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Structure of simian virus 40 II. Symmetry and components of the virus particle
VIROLOGY
32, 511-523 (1967)
Structure II. Symmetry F. A. AKDERER,
of Simian
and Components
Virus
40
of the Virus Particle
H. D. SCHLCMBERGER, ill. A. KOCH,’ H. FRAn-I<, AKD H. J. EGGERS’
Xax-Planck-Institut
jiir
Virusjorschung,
Accepted April
Tiibingen,
Germany
I.$, 1967
The symmetry of Sk'40 particles was examined and proved to be of the 2’ = 7 (dextro) icosahedral surface lattice. Evidence is presented that t,he three types of particle shells of smaller size are derived from reassembled morphological units of disintegrated normal particle shells. Examination of the sllrface structure of the smaller particles strongly suggested icosahedral surface lattices of T = 1, T = 3, and T = 4 symmet,ry classes. Three different polypeptide chains with an average molecular weight of 16,350 f 100/d were identified in the protein moiety of the virus particle. The proportion was determined as 45.5 : 45.5 : 9. Four different approaches were used to determine the size of the virus DSA. A molecular weieht of 2.25 X 106 f 15% was established. A calculation of the particle weight from the size and number of the components yielded 17.3 X IO6 f 10yO.
several authors. Mayor et al. (1963) calculated a molecular weight of 4.0 to 5.2 X 106, Crawford and Black (1964) found molecular weights of 2.6 X 10” and 3.2 X lo6 for the virus DKA. The st’ructural details of the SV40 DNA have been only interpreted in analogy to polyoma virus DNA (Vinograd et al., 1965). This paper present’s our findings on the symmetry of bhe particle shell. Furthermore, the size, number, and chemical nature of t,he viral components were determined.
INTRODUCTION
In Part I of this series, a simple method for t,he propagation and purification of SV40 virus was described (Koch et al., 1967). The diameter of purified full SV40 particles was found to be 41.1 f 0.6 rnp, and the molecular weight of the part’icles was calculated to be 17.3 X 106. The number of the morphological units of the icosahedral SV40 shell has been reported to be 42 (Mayor et al., 1963). Klug (1965) however, predicted in analogy to human wart virus (Klug and Finch, 1965) and rabbit papilloma virus (Finch and Klug, 196.5) for SV40 the symmetry of the T = 7 icosahedral surface lattice which is composed of 72 morphological unit’s. No determinat,ions of the exact number and size of the constituting polypeptide chains have been published. Investigations on the size and st,ructure of the virus DNA have been reported by 1 Present address: Inst.itut fiir Virologie, Liebig-UniversitLt, Giessen, Germany.
Justus
MATERIALS
SND
METHODS
Electron microscopy. For t’he determination of particle symmetry a small drop of 2% phosphotungstate, adjusted to pH 7.2, was placed on a copper grid covered with carbon-coated Formvar film and dried by infrared light’. One drop of a virus solution containing l-2 mg. of virus/ml (0.1.5 M NaCI-0.02 M Tris-HCI) pH 7.2 was brought on this grid and withdrawn immediately by touching the edge of the grid with filter
511
512
ANDERER,
l)apcr. One should hc careful that the phosphotungstate film does not dissolve completely and mix with the drop of the virus solution. Therefore all manipulations have to be performed rapidly if one wants t,o obtain “OIIC side” particle images. Further negative staining t,echniques were either t,o mix the virus solutions with 2%. phosphotungstat,e before placing the mixture on a grid or to place the virus solution directly on the support’ followed by 1 or 2 washings with 2 % phosphohungstate. A Sicmcns Elmiskop IA was used at a magnification of 37,000 with double-condensor illumination, operating at SO kV. iLlagnification calibration of the electron microscope was performed wit,h carbon grat,ing replicas (E. Ii. Fulham, Inc., Schenectady, New York). Replicas of diffraction grat)ings ruled to 54,800 lines/inch were used. The grids prepared for determination of particle symmetry were always inserted in t.he electron microscrope wit’h t)he specimen side away from the electron source and for the photographic printing the elect)ron microscope plates were always insert,ed into the enlarger with the emulsion side away from the bromide paper. For the det’ermination of the strand length of ST40 DKA the protein monolayer t’echniqur of Kleinschmidt et ab. (1959) was used. Cytochrome c solutions, at’ a conccn tration of 0.1 mg/ml, were prepared wit’h the following solver~t~s: (,4) 1 A/ ammonium acet,ate adjusted to pH 6.0 with acetic acid, (B) 1 II1 ammonium acet,ate-0.5 % formaldehyde, pH 6.0, (C) 1 nZ ammonium acetate adjusted to pH S.0 with 1 ill NaOH. The solutions for spreading DXA with prot’ein contained 1-3 pg of SV40-DXA/ml, freshly diluted from tenfold DNA concentrat~ions. The hypophase for spreading t,he DNA in solvent (A) was 0.3 df ammonium acetate, pH 7.1; for spreading the DNA in solvent (B), it was 0.3 M ammonium acetate-0.5% formaldehyde, pH 6.0; and for spreading the DKA in solvent (C), it was 0.015 31 ammonium acct~atc, pH S.0 (Weil and 1’inograd, 196X). Carbon-coated Formvar films were used as support,s for t,he DNA-protein monolayers. Rotary shadowing of t.he specimens was performed with vaporized Pt-Pd-Au-
ET AL.
Ag alloy at a grazing angle of 10”. ?tIicrographs of these preparations were obt)ained with a Siemens Elmiskop IA operating at SO kV, with double-condenser illumination. Magnification calibration of the elect,ron microscope was performed with carbon grating replicas (E. P’. Fulham, Inc., Schenectady, New York). ,411 exposures were made at a magnification of X 19,200 with constant strength of the current of the objective lens (459 m-4) and with constantly comrolled positions of the projcctivc lens and intermediat,e lens. Contour length measurements of twodimensional images of DNA at a final magnification of X 192,000 were carried out with a calibrated map ruler. All measurements were done by t’hree persons to compensate for individual errors. The maximum deviations were f 1.5 %$. Determinations of the molecular weight of the polypepticle chains. For the determination of t,he partial specific volume of SV40 protein a L-ml pycnomeber cell was used. A sample of empty particles twice purified by densit,y gradient centrifugation [see Part I (Koch et al., 1967)] was dried in vucuo at SO” for 48 hours and dissolved in 4 M guanidinium hydrochloride-O.1 M Tris-HCl-0.01 M fi-mercaptoethanol, pH 8.0, and t)he extinction at 280 rnp was determined after appropriate dilution. After dialysis against, the same buffer for 78 hours at room temperature measurements were performed with solutions containing 4.32, 3.16, 2.37, 1.48, 0.925, and 0.465 mg/ml, as derived from OD measurements. All measurements were corrected for zero time of correct filling by following the decrease in weight for 200 seconds during t,he weighing. The t’emperature was 20.0 f 0.4”. Sedimentation and diffusion experiments were carried out, with protein solutions (same buffer as above) using a Spinco model E ultracentrifuge with schlieren optics. Schlieren pictures were measured with a t,raveling microscope. Sedimentation was performed with protein concentratJions of 8.0, 6.0, 1.0, and 2.0 mg/ml in the AX-D rotor (capillary cell assembly) at 42,040 rpm. All measurements were made at 20.0 f 0.01”. The diffusion patterns were evaluated according to Elias (1961). The determina-
STRUCTURE
OF SIMIAN
tion of the average molecular weight of the polypeptide chains, using the modified Archibald method (Elias, 1961), was performed in t’he AN-D rotor with double sector cell assembly at 42,040 rpm. Electyophoresis of virus proteins. A Beckman Model R-190 microzone electrophoresis and cellulose acetate membranes (RIillipore Filter Corp.) were used. The membranes were soaked in 8 M urea-O.1 M Tris-HCl0.01 M P-mercaptoethanol, pH 5.6. The cell was filled with the same buffer. Lyophilized full, as well as empty, part’icles were dissolved in this buffer (concentration of @-mercaptoethanol: 0.1 111) to give 1% protein solutions. The samples mere run at room temperature with 13-17 volts/cm for 6G-120 minutes. The wet membrane strips were st,ained with Ponceau S; after removal of t#he excess stain bhe membrane strips were dried t,o make them transparent. For the evaluation of the electropherograms the Beckman Model RB (Analytrol) with Jlodel R-202 microzone scanning attachment, was used. Phot’onegatives of the electropherograms were prepared using a blue filter and t’hen scanned with a Joyce-Loebl MI< IIIB microdensitometer. Tryptic degradation of the fdl virus particles. Samples of 1.6 mg SV40 in 1 ml 0.02 M phosphate buffer, pH 7.5, were digested at 37” with 5 X crystallized trypsin (enzyme: substrate ratio, 1 :lOO). After 30, 60, 120, and 240 minutes of incubation, the trypsin action was st)opped in individual samples by addition of equivalent amounts of kallikrein inactivator (identical with bovine pancreatic trypsin inhibitor). The samples were treated wit)h DBase overnight folowed by dialysis against water. The lyophilized samples were subject’ed to microzone electrophoresis. Determination of the amount of DNA per virus particle. Four virus samples cont,aining 0.450 or 0.475 mg virus in 0.15 ml of 0.15 M NaCl-0.02 Af Tris-HCI buffer, pH 7.2, were used for phosphorus analysis. For the determination of the deoxyribose content the Burton (1956) modification of the Dische diphenylamine reaction was used. The color intensities were standardized with solutions of calf t,hymus DXA (Calbiothem.) with krlowr~ phosphorus content.
VIRUS
513
40. II.
Controls performed with thymus DNA in mixtures with variable concentrations of bovine serum albumin did not show significant deviations of DSA-specific color intensities. Sedimentation of SIT40 DNA. In order t’o obtain protein-free SV40 DNA, the virus was dissociated in pH 10.5 buffer at 4” for 20 hours. The DNA was separat,ed from the prot,ein in a self-generating CsCl density gradient, which was prepared by a 2 ml overlay of dissociated virus on 3 ml of CsCl solution, saturated at room temperature. Aft)er fractionat,ion and dialysis again& 0.15 Al XaCl-0.05 A1 Tris-HCl-10P3 M EDTA, pH 7.2, t’he ratio E26,JE280was l.S3. Sedimentation was performed with DNA solutions containing 0.8 OD260 &ml. A Spinco Rlodel E ultracentrifuge with UVabsorption optics was used. All measurements were made at’ 20.0 f 0.01‘. The rotor speed of t,he AN-H rotor was eit,her 56,100 rpm or 39,460 rpm. UV-absorption pictures were scanned with a Joyce-Loebl MT< III B microdensitometer. RESULTS
Examination Shells
of the Symmetry of the Particle
Caspar and Iilug (1962) have proposed a theory for the const,ruction of protein shells built from ident’ical protein subunits. For isodimensional particles, the icosahedral symmet’ry is preferred. The authors enumerated all the possible icosahedral surface lattices, the numbers of morphological units and the number of structural units involved in each case. This theory provides a classification of icosahedral virus prot,ein shells. The best technique available to obtain virus part)icle images of high resolution is negative staining with heavy metal salts. To determine the arrangement, of morphological units one is restricted t’o those particles which are contrasted on one side only. From the results of the work of Finch and Mug (1965), we know that the dominantly contrasted side is the side near t,o the carbon substrate. The examination of ‘(one side” particle images of negatively stained SV40 revealed the symmetry of the T = 7 (dextro) ico-
FIG. la-d. Selected “one side” particle images of negatively stained SVlO particles together with duplicates where the 5-coordinated morphological rmits arc marked .r and the (i-coortlim~ted tInits which determine the “72d path” are marked with a black dot. Magnification : X 380,000.
sahedral surface IaUice which is composed of 72 morphological urms. Several selected particle images are shown in I’igs. la-d, and t,hese have at least two unequivocal S-coordinated morphological uuits which are marked with X in the duplicate image of the same part’icle. The A-coordinated morphological units, which determine the t#ypical 72 d path between the 5-coordinated units are marked by black dots. No parMe images with T = 7 (levo) symmet,ry were observed. The hand of 7’ = 7 symmet,ry is determined by t)he convention of specimen mounting and printing given by Pinch and Klug (196ri). Higher yields of “one side” particle images as well as sharper contrast, as compared wit,h conventional negative st’aining t,echniques, were obtained when the carbon substrate was coated first, with a solid phosphotungst8at,e film. This modification of negative staining excludes the possibility that particles will be stained only on the “far” side wit,h respect, to the carbon substrate. The yield of “one side” images with at least two unequivocal %coordinxted morphological units was about 2-5 ci;, though the majority of particle images could not be well examined because of a contin uously dark background of phosphotung stat,e. Scarcely any ‘%wo side” particle
images were detectable. The “two side” images obtained by conventional negative staining techniques were in agreement with shadowgraphs of T = 7 Geodestix models (Mug and P’inch, 1965). Some of these part,icles show a dist’inct superposition displaying “eyes” or terraced structures (I:igs. 2a-e). III the first communication (Koch et al., 1967) it was report,ed that the highly puriticd empty particle pool contained not only particle shells of normal size, but also appreciable amounts of smaller-size shells. We designated these smaller particles as I, II, and III. These have t,hree distinctly different diamet,ers; size I = 31.5 =I= 1.7 rnp, size II = 27.4 f 0.9 mp, and size III = 16.S f 1.0 rnp. The diameters were measured on particles well embedded in phosphotungst,at,e. The examination of the surface structure of the small particles III, the most frequently occurring (7-S %‘), st’rongly suggests a symmetry of the T = 1 icosahedral surface lattice. Figures 3a and 3c show selected “one side” particle images with two 5-coordinated morphological units. A photograph of a T = 1 model in the same oriernation is depicted in Fig. 3b. Figure 3d shows a particle only partly embedded
STRUCTUI:E
OF SIhlIrlN
VIRUS
515
10. II.
FIG. 2a-e. Selected “t,wo side” particle images which show a distinct or terraced strrlctrlres. Maguificatioll: X 380,000.
srlperposition
displaying
“eyes”
FIG. 3. Selected “one side” images of small particle shell III with two j-coordinated morphological units (a, c), and particle images viewed along the 5-fold axis (d, f). Photographs of two different views (b, e) of a T = 1 model are added as a visual aid. Magnification: X 380,000.
in phosphotungstate viewed close to a fivcfold axis. The part’icle shown in Fig. 3f is covered with phosphotungstatc and, because of the superposition of the morphological units which occurs in views along the fivefold axis, the particle appears as a ring of 10 “beads.” A phot’ograph of a T = 1 model viewed also along the fivefold axis is shown in Fig. 3e to facilitate the interpretation of the electron micrographs. Three ‘Lone side” images of small par-
II are preserited in E’igs. 4a-c. These particles show strong evidence for the symmetry of the T = 3 icosahedral surface lattice. In duplicates of the same images, the G-coordinated morphological units are marked 5 and the (i-coordinated are marked with black dots. In addition, photographs of 2’ = 3 models viewed in about t’he same position are presented. The examination of the surface structure of small part’icles I is hampered by t’heir Ccles
516
4a
FIG. 4. Selected “one side” images of small particle shell II (a, b) are shown i,ogether with dllplicates where the 5.coordinated morphological units arc marked L alrd the (i-coordinated llnits which determine the T = 3 arrangement are marked with black dots; (c) particle viewed along a 5-fold axis. l’holographs of T = 3 models viewed from corrrspotldillg posit ions are added. ?vlagttificat ioll: X 380,000.
low frequency (less than 0.5 ‘2 ). One selected particle image suggesting a symmetry of the T = 4 icosahedral surface lattice is presented in Fig. 5 together with a duplicate for which the 5coordinat,ed morphological units are marked .c and t,he li-coordinated morphological unit is marked with a dot,. A T = 4 model placed tentatively in the same position illustrat,cs the arrangement of t,he morphological units. If the smaller-size particle shells are built, from the same structural u&s as the S\?40 particle and the symmetry classes of particles I, II, and III, as evaluat’ed above, are correct, t.he diameters of tjhe resulting shells can be calculated. The ratio of the diameters of such shells is 1 (T = 7)/O. 76 (T = 4)/ 0.65 (T = 3)/0.3S (1’ = l), which is in good agreement with the rat’io of the actually observed diameters, when t,he value for the diameter of well-embedded empty shells of normal size is 12.0 f 1.7 mp.
Size
and
Number oj’ the I’olypeptitle
Chains
The structural u&s of :I protjein shell mav consist) of one or several polypeptidc chiins, which in t,he second cast need not, be idenGca1 nor need t,hey be different, since the icosahedral symmct’ry requires only t)hat groups of 60 1’ be cquivalcnt. Determination of thr size and number of polvpeptidr chains of t,hr S\‘40 protein moiety was performed under conditions known to dissociate an assembly of polgpept,ide chains into monomers. I’or the determinat,ion of the molecular weight) of the polgpeptide chains, the lyophilized fraction of empty particles (Koch et al., 1967) dissolved in 4 :1L guanidinium hydrochloride-0.1 Al Tris-HCl-0.01 N p-mercaptoet,hanol, pH 8.0, was used. The partial specific volume of the polypept,ide chains in this solvent, was determined pycnomctrically at, various conccrltrabiorls. The dc-
,517
FIG. 5. Image of small particle shell I sllggesting a 7’ = 4 symmetry of the icosahedral surface lattice. The 5- and (i-coordinated units are marked in analogy t,o Fig. 4. The photograph of a 7’ = 4 model is placed in approximately t,he same orientat ion as the particle. Magnifirat ion: X 380,000.
pendence of density of these protein solutions on the prot,ein concentrations is given in Vig. 6. The data fit a straight, line. The slope of t,his line corresponds directly to (1 - z’pO)(Elias, 1961). E’rom (1 - tip”) = 0.230 and p0 = 1.0989 t)he partial specific volume of the virus protein in this solvent is found to be fi = 0.70. The sedimentation pattern showed a single symmetrical gradient which sedimentatjed wit,h SZO = 0.75, without dependence on prot,ein concentrations bet,ween 2 and S mg’ml. The diffusion const~ant was evaluated from sedimentat,ion gradicnt8s. The corlcent8ration dependence of the diffusion is given in Fig. 7. Graphic extrapolation to zero concentration yields a value D20 = 4.S.5 X 10e7 cm2/sec. The average molecular weight (&I) of the polypcpt’ide chains calculat)ed from SpO = 0.75, I)?” = 4.S.5 X 10m7 cm2/sec, and (1 - tip,) = 0.230, is Ms,u = 16,350. When the dcterminatjion of the molecular weight was performed wit)h the modified Archibald method, a value M APP = 16,100 was obtjained. The larger scatter of thcsc values compared with the values used for the dls,D calculation does not, allow the detection of a concentration dependency between 4 and S mg. So significant, het’erogcneity was detectable from the shape of t,he concentration gradients. Thus we might conclude that all polypept,idc chains, chemically identical or not,, have about’ the same size. On the basis of the observed scat,t,er in the various parameters and of reasonable estimat’cs for other sources of error the average molecular weight is probably good within 10 % . To establish t,hc number and proportion of polypeptide chains in the full as well as in
I
1 2 3 - 1O’g Protein/ml FIG. (i. Ikpetldrnce of densit,y of SV40 protein sollltions o!k protein concentraliolr at. 20 + 0.4” (4 dl guanidinillm h,vdrochloride-0.1 M Tris-IICIL 0.01 .lf 8-mrrcaptoethar~ol, pII 8.0).
“1
2
4
6 = t03g Protein/ml
FIG. 7. 1)iffusion constant of SVdO at variorw protein concrntr.:itions (buffer as in Fig. 6).
the empt,\. virus particles, microzone electrophoresis in S M urea-O.01 dl p-mercaptoethanol between pH 2 and pH 9 was used. At any one of these conditions IKI more t,han t)hree different’ componems were observed. Best separation of the pept,ide chains was obtained in S dl urea-O.1 III Tris-HCI0.01 :I/ P-nlcrc:lptoc~thaIlol at pH S.6. k’igur(~
8
STRUCTUI
OF SIMIAN
s shows the pattern of the polypeptjide chain bands, designated A, B, and C. This pattern is qualitatively and quantitatively the same for full and empty particles. Two methods were applied to determinr the cluantitjative proportion of the polypcpt,ide chains rcprcscnted by the three bands. Calorimetric measurements with the Analytrol equipment y,icldcd in 9 runs with full particles average values of 45.3 f 1.S ‘< for band A, 35.6 f 3.O’Z for band R, and !I.1 f 2.2% for band C. III 13 runs with empty part,icles average values of 46.0 f 2.2% for band A, 45.6 f 2.9rl for band B, and S.4 f 2.6 % for band C wcrc obtained. When the evaluation of the electropherograms was done by microderlsitometrv of the photonegatives, the average values for the full part,iclcs were 45.0 f 2.4 ‘ii (A band), 4G.S f 2.1 “:’ (B band), S.2 f l..i? CC band) and for t,hc empty particles 45.S f 1.9% (-4 band), 45.1 f 2.4’;’ (13 band), 9.1 f 2.3 5: (C band). All the values calculated reprcseru t)he amoum of stain absorbed. When we assume that, the acidic stain (I’onceau S) adsorbed preferentjially to positively charged groups, A and B bands must have t,he same specific absorbing capacit)y. They migrate together in the clcct,ric field at, pH 2 and must, t,hercforr have about the same number of positive charges. The average values for A and 13 bands calculated from the data prescnt,cd are 45.5 and 45.6 % ; this means that these polypcptide chains are present in cquimolecular amoums. The C band is more basic and might therefore have a higher specific absorbing capaciky. This means that the weight proportion of C polypeptide chains in t>hevirus particle might be smaller than t,he average value of S.7 7;) which can lw calculated from t,he data presented. PI correction of this value might be necessary when the amino acid composition of the individual chains is krIow~I. .4n A ppyoach to Localize the Polypeptirle Chains in th.e Virus Partick We found that extended a&ion of trypsin digests t,he protein moiet’y of the full and empty SV40 particles completely; i.e., none of the t’hree polypeptide bands was detectable by our standard ele&ophoresis tech-
VIRUS 40. II.
519
nique. It has to be expected that stericall) exposed polypeptide chains are more accessable to tryptic digestion than t’hose chains which are not localized direct,ly on the surface of the particles. ParM tryptic digest,ion of full as well as of empty particlcs showed that A and B polypeptide chains mere more rapidly degraded than C chains. The quantitative cv:tluat,ion is difiicult to imerprct since nondial~~zable degradation products of the chains interfere in part’ with a correct dcternlirl:tt,ioll of the amount 01 the chains. I:urther work is in progress.
The amount of DS,4 per virus particle be determined from the phosphorus :IIK~ deoxyribosc content. l;or analyt’ical calculust,ions one has to consider that the 01): weight ratio = 3.G OD&mg virus (Koch et al., 1967) was determined for the ammonium salt of the complete virus. Thenfore all calculations are based on the am monium form of the DNA (phosphorus can
content,
!).r, ‘; ).
In 4 experiments the average value of the phosphorus content of N-10 (full part,iclrs) was 1.19 f 0.02’2, which equals :I DNtZ content of 12.5 f 0.2 3. On the basis of the molecular weight, .lI = 17.3 X 10” for the SV40 partjiclc, t,hc virus DSA has a molccu lar weight of 2.2 X 10fi. l;or the determination of the deoxyribosc content the Burton (1933) modification of the Dische diphenylamine reaction was used t’o relate the virus samples to a standard thymus DSA sample v&h known phosphorus content. I’rom the results of 5 experiments the average DSA content of SY-10 particles was calculated to be 11.4 f 0.4 “‘4, this corresponds to a molecular weight kf = 2.0 X 106 for SV40 DIVA, considering a particle weight of 17.3 X 10”. This value is not as reliable as that calculated from the phosphorus content, since different color imensit,ies might arise because of differences in the base ratios of t#he two samples conpared. A t,hird method for the calculat~ion of the molecular weight, of the viral DNA is based on the dctcrmination of the sedimematiou constant. The molecular weight can then be obt,:rincd with the Eigner-Doty ( 19(G)
520
ASI)E:ltEII.
cquat~ion S = 0.116 X JI"."2:'. 'his quat ion however, applies only to troucircular double - skanded DSA with ~nolcwhr weights less than 4 X 10”. The> original form of the DSA in the SV40 p:nticlc is most’ likely the twisted circuhtr doublcstranded form. Analogous to the findings of Vinograd et al. (1965) with polgom:\ l>NA, it. leas found thnt n limited numhcr of brwks produces 3 mixture of twist& cGcul:ir, doublcopt’” circulkw, a11 d tloucircuhw stranded forms. Such mixtures have bccri used to determine the sedimentation cow st,>tnts of these three forms of S\‘10 1>SL4. Sprcitil c:trc wns t:ken to l
ET AI.
shorter st pH (i th:m at pH S or ut pH 6 with formaldehyde trcatmcnt, although all PXperiments were pcrfornwd with the s:tmc DKA sample. Sinw :~ll clect~rotl opt,ic:~l cxposures were made under the sumc coudtions :md the error of leugth measurrmcr1t.s was :tbout 3 ( ;x, thcsc diffcreuces c:m bc best explained by wrinblc stacking dist:mccs in the different forms of the I)NA structure. When \YP compare the length distribution of noncircul:w :md open circuhu forms of SVGDlnA obtaitwd by sprcuding at pH (i (Fig. 11) WC rccogriizc il dist,iuct ni:~ximum at, 1.4 p for the noncirculw form, :md for t hr opeu circul:w form :i m:iiu ni:kxitnuni of I .(i p :tnd :L less signifiwnt, otw :rt 1.55 p. This differctw of mow thtm IO’; for the two forms of the S\:40 I)NA might, :dso tw due: to diffrrcnt stacking distsucw in the DNA structure. Spre:lding :lt pH S giws length distributions with :t wry t)ro;Ld maximum betwwu 1.&5 p :md 1.(i k for t hc twncircular form :mtl two sharp m:tximn :Lt 1.65 p :md 1.69 ~1for the open c+irwlar form (I<&. 12). The lcugt,h distributions of the formaldehydctreakd sample is cwngrucut with t tic> dist ri butiori at, pH S. Since formuldchyde is l~nowr~ to induw :Lt, lenst ptirti:ll dcwlt~ulatiorl of double~st,r:lrlded DKA, w tend to :I simil:w irit,crl)rc~t:lt,iorl of the pH S slnwdiug c#ccts. Cudcr both of these conditions, the lcngt,h dist,ributiorls for the noncircul:u :IS ~~~11:LS for the opw circular form :w congruent,. Thcb l(qth dist,ributiotl for the noncirculur form h:w RIO discrete m;Lximum for clithcr OIW of the two spreading conditions. The qq)car:mcc of thcl twisted circular forms is the same for bot’h spreading twhniqucs ( l’igs. 10, b, c). It should bc st rcssctl t,hat for these detjerminnlions ow :md the wnic I)NA prcpw:~t,iott \vas used. The incrcwe in IengSth of r&hit p:tpillomtl virus DNA due to formuldchyde det&urntion has been reportt>d t,J. Iileit~schmidt~ ~1 al. (,19&j). Similw effects wcrc observed t)!, Ilang ef al. (1967) dw to low ionic kength of the hypophtlse. These condit.ions :~lso apply to our spreading :kt pH S. Thcrcforc WC do not, feel justified t,o USCthe length measuremcnt,s obtjained at, pH S for t>he determn:itiotl of the tot:J m:~ss of thr SV40 D5.i molcculcs.
STI:l;CTI:I:E non - circular
OF SIMIAN
farm
FIG. 11. Lellgth dist,ribuliou of SVdO IIN. spread at pH 6.0. h’oucircular form: 229 illdivid1131s;open circular form: 319 iridividllals non -circular
form
pH6 open ch-culw form J1
L
1
1
i2
1.5
1.7 /I
FIG. 12. Lellgth distribtlt ioll of SF7-I0 IIN. spread at 111% 8.0. Noucircular form: 20%iudiviclr~:~ls;open rircular form: 165 irldividrl:lls.
lkfore calculating the molecular weight on the basis of the mass per unit length, all the available data on DNA should be con sidercd. LY-ray diffraction patterns of many kinds of double-stranded DIVA show several diffcrent~ crystalline and semicrystallinc forms of DNA which have different masses per unit length (for reference see T,angridgc cl nl., 1960). All those measurements were performed on fibers of noncircular doublestranded DNA. Therefore only the length distribution of t,he noncircular form, in our case obt,ained at pH 6, can be used for such calculations.
VII:I:S
10. II.
.521
The length distribution in this case is asymmetric with respect to t,he maximum at 1.4 p (Pig. 11). The distribution of t,he steep slope on the side of the lower length values is most likely due to furt,hcr degradation after splitting of the DSA ring. On the side of the higher values, :I continuous tran&ion to the lengths of t,he open circular forms can bc observed. A small maximum of the noncircular forms at 135 p corresponds to the first,, less significant, maximum, of the opctn circular form. This continuous transition can bc interpreted in terms of diffcrcnt forms of the DSA structure, possibly aready present within one and the same molecule. Thus the maximum at 1.1 p might rcprcsem DLKA molecules with an cncrgeticnlly favored base stacking. When w assume that, the base stacking is smulur to, or the same as, that, in semicryst,alline H forms of DNA fibers, the molecular weight of the S\‘-lO DS:I is ill = 2.53 X lo”, as cnlculat~cd from :I length of 1.4 p, from an average stackirlg distance of 3.12 A, ir,ntl from an avcragc molecular wight of 61A for a base pair. I’inally, WChave to consider the possibilit? that further heterogcncity in lengt,h might arise from changes in the DKA structjure in duced by additional hits in the open circular as well as in the noncircular form which do not induce chain scission. The effect, should result, in an incrcnsc in length and might be in part rcsponsiblc for the presence of the two maxima in length of the open circular fornu and might also be in part the cause for the higher Ictrgth values of the noncirculat forms. The values for the molcculnr weight of the SVdO DXA calculated from four different parameters are in agreement within f lo!‘;. A fifth method applied by Crawford and Black (1964) is equilibrium densit)y gradient wntrifugat~ion. l’rom the band width thq calculated thr molecular weight of S\‘40 DSA to bc 2.6 X IOfi, which is in agreement with our findings. Calculatim oj S l’-$O l’artide Weight jro~t~ Number and Size uj” Courpmenfs. The analysis of the particle shell of S\‘40 proved that the shell has the symmetry of
522
A~l)EREIt,
the ‘I’ = 7 (dexko) icosahedral surface lattice which is composed of 72 morphological units. A shell of this typo is built of 420 structural units, each of which might cow sist) of one or several, possibly different8 types of polypeptjidc chains. Our investig:Ltwns on the protein moiety of the virus particle showed the presence of three different types of polypeptidc chains all of about the same size. Only A :md I3 chains arc prcscnt in cquimolecular proportions, and thus we conclude that the structur:~l units of t.he virus shell arc constituted orllv of A and I3 chains. This is in agreement. \iith the tentative localization of the C polypeptidc chains inside the particle by partial tryptic digestion. In the cast that OIIC st,ructurul unit COIItains one ,A and OIW B chain, the weight of the protein shell of one particlr can he calculated to he 330 x 2 x 16,350 = 13.7% X IOfi. This value represents 91.1 2 of the total virus prot,ein. When wc include the proportion of C polypeptidc chains the result,ing weight of the total protein moict)y of OIIC virus particle is 1,5.05 X 10”. The most reliable values for the molecular weight of S\‘40 11SA are those obtained from the determination of the phosphorus content and from sedimentation data. On this basis a wlue of 17.2 to 17.3 x 10Gcan be calculated for the molecular weight, of the virus particle. This is in good agreement, with t,he wlue derived from scdimcnt:~tion and diffusion measurements performed wit)h full SV40 particles, even if our wtimated wror of 10 ‘2 is taken into account.
ET
AT,.
moved the smallest particles. l~urtjhcrmort~ we found that smaller p&icles of t#hc S:U~~C size (I, II, and III) and nppcarancc c:m bc oht,aincd from 8\‘10 particles by partial tryptic digest,ion or treatment, with nkdine buffers. (2) The smaller-size pnrticles cannot, repr(~sent an inner prot,ein shell normally prcwut~ inside t.hc complete virus part.iclc. We h:tvc: found particles with three distinctly different diameters and surface st’ructurcs. Thaw: particles are most probably members of th(x 7’ = 1, 7’ = :%,and T = 1 clusscs of kos:lhedral symmctr~-. All three cannot he tit tcvl to&her into one particle wit,h T = 7 synmctry. The molecular weight of that S\‘10 particle as calculated from the number and size of the components does not tolcratc t IIV presence of the small part,icle shells. c13) The third possibility discussed by I;inch and I\lug (19(G) for the origin of the sm:dI~~r parbiclcs has :l high probxbilit,!. of twing correct. Thwc particles cm I-JC corlsidcwtl t () originate b’, rwsscmblg of norm:d morphological umts derived from drsintegratc~d normal sizcl pnrticlrs, but, in a11 :~bnorm:ll arrangemerit~. Our data .show that an exact detcrmiIw tion of the particle weight of an icosahcdr:~l virus together with a11 exact determination of the size and number of the componcwts makes it possible to predict’ the s>wm(+rJ class of this virus particle. We used thcb established particle symmetry to check the previously det,ermined p&icle weight i I’:trt I of t,his series) on the basis of the e(lumolecular proportion of the 11 and 13 polypeptidc chains and other determined p:w:rneters of the constituents. For th(L determination of the amount of As Mug (19ti.i) predicted, the SV-10 par- I1SA per particle, four different paramctew ticle has a symmetry of the T = 7 icosa- of the DNA were investigated. The values hcdral surface lattice. The hand of the obt,ained do not vary from t,hc mean v:dut: by more than 10 %. The results cst~ablish skewness proved to he dextro. OIW J>D;A molecule is contained The presence of smaller size empty shells that only in the full particle. Ko evidence was obtained was not surprising since similar observations t,hat any additional DiY.4 fragments arc with rabbit papilloma virus were reported by Finch and Klug (196.5). Of t,he t,hree pos- present in the particles. The contour lengths measurements of sibilities for t,he origin of these small shells discussed by Finch and Khg (1965) WC can DKA molecules yielded different, values fol exchlde two: (1) The small-size shells do not the open circular and t,he noncircular forms. represent, a c,oIltnmillntiorl by extraneous par- WC must assume therefore that the stacking t’clcs in the crude virus suspension because distances which dctcrmine the mass ~JW L+c method of purification would have re- Illlit Ien~th o tliffor for the two forms.
The cont,our lengths of open circular S\‘10 I>SA molecules reported by Crawford rotal. ( 1966) were obtained from pH S spreading. Their result,s are in agreement with ours obtained under the same spreading conditions, originally used by Stocckcnius (Appndix to Weil and Vinograd, 1963). About) SO C polypepbidc chains we co11taincd in OIIC’ virus particle, as calculated from the amount and size of the C chains. This number does not fit into a11 icosahedral shell with the symmetry of 7’ = 7 which is built, from 420 ‘ident,ical structural wits as proposed by C&par and Klug (1962). The wsultjs from partial tryptic digestion of full S\‘40 particles indicate that the C plypeptide chains are probably inside the particle. Considering the more basic character of the c‘ polyprptide chains we assume that they play a role in orient,ing the virus IISA inside tho p:irt,iclc. Work is in progress to obtain final proof for this assumption.
I~IGNEII, J., atld 1)o.r~. t’. (19li5). The native, denatltred atld remttrrred states of deoxyrihonucleic acid. J. Mol. Riol. 12, 549-580. ~‘:LIAH, H. G. (1961). “l:ltraeerlt riftlgell-Met hoden,” 2nd rev. ed. Ueckmalr Instrllrnetlts Gmhlf, ibllinich, \Vrsl (;ermany. FINCH, J. T., and KLI-G, ii. (1965). Strllct~lrc of virllses of the I)apillorn:~-l)ol~-o~~~~ type. I II. Strrlct IIrc of rabbit papilloma virrls. .J. .Ilo/. Hid. 13, l-12. KLEISR(‘HMII~,
A..
S(:HI,I-RIBEI((;Z:Il,
Thr :tlIthors wo111d like to t haIlk Miss II. voll Schiitz, Miss M. I
K. (195(i). A sttidy of the conditions alld mechallisrn of the diphenylamine react ion for I he calorimetric estimatioil of tfeos~riboirlicleic arid. Hioche~. J. 62, 315. (‘.\w.\R, 1). L. I)., and KLPG, A. (19(Z). Physical principles in the construction of regrdar virllses. (‘old Spring Harbor Sgmp. Quanl. Bid. 27, l-24. (ill.\\\-FORD, L. V., and RL.\CK, I’. Il. (196-l). The nrlcleic acid of simian virlls 10. I’i,ology 2-E, 388392. C’~t.\\~mto, L. V., FOI~TI’, E. 4. C., a11d CRAW FORI), E. ?*I. (1966). An electron microscopic stlldy of I)NS from i hrre t llmor virrlsrs. J.Mieroscop. 5, 597-601. I~I~I~ToN,
IANC:.
I).,
:rl~tl
%.\HN,
1:.
K.
(1959). j’brr I)rsox~riho~~~~klei~~~~~~re-nlolk~l~~ ii1 I’rotcitl-~Iisrhflmrll. %. .\~a/ur~or.sch. lit,, 770-779. KI~EISSVHMIIYI’~ A. Ii., K\ss, S. J., WILLI.\MS, II. c., Rlld KNIGHT. (1. A. (1965). Cyclic I>NA of Shape papilloma virlls. .I. Mol. Hiol. 1:3, 7-%75ti. KI,~-c:, A. (1965). St rllctrlre of virhlses of the pul)illom:t-~~ol~or~r;l type. TI. Commellts on other work. ./. .Ilo(. Bid. Ii, 121-131, Kr.cc,, A., anti VINl’H, d. T. (19;i5). St rII(:tllrP of virllses of t hca ~~;~pillorna-pc~l~o~~i:~ type. I. IIrtmn~~ wart virlis. ./. .Ilol. Riol. 11, #X+423. KOVH. hl. it.. ~~;c:c:~lts, 11. J.. 24NI)EI
I)..
:llld
Fll
\SK,
11.
(ltjti?).
Si riict1irp of simiatl vir\Is -10. I. I’tirification at~tl physical characterixat ioil of the virrls particle. lTirdog!/ 32, 503 -510. L\SG, I)., I3r’.r.\ltI,, II., \I’OLFF, B., 1Llld I:I.ssELL, I). (lD!ii). 121crt 1’011microsc:)py of size and shape of viral IjN.4 ill sollitions of different iotlic strengths. ./. Mol. Viol. 23, l(i:I-~181. T,\xcar~m, I:., W~r,w,u, II. I:.. Ifoomx. C. \V., W’ILKINS, hr. IT. F., aud H.\MII,~IY)s, I,. I). (19liO). The moleclllar collfigllrat ioll of deosyrihotlrlcleic acid. .1. Mol. Rio/. 2, 19G37. hl.\~m, 11. 11.. ,r.\MISOS, 9. &I., and JoKI).\s, I,. II:. (19(X3). Biophpsical stltdies on the natrlre of the sinGail papova virris particle (vacriolat ing SYXI virlls). I’irolog!/ 19, 359Mtiti. ~INOC;I~.U),
.I.,
LEBO\\~ITZ,
J.,
It.\ULOFF,
SON, I:., alld LUE~IS, E’. (1965). The ciilar form of polyoma viral I)N’A. .Icad. hsci. I’.S. 34, 11OG1111. WEIL, II., and T~rsoc:r~ \I), J., Appendix ESIITS, W. (1963). Thr cyrlic helix roil forms of polyoma viral DNA. .I cad. Sci. I’.S. .X, 7X- 738.
It...
\v.\‘r-
twisted cirProc. .\:ntl. by STOECKand c,vclic Proc. .\-o/l.
32, 511-523 (1967)
Structure II. Symmetry F. A. AKDERER,
of Simian
and Components
Virus
40
of the Virus Particle
H. D. SCHLCMBERGER, ill. A. KOCH,’ H. FRAn-I<, AKD H. J. EGGERS’
Xax-Planck-Institut
jiir
Virusjorschung,
Accepted April
Tiibingen,
Germany
I.$, 1967
The symmetry of Sk'40 particles was examined and proved to be of the 2’ = 7 (dextro) icosahedral surface lattice. Evidence is presented that t,he three types of particle shells of smaller size are derived from reassembled morphological units of disintegrated normal particle shells. Examination of the sllrface structure of the smaller particles strongly suggested icosahedral surface lattices of T = 1, T = 3, and T = 4 symmet,ry classes. Three different polypeptide chains with an average molecular weight of 16,350 f 100/d were identified in the protein moiety of the virus particle. The proportion was determined as 45.5 : 45.5 : 9. Four different approaches were used to determine the size of the virus DSA. A molecular weieht of 2.25 X 106 f 15% was established. A calculation of the particle weight from the size and number of the components yielded 17.3 X IO6 f 10yO.
several authors. Mayor et al. (1963) calculated a molecular weight of 4.0 to 5.2 X 106, Crawford and Black (1964) found molecular weights of 2.6 X 10” and 3.2 X lo6 for the virus DKA. The st’ructural details of the SV40 DNA have been only interpreted in analogy to polyoma virus DNA (Vinograd et al., 1965). This paper present’s our findings on the symmetry of bhe particle shell. Furthermore, the size, number, and chemical nature of t,he viral components were determined.
INTRODUCTION
In Part I of this series, a simple method for t,he propagation and purification of SV40 virus was described (Koch et al., 1967). The diameter of purified full SV40 particles was found to be 41.1 f 0.6 rnp, and the molecular weight of the part’icles was calculated to be 17.3 X 106. The number of the morphological units of the icosahedral SV40 shell has been reported to be 42 (Mayor et al., 1963). Klug (1965) however, predicted in analogy to human wart virus (Klug and Finch, 1965) and rabbit papilloma virus (Finch and Klug, 196.5) for SV40 the symmetry of the T = 7 icosahedral surface lattice which is composed of 72 morphological unit’s. No determinat,ions of the exact number and size of the constituting polypeptide chains have been published. Investigations on the size and st,ructure of the virus DNA have been reported by 1 Present address: Inst.itut fiir Virologie, Liebig-UniversitLt, Giessen, Germany.
Justus
MATERIALS
SND
METHODS
Electron microscopy. For t’he determination of particle symmetry a small drop of 2% phosphotungstate, adjusted to pH 7.2, was placed on a copper grid covered with carbon-coated Formvar film and dried by infrared light’. One drop of a virus solution containing l-2 mg. of virus/ml (0.1.5 M NaCI-0.02 M Tris-HCI) pH 7.2 was brought on this grid and withdrawn immediately by touching the edge of the grid with filter
511
512
ANDERER,
l)apcr. One should hc careful that the phosphotungstate film does not dissolve completely and mix with the drop of the virus solution. Therefore all manipulations have to be performed rapidly if one wants t,o obtain “OIIC side” particle images. Further negative staining t,echniques were either t,o mix the virus solutions with 2%. phosphotungstat,e before placing the mixture on a grid or to place the virus solution directly on the support’ followed by 1 or 2 washings with 2 % phosphohungstate. A Sicmcns Elmiskop IA was used at a magnification of 37,000 with double-condensor illumination, operating at SO kV. iLlagnification calibration of the electron microscope was performed wit,h carbon grat,ing replicas (E. Ii. Fulham, Inc., Schenectady, New York). Replicas of diffraction grat)ings ruled to 54,800 lines/inch were used. The grids prepared for determination of particle symmetry were always inserted in t.he electron microscrope wit’h t)he specimen side away from the electron source and for the photographic printing the elect)ron microscope plates were always insert,ed into the enlarger with the emulsion side away from the bromide paper. For the det’ermination of the strand length of ST40 DKA the protein monolayer t’echniqur of Kleinschmidt et ab. (1959) was used. Cytochrome c solutions, at’ a conccn tration of 0.1 mg/ml, were prepared wit’h the following solver~t~s: (,4) 1 A/ ammonium acet,ate adjusted to pH 6.0 with acetic acid, (B) 1 II1 ammonium acet,ate-0.5 % formaldehyde, pH 6.0, (C) 1 nZ ammonium acetate adjusted to pH S.0 with 1 ill NaOH. The solutions for spreading DXA with prot’ein contained 1-3 pg of SV40-DXA/ml, freshly diluted from tenfold DNA concentrat~ions. The hypophase for spreading t,he DNA in solvent (A) was 0.3 df ammonium acetate, pH 7.1; for spreading the DNA in solvent (B), it was 0.3 M ammonium acetate-0.5% formaldehyde, pH 6.0; and for spreading the DKA in solvent (C), it was 0.015 31 ammonium acct~atc, pH S.0 (Weil and 1’inograd, 196X). Carbon-coated Formvar films were used as support,s for t,he DNA-protein monolayers. Rotary shadowing of t.he specimens was performed with vaporized Pt-Pd-Au-
ET AL.
Ag alloy at a grazing angle of 10”. ?tIicrographs of these preparations were obt)ained with a Siemens Elmiskop IA operating at SO kV, with double-condenser illumination. Magnification calibration of the elect,ron microscope was performed with carbon grating replicas (E. P’. Fulham, Inc., Schenectady, New York). ,411 exposures were made at a magnification of X 19,200 with constant strength of the current of the objective lens (459 m-4) and with constantly comrolled positions of the projcctivc lens and intermediat,e lens. Contour length measurements of twodimensional images of DNA at a final magnification of X 192,000 were carried out with a calibrated map ruler. All measurements were done by t’hree persons to compensate for individual errors. The maximum deviations were f 1.5 %$. Determinations of the molecular weight of the polypepticle chains. For the determination of t,he partial specific volume of SV40 protein a L-ml pycnomeber cell was used. A sample of empty particles twice purified by densit,y gradient centrifugation [see Part I (Koch et al., 1967)] was dried in vucuo at SO” for 48 hours and dissolved in 4 M guanidinium hydrochloride-O.1 M Tris-HCl-0.01 M fi-mercaptoethanol, pH 8.0, and t)he extinction at 280 rnp was determined after appropriate dilution. After dialysis against, the same buffer for 78 hours at room temperature measurements were performed with solutions containing 4.32, 3.16, 2.37, 1.48, 0.925, and 0.465 mg/ml, as derived from OD measurements. All measurements were corrected for zero time of correct filling by following the decrease in weight for 200 seconds during t,he weighing. The t’emperature was 20.0 f 0.4”. Sedimentation and diffusion experiments were carried out, with protein solutions (same buffer as above) using a Spinco model E ultracentrifuge with schlieren optics. Schlieren pictures were measured with a t,raveling microscope. Sedimentation was performed with protein concentratJions of 8.0, 6.0, 1.0, and 2.0 mg/ml in the AX-D rotor (capillary cell assembly) at 42,040 rpm. All measurements were made at 20.0 f 0.01”. The diffusion patterns were evaluated according to Elias (1961). The determina-
STRUCTURE
OF SIMIAN
tion of the average molecular weight of the polypeptide chains, using the modified Archibald method (Elias, 1961), was performed in t’he AN-D rotor with double sector cell assembly at 42,040 rpm. Electyophoresis of virus proteins. A Beckman Model R-190 microzone electrophoresis and cellulose acetate membranes (RIillipore Filter Corp.) were used. The membranes were soaked in 8 M urea-O.1 M Tris-HCl0.01 M P-mercaptoethanol, pH 5.6. The cell was filled with the same buffer. Lyophilized full, as well as empty, part’icles were dissolved in this buffer (concentration of @-mercaptoethanol: 0.1 111) to give 1% protein solutions. The samples mere run at room temperature with 13-17 volts/cm for 6G-120 minutes. The wet membrane strips were st,ained with Ponceau S; after removal of t#he excess stain bhe membrane strips were dried t,o make them transparent. For the evaluation of the electropherograms the Beckman Model RB (Analytrol) with Jlodel R-202 microzone scanning attachment, was used. Phot’onegatives of the electropherograms were prepared using a blue filter and t’hen scanned with a Joyce-Loebl MI< IIIB microdensitometer. Tryptic degradation of the fdl virus particles. Samples of 1.6 mg SV40 in 1 ml 0.02 M phosphate buffer, pH 7.5, were digested at 37” with 5 X crystallized trypsin (enzyme: substrate ratio, 1 :lOO). After 30, 60, 120, and 240 minutes of incubation, the trypsin action was st)opped in individual samples by addition of equivalent amounts of kallikrein inactivator (identical with bovine pancreatic trypsin inhibitor). The samples were treated wit)h DBase overnight folowed by dialysis against water. The lyophilized samples were subject’ed to microzone electrophoresis. Determination of the amount of DNA per virus particle. Four virus samples cont,aining 0.450 or 0.475 mg virus in 0.15 ml of 0.15 M NaCl-0.02 Af Tris-HCI buffer, pH 7.2, were used for phosphorus analysis. For the determination of the deoxyribose content the Burton (1956) modification of the Dische diphenylamine reaction was used. The color intensities were standardized with solutions of calf t,hymus DXA (Calbiothem.) with krlowr~ phosphorus content.
VIRUS
513
40. II.
Controls performed with thymus DNA in mixtures with variable concentrations of bovine serum albumin did not show significant deviations of DSA-specific color intensities. Sedimentation of SIT40 DNA. In order t’o obtain protein-free SV40 DNA, the virus was dissociated in pH 10.5 buffer at 4” for 20 hours. The DNA was separat,ed from the prot,ein in a self-generating CsCl density gradient, which was prepared by a 2 ml overlay of dissociated virus on 3 ml of CsCl solution, saturated at room temperature. Aft)er fractionat,ion and dialysis again& 0.15 Al XaCl-0.05 A1 Tris-HCl-10P3 M EDTA, pH 7.2, t’he ratio E26,JE280was l.S3. Sedimentation was performed with DNA solutions containing 0.8 OD260 &ml. A Spinco Rlodel E ultracentrifuge with UVabsorption optics was used. All measurements were made at’ 20.0 f 0.01‘. The rotor speed of t,he AN-H rotor was eit,her 56,100 rpm or 39,460 rpm. UV-absorption pictures were scanned with a Joyce-Loebl MT< III B microdensitometer. RESULTS
Examination Shells
of the Symmetry of the Particle
Caspar and Iilug (1962) have proposed a theory for the const,ruction of protein shells built from ident’ical protein subunits. For isodimensional particles, the icosahedral symmet’ry is preferred. The authors enumerated all the possible icosahedral surface lattices, the numbers of morphological units and the number of structural units involved in each case. This theory provides a classification of icosahedral virus prot,ein shells. The best technique available to obtain virus part)icle images of high resolution is negative staining with heavy metal salts. To determine the arrangement, of morphological units one is restricted t’o those particles which are contrasted on one side only. From the results of the work of Finch and Mug (1965), we know that the dominantly contrasted side is the side near t,o the carbon substrate. The examination of ‘(one side” particle images of negatively stained SV40 revealed the symmetry of the T = 7 (dextro) ico-
FIG. la-d. Selected “one side” particle images of negatively stained SVlO particles together with duplicates where the 5-coordinated morphological rmits arc marked .r and the (i-coortlim~ted tInits which determine the “72d path” are marked with a black dot. Magnification : X 380,000.
sahedral surface IaUice which is composed of 72 morphological urms. Several selected particle images are shown in I’igs. la-d, and t,hese have at least two unequivocal S-coordinated morphological uuits which are marked with X in the duplicate image of the same part’icle. The A-coordinated morphological units, which determine the t#ypical 72 d path between the 5-coordinated units are marked by black dots. No parMe images with T = 7 (levo) symmet,ry were observed. The hand of 7’ = 7 symmet,ry is determined by t)he convention of specimen mounting and printing given by Pinch and Klug (196ri). Higher yields of “one side” particle images as well as sharper contrast, as compared wit,h conventional negative st’aining t,echniques, were obtained when the carbon substrate was coated first, with a solid phosphotungst8at,e film. This modification of negative staining excludes the possibility that particles will be stained only on the “far” side wit,h respect, to the carbon substrate. The yield of “one side” images with at least two unequivocal %coordinxted morphological units was about 2-5 ci;, though the majority of particle images could not be well examined because of a contin uously dark background of phosphotung stat,e. Scarcely any ‘%wo side” particle
images were detectable. The “two side” images obtained by conventional negative staining techniques were in agreement with shadowgraphs of T = 7 Geodestix models (Mug and P’inch, 1965). Some of these part,icles show a dist’inct superposition displaying “eyes” or terraced structures (I:igs. 2a-e). III the first communication (Koch et al., 1967) it was report,ed that the highly puriticd empty particle pool contained not only particle shells of normal size, but also appreciable amounts of smaller-size shells. We designated these smaller particles as I, II, and III. These have t,hree distinctly different diamet,ers; size I = 31.5 =I= 1.7 rnp, size II = 27.4 f 0.9 mp, and size III = 16.S f 1.0 rnp. The diameters were measured on particles well embedded in phosphotungst,at,e. The examination of the surface structure of the small particles III, the most frequently occurring (7-S %‘), st’rongly suggests a symmetry of the T = 1 icosahedral surface lattice. Figures 3a and 3c show selected “one side” particle images with two 5-coordinated morphological units. A photograph of a T = 1 model in the same oriernation is depicted in Fig. 3b. Figure 3d shows a particle only partly embedded
STRUCTUI:E
OF SIhlIrlN
VIRUS
515
10. II.
FIG. 2a-e. Selected “t,wo side” particle images which show a distinct or terraced strrlctrlres. Maguificatioll: X 380,000.
srlperposition
displaying
“eyes”
FIG. 3. Selected “one side” images of small particle shell III with two j-coordinated morphological units (a, c), and particle images viewed along the 5-fold axis (d, f). Photographs of two different views (b, e) of a T = 1 model are added as a visual aid. Magnification: X 380,000.
in phosphotungstate viewed close to a fivcfold axis. The part’icle shown in Fig. 3f is covered with phosphotungstatc and, because of the superposition of the morphological units which occurs in views along the fivefold axis, the particle appears as a ring of 10 “beads.” A phot’ograph of a T = 1 model viewed also along the fivefold axis is shown in Fig. 3e to facilitate the interpretation of the electron micrographs. Three ‘Lone side” images of small par-
II are preserited in E’igs. 4a-c. These particles show strong evidence for the symmetry of the T = 3 icosahedral surface lattice. In duplicates of the same images, the G-coordinated morphological units are marked 5 and the (i-coordinated are marked with black dots. In addition, photographs of 2’ = 3 models viewed in about t’he same position are presented. The examination of the surface structure of small part’icles I is hampered by t’heir Ccles
516
4a
FIG. 4. Selected “one side” images of small particle shell II (a, b) are shown i,ogether with dllplicates where the 5.coordinated morphological units arc marked L alrd the (i-coordinated llnits which determine the T = 3 arrangement are marked with black dots; (c) particle viewed along a 5-fold axis. l’holographs of T = 3 models viewed from corrrspotldillg posit ions are added. ?vlagttificat ioll: X 380,000.
low frequency (less than 0.5 ‘2 ). One selected particle image suggesting a symmetry of the T = 4 icosahedral surface lattice is presented in Fig. 5 together with a duplicate for which the 5coordinat,ed morphological units are marked .c and t,he li-coordinated morphological unit is marked with a dot,. A T = 4 model placed tentatively in the same position illustrat,cs the arrangement of t,he morphological units. If the smaller-size particle shells are built, from the same structural u&s as the S\?40 particle and the symmetry classes of particles I, II, and III, as evaluat’ed above, are correct, t.he diameters of tjhe resulting shells can be calculated. The ratio of the diameters of such shells is 1 (T = 7)/O. 76 (T = 4)/ 0.65 (T = 3)/0.3S (1’ = l), which is in good agreement with the rat’io of the actually observed diameters, when t,he value for the diameter of well-embedded empty shells of normal size is 12.0 f 1.7 mp.
Size
and
Number oj’ the I’olypeptitle
Chains
The structural u&s of :I protjein shell mav consist) of one or several polypeptidc chiins, which in t,he second cast need not, be idenGca1 nor need t,hey be different, since the icosahedral symmct’ry requires only t)hat groups of 60 1’ be cquivalcnt. Determination of thr size and number of polvpeptidr chains of t,hr S\‘40 protein moiety was performed under conditions known to dissociate an assembly of polgpept,ide chains into monomers. I’or the determinat,ion of the molecular weight) of the polgpeptide chains, the lyophilized fraction of empty particles (Koch et al., 1967) dissolved in 4 :1L guanidinium hydrochloride-0.1 Al Tris-HCl-0.01 N p-mercaptoet,hanol, pH 8.0, was used. The partial specific volume of the polypept,ide chains in this solvent, was determined pycnomctrically at, various conccrltrabiorls. The dc-
,517
FIG. 5. Image of small particle shell I sllggesting a 7’ = 4 symmetry of the icosahedral surface lattice. The 5- and (i-coordinated units are marked in analogy t,o Fig. 4. The photograph of a 7’ = 4 model is placed in approximately t,he same orientat ion as the particle. Magnifirat ion: X 380,000.
pendence of density of these protein solutions on the prot,ein concentrations is given in Vig. 6. The data fit a straight, line. The slope of t,his line corresponds directly to (1 - z’pO)(Elias, 1961). E’rom (1 - tip”) = 0.230 and p0 = 1.0989 t)he partial specific volume of the virus protein in this solvent is found to be fi = 0.70. The sedimentation pattern showed a single symmetrical gradient which sedimentatjed wit,h SZO = 0.75, without dependence on prot,ein concentrations bet,ween 2 and S mg’ml. The diffusion const~ant was evaluated from sedimentat,ion gradicnt8s. The corlcent8ration dependence of the diffusion is given in Fig. 7. Graphic extrapolation to zero concentration yields a value D20 = 4.S.5 X 10e7 cm2/sec. The average molecular weight (&I) of the polypcpt’ide chains calculat)ed from SpO = 0.75, I)?” = 4.S.5 X 10m7 cm2/sec, and (1 - tip,) = 0.230, is Ms,u = 16,350. When the dcterminatjion of the molecular weight was performed wit)h the modified Archibald method, a value M APP = 16,100 was obtjained. The larger scatter of thcsc values compared with the values used for the dls,D calculation does not, allow the detection of a concentration dependency between 4 and S mg. So significant, het’erogcneity was detectable from the shape of t,he concentration gradients. Thus we might conclude that all polypept,idc chains, chemically identical or not,, have about’ the same size. On the basis of the observed scat,t,er in the various parameters and of reasonable estimat’cs for other sources of error the average molecular weight is probably good within 10 % . To establish t,hc number and proportion of polypeptide chains in the full as well as in
I
1 2 3 - 1O’g Protein/ml FIG. (i. Ikpetldrnce of densit,y of SV40 protein sollltions o!k protein concentraliolr at. 20 + 0.4” (4 dl guanidinillm h,vdrochloride-0.1 M Tris-IICIL 0.01 .lf 8-mrrcaptoethar~ol, pII 8.0).
“1
2
4
6 = t03g Protein/ml
FIG. 7. 1)iffusion constant of SVdO at variorw protein concrntr.:itions (buffer as in Fig. 6).
the empt,\. virus particles, microzone electrophoresis in S M urea-O.01 dl p-mercaptoethanol between pH 2 and pH 9 was used. At any one of these conditions IKI more t,han t)hree different’ componems were observed. Best separation of the pept,ide chains was obtained in S dl urea-O.1 III Tris-HCI0.01 :I/ P-nlcrc:lptoc~thaIlol at pH S.6. k’igur(~
8
STRUCTUI
OF SIMIAN
s shows the pattern of the polypeptjide chain bands, designated A, B, and C. This pattern is qualitatively and quantitatively the same for full and empty particles. Two methods were applied to determinr the cluantitjative proportion of the polypcpt,ide chains rcprcscnted by the three bands. Calorimetric measurements with the Analytrol equipment y,icldcd in 9 runs with full particles average values of 45.3 f 1.S ‘< for band A, 35.6 f 3.O’Z for band R, and !I.1 f 2.2% for band C. III 13 runs with empty part,icles average values of 46.0 f 2.2% for band A, 45.6 f 2.9rl for band B, and S.4 f 2.6 % for band C wcrc obtained. When the evaluation of the electropherograms was done by microderlsitometrv of the photonegatives, the average values for the full part,iclcs were 45.0 f 2.4 ‘ii (A band), 4G.S f 2.1 “:’ (B band), S.2 f l..i? CC band) and for t,hc empty particles 45.S f 1.9% (-4 band), 45.1 f 2.4’;’ (13 band), 9.1 f 2.3 5: (C band). All the values calculated reprcseru t)he amoum of stain absorbed. When we assume that, the acidic stain (I’onceau S) adsorbed preferentjially to positively charged groups, A and B bands must have t,he same specific absorbing capacit)y. They migrate together in the clcct,ric field at, pH 2 and must, t,hercforr have about the same number of positive charges. The average values for A and 13 bands calculated from the data prescnt,cd are 45.5 and 45.6 % ; this means that these polypcptide chains are present in cquimolecular amoums. The C band is more basic and might therefore have a higher specific absorbing capaciky. This means that the weight proportion of C polypeptide chains in t>hevirus particle might be smaller than t,he average value of S.7 7;) which can lw calculated from t,he data presented. PI correction of this value might be necessary when the amino acid composition of the individual chains is krIow~I. .4n A ppyoach to Localize the Polypeptirle Chains in th.e Virus Partick We found that extended a&ion of trypsin digests t,he protein moiet’y of the full and empty SV40 particles completely; i.e., none of the t’hree polypeptide bands was detectable by our standard ele&ophoresis tech-
VIRUS 40. II.
519
nique. It has to be expected that stericall) exposed polypeptide chains are more accessable to tryptic digestion than t’hose chains which are not localized direct,ly on the surface of the particles. ParM tryptic digest,ion of full as well as of empty particlcs showed that A and B polypeptide chains mere more rapidly degraded than C chains. The quantitative cv:tluat,ion is difiicult to imerprct since nondial~~zable degradation products of the chains interfere in part’ with a correct dcternlirl:tt,ioll of the amount 01 the chains. I:urther work is in progress.
The amount of DS,4 per virus particle be determined from the phosphorus :IIK~ deoxyribosc content. l;or analyt’ical calculust,ions one has to consider that the 01): weight ratio = 3.G OD&mg virus (Koch et al., 1967) was determined for the ammonium salt of the complete virus. Thenfore all calculations are based on the am monium form of the DNA (phosphorus can
content,
!).r, ‘; ).
In 4 experiments the average value of the phosphorus content of N-10 (full part,iclrs) was 1.19 f 0.02’2, which equals :I DNtZ content of 12.5 f 0.2 3. On the basis of the molecular weight, .lI = 17.3 X 10” for the SV40 partjiclc, t,hc virus DSA has a molccu lar weight of 2.2 X 10fi. l;or the determination of the deoxyribosc content the Burton (1933) modification of the Dische diphenylamine reaction was used t’o relate the virus samples to a standard thymus DSA sample v&h known phosphorus content. I’rom the results of 5 experiments the average DSA content of SY-10 particles was calculated to be 11.4 f 0.4 “‘4, this corresponds to a molecular weight kf = 2.0 X 106 for SV40 DIVA, considering a particle weight of 17.3 X 10”. This value is not as reliable as that calculated from the phosphorus content, since different color imensit,ies might arise because of differences in the base ratios of t#he two samples conpared. A t,hird method for the calculat~ion of the molecular weight, of the viral DNA is based on the dctcrmination of the sedimematiou constant. The molecular weight can then be obt,:rincd with the Eigner-Doty ( 19(G)
520
ASI)E:ltEII.
cquat~ion S = 0.116 X JI"."2:'. 'his quat ion however, applies only to troucircular double - skanded DSA with ~nolcwhr weights less than 4 X 10”. The> original form of the DSA in the SV40 p:nticlc is most’ likely the twisted circuhtr doublcstranded form. Analogous to the findings of Vinograd et al. (1965) with polgom:\ l>NA, it. leas found thnt n limited numhcr of brwks produces 3 mixture of twist& cGcul:ir, doublcopt’” circulkw, a11 d tloucircuhw stranded forms. Such mixtures have bccri used to determine the sedimentation cow st,>tnts of these three forms of S\‘10 1>SL4. Sprcitil c:trc wns t:ken to l
ET AI.
shorter st pH (i th:m at pH S or ut pH 6 with formaldehyde trcatmcnt, although all PXperiments were pcrfornwd with the s:tmc DKA sample. Sinw :~ll clect~rotl opt,ic:~l cxposures were made under the sumc coudtions :md the error of leugth measurrmcr1t.s was :tbout 3 ( ;x, thcsc diffcreuces c:m bc best explained by wrinblc stacking dist:mccs in the different forms of the I)NA structure. When \YP compare the length distribution of noncircul:w :md open circuhu forms of SVGDlnA obtaitwd by sprcuding at pH (i (Fig. 11) WC rccogriizc il dist,iuct ni:~ximum at, 1.4 p for the noncirculw form, :md for t hr opeu circul:w form :i m:iiu ni:kxitnuni of I .(i p :tnd :L less signifiwnt, otw :rt 1.55 p. This differctw of mow thtm IO’; for the two forms of the S\:40 I)NA might, :dso tw due: to diffrrcnt stacking distsucw in the DNA structure. Spre:lding :lt pH S giws length distributions with :t wry t)ro;Ld maximum betwwu 1.&5 p :md 1.(i k for t hc twncircular form :mtl two sharp m:tximn :Lt 1.65 p :md 1.69 ~1for the open c+irwlar form (I<&. 12). The lcugt,h distributions of the formaldehydctreakd sample is cwngrucut with t tic> dist ri butiori at, pH S. Since formuldchyde is l~nowr~ to induw :Lt, lenst ptirti:ll dcwlt~ulatiorl of double~st,r:lrlded DKA, w tend to :I simil:w irit,crl)rc~t:lt,iorl of the pH S slnwdiug c#ccts. Cudcr both of these conditions, the lcngt,h dist,ributiorls for the noncircul:u :IS ~~~11:LS for the opw circular form :w congruent,. Thcb l(qth dist,ributiotl for the noncirculur form h:w RIO discrete m;Lximum for clithcr OIW of the two spreading conditions. The qq)car:mcc of thcl twisted circular forms is the same for bot’h spreading twhniqucs ( l’igs. 10, b, c). It should bc st rcssctl t,hat for these detjerminnlions ow :md the wnic I)NA prcpw:~t,iott \vas used. The incrcwe in IengSth of r&hit p:tpillomtl virus DNA due to formuldchyde det&urntion has been reportt>d t,J. Iileit~schmidt~ ~1 al. (,19&j). Similw effects wcrc observed t)!, Ilang ef al. (1967) dw to low ionic kength of the hypophtlse. These condit.ions :~lso apply to our spreading :kt pH S. Thcrcforc WC do not, feel justified t,o USCthe length measuremcnt,s obtjained at, pH S for t>he determn:itiotl of the tot:J m:~ss of thr SV40 D5.i molcculcs.
STI:l;CTI:I:E non - circular
OF SIMIAN
farm
FIG. 11. Lellgth dist,ribuliou of SVdO IIN. spread at pH 6.0. h’oucircular form: 229 illdivid1131s;open circular form: 319 iridividllals non -circular
form
pH6 open ch-culw form J1
L
1
1
i2
1.5
1.7 /I
FIG. 12. Lellgth distribtlt ioll of SF7-I0 IIN. spread at 111% 8.0. Noucircular form: 20%iudiviclr~:~ls;open rircular form: 165 irldividrl:lls.
lkfore calculating the molecular weight on the basis of the mass per unit length, all the available data on DNA should be con sidercd. LY-ray diffraction patterns of many kinds of double-stranded DIVA show several diffcrent~ crystalline and semicrystallinc forms of DNA which have different masses per unit length (for reference see T,angridgc cl nl., 1960). All those measurements were performed on fibers of noncircular doublestranded DNA. Therefore only the length distribution of t,he noncircular form, in our case obt,ained at pH 6, can be used for such calculations.
VII:I:S
10. II.
.521
The length distribution in this case is asymmetric with respect to t,he maximum at 1.4 p (Pig. 11). The distribution of t,he steep slope on the side of the lower length values is most likely due to furt,hcr degradation after splitting of the DSA ring. On the side of the higher values, :I continuous tran&ion to the lengths of t,he open circular forms can bc observed. A small maximum of the noncircular forms at 135 p corresponds to the first,, less significant, maximum, of the opctn circular form. This continuous transition can bc interpreted in terms of diffcrcnt forms of the DSA structure, possibly aready present within one and the same molecule. Thus the maximum at 1.1 p might rcprcsem DLKA molecules with an cncrgeticnlly favored base stacking. When w assume that, the base stacking is smulur to, or the same as, that, in semicryst,alline H forms of DNA fibers, the molecular weight of the S\‘-lO DS:I is ill = 2.53 X lo”, as cnlculat~cd from :I length of 1.4 p, from an average stackirlg distance of 3.12 A, ir,ntl from an avcragc molecular wight of 61A for a base pair. I’inally, WChave to consider the possibilit? that further heterogcncity in lengt,h might arise from changes in the DKA structjure in duced by additional hits in the open circular as well as in the noncircular form which do not induce chain scission. The effect, should result, in an incrcnsc in length and might be in part rcsponsiblc for the presence of the two maxima in length of the open circular fornu and might also be in part the cause for the higher Ictrgth values of the noncirculat forms. The values for the molcculnr weight of the SVdO DXA calculated from four different parameters are in agreement within f lo!‘;. A fifth method applied by Crawford and Black (1964) is equilibrium densit)y gradient wntrifugat~ion. l’rom the band width thq calculated thr molecular weight of S\‘40 DSA to bc 2.6 X IOfi, which is in agreement with our findings. Calculatim oj S l’-$O l’artide Weight jro~t~ Number and Size uj” Courpmenfs. The analysis of the particle shell of S\‘40 proved that the shell has the symmetry of
522
A~l)EREIt,
the ‘I’ = 7 (dexko) icosahedral surface lattice which is composed of 72 morphological units. A shell of this typo is built of 420 structural units, each of which might cow sist) of one or several, possibly different8 types of polypeptjidc chains. Our investig:Ltwns on the protein moiety of the virus particle showed the presence of three different types of polypeptidc chains all of about the same size. Only A :md I3 chains arc prcscnt in cquimolecular proportions, and thus we conclude that the structur:~l units of t.he virus shell arc constituted orllv of A and I3 chains. This is in agreement. \iith the tentative localization of the C polypeptidc chains inside the particle by partial tryptic digestion. In the cast that OIIC st,ructurul unit COIItains one ,A and OIW B chain, the weight of the protein shell of one particlr can he calculated to he 330 x 2 x 16,350 = 13.7% X IOfi. This value represents 91.1 2 of the total virus prot,ein. When wc include the proportion of C polypeptidc chains the result,ing weight of the total protein moict)y of OIIC virus particle is 1,5.05 X 10”. The most reliable values for the molecular weight of S\‘40 11SA are those obtained from the determination of the phosphorus content and from sedimentation data. On this basis a wlue of 17.2 to 17.3 x 10Gcan be calculated for the molecular weight, of the virus particle. This is in good agreement, with t,he wlue derived from scdimcnt:~tion and diffusion measurements performed wit)h full SV40 particles, even if our wtimated wror of 10 ‘2 is taken into account.
ET
AT,.
moved the smallest particles. l~urtjhcrmort~ we found that smaller p&icles of t#hc S:U~~C size (I, II, and III) and nppcarancc c:m bc oht,aincd from 8\‘10 particles by partial tryptic digest,ion or treatment, with nkdine buffers. (2) The smaller-size pnrticles cannot, repr(~sent an inner prot,ein shell normally prcwut~ inside t.hc complete virus part.iclc. We h:tvc: found particles with three distinctly different diameters and surface st’ructurcs. Thaw: particles are most probably members of th(x 7’ = 1, 7’ = :%,and T = 1 clusscs of kos:lhedral symmctr~-. All three cannot he tit tcvl to&her into one particle wit,h T = 7 synmctry. The molecular weight of that S\‘10 particle as calculated from the number and size of the components does not tolcratc t IIV presence of the small part,icle shells. c13) The third possibility discussed by I;inch and I\lug (19(G) for the origin of the sm:dI~~r parbiclcs has :l high probxbilit,!. of twing correct. Thwc particles cm I-JC corlsidcwtl t () originate b’, rwsscmblg of norm:d morphological umts derived from drsintegratc~d normal sizcl pnrticlrs, but, in a11 :~bnorm:ll arrangemerit~. Our data .show that an exact detcrmiIw tion of the particle weight of an icosahcdr:~l virus together with a11 exact determination of the size and number of the componcwts makes it possible to predict’ the s>wm(+rJ class of this virus particle. We used thcb established particle symmetry to check the previously det,ermined p&icle weight i I’:trt I of t,his series) on the basis of the e(lumolecular proportion of the 11 and 13 polypeptidc chains and other determined p:w:rneters of the constituents. For th(L determination of the amount of As Mug (19ti.i) predicted, the SV-10 par- I1SA per particle, four different paramctew ticle has a symmetry of the T = 7 icosa- of the DNA were investigated. The values hcdral surface lattice. The hand of the obt,ained do not vary from t,hc mean v:dut: by more than 10 %. The results cst~ablish skewness proved to he dextro. OIW J>D;A molecule is contained The presence of smaller size empty shells that only in the full particle. Ko evidence was obtained was not surprising since similar observations t,hat any additional DiY.4 fragments arc with rabbit papilloma virus were reported by Finch and Klug (196.5). Of t,he t,hree pos- present in the particles. The contour lengths measurements of sibilities for t,he origin of these small shells discussed by Finch and Khg (1965) WC can DKA molecules yielded different, values fol exchlde two: (1) The small-size shells do not the open circular and t,he noncircular forms. represent, a c,oIltnmillntiorl by extraneous par- WC must assume therefore that the stacking t’clcs in the crude virus suspension because distances which dctcrmine the mass ~JW L+c method of purification would have re- Illlit Ien~th o tliffor for the two forms.
The cont,our lengths of open circular S\‘10 I>SA molecules reported by Crawford rotal. ( 1966) were obtained from pH S spreading. Their result,s are in agreement with ours obtained under the same spreading conditions, originally used by Stocckcnius (Appndix to Weil and Vinograd, 1963). About) SO C polypepbidc chains we co11taincd in OIIC’ virus particle, as calculated from the amount and size of the C chains. This number does not fit into a11 icosahedral shell with the symmetry of 7’ = 7 which is built, from 420 ‘ident,ical structural wits as proposed by C&par and Klug (1962). The wsultjs from partial tryptic digestion of full S\‘40 particles indicate that the C plypeptide chains are probably inside the particle. Considering the more basic character of the c‘ polyprptide chains we assume that they play a role in orient,ing the virus IISA inside tho p:irt,iclc. Work is in progress to obtain final proof for this assumption.
I~IGNEII, J., atld 1)o.r~. t’. (19li5). The native, denatltred atld remttrrred states of deoxyrihonucleic acid. J. Mol. Riol. 12, 549-580. ~‘:LIAH, H. G. (1961). “l:ltraeerlt riftlgell-Met hoden,” 2nd rev. ed. Ueckmalr Instrllrnetlts Gmhlf, ibllinich, \Vrsl (;ermany. FINCH, J. T., and KLI-G, ii. (1965). Strllct~lrc of virllses of the I)apillorn:~-l)ol~-o~~~~ type. I II. Strrlct IIrc of rabbit papilloma virrls. .J. .Ilo/. Hid. 13, l-12. KLEISR(‘HMII~,
A..
S(:HI,I-RIBEI((;Z:Il,
Thr :tlIthors wo111d like to t haIlk Miss II. voll Schiitz, Miss M. I
K. (195(i). A sttidy of the conditions alld mechallisrn of the diphenylamine react ion for I he calorimetric estimatioil of tfeos~riboirlicleic arid. Hioche~. J. 62, 315. (‘.\w.\R, 1). L. I)., and KLPG, A. (19(Z). Physical principles in the construction of regrdar virllses. (‘old Spring Harbor Sgmp. Quanl. Bid. 27, l-24. (ill.\\\-FORD, L. V., and RL.\CK, I’. Il. (196-l). The nrlcleic acid of simian virlls 10. I’i,ology 2-E, 388392. C’~t.\\~mto, L. V., FOI~TI’, E. 4. C., a11d CRAW FORI), E. ?*I. (1966). An electron microscopic stlldy of I)NS from i hrre t llmor virrlsrs. J.Mieroscop. 5, 597-601. I~I~I~ToN,
IANC:.
I).,
:rl~tl
%.\HN,
1:.
K.
(1959). j’brr I)rsox~riho~~~~klei~~~~~~re-nlolk~l~~ ii1 I’rotcitl-~Iisrhflmrll. %. .\~a/ur~or.sch. lit,, 770-779. KI~EISSVHMIIYI’~ A. Ii., K\ss, S. J., WILLI.\MS, II. c., Rlld KNIGHT. (1. A. (1965). Cyclic I>NA of Shape papilloma virlls. .I. Mol. Hiol. 1:3, 7-%75ti. KI,~-c:, A. (1965). St rllctrlre of virhlses of the pul)illom:t-~~ol~or~r;l type. TI. Commellts on other work. ./. .Ilo(. Bid. Ii, 121-131, Kr.cc,, A., anti VINl’H, d. T. (19;i5). St rII(:tllrP of virllses of t hca ~~;~pillorna-pc~l~o~~i:~ type. I. IIrtmn~~ wart virlis. ./. .Ilol. Riol. 11, #X+423. KOVH. hl. it.. ~~;c:c:~lts, 11. J.. 24NI)EI
I)..
:llld
Fll
\SK,
11.
(ltjti?).
Si riict1irp of simiatl vir\Is -10. I. I’tirification at~tl physical characterixat ioil of the virrls particle. lTirdog!/ 32, 503 -510. L\SG, I)., I3r’.r.\ltI,, II., \I’OLFF, B., 1Llld I:I.ssELL, I). (lD!ii). 121crt 1’011microsc:)py of size and shape of viral IjN.4 ill sollitions of different iotlic strengths. ./. Mol. Viol. 23, l(i:I-~181. T,\xcar~m, I:., W~r,w,u, II. I:.. Ifoomx. C. \V., W’ILKINS, hr. IT. F., aud H.\MII,~IY)s, I,. I). (19liO). The moleclllar collfigllrat ioll of deosyrihotlrlcleic acid. .1. Mol. Rio/. 2, 19G37. hl.\~m, 11. 11.. ,r.\MISOS, 9. &I., and JoKI).\s, I,. II:. (19(X3). Biophpsical stltdies on the natrlre of the sinGail papova virris particle (vacriolat ing SYXI virlls). I’irolog!/ 19, 359Mtiti. ~INOC;I~.U),
.I.,
LEBO\\~ITZ,
J.,
It.\ULOFF,
SON, I:., alld LUE~IS, E’. (1965). The ciilar form of polyoma viral I)N’A. .Icad. hsci. I’.S. 34, 11OG1111. WEIL, II., and T~rsoc:r~ \I), J., Appendix ESIITS, W. (1963). Thr cyrlic helix roil forms of polyoma viral DNA. .I cad. Sci. I’.S. .X, 7X- 738.
It...
\v.\‘r-
twisted cirProc. .\:ntl. by STOECKand c,vclic Proc. .\-o/l.