THE DENSITY AND SIZE OF THE RABBIT PAPILLOMA VIRUS*

BY D. G. SHARP, A. R. TAYLOR, AND J. W. BEARDt

(From the Department of Surgery, Duke University School of Medicine, Durham, North Carolina)

(Received for publication, November 9,1945)

A direct method of measuring the density of virus particles in suspension consists of sedimentation velocity studies of the virus in media of various densities (14). Unfortunately, this method is unsatisfactory when the density of the suspending medium is va.ried with materials such as NaCl, sucrose, urea, or glycerol because of alteration in the density of the particle under study. As a consequence, there occurs a breakdown of the linear relationship which should exist between the sedimentation rate of the virus and the density of the medium, and the resulting values of particle density are too high. This effect has been attributed to the osmotic pressure of the denser media (4).

A means for lessening the possible osmotic influence is by use of bovine serum albumin, which is of relatively high molecular weight, for varying the density of the suspending medium. Use of this material has given satis- factory results in studies on the density of the three types of influenza virus (5, 6) and, in more recent experiments, has been employed in a study of the density of the rabbit papilloma virus in aqueous suspensipn. The results thus obtained are given in the present report. For purposes of computation it was desirable first to determine the relation of sedimenta- tion velocity to virus concentration, and the findings in this preparatory work are likewise described in the present paper.

Material and Methods

The papilloma virus was procured from extracts of cottontail rabbit warts by centrifugal concentration and purification. Some of the virus was purified in the usual way (7) by means of the air-driven quantity ultra- centrifuge; most of it, however, was first concentrated in the Sharples

* This work was supported through the Commission on Influenza and the Com- mission on Epidemiological Survey, Board for the Investigation and Control of In- fluenza and Other Epidemic Diseases in the Army, Praventive Medicine Service, Office of the Surgeon General, United States Army. The work was aided also in part by a grant to Duke University from the Lederle Laboratories, Inc., Pearl River, New York.

t Consultant to the Secretary of War and a Member of the Commission on Acute Respiratory Diseases,Board for the Investigation and Control of Influenza and Other Epidemic Diseases in the Army, Preventive Medicine Service, Office of the Surgeon General, United St’ates hrmy.

289

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290 VIRUS DEXSITY AND SIZE

centrifuge and purified finally by repeated sedimentation in the quantity ultracentrifuge. The procedures involving use of the Sharples centrifuge and the product thus obtained have been described in a separate report (8).

Severa, different batches of concentrates mere used separately for the present studies. The purified virus was suspended in concentrations of 6.3 to 9.6 mg. of virus per ml. in a solution of 0.13 M NaCl and 0.05 M phos- phate buffer of pH 6.5. Estimation of virus cont.ent was made from Kjeldahl nitrogen determinations with a conversion factor (9) of 6.66. The character of the concentrates may be judged from the single sharp boundaries of the sedimentation velocity diagrams described elsewhere (8).

0-Cdm*lntD,r-a, q99999990 __---- -2

Density Of Suspending Medium

FIG. 1. Sedimentation of the rabbit papilloma virus in aqueous solutions of bovine serum albumin of various densities. The ordinate of the continuous line is the product of observed virus sedimentation rate and the viscosity of wat.er at the rotor temperature and that of the broken line is the relative viscosity of the suspending media at 25”.

The bovine serum albumin was a crystalline fraction which Dr. Hans Neurath obtained from the Armour Laboratories, Chicago, Illinois, through the courtesy of Dr. E.‘J. Cohn and Djr. H. B. Vickery. The albumin was dissolved in approximately 25 per cent concentration in a solution con- taining 0.13 M NaCl and 0.05 M phosphate buffer of pH 6.5, which was identical with the solution used for suspending the virus. The stock al- bumin solution had a density of 1.0686 and the results of viscosity measure- ments’ made on it at 25” gave the data shown in Fig. 1. The relative viscosity of the buffered saline solution, measured also at 25”, was 1.025.

For the studies on the relation of sedimentation velocity to virus con-

The measurements of viscosity were very generously made by Dr. John 0. Erickson.

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SHARP, TAYLOR, AND BEARD 291

centration, the virus preparations were diluted to the desired virus content with appropriate volumes of the buffered saline solution.

The virus-albumin preparations for the density studies mere made by diluting the stock virus suspensions with the buffered saline solution and mixing with the stock albumin solution described above. These mixtures contained, in all instances, 2 mg. of virus 7er ml. a.nd the respective amounts of bovine serum albumin required to vary the density from 1.0083 (the density of t,he buffer-saline solution) to 1.0686 in a series of five steps. It was necessary to mix fairly small volumes of the virus preparations in order to conserve the supply. Small total samples of 0.8 to 1.0 ml. could not be made up by pipette from the three ingredients with sufficient accuracy for the work and, consequently, the mixtures were made by weighing to 0.1 mg. the stock solution of albumin of previously measured density. Virus in buffered saline was added and the total sample weighed to 0.1 mg. The density of the suspending medium surrounding the virus particles was

FIG. 2. A series of typical schlieren photographs showing the progress, at 3 minute intervals, of the papilloma virus boundary as it moves down the ultracentrifuge cell. The radial distances measured to the different boundary positions yielded data for analysis such as that shown in Fig. 3. The concentration of virus was 3 mg. per ml.

then calculated from these values. To prepare the sample containing the highest concentration of albumin, the virus was sedimented in the ultra- centrifuge and resuspended in the undiluted albumin solution. When a sample was prepared, about half of it was placed in the rotor cell of the ultracentrifuge immediately, and its sedimentation characteristics were recorded. Most of the remainder of the sample was similarly studied the following day, and the residue was employed for pH measurement.

Sedimentation velocity measurements were made in an air-driven ultra- centrifuge carrying a 4” sector-sha,ped cell 12 mm. high at a mean radius of 6.5 cm. The cell thickness n-as 5 mm. Photographic record of the sedimenting boundary was made by virtue of its accompanying refractive index gradient. Schlieren pictures mere taken with a lens system giving 2 X magnification in the direction of sedimentation. Because of the high degree of homogeneity of the papilloma virus and the short time of sedi- mentation, little change in the position of the “schlieren cutter” was neces- sary during the runs. In Fig. 2 there is shown a representative series of

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292 VIRUS DENSITY AND SIZE

photographs2 made at 3 minute intervals while the rotor turned 210 R.P.S.

Measurements to locate the boundary positions on the negatives were made with a traveling microscope equipped with low power lenses. In each case the mid-point of the shadow was taken as the position of the boundary. Common logarithms of the radii obtained from this plate are shown in Fig. 3 plotted against time, giving the line whose slope was used for sedimenta- tion velocity calculations as follows :

S= 2.303(logl,, Rz - log,, R,)

6Od (T’s - I;) = K(slope)

where RI and Rg are radial distances in cm. to the boundary at times T1 and Tz measured in minutes; w is the angular velocity; a.nd K is the constant for a given rotor speed.

0 3 6 9 12 15 I8 21 24 27 30 33

Time In Minutes

FIG. 3. Analysis of a series of schlieren photographs showing the log,0 of the radial distances plotted against time. The sldpe of the line is proportional to the sedimen- tation rate.

In all of the determinations, care was taken to have the temperature of the sample the same as that of the rotor. The temperature of the rotor was measured before and after each run to provide a mean value from which density and viscosity corrections were calculated.

Results

The findings concerned with the dependence of sedimentation velocity on virus concentration are illustrated in Fig. 4. The sedimentation con- stants (A!!&) of a single virus preparation at various virus concentrations fall on a straight line inclined only slightly to the horizontal, showing a

2 These photographs supplied the data for the point of Fig. 4 corresponding to 3 mg. of virus per ml.

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SHARP, TbYLOR, AND BEaRD 293

small but definite effect of concentration on AS,,. for this preparation. The results with a second preparation at two different concentrations are also shown in Fig. 4. The straight line was drawn through all of the points by the method of least squares. From these results, it appears t’hat little change occurred in S 200 of the papilloma virus at concentrations up to 6 mg. per ml. The value of Szoo used for estimation of particle radius in subse- quent calculat,ions was obtained by extrapolating the relation to zero virus concentration. This value was A% = 297 Svedberg units.

The results of the studies on density of the virus particles in the bovine serum albumin are given in Figs. 1 and 5. In Fig. 1 there a.re shown the observed sedimentation rates, multiplied by the absolute viscosity of water, $, at the temperature of the run in relation to the density of the al- bumin solutions. It is seen that the sedimentation rate of the virus de- creases very rapidly with the increase in the density of the albumin

200 01234567

Virus Goncentration-mghl.

FIG. 4. The dependence of sedimentation constant (8200) on papilloma virus con- centration is shown extrapolated to infinite dilution, indicating a limiting value of the sedimentation constant of 297 Svedberg units. The open circles represent the sedimentation constants of a single preparation at various virus concentrations. A second preparation at two different concentrations is shown by the square symbols.

solution. The increase in t,he viscosity of this suspending medium associ- ated with increase in the concentration of albumin is also indicated in Fig. 1. It is seen, further, that at the higher densities of the media the virus sedimented at speeds (&S = (28 X 10-15)) so low that the sedimenta- tion rate of bovine albumin itself (~$3, = about 5 X 10-15) at the tempera- tures of the studies contributed significantly to the observed rate of sedimentation of the virus. Thus, if the rotor speed and albumin concen- tration could be increased indefinitely, the rate of virus sedimentation would never reach zero ‘but would approach asymptotically that of the albumin. This approach would be exceedingly slow because of the rapid increase in the viscosity of the albumin solution through which the virus must sediment.

In order to estimate the influence of the density of the suspending medium alone on the sedimentation rate, therefore, two corrections must be made. First, the sedimentation velocity of bovine serum albumin must be cal-

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294 VIRUS DENSITY AND SIZE

culated from its sedimentation constant at each t’emperature used and sub- tracted from the observed velocity of the sedimenting virus boundary; and, second, this difference must be multiplied by the relative viscosity of the suspending medium (assumed in this experiment to be constant over the temperature range used, namely 23.4-27.0”),3 and by the absolute viscosity of water at the temperature of the experiment.

The resulting sedimentation velocity values corrected as described are given in Fig. 5, plotted against the density of the suspending medium. One set of the values was obtained from the results of examination of the virus- albumin mixtures immediately after preparation and another group from examination of the same mixtures after 24 hours. There was no evidence

Density Of Suspending Medium

FIG. 5. The dependence of sedimentation rate of the papilloma virus on the density of the bovine albumin suspending medium. Correction of the data of Fig. 1 for the effects of viscosity and finite sedimentation of the albumin yielded the linear relation- ship shown here. These data indicate a virus particle density of 1.133 under the conditions of the experiment. The closed circles represent the results obtained immediately after preparation of the virus-albumin mixtures; the open circles, the results for the same mixtures after 24 hours.

of systematic influence of time, and the straight line shown was con- structed through all of the points by the method of least squares. The results reveal no evidence of any consistent deviation of the points from the linear relationship. It may be seen that the accuracy of repeated estimates on a given preparation is definitely greater than that from preparation to prepa.ration, as judged from the closeness of points to each other and to the line. Residua.1 weighing errors may account for this.

The sedimentation rate of a particle is determined by the strength of the acceleration field in which it lies, by the increment of unbalanced mass after buoyancy allowance has been made, and by t,he frictional resistance

3 The assumptions involved in these corrections lead to errors smaller than the ran- dom errors of the experiments.

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SHARP, TAYLOR, AND BEARD 295

to sedimentation offered by the fluid through which movement is imminent. The sedimentation constant, as defined by Svedberg for proteinmolecules, is the sedimentation rate corrected to the density and viscosity of water at 20” (Xzoo). These corrections are applied as follows:

(2)

where the symbols have the following meanings: at = the viscosity of water at the temperature of the centrifuge run; $0. = the viscosity of water at

20”; l?st = the viscosity of the suspending medium at the temperature of the centrifuge run; ptoo = the density of water at 20”; pt = the density of the suspending medium at the temperature of the centrifuge run; VzoO = the partial specific volume of the virus at 20”; V, = the pa,rtial specific volume of the virus at the temperat.ure of the centrifuge run; and S = the observed sedimentation rate.

This is a convenient form of the equation, for the first parentheses hold the temperature correction for the viscosity of water which varies from run to run; the second hold the correction for relative viscosity of the buffer solution, which is constant over a fairly great temperature range; and the third hold the correction for buoyancy. The relationship between the fundamental quantities influencing sedimentation can be indicated as follows:

ClR (Particle volume) (pV - pt) we R = f -

at (3)

where u2R is the centrifugal field strength;‘the particle volume times the difference between the density of the virus (p,,) and that of the suspending medium (pt) is the unbalanced mass; and f is the frictional resistance per unit velocity dR/dt in the R direction. If the particle is a sphere, as indi- cated by electron micrographs (lo), then its volume is knoa-n in terms of its radius. Its frictional coefficient, according to Stokes, is known also in terms of its radius, r. The relationship (Equation 3) now becomes

4 - 3@ CPU - 3

ph2R = 6~77~‘ T z

therefore,

by definition of S,

r=3 d

liar s 2bJ - A%)

(4)

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296 VIRUS DENSITY AND SIZE

The linear relationship between Q$’ and pt or (pv - ,Q) predicted by these equations is borne out in the present experiments, as is shown in Fig. 5. Assuming that particle size, shape, and density of the virus are unaltered a.s the bovine albumin density around it changes (a condition suggested but not proved by the linearity of the relationship), the virus density calculated from these dat,a is 1.133 for the region under investiga- tion. In order to obtain virus part.icle size, the value of X2,,. at infinite virus dilution, Fig. 4, is necessary and yields, with appropriate substitution in Equation 4, the value of the radius r = 33 X lo-’ cm., or 33 mp.

If the virus particle is thought to be composed of a substance whose dry density is l/V and some quantity of water, this quantity can be calcu- lated from V and pV. The partial specific volume of the papilloma virus determined in previous work (11) was 0.754; pycnometric measurements on the material employed in the present studies-gave 0.761. When the value 0.761 is used, the calculated amount of water associated with the virus particle is 58 per cent by volume, or 1.04 gm. of water per gm. of dry virus. If the liquid part of the sedimenting virus unit is not water but buffer salt solution (p = 1.0083), the ralue is 59 per cent by volume.

DISCUSSIOK

In the present studies, the sedimentation velocity of the papilloma virus in bovine serum albumin solutions, corrected for known conditions of the experiment, was found to be related in a linear fashion t.o the density of the suspending albumin solution. With the data thus obtained, the density of the virus particles was found to be P.133 in the region investigated. With respect to t,he character of the relationship between density of the suspending medium and the sediment,ation velocity of the virus, the results with the papilloma virus were a.nalogous to the findings with influenza viruses A and B and the swine influenza. virus (5, 6). In the work with the papilloma virus, greater accuracy in estimating sedimentation velocity was necessary in order to attain a comparable degree of precision in the final value of density because the slope of the line, 7s versus (pV - pt), for the papilloma virus is less than the analogous slopes for the influenza virus. The smallness of size and the relatively great density of the papilloma virus account for this and necessitate the weighing of samples and correction for appreciable sedimentation of the bovine serum albumin itself.

The density value, 1.133, considered with the partial specific volume 0.761, reveals the presence of a large amount of water associated with the papilloma virus particle, and the diameter of a spherical particle of this sort (lo), 66 rnl.c, is much greater than the size, 44 mp, observed from elec- tron micrographs (10). Removal of the 58 per cent water, if it were ac- companied by 58 per cent shrinkage in the volume of the particle without

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SHARP, TAYLOR, AND BEARD 297

change in shape, would reduce the diameter of the particle to 48 mp. The significance of such a. value, however, is questionable, for the shrinkage, if any, might not be as much as 58 per cent. In the initial measurement-s in electron micrographs it was realized (10, 12) that the values for the di- ameter might be low because of the obvious diffuseness in particle limits. It is possible that the difference between the values 66 and 44 rnp may be associated with both difficulty in measurement in electron micrographs and shrinkage of the particle on drying. No evidence has been found for shrinkage of bacteria (13), but the diameters of influenza virus particles observed in electron micrographs (6) are generally significantly lower than those calculated from observed density and sedimentation velocity data.

In previous work (11) direct mea.surements were made on the rate of diffusion of a preparation of papilloma virus which was characterized by a sedimentation constant of 280 X lo-l3 and a dry density of 0.754. The diffusion constant of the material was 6.65 X 1OP. Markham, Smith, and Lea (14), in calculations from the above data together with the electron micrographic evidence of the virus particle shape (lo), predicted the value 73.4 rnp for the particle diameter and 1.88 gm. of water in association with 1 gm. of the dry virus. From the data of the present work, the diameter of the particle in aqueous media was 66 rnp, the amount of associated water 1.04 gm. per gm. of dry virus, and the calculated diffusion constant 7.2 X lo-* cm.’ per second. On consideration of the many factors possibly involved, the two sets of data and calculated values are not necessarily in- consistent. Measurement of diffusion constants, especially of such large particles, may be subject to considerable variation. It should be noted that small differences in the rate of diffusion of particles in this range of size and degree of hydration reflect relatively great differences in both size and water content. The same is true, also, for variation in the values of the sedimentation constant, but measurements of sedimentation velocity for this size range are of far greater accuracy than diffusion measurements. There is reason to believe, however, that the differences between the pre- vious data and those of the present work may reflect, at least in part, actual variation in the characters of the papilloma virus from one preparation to another. It is well to recall that the sedimentation velocity studies of the previous work (11) were made on twelve different purified virus prepara- tions and that the values observed varied from 266 to 288 about a mean value of 278.3 Svedberg units. Such variation is well outside the limits of error of the method of measurement and might be accounted for either on the basis, possibly, of dissymmetry in shape or, more probably, of the amount of water associated with the particle. It is unlikely that, the chemical constitution of t,he virus is subject to great differences, since the values of dry density, 0.754 for the material of the previous work and 0.761

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298 VIRUS DENSITY AND SIZE

for that used here, show small variation. If the papilloma virus is assumed to be molecular, variation in experimental data is difficult to explain. On the contrary, variation among living organisms is to be expected.

The interpretation of the data relative to diffusion, size, and shape of the papilloma virus previously obtained (11) obviously was greatly in error. These interpret,ations were based on the judgment that the pre- ponderance of evidence then available indicated that the papilloma virus was a molecular nucleoprotein. Recent experience in the study of animal viruses (12), especially with the electron microscope, renders this position untenable at the present time. Electron micrographic studies on the influenza (l&17), vaccinia (IS), and equine encephalomyelitis viruses (19) have revealed variations in structure, shape, or size which definitely remove these agents from the category of molecular materials. Because of the low contrast in the electron micrographic images of the papilloma virus (lo), unequivocal evidence of morphological variation in the papilloma virus has not yet .been obtained. The conclusion cannot be evaded, how- ever, that the properties and behavior of the papilloma virus revealed in electron micrographs and in the present work are much more closely similar to those of the viruses mentioned above and to living matter in general than to the characters expected of molecules.

SUMMARY

Studies were made on the sedimentation velocity of purified papilloma virus in bovine serum albumin solutions of various concentrations and densities. The relation of sedimentation velocity to the density of the medium was linear in the region studied when suitable corrections were made for viscosity of the medium and the sedimentation of the albumin molecules occurring in the centrifugal fields employed. From this relation- ship, calculations of the density of the virus particle in aaqueous suspension gave the value of 1.133. The partial specific volume of the virus material studied was 0.761, which is in satisfactory agreement with previous find- ings, 0.754. By extrapolation of the linear relationship of sedimentation velocity to virus concentration, the sedimentat,ion constant at infinite virus dilution was found to be 297 X lo-13. From the sedimentation velocity data and the density of the particle with associated water, the diameter of the hydrated spherical particle was 65.6 rnp. With the reciprocal of the partial specific volume, l/0.761, as the dry density and the value 1.133 as the wet density, the amount of water associated with the virus was calculated to be 58 volumes per cent, or 1.04 gm. of water per gm. of dry virus. The calculated diffusion constant for particles of these characters was 7.2 X 10m8.

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BIBLIOGRAPHY

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D. G. Sharp, A. R. Taylor and J. W. BeardRABBIT PAPILLOMA VIRUS

THE DENSITY AND SIZE OF THE

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