The chemical composition and particle weight of Tipula iridescent virus

VIROLOGY

14, 240-252

The

(1961)

Chemical

Composition of Tipula

and

Iridescent

RICHARD Virus

Laboratory,

Weight

Virus’

S. THOMAS”

University Accepted

Particle

of California, February

Berkeley,

California

27, 1961

Tipula iridescent virus is shown to contain 12.4% deoxyribonucleic acid (DNA) and 5.2% lipid, most or all of which is phospholipid. No ribonucleic acid (Rh’A), neutral polysaccharide, or amino polysaccharide can be detected, however, and indirect evidence indicates the absence of acid-soluble phosphorus compounds. The remainder of the virus material appears to consist mostly, or perhaps entirely, of protein, including some phosphoprotein. The DNA of the virus contains only the usual four bases, with the molar amount of adenine (A) equal to thymine (T) and of guanine (G) equal to cytosine (C). The molar dissymmetry ratio (A + T)/(G + C) = 2.20. The particle weight is 1.22 X IO’, and the diameter of the frozen-dried particle is 130 mp. The two figures are consistent with one another upon assuming a particle density of 1.39, which can be estimated from the chemical composition of the virus. The results are discussed with reference to other insect viruses and viruses in general.

to be a record yield for all virus diseases except perhaps some bacteriophage infections of Escherichia coli (Williams and Smith, 1957). Purified TIV particles are about 1300 A in diameter, are icosahedral in shape, and appear to be completely uniform morphologically (Williams and Smith, 1957, 1958).‘No other known virus of comparable size has such a high degree of structural symmetry or of uniformity. Purified TIV is physically and biologically stable when stored in the cold room and is probably stable even at room temperature (K. M. Smith, 1960, private communication) . The above-described properties of TIV render it exceptionally suitable for a wide variety of experimental investigations. In biological studies, the distinctive morphology of the particles and the iridescence of the crystals serve as markers by which the virus can be unmistakably recognized in vivo. Thus, Smith and Rivers (1959) recently showed that the virus will grow in a wide range of insect species; in fact, TIV is

INTRODUCTION

A rather unusual virus affecting the European crane fly Tipula paludosa Meig. was discovered in 1954 at the Agricultural Research Council Virus Research Unit at Cambridge (Xeros, 1954; Smith, 1955). It has the unique property of spontaneously forming crystals, either in vitro or in vivo, which when viewed in reflected white light are iridescent. Because of this it has been named Tipula iridescent virus (TIV) (Williams and Smith, 1957; Smith and Williams, 1958). TIV is found free (unencapsulated) within the cytoplasm of infected larvae, from which it may be extracted and purified quite readily (Xeros, 1954; Smith, 1955; Smith and Williams, 1958). A moribund larva may contain virus to an amount of 25% of its dry weight, which is believed ’ Aided by cer Institute, ’ Present Laboratory, ture, Albany,

Grant C-2245 from the National CanNational Institutes of Health. address : Western Regional Research United States Department of AgriculCalifornia. 240

TIV

COMPOSITION

AND

the first insect virus known to .infect three different orders, Diptera, Lepidoptera, and Coleoptera. The intracellular development of the virus has also been studied (Smith, 1958; Smith and Hills, 1959). The ease of purification of TIV, together with its stability, great morphological uniformity, and other physical properties, makes it an appealing object for detailed physical and chemical studies. Furthermore, its large size permits cytochemical studies for the localization of chemical constituents within the particles. Only a few investigations along physical and chemical lines have been conducted so far. In addition to the determination of size and shape, alluded to above, a complex internal structure of the particles has been demonstrated with the electron microscope (Smith, 1956; Williams and Smith, 1957; Smith and Williams, 1958; Smith and Hills, 1959). The particles in aqueous suspension were found to have a hydrated diameter of about 1800 A, by X-ray scattering (Arndt and Beeman, 1958)) and their manner of packing in crystals, including lattice dimensions, was determined by diffraction of visible light (Klug et al., 1959). Xeros (1954) showed with the Feulgen reaction that TIV contains deoxyribonucleic acid (DNA), and later, J. D. Smith (1958) reported that the relative amount of this constituent is about 15%, but no details were given. No other information has been available on the chemical composition of this virus. In the present investigation, TIV was analyzed for DNA, ribonucleic acid (RNA), neutral polysaccharide, amino polysaccharide, total lipid, phospholipid, phosphoprotein, total phosphorus, and total nitrogen; in addition, the base composition of the DNA was determined. To allow the relative amounts of the chemical constituents to be expressed in absolute terms, the particle weight was also determined. An internal check on the rationality of the particle weight was obtained by comparing the particle diameter calculated from the particle weight and density (estimated from the chemical composition of the virus) with the diameter measured on frozen-dried particles in the electron microscope. Micromethods were used throughout the

PARTICLE

241

W-EIGHT

chemical investigation since the amount of virus available for this purpose was limited. MATERIALS

AND

METHODS

Virus. The available TIV sample had been extracted from deceased larvae of Tipula paludosa and purified by 5 cycles of differential centrifugation in distilled water as described by Williams and Smith (1958). The last 2 cycles immediately preceded the chemical analyses. The final purified virus pellet was resuspended (approximately 10 mg TIV per milliliter) in 0.003 M ammonium bicarbonate. A low concentration of salt was found necessary to prevent spontaneous crystallization which occurs in distilled water (Smith and Williams, 1958; Klug et al., 1959). The preparation showed no visible impurities in the electron microscope by the spray droplet technique (see below). Dry weight. To determine the dry weight concentration of the stock virus suspension, lo-30 ~1 was plated with calibrated micropipettes onto small, tared aluminum-foil planchettes (5 x 7 mm, weight about 2.5 mg), and the water and ammonium bicarbonate were removed by evaporation in a vacuum desiccator at room temperature. The seemingly dry planchettes were weighed in vacua on a quartz helix microbalance (range O-5 mg, sensitivity 0.44 pg/O.Ol mm j (Kirk and Schaffer, 1948) which was connected to a vapor trap at -78”. Constant weight was attained in 2 hours at room temperature. After this time, heating the specimens on the evacuated balance to 80” did not drive off any additional moisture. The over-all internal precision of the dry-weight determinations was +0.3%. For blank determinations, aliquots of the suspending medium, 0.003 M ammonium bicarbonate, were dried and weighed as for the virus suspension. Nonvolatile residue was negligible. Phosphorus. Determination of phosphorus was essentially as described by Nakamura (1952). The range of the method was 0.4 to 4 pg P and the precision was +0.02 pg P. Nitrogen. This was determined by a Nesslerization procedure as described by Miller (1944). The range of the method was 20--60 pg N and the precision was about ir2 pg N.

242

THOMAS

Neutral polysaccharide and RNA. Sugar chromatography was used as a qualitative method for the simultaneous detection of neutral polysaccharide and RNA ribose. Aliquots (100-200 ~1) of stock suspension containing l-2 mg TIV were hydrolyzed in 1 N HCl for 5 hours at 100” with stirring in stoppered microcentrifuge tubes. Subsequent procedure followed that of Frommhagen et al. (1959)) but with appropriate reduction in scale. The final deionized sugar residue was reconst,ituted in 10 ~1 distilled water and applied, about 0.25 ~1 at a time, to a single spot on Whatman no. 1 paper. This was chromatographed 15-20 hours in butanol-acetic acid-water, as described by Putman (1957). The color of sugar spots was developed with p-anisidine hydrochloride. As little as 3 yg of a test sugar (glucose, fructose, ribose) could easily be detected by the method. Alrhino polysaccharide. This could not be detected by the previous method since amino sugars were excluded from the hydrolyzate by the necessary deionization step. A modified Elson and Morgan assay for amino sugars was used to test for this substance. Aliquots (100-200 ~1) of stock TIV suspension (l-2 mg) were hydrolyzed in 8 S HCl for 15 hours at 100” with stirring, and insoluble residues were centrifuged out. The subsequent procedure was as described by Ashwell (1957)) except that all reagent volumes were reduced tenfold. Less than 2 pg glucosamine could easily be detected. Total lipid and phospholipid. Virus total lipid was determined by extraction and weighing, and phospholipid was estimated by phosphorus assay of the extracted material. The microextraction apparatus was similar to that described by Schaffer et al. (1954). Aliquots (100-200 ~1) of stock suspension were delivered to microcentrifuge tubes and the TIV (l-2 mg) was pelleted for 30 minutes at 8000 g (Servall SS-1 centrifuge). After careful withdrawal of the supernatant solutions, the pellets were frozendried in the tubes. The pellets were twice extracted for 15 minutes with lOO-~1 portions of 1:l methanol-ethyl ether at 50”

and washed twice with 50-~1 portions of the same solvent. For total lipid determination the combined extracts and washes from each tube were evaporated down under nitrogen and the lipid was reextracted six times with 4O-~1portions of petroleum ether. A small residue remained behind. The six successive portions were plated 10 ~1 at a time into a small, tared, aluminum-foil cup and allowed to evaporate. The cup contained a small piece of tissue paper to prevent creeping of the lipid as it dried (total weight of cup and paper: about 3 mg) . The lipid was thoroughly dried in a vacuum desiccator over paraffin chips. After allowing the cup to re-equilibrate with the humidity of the room, it was weighed in air on the quartz helix microbalance. Slight increments in weight due to nonvolatile residues in the solvents and variable moisture content of the specimenswere controlled by appropriate blanks. To test the over-all efficiency of the method exclusive of the initial extraction, known amounts of lipid (lard) dissolved in 1: 1 methanol-ethyl ether were processed through the procedure to final weighing. Recovery of 100 pg sampleswas 98-103s. The efficiencies of the initial methanol-ether extraction of TIV and the re-extraction of TIV lipid into petroleum ether were tested by repeating these steps on previously extracted or re-extracted material. No additional lipid was recovered. To show that the method was free from interference by nonlipid materials, it was applied to a 1.60-mg sample of tobacco mosaic virus, known to be free of lipid. The weight of material recovered was nil. For phospholipid assay the combined methanol-ethyl ether extracts were made to a standard volume and aliquots were taken for re-extraction into petroleum ether, as above. Phosphorus was determined on both the methanol-ethyl ether extracts and the petroleum ether re-extracts, for comparison, but only the latter phosphorus was taken as a reliable measure of phospholipid. Phospholipid was calculated as weight of petroleum ether-soluble phosphorus times 25. IlAVA. The amount of DNA, as well as the ratio of DNA bases, was determined by

TIV

COMPOSITION

AND

phosphorus analysis and base chromatography after extraction of the DNA from the virus. Lipid-free residues from 2-3 mg TIV were extracted three times with 5% trichloroacetic acid (TCA) (100 pl, 50 pl, 50 ~1) for 15 minutes at 90” with stirring. The pooled extracts from each sample were made to volume and small aliquots were taken for phosphorus analysis. The remainder was heated at 95” for 2 hours to decompose the TCA which would interfere with subsequent steps (Hershey et al., 1953). The TCA-free solution was evaporated to dryness under reduced pressure with a dry nitrogen jet and the residue was then hydrolyzed with 400 ~1 90% formic acid in a nitrogen-filled, sealed tube at 175” for 30 minutes (Wyatt and Cohen, 1953). The hydrolyzate was evaporated to dryness, as before, and the final residue was reconstituted in 40 ~1 1 N HCl. Two 15~1 aliquots were taken for chromatography and the remainder was used for duplicate phosphorus assay. The detailed chromatographic procedure and quantitative determination of the bases were as described by Bendich (1957). Isopropanol-2 N HCI was used as the developing solvent. The base spots were each eluted in 3 ml 0.1 N HCI. Complete spectra were determined on the eluates to confirm the identity and purity of the bases, and concentrations were determined by the differential extinction technique, using a Beckman DU spectrophotometer. The alignment and calibration of the instrument were checked with standard solutions of the bases” and were found to agree within 1% of Bendich’s values. To determine the efficiency of the whole procedure, a mixture containing 85% bovine plasma albumen and 15% calf thymus sodium deoxyribonucleate was used as a test substance. The deoxyribonucleate was prepared by the method of Crampton et al. (1954) .4 Protein residue. To characterize the pro’ Adenine, thymine, guanine, and cytosine were obtained from California Corporation for Biochemical Research. ‘The purified calf thymus sodium deoxyribonucleate was kindly supplied by Miss Sue Ha&n.

PARTICLE

WEIGHT

243

tein residue remaining from lipid and nucleic acid extraction, it was dissolved in a small amount of 1 N NaOH at 40” and made to a standard volume; aliquots were taken for phosphorus analyses and ultraviolet absorption spectra (after suitable dilution with distilled water). For comparison, it was desirable to take spectra on whole TIV at different pH’s. To solubilize the virus for this purpose, a dilute suspension (1.4 mg TIV per milliliter) was heated for 10 minutes at 100” in 0.85% sodium dodecyl sulfate. It was then diluted still further with distilled water and brought to the proper pH by addition of NaOH. To investigate the nature of phosphorus found in the protein residue, the lipid-free virus was subjected to a simplified SchmidtThannhauser procedure fractionation (Schmidt and Thannhauser, 1945). The dry, lipid-free residue from about 1 mg TIV was hydrolyzed at 37” overnight with 50 ~1 1 N NaOH. The hydrolyzate was neutralized with HCl and perchloric acid was added to a concentration of 5%. The precipitate was centrifuged down at 8000 g for 10 minutes. The supernatant liquid was pipetted off and the sediment was washed twice with 30 ~1 5% perchloric acid, using low speed centrifugation. The sediment was dissolved in 1 N NaOH at 37”. The dissolved sediment and combined supernatant and washes were made to standard volumes for phosphorus analysis. Particle weight. The particle weight of TIV was calculated simply by dividing the known dry-weight concentration of virus in stock suspensions by the number of virus particles per unit volume. The latter was determined by counting particles in spray droplets with the electron microscope (RCA EMU-3; final magnification, 8000 x) as described by Williams and Backus (1949). The stock suspensions were diluted 1:400 with 0.003 M ammonium bicarbonate, giving a concentration of about 0.025 mg TIV per milliliter for spraying; it contained, in addition to the virus, polystyrene latex (PSL) particles, at a final concentration of 1.50 X lOlo particles per milliliter for reference, and plasma albumen, 1 mg/ml, to prevent clumping of the particles on the electron microscope specimen grids.

244

THOMAS

The standard error of the number concentration of TIV particles was calculated as described by Luria et al. (1951). Possible errors in the virus dilution were neglected, but the number concentration of the reference PSL part,icles was assumed to have a standard error amounting to 5% (Williams and Backus, 1949). In calculating the standard error of the particle weight, any possible error in TIV weight concentration was neglected, since it was so much lessthan the other errors. Particle diameter. The minimum breadth of the hexagonal profiles of 50 unshadowed, frozen-dried TIV particles was measured on electron micrograph prints at a magnification of 46,000 X. The scale of length was calibrated by reference to the profiles of polystyrene latex spheres. These particles are quite uniform, with a diameter of 259.0 ” 2.5 mp (Backus and Williams, 1949). RESULTS

Test for ,Veutral Polysaccharide and RNA by Sugar Chromatography A chromatogram of hydrolyzed TIV, together with sensitivity controls, is represented in Fig. 1. The virus showed no clearcut indications of any sugar. There was a faint spot, which from its location might be glucose, but it was so extremely light that it is of doubtful significance. The same may be said for a faint streak which preceded the spot. It might seem paradoxical, since DNA is known to be present in the virus, that no deoxyribose spot was seen.Actually, this could be expected since deoxyribose is an exceptionally labile sugar which is destroyed by ordinary hydrolysis procedures (Overend and Stacey, 1955). Note that the deoxyribose spot was also missing in the test mixture control (see below, and Fig. 1). To demonstrate that the complete procedure had adequate sensitivity of detection, it was applied to a test mixture of substances designed to represent a possible chemical composition of TIV, and consisting of 83% bovine plasma albumen, 15% DNA (sodium salt), 0.7% RNA (sodium salt), and 0.8% glycogen. In this case a ribose spot from RNA and a glucose spot from glycogen could be easily seen. The glucose

spot was, in fact, quite intense. As a further demonstration of sensitivity, the test procedure was applied to a sample of TIV to which had been added RNA, 0.008 mg/mg TIV, and glycogen, 0.009 mg/mg TIV. The chromatogram looked very similar to that of the test mixture, with glucose and ribose spots clearly in evidence. These results indicate that if the virus contains any neutral polysaccharide or RNA at all, the relative amounts must be far less than 1%. Test for RNA by Base Chromatography Confirming evidence for the absence of RNA in the virus was easily obtained in the course of analyzing for DNA bases (see below) by noting the complete absence of uracil spots on the chromatograms. To demonstrate that a trace of RNA could be detected by this means, the complete procedure was applied to an appropriate amount (about 1 mg) of the test mixture. The uracil from the 0.7% content of RNA in the test mixture could easily be detected. Test for Amino Polysaccharide The absence of detectable neutral sugars in the virus excluded the presence of any significant amount of neutral or mixed polysaccharide. However, a pure amino polysaccharide would have escaped detection. It was not unthinkable that TIV might contain this substance, since the host for the virus is a chitin-producing insect, and no insect viruses heretofore have been examined for amino polysaccharide content. As before, the sensitivity of the test procedure was verified by applying it to a model mixture, This was the same as that used for sugar chromatography but with the addition of chitin to a content of 0.5%. Glucosamine from the chitin could very easily be detected in 1-mg samples; in fact, much smaller samplescould have been used. The test on l-2-mg samples of TIV was completely negative. When chitin was added to a 1-mg sample of TIV to a content of 0.5%, this could easily be detected, just as for the test mixture. Apparently TIV contains no significant amount of any polysaccharide.

TIV

COMPOSITION

-

AND PARTICLE

245

WEIGHT

FRONT L

1. Sugar chromatogram of hydrolyzed TIV and two control mixtures. The control labeled test mixture consisted of 83% bovine plasma albumen, 15% DNA (sodium salt), 0.7% Rh‘A (sodium salt), and 0.8% glycogen. The control labeled TIV + RNA + glycogen consisted of TIV to which had been added 0.008 mg RNA per milligram TIV and 0.009 mg glycogen per milligram. One milligram of the preparation was used in each case. Descending development was on Whatman no. 1 paper with n-butanol (52 vol. %)-acetic acid (13 vol. %)-water (35 vol. %) for 15 hours. Legend: G, glucose; F, fructose; R, ribose; D, deoxyribose ; FL, fluorescent; &, UV quenching. The density of crosshatching on the spots indicates the approximate relative intensity. FIG.

TABLE

Total Lipid and Phospholipid Content In contrast to the negative tests for polysaccharide and RNA, TIV did prove to contain some petroleum ether-soluble lipid, accounting for slightly more than 5% of the mass of the virus. The results of several quantitative determinations are shown in Table 1. In addition to analysis on a standard preparation of the virus, determinations

TOTAL

Preparation of TIV Standard Repurified

LIPID

1 IN

TI\

Amount of TIV bs)

Total lipid content (Wmg TIV)

1.12 1.68 0.92 1.38

0.051 0.051 0.053 0.052

246

THOMAS TABLE 2 PHOSPHOLIPID

IN

TIV

Amount of TIV 1.71 mg Methanol ether-soluble 2.14 pg/mg TIV phosphorus Petroleum ether-soluble 1.80, 1.88 ,ug/mg TIV phosphorus (duplicate re-extractions) Recovery of lipid phos’ 84%, 88% phorus in re-extraction petroleum ether extract P x 100 methanol ether extract P 1 ( petroleum 0.045, 0.047 mg/mg Calculated TIV ether-soluble phospholipid content of TIV

were made also on a repurified preparation which had been subjected to two additional cycles of high speed and low speed centrifugation. This was to investigate the remote possibility that the lipid represented a particulate contaminant in the standard preparation. As may be seen in Table 1, the repurification did not produce any significant change in the lipid content. This strongly suggests that the lipid is truly associated with the virus particles. Results of a phosphorus analysis of the lipid are summarized in Table 2. Most, but not all, of the methanol ether-soluble phosphorus could be re-extracted into petroleum ether. Whether the insoluble residual phosphorus represented nonlipid material or petroleum ether-insoluble phospholipid is not known. If it is assumed that the petroleum ethersoluble phosphorus represents phospholipid with a 4% phosphorus content, the petroleum ether-soluble phospholipid content of TIV is calculated to be about 4.6%. This is only slightly less than the figure for total lipid content. Apparently all, or nearly all, of the lipid in the virus is phospholipid. DNA

Content

and Composition

Chromatograms of TIV nucleic acid hydrolyzates showed only the four bases usually found in DNA: adenine, thymine, guanine, and cytosine. Methylcytosine, which is found in some DNA’s (Chargaff,

1955), would have been resolved on the chromatograms but was not seen; only a trace, if any, could have been present. 5Hydroxymethylcytosine, which is found in large amount in T-even bacteriophage (Wyatt and Cohen, 1953), could likewise have been present only in trace amount. Although it could not be resolved from the cytosine spot on the chromatograms, the spectrum of eluted cytosine was identical with that of pure cytosine. Quantitative recovery of the four bases in terms of the phosphorus in the hydrolyzate confirmed the fact that no appreciable amount of exotic bases lay undetected on the chromatograms. As may be seen in Table 3, the sum of moles of bases recovered from the hydrolyzate in two experiments equaled 97% of the moles of phosphorus. It will also be seen in Table 3 that the TIV DNA bases are paired, adenine equal to thymine and guanine equal to cytosine, as with most other DNA’s. The DNA from TIV is unusual, however, in that the dissymmetry ratio, adenine plus thymine : guanine plus cytosine, is extremely high-2.20. This will be discussed later. The reliability of the base ratio data for TIV is shown by results obtained by the same procedure on a test system containing calf thymus DNA. Here, the base ratios are in close agreement with the average values from the literature (Chargaff, 1955) . Since essentially all the phosphorus in the hydrolyzed TCA extracts was accounted for by the bases, the TCA-extracted phosphorus can be taken as a valid measure of DNA. The amount per milligram of TIV, a closely reproducible value, is shown in Table 3, together with the phosphorus content of the DNA calculated from base ratios. From this, the DNA content of TIV is calculated to be 12.4%. This is slightly less than the.previously reported value, but may fall within its limit of accuracy (Smith, 1958). Possibility of Undetected Substances: Characterization of Protein Residue and Total Nitrogen and Phosphorus Content of TIV The total nitrogen content of TIV was found to be about 17%. This is the expected

TIV

COMPOSITION

AND

PARTICLE

TABLE DNA

COMPOSITION

Source of DNA

TIV TIV Test system (8570 bovine plasma albumen, 15% calf thymus sodium deoxyribonucleate) Calf thymus (from the literature) b Amount Calculated Amount

AND AMOUNT

IN TIV

Hydrolyzate phosphorus &moles)

Recovery (sum of bases/phosphorus)

1.14 0.768 1.15

-

I

247

WEIGHT

3 (CALF

Ratios

THYMUS

(base/sum

DNA

FOR COMPARISON)

of bases)” A/T

G/C

AS-T ___ G+C

A ______

T

G

C _____

0.97 0.97 0.98

0.351 0.349 0.291

0.334 0.349 0.286

0.156 0.148 0.213

0.159 0.154 0.210

1.06 1.00 1.02

0.98 0.97 1.02

2.17 2.20 1.36

0.93

0.290

0.285

0.212

0.212

1.02

1.00

1.36

of DNA (TCA extract) phosphorus content of DNA in TIV: 0.124

phosphorus in TIV: 12.4 f 0.2 rg/mg of TIV DNA: 0.100 mg/mg DNA mg/mg TIV

0 Adenine, A; thymine, T; guanine, G; cytosine, C. b Data taken from Table 5, p. 352 in Chargaff (1955).

value for protein and nucleic acid, and hence it seems unlikely that, aside from the 5% lipid content, the virus contains much of anything except nucleoprotein. If undetected substances do exist in TIV, they most likely would be found in the protein residue remaining after lipid and nucleic acid extraction. Only a strict accounting of the mass of the residue in terms of amino acids could determine their presence or absence unambiguously. Amino acid analyses were not undertaken in the present investigation, but as a first and simple test, t,he UV spectrum of the residue was determined (see Fig. 2). At pH 12 (0.01 N NaOH used to dissolve the residue), two high absorption regions are seen: at about 245 rnp and at about 285 rnp, The double-peaked spectrum is typical of many tyrosine-rich proteins when they are measured at high pH, the 245 rnp peak being due to the ionization of tyrosine (pK % 11 in protein) (Doty and Geiduschek, 1953). There does not appear to be any indication of UV-absorbing nonprotein material in the residue. Confirmation of the nature of the 245 rnp absorption is shown by spectra on the whole virus taken at both high and low pH (Fig. 2). The 245 rnp absorption is present at pH 12 but absent at pH 5. It is interesting to note that the calculated difference spectrum

The

figures

are the average

TIV

for 21 preparations.

between the whole virus and the protein residue at pH 12 has a peak at 260 rnp and is simply that of nucleic acid, as it should be. This confirms that the two spectra, resi-

;: R

2.

1.8 I.6

:

230 240

250

-WHOLE TIV, pH I2 .------WHOLE TIV, PH 5 --.-TIV PROTEIN RESIDUE, ----DIFFERENCE SPECTRUM, WHOLE TIV-PROTEIN RESIDUE. pH 12

pH I2

260

310 3;-

270

260

290

300

i(rnFL)

FIG. 2. Spectra of TIV protein residue and whole TIV, and calculated difference spectrum. The concentration of whole TIV and of TIV from which the protein residue was derived is 0.285 mg/ml.

248

THOMAS TABLE PARTITION

OF PHOSPHORUS

-

Simplified Chemical

IN TIV

Schneider

Lipid DNA

Methanol extract Hot TCA

Phosphoprotein

Protein

-

Simplified

Phosphorus (rglmg TIV) 2.18

ether

12.4

extract

1.03

residue

Recovery

15.6 15.9

-

sum

of fractions

whole

TIV

phosphorus phosphorus

: 0.98

& 0.04 f

0.2

f

0.10

f f

0.3 0.3

Methanol ether extract Alkaline digest precipitate Alkaline digest supernatant

Number of experi merits

Phosphorus (rgl mg TIV)

1 1

12.6 0.87

42 0.04

>

L

due and whole virus, are consistent with one another. The protein residue was found to contain a small amount of phosphorus, but it apparently represents phosphoprotein rather that someundetected classof substance. The amount of residue phosphorus per milligram of TIV is included in Table 4 (simplified Schneider procedure), which is a balance sheet for all the phosphorus in the virus. With the inclusion of the residue, essentially all the phosphorus of the virus is accounted for. The probable protein nature of the residue phosphorus was demonstrated by subjecting the lipid-free virus to a simplified Schmidt and Thannhauser (1945) fractionation procedure. The results are shown in Table 4 (simplified Schmidt-Thannhauser procedure). The amount of alkaline digest supernatant phosphorus, which in a lipid, acid-soluble,6 and RNA-free preparation can be ascribed to phosphoprotein, is in good agreement with the phosphorus of the protein residue. It will be noted also that the phosphorus of the alkaline digest precipitate, ascribable to DNA, is in close agreement with the DNA phosphorus extracted with hot TCA. ‘Evidence for the absence of acid-soluble pounds in TIV will be discussed below.

Schmidt-Thannhauser procedure

Fraction

.Sum of fractions Whole TIV

PROCEDURES

.I-

Number of experiments

Fraction

EXTRACTION

procedure

T

fraction

4 BY Two

com-

Particle Weight The particle weight of TIV was determined on two suspensions(the standard and repurified preparations for lipid analysis) which differed in concentration; this afforded a check on the precision of the complete procedure including both weighing and counting. Also, since the second suspension had undergone additional centrifugal purification relative to the first, this allowed a check, beyond that mentioned earlier, for the presence of any particulate contamination of the virus preparation. The particle weight determination by the present method is rather sensitive to contamination, since such material is weighed but is not counted. The weight concentration and counting data are shown in Table 5, together with the calculated particle weights. The small difference between the two values for the particle weight is well within the theoretical standard errors for the numbers of particles counted. To be certain of the validity of the counts, it was necessary to demonstrate that the polystyrene and virus particles were distributed independently and at random (Poisson) in the spray droplets. Hence, the intercept of the regression line for the correlation between the counts of the two kinds of particles in individual drop pat-

TIV

COMPOSITION

AND

PARTICLE

TABLE DETERMINATION

I Preparation of TIV

Standard Repurified

OF

I

TIV

PARTICLE

~

2.81 2.31

13 17

r*

357 j 600

Mean

249

5

WEIGHT

Counting data

Weight concentration of TIV (g/ml) Droplets examined

WEIGHT

0.911 0.997

BY

WEIGHING

AND

COUNTING

Number concentration of TIV (particles/ml) x 10’0 1.36 f 0.12 l.lG f 0.09

Particle weight

Grams

Mol. wt. units

x 10-15 2.07 f 0.18 1.99 f 0.15

x 109 1.25 f 0.11 1.20 f 0.09

2.03 f

1.22 zk 0.07

0.12

a PSL = polystyrene latex particles. * r = correlation coefficient.

was calculated together with the correlation coefficient (Luria et al., 1951). In both determinations, the correlation coefficient (r, shown in Table 5) was highly significant (99% probability) and the regression line passed through an intercept not significantly different from the origin. These results are considered to be an adequate demonstration of independent, random distribution.

terns

Density, Diameter, and Other Characteristics of Xingle Particles

probable that the particles are rather solid, instead of spongelike, and consequently their density is probably not very much different from the reciprocal of their partial specific volume. The latter can be estimated from the chemical composition of the particles by assuming reasonable values for the partial specific volumes of the constituents: protein, 0.74; DNA, 0.55; lipid, 1.00. The estimated density and resultant calculated particle diameter are shown in Table 6, together with the measured diameter. It will be seen that the two figures for the diameter are not significantly different. The measured value, 130 rnp, is in agreement with that previously reported (Smith and Williams, 1958). For purposes of comparison and discussion (seebelow) it is useful to calculate the surface area7 of a single particle and also the total absolute weight of lipid and DNA which it contains. These figures are included in Table 6.

To show that the particle weight given in Table 5 is reasonable, it is instructive to assumea likely value for the density of the particles, use this and the particle weight to calculate particle volume and diameter,6 and then compare the latter figure with direct measurements of diameter on frozendried particles in the electron microscope. In estimating the density of the particles it is important to note that air-dried TIV particles show only slight distortion as a DISCUSSION result of the large surface tension forces The analyses of TIV indicate that it conwhich act on them while they are drying tains 12.4% DNA and 5.2% lipid, most, if out of a water droplet (Williams and Smith, 1958). They are almost as well preserved not all, of which is phospholipid, but it does as frozen-dried particles. Thus it seems not contain any appreciable amount of polysaccharide or RNA. The remainder of the ‘The diameter is taken to be the minimum virus, 82.4%, appears to be all, or nearly breadth of the hexagonal profile of an icosahedron of the proper volume, corresponding to the dimension measurable in the electron microscope. This diameter is about 1.56 times the length of the edge which appears in the well-known formula for the volume of an icosahedron.

‘Surface area is calculated for an using the measured minimum breadth agonal profile of the particle and the to obtain the icosahedral edge from breadth.

icosahedron of the hexfactor 1.56 the profile

THOMAS

250 TABLE

6

ing perfect recovery, only this amount at most, could be ASP. The absence of RNA in TIV is similar to the finding for other insect viruses, and Particle weight (from Table 5) 2.03 X 1C16 g Density (reciprocal of partial 1.39 g/cm3 probably for all true viruses, that only one specific volume estimated kind of nucleic acid is present (Bergold, from chemical composition)R 1958; Schgfer, 1959). The absence of polyVolume (calculated from par- 1.46 X 10-15cm3 saccharide also is consistent with previous title weight and density) results on insect viruses, but it must be Diameter (profile minimum 137 rnp noted that only two insect viruses, the nubreadth, calculated from volclear polyhedral inclusion particles of Bomume) byx: mori L. (Bergold, 1958) and Porthetria Diameter (profile minimum 130 f 5 mp &spar L. (Wyatt, 1950), have been exbreadth, measured on frozendried particles in electron miamined for the presenceof nonnucleoprotein croscope) constituents. Apparently, only the myxoSurface area (calculated from 6.0 X 10-l” cm2 viruses are known for certain to contain measured diameter) polysaccharide (SchSifer, 1959). Total weight of lipid in particle 6.3 X lo7 mol. The presence of a small amount of lipid wt. units (calculated from chemical in TIV is similar to the finding for B. mori composition and particle virus (=4% lipid) (Bergold, 1958). P. disweight) par virus, on the other hand, is apparently Total weight of DNA in particle 1.51 X 108 mol. free of lipid (Wyatt, 1950). The fact that wt. units (calculated from chemical most of the lipid in TIV is phospholipid sugcomposition and particle weight) gests a structural role for this constituent, perhaps in membrane formation. However, a The partial specific volumes of the constituother considerations lead to speculation ents were assumed to be: protein, 0.74; DNA, that the lipid is extraneous. It almost cer0.55; lipid, 1.00. tainly is not a particulate contaminant mixed with the virus in suspension, as all, protein, including some phosphoprotein. shown by the repurification experiment, but There could, of course, be small amounts of it could be adsorbed on the surface of the other constituents present since the protein particles. This is suggestedby the following fraction was not completely characterized facts: (1) the particles arise in the fat-body and exhaustive tests for minor constituents of the host insect larvae; (2) the absolute were not conducted. amount of lipid, if calculated as a typical A group of minor constituents which can phospholipid such as lecithin, is just about probably be excluded, even though it was right to coat the surface of the particle with not assayed, is cold-acid-soluble phosphorus a monomolecular film*; and (3) the relative compounds. In the extraction (ASP) amount of lipid is much less than in viruses scheme used here, which did not include an such as those of equine encephalomyelitis initial cold TCA extraction, any ASP com- (=50%) and influenza (=20%), where the pounds present in TIV would have been ex- essential structural role of lipid has been tracted either into the methanol-ethyl clearly demonstrated (SchZfer, 1959). ether or hot, TCA fractions. In the former That TIV contains DNA rather than fraction, however, there could be little RNA is in line with findings for most other ASP, since at least 90% of the phosphorus CHARACTERISTICS

OF SINGLE

TIV

PARTICLES

* The cross-sectional area of the lecithin molecule in a monomolecular film is about 1.13 X lo-” cm2 extraction into petroleum ether. There could (Wittcoff, 1951). Taking a molecular weight of 808 likewise be very little ASP in the latter fracfor lecithin (distearyl lecithin), the number of tion since here at least 97% of the phos- molecules in a TIV particle would be 7.8 x IO’. phorus was identified with DNA. It seems Hence the area of the film that could be formed that only about 4% of the total phosphorus is 8.8 X10-” cm*. The surface area of a TIV particle in the virus is unidentified, and so, assum- is about 6.0 XIOdl’ cm2 (Table 6). could

be identified

as lipid-bound

by its re-

TIV

COMPOSITION

AND

well-characterized insect viruses (Bergold, 1958). However, these other DNA viruses develop in the nucleus. Thus it is interesting to note that TIV is a DNA virus which seems to develop entirely in the cytoplasm (Williams and Smith, 1957). The relative amount of nucleic acid in TIV, 12.4%, is similar to the figure for other insect viruses (Bergold, 1958; Wyatt, 1952). The absolute amount, 1.5 x lo* mol. wt. units, is probably somewhat greater, since TIV appears to have a greater particle weight than that of any other insect virus investigated so far.Q The absolute amount is of the same order as found in T2 bacteriophage (Evans, 1959). In containing only adenine, guanine, thymine, and cytosine, and no methylcytosine or 5-hydroxymethylcytosine, TIV DNA is apparently like other insect virus DNA’s, although with the latter, the absence of 5hydroxymethylcytosine has not been demonstrated (Wyatt, 1952). The pairing of the bases, adenine equal to thymine and guanine equal to cytosine, presumably means that TIV DNA is the common doublestranded type. However, TIV DNA is uncommon in that the dissymmetry ratio, adenine plus thymine : guanine plus cytosine, equal to 2.20, is one of the highest reported (see Lee, et al., 1956,.for other high values). This may make TIV interesting from the standpoint of the DNA-protein coding problem (Crick, 1959) ; i.e., does the unusual ratio of bases in the DNA bring about an unusual ratio of amino acids in the virus protein? Further work will be necessary to answer this question. The DNA accounts for only about 80% of the total phosphorus in TIV; the remainder is associated with phospholipid and phosphoprotein. A lack of agreement between total phosphorus and DNA content has also been found in other insect viruses, but the non-DNA phosphorus was not characterized (Wyatt, 1952). TIV may ’ Accurate particle weights are not available for other insect viruses owing to the polydispersity of the preparations. Particle weights have been calculated from particle volumes determined from electron mircoscope measurements by assuming a figure for the density (Bergold, 1953).

PARTICLE

WEIGHT

251

be the first virus for which there is definite evidence for the presence of phosphoprotein. Among viruses that are highly uniform in size and shape, TIV appears to have the largest particle weight ever determined. Vaccinia has a greater particle weight, ~3.2 x log, but the particles vary somewhat in size and shape (Schafer, 1959). Large as the figure for TIV is, it is nevertheless consistent with the diameter of these unusual particles. ACKNOWLEDGMENTS I wish to thank Professor Robley C. Williams for continued support and encouragement during the course of this work and also for supplying the TIV which had been initially purified in the laboratory of Dr. Kenneth M. Smith. Mrs. Helen Weaver and Mr. Joseph Toby were of invaluable assistance in conducting the nitrogen analyses and electron microscopy, respectively. I am also indebted to Professor C. Arthur Knight, Dr. Milton P. Gordon, Dr. E. W. Putman, Dr. K. K. Reddi, Miss Marion Sandomire, and Dr. F. L. Schaffer for indispensable technical advice and discussion. REFERENCES ARNDT, U. W., and BEEMAA-, W. W. (1958). Private communication. ASEIWELL, G. (1957). Calorimetric analysis of sugars. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, pp. 73-105. Academic Press, New York. BACKUS, R. C., and WILLI& R. C. (1949). Small sperical particles of exceptionally uniform size.

J. Appl. Phys. 20, 224-225. BESDICH, A. (1957). Method for characterization of nucleic acids by base composition. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, pp. 715-723. Academic Press, New York. BERGOLD, G. H. (1953). Insect viruses. Advances in

Virus Research 1,91-139. BERGOLD, G. H. (1958). Viruses of insects. In “Handbuch der Virusforschung” (C. Hallauer and K. F. Meyer, eds.), Vol. 4, pp. 66-142. Springer, Vienna. CHARGAFF, E. (1955). Isolation and composition of the deoxypentose nucleic acids and of the corresponding nucleoproteins. In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, pp. 307-372. Academic Press, New York. CRAMPTON, C. F., LIPSHITZ, R., and CHARGAFF, E. (1954). Studies on nucleoproteins. I. Dissociation and reassociation of the deoxyribonucleohistone of calf thymus. J. Biol. Chem. 206, 499510.

THOMAS

252

F. H. C. ( 1959). The present position of the coding problem. Brookhaven Symposia in Biol.

CRICK,

No. 12,35-39.

P., and GEIDUSCHEK, E. P. (1953). Optical properties of proteins. In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 1, Part A, pp. 393-460. Academic Press, New York. EVANS, E. A. (1959). The comparative chemistry of infective virus particles and their functional activity : T2 and other bacterial viruses. In “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. 1, pp. 459-473. Academic Press, New York. FROMMHAGEN, L. H., KNIGHT, C. A., and FREEMAN, N. K. (1959). The ribonucleic acid, lipid, and polysaccharide constituents of influenza virus preparations. Virology 8, 176-197. HERSHEY, A. D., DIXON, J., and CHASE, M. (1953). Nucleic acid economy in bacteria infected with bacteriophage T2. I. Purine and pyrimidine composition. J. Gen. Physiol. 36,777-789. KIRK, P. L., and SCHAFFER, F. L. (1948). Construction and special uses of quartz helix balances. Rev. Sci. Znstr. 19,785-790. KLUC, A., FRANKLIN, R., and HUMPHREYS-OWEN, S. P. F. (1959). The crystal structure of Tipula iridescent virus as determined by Bragg reflection of visible light. Biochim. et Biophys. DOTY,

Acta

32,203-219.

K. Y., WAHL, R., and BARBU, E. (1956). Contenu au bases puriques et pyrimidiques des acides desoxyribonucleiques des bacthries. Ann. inst. Pasteur 91,2X2-224. LURIA, S. E., WILLIAMS, R. C., and BACKUS, R. C. (1951). Electron micrographic counts of bacteriophage particles. J. Bacterial. 61,179-188. MILLER, G. L. (1944). A study of conditions for optimum production of PR8 influenza virus in chick embryos. J. Exptl: Med. 79, 173-183. NAKAMURA, G. R. (1952). Microdetermination of phosphorus. Anal. Chem. 24,1372. OVEREND, W. G., and STACEY, M. (1955). Chemistry of ribose and deoxyribose. In “The Nucleic $cids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, pp. 9-80. Bcademic Press, New York. PUTMaN, E. W. (1957). Paper chromatography of sugars. Zn “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, pp. 62-72. Academic Press, New York. SCHAFER, W. (1959). The comparative chemistry of infective virus particles and of other virusspecific products: animal viruses. In “The Vi-

LEE,

ruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. 1, pp. 475-504. Academic Press, New York. SCHAFFER, F. L., GRUNBAUM, B. W., and KIRK, P. L. (1954). The composition of the saponifiable lipides of cultured tissue cells. A microfractionation study of Earle’s strain L. Arch. Biochem. Biophys.

50,188198.

G., and THANNHAUSER, S. J. (1945). A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem. 161, 8389. SMITH, J. D., unpublished. Quoted by Smith, K. M.

SCHMIDT,

(1958).

Parasitology

48,459.

K. M. (1955). What is a virus? Nature 175,12-15. SMITH, K. M. (1956). The structure of insect virus particles. J. Biophys. Biochem. Cvtol. 2, 301306. SMITH, K. M. (1958). A study of the early stages of infection with the Tipula iridescent virus. Parasitology 48,459-462. SMITH, K. M., and HILLS, G. J. (1959). Further studies on the electron microscopy of the Tipula iridescent virus. J. Mol. Biol. 1,277-280. SMITH, K. M., and RIVERS, C. F. (1959). Crossinoculation studies with the Tipula iridescent virus. Virology 9,140-141. SMITH, K. M., and WILLIAMS, R. C. (1958). Insect viruses and their structure. Endeuvour 17, 1%21. WILLIAMS, R. C., and BACKUS, R. C. (1949). Macromolecular weights determined by direct particle counting. I. The weight of the bushy stunt virus particle. J. Am. Chem. Sot. 71,405%4057. WILLIAMS, R. C., and SMITH, K. M. (1957). A crystallizable insect virus. Nature 179,119-120. WILLIAMS, R. C., and SMITH, K. M. (1958). The polyhedral form of the Tipula iridescent virus. SMITH,

Biochim.

et Biophys.

Acta

28,464-469.

H. (1951). “The Phosphatides,” p. 69. Reinhold, New York. WYATT, G. R. (1950). Studies on insect viruses and nucleic acids. Thesis, University of Cambridge, Cambridge, England. Quoted by Bergold, G. H. (1953). Advances in V&s Research 1,119. WYATT, G. R. (1952). The nucleic acids of some insect viruses, J. Gen. Physiol. 36, 201-205. WYATT, G. R., and COHEN, S. S. (1953). The bases of the nucleic acids of some bacterial and animal viruses : the occurrence of B-hydroxymethylcytosine. Biochem. J. 55, 774-782. XEROS, N. (1954). A second virus disease of the leather-jacket, Tipula paludosa. Nature 174, 562-563. WITTCOFF,