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The characterization of a mosquito iridescent virus II. Physicochemical characterization
IOURNAI,
OF
INVERTEBRATE
PATHOLOGY
16,
157-164
(1970)
The Characterization of a Mosquito Iridescent II. Physicochemical Characterization1
Virus
JAMES F. MATTA? Department
of Entomology and Gainesville, Received
Nematology, Unioersity Florida 32601
October
of Florida,
10, 1969
A procedure for the purification of the R type of a mosquito iridescent virus is presented. The amino acid composition and ultraviolet absorption spectrum of the purified virus are also presented, The Eig for the virus was determined to be 100 and the
El% was determined
to be 10.8.
The
S20,W
was 4458,
and the density, determined
b;qnilibrium ultracentrifugation in CsCl, was 1.354 g/ems. The average diameter was determined to be 180 rnp by electron-micrograph measurements. The particle weight, calculated from density and diameter measurements, was 2.486 X 109 daltons was distinctly different from the and the particle contained 15.97% DNA. The RMIV other iridescent viruses in terms of several physical parameters, and it would be difficult to justify considering it as a strain of these viruses. There was, however, a indicating that they have a common striking similarity in amino acid composition, phylogeny.
adenovirus group, it appears necessary to have all members of the group characterized to determine the diversity of the group. Kalmakoff and Tremaine (1968) have suggested that all iridescent viruses are strains of a single virus’ species. However, in the absence of physical or chemical information concerning the mosquito iridescent viruses this statement seemssomewhat precipitant. This statement, based in great part on supposition, serves to point out the need for the detennination of the basic physical and chemical properties of the mosquito iridescent viruses. Glitz et al. ( 1968) presented evidence which indicated that Sericesthis iridescent virus and the Tipula iridescent virus have identical physical characteristics and very similar amino acid compositions; while differing significantly in biological properties. The biological characteristics of the Rtype MIV have been discussedin a previous
INTRODU~~ON
The relative ease with which large quantities of purified material can be obtained has stimulated several studies on the physical and chemical properties of the iridescent viruses (Thomas, 1961; Kalmakoff and Tremaine, and Glitz
1968; Bellett et al., 1968).
and Inman, 1967; There is, however,
no published information about the physical or chemical properties of the mosquito iridescent viruses, other than a determination of the DNA content by Faust et al. (1968). Since the iridescent viruses apparently represent a new group of animal viruses, which can be associated in some respects with the 1 This study was conducted at the Insects Affecting Man and Animals Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FIorida 32601; Florida Agricultural Experiment Station Journal Series No. 3471. 2 Present address: Department of Biology, Old Dominion University, Norfolk, Virginia 23508. Copyright
0
1970
by
Academic
Press
Inc. 157
I58
MATTA
paper ( Matta and Lowe, 1970) ; this paper will deal with the physical and chemical characteristics of the virus. It is hoped that the characterization of this virus will make the relationship between the iridescent viruses somewhat clearer. METHODS
AND
MATERIALS
Purification. The technique of mass production of virus-infected larvae has been discussed in a previous paper (Matta and Lowe, 1970). For purification approximately 500 infected larvae were triturated in a 15-ml glass tissue grinder in 0.01 M phosphate buffer (pH 7.0). The homogenate was then diluted to 100 ml with buffer and subjected to two cycles of differential centrifugation, 4000 r-pm for 5 min and 15,000 r-pm for 25 min, in a Beckman model L preparative ultracentrifuge using a type50 angle-head rotor. After differential centrifugation the virus suspension was further purified by means of sucrose density-gradient centrifugation. To prepare the gradients, a stock 54% (w/v) sucrose solution was diluted with buffer to yield five final solutions of 54%, 44%, 30%, 16%, and 6% sucrose. With the densest solution first, 0.9 ml of each solution was layered into 5 ml nitrocellulose tubes and these were allowed to stand overnight at 4°C to allow the gradient to become established. It was found that these gradients could be kept for 2 days before they deteriorated to the point where their use influenced results. One half-milliliter aliquots of the virus suspension were layered on top of each gradient and these were centrifuged immediately at 12,000 r-pm for 20 min in a SW 39L swinging-bucket rotor. The bands were then removed with an ISCO mode1 D density-gradient fractionator. The fractions separated by the fractionator were centrifuged at 30,000 rpm for 30 min in a type-50 angle-head rotor, the pellets resuspended,
and the sucrose-gradient centrifugation and separation repeated. The resulting material was dialyzed exhaustively against 0.055 M phosphate buffer to remove the sucrose, and the purified virus was stored at -70°C until needed. Spectrophotometry. The ultraviolet absorption spectrum of the virus at pH 7.0 was determined with a Perkin-Elmer Model 139PM spectrophotometer which had been calibrated with the 656.1 spectra1 emission band of the deuterium lamp. The pH of the virus suspension was adjusted to 7.0 by dialysis against 0.055 M phosphate buffer. Extinction coeficient. A sample of highly purihed virus was lyophilized in a commercial freeze-drying apparatus (Virtis ), and a portion of the lyophilized virus was dried overnight in a CaCl? desiccator under vacuum. A 2.32-mg fraction of this dried virus was added to 3 ml of 0.055 M phosphate buffer and allowed to dissolve, with frequent agitation, for 4 hr. Appropriate dilutions were made in the cuvettes, and the absorption at each dilution was recorded at wavelengths of 254 mn, 280 rnp, 436 rnp, and 700 rnp. Sedimentation coeficient. The sedimentation coefficient was determined in a Beckman Model E analytical uhracentrifuge equipped with Schleirin optics. An AN-D rotor with a 4” standard cell was used, and the bar angle was set at 60”. The solvent system was 0.055 M phosphate buffer at a pH of 7.2, and the temperature was allowed to stabilize at 20°C before each run. The viscosity of the buffer was determined with a Cannon-Fenske capillary viscometer. The positions of the peaks were measured with a microcompareter on the original plates. Diameter measurements. The diameter of the virus was measured from electron micrographs of infected tissue sections. The tissue was fixed in glutaraldehyde and embedded in Luft’s Epon. Sections were cut to a thickness of 90 A with a MT-2 Porter-
PHYSICOCHEMICAL
CHARACTERIZATION
Blum ultramicrotome. The sections were placed on a l?ormvar-coated grid and stained with uranyl acetate and lead citrate. The virus was measured by projecting a picture of a typical field on a screen and measuring the projected images. The measuremcnts were corrected for magnification due to the projection and to the electron microscope. The magnification of the electron microscope was determined from a picture of a diffraction grating taken at the same maignification step as the tissue section. Quantitatke determination of DNA. The virus concentration of a known volume of stock virus suspension was determined using the extinction coefficient at 700 mp. An equal volume of 10% trichloroacetic acid was added to the virus suspension and the mixture was maintained at 95°C for 30 min. The material was centrifuged at 1000 rpm for 15 min in an IEC (International Equipment Co.) Pr-6 refrigerated centrifuge equipped with a swinging-bucket rotor. The supernatant fluid was removed, and the pellet was reextracted with an appropriate volume of 5% trichloroacetic acid. After centrifugation the two supernatant fluids were pooled and the pellets were discarded. The same procedure was performed on a weighed sample of salmon sperm DNA with a purity of 76.2% (Mann, Co.), which was used as a standard for the diphenylamine reaction. The diphenylamine test was performed essentially by the method of Giles and Myers (1965) except that the diphenylamine reagent (4 g of diphenylamine in 100 ml of glacial acetic acid) was modified by the addition of 5 ml of concentrated sulfuric acid to prevent precipitation of the diphenylamine upon addition of the test mixture. Two-milliliter aliquots of the diphenylamine reagent were placed in screw-capped tubes and 2 ml of the DNA extract were added to each tube. The tubes were agi-
OF
RhII\
1%
tated and 0.1 ml of acetaldehyde solution (1.6 mg/ml) was added to each tube. The tubes were incubated at 30°C for 12 hr. and the optical densities were read at 59.5 rnp and 700 rnp using a blank of reagent plus 5% trichloroacetic acid. Equilibrium ultracentrifugation. The density of the virus was determined bv equilibriunl ultracentrifugation in a cesium chloride gradient, using the basic principles of Vinograd and Hearst ( 1962). Two-millilittr aliquots of a CsCl solution, with an average density of 1.6 g/ml, was placed in three centrifuge tubes, with a 5-ml capacity, and 0.5 ml of virus solution (0.4 mg/ml) w;\s layered on top. The two solutions in one tube were mixed and the other two tubcss wcrc left banded. To prevent the collapse of the tubes 2.5 ml of mineral oil were lavered on each tube, and they were ccntrifuged in a Spinco SW-39L rotor at 35.000 rpm for 48 hr at 25°C. It was detcrmincbd that the solutions had reached equilibrium when the virus band in the mixed tube was in the same position as the virus band in the lawrcd tubes. The tubes were punctured -at the bottom and 2-drop fractiotls were collected. Densitv was determined bv drawing each fraction into a 50-p] pipette (of a known weight) and weighing it. The fraction was then diluted to 3 ml and the optical density at 280 rnp was recorded. Antibody production. To produce antibody to the RMIV, rabbits were inocldatcd intramuscularlv with 1 ml of a virus mi Yture injected into the hip. The inoculum was a mixture containing equal parts of Freund’s complete adjuvent (Difco) and an RMIV solution containing 5 mg/ml. One month later the rabbits were given an intravenous injection of 2.5 mg of RMIV in 0.5 ml of saline. One week after the secor~d injection the rabbits were bled bv cardiac puncture, and the blood was allowed to clot in glass petri dishes held at room temperature for 2 hr. The dishts were th(,lt incubated at 4°C for 24 hr to allow the clot
160
MATl-A
to contract, and the serum was collected, placed in tubes, and frozen at -22°C until needed. Light-scattering. Light-scattering was measured with a Brice-Phoenix light-scattering photometer, and the refractive index measurements were made with a BricePhoenix differential refractometer. The refractometer was calibrated with a standard potassium chloride solution (1.4911 g of KC1 brought to 100 ml with distilled water) which had a refractive index difference of 2093 X 10m6 when compared to distilled water. Amico acid analysis. A 1.2-mg sample of highly purified RMIV was hydrolyzed in 6 N HCl for 24 hr and analyzed for amino acid composition using a Beckman-Spinco Automatic analyzer. RESULTS
AND
DISCUSSION
Purification. After two cycles of differential centrifugation the material appeared to contain two distinct components (Fig. 1) when observed in the analytical ultracentrifuge. Three light-scattering bands were observed in most of the first-run sucrose-gradient tubes; however, it was found that the lower band could be completely eliminated if sufficient care was taken in resuspending the virus pellets. Since minor contamination occurred during the separa-
FIG. 1. Schleiren pattern of RMIV after two cycles of differential centrifugation. Note the presence of both top component and virus.
tion of the bands, both fractions were rerun on sucrose gradients and refractionated. The top band, which was designated “top component” appeared as an amorphous black mass when pelleted, and the bottom component appeared as a green-to-red iridescent pellet. The top component was not infectious; however, the bottom component did produce infected larvae. No attempt was made to compare the infectivity of purified and non-puriiled virus because of the difficulty in accounting for all the virus from a single batch of larvae and because of the value of purified virus. Ultraviolet-absorption spectrum. The ultraviolet-absorption spectrum and the absorption spectrum corrected for light-scattering by the method of Bonhoeffer and Schachman ( 1960) are presented in Fig. 2. The ultraviolet-absorption spectrum, once corrected for light-scattering, corresponds to the absorption spectrum of TIV and SIV published by Glitz et al. (1968) in most respects. However, it should be noted that the corrected absorption curve does not show a peak at 280 mu as would be expected of a virus particle. This is probably because the light-scattering is so great that the corrected curve is relatively insensitive to small changes in absorption. Extinction coeficients. The extinction coefficients were calculated for wavelengths of 254, 280, 436, and 700 mu. The ratio of absorption to concentration is constant at 700 my; however, at 436 mu the ratio is not constant for concentrations above 0.5 mg/ ml. At 280 my the ratio is not constant for concentrations above 0.08 mg/ml, and for 254 mu the ratio is not constant for concentrations above 0.05 mg/ml. The extinction coefficients are Eiz = 118.3, EiE = 100.0 Eir6 = 27.2, and El% = 10.8. 700 The sedimenSedimentation coeficient. tation coefficient, calculated from the Schleiren patterns derived from a sedimentation run at 2994 r-pm, was 4.4578 X lo-lo. Since the viscosity of the buffer was
PHYSICOCHEMICAL
CHARACXERIZATION
01 220
230
240
260
250 Wavelength
FIG.
corrected
2. for
The ultraviolet light-scattering
absorption spectrum of RMIV. by the method of Bonhoeffer
very close to that of water, and the virus concentrations used were low, correction factors for these properties would have little effect on this system. Therefore, this value can be taken as a valid approximation of the Szo,w and the RMIV has an S value of 4458. Quantitative determination of DNA. The results of the diphenylamine test indicate that there are 170.25 t 2.5 pg of DNA in 1.066 mg of virus. This yields a DNA content of 15.97 i- 0.29%. Faust et al. (1968) reported a DNA content for RMIV of 11.7%, and the probable reason for the lower value is that the purification procedure was not rigorous enough to exclude some proteins, probably the top component. This would increase the weight of the preparation being tested without increasing the DNA content, thus resulting in a lower figure for the DNA content. Assuming a particle weight for the virus of 2.486 X log daltons, the figure of 15.97% corresponds to a DNA molecule with a molecular weight of 397 million, which is considerably more than the values of 130155 million reported for other iridescent viruses, Hyde et al. (1967) gave a value of
OF
270 ih
---and
161
RMIV
280
290
mp
uncorrected values; Schachman ( 1960).
___
values
approximately 200 X 10fi daltons for the molecular weight of fowlpox DNA and stated that fowlpox contains more DNA than any other virus. The RMIV has almost twice as much DNA as fowlpox virus and, therefore, contains more DNA than anrv other virus as yet discovered. Equilibrium ultracentrifugation. The density and optical densities of the drop-collected fractions from the equilibrium ultracentrifugation are presented in Fig. 3. The average density of the hydrated virus particle was 1.354 g/cm”. . Gel diffusion. The pattern of precipitation for the gel-diffusion plates used in these studies is shown in Fig. 4. Material which banded in sucrose gradients in the same position as the top component was isolated from normal larvae and compared with the top component from virus-infected larvae. There was no antigenic reaction bctween the normal top component and the antiserum produced in rabbits inoculated with the virus. There was, however, a r(‘action of identity between the top component isolated from infected larvae and the virus, indicating an antigenic relationship. It is possible that the top component ac-
162
MATTA
1.35 Density IO Drop-collected
FIG. 3. centrifugation
The
densities and tubes containing
optical kMIV.
densities
from
tually consists of hollow virus particles as has been inferred by Glitz et al. (1968); however, since the top component does not form an iridescent pellet, it is probable that in some the protein ‘shell” is deformed manner and is incapable of crystal formation. This hypothesis is supported by electron micrographs of the top component of TIV by Glitz et al. ( 1968), which showed the top component to be similar to deformed and collapsed virus particles.
FIG. 4. Gel-diffusion pattern of RMIV (R), top component (TC), and normal host tissues ( NTC ). The antiserum (AS ) was induced in rabbits against whole RMIV.
in
1.40 g/cm3 15
20
25
fraction
drop-collected
fractions
of CsCl
equilibrium
ultra-
The top component occurs in relatively large quantities and it would be interesting to determine its exact relationship to the virus. If it indeed consists of empty virus particles, is it only the DNA which is missing or are there also internal proteins missing? The study of the composition of the top component in relation to the virus could offer a chance to determine whether the shape of the virus particle is influenced by the DNA, or if the loss of iridescence is simply due to missing internal proteins. Amino acid analysis. The results of the amino acid analysis are presented in Table 1. A single analysis was run because of the limited access to the amino acid analyzer. The partial specific volume of RMIV was calculated from the amino acid composition and the percentage of DNA by adding the weighted partial specific volumes of each of the amino acids and the DNA, This gave a partial specific volume for the RMIV of 0.70763 cm3/g. The amino acid composition is very similar to that of the other iridescent viruses. In fact, the differences between the two published analyses of TIV appear to be greater than the differences between TIV and RMIV. These similarities invite speculation as to the phylogenetic relationship of the iridescent viruses which would allow
PHYSICOCHEMXCAL
TABLE .4xrmo
ACID
CHARACI’ERIZATION
1
COhiPOSITION
OF
RMIV
Amino acid residues Amino acid Lysine Histidine Arginine Aspartic
2.312
9.390 acid
Thrcwnine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isolencine
Leucine Tyrosine Phenylalanine -
Totals
M 8.832
13.830
8.195 8.618 6.474 7.323 6.354 5.827
per cent/100 g 7.24 1.90 7.70 11.34 6.71 7.06 5.31 6.00 5.21 4.78
2.163
1.77
7.837 3.372 9.931
6.42 2.76 8.14
9.604 5.961 5.881 121.994
7.95 4.89 4.82
100.00
such similarities between the viruses in the face of such gross physical differences. Light-scattering. SeveraI attempts were made to determine the molecular weight of tht> virus by the light-scattering method, using absolute turbiditv and refractive index. It was found that the size of the virus particle was beyond the upper limit of this system, and the molecular weight couId not be determined by this method. Electron-micrograph measurements. The measurements of the RMIV particles made from corner to comer, across the largest diameter of the particle, averaged 200 mu. Measurements made from the center of a face to the center of the opposite face averaged 170 mu. An icosahedron has a maximum diameter (twice the distance from the center to a corner) which is 1.18 times the diameter of a sphere of the same volume, and a minimum diameter (twice the distance from the center to the center of a face) which is 0.94 times the diameter of a sphere of the same volume (Markham, 1967). Applying this rule, the minimum diam-
OF
HSll\’
16:.?
eter measurements yield a sphere of 180 my in diameter while the maximum diameter measurements yield a sphere with a diameter of 170 my. Since the corner-tocorner measurements are more susceptibk: to error, due to the orientation of the particle in the tissue section, it is felt that the estimate of 180 my is the best. Molecular zceiglrt. The molecular weight was caIcuIatcd from the diameter and th
Purified RMIV offers several conveniently measured physical parameters which differentiate it from the other iridescent viruses. The sedimentation coefficient of 4458 is approximately twice that of SIV and TIV. Published data indicates that the diameter of RMIV is at least 20 mu larger than anv of the other iridescent viruses. This is . supported bv the measurements made on virus particles in tissue sections of A. taeniorh yncllus mosquito larvae which showed that the particle had an average diameter of 180 mu. RMIV has a molecular weight of 2.486 X 1Oa daltons which is approximately twice that recorded for TIV and SIV. The partial specific volume of 0.70763, density of 1,354, and ultraviolet-absorption ratio (260 mu over 260 mu) of 1.19 are all quite similar to the values given for the other iridescent viruses. This is as expected since nucleaproteins, of approximately the same size and with approximately the same proportions of DNA to protein would be similar with respect to these parameters. It proved impossible to measure the diffusion coefficient because the equipment necessary for this measurement was not available. However, since figures for the molecular weight, sedimentation coefficient, and partial specific volume were known. it
164
MAT-J.-A
was possible to calculate efficient by the following
the diffusion coformula (Mark-
ham, 1967) : D = RTS/M( 1-VP), where R is the gas constant, T is the absolute temperature, S is the sedimentation coefficient, v is the partial specific volume, A4 is the molecular weight, and p is the density of the suspending medium. Using the values obtained for RMIV, the diffusion coefficient was found to be I.488 X lo-*m which is comparable to the values published for TIV and SIV (Glitz et al. 1968), although, as would be expected, it is somewhat smaller because of the larger size of the RMIV particle. It would appear that the statement made by Kalmakoff and Tremaine (1968) concerning the strainal relationship of the iridescent viruses is no longer acceptable, since viruses so different in physical characteristics as RMIV and TIV could not be strains of the same species. It is impossible at this time to say anything definite about the phylogenic relationships of the known iridescent viruses; however, the rash of discoveries of new iridescent viruses in recent years indicates that this is probably a diverse group and that there will be more material on which to base speculation in the future. The RMIV has many interesting physical and biological properties which make it worthy of further study and with the availability of large quantities of this virus it should make a very profitable research tool. ACKNOWLEDGMENTS The author ciation to Dr. encouragement
wishes Ronald during
to express his deep appreE. Lowe for his help and the course of this work.
REFERENCES BELLETT, A. J, D., AND INMAN, R. B. 1967. Some properties of deoxyribonucleic acid preparations from Chile, Sericesthis and Tipula iridescent viruses. J. Mol. Biol., 25, 425432. BONHOEFFER, F., AND SCHACHMAN, H. K. 1960. Studies on the organization of nucleic acids within nucleoproteins. Biochem. Biqphys. Res. Commun., 2, 366-371. FAUST, R. M., DOUGHERTY, E. M., AND ADAMS, J. R. 1968. Nucleic acid in the blue-green and orange mosquito iridescent viruses (MIV) isolated from larvae of Aedes tueniorhynchw. J. Invertebr. Puthol., 10, 160. GILES,
K. proved mation 93.
W., AND MYERS, A. 1965. An imdiphenylamine method for the estiof deoxyribonucleic acid. Ndure, 206,
GLITZ,
D. G., HILLS, G. H., AND RIVERS, G. F. 1968. A comparison of the Tip& and Sericesthis iridescent viruses. J. Gen. Viral., 3, 209-220. HYDE, J. M., GAFFORD, L. G., AND RANDALL, C. C. 1967. Molecular weight determination of fowl pox virus DNA by electron microscopy. J. Viral., 33, 112-120. KALMAKOFF, J., AND TREZMAINE, J, H. 1968. Physiochemical properties of TipuZu iridescent virus. J. Virol., 2, 738-744. MARKHAM, R. 1967. Diffusion. In “Methods in Virology,” ( Maramorosch, K. and Koprowski, H., eds.) Vol. 2, pp. 275-302. Academic Press. MATTA, J. F., AND LOWE, R. E. 1970. The characterization of a mosquito iridescent virus (MIV). I. Biological characteristics, infectivity and pathology. J. Inuertebr. Pathol., 16, 3841, in press. THOMAS, R. S. 1961. The chemical composition and particle weight of Tip&z iridescent virus. I. Viral., 14, 240-252. VINOGRAD, J., AND HEARST, J. E. 1962. Equilibrium sedimentation of macromolecules and viruses in a density gradient. Fortschr. Chem. Org. Naturst., 20, 372-422.
OF
INVERTEBRATE
PATHOLOGY
16,
157-164
(1970)
The Characterization of a Mosquito Iridescent II. Physicochemical Characterization1
Virus
JAMES F. MATTA? Department
of Entomology and Gainesville, Received
Nematology, Unioersity Florida 32601
October
of Florida,
10, 1969
A procedure for the purification of the R type of a mosquito iridescent virus is presented. The amino acid composition and ultraviolet absorption spectrum of the purified virus are also presented, The Eig for the virus was determined to be 100 and the
El% was determined
to be 10.8.
The
S20,W
was 4458,
and the density, determined
b;qnilibrium ultracentrifugation in CsCl, was 1.354 g/ems. The average diameter was determined to be 180 rnp by electron-micrograph measurements. The particle weight, calculated from density and diameter measurements, was 2.486 X 109 daltons was distinctly different from the and the particle contained 15.97% DNA. The RMIV other iridescent viruses in terms of several physical parameters, and it would be difficult to justify considering it as a strain of these viruses. There was, however, a indicating that they have a common striking similarity in amino acid composition, phylogeny.
adenovirus group, it appears necessary to have all members of the group characterized to determine the diversity of the group. Kalmakoff and Tremaine (1968) have suggested that all iridescent viruses are strains of a single virus’ species. However, in the absence of physical or chemical information concerning the mosquito iridescent viruses this statement seemssomewhat precipitant. This statement, based in great part on supposition, serves to point out the need for the detennination of the basic physical and chemical properties of the mosquito iridescent viruses. Glitz et al. ( 1968) presented evidence which indicated that Sericesthis iridescent virus and the Tipula iridescent virus have identical physical characteristics and very similar amino acid compositions; while differing significantly in biological properties. The biological characteristics of the Rtype MIV have been discussedin a previous
INTRODU~~ON
The relative ease with which large quantities of purified material can be obtained has stimulated several studies on the physical and chemical properties of the iridescent viruses (Thomas, 1961; Kalmakoff and Tremaine, and Glitz
1968; Bellett et al., 1968).
and Inman, 1967; There is, however,
no published information about the physical or chemical properties of the mosquito iridescent viruses, other than a determination of the DNA content by Faust et al. (1968). Since the iridescent viruses apparently represent a new group of animal viruses, which can be associated in some respects with the 1 This study was conducted at the Insects Affecting Man and Animals Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FIorida 32601; Florida Agricultural Experiment Station Journal Series No. 3471. 2 Present address: Department of Biology, Old Dominion University, Norfolk, Virginia 23508. Copyright
0
1970
by
Academic
Press
Inc. 157
I58
MATTA
paper ( Matta and Lowe, 1970) ; this paper will deal with the physical and chemical characteristics of the virus. It is hoped that the characterization of this virus will make the relationship between the iridescent viruses somewhat clearer. METHODS
AND
MATERIALS
Purification. The technique of mass production of virus-infected larvae has been discussed in a previous paper (Matta and Lowe, 1970). For purification approximately 500 infected larvae were triturated in a 15-ml glass tissue grinder in 0.01 M phosphate buffer (pH 7.0). The homogenate was then diluted to 100 ml with buffer and subjected to two cycles of differential centrifugation, 4000 r-pm for 5 min and 15,000 r-pm for 25 min, in a Beckman model L preparative ultracentrifuge using a type50 angle-head rotor. After differential centrifugation the virus suspension was further purified by means of sucrose density-gradient centrifugation. To prepare the gradients, a stock 54% (w/v) sucrose solution was diluted with buffer to yield five final solutions of 54%, 44%, 30%, 16%, and 6% sucrose. With the densest solution first, 0.9 ml of each solution was layered into 5 ml nitrocellulose tubes and these were allowed to stand overnight at 4°C to allow the gradient to become established. It was found that these gradients could be kept for 2 days before they deteriorated to the point where their use influenced results. One half-milliliter aliquots of the virus suspension were layered on top of each gradient and these were centrifuged immediately at 12,000 r-pm for 20 min in a SW 39L swinging-bucket rotor. The bands were then removed with an ISCO mode1 D density-gradient fractionator. The fractions separated by the fractionator were centrifuged at 30,000 rpm for 30 min in a type-50 angle-head rotor, the pellets resuspended,
and the sucrose-gradient centrifugation and separation repeated. The resulting material was dialyzed exhaustively against 0.055 M phosphate buffer to remove the sucrose, and the purified virus was stored at -70°C until needed. Spectrophotometry. The ultraviolet absorption spectrum of the virus at pH 7.0 was determined with a Perkin-Elmer Model 139PM spectrophotometer which had been calibrated with the 656.1 spectra1 emission band of the deuterium lamp. The pH of the virus suspension was adjusted to 7.0 by dialysis against 0.055 M phosphate buffer. Extinction coeficient. A sample of highly purihed virus was lyophilized in a commercial freeze-drying apparatus (Virtis ), and a portion of the lyophilized virus was dried overnight in a CaCl? desiccator under vacuum. A 2.32-mg fraction of this dried virus was added to 3 ml of 0.055 M phosphate buffer and allowed to dissolve, with frequent agitation, for 4 hr. Appropriate dilutions were made in the cuvettes, and the absorption at each dilution was recorded at wavelengths of 254 mn, 280 rnp, 436 rnp, and 700 rnp. Sedimentation coeficient. The sedimentation coefficient was determined in a Beckman Model E analytical uhracentrifuge equipped with Schleirin optics. An AN-D rotor with a 4” standard cell was used, and the bar angle was set at 60”. The solvent system was 0.055 M phosphate buffer at a pH of 7.2, and the temperature was allowed to stabilize at 20°C before each run. The viscosity of the buffer was determined with a Cannon-Fenske capillary viscometer. The positions of the peaks were measured with a microcompareter on the original plates. Diameter measurements. The diameter of the virus was measured from electron micrographs of infected tissue sections. The tissue was fixed in glutaraldehyde and embedded in Luft’s Epon. Sections were cut to a thickness of 90 A with a MT-2 Porter-
PHYSICOCHEMICAL
CHARACTERIZATION
Blum ultramicrotome. The sections were placed on a l?ormvar-coated grid and stained with uranyl acetate and lead citrate. The virus was measured by projecting a picture of a typical field on a screen and measuring the projected images. The measuremcnts were corrected for magnification due to the projection and to the electron microscope. The magnification of the electron microscope was determined from a picture of a diffraction grating taken at the same maignification step as the tissue section. Quantitatke determination of DNA. The virus concentration of a known volume of stock virus suspension was determined using the extinction coefficient at 700 mp. An equal volume of 10% trichloroacetic acid was added to the virus suspension and the mixture was maintained at 95°C for 30 min. The material was centrifuged at 1000 rpm for 15 min in an IEC (International Equipment Co.) Pr-6 refrigerated centrifuge equipped with a swinging-bucket rotor. The supernatant fluid was removed, and the pellet was reextracted with an appropriate volume of 5% trichloroacetic acid. After centrifugation the two supernatant fluids were pooled and the pellets were discarded. The same procedure was performed on a weighed sample of salmon sperm DNA with a purity of 76.2% (Mann, Co.), which was used as a standard for the diphenylamine reaction. The diphenylamine test was performed essentially by the method of Giles and Myers (1965) except that the diphenylamine reagent (4 g of diphenylamine in 100 ml of glacial acetic acid) was modified by the addition of 5 ml of concentrated sulfuric acid to prevent precipitation of the diphenylamine upon addition of the test mixture. Two-milliliter aliquots of the diphenylamine reagent were placed in screw-capped tubes and 2 ml of the DNA extract were added to each tube. The tubes were agi-
OF
RhII\
1%
tated and 0.1 ml of acetaldehyde solution (1.6 mg/ml) was added to each tube. The tubes were incubated at 30°C for 12 hr. and the optical densities were read at 59.5 rnp and 700 rnp using a blank of reagent plus 5% trichloroacetic acid. Equilibrium ultracentrifugation. The density of the virus was determined bv equilibriunl ultracentrifugation in a cesium chloride gradient, using the basic principles of Vinograd and Hearst ( 1962). Two-millilittr aliquots of a CsCl solution, with an average density of 1.6 g/ml, was placed in three centrifuge tubes, with a 5-ml capacity, and 0.5 ml of virus solution (0.4 mg/ml) w;\s layered on top. The two solutions in one tube were mixed and the other two tubcss wcrc left banded. To prevent the collapse of the tubes 2.5 ml of mineral oil were lavered on each tube, and they were ccntrifuged in a Spinco SW-39L rotor at 35.000 rpm for 48 hr at 25°C. It was detcrmincbd that the solutions had reached equilibrium when the virus band in the mixed tube was in the same position as the virus band in the lawrcd tubes. The tubes were punctured -at the bottom and 2-drop fractiotls were collected. Densitv was determined bv drawing each fraction into a 50-p] pipette (of a known weight) and weighing it. The fraction was then diluted to 3 ml and the optical density at 280 rnp was recorded. Antibody production. To produce antibody to the RMIV, rabbits were inocldatcd intramuscularlv with 1 ml of a virus mi Yture injected into the hip. The inoculum was a mixture containing equal parts of Freund’s complete adjuvent (Difco) and an RMIV solution containing 5 mg/ml. One month later the rabbits were given an intravenous injection of 2.5 mg of RMIV in 0.5 ml of saline. One week after the secor~d injection the rabbits were bled bv cardiac puncture, and the blood was allowed to clot in glass petri dishes held at room temperature for 2 hr. The dishts were th(,lt incubated at 4°C for 24 hr to allow the clot
160
MATl-A
to contract, and the serum was collected, placed in tubes, and frozen at -22°C until needed. Light-scattering. Light-scattering was measured with a Brice-Phoenix light-scattering photometer, and the refractive index measurements were made with a BricePhoenix differential refractometer. The refractometer was calibrated with a standard potassium chloride solution (1.4911 g of KC1 brought to 100 ml with distilled water) which had a refractive index difference of 2093 X 10m6 when compared to distilled water. Amico acid analysis. A 1.2-mg sample of highly purified RMIV was hydrolyzed in 6 N HCl for 24 hr and analyzed for amino acid composition using a Beckman-Spinco Automatic analyzer. RESULTS
AND
DISCUSSION
Purification. After two cycles of differential centrifugation the material appeared to contain two distinct components (Fig. 1) when observed in the analytical ultracentrifuge. Three light-scattering bands were observed in most of the first-run sucrose-gradient tubes; however, it was found that the lower band could be completely eliminated if sufficient care was taken in resuspending the virus pellets. Since minor contamination occurred during the separa-
FIG. 1. Schleiren pattern of RMIV after two cycles of differential centrifugation. Note the presence of both top component and virus.
tion of the bands, both fractions were rerun on sucrose gradients and refractionated. The top band, which was designated “top component” appeared as an amorphous black mass when pelleted, and the bottom component appeared as a green-to-red iridescent pellet. The top component was not infectious; however, the bottom component did produce infected larvae. No attempt was made to compare the infectivity of purified and non-puriiled virus because of the difficulty in accounting for all the virus from a single batch of larvae and because of the value of purified virus. Ultraviolet-absorption spectrum. The ultraviolet-absorption spectrum and the absorption spectrum corrected for light-scattering by the method of Bonhoeffer and Schachman ( 1960) are presented in Fig. 2. The ultraviolet-absorption spectrum, once corrected for light-scattering, corresponds to the absorption spectrum of TIV and SIV published by Glitz et al. (1968) in most respects. However, it should be noted that the corrected absorption curve does not show a peak at 280 mu as would be expected of a virus particle. This is probably because the light-scattering is so great that the corrected curve is relatively insensitive to small changes in absorption. Extinction coeficients. The extinction coefficients were calculated for wavelengths of 254, 280, 436, and 700 mu. The ratio of absorption to concentration is constant at 700 my; however, at 436 mu the ratio is not constant for concentrations above 0.5 mg/ ml. At 280 my the ratio is not constant for concentrations above 0.08 mg/ml, and for 254 mu the ratio is not constant for concentrations above 0.05 mg/ml. The extinction coefficients are Eiz = 118.3, EiE = 100.0 Eir6 = 27.2, and El% = 10.8. 700 The sedimenSedimentation coeficient. tation coefficient, calculated from the Schleiren patterns derived from a sedimentation run at 2994 r-pm, was 4.4578 X lo-lo. Since the viscosity of the buffer was
PHYSICOCHEMICAL
CHARACXERIZATION
01 220
230
240
260
250 Wavelength
FIG.
corrected
2. for
The ultraviolet light-scattering
absorption spectrum of RMIV. by the method of Bonhoeffer
very close to that of water, and the virus concentrations used were low, correction factors for these properties would have little effect on this system. Therefore, this value can be taken as a valid approximation of the Szo,w and the RMIV has an S value of 4458. Quantitative determination of DNA. The results of the diphenylamine test indicate that there are 170.25 t 2.5 pg of DNA in 1.066 mg of virus. This yields a DNA content of 15.97 i- 0.29%. Faust et al. (1968) reported a DNA content for RMIV of 11.7%, and the probable reason for the lower value is that the purification procedure was not rigorous enough to exclude some proteins, probably the top component. This would increase the weight of the preparation being tested without increasing the DNA content, thus resulting in a lower figure for the DNA content. Assuming a particle weight for the virus of 2.486 X log daltons, the figure of 15.97% corresponds to a DNA molecule with a molecular weight of 397 million, which is considerably more than the values of 130155 million reported for other iridescent viruses, Hyde et al. (1967) gave a value of
OF
270 ih
---and
161
RMIV
280
290
mp
uncorrected values; Schachman ( 1960).
___
values
approximately 200 X 10fi daltons for the molecular weight of fowlpox DNA and stated that fowlpox contains more DNA than any other virus. The RMIV has almost twice as much DNA as fowlpox virus and, therefore, contains more DNA than anrv other virus as yet discovered. Equilibrium ultracentrifugation. The density and optical densities of the drop-collected fractions from the equilibrium ultracentrifugation are presented in Fig. 3. The average density of the hydrated virus particle was 1.354 g/cm”. . Gel diffusion. The pattern of precipitation for the gel-diffusion plates used in these studies is shown in Fig. 4. Material which banded in sucrose gradients in the same position as the top component was isolated from normal larvae and compared with the top component from virus-infected larvae. There was no antigenic reaction bctween the normal top component and the antiserum produced in rabbits inoculated with the virus. There was, however, a r(‘action of identity between the top component isolated from infected larvae and the virus, indicating an antigenic relationship. It is possible that the top component ac-
162
MATTA
1.35 Density IO Drop-collected
FIG. 3. centrifugation
The
densities and tubes containing
optical kMIV.
densities
from
tually consists of hollow virus particles as has been inferred by Glitz et al. (1968); however, since the top component does not form an iridescent pellet, it is probable that in some the protein ‘shell” is deformed manner and is incapable of crystal formation. This hypothesis is supported by electron micrographs of the top component of TIV by Glitz et al. ( 1968), which showed the top component to be similar to deformed and collapsed virus particles.
FIG. 4. Gel-diffusion pattern of RMIV (R), top component (TC), and normal host tissues ( NTC ). The antiserum (AS ) was induced in rabbits against whole RMIV.
in
1.40 g/cm3 15
20
25
fraction
drop-collected
fractions
of CsCl
equilibrium
ultra-
The top component occurs in relatively large quantities and it would be interesting to determine its exact relationship to the virus. If it indeed consists of empty virus particles, is it only the DNA which is missing or are there also internal proteins missing? The study of the composition of the top component in relation to the virus could offer a chance to determine whether the shape of the virus particle is influenced by the DNA, or if the loss of iridescence is simply due to missing internal proteins. Amino acid analysis. The results of the amino acid analysis are presented in Table 1. A single analysis was run because of the limited access to the amino acid analyzer. The partial specific volume of RMIV was calculated from the amino acid composition and the percentage of DNA by adding the weighted partial specific volumes of each of the amino acids and the DNA, This gave a partial specific volume for the RMIV of 0.70763 cm3/g. The amino acid composition is very similar to that of the other iridescent viruses. In fact, the differences between the two published analyses of TIV appear to be greater than the differences between TIV and RMIV. These similarities invite speculation as to the phylogenetic relationship of the iridescent viruses which would allow
PHYSICOCHEMXCAL
TABLE .4xrmo
ACID
CHARACI’ERIZATION
1
COhiPOSITION
OF
RMIV
Amino acid residues Amino acid Lysine Histidine Arginine Aspartic
2.312
9.390 acid
Thrcwnine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isolencine
Leucine Tyrosine Phenylalanine -
Totals
M 8.832
13.830
8.195 8.618 6.474 7.323 6.354 5.827
per cent/100 g 7.24 1.90 7.70 11.34 6.71 7.06 5.31 6.00 5.21 4.78
2.163
1.77
7.837 3.372 9.931
6.42 2.76 8.14
9.604 5.961 5.881 121.994
7.95 4.89 4.82
100.00
such similarities between the viruses in the face of such gross physical differences. Light-scattering. SeveraI attempts were made to determine the molecular weight of tht> virus by the light-scattering method, using absolute turbiditv and refractive index. It was found that the size of the virus particle was beyond the upper limit of this system, and the molecular weight couId not be determined by this method. Electron-micrograph measurements. The measurements of the RMIV particles made from corner to comer, across the largest diameter of the particle, averaged 200 mu. Measurements made from the center of a face to the center of the opposite face averaged 170 mu. An icosahedron has a maximum diameter (twice the distance from the center to a corner) which is 1.18 times the diameter of a sphere of the same volume, and a minimum diameter (twice the distance from the center to the center of a face) which is 0.94 times the diameter of a sphere of the same volume (Markham, 1967). Applying this rule, the minimum diam-
OF
HSll\’
16:.?
eter measurements yield a sphere of 180 my in diameter while the maximum diameter measurements yield a sphere with a diameter of 170 my. Since the corner-tocorner measurements are more susceptibk: to error, due to the orientation of the particle in the tissue section, it is felt that the estimate of 180 my is the best. Molecular zceiglrt. The molecular weight was caIcuIatcd from the diameter and th
Purified RMIV offers several conveniently measured physical parameters which differentiate it from the other iridescent viruses. The sedimentation coefficient of 4458 is approximately twice that of SIV and TIV. Published data indicates that the diameter of RMIV is at least 20 mu larger than anv of the other iridescent viruses. This is . supported bv the measurements made on virus particles in tissue sections of A. taeniorh yncllus mosquito larvae which showed that the particle had an average diameter of 180 mu. RMIV has a molecular weight of 2.486 X 1Oa daltons which is approximately twice that recorded for TIV and SIV. The partial specific volume of 0.70763, density of 1,354, and ultraviolet-absorption ratio (260 mu over 260 mu) of 1.19 are all quite similar to the values given for the other iridescent viruses. This is as expected since nucleaproteins, of approximately the same size and with approximately the same proportions of DNA to protein would be similar with respect to these parameters. It proved impossible to measure the diffusion coefficient because the equipment necessary for this measurement was not available. However, since figures for the molecular weight, sedimentation coefficient, and partial specific volume were known. it
164
MAT-J.-A
was possible to calculate efficient by the following
the diffusion coformula (Mark-
ham, 1967) : D = RTS/M( 1-VP), where R is the gas constant, T is the absolute temperature, S is the sedimentation coefficient, v is the partial specific volume, A4 is the molecular weight, and p is the density of the suspending medium. Using the values obtained for RMIV, the diffusion coefficient was found to be I.488 X lo-*m which is comparable to the values published for TIV and SIV (Glitz et al. 1968), although, as would be expected, it is somewhat smaller because of the larger size of the RMIV particle. It would appear that the statement made by Kalmakoff and Tremaine (1968) concerning the strainal relationship of the iridescent viruses is no longer acceptable, since viruses so different in physical characteristics as RMIV and TIV could not be strains of the same species. It is impossible at this time to say anything definite about the phylogenic relationships of the known iridescent viruses; however, the rash of discoveries of new iridescent viruses in recent years indicates that this is probably a diverse group and that there will be more material on which to base speculation in the future. The RMIV has many interesting physical and biological properties which make it worthy of further study and with the availability of large quantities of this virus it should make a very profitable research tool. ACKNOWLEDGMENTS The author ciation to Dr. encouragement
wishes Ronald during
to express his deep appreE. Lowe for his help and the course of this work.
REFERENCES BELLETT, A. J, D., AND INMAN, R. B. 1967. Some properties of deoxyribonucleic acid preparations from Chile, Sericesthis and Tipula iridescent viruses. J. Mol. Biol., 25, 425432. BONHOEFFER, F., AND SCHACHMAN, H. K. 1960. Studies on the organization of nucleic acids within nucleoproteins. Biochem. Biqphys. Res. Commun., 2, 366-371. FAUST, R. M., DOUGHERTY, E. M., AND ADAMS, J. R. 1968. Nucleic acid in the blue-green and orange mosquito iridescent viruses (MIV) isolated from larvae of Aedes tueniorhynchw. J. Invertebr. Puthol., 10, 160. GILES,
K. proved mation 93.
W., AND MYERS, A. 1965. An imdiphenylamine method for the estiof deoxyribonucleic acid. Ndure, 206,
GLITZ,
D. G., HILLS, G. H., AND RIVERS, G. F. 1968. A comparison of the Tip& and Sericesthis iridescent viruses. J. Gen. Viral., 3, 209-220. HYDE, J. M., GAFFORD, L. G., AND RANDALL, C. C. 1967. Molecular weight determination of fowl pox virus DNA by electron microscopy. J. Viral., 33, 112-120. KALMAKOFF, J., AND TREZMAINE, J, H. 1968. Physiochemical properties of TipuZu iridescent virus. J. Virol., 2, 738-744. MARKHAM, R. 1967. Diffusion. In “Methods in Virology,” ( Maramorosch, K. and Koprowski, H., eds.) Vol. 2, pp. 275-302. Academic Press. MATTA, J. F., AND LOWE, R. E. 1970. The characterization of a mosquito iridescent virus (MIV). I. Biological characteristics, infectivity and pathology. J. Inuertebr. Pathol., 16, 3841, in press. THOMAS, R. S. 1961. The chemical composition and particle weight of Tip&z iridescent virus. I. Viral., 14, 240-252. VINOGRAD, J., AND HEARST, J. E. 1962. Equilibrium sedimentation of macromolecules and viruses in a density gradient. Fortschr. Chem. Org. Naturst., 20, 372-422.