"Science, Faculty of"@en . "Microbiology and Immunology, Department of"@en . "DSpace"@en . "UBCV"@en . "Kalmakoff, James"@en . "2011-08-17T22:38:24Z"@en . "1968"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "An investigation into the physical and chemical properties of two icosahedral viruses was undertaken, one a plant virus, carnation ringspot virus (CRSV), and the other an insect virus, Tipula iridescent virus (TIV). These viruses were studied using accepted chemical and biophysical methods and parameters such as amino acid composition, nucleotide composition, sedimentation coefficients, diffusion coefficients, molecular weights, hydration, and morphology in the electron microscope were determined.\r\nCRSV could be purified by frontal elution from AG1-X8 quaternary ammonium anion exchange resin with a NaCl-phosphate buffer, pH 6.8. The composition of CRSV based on nucleotides and amino acids recovered was 20.48% and 79.52% protein. Amino acid analyses indicated that the subunit protein was composed of approximately 347 amino acid residues with a subunit molecular weight of 38,000. The sedimentation and diffusion coefficients were 132 x 10\u00C2\u00AF\u00C2\u00B9\u00C2\u00B3 sec and 1.48 x 10\u00C2\u00AF\u00E2\u0081\u00B7 cm\u00C2\u00B2 /sec, respectively. A molecular weight based on the above two parameters was 7.07 x 10\u00E2\u0081\u00B6. An uncorrected extinction coefficient at 260 m\u00C2\u00B5 for the virus was found to be 6.46 cm\u00C2\u00B2 /mg virus. The frictional ratio calculated from the hypothetical diffusion coefficient of the anhydrous particle and the observed diffusion was 1.16. Assuming the virus particle to be spherical the degree of hydration was O.38 g water/g of virus.\r\nTIV was purified by sedimentation through a 5-40% sucrose density gradient. The particle molecular weight of TIV based on a sedimentation coefficient of 2200 x 10\u00C2\u00AF\u00C2\u00B9\u00C2\u00B3 sec and a diffusion coefficient of 3.3 x 10\u00C2\u00AF\u00E2\u0081\u00B8 cm\u00C2\u00B2/sec was 5.51 x 10\u00E2\u0081\u00B8 g/mole. A frictional ratio of 1.22 was calculated and the value of 0.57 g water/g virus was obtained assuming the deviation of the frictional ratio from unity was due to hydration.\r\nThe DNA content based on total inorganic phosphorous liberated was 19 \u00C2\u00B1 0.2%. At 260 m\u00CE\u00BC the virus gave an uncorrected absorbance of 18.2 cm\u00C2\u00B2 /mg virus and a light scattering corrected absorbance of 9.8 cm\u00C2\u00B2 /mg virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus (SIV), suggesting a strain relationship. The relative amounts of all the amino acids of these two viruses, with the exception of arginine, are within experimental error. The possibility that the four iridescent insect viruses Sericesthis iridescent virus (SIV), mosquito iridescent virus (MIV), and Chilo iridescent virus (CIV) bear a strain relationship is discussed."@en . "https://circle.library.ubc.ca/rest/handle/2429/36747?expand=metadata"@en . "SOME PHYSICO-CHEMICAL STUDIES ON TWO ICOSAHEDRAL VIRUSES BY JAMES KALMAKOFF B.Sc. (Microbiology), University of B r i t i s h Columbia, 1 9 6 5 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1 9 6 8 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r equ i r emen ts f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree tha t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r an t ed by the Head o f my Department o r by h i s r e p r e s e n -t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l owed w i t hou t my w r i t t e n p e r m i s s i o n . Department n f Microbiology The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date July 3rd, 1968 i i ABSTRACT An investigation into the physical and chemical properties of two icosahedral viruses was undertaken, one a plant v i r u s , carnation ringspot virus (CRSV), and the other an insect v i r u s , Tipula iridescent virus (TIV). These viruses were studied using accepted chemical and biophysical methods and parameters such as amino acid composition, nucleotide composition, sedimentation c o e f f i c i e n t s , d i f f u s i o n c o e f f i c i e n t s , molecular weights, hydration, and morphology in the electron microscope were determined. CRSV could be purified by frontal elution from AG1-X8 quaternary ammonium anion exchange resin with a NaCl-phosphate buffer, pH 6.8. The composition of CRSV based on nucleotides and amino acids recovered was 20.48% and 79.52% protein. Amino acid analyses indicated that the subunit protein was composed of approximately 347 amino acid residues with a subunit molecular weight of 38,000. The sedimentation and -13 -7 d i f f u s i o n c o e f f i c i e n t s were 132 x 10 sec and 1.48 x 10 2 cm /sec, respectively. A molecular weight based on the above two parameters was 7.07 x 10^. An uncorrected extinction c o e f f i c i e n t 2 at 260 my for the virus was found to be 6.46 cm /mg virus. The f r i c t i o n a l r a t i o calculated from the hypothetical diffusion c o e f f i c i e n t of the anhydrous p a r t i c l e and the observed diffusion was 1.16. Assuming the virus p a r t i c l e to be spherical, the degree of hydration was O.38 g water/g of virus. i i i TIV was purified by sedimentation through a 5-^0% sucrose density gradient. The p a r t i c l e molecular weight of TIV based on -13 a sedimentation c o e f f i c i e n t of 2200 x 10 sec and a diffusion -8 2 8 coef f i c i e n t of 3.3 x 10 cm /sec was 5.51 x 10 g/mole. A f r i c t i o n a l r a t i o of 1.22 was calculated and the value of 0.57 g water/g virus was obtained assuming the deviation of the f r i c t i o n a l r a t i o from unity was due to hydration. The DNA content based on total inorganic phosphorous liberated was 19.\u00C2\u00B1 0.2%. At 260 my the virus gave an 2 uncorrected absorbance of 18.2 cm /mg virus and a l i g h t 2 scattering corrected absorbance of 9.8 cm /mg virus. Amino acid analyses of the virus protein revealed a remarkable s i m i l a r i t y to Sericesthis iridescent virus (SIV), suggesting a s t r a i n relationship. The rel a t i v e amounts of a l l the amino acids of these two viruses, with the exception of arginine, are within experimental error. The p o s s i b i l i t y that the four iridescent insect viruses Sericesthis iridescent virus (SIV), mosquito iridescent virus (MIV), and Chilo iridescent virus (CIV) bear a s t r a i n relationship is discussed. i v TABLE OF CONTENTS Page INTRODUCTION MATERIALS AND METHODS Virus p u r i f i c a t i o n I. CRSV k I I . TIV. \u00E2\u0080\u00A2 . \u00E2\u0080\u00A2 5 Amino Acid Analysis I. CRSV . , 6 I I. TIV 8 Nucleic Acid Analysis I. CRSV 8 II . TIV 9 Electrophoresis and Diffusion 9 Sedimentation 9 P a r t i a l Specific Volume 9 Serology 10 Ult r a v i o l e t Absorbance 10 Electron Microscopy 11 RESULTS Virus P u r i f i c a t i o n I. CRSV \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 12 II . TIV 18 Ul t r a v i o l e t Absorbance I. CRSV 21 II . TIV 21 V Page Amino Acid Composition I. CRSV 25 I I. TIV 28 Nucleic Acid Composition I. CRSV 33 11. TIV 33 Physical Properties I. CRSV 34 I I . TIV 35 DISCUSSION General Remarks 4 0 I. Carnation ringspot virus 41 11. Tipula i ridescent virus 42 LITERATURE CITED 47 vi LIST OF TABLES Page Table I. Amino acid residue recoveries from CRSV protein 26 Table I I . Composition and molecular weight of CRSV protein 29 Table I I I . Amino acid residue recoveries from TIV protein 30 Table 'IV. Amino acid composition from hydrolysis of whole TIV virus 31 Table V. Composition and molecular weight of TIV protein 32 V I I LIST OF FIGURES Page Fig. 1. Ultracentrifuge patterns of p a r t i a l l y p urified CRSV at 20,410 rpm 13 Fig. 2. Elution p r o f i l e of 6 ml fractions of CRSV from a 1.5 x 16 cm AG1-X8 quaternary ammonium anion exchange column 16 Fig. 3. Electron micrographs of CRSV 17 Fig. 4. Ultracentrifuge patterns of pure CRSV 19 Fig. 5. Sucrose density gradient fractionation and i n f e c t i v i t y of CRSV 20 Fig. 6. P u r i f i c a t i o n and components of TIV 22 Fig. 7. U l t r a v i o l e t spectrum of CRSV in disti11ed water 23 Fig. 8. U l t r a v i o l e t spectrum of TIV in d i s t i 1 led water 2k Fig. 9. Plot of hydrolysis time with amino acids recovered for CRSV 27 Fig. 10. Plot of D . t 1 vs_ t 1 values from dif f u s i o n experiment for CRSV 36 Fig. 11. Plot of D . t 1 vs_t' values from dif f u s i o n experiment for TIV 37 Fig. 12. Plot of D vs_ 1/t' of di f f u s i o n experiment for TIV 39 ACKNOWLEDGEMENTS I wish to express thanks to Dr. R.E. Fi t z p a t r i c k , Director of the Research Station, Canada Department of Agriculture, for making the f a c i l i t i e s of the Research Station available; to Mr. J.H. Severson for the photography, and to Dr. J.J.R. Campbell, professor and head of the Department of Microbiology, University of B r i t i s h Columbia, for his kind cooperation. I also wish to thank my supervisor, Dr. J.H. Tremaine, whose patience and understanding lead me through the biophysical maze and whose constant encouragements made this investigation possible. Thanks are also expressed to Mr. A. Valcic for technical advice and consultation. This work was supported, in part, by National Research Council Studentships. Part of this thesis has been published in Virology 33.: 10-16 (1967). INTRODUCTION A j u s t i f i c a t i o n for studying the fundamental properties of vi ruses per se is that they represent a highly i n t r i c a t e form of biological organization and the dynamic principles involved in virus organization are l i k e l y to apply to other organized c e l l u l a r components. Since there appear to be general principles upon which viruses are assembled, this knowledge of molecular architecture should contribute to our understanding of the functional organization of the parts of a l i v i n g c e l l . An investigation into the biophysical properties of two icosahedral viruses was undertaken and is the subject of this thesis. One of these is a plant v i r u s , carnation ringspot virus (CRSV) and the other is an insect vi rus, Tipula iridescent virus (TIV). These viruses d i f f e r widely in their biological and biophysical properties, the former representing a single-6 stranded RNA plant virus of 7.1 x 10 molecular weight, while 8 the l a t t e r is a double-stranded DNA insect virus of 5.5 x 10 molecular weight. The development of good p u r i f i c a t i o n procedures made i t possible to study the physical and chemical properties of these two viruses. Parameters such as amino acid composition, nucleotide composition, sedimentation c o e f f i c i e n t s , d i f f u s i o n c o e f f i c i e n t s , molecular weights and morphology in the electron microscope were investigated. Carnation ringspot virus was described by Kassanis (1955), and has been reported since from countries where carnations are extensively grown. Kemp (1964) detected CRSV in commercial carnation varieties in Canada. Tremaine (1961) separated CRSV from cowpea host antigens by column chromatography on a basic ion-exchange resin. Hoi lings and Stone (1965) showed that CRSV was s e r o l o g i c a l l y d i s t i n c t from other icosahedral viruses tested having similar properties, e.g. broad bean mottle, carnation mottle, carnation I t a l i a n ringspot, cymbidium ringspot, pelargonium leaf c u r l , raspberry ringspot, tobacco necrosis, tobacco ringspot, tomato bushy stunt and turnip c r i n k l e . Tipula iridescent virus was discovered by Xeros (1954) at the Virus Research Unit at Cambridge, England, and reports of other iridescent viruses have appeared in the l i t e r a t u r e in recent years. Sericesthis iridescent virus (SIV) was found (Steinhaus and Leutenegger, 1963) in a Scarab beetle larva in A u s t r a l i a , mosquito iridescent virus (MIV) was isolated in Florida (Clark e_taj_., 1965) and'Chi lo iridescent virus (CIV) was found in the rice stem borer moth in Japan (Fukaya and Nasu, 1966). These viruses are closely similar in most physical and biological properties, but are not i d e n t i c a l . Day and Mercer (1964) reported that TIV and SIV are s e r o l o g i c a l l y related and B e l l e t t and Inman (1967),studying the DNA of CIV, SIV, and TIV found that the nucleic acids of these viruses were very s i m i l a r , but not i d e n t i c a l . These viruses have icosahedral symmetry with reported diameters ranging from 1800 A for MIV to 1300 A for TIV. Extensive chemical analyses of TIV were carried out by Thomas (1961) , 3 and an amino acid analysis was done on SIV by Day and Mercer. Recently Smith (1967) in his monograph on insect viruses summarized the properties of TIV and SIV. Considering the wide experimental and natural host and geographical range of TIV (Smith et_ a_l_., 1961) and the similar properties of a l l the iridescent viruses, i t is probable that these insect viruses represent strains of a single iridescent vi rus. 4 MATERIALS AND METHODS Virus p u r i f i c a t i o n . I. Carnation ringspot virus. CRSV and i t s antiserum were obtained from W.G. Kemp, Research Station, Canada Department of Agriculture, Vineland, Ontario. The virus was increased in cowpea (V i gna s i ties i s End 1., variety Blackeye). A clone for amino acid analysis was established by picking a local 1esion from Chenopodium amarahticolor, Coste and Reyn. Cowpea seedlings were infected by mechanical inoculation with sap. About 10 days after inoculation, the infected plants were harvested and frozen. Samples of the virus-infected leaves were ground in a meat grinder and the sap was expressed from the pulp by squeezing, through cheesecloth. To determine the best p u r i f i c a t i o n procedure, the sap from 1000 g of leaves (wet weight) was divided into four aliquots and each c l a r i f i e d by adding one of the following: (NH^SO^, 10% by weight; chloroform, 30% by volume; ethanol, 20% by volume; HCL to pH 5. After low speed centrifugation, the virus was pelleted in a No. 21 rotor in the Spinco Model L at 20,000 rpm for 3 hr. The pellets were dissolved in 3.0 ml of 0.008 M_ sodium phosphate buffer pH 6.7 containing 0.063 M_ sodium chloride (buffer A). Sedimentation patterns of the dissolved pellets were studied in a Spinco Model E analytical ultracentrifuge at 20,410 rpm. The dissolved pellets 5 were pooled and chromatographed on a 1.5 x 16 cm column of AG1-X8 quaternary ammonium anion exchange resin equilibrated to pH 6.7 with buffer A. To establish that the material isolated was in fact the e t i o l o g i c a l agent, an i n f e c t i v i t y study was carried out. The pur i f i e d virus was centrifuged in a Spinco SW25 rotor at 23,000 rpm for 1 hr in a smooth sucrose density gradient of 5-30% prepared by the method of Stace-Smith (1965). The gradient was fractionated on an ISCO model D density-gradient fractionater. The optical density of the fractions was measured at 260 my with a Beckman DU Spectrophotometer. Appropriate fractions were diluted 1:10 with d i s t i l l e d water and tested for i n f e c t i v i t y by local lesion assay on C_. amaranticolor, using the incomplete block method of 8 ha If-leaves per 0.25 ml f r a c t i o n . I I. Tipula i ridescent vi rus. TIV was obtained from K.M. Smith of the University of Texas, Austin, Texas, U.S.A. , and from C F . Rivers of the Agricultural Research Council Virus Research Unit, Cambridge, England. Both virus preparations proved infectious when inoculated into larvae of TipUla palUdosa, Meigen, giving the c h a r a c t e r i s t i c symptomatology (Smith, 1967). The culture obtained from K.M. Smith was passaged in the laboratory by inoculation Q of 10 yl of a virus suspension containing about 1.8 x 10 virus pa r t i c l e s with 250 units penici11 in and 0.25 mg streptomycin per ml. In the mass production of TIV, the larvae were cut into fine 6 pieces with scissors 6-10 days after inoculation. The pieces of larvae in d i s t i l l e d water were s t i r r e d magnetically at 4\u00C2\u00B0 C overnight. The resulting suspension was c l a r i f i e d at 1000 rpm for 10 min in a Sorvall SS-1 rotor, followed by centrifugation in a No. 30 rotor in the Spinco Model L centrifuge at 20,000 rpm for 30 min. The pellets were dissolved in 0.01 M_ phosphate buffer pH 7.4 or in d i s t i l l e d water. The resuspended virus was further purified by centrifugation in a Spinco SW-25. rotor at 15,000 rpm for 20 min through a smooth sucrose density gradient.of 5-40% prepared by the method of Stace-Smith (1965). The gradient was fractionated on an ISC0 Model D density gradient fractionator, and the optical density of the fractions was measured at 260 my with a Beckman DU Spectrophotometer. The virus fractions were dialyzed against buffer and concentrated by high speed centrifugation i f necessary. The virus was repurified by sucrose density gradient prior to use for physical studies, since i t was found that virus suspensions tended to aggregate upon standing. Amino Acid Analyses I. Carnation ringspot virus. Analyses were performed on purified virus obtained from clone-infected plants. The protein was separated from the nucleic acid by mixing 5 ml of a 5 mg/ml virus preparation with 1 ml 6 N HCI. This mixture was allowed to stand for 24.hrs 7 at room temperature. The denatured protein was separated by centrifuging at .15,000. rpm for 15 min, then washing twice with 1 N_ HC1. The supernatants were pooled and used for nucleotide analysis. The protein p e l l e t was dissolved in 5 ml of .'12. N_ HC1 and 1.5 ml aliquots were pipetted into 3 hydrolysis tubes. An equal amount of d i s t i l l e d water was added to each tube. The tubes were evacuated, sealed and hydrolyzed at 107\u00C2\u00B0 C f o r 1 2 , 2k and 72 hrs. Excess HC1 was removed by flash evaporation at 35\u00C2\u00B0 C and the residues were dissolved in c i t r a t e buffer pH 2. One ml aliquots containing approximately 0.^5 mg protein were analyzed in t r i p l i c a t e on a Spinco amino acid analyzer. The micromoles of amino acids recovered were compared with a Spinco c a l i b r a t i o n mixture. Cysteine content was determined on a separate sample of virus protein oxidized with.performic acid. Excess performic acid from the sample was removed by d i a l y s i s . The sample was evaporated in vacuo and hydrolyzed for 2k hrs. The cysteic acid content was estimated by reference to aspartic acid and alanine which were determined in the same analysis. The results were corrected for 10% loss of cysteic acid. Tryptophan content was determined on the undegraded virus by reaction with p-aminobenzaldehyde (Spies and Chambers, 1949). The amount of virus protein in the assay was calculated from the extinction c o e f f i c i e n t of the virus. A correlation of protein content and absorbance at 260 my 8 was made by placing an aliquot of whole virus in a hydrolysis tube with 6 N_ HCI, hydrolyzing for 24 hrs and analyzing i t in the same manner as the virus protein. Adjustments for glycine, tryptophan, cysteine, threonine, and serine were made by comparison with values obtained for the virus protein. I I. T i pula i ridescent virus. Analyses were performed on purified vi rus f roifi Tipula larvae inoculated with a pool of material received from CF. Rivers and K.M. Smith. The protein was separated from the nucleic acid by mixing 5 ml of a 2 mg/ml virus preparation with 1 ml of 6 N HCI. The denatured protein was separated by centrifugation and the protein p e l l e t was hydrolyzed as described for CRSV using 2k- and 72-hr hydrolysis times. Nucleic Acid Analysis I. Carnation ringspot virus. The supernatants\u00E2\u0080\u00A2of the denatured protein pel lets (described above for amino acid analysis) were pooled and brought up to 12 ml with 1 N_ HCI. Six ml were sealed in a test tube, heated for 1 hr in a boili n g water bath, and flash evaporated at 35\u00C2\u00B0 C The residue was dissolved in a minimal amount of 0.1 N_ HCI and transferred quantitatively to Whatman No. hO f i l t e r paper. The purine bases and pyrimidine nucleotides were separated by descending chromatography and quantitatively determined as described by Markham (1955). 9 I I Tjpula i ridescent vi rus. An attempt was made to analyze the nucleotide ratios of the DNA by using the 70% perchloric acid technique described by Knight (I963) and descending paper chromatography. When analyses were run in t r i p l i c a t e anomolous results were obtained indicating a loss of nucleotides. A phosphorus analysis was used to determine the amount of DNA present. Inorganic phosphorus liberated from samples of whole virus by heating at 190 - 200\u00C2\u00B0 C for 15 min with 70% perchloric acid was determined by the method of Chen e_t a_j_. (1956). The virus had previously been dialyzed at k\u00C2\u00B0 C against 0.02 M Tris-HCl buffer pH 8.1 for 3 weeks. Electrophoresis and Diffus ion. These were determined in the Spinco Model H electro-phores i s - d i f f u s ion apparatus using the 11 ml c e l l at 4\u00C2\u00B0 C with virus in 0.02 ionic strength buffer. The diffusion coefficients were calculated from photographs of the Rayleigh interference fringes taken over a period of 6-18 days (Schachman, 1957). Measurements of fringe positions were made with a Gaertner two-dimensional microcomparator. Sedimentation Sedimentation velocity was determined at 0.02 ionic strength in a Spinco Model E analytical ultracentrifuge at 21,740 rpm for CRSV and 6,995 for TIV, the rotor was regulated at 20\u00C2\u00B0 C. Par t i a l Spec i f i c Vo1ume The p a r t i a l s p e c i f i c volume of the viruses was calculated from their composition using the additive procedure described by Cohn and Edsall (1943). Therefore, V = < V.W. + V N A- W N A-Where V. is the p a r t i a l s p e c i f i c volume of amino acid residue i ; V ^ is the p a r t i a l s p e c i f i c volume of nucleic acid,DNA (0.59 ml/gm), RNA (0.53 ml/gm); W. and are the percentage composition of each of the amino acid residues and the nucleic acid, respectively. Serology Antiserum was prepared by injecting a rabbit i n t r a -muscularly with 2 mg of purified virus emulsified with incomplete Freund's adjuvant. After 24 days the animal was given a si m i l a r inoculation and then bled 21 days after the second injection. The serum was mixed with an equal volume of glycerol and stored at -20\u00C2\u00B0 C u n t i l used. Serology was carried out by diffusion in 0.75% agar gel against extracts of healthy and infected tissues. For TIV, p r e c i p i t i n ring tests were carried out as described by Whitcomb and Black (1961). U l t r a v i o l e t Absorbance Measurements of u l t r a v i o l e t absorbance were routinely performed in a Beckman DU Spectrophotometer. Since large particles l i k e virus particles have 1ight scattering properties, the observed UV absorbance was corrected (Bonhoeffer and Schachman, I960).by plotting the log of absorbance at wave lengths (A ) between 320 my to 700 my against log ( A ) . The straight line portion was extrapolated to 240 my and the resultant values were subtracted to give the true u l t r a v i o l e t absorbance. Electron Microscopy Virus preparations were dialyzed against d i s t i 1 led water for 48 hrs and negatively stained with 2% uranyl acetate or \\u00C2\u00B0/0 phosphotungstic acid or shadowed with Pd, and examined in a Ph i l i p s 200 electron microscope. RESULTS Virus P u r i f i c a t i o n I. Carnation ringspot virus. Sedimentation patterns of the p a r t i a l l y purified virus in the analytical ultracentrifuge are given in Figure 1. The major peak sedimenting at 135 S is the virus. A peak sedimenting at 18 S is probably the enzyme, 1,5 ribulose diphosphate cocarboxylase (Weissbach et a l . , 1956), a component of infected and healthy sap. Twenty percent ethanol appears almost completely to remove this component from the sap (Fig. 1C). Another peak, sedimenting at k-S S is Fraction II, described by Singer et_aj_. (1952). For comparative studies the virus concentration was calculated from the area under the schlieren peak, as described by Schachman (1957) from the following equation: Where A = measured area (cm ); 6 = angle of the schlieren diaphragm; a =? c e l l thickness along optical path; b, m , m = X c optical constants of analytical centrifuge; An = s p e c i f i c refractive increment (at approximately mg/ml this was taken to (D 13 Figure 1: Ultracentrifuge patterns of p a r t i a l l y purified CRSV at 20,410 rpm, c l a r i f i c a t i o n by: A. 10% (NH|()2S02t, B. 30% chloroform, C. 20% ethanol, D. HC1 to pH 5.0. be 0.000186); (x/xQ) = position of boundary at zero time (x ) and position (x) at the time of the picture. The virus peaks represent 1.1 mg/ml for (NH^J^SO^, 0.9 mg/ml for chloroform, 0.6 mg/ml for ethanol, and 0.7 mg/ml for HCI treatment. Although the ethanol treatment gave the lowest y i e l d , i t was taken as the method of choice since i t contained the lowest concent rat ion of jmpuri t ies, thereby elimi nat i ng subsequent steps in p u r i f i c a t i o n . For routine p u r i f i c a t i o n of large volumes, k0% (NH^^SO^ was added to the plant sap, which precipitated the virus and large amounts of h-5 S proteins. These Fraction I I proteins were not completely removed when 20% ethanol was added to the suspensions of the (NHj^SO^ pellet (removal of insoluble material from the suspension before the addition of ethanol resulted in appreciable loss of the vir u s , suggesting that the virus was adsorbed to the (NH^J^SO^ p e l l e t ) . The virus and denatured Fraction If proteins were concentrated by centrifugation in a No. 30 rotor at 28,000 rpm for 90 min. When such a preparation was further p u r i f i e d on AG1-X8 quaternary ammonium anion exchange resin eluted with buffer A pH 6.7, as shown in Figure 2, the virus was f r o n t a l l y eluted followed by nucleic acid. Subsequent elution with 1 M_ NaCl and 1 N_ HCI resulted in release.of some protein, however, most of the Fraction II protein appeared to be ir r e v e r s i b l y bound to the resin. The pure virus gave a charac t e r i s t i c 280/260 r a t i o of 0.62 which was constant when the virus was rechromatographed on another column of AG1-X8. Electron micrographs (Fig. 3A) of the effluent, negatively stained with \% phosphotungstic acid, revealed homogeneous polyhedral particles of 31.5 my diameter. This agrees with the sizes reported by Tremaine (I96I) and Hoi lings and Stone (1965). An electron micrograph of particles stained with 2% uranyl acetate (Fig. 3B) suggests that CRSV probably has the 5^1^ 5 capsomere arrangement that is cha r a c t e r i s t i c of a 2-fold axis view for a 32 capsomere v i r u s , similar to that described for cowpea chlorotic mottle virus (Bancroft et a l . , 1967) and broad bean mottle virus (Finch and Klug, 1967). In the analytical ultracentrifuge the purified virus showed a single homogeneous peak (Fig. 4). Electrophoresis carried out at pH 4.0 indicated the presence of a single virus component. It was necessary to show that the particles of the electron micrograph were in fact the infectious entity. The p o s s i b i l i t y that the e t i o l o g i c a l agent is an undetected contaminant presents a problem in those biological systems in which merely trace amounts are necessary for biological a c t i v i t y . Figure 5 represents fractionation of a sucrose density gradient of the purified virus as measured at 260 my and local lesion assay of appropriate fractions oh C. amaranticolor. The number of local lesions is the total on 8 half-leaves from a 0.25 ml frac t i o n . The i n f e c t i v i t y curve shows good agreement with the major component of the density gradient indicating that the particles isolated were indeed the infectious entity. A clone started from a local lesion reacted with CRSV antiserum and gave typical symptoms on the cowpea plants. 16 8.0-, FRACTION NUMBER Figure 2: Elution p r o f i l e of 6 ml fractions of CRSV from a 1.5 x 16 cm AG1-X8 quaternary ammonium anion exchange column, eluted with buffer A, followed by 1 N_ NaCl and 1 N_HC1. The frontal ly eluted peak is CRSV. l A B F i g u r e 3: E l e c t r o n m i c r o g r a p h s o f CRSV. A. P a r t i c l e s n e g a t i v e l y s t a i n e d w i t h 1% p h o s p h o t u n g s t i c a c i d a t m a g n i f i c a t i o n 219,000 x. B. P a r t i c l e s s t a i n e d w i t h 2% u r a n y l a c e t a t e a t m a g n i f i c a t i o n 1,095,000 x. The p a r t i c l e denoted by t h e arrow c o u l d r e p r e s e n t a 2 - f o l d a x i s view t y p i c a l o f an i c o s a h e d r a l s u r f a c e l a t t i c e T=3. I I Tlpula i ridescent virus. A virus preparation purified by one cycle of u l t r a -centrifugation was separated by sucrose density gradient into at least three components (Fig. 6A). This pattern was not s i g n i f i c a n t l y altered by use of: 0.01 M_ phosphate pH 6.7, borate at pH 7 . 5 (Day and Mercer, 1964), 0.01 M Tris HC1 pH 8.1, d i s t i l l e d water, or by extraction of the virus with 5% chloro-form at 4 \u00C2\u00B0 C. When the main virus component was concentrated by centrifugation and subjected to another density gradient, the main and bottom components were present, the top component was absent. The nature of these components was studied by dialyzing appropriate fractions against d i s t i 1 led water and examining them in a Phi l i p s 200 electron microscope after negative staining with 2% uranyl acetate (Fig. 6 B and 6C). The main component (M_) consisted of virus particles with diameters of 1300. A showing icosahedral shape (Fig. 6B). The appearance of chloroform treated virus was not s i g n i f i c a n t l y different from that of untreated virus. Many aggregated virus particles were found in the bottom component (B) (Fig. 6C). The view that this component consists of aggregates is supported by i t s formation upon pelleting the main virus component (M_) and repur i f i c a t i o n on a sucrose gradient. The particles in the top component did not reveal any unique sizes or other detectable structural changes but may have a different DNA or protein composition from that of the virus. No further attempt was made to characterize the top component. Figure 6D shows the sedimentation pattern of the Figure 4: Ultracentrifuge patterns of CRSV (2.3 mg/ml), photographs taken 4 and 8 min after reaching 35,600 rpm. 20 Figure 5: Sucrose density gradient fractionation and i n f e c t i v i t y of CRSV. Fractions collected in 0.25 ml amounts and diluted 1/10. Local lesions are the number on 8 half-leaves of C_. amaranticolor per fract i o n . optical density; x x x local lesions. purified main component in the analytical ultracentrifuge. The schlieren optical patterns were v i s i b l e only at concentrations below 0.5 mg virus/ml because of the large light scattering property of this virus. Inoculation into a rabbit, of virus purified in this manner did not induce antibodies to healthy larval extracts. U l t r a v i o l e t Absorbance I. Carnation ringspot virus. The u l t r a v i o l e t spectrum of CRSV (Fig. 7) is ch a r a c t e r i s t i c of nucleoproteins with a maximum at 260 my and a trough at 240 my. The corrected absorbance at 260 my was 14% lower, corresponding to a value of 11% reported for tomato bushy stunt virus (Bonhoeffer and Schachman, 1960). I I. Tjpula i ridescent vi rus. The spectrum of TIV (Fig. 8) is not characteristic of a nucleoprotein since i t gave no trough at 240 my and no peak at 260 my. The li g h t scatterings correction corresponded to 5h% of the observed optical density at 260 my; this is similar to the 66% reported by Day and Mercer (1964) for Sericesthis iridescent virus (SIV), another iridescent virus. The spectrum of TIV is almost identical to that for SIV and the rati o of absorbance of 260 my to 280 my for TIV was 1.24, similar to the value of 1.23 reported for SIV. 22 Figure 6: P u r i f i c a t i o n and components of TIV. A. Photograph of a 5-40% sucrose density showing separation of TIV into (T) top, (M) main and (B) bottom components. B. Electron micrographs of TIV from main component negatively stained with 2% uranyl acetate, magnification 66,400 x. C. TIV from bottom component stained with uranyl acetate, magnification 66,300 x. D. Ultracentrifuge pattern of TIV (0.5 mg/ml) in 0.02 ionic strength Tris-HCl pH 8.1. Photographs taken 10 and 14 min after reaching 6,995 rpm, note the li g h t scattering effect of the sedimenting virus. Figure 7: U l t r a v i o l e t spectrum of CRSV in d i s t i 1 led water. uncovered spectrum; corrected for l i g h t scattering. 24 Figure 8: U l t r a v i o l e t spectrum of TIV in d i s t i l l e d water. uncorrected spectrum; corrected for 1ight scattering. 25 Am i no Ac i d Compos 11 i on I. Carnation ringspot virus. The analytical data from 12-, 2k-, and 72-hr hydrolyzates are presented in Table I. The values obtained from the three hydrolysis times are included in the average except where otherwise indicated. Threonine and serine were calculated by extrapolating the values from the 12-, 2k-, and 72 hr hydrolyzates to zero hydrolysis time (Fig. 9)-. The 72-hr values were used in the average for isoleucine, leucine and valine. The increase of these amino acids with extended hydrolysis agrees with reports of greater s t a b i l i t y of peptide bonds involving these amino acids ( H i l l et a l . , 1959). The increase of h i s t i d i n e with hydrolysis time suggests a peptide bond with one or two of isoleucine, valine or leucine. A decrease in methionine in the 24-hr hydrolyzate was correlated with an increase in ninhydrin reactive components, presumably methionine sulfoxides, eluted from the 60 cm column in a position preceding aspartic'acid. The same components were present to a lesser extent in the 12- and 72-hr hydrolyzates. The value for methionine was corrected for the 5% loss reported by Moore et_al_. (1958). Amino acid analyses of separated virus protein indicated a k% loss in protein during separation from whole virus. The minimal molecular weights of the subunit protein calculated from the percentage composition are presented in Table II. The lowest molecular weight giving the best f i t for the protein was calculated to be 38,000. The average molecular Table I: Amino acid residue recoveries from CRSV protein. Ave r*9Q6 Amino Acid Residues in g/100 g recovered Values Amino Acid 1 0 , 0. , , 12 hr 24 hr 72 hr Lysine d 4.53 4.65 4.70 4.80 4.78 4.76 4.82 4.93 4.86 4.82 H i s t i d i n e b 0.44 0.42 0.46 0.55 0.57 0.57 0.71 0.73 0.71 0.71 Arginine 6.39 6.23 6.39 6.81 6.75 6.94 6.69 6.71 6.52 6.60 Aspartic Acid 10.17 10.29 10.25 10.29 10.25 10.17 10.23 10.23 10.21 10.23 Threonine 3 9.71 9.77 9.81 9.66 9. 69 9.85 9.64 9.43 9.54 9.81 Serine 8.32 8.39 8.41 8.13 8. 07 8.22 7.40 7.38 7.36 8.57 Glutamic Acid 7.55 7.63 7.63 7.80 7. 57 7.65 7.82 7.65 7.82 7.67 Proline 5.01 5.05 4.82 5.41 5. 26 5.26 5.26 5.09 5.22 5.16 Glycine 3.17 3.19 3.19 3.23 3. 14 3.21 3.21 3.14 3.14 3.17 Alani ne 4.53 4.57 4.57 4.63 4. 55 4.61 4.63 4.53 4.51 4.57 V a l i n e C 6.92 7.06 7.11 8.47 8. 28 8.43 9.37 9.48 9.48 9.43 Methionine'5 2.10 2.10 2.08 1.59 1. 61 1.61 2.30 2.30 2.41 2.49 1soleuci ne C 3.73 3.17 3.77 4.09 4. 17 4.15 4.67 4.78 4.82 4.76 Leuci ne C 7.61 7.53 7.53 7.74 7. 57 7.74 7.78 7.74 7.86 7.80 Tyrosine 6.71 6.88 6.88 6.77 6. 77 6.92 7.09 7.21 . 7.40 6.81 Phenylalanine 0' 4.47 4.51 4.47 4.78 4. 76 4.91 4.76 4.72 4.88 4.80 - Average-obtained by extrapolation to zero hydrolysis time; k - In text; - 72-hr values used in average; ^ - 24- and 72-hr values used in average; 6 - 12-hr values used in average. 27 Figure 9 : Plot of hydrolysis time with amino acids for CRSV. o \u00C2\u00B0 threonine; x \u00E2\u0080\u0094 recovered \u00E2\u0080\u0094x serine. weights for al1 amino acids was 37,964 for the protein. The estimated number of residues of each amino acid was calculated on the basis of this average molecular weight. The assumed number of residues for CRSV protein was 347. I I Tipula iridescent virus. The results of the amino acid analyses of virus protein are presented in Table I I I . The values obtained in the 24 hr hydrolysis were used in calculation of the average value unless otherwise indicated. Threonine and serine were calculated by extrapolating the values from the 24- and 72-hr hydrolyzates to zero hydrolysis time. These values compared favourably with the 10.6% loss for serine and 0.8% loss of threonine at 24 hrs observed for carnation ringspot virus protein (Fig. 9). The 72-hr hydrolysis values were used in calculating the average for isoleucine, leucine and valine because of the greater s t a b i l i t y of their peptide bonds. The 24-hr hydrolysis value for methionine was corrected for the 5% loss reported by Moore e_taj_. (1958). A decrease of methionine in the 72-hr hydrolyzate was correlated with an increase in ninhydrin-reactive components, presumably methionine sulfoxides, eluted in a position preceding aspartic acid. Table IV gives the uncorrected average value from a 24-hr hydrolysis of whole virus and the average value obtained by Kawase and Hukuhara . (1967) from an 18-hr hydrolysis of whole virus. The whole virus and virus protein in the analyses are similar except for glycine which is higher for the whole virus; there are also the expected variations in those amino Table I I : Composition and molecular weight of CRSV protein. . . ... Amino Acid Minimal . , Calculated \u00E2\u0080\u009E , , . . Amino Acid . , . . Assumed , , Calculated residue molecular . , molecular . , c . . ^ residues . , ^ residues t'/mn ,\a weight weight (g/100 g recovered) \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Lys i ne 4.82 2 , 6 5 8 14 3 7 , ' 2 1 2 14 .28 + 0 . 1 6 Histidine 0 . 7 1 1 9 , 3 1 0 2 3 8 , 6 2 0 1 . 9 7 + 0.09 Arginine 6.60 2 , 3 6 7 16 3 7 , 8 7 2 1 6 . 0 4 + 0 . 3 6 Aspartic Acid 1 0 . 2 3 1 , 1 2 5 3 4 3 8 , 2 5 0 3 3 . 7 4 + 0 . 0 7 Threoni ne 9.81 1,031 37 3 8 , 1 4 7 3 6 . 8 4 Seri ne 8 . 5 7 1 , 0 1 6 37 3 7 , 5 9 2 3 7 . 3 5 Glutamic Acid 7 . 6 7 1 , 6 3 8 23 3 8 , 7 0 9 2 2 . 5 5 +.0.18 Proline 5 . 1 6 1 , 8 8 2 20 3 7 , 6 4 0 2 0 . 1 7 + 0 . 2 3 Glyci ne 3.17 1 , 8 6 4 20 3 7 , 2 8 0 2 0 . 3 6 + 0 . 1 6 Alanine 4 . 5 7 1 , 5 5 6 24 3 7 , 3 4 4 24 .40 + 0 . 1 2 Cysteine' 3 0.82 1 2 , 5 8 5 3 3 7 , 7 5 5 3 . 0 2 Va1i ne 9 . 4 3 1,051 36 3 7 , 8 3 6 3 6 . 1 3 + 0 . 5 2 Meth ioni ne 2 . 4 9 5 , 2 6 9 7 3 6 , 8 8 3 7 .21 1soleuci ne 4 . 7 6 2 , 3 7 8 16 3 8 , 0 4 8 1 5 . 9 6 + 0 . 3 9 Leuci ne 7.80 1,450 26 3 7 , 7 0 0 2 6 . 1 6 + 0 . 2 0 Tyros i ne 6.81 2 , 3 9 6 16 3 8 , 3 3 6 1 5 . 8 4 + 0 . 4 4 Phenylalanine 4.80 3 , 0 6 7 12 3 6 , 8 0 4 1 2 . 3 8 + 0 . 2 0 Tryptophan'3 .1 .80 1 0 , 3 3 3 4 4 1 , 3 3 2 3 . 6 7 Totals 1 0 0 . 0 2 347 3 7 , 9 6 4 3 4 8 , 0 7 Average - From Table I; k - As described in text; C - Includes t s ~ at P = 0.05. x Table I I I : Amino acid residue recoveries from TIV protein. Amino Acid Residues (g/100 g recovered) Amino Acid 24 hrs 72 hrs Average Values^ Lys i ne 4 . 7 7 4 . 6 3 4 . 6 7 4 . 7 1 4.71 4 , 7 7 4 . 6 9 Histidine 1 . 7 8 1 . 7 4 1 . 7 8 1 . 6 4 1 . 6 4 1 . 6 4 1 . 7 6 Arginine 5 . 4 0 5 . 4 0 5 . 5 2 5 . 3 4 5 . 4 0 5 . 4 0 5 . 4 4 Aspartic Acid 1 1 . 4 3 1 1 . 7 7 1 1 . 6 1 11.71 11.51 1 1 . 6 1 1 1 . 6 1 Threonine 3 6 . 9 0 6 . 8 8 6 . 9 2 6 . 8 4 6 . 8 2 6 . 8 4 6 . 9 4 Serine 7 . 0 2 7 . 1 0 7 . 0 4 6 . 6 3 6 . 4 9 6 . 5 5 7 . 4 0 Glutamic Acid 7 . 4 2 7 . 5 0 7 . 4 6 7 . 5 4 7 . 6 9 7 . 4 4 7 . 4 6 P r o l i ne 6 . 5 7 6 . 6 3 6 . 5 3 6 . 9 0 6 . 9 4 6 . 7 0 6 . 5 7 Glyci ne 4 . 3 7 4 . 3 5 4 . 4 7 4 . 7 1 4 . 6 9 4 . 5 9 4 . 3 9 Alanine 5 . 3 0 5 . 3 2 5 . 4 0 5 . 4 0 5 . 4 0 5 . 2 0 5 . 3 4 Vali ne\u00C2\u00B0 5 . 2 6 5 . 3 8 5 . 3 4 6 . 3 5 6 . 2 7 6 . 1 5 6 . 2 5 Methionine 1 . 8 8 1 . 8 8 1 . 8 8 -trace-- 1 . 9 8 1soleuci ne* 5 . 5 0 5 . 5 4 5 . 6 0 6 .61 6 . 3 9 6 .41 6 . 4 5 . . . b Leucine 7 . 9 5 7.91 8 . 0 9 8 . 3 3 8.29 8.29 8 . 3 0 Tyrosine 5 . 7 0 5 . 8 5 5.81 4.98 5 . 0 6 4 . 9 2 5 . 7 8 Phenylalanine 5 . 8 9 6 .01 5 . 9 5 6 . 0 5 6 .11 5 . 8 9 5 . 9 5 a - Average obtained by extrapolation to zero hydrolysis time and comparison to CRSV losses; k - 72-hr values used in average; C - 24-hr values corrected for 5% loss; ^ - 24-hr values used in calculation of average unless otherwise i nd icated. Table IV: Amino acid TIV vi rus. composition from hydrolysis of whole Amino Acid Residues (g/100 g recovered) Amino Acid Kawase and Hukuhara3 Kalmakoff'3 Lys i ne 6.60 5 . 1 3 Histidine 2 . 8 5 1 . 9 6 Arg i ni ne 9 . 6 5 5 . 9 5 Aspartic Acid 1 0 . 3 5 1 2 . 8 0 Threoni ne 6 . 1 0 6.85 Seri ne 8 . 3 5 9 . 1 7 Glutamic Acid 7-10 8 .11 P ro1ine 7 . 4 5 8 . 1 9 Glyci ne 4 . 9 0 5 . 3 0 Alani ne 5 . 6 0 5 . 8 3 Cystei ne trace trace Vali ne 4 . 8 5 4 . 6 9 Methionine 1 . 4 5 1 . 6 7 1soleuci ne 4 . 7 0 4 . 5 6 Leuci ne 6 . 6 5 7 . 6 6 Tyros i ne 5 . 7 0 6.23 Phenylalanine 5 . 1 5 5 . 4 6 Ammon i a 2 . 7 0 \u00E2\u0080\u0094 Totals 1 0 0 . 1 5 9 9 . 5 6 -Average from 18-hr hydrolysis carried out by Kawase and Hukuhara ( 1 9 6 7 ) . - Uncorrected average from 24-hr hydrolysis of virus in this investigation. Table V: Composition and Molecular weight of TIV protein. Amino acid residue (g/100 g re-covered) 3 M i n i ma 1 mole-cular weight Assumed res idues Calculated molecular weight TIV residues SIV res idues TIV 1 res idues Lys i ne 4.69 2,731 >. 10 27,310 10.3 + 0.6 11.7 11.2 Histidine 1.76 7,790 4 31,160 3.6 + 0.04 4.0 3.9 Arginine 5.44 2,871 10 28,710 9.8 + 0.2 18.1 10.6 Aspartic Acid 11.61 991 28 27,748 28.3 + 0.7 29.2 30.7 Threoni ne 6.94 1,457 19 27,683 19.3 18.5 21.0 Seri ne 7.40 1,177 24 28,248 23,8 26.4 25.8 Glutamic Acid 7.46 1,731 16 27,696 16.2 + 0.2 16.2 17.6 Pr o l i ne 6.57 1,478 19 28,082 19.0 + 0.3 23.9 20.6 Glycine 4.39 1,346 21 28,266 20.8 + 0.5 23.7 22.6 Alanine 5.34 1,331 21 27,951 21 .1 + 0.4 21.9 22.9 r 4. \u00E2\u0080\u00A2 b Cysteine 1.11 9,297 3 27,891 3.0 1.9+ 3.3 Vali ne 6.25 1,586 18 28,548 17.7 + 0.5 19.3 19.2 Meth ioni ne 1.98 6,629 4 26,516 4.2 5.0 4.6 Isoleuci ne 6.45 1,775 16 28,080 16.0 + 0.5 15.5 17.4 Leuci ne 8.30 1,366 20 27,320 20.5 + 0.1 19.7 22.3 Tyros i ne 5.78 2,826 10 28,260 9.9 + 0.2 10.7 10.8 Phenylalani ne 5.95 2,474 11 27,214 11.3 + 0.2 11.0 12.3 Tryptophan 0 2.63 7,072 4 28,288 4.0 \u00E2\u0080\u0094 --Totals 100.05 258 28,054 (Average) 258.8 276.7 276.8 - From Table I; - As described in text; 6 - Amino acid analysis of SIV, in Smith (1967); By performic acid analysis plus 10% loss; Residues calculated from minimum molecular weight of 28,054.. Includes t s x at P = 0.05, (n-1) degrees Gf freedom; Residues calculated from minimum molecular weight of 30,460. a c i d s w h i c h depend on h y d r o l y s i s t i m e . The r e s u l t s o f t h e o t h e r w o r k e r s a r e d i s c u s s e d l a t e r . The minimal m o l e c u l a r w e i g h t s o f t h e s u b u n i t p r o t e i n c a l c u l a t e d from t h e p e r c e n t a g e c o m p o s i t i o n a r e p r e s e n t e d i n T a b l e V. The l o w e s t m o l e c u l a r w e i g h t g i v i n g a good f i t f o r t h e p r o t e i n was c a l c u l a t e d t o be 28,054. T h i s p r o t e i n m o l e c u l a r w e i g h t would be i n error i f two o r more p r o t e i n s p e c i e s were p r e s e n t . C a l c u l a t i o n o f amino a c i d r e s i d u e s based on a minimum m o l e c u l a r w e i g h t . o f 30,460 and t h e amino a c i d a n a l y s i s f o r SIV as p u b l i s h e d i n Smith (1967) a r e i n c l u d e d i n T a b l e V f o r c o m p a r i s o n . N u c l e i c A c i d Compos i t i o n I. C a r n a t i o n r i n g s p o t v i r u s . The c o m p o s i t i o n o f n u c l e o t i d e s r e c o v e r e d from t h e n u c l e i c a c i d o f CRSV were G = 2 5 . 8 5 + 0 . 7 2 % ; A = 2 7 . 2 6 + 0 . 5 5 % ; C = 2 2 . 6 8 + 0 . 2 3 % ; U = 2 4 . 1 8 + 0 . 6 8 % . These r e p r e s e n t average v a l u e s o f f o u r d e t e r m i n a t i o n s showing t h e l a r g e s t d i f f e r e n c e . The c o m p o s i t i o n o f n u c l e i c a c i d i n CRSV c a l c u l a t e d from r e c o v e r e d n u c l e o t i d e s and p r o t e i n was 20.48%. An e x t i n c t i o n c o e f f i c i e n t 2 o f 6.46 cm /mg a t 260 my was c a l c u l a t e d from t h e o p t i c a l d e n s i t y o f t h e p u r i f i e d v i r u s b e f o r e h y d r o l y s i s and t h e y i e l d o f amino a c i d s and n u c l e o t i d e s a f t e r h y d r o l y s i s . I I. T i p u l a i r i d e s c e n t v i r u s . I n o r g a n i c phosphorus l i b e r a t e d by 70% p e r c h l o r i c a c i d was a n a l y z e d from e i g h t samples o f whole v i r u s . The q u a n t i t y o f v i r u s p r o t e i n was d e t e r m i n e d by measuring t h e o p t i c a l d e n s i t y -at 260 my o f t h e whole v i r u s and t h e y i e l d o f amino a c i d s a f t e r hydrolysis of the whole virus. Using the optical density of 2 22.5 cm /mg virus protein, an average value of 0.02395 \u00C2\u00B1 0.0002 g of phosphorus per g of virus protein was obtained. If TIV is composed only of protein and DNA and the average nucleotide residue weight is 309g/mole, the virus contains 19 - 0.2% DNA. Phys i ca1 P rope rt i es I. Carnation ringspot virus. The molecular weight M was determined from the equation: D20. where R = gas constant, T = absolute temperature, V = p a r t i a l s p e c i f i c volume, p = density of water at 20\u00C2\u00B0 C, S^Q and = sedimentation and diffusion coefficients corrected to standard state. Studies of the sedimentation rate at concentrations from 0.2 to 2 mg/ml did not indicate that the sedimentation -13 rate depended on concentration; a value of 132 x 10 sec was obtained in 0.02. ionic strength buffer. Furthermore, there was no detectable difference in sedimentation rate measured at pH 4.1 or at pH 7.6. A diffusion run at 1 mg/ml was made over a period of 5 days. The apparent diffusion coefficients were calculated from the Rayleigh fringes. Since particles with large molecular weights have a considerable time correction, the relationship given by Gosting (1-956) was used: DA = D(1 + At ) (3) a \u00E2\u0080\u0094 where At = time correction, t 1 = observed time, D = apparent 3 35 diffusion c o e f f i c i e n t , P = d i f f u s i o n c o e f f i c i e n t . A plot of D t' vs t' (Fig. 10) gave a linear relationship and D was a calculated from the slope. -7 2 A value of D = 1.48 x 10 cm /sec was obtained after correction to standard state. The p a r t i a l s p e c i f i c volume of CRSV calculated from i t s composition was V = 0.693 ml/gm. The assumption was made that the sedimentation and d i f f u s i o n c o e f f i c i e n t have a similar concentration dependence; the values observed at 1 mg/ml were used. The calculated molecular weight 6 for the virus was M = 7.07 x 10 . The f r i c t i o n a l r a t i o calculated from the hypothetical di f f u s i o n c o e f f i c i e n t of the anhydrous p a r t i c l e and the observed diff u s i o n was 1.16. Assuming the virus p a r t i c l e to be spherical, the degree of hydration was 0.38 water/g of virus. I I. Tipula i ridescent vi rus. A sedimentation c o e f f i c i e n t of S ^ Q - 2147 X 10 ^ sec was obtained in Tris-HCl pH 8.1 at 0.02 ionic strength buffer. This value does not d i f f e r s i g n i f i c a n t l y from the reported value of 2200 S (Bellett and Inman, 1967; Weber e_taj_., 1963). Several di f f u s i o n experiments were done in different buffers since TIV aggregated spontaneously in d i s t i l l e d water (Smith and Williams, 1958; Kl ug e_t: al_. , 1959) and SIV precipitated in phosphate buffer (Day and Mercer, 1964). A d i f f u s i o n experiment was carried out at pH 8.1 in Tris-HCl 0.02 ionic strength buffer over a period of 10 days. A plot of D . t 1 vs t 1 (Fig. 11) gave a 1inear relationship and D was a ..calculated from the slope. JL 4 JL. 8 mi I I I . I I 1 \u00C2\u00BB 12 16 2 0 2 4 2 8 3 2 3 6 4 0 \u00E2\u0080\u00A2f'x 10* S E C O N D S Figure 10: ,Plot of D . t ' vs t' values from di f f u s i o n experiment for CRSV at 4\u00C2\u00B0 C in 0.02 ionic strength acetate buffer pH 4.0 where D is the apparent d i f f u s i o n c o e f f i c i e n t 3 and t ' i s the observed time. Diffusion c o e f f i c i e n t was calculated from the slope. 37 12-I O \u00E2\u0080\u00A2 XlO-t X \0 SECONDS 9 10 Figure 11:,Plot of D . t ' vs t' values from diffusion experiment for TIV carried out at 3.7 P C in 0.02 ionic strength Tris-HCl pH 8.1 where D is the apparent dif f u s i o n c o e f f i c i e n t and t' is the observed time. Diffusion c o e f f i c i e n t was calculated from the slope. A value of D^Q = 3.27 x 10 cm /sec was obtained after correction to standard state. A di f f u s i o n experiment was carried out at pH 7-4 in phosphate buffer 0.02 ionic strength, since a molecular weight for TIV was determined by Weber et a l . , (1-963). in phosphate buffer. A plot of D vs 1/t1 (Fig. 12) shows that the virus aggregated rapidly after 5 days in phosphate buffer and extremely low diffusion coefficients were obtained. Extrapolating the D values obtained in the f i r s t a -8 2 5 days (Fig. 12) gave a value of 1.03.x 10 cm /sec, which -8 2 is s imilar to the diffusion c o e f f i c i e n t of 1.07.x 10 cm /sec calculated from the molecular weight and sedimentation coe f f i c i e n t determined by Weber et a l . (1963) in an 18-day experiment. Using the diffusion c o e f f i c i e n t obtained in Tris-HCl buffer, the molecular weight, M, was calculated from the equation (2). The p a r t i a l s p e c i f i c volume of TIV calculated from i t s composition was V = 0.703. A molecular weight of g 5.51 x 10 was calculated from the data at 0.5 mg virus/ml. A f r i c t i o n a l r a t i o of 1.22 was calculated in a similar manner as for CRSV and a value of 0.57 g water/g virus was obtained for TIV assuming the deviation of the f r i c t i o n a l r a t i o from unity was due to hydration. The hydration.of T-2 bacteriophage which is similar in size to TIV is 0.60 g water/g virus (Lauffer and Bendet, 1954). 39 gure 12: Plot of D vs 1/t' of di f f u s i o n experiment for TIV a 1 carried out at 3.7\u00C2\u00B0 C in 0.02 ionic strength phosphate buffer pH 7.k. Point P_ is the diffusion c o e f f i c i e n t at : 18 days when D for f i r s t 5 days is extrapolated. The downward curvature after 5 days indicates d i f f u s i o n coefficients for aggregates of TIV. DISCUSSION General Remarks Comparison of physical properties of two viruses, which vary in mass by almost two orders of magnitude, is somewhat t r i v i a l . CRSV, being a small v i r u s , lends i t s e l f well to recently developed biophysical methods. However, TIV, having such a large molecular weight, deviates from i d e a l i t y since there is p a r t i c l e -p a r t i c l e interaction and the force of the earth's gravity acts upon suspensions of the virus, in fact, a molecular weight by Weber et a l . (1963) was obtained by sedimentation-equilibrium under the force of gravity. Since most biophysical methods are dependent on optical methods for measurement, TIV presents additional problems due to i t s large 1ight scattering properties, i.e. 54% as compared with 14% for CRSV. Parameters such as hydration and p a r t i a l s p e c i f i c volume of these viruses are similar but this is mainly because these parameters do not vary s i g n i f i c a n t l y with icosahedral viruses. The chemical composition of 20.5% nucleic acid for CRSV and 19% for TIV are s i m i l a r , but when one considers that this 6 represents a single-stranded RNA of molecular weight 1.45 x 10 for CRSV and a double-stranded DNA of molecular weight 8 1.05 x 10 for TIV, this s i m i l a r i t y is fortuitous. The amino acid composition of these two viruses is similar in that there is a r e l a t i v e l y high amount of threonine, serine, isoleucine and leucine and a low amount of cysteine, methionine, tryptophan, and h i s t i d i n e . This pattern of amino acid composition may refle c t some fundamental structural feature of icosahedral viruses and a recent paper by Tremaine and Goldsack (1968) proposes such a hypothesis. I. Carnation ringspot virus. Although CRSV is serologically d i s t i n c t from other icosahedral plant viruses, i t has simi l a r physical and chemical properties. Haselkorn (1966) arranged the plant viruses into s i x groups based on morphology, nucleic acid composition, molecular weight, existence of nucleic acid deficient particles and serology. CRSV does not appear to f i t into any of these groups, since i t does not have \"empties\" ( i . e . nucleic acid deficient particles) and i t has a r e l a t i v e l y high molecular weight. From comparison of electron micrographs of CRSV to cowpea chlorotic mottle virus (Bancroft e_taj_., 1967), i t would appear that CRSV has a 32 capsomere structure and this infers 180 chemical subunits. Taking the protein molecular weight of 5.62 x 10^ for CRSV and dividing by 180, one obtains a protein subunit molecular weight of 31 ,200. However, calculations from the amino acid composition indicates a subunit molecular weight of 38,000. In order to resolve this discrepancy, several attempts were made to obtain protein subunits of CRSV so that a molecular subunit weight could be determined. D i f f i c u l t y arose in obtaining soluble subunits of CRSV, the following methods were t r i e d : the cold s a l t method using 1 M_CaCl_ (Knight, 1963); cold acetic acid method using 67% acetic acid (Fraenkel-Conrat, 1957); the mild a l k a l i method for tobacco mosaic virus (Fraenkel-Conrat and Williams, 1955) and modifications of these methods using mercaptoethanol and 6 M_ urea. In a l l cases, except the 1 M^CaCl^ method, an insoluble gelatinous precipitate was obtained. The 1 M_ CaC^ method did not appear to,affect the virus in any way. Although conditions for the production of subunits were not found, i t is l i k e l y that such conditions do ex i s t . Since the appearance of CRSV in the electron microscope was sim i l a r to cowpea chl o r o t i c mottle virus (CCMV), and the p o s s i b i l i t y that the RNA of CRSV might be susceptible to pancreatic ribonuclease, the system of Bancroft et a l \u00E2\u0080\u00A2 (1967) was t r i e d . There were no observed differences in sedimentation rates at pH 4.0 or pH 8.1 for CRSV, neither did incubation with ribonuclease at 37\u00C2\u00B0 C y i e l d any virus products. Electro-phoresis carried out in Spinco Model H, electrophoresis-diffusion apparatus at pH 6.8 or higher gave anomolous boundary spreading. Further studies on this virus could prove interesting. I I. Tipula iridescent virus. The properties of TIV are in many respects similar to those of other iridescent insect viruses, indicating that a l l the iridescent viruses may be strains of a single virus species. Due to the wide geographic d i s t r i b u t i o n of the discoveries of the i s o l a t e s , and the wide experimental host range, i t would be surprising i f many more isolates of iridescent viruses are not reported. An objection to the single virus theory is the wide variation in reported sizes, from 1300-1800 A. This does not present so formidable an obstacle as i t would seem, since Hosaka (1965) pointed out that the diameter of the outline of an icosahedron can range from 0.851 to 1, depending on the orientation of the p a r t i c l e , and whether the measurements are maximum or minimum diameters. Moreover, Smith et a l . (1961), in t h e i r studies on the inoculation of TIV into different hosts, suggested that there might be a s l i g h t variation in p a r t i c l e size with host species. These factors and different techniques in electron microscopy could account for the apparent variabi 1ity in the size of the iridescent viruses. The s i m i l a r i t y of amino acid composition for TIV and SIV (Table V) strongly suggests a st r a i n relationship. If the residues are calculated on the basis of a minimum molecular weight of 30,460, the values for a l l ami no acids, with:the exception of arginine, are within experimental error. The higher arginine in SIV may re f l e c t a host modification of the v i r u s , since the moth larva, G a l l e r i a mellonella (L), was used for virus passage (Smith, 196?). There is some precedent for host modification of viruses from work on bacteriophages as reviewed by Arber (1965). The data of Kawase and Hukuhara (1967), although not so r e l i a b l e , show a s i m i l a r i t y of composition between their TIV and the virus used in this investigation (Table IV). Their virus had a content of arginine s i m i l a r to that in SIV and higher than in this investigation. It is interesting that the host modification concept is supported by the results of Kawase and Hukuhara (1967), since they also used G a l l e r i a mel lone! la (L) as host for TIV. It is l i k e l y that there was a host selection of strains already present in the TIV inoculum, rather than host modification. Various estimates of DNA content for iridescent viruses have been reported: for TIV, Smith (1958) reported 15%; Thomas (1961) reported 12.4%, and Al1ison and Burke (1962). reported 16%, while for SIV, Day and Mercer (1964) found 17.6%. Any estimation of DNA content depends d i r e c t l y on the accuracy of virus or protein weight determination. Since extraction with chloroform for TIV or with chloroform and ether for SIV (Day and Mercer, 1964) did not a l t e r these viruses, the 5.2% l i p i d reported in TIV by Thomas (1961) was probably a host contaminant and would have affected his reported DNA percentage. The estimate of 19 + 0.2% DNA based on amino acid content should be v a l i d because Thomas (1961), after extensive tests, did not detect major components other than DNA and protein. Recently B e l l e t t and Inman (1967) studied DNA isolated from CIV, SIV, and TIV and found that DNA preparations from these viruses had sedimentation coefficients of about 62 S 8 and molecular weights of 1.30 x 10 . Using the molecular 8 weight of 5.51 x 10 for TIV, a value of 23% DNA is obtained. Considering that the molecular weights of DNA were calculated using an empirical formula, 23% DNA is in good agreement with the chemical estimation o f 1 9 % DNA. 8 The molecular weight reported here of 5-51 x 10 is 8 lower than the values of 10.5 x 10 (Weber et a l . , 1963) and 12.2 x 10 (Thomas, 1961). If we assume the particles are spherical, the hydrodynamic diameter can be calculated from the molecular weight and f r i c t i o n a l coefficient (Schachman, 1959) ... Nfs M = - \u00E2\u0080\u0094 ' (4) (1 \" Vp) f = 6 n n r (5) where N = Avogadro's number, f = f r i c t i o n a l c o e f f i c i e n t , n = v i s c o s i t y of water at 20\u00C2\u00B0 C, r = hydrated radius of the p a r t i c l e . The other symbols were previously described. Hydrated diameters of 1310 A, 2500 A and 2910 A were 8 8 calculated from molecular weights of 5.5 x 10 , 10.5 x 16.'., 8 and 12.2 x 10.., respectively. The value of 1310 A is s l i g h t l y greater than 1300 A, the diameter established for the frozen-dried p a r t i c l e by electron microscopy (Klug,'et'al., 1959). The value of 1310 A is reasonable, since Harrison and Klug (1966) pointed out that much of the water of hydration probably occupies the depressions on the surface of virus p a r t i c l e s . If the frozen-dried diameter of 1300 A is taken as the anhydrous diameter, the diameters of 2500 A and 2910 A, -8 8 representing molecular weights of 10.5 x 10\" and 12.2 x 10.., would give hydration values of 4.3 g water/g virus and 7.2 g water/g v i r u s , respectively. Since most proteins have a total hydration not greater than about 1 g water/g protein, the large hydration values obtained are improbable and hence the molecular weights used in the i r calculation are also improbable. Considering that many of the properties of CIV, MIV, SIV and TIV are s i m i l a r , i f not i d e n t i c a l , the molecular weight 8 of 5.51 x 10 for TIV is probably a good estimate of the molecular weight of these other iridescent viruses. 47 LITERATURE CITED 1. A l l i s o n , A.G., and Burke, D.G. 1962. The nucleic acid content of viruses. J. Gen. Microbiol. 27_: 181-194. 2. Arber, W. 1965. Host-controlled modification of bacterio-phage. Ann. Rev. Microbiol. ]S_: 3 6 5 - 3 7 8 . 3 . 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A p r e c i p i t i n ring time for estimation of re l a t i v e soluble-antigen concentrations. Virology 15: 507 -508. 44. Xeros, N. 1954. A second virus disease of the leather-jacket , Xi\u00C2\u00A3ul^ P^JjJdosa_, Nature 174: 562. "@en . "Thesis/Dissertation"@en . "10.14288/1.0093629"@en . "eng"@en . "Microbiology and Immunology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Some physico-chemical studies on two icosahedral viruses"@en . "Text"@en . "http://hdl.handle.net/2429/36747"@en .