . At the lower pH, the reaction products sedimented with about the same rates, but exhibited increased polydispersity (cf. insets, Fig. 4) . When the reaction time was extended until almost all the available lysyl residues had reacted, the polydispersity increased (cf. Fig . 8) . This was caused by an increased tendency toward aggregation of the reaction products . That even prolonged TNBS reaction with CMV did not yield free protein and RNA was demonstrated by taking a 24-hr reaction mixture, and centrifuging it at pH 8 under conditions of CMV-RNA rate zonal centrifugation (cf. Fig . 1C) . The resulting sedimentation pattern of the reaction mixture only revealed the yellow, nonsedimenting, uv-absorbing zone of TNBS excess at the top of the gradient. However, there was a yellow pellet at the bottom of the tube . This material was redissolved in pH 8 buffer containing 0 .1% SDS, and recentrifuged simultaneously with CMV-RNA as a control . Both tubes exhibited typical CMV-RNA patterns, except that there was a yellow trailing zone in the TNBStreated sample which represented the trinitrophenylated protein . Identification of the RNA components in the nucleoprotein products of the pH 9 re-

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action was carried out by fractionation of a scaled-up (10x) reaction mixture (8 .7 TNP per subunit) centrifuged on sucrose gradients, and analysis of the fractions with SDS-polyacrylamide electrophoresis . It was found that the zone at 0 .76 CMV depth contained primarily RNA 1 and RNA 2, the shoulder at 0 .63 CMV depth RNA 3, and that RNA 4 was in the peak at 0 .45 CMV depth . Effect of trinitrophenylation on the reassembly of CMV . In the beginning of this subsection it was shown that the reaction of CMV-protein in 2 .0 M LiCI with TNBS led to the formation of a precipitated reaction product . This made it impossible to quantitate the trinitrophenylation, and to investigate its effect on the reassembly capacity of CMV-protein . An alternative approach would be to have virions react with TNBS to different degrees of amino-substitution, isolate the protein, and then attempt reassembly with CMV-RNA . A potentially interesting degree of trinitrophenylation seemed to be that level which brings the virus to the beginning of structural transformation as shown, for instance, in the sedimentation diagram c8 of Fig . 4 . For this purpose, a scaled-up (10x) version of the pH 8 TNBS reaction was carried out so that an amino-substitution level of 3 .2 was reached . The reaction mixture was ultracentrifuged on pH 7 .2 sucrose gradients, and the middle third of the 3TNP-CMV zone was collected, dialysed overnight at 4° against distilled water, and concentrated by ultracentrifugation . Ultraviolet spectra of 3TNP-CMV had an absorbance maximum at 345 urn, typical for TNP-lysyl compounds (Goldfarb, 1966) . A change in the 260 nm/250 nm ratio, due to the 255-nm absorbance maximum of TNP substituents, was also noted . In the analytical ultracentrifuge 3TNPCMV sedimented somewhat less monodisperse than CMV. Protein was prepared from 3TNP-CMV with the 2 .0 M LiCI dissociation method . No precipitation problems were encountered with the modified protein . Ultraviolet spectra of 3TNP-protein and regular CMV-protein are displayed in Fig . 6 . Again, the effect of the TNP substituents



J . M. KAPER

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Comparison of ultraviolet spectra of (A) CMV-protein, and (B) 3TNP-CMV-protein in 2 .0 M LiCl . Spectra are adjusted to approximately similar optical densities in the 270-280-nm range . FIG . 6 .

on the shape of the spectrum is best noticeable at wavelengths higher than 300 rim, and in the region below 270 nm. Both protein preparations were used for reassembly with CMV-RNA at pH 7 .2 . Unmodified protein reassembled with CMV-RNA in a normal fashion (Kaper and Geelen, 1971), but 3TNP-CMV-protein recombined with the RNA in a nonspecific way . In the electron microscope, a mixture of fibrous structures, particles of undefined structure and size, and occasionally an imperfectly reassembled virus particle was seen . This was corroborated by the sedimentation polydispersity observed in the analytical ultracentrifuge . Thus, an average level of three modified lysyl a-amino groups appeared to be sufficient to prevent regular reassembly of CMV-protein and RNA . An attempt was also made to prepare protein from CMV with nine TNP-lysyl residues per subunit, and to test its reassembly properties with CMV-RNA . The reaction was carried out as described for isolation of the TNP-CMV reaction products and their RNAs (see above) . The nucleoprotein fractions were isolated after sucrose gradient centrifugation . After dialysis and concentration by ultracentrifugation, the pellets were resuspended in distilled water without difficulty . However, this TNP-nucleoprotein could not be degraded by 2 .0 M LiCl, and further reassembly attempts had to be abandoned . Reaction of TNBS with CMV-D and Viruses of the Bromogroup

A preliminary investigation of the TNBS reaction with CMV-D, BMV, CCMV, and BBMV was begun to see

whether lysyl residues also played an important structural role in other viruses stabilized by protein-RNA linkages (cf . Kaper, 1972, 1973) . The D-strain of CMV (Lot et al., 1974) was selected for these tests, because a recent serological classification (Devergne and Cardin, 1973) suggests that its antigenic structure is significantly different from that of CMV-S . Also, a comparative study of the physical-chemical properties of several CMV strains has shown CMV-D to differ markedly from CMV-S (Lot and Kaper, in preparation) . Figure 7 shows the time-course of the reaction of TNBS with CMV-D (and CMV-S, for comparison) at pH 9 . The reaction is much slower, and unlike CMV-S, there is no obvious evidence of a structural transition (no clear inflections in the curve) . Ultracentrifugal analysis (see insets of Fig . 7) confirms this difference between the two strains . Diagrams 1S and 1D represent the reaction mixtures sampled at the same time of reaction ; 1S and 3D were withdrawn at the same level of trinitrophenylation . The structural transformation as represented by diagram 1S is almost complete (see diagram 3S for comparison), while in diagram 1D none is visible . Diagram 3D represents approximately 50% transformation of the D strain . Figure 8 shows the time-courses of the reactions of TNBS with BMV, CCMV, and BBMV at pH S . CMV-S was included in the same experiment as a reference . The sedimentation diagrams of the reaction mixtures (insets) show that all viruses, except BMV, had degraded in their entirety after 19 hr of reaction . BMV had only partially degraded . No sharply sedimenting bands could be recognized in the completely transformed mixtures ; all had a yellow pellet in the bottom of the tubes, obviously as a result of severe aggregation at later stages of the reaction . The virus controls held under the same conditions in the absence of TNBS, were all virtually undegraded . At the present stage of the investigation, the time-course of structural transformation of these viruses has not yet been analyzed . However, it is suspected that such an analysis may reveal



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CMV-S and different bromoviruses at FIG . 8 . Comparison of time-courses of the reactions of TNBS withCMV-S pH 8 and 21°. Stock solutions of the bromoviruses (kept at pH 5 .5-6 .0) were diluted directly into the pH 8 buffer, and were not predialysed for conversion into the swollen form of these viruses . Insets are rate zonal centrifugation patterns of the reaction mixtures after 19 hr .

that BBMV, with a weaker capsid strucDISCUSSION The present work on the anomalous ture, will show a closer relationship to CMV-S than, for instance, BMV . This reassociation of CMV-protein and CMVseems already evident from the rate of RNA has demonstrated that the pH 10.3 modification of either constituent is essenreaction shown in Fig . 8.

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tially reversible, and of no consequence to their reassembly capacity at pH 7 . This suggests that simple deprotonation events are responsible for the anomalous behavior at pH 10 or higher . Uridine and guanosine phosphate residues of the RNA have a pK of about 9 .5 (Steiner and Beers, 1961), while tyrosyl and lysyl residues in proteins have a pK of about 10 (Tanford, 1962) . Thus, as suggested previously (Kaper and Geelan, 1971), the deprotonation of each of these types of groups, all of which occur in relative abundance in CMV, may contribute to the pH 10 .3 degradation and anomalous reassociation of CMV . However, since the deprotonation of lysyl groups could involve the removal of potential interaction sites with the RNA phosphate residues, a closer study of the role of lysyl residues was undertaken . The experiments with TNBS support the notion that lysyl residues are important in the stabilization of CMV, although not necessarily in a role of interaction partner with the RNA . The bimodal character of the amino group reactivity of CMV-S was demonstrated to be directly related to a structural transformation of the virions into a set of nucleoproteins of ill-defined structure (Fig . 5) . The RNA component distribution found in the separable fractions of the TNP-nucleoproteins suggested a resemblance with the nucleoproteins resulting from the pH 10 .3 reassociation (Fig. 2A). The former displayed a somewhat stronger tendency to aggregate, particularly in the pH 8 reaction, which explains their "clustered" appearance in sedimentation diagrams (Fig . 4) . Probably this is because in the TNBS reaction the nucleoproteins arise from a degradative transformation of intact virus particles, where certain features of the original structure may have been retained (Fig . 5) . In the pH 10 .3 reassociation, on the other hand, there is a de novo formation of linkages that seem at least partially different from those in the original virion . It is not known whether in the pH 10 .3 reassociation reaction the protein subunits with largely deprotonated lysyl residues, upon recombination with the RNA components, have or assume a conformation

which is different from the one they had at pH 7 . If this is the case, the conformational change is a reversible one, since reversal of the pH to neutrality, either prior to pH 10 .3 reassociation with the RNA, or after reisolation from the pH 10 .3 nucleoproteins, restored the protein's capacity for normal reassembly into virions . However, it is certain that the pH 10 .3 nucleoproteins bear no obvious resemblance to CMV's original structure . Since at pH 10 .3 the RNA components have probably lost most of their tertiary structures, their recombination with deprotonated protein subunits, regardless of the latter's conformation, would probably result in nucleoproteins with less specific structures . Therefore, the best picture of these nucleoprotein structures, that emerges from this work, is that of aggregates resulting from a nonspecific electrostatic union between two types of polyelectrolyte, an RNA polyanion, and several protein polycations . In such a structure the optimal size of the nucleoprotein would be determined, to a greater or lesser extent, by the size of the RNA . This would explain the observed sedimentation distribution according to RNA size . Both the TNBS-induced degradation, as well as the failure of 3TNP-CMV-protein to reassemble correctly with CMV-RNA, suggest that of a total of 14-15 lysyl residues, three play an important role in the maintenance of the native conformation of CMV-S . This notion has been supported by recent experiments (unpublished work), in which it could be shown that at pH 8 or 9 the lysyl amino groups of the pH 10 .3 nucleoproteins did not exhibit the characteristic biphasic reactivity with TNBS that is displayed by the intact virions (Fig . 4) . Instead, a more rapid initial reaction rate (without inflection) was followed by a time-course which was parallel to that of the virion reaction, after a trinitrophenylation level of about 10 amino groups per subunit had been reached . As to the lysyl role in the stabilization of CMV, two possibilities, or a combination of them, should be considered . One was suggested previously (Kaper and Geelen, 1971), and entails specific lysyl residues

ROLE OF LYSYLS IN CMV STABILIZATION functioning as the sites of interaction of protein subunits with the RNA in the original virion structure . However, equally likely is the possibility that deprotonation or trinitrophenylation of certain lysyl residues perturbs the protein subunit conformation and/or the stabilizing interactions sufficiently so as to cause virions to be structurally transformed into the nucleoproteins described, and to prevent protein subunits from recombining with RNA in a normal fashion . This scheme of events would also be in better agreement with the results obtained in the trinitrophenylation reaction with the more stable D strain of CMV (Fig . 7) . Amino acid analyses of the coat proteins of CMV (Van Regenmortel et al., 1972), tomato aspermy virus (TAV) (State-Smith and Tremaine, 1973 ; Habili and Francki, 1974), and peanut stunt virus (PSV) (S . Boatman, personal communication), all of which are considered to be distantly related viruses of the cucumovirus group (Harrison et al ., 1971), show a relatively high lysine content . The proteins of the bromoviruses (cf. Lane, 1974) and alfalfa mosaic virus (AMV) (Kruseman et al ., 1971) also have high lysine contents . Some members of this group of structurally related viruses have been shown to be susceptible to protease digestion . During tryptic digestion of CCMV, a basic N-terminal section, comprising 25 amino acid residues (of which 3 are lysyls and 5 arginyls), is split from the protein subunits, causing the virus particle to disintegrate (Chidlow and Tremaine, 1971 ; Tremaine et al., 1972) . A basic (8 arginyls and 1 lysyl) N-terminal section of 25 amino-acid residues can also be removed by the action of trypsin on BMV [J . H . Tremaine et al ., as quoted by Pfeiffer and Hirth (1975)] . These workers postulate that this section of the polypeptide chain is directly involved in the protein-RNA interactions of these two viruses . Bol et al. (1974) have demonstrated that in the case of AMV, tryspin treatment also caused the release of a basic (5 lysine and 2 arginine) 27-amino acid section from the protein subunit . However, with CMV-S they found no effect of trypsin . With BBMV, Agrawal and Tre-

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maine (1972) reported a more random susceptibility to proteases . These observations, made on several viruses from different groups (cf. Harrison et al ., 1971), but which have as a common denominator the fact that they derive much of their stabilization from proteinRNA interactions (Kaper, 1972, 1973), suggest an important structural role for their basic amino-acid residues . The reactivity of several of the bromoviruses with TNBS, and their subsequent structural transformation, demonstrated in the present work, seems to confirm this for the lysyl residues . Recently it has been shown for BMV that the pH-induced susceptibilities to proteases and RNase are directly synchronous to each other (Pfeiffer and Hirth, 1975) . This would mean that in the swollen state, RNA, and perhaps also the proteinRNA interaction sites, become exposed . These conclusions can be extended directly to CCMV, and probably also to CMV, which is RNase-sensitive regardless of pH (Kaper and Geelen, 1971) . However, further work is required to clarify the precise relationships of the lysyl residues to their possible role as interaction partners with the RNA and/or with other amino-acid residues in the intact virion . ACKNOWLEDGMENTS At different stages of this investigation Carolyn West, Rita Tickel, and Marie Tousignant provided expert technical assistance . Mike Moseley and Eric Erbe carried out the electron microscopy . REFERENCES and TREMAINE, J . H . (1972) . Proteins of cowpea chlorotic mottle, broad bean mottle, and brome mosaic viruses . Virology 47, 8-20. BOATMAN, S ., and KAPER, J . M . (1976) . Molecular organization and stabilizing forces of simple RNA viruses . IV . Selective interference with proteinRNA interactions using sodium dodecyl sulfate . Virology, in press . BOCxsTAHLER, L . E ., and KAESBERG, P . (1962) . The molecular weight and other biophysical properties of bromegrass mosiac virus . Biophys . J . 2, 1-9 . BOL, J . F ., KRAAL, B ., and BREDERODE, F . TH . (1974) . Limited proteolysis of alfalfa mosiac virus: Influence on the structural and biological function of the protein . Virology 58, 101-110, CHIDLOW, J ., and TREMAINE, J . H . (1971) . Limited hydrolysis of cowpea chlorotic mottle virus by AGRAWAL, H . 0 .,

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trypsin and chymotrypsin . Virology 43, 267-278 . DEVERGNES, J . C ., and CAamN, L . (1973) . Contribution a I'etude du virus de Is mosaique du concombre (CMV) . IV . Easai de classification de plusieurs isolats sur la base de leur structure antigenique . Ann . Pytopathologie 5, 409-430 . GOLDFARB, A. R . (1966) . A kinetic study of the reactions of amino acids and peptides with trinitrobenzenesulfonic acid . Biochemistry 5, 2570-2574 . HABILI, N ., and FRANCKI, R . I . B . (1974) . Comparative studies on tomato aspermy and cucumber mosaic viruses . I . Physical and chemical properties . Virology 57, 392-401 . HARRISON, B . D ., FINCH, J . T ., Glsas, A . J ., ROLL ING$, M ., SHEPHERD, R. J ., VALENTA, V ., and WETTER, C . (1971) . Sixteen groups of plant viruses . Virology 45, 356-363 . KAPER, J . M ., DIENER, T . 0 ., and SCOTT, H . A . (1965) . Some physical and chemical properties of cucumber mosaic virus (strain Y) and of its isolated ribonucleic acid . Virology 27, 54-72 . KAPER, J . M . (1969) . Reversible dissociation of cucumber mosaic virus (strain S) . Virology 27, 134139 . KAPER, K . 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 ., and GEELEN, J. L . M . C . (1971) . Studies on the stabilizing interactions of simple RNA viruses. II . Stability, dissociation, and reassembly of cucumber mosaic virus . J . Mod . Biol. 56, 277294 . 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 .), FEBS Symp . 27, 19-41 . 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 . KAPER, J . M . (1973) . Arrangement and identification of simple isometric viruses according to their dominating stabilizing interactions . Virology 55, 299-304 . KAPER, J . M . (1975). "The Chemical Basis of Virus Structure, Dissociation and Reassembly ." North

Holland/American Elsevier, Amsterdam . KRUSEMAN, J ., KRAAL, B ., JASPARS, E . M . J ., BOL, J . F., BREDERODE, F . TH ., and VELDSTRA, H . (1971) . Molecular weight of the coat protein of alfalfa mosaic virus, Biochemistry 10, 447-455 . LANE, L. C . (1974) . The bromoviruses . Ado Virus Res . 19, 151-220 . LOT, H ., MAECHOOx, G., MAEROU, J ., KAPER, J . M., WEST, C . K ., VAN VLOTEN-DOTING, L ., and HULL, R . (1974) . Evidence for three functional RNA species in several strains of cucumber mosaic virus . J . Gen . Virol . 22, 81-93 . LOT, H ., and KAPER, J . M. (1973) . Comparison of the buoyant density heterogeneity of cucumber mosaic virus and brome mosaic virus . Virology 54, 540-543 . MEANS, G. E ., and FEENEY, R . E . (1971) . "Chemical Modification of Proteins ." Holden-Day, San Francisco . PFEIFFER, P ., and HIRTH, L . (1975) . The effect of conformational changes in brome mosaic virus upon its sensitivity to trypsin, chymotrypsin and ribonuclease . FEES Letters 56, 144-148 . RE, G. G_, and KAPER, J . M . (1975) . Molecular organization and stabilizing forces of simple RNA viruses . III . Chemical accessibility of tyrosyl and lysyl residues in turnip yellow mosaic virus capsids . Biochemistry 14, 4492-4497 . SCHEELE, R . B ., and LAUFFER, M . A . (1969) . Restricted reactivity of the epsilon amino groups of tobacco mosaic virus protein toward trinitrobenzenesulfonic acid . Biochemistry 8, 3597-3603 . SrACE-SMITH, R ., and TREMAINE, J . H . (1973). Biophysical and biochemical properties of tomato aspermy virus . Virology 51, 401-408. STEINER, R . F ., and BEERS, R . F . (1961) . "Polynucleotides ." Elsevier, Amsterdam . TANFORD, C . (1962) . The interpretation of hydrogen ion titration curves of proteins . Ado . Prot. Chem . 17, 69-165 . TREMAINE, J . H ., ACRAWAL, H . 0 ., and CHIDLOw, J . (1972). Partial sequence of the N-terminal portion of the protein of cowpea chlorotic mottle virus . Virology 48, 245-254 . VAN REGENMORTEL, M . H . V ., HENDRY, D . A ., and BALTZ, T. (1972) . A reexamination of the molecular size of cucumber mosaic virus and its coat protein . Virology 49, 647-653 .