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Isolation and partial characterization of a virus-like particle from a eucaryotic alga Cole, Anabel 1978

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ISOLATION AND PARTIAL CHARACTERIZATION OF A V I R U S - L I K E PARTICLE FROM A EUCARYOTIC ALGA by ANABEL COLE B.A., Reed C o l l e g e , 1973 A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA J u n e , 1978-| j . A n a b e l C o l e , 197 8 In presenting th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representa t ives . It is understood that copying or pub l ica t ion of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of Plant Science The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 19 May 1978 ABSTRACT Large i c o s a h e d r a l v i r u s - l i k e p a r t i c l e s (VLP's) have been i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d from a e u c a r y o t i c freshwater green al g a . The a l g a l host, Uronema gicras. was reported to contain VLP's i n an e a r l i e r u l t r a s t r u c t u r a l study. Subcultures of t h i s alga were found t o r e l e a s e VLP's i n t o the l i q u i d medium i n which the alga was grown, from which the VLP's were p u r i f i e d without homogenization of i n t a c t c e l l s . The VLP's i n s i t u w i t h i n i n f e c t e d c e l l s , or when p u r i f i e d and f i x e d i n glutaraldehyde, have a diameter of approximately 400 nm. Sectioned VLP's e x h i b i t an e x t e r n a l membrane-like s h e l l e n c l o s i n g some regions of d e n s e l y - s t a i n i n g , homogeneous m a t e r i a l and other regions of f i b r i l l a r m a t e r i a l . About 10% of the p a r t i c l e s are t a i l e d , the t a i l s measure up to one micron i n length and are attached to one vertex of the icosahedron. The development of the VLP's appears to i n v o l v e the nucleus, and a l g a l c e l l s show extensive d i s o r g a n i z a t i o n of a l l c e l l u l a r membrane systems as the formation of the VLP's progresses. T a i l e d p a r t i c l e s have been observed forming w i t h i n a l g a l c e l l s . The VLP's are never rele a s e d i n high c o n c e n t r a t i o n s , so a l l biochemical c h a r a c t e r i z a t i o n s have been hampered by the l i m i t e d amount of m a t e r i a l . P u r i f i e d VLP's from U.. gigas have a d e n s i t y of approximately 1.32 g/ml i n sucrose, a sedimentation c o e f f i c i e n t of 6300 S, and are h i g h l y l i g h t s c a t t e r i n g . The polypeptides from the VLP's were analyzed by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . Ten p r o t e i n bands were detected; the major species had a molecular weight of 45,000 d a l t o n s . The n u c l e i c a c i d contained w i t h i n the i n t a c t VLP was determined to be double-stranded DNA by the f o l l o w i n g methods: a c r i d i n e orange s t a i n i n g , diphenylamine and o r c i n o l t e s t s , and s p e c i f i c enzyme d i g e s t i o n s of VLP p e l l e t s prepared f o r e l e c t r o n microscopy. P u r i f i e d DNA from the VLP 1s was found to have a buoyant d e n s i t y of 1.719 g/ml i n cesium c h l o r i d e , corresponding to a molar f r a c t i o n of c y t o s i n e plus guanine of about 60%. The DNA appeared to be l i n e a r and double-stranded using e l e c t r o n m i c r o s c o p i c a l techniques. Length measurements of the DNA were v a r i a b l e , r e p r e s e n t i n g 6 6 DNA molecular weights of 8 x 10 to 72 x 10 d a l t o n s , although the accuracy of the technique was confirmed w i t h DNA's of known lengths. A l g a l germlings seemed to be the most s u s c e p t i b l e stage i n the l i f e c y c l e of U. gigas f o r synthesis and r e l e a s e of VLP's. No p a r t i c l e s were observed i n t h i n s e c t i o n s of elongated c e l l s of the mature f i l a m e n t . Scanning e l e c t r o n m i c r o s c o p i c a l views of la r g e germling populations showed that a c e r t a i n percentage of the c e l l s contained opaque spheres of a diameter s i m i l a r to the VLP, and by x-ray m i c r o a n a l y s i s these spheres were found to contain more phosphorus than the surround-i n g a l g a l cytoplasm. A heat shock of 38 C administered i n the dark, during the p e r i o d of zoospore s e t t l i n g , seemed to g r e a t l y increase the y i e l d of VLP' s. Every c u l t u r e of TJ. gigas examined contained VLP's; a demonstration of the i n f e c t i v i t y of t h i s VLP has not been p o s s i b l e without a healthy c u l t u r e . - S t r u c t u r a l and biochemical aspects of the VLP from U. gigas are unusual. The p a r t i c l e does not appear to be r e l a t e d to any e x i s t i n g group of v i r u s e s , i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES ...v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS X LITERATURE REVIEW 1 MATERIALS AND METHODS 17 Uronema gigas — sources and culture techniques 17 Chlorophyll ,a_ determination 18 Virus p u r i f i c a t i o n 20 I. Uronema gigas VLP 20 II. Tipula i r i d e s c e n t virus 22 II I . Cauliflower mosaic virus 23 Electron microscopy 24 Scanning electron microscopy 27 Light scattering properties of UGV 28 Sedimentation c o e f f i c i e n t of UGV 29 Measurements of UGV dimensions 29 Acridine orange staining..,' 30 Diphenylamine and o r c i n o l tests 31 Protein electrophoresis 32 Lipids 34 DNA i s o l a t i o n and p u r i f i c a t i o n 36 Buoyant density determinations 37 Electron microscopy of DNA 39 Radioisotope l a b e l l i n g of UGV nucleic acid 40 Heat shock experiments 42 V Page RESULTS 44 I . I n t e r a c t i o n o f UGV w i t h Uronema g i g a s 44 A. M o r p h o l o g y o f UGV and i t s h o s t a l g a 4 4 B. C y t o p a t h o l o g y 48 C. S c a n n i n g e l e c t r o n m i c r o s c o p y o f g e r m l i n g s 50 I I . I s o l a t i o n and p u r i f i c a t i o n o f UGV 54 A. Growth o f t h e h o s t a l g a 54 B. P u r i f i c a t i o n o f t h e VLP ' s 55 C. H e a t s h o c k e f f e c t s on VLP y i e l d 63 I I I . P a r t i a l c h a r a c t e r i z a t i o n o f UGV and i t s components 66 A. D i m e n s i o n s o f UGV 6 6 B. E l e c t r o n m i c r o s c o p y and enzyme d i g e s t i o n o f p e l l e t e d VLP ' s 6 8 C. L i g h t s c a t t e r i n g c o r r e c t i o n 73 D. S e d i m e n t a t i o n c o e f f i c i e n t 75 E. D e n s i t y i n s u c r o s e o f UGV 75 F. UGV p r o t e i n s 78 G. N u c l e i c a c i d c h a r a c t e r i z a t i o n 81 H. N u c l e i c a c i d p u r i f i c a t i o n 83 I . B u o y a n t d e n s i t y o f UGV-DNA 84 J . N u c l e i c a c i d v i s u a l i z a t i o n by t h e K l e i n s c h m i d t t e c h n i q u e 87 K. R a d i o i s o t o p e l a b e l l i n g o f UGV-DNA........ 91 DISCUSSION 96 I . I n t r o d u c t i o n . . 96 v i Page II. Morphology of the p a r t i c l e 96 II I . I n t r a c e l l u l a r development 102 IV. P u r i f i c a t i o n and characterization 106 V. B i o l o g i c a l considerations 117 LITERATURE CITED 122 - v i i -LIST OF TABLES TABLE PAGE I Reported v i r u s - l i k e p a r t i c l e s i n eucaryotic algae. 5 II Characteristics of single c e l l i s o l a t e s of Uronema gigas 13 III Composition of Beijerinck's mineral medium 19 IV Electron microscopic counting technique for assay of VLP concentration 56 V Release of UGV p a r t i c l e s i n r e l a t i o n to a l g a l growth (measured as chlorophyll a content per ml of culture medium) 56 VI Comparison of concentration and p u r i f i c a t i o n methods of UGV 5 8 VII C e l l disruption methods assayed for JJ. gigas 64 VIII Heat shock ef f e c t s on UGV release 64 IX Measurements of the UGV p a r t i c l e 67 X Sedimentation c o e f f i c i e n t of UGV 77 XI UGV polypeptides detected by SDS-polyacrylamide gel electrophoresis 77 XII Nucleic acid colorimetric tests 82 XIII Buoyant density of UGV-DNA 86 XIV Guanine plus cytosine molar f r a c t i o n of UGV-DNA.. 86 XV Characterization of UGV nucleic acid 109 XVI Comparison of the properties of ICDV's, cyanophages, and UGV , 116 - v i i i -LIST OF FIGURES FIGURE PAGE 1 Thin sections of healthy and VLP-containing c e l l s examined by transmission electron microscopy 45 2 P u r i f i e d UGV p a r t i c l e s negatively stained, examined by transmission electron microscopy. Size comparisons with TMV and TIV 46 3 • Thin sections of UGV p a r t i c l e s ; t a i l e d UGV p a r t i c l e s within c e l l s ; early stages of i n f e c t i o n with UGV 47 4 Scanning electron microscopy of U_. gigas germlings; x-ray microanalysis of a l g a l cytoplasm and possible UGV p a r t i c l e s 52 5 Growth of U. gigas and release of UGV p a r t i c l e s i n two culture media, Beijerinck's and B r i s t o l ' s . 53 6 F i n a l p u r i f i c a t i o n procedure for UGV p a r t i c l e s . . . 60 7 Sucrose density gradient centrifugation of UGV and TIV ' 6 2 8 Enzyme digestion treatments of embedded UGV p a r t i c l e s : pronase, RNAse, and DNAse 7 0 9 Sectioned p e l l e t s of UGV p a r t i c l e s , pre-treated with organic solvents 72 10 Light scattering correction curve for one preparation of UGV p a r t i c l e s 7 4 11 Equilibrium centrifugation of UGV, TIV, and BSMV in sucrose 76 12 Polyacrylamide gel electrophoresis of UGV polypeptides 7 9 - i x -FIGURE PAGE 13 Microdensitometer scan of gel shown i n Figure 12 80 14 Equilibrium centrifugation of UGWDNA with marker DNA's i n cesium chloride and cesium sulfate 85 15 Molecules of DNA from UGV and CaMV vi s u a l i z e d by the Kleinschmidt technique 8 9 16 D i s t r i b u t i o n of lengths of DNA molecules measured of UGV-DNA and CaMV-DNA, vi s u a l i z e d by the Kl e i n -schmidt technique 9 0 17 Attempted radioisotope l a b e l l i n g of UGV nucleic acid 94 -x-ACKNOWLEDGEMENTS I would l i k e to express my g r a t i t u d e to the f o l l o w i n g people who have given me advice and help i n the pr e p a r a t i o n of t h i s t h e s i s . Dr. J . A. Dodds, who i n i t i a t e d t h i s research p r o j e c t and generously shared i t w i t h me, has been a co n t i n u i n g source of i n t e l l e c t u a l s t i m u l a t i o n . I am very g r a t e f u l to Dr. Marvin Weintraub, D i r e c t o r , A g r i c u l t u r e Canada Research S t a t i o n , Vancouver, f o r the use of the e x c e l l e n t f a c i l i t i e s at h i s s t a t i o n and f o r h i s i n t e r e s t i n my progress. Many members of the s t a f f of the A g r i c u l t u r e Canada Research.Station, Vancouver, have helped me, g i v i n g of t h e i r time and e x p e r t i s e ; i n p a r t i c u l a r , Bea Schroeder, B i l l Ronald and Connie N i c h o l s i n s t r u c t e d me i n e l e c t r o n microscopy, a n a l y t i c a l u l t r a c e n t r i f u g a t i o n , and g e l e l e c t r o p h o r e s i s . I am e s p e c i a l l y indebted to Wes MacDiarmid, the Research S t a t i o n photographer, f o r h i s help w i t h the f i n a l photographs. The members of my graduate committee have a l l c o n t r i b u t e d h e l p f u l ideas over the course of t h i s t h e s i s . I would l i k e to thank Dr. A.J. Warren, Department of M i c r o b i o l o g y , U n i v e r s i t y of B r i t i s h Columbia (U.B.C.), Dr. Beverly Green, Department of Botany, U.B.C., Dr. J.H, Tremaine, A g r i c u l t u r e Canada Research S t a t i o n , Vancouver and Dr. V.C. Runeckles, Department of P l a n t Science, U.B.C., f o r t h e i r advice. I am e s p e c i a l l y g r a t e f u l to Dr. R.I, Hamilton, my su p e r v i s o r , f o r h i s con t i n u i n g guidance, encouragement, and support over the three years I have spent i n h i s l a b . - x i -I have been f i n a n c i a l l y of t h i s project by a Postgraduate Research Council of Canada, which supported for the duration Fellowship from the National i s also g r a t e f u l l y acknowledged. -1-LITERATURE REVIEW V i r a l i n f e c t i o n s or accumulations of v i r u s - l i k e p a r t i c l e s have been reported i n a l l forms of l i f e , p r o c a r y o t i c and e u c a r y o t i c . Many bacteriophages and v i r u s e s of higher p l a n t s and animals have been i s o l a t e d and e x t e n s i v e l y c h a r a c t e r i z e d , the research i n i t i a l l y motivated by the need to c o n t r o l diseases a f f e c t i n g humans and domesticated animals and p l a n t s . Taxonomic groups i n which few v i r a l i n f e c t i o n s have been reported, such as the algae, f u n g i , and protozoa, probably f u n c t i o n as hosts to as many v i r u s e s as the more i n t e n s i v e l y s t u d i e d groups, but they simply have not been the focus of as much research e f f o r t . This t h e s i s describes the i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of a new v i r u s - l i k e p a r t i c l e (VLP) from a e u c a r y o t i c a l g a , Uronema gigas. Cyanophages--the v i r u s e s of p r o c a r y o t i c blue-green algae--were discovered i n 1963 w i t h the i d e n t i f i c a t i o n of LPP-1 v i r u s from a waste s t a b i l i z a t i o n pond i n Indiana (Safferman and Morris 1963). Understandably, sewage treatment ponds provided the f i r s t examples of cyanophages since they all o w f a s t and continuous growth of the b a c t e r i a l hosts to pop u l a t i o n d e n s i t i e s which magnify f l u c t u a t i o n s . In the years since the f i r s t i s o l a t i o n at l e a s t four d i f f e r e n t types of cyanophages have been c h a r a c t e r i z e d , and extensive research has been done concerning the v i r a l i n f e c t i o n c y c l e , e f f e c t s on host metabolism, and e c o l o g i c a l i n t e r a c t i o n s . E x c e l l e n t reviews of -2-cyanophage research have been published recently (Brown 1972, Padan and Shilo 1973, and Safferman 1973) and progress i n understanding these viruses has been rapid (Safferman and Morris 1977). Cyanophages are similar to other bacteriophages in structure and biochemistry, as one would expect considering the s i m i l a r i t i e s of t h e i r procaryotic hosts. One group, the LPP cyanophages, includes p a r t i c l e s which are polyhedral, 60 nm in diameter, with a short non-contractile t a i l , resembling phages T7 and T3. A second group (SM-1 cyanophages) contains icosahedral p a r t i c l e s , about 67 nm i n diameter with a short c o l l a r and a thin appendage. The N-l group, similar to type A bacteriophages (Bradley 1967) , was o r i g i n a l l y i s o l a t e d from the filamentous Nostoc muscorum, and has an icosahedral capsid with a diameter of 55 nm and a t a i l of about 110 nm attached to one vertex of the head (Adolph and Haselkorn 1971). The largest cyanophage discovered thus far i s AS-1, from Anacystis nidulans and Synechococcus cedrorum, with an icosahedral head 9 0 nm i n diameter and a c o n t r a c t i l e t a i l , approximately 245 nm long by 23 nm wide (Safferman et a l . 1972). A l l four groups contain lin e a r double-stranded deoxyribonucleic acid (DNA), of molecular g weights between 27 and 62 x 10 daltons (Padan and Shilo 1973). The SM-1 group of cyanophages has a complete dependence on the continuing photosynthesis of the host c e l l during the i n f e c t i o n cycle. Cyanophages of the N-l and LPP groups have no such requirement for host photosynthesis, moreover, both of these phage types cause extensive disruption of the host's photosynthetic lamellae, early i n the i n f e c t i o n cycle, l i m i t i n g - 3 -host p h o t o s y n t h e s i s (Sherman and Haselkorn 1970, Padan and S h i l o 1 9 7 3 ) . The p h y s i o l o g y of AS-1 i n f e c t i o n s has not been c h a r a c t e r i z e d . The e x i s t e n c e of v i r u s e s which i n f e c t the e u c a r y o t i c algae was p o s t u l a t e d i n 1958 by Z a v a r z i n a and Protsenko, who observed l y s i s of C h l o r e l l a pyrenoidosa (Zavarzina 1961, 1 9 6 4 ) . F u r t h e r i n v e s t i g a t i o n of the i n f e c t e d C h l o r e l l a c u l t u r e s suggested t h a t a phage which a t t a c k s a p r o c a r y o t i c c e l l w a l l symbiont of the a l g a may be r e s p o n s i b l e f o r l y s i s (Mamkaeva 1966, Tikhonenko and Z a v a r z i n a 1966, Gromov and Mamkaeva 1 9 7 2 ) , but the p o s s i b i l i t y s t i l l e x i s t s t h a t the observed v i r u s - l i k e p a r t i c l e can i n f e c t the a l g a l c e l l (Lemke 19 7 6 ) . No f u r t h e r d e s c r i p t i o n of v i r u s -l i k e p a r t i c l e s a s s o c i a t e d w i t h higher algae appeared u n t i l the e a r l y 1970's. Since 1971 a t l e a s t t h i r t e e n r e p o r t s have been p u b l i s h e d d e s c r i b i n g v i r u s - l i k e i n c l u s i o n s i n a l g a l c e l l s ; a summary of the r e p o r t s i s presented i n Table 1. A l l s t u d i e s have d e a l t with the u l t r a s t r u c t u r a l changes i n i n f e c t e d c e l l s o n l y ; to date no p u b l i c a t i o n has d e s c r i b e d s u c c e s s f u l i s o l a t i o n and p u r i f i c a t i o n of the p o l y h e d r a l VLP's. (An e x c e p t i o n a l i n s t a n c e of a t u b u l a r p a r t i c l e t h a t has been c h a r a c t e r i z e d i s d i s c u s s e d s e p a r a t e l y . ) A c c o r d i n g to Safferman and M o r r i s ( 1 9 7 7 ) , i n comparison wi t h e a r l y work on cyanophages, i s o l a t i o n of e u c a r y o t i c a l g a l v i r u s e s has presented "a formidable o b s t a c l e to r e s e a r c h e r s . " In a l l cases the d i s r u p t i o n of c e l l u l a r o r g a n i z a t i o n observed by e l e c t r o n microscopy c o n s t i t u t e s the e n t i r e evidence f o r the p a t h o g e n i c i t y -4-of these p a r t i c l e s . Deviations from normal c e l l u l t r a s t r u c t u r e are always observed i n c e l l s which contain VLP's, so the p a r t i c l e s appear to cause a c y t o p a t h o l o g i c a l c o n d i t i o n , but without proof of i n f e c t i v i t y the pathogenic e f f e c t s of VLP synthesis w i t h i n the a l g a l c e l l s remain presumptive. Throughout t h i s t h e s i s the p a r t i c l e s s t u d i e d w i l l be r e f e r r e d to as VLP's, to emphasize the lack of i n f o r m a t i o n about p a t h o g e n i c i t y . When the p a r t i c l e studied i s r e f e r r e d to as UGV (Uronema gigas v i r u s ) , i t i s simply to r e f e r s p e c i f i c a l l y to the host a l g a , r a t h e r than to imply t h a t the true v i r a l nature of t h i s VLP has been demonstrated. The host algae l i s t e d i n Table I- are from d i v e r s e taxonomic groups re p r e s e n t i n g the green, brown and red algae, and s e v e r a l p l a n k t o n i c and u n i c e l l u l a r f l a g e l l a t e s , from both marine and freshwater environments. A l l but one of the VLP's described are p o l y h e d r a l ; p a r t i c l e o u t l i n e i n cross s e c t i o n i s hexagonal or pentagonal, suggesting the 5-3-2 symmetry of an icosahedron (Home 197 4). P a r t i c l e diameters range from 25 nm (Manton and Leadbeater 197 4) to 3 00 nm (Mattox et al_. 1972) , w i t h p o s s i b l e c l u s t e r s at 50-60 nm (Lee 1971, Chapman and Lang 1973, Pearson and N o r r i s s 1974, and Pienaar 1976), 100 nm (Pienaar 1976), 130-170 nm (Toth and Wilce 1972, C l i t h e r o e and Evans 1974, Markey 1974, Moestrup and Thomsen 1974, and Pienaar 1976), and 200-240 nm (Pickett-^Heaps 1972, Swale and Belcher 1973, Hoffman and Stanker 1975). Size measurements are approximate since d i f f e r e n t authors have used TABLE I -- Reported v i r u s - l i k e p a r t i c l e s i n eucaryotic algae Al g a l host Sirodotia  tenuissima Uronema gigas C l a s s i f i c a t i o n red alga, freshwater green algae, StigeocIonium sp. freshwater Coleochaete scutata  Radiofilum sp. Oedogonium sp, green alga, freshwater Chorda tomentosa brown alga, marine Porphyridium  purpureum Aulacomonas SP. red alga, marine colorless f l a g e l l a t e , green alga r e l a t i v e P a r t i c l e Shape Size poly-hedral poiy~ hedral poly-hedral poly-hedral poly-hedral poly-hedral with t a i l 50-60 nm not stated, 50-300 nm from micro -graphs. 240 nm 17 0 nm 40 nm 200-230 150-200 C e l l type infected and cytopathology caused ___ Apical c e l l only; nucleus and cytoplasmic membrances degenerate. Disorganized nucleus and cytoplasmic degeneration; c e l l types not s p e c i f i e d . Germlings only; cytoplasm and organelles disrupted. S e t t l i n g spores; general organellar degeneration. Tubular nuclear inclusions; general organellar degeneration. Loss of nuclear membrane; disorganized organelles. Reference Lee 1971 Mattox et a l 1972 Pickett-Heap 1972 Toth and Wil 1972 Chapman 197 2 1973; Chapman and Lang 1973 Swale and Belcher 1973 A l g a l host Ectocarpus  fasciculatus C l a s s i f i c a t i o n brown alga, marine P a r t i c l e Shape Size poly- 17 0 nm hedral Chrysochromulina  mantoniae P y l a i e l l a l i t t o r a l i s phytoplankton brown alga, marine poly- 22 nm hedral poly- 130-170 hedral Pyramimonas  o r i e n t a l i s Platymonas sp. f l a g e l l a t e , green alga green alga, marine poly- 160-170 hedral poly- 56-58 hedral Chara c o r a l l i n a green alga tubular 53 2 nm Hymenomonas car- phytoplankton terae Micromonas p u s i l l a e  Cryptomonas sp. poly- 65 nm hedral poly- 13 0 nm hedral poly- 100 nm hedral C e l l type.infected and cytopathology caused  Sporangial c e l l s only; loss of nuclear membrane, VLP's and tubular inclusions i n nucleus. Disorganized nucleus. Young sporangia only; nuclear disorganisation and tubular inclusions; degenerating organelles. Loss of nuclear Moestrup and membrane. Thomsen 197 4. VLP's i n nucleus only; Pearson and cytoplasmic Norriss 1974. degeneration. Apical c e l l s ; Gibbs e_t a l . cytoplasmic inclusions 1975. of v i r u s . Degenerating nucleus, Pienaar 1976. VLP's intranuclear; cytomplasmic accumulation. Reference Clitheroe and Evans 1974. Manton and Leadbeater 1974. Markey 1974. VLP's intranuclear. A l g a l host Cylindrocapsa  geminella C l a s s i f i c a t i o n green alga, freshwater P a r t i c l e Shape Size poly-hedral 200-230 C e l l type infected and cytopathology caused  Loss of nuclear membrane; germlings only. Other VLP's of similar morphology i n fungi and protozoans Aphelidium sp. Paramoebidium  arcaturn Naegleria gruberi Entamoeba  h i s t o l y t i c a phycomycete, green al g a l parasite. trichomycete, arthropod parasite. amoebo-f l a g e l l a t e amoeba poly-hedral 2 00 nm poly- 105-110 hedral poly-hedral poly-hedral 100 nm 70-75 Inside lys i n g fungal protoplasts; cytoplasmic accumu-l a t i o n . Limited to nucleus. Nuclear o r i g i n ; transport to cytoplasm. Development i n perinuclear cytoplasm. Reference Hoffman and Stanker 1975. Schnepf et a l 1970. Manier et a l . 1971. Schuster and Dunnebacke, 1971. Mattern .et a l . 1972, 1974. i i -8-v a r i o u s f i x a t i o n and s t a i n i n g procedures and in c l u d e d i f f e r e n t i n t e r n a l s i z e markers. The only t a i l e d p a r t i c l e was reported i n the f l a g e l l a t e Aulacomonas (Swale and Belcher 1973). D e s c r i p t i o n s of the VLP's, e s p e c i a l l y those over 50 nm, are remarkably c o n s i s t e n t . E l e c t r o n micrographs show a r i g i d outer s h e l l of recognizably p o l y h e d r a l o u t l i n e , 5- or 6-sided, surrounding a dense core of h e a v i l y s t a i n i n g m a t e r i a l , which may completely f i l l the i n t e r i o r of the s h e l l or occupy only a p a r t of the i n t e r n a l space. The shell has a t r i - l a m i n a t e appearance, very s i m i l a r to the c l a s s i c a l u n i t membrane i n c e l l s . In a d d i t i o n to the electron-dense core r e g i o n s , f i b r i l l a r m a t e r i a l i s o f t e n seen i n "emptier" regions i n t h i n s e c t i o n s of the VLP's. The p a r t i c l e s are u s u a l l y found i n a p i c a l c e l l s of f i l a m e n t s , i n c l u d i n g t e r m i n a l sporangia, or i n zoospores or young germlings. No repor t s of VLP's from mature elongated c e l l s of any alga have appeared; perhaps r e f l e c t i n g the mode of tran s m i s s i o n of the e u c a r y o t i c a l g a l v i r u s e s (Andrews 1976). I f the VLP's occur as e x t r a c e l l u l a r i n f e c t i o u s agents, adsorption or p i n o c y t o s i s or entrance through wounds would be most l i k e l y i n the zoospore which does not have a c e l l w a l l . Sporangial and a p i c a l c e l l i n f e c t i o n s a l s o suggest a tra n s m i s s i o n p a t t e r n s i m i l a r to seed transm i s s i o n i n higher p l a n t s : c e l l s adapted f o r d i s p e r s a l might be most l i k e l y to become i n f e c t e d , p r o v i d i n g a means of spreading the i n f e c t i o n along w i t h a l g a l growth. I n t r a c e l l u l a r development of the VLP's appears to in v o l v e the nucleus and the cytoplasm. V i r u s - l i k e p a r t i c l e s -9-u s u a l l y accumulate i n the cytoplasm, but a nuclear r o l e i n the r e p l i c a t i o n c y c l e i s always i m p l i e d : by a cytoplasmic s i t e of VLP assembly at the periphery of the nucleus; by the frequent observation of the l o s s of the nuclear membrane; and by the presence of membranous i n c l u s i o n s i n the n u c l e i of s e v e r a l of the i n f e c t e d algae ( P y l a i e l l a , Ectocarpus, Porphyridium). The nuclear i n c l u s i o n s appear to be long c y l i n d e r s , of a diameter i d e n t i c a l to th a t of the VLP's, and o c c a s i o n a l l y w i t h p o l y h e d r a l ends of a s i z e and appearance s i m i l a r to the VLP's ( C l i t h e r o e and Evans 1974). An e x c e p t i o n a l v i r u s of a e u c a r y o t i c a l g a i s the 532 nm tu b u l a r p a r t i c l e i s o l a t e d from Chara c o r a l l i n a (Gibbs et a l . 1975 and S k o t n i c k i e_t a l . 1976). I n f e c t i v i t y has been demonstrated f o r t h i s v i r u s , and the p u r i f i e d p a r t i c l e has been e x t e n s i v e l y c h a r a c t e r i z e d . The p a r t i c l e i s s e r o l o g i c a l l y r e l a t e d to tobacco mosaic v i r u s . This v i r u s i s unusual among the eu c a r y o t i c a l g a l VLP's i n i t s shape and cl o s e r e l a t i o n s h i p to the v i r u s e s of higher p l a n t s , perhaps r e f l e c t i n g the r e l a t i v e complexity of the host alga Chara as compared to the lower algae. Four other r e p o r t s of VLP's are included i n Table 1, one from a phycomycete p a r a s i t e on a green a l g a l host (Schnepf _et a l . 1970), one of a trichomycete p a r a s i t e of an arthropod (Manier est a l . 1971) , and two from amoebae (Schuster and Dunnebacke 1971, and Mattern et a l . 1972). A l l three rep o r t s describe p a r t i c l e s w i t h a morphology and i n t r a c e l l u l a r development s i m i l a r to the a l g a l VLP's. On the b a s i s of s t r u c t u r e and c y t o p a t h o l o g i c a l e f f e c t s , these VLP's may represent a new v i r u s group found i n the r e l a t i v e l y p r i m i t i v e eucaryotes. -10-There i s an e x i s t i n g grouping of w e l l - c h a r a c t e r i z e d v i r u s e s , the i c o s a h e d r a l cytoplasmic deoxyriboviruses (ICDV's) which have s t r u c t u r e s s i m i l a r to those of the l a r g e p o l y h e d r a l a l g a l v i r u s e s . S t r u c t u r a l aspects of ICDV's have been discussed by S t o l t z (1971 and 1973), biochemical development by McAuslan and Armentrout (1974), and c h a r a c t e r i s t i c s of the group as a whole were reviewed by K e l l y and Robertson (197 3). The group inc l u d e s v i r u s e s which i n f e c t mammals, amphibians, r e p t i l e s , f i s h , i n s e c t s , and one mollusc. The best described of the group are the i r i d e s c e n t v i r u s e s of i n s e c t s , lymphocystis v i r u s of f i s h , f r o g v i r u s 3, and A f r i c a n swine fever v i r u s . The u n i f y i n g c h a r a c t e r i s t i c s of the v i r u s e s i n t h i s group are as suggested by the name: an i c o s a h e d r a l shape, of diameter ranging from 130 nm f o r the smaller i r i d e s c e n t v i r u s e s to 300 nm f o r lymphocystis v i r u s ; v i r u s assembly i n the cytoplasm, and double-stranded DNA as the v i r a l n u c l e i c a c i d . The two higher p l a n t v i r u s e s which might be included i n t h i s g r o u p - - c a u l i f l o w e r mosaic v i r u s and d a h l i a mosaic v i r u s — a r e much smaller (50 nm diameter) and seem to have c l e a r d i f f e r e n c e s i n chemical composition, e s p e c i a l l y l i p i d content ( S t o l t z 1973). Frog v i r u s 3 and A f r i c a n swine fever v i r u s are enveloped by a true membrane, derived from the host c e l l ( K e l l y and Robertson 1973). Two i n s e c t v i r u s e s , T i p u l a i r r i d e s c e n t v i r u s and Chironomus i r i d e s c e n t v i r u s , have been shown to c o n tain approximately 9% l i p i d ( K e l l y and Vance 1973), p o s t u l a t e d by S t o l t z (1973) to be l o c a t e d i n s i d e the hexagonal s h e l l , as a s i n g l e b i l a y e r covering the v i r a l core. Previous workers had disputed the presence of l i p i d , a t t r i b u t i n g the small q u a n t i t i e s -11-detected to contamination by host membranes during e x t r a c t i o n and p u r i f i c a t i o n . The i n t e r n a l l o c a t i o n of the l i p i d e x p l a i n s r e p o r t s t h a t the v i r u s e s are not e t h e r - s e n s i t i v e . The controversy i s reviewed by K e l l y and Vance (197 3). The u l t r a s t r u c t u r a l s i m i l a r i t i e s between ICDV's and the l a r g e p o l y h e d r a l a l g a l v i r u s e s have been noted by s e v e r a l authors (Pickett-Heaps 1972, Toth and Wilce 1972, C l i t h e r o e and Evans 1974, Markey 1974, and Hoffman and Stanker 1976). The r e p o r t i n 1972 by Mattox, Stewart and F l o y d , c i t e d i n Table 1, described VLP's present i n four genera of freshwater filamentous green a l g a , a l l of the order U l o t r i c h a l e s . The VLP's were observed by t h i s team of p h y c o l o g i s t s i n the course of an u l t r a s t r u c t u r a l survey of the order; these workers had no v i r o l o g i c a l e x p e r t i s e themselves, and proposed t h a t some of the VLP's could be p u r i f i e d and studied f u r t h e r by other workers. Subsequently Dr. J.A. Dodds, p o s t - d o c t o r a l f e l l o w , Department of Botany, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, sent f o r a sub-culture of the c o l l e c t i o n of Uronema qicras reported by Mattox and co-workers to contain VLP's. U l t r a s t r u c t u r a l examination confirmed the presence of the VLP's, and f r e e p a r t i c l e s were detected i n n e g a t i v e l y s t a i n e d a l i q u o t s of the c u l t u r e medium. A simple VLP i s o l a t i o n was worked out, i n v o l v i n g f i l t r a t i o n to remove a l g a l c e l l s followed by c e n t r i f u g a t i o n to concentrate the VLP's. R e l a t i v e amounts of a l g a l growth per f l a s k or t e s t tube were estimated by an assay of c h l o r o p h y l l a. (Hansmaan 1973) . In subsequent work, Dr. Dodds i s o l a t e d 49 subcultures by s i n g l e zoospore t r a n s f e r s , and q u a n t i f i e d VLP r e l e a s e i n each -12-of the i s o l a t e s . The q u a n t i t y of VLP's rele a s e d from each i s o l a t e v a r i e d w i d e l y , but a l l subcultures released VLP's. In the search f o r a healthy i s o l a t e of U. gigas a second c o l l e c t i o n of the alga r e c e i v e d from the Cambridge U n i v e r s i t y C u l t u r e C o l l e c t i o n . This sample proved to contain VLP's as w e l l ; on f u r t h e r i n v e s t i g a t i o n we learned t h a t the Indiana C o l l e c t i o n had been derived by subculture from the Cambridge C o l l e c t i o n (Dr. J . S t e i n , Department of Botany, U n i v e r s i t y of B r i t i s h Columbia, personal communication). To date no other c o l l e c t i o n of t h i s a l g a l species has been deposited i n world c o l l e c t i o n s ; thus i t appears t h a t a l l known U_. gigas c u l t u r e s c o n t a i n VLP's. A healthy c u l t u r e — w i t h no V L P ' s — i s needed f o r a demonstration of i n f e c t i v i t y . In other work, Dr. Dodds observed a c o r r e l a t i o n between growth of very long f i l a m e n t s i n some s i n g l e - c e l l - d e r i v e d c u l t u r e s and reduced VLP y i e l d (Table I I ) , other c u l t u r e s contained only r e l a t i v e l y short f i l a m e n t s and released VLP's i n greater amounts. A working hypothesis was developed t h a t fragmentation of the f i l a m e n t r e s u l t e d from VLP synthesis and subsequent r e l e a s e of VLP's by i n d i v i d u a l c e l l s along the f i l a m e n t . Highly i n f e c t e d f i l a m e n t s would be broken i n t o many short fragments. Three c u l t u r e s which were h i g h - y i e l d e r s of VLP's and three l o w - y i e l d e r s were s e l e c t e d and assayed f o r c h l o r o p h y l l a, content. The high V L P - y i e l d i n g c u l t u r e s contained l e s s than h a l f as much c h l o r o p h y l l a. as the low y i e l d e r s . In other experiments Dr. Dodds determined t h a t a c u l t u r e p e r i o d of 6-8 weeks was necessary f o r maximal y i e l d of VLP's (Dodds et a l . 1975) . -13-TABLE I I . Characteristics of single c e l l i s o l a t e s of Uronema cricrasa Culture appearance'3 VLP index Fragmented Long filaments Greater than 5 20/23 5/26 Less than 5 3/23 21/26 Data from J.A. Dodds, personal communication. Total of 49 single c e l l i s o l a t e s . -14-Uronema gigas i s a U l o t r i c h a l e a n green a l g a , of long unbranched f i l a m e n t s , w i t h a s p e c i a l i z e d b a s a l h o l d f a s t c e l l and a pointed or accuminate t e r m i n a l c e l l . Except f o r the presence of these two d i s t i n g u i s h i n g types of c e l l s , a l l species of Uronema are v i r t u a l l y i d e n t i c a l to species of U l o t h r i x (Ramanathan 1 9 6 4 ) . Taxonomists have disagreed about the v a l i d i t y of separating Uronema from U l o t h r i x on t h i s b a s i s along ( F r i t s c h 1 9 5 6 ) , but the c h a r a c t e r i s t i c s appear s t a b l e (although w i t h some f l u c t u a t i o n i n response to d i f f e r e n t environmental c o n d i t i o n s ) , and i n general the two groupings have been approved (Ramanathan 1 9 6 4 ) . JJ. gigas was c o l l e c t e d once, i n Switzerland i n 1933 by V i s c h e r , growing on the surface of a small trough i n the B o t a n i c a l Gardens i n B a s e l . The species seems to grow best i n s t i l l or only s l i g h t l y moving water, w i t h optimal growth i n s p r i n g and f a l l (Vischer 1933 as reported by Ramanathan 1 9 6 4 ) . Asexual reproduction by fragmentation i s r e p o r t e d l y l e s s common than i n other members of the U l o t r i c h a l e s , but a l l species of Uronema r e a d i l y produce and r e l e a s e asexual zoospores, induced by changes i n the environment r a t h e r than as a c u l m i n a t i o n of c e l l u l a r maturity (Mitra 1945, 1 9 4 7 ) . The b a s a l c e l l and the t e r m i n a l c e l l , as w e l l as the c y l i n d r i c a l c e l l s of the f i l a m e n t , can r e l e a s e the q u a d r i f l a g e l l a t e zoospores. M i t r a (1947) suggests th a t continuous c y c l e s of zoospore production account f o r the presence of many short f i l a m e n t s , r a t h e r than d i r e c t fragmentation of the f i l a m e n t s . Very long f i l a m e n t s of up to 30 cm can be found i n c u l t u r e d Uronemas, but they do not occur i n nature. Zoospore production i s r e a d i l y induced i n c u l t u r e by t r a n s f e r of f i l a m e n t s to d i s t i l l e d water or f r e s h medium or -15-merely by d i l u t i o n of the e x i s t i n g medium. Sexual reproduction has not been reported f o r any Uronema speci e s . In c o n t r a s t to the l i m i t e d d i s t r i b u t i o n of U_. g i g a s , y_. confervicolum, which d i f f e r s only i n c e l l u l a r dimensions, 3.5 u to 8 u broad versus 8 to 11 u broad (Ramanathan 1964), i s found i n Europe, A f r i c a , the Americas, and A s i a , growing e p i p h y t i c a l l y on other algae and submerged p l a n t s . Other members of the genus a l s o have world-wide d i s t r i b u t i o n s . In August 1975 I began research on the VLP from y_. gigas w i t h the plan of concentrating on i t s biochemical and b i o p h y s i c a l c h a r a c t e r i z a t i o n , r a t h e r than anal y z i n g b i o l o g i c a l aspects of the i n t e r a c t i o n w i t h the host a l g a . I t was c l e a r t h a t l i m i t a t i o n s on what could be done would be imposed by the minimal amount of VLP's a v a i l a b l e f o r experimentation. The f i r s t area of study i n v o l v e d f i n d i n g an improved p u r i f i c a t i o n technique.. The major d i f f i c u l t y w i t h the simple f i l t r a t i o n and c o n c e n t r a t i o n procedure that had been used was the presence, i n a l l p u r i f i e d p r e p a r a t i o n s , of dense spheres, about 100-130 nm i n diameter, which were almost i d e n t i c a l to the VLP's i n d e n s i t y and were of unknown o r i g i n and composition. The other obvious problem was the l i m i t e d amount of VLP's produced even i n the highest y i e l d i n g c u l t u r e s . Some biochemical methods would be u s e f u l only when done i n conjunction w i t h e l e c t r o n microscopy, because of the l i m i t e d s t a r t i n g m a t e r i a l . Two v i r u s e s were s e l e c t e d f o r use as markers i n e l e c t r o n microscopy and f o r b i o p h y s i c a l c h a r a c t e r i z a t i o n s of UGV: T i p u l a i r i d e s c e n t v i r u s ( TIV—an ICDV) and c a u l i f l o w e r mosaic v i r u s (CaMV). P r e l i m i n a r y sucrose d e n s i t y gradient a n a l y s i s i n d i c a t e d that TIV approximated UGV i n d e n s i t y , and could t h e r e f o r e be used as a v a l u a b l e marker f o r d e n s i t y g r a d i e n t s , as w e l l as f o r comparisons of s t r u c t u r e s , as discussed above. CaMV was included as an example of a higher p l a n t v i r u s which contains DNA. The general o u t l i n e of my p r o j e c t included c h a r a c t e r i z a t i o n of the i n t a c t VLP i n terms of such p r o p e r t i e s as sedimentation c o e f f i c i e n t , l i g h t absorbing c h a r a c t e r i s t i c s , and d e n s i t y ; a n a l y s i s of VLP components: type of n u c l e i c a c i d , number and molecular weight of p r o t e i n s , p o s s i b l e presence of l i p i d ; and some i n v e s t i g a t i o n of the u l t r a s t r u c t u r e of the i n f e c t e d c e l l and of VLP's i n s i t u and i n v i t r o . C h a r a c t e r i z a t i o n s of t h i s k i n d must be done f o r the p o l y h e d r a l VLP's of e u c a r y o t i c algae before they can be r e l i a b l y assigned to an e x i s t i n g group of v i r u s e s or e s t a b l i s h e d as a new group. -17-MATERIAL5 AND METHODS  Uronema gigas—sources and culture techniques Uronema gigas cultures were obtained from the Indiana Type Culture C o l l e c t i o n , accession number 174, the source of the culture studied by Mattox et. a l . (197 2) . Another culture of JJ. gigas maintained at Cambridge University was also obtained, but on investigation we found that the Indiana culture had been derived from the Cambridge c o l l e c t i o n , so the two cultures are g e n e t i c a l l y i d e n t i c a l unless changes have occurred i n culture. Both cultures release UGV p a r t i c l e s , and show similar cytopathologies (Dr. J . Allan Dodds, personal communication). JJ. gigas was grown i n Beijerinck's mineral medium (Stein 1973). The procedure for making t h i s medium i s shown i n Table I I I . Stock solutions were stored i n d e f i n i t e l y at 4C; d i l u t e d medium was prepared as needed and s t e r i l i z e d by autoclaving at 121 C for 20 minutes. Beijerinck's medium s o l i d i f i e d with 1% agar was used for a l g a l growth on test tube slants for storage of cultures. Subcultures were begun by single zoospore transfer to 10 ml of fresh, s t e r i l e medium i n 25 ml culture tubes. JJ_. gigas zoospore formation was e a s i l y induced by one night i n fresh medium or d i s t i l l e d water. Young germlings,'3 to 4 c e l l s i n length, were easier to see and to manipulate under a dissecting microscope for transfer through several drops of medium to r i d a culture of contaminants, as described by Stein (1973). Stock cultures maintained on agar slants were "cleaned" every six -18-months by i n d u c i n g z oospores o v e r n i g h t i n f r e s h medium, washing i n d i v i d u a l g e r m l i n g s on the f o l l o w i n g day by the t e c h n i q u e j u s t m entioned, and t r a n s f e r r i n g t h e g e r m l i n g s t o f r e s h l y p r e p a r e d agar s l a n t s o r seeder t e s t tubes c o n t a i n i n g 10 ml o f l i q u i d medium. The seeder tubes c o u l d be used f o r i n o c u l a t i o n o f l a r g e growth f l a s k s a f t e r two weeks t o a month o f growth. S t e r i l e t e c h n i q u e was used t h r o u g h o u t a l l c u l t u r i n g o p e r a t i o n s . C u l t u r e s were grown a t 20 t o 23 C a t 4800 t o 5400 l u x and a 16:8 hour c y c l e o f l i g h t and dark. U n l e s s o t h e r w i s e noted c u l t u r e s were s t a t i o n a r y and no e x t r a a e r a t i o n was s u p p l i e d . LL» cricras growth f o r v i r u s p u r i f i c a t i o n was i n one l i t e r f l a s k s c o n t a i n i n g 400 ml o f B e i j e r i n c k ' s medium. E i g h t t o t h i r t y f l a s k s were i n o c u l a t e d a t one t i m e . Maximum v i r u s c o n c e n t r a t i o n was reached a f t e r about two months o f gro w t h , under normal c o n d i t i o n s . S h o r t e x p e r i m e n t s t o t e s t t h e e f f e c t s of v a r i o u s t r e a t m e n t s on v i r u s r e l e a s e were more p r a c t i c a l l y p erformed u s i n g 125 ml f l a s k s c o n t a i n i n g 40 ml o f medium, which c o u l d be c o n t i n u o u s l y shaken. Growth i s f a s t e r under t h e s e c o n d i t i o n s ; e x p e r i m e n t s c o u l d be t e r m i n a t e d a f t e r two weeks. C h l o r o p h y l l a d e t e r m i n a t i o n C h l o r o p h y l l a c o n t e n t v/as used as a measure o f t o t a l a l g a l growth (Hansmaan 1973). A l g a l c e l l s were washed o f f f i l t e r p a pers u s i n g a known volume o f d i s t i l l e d w a t e r . Acetone was added t o b r i n g the f i n a l c o n c e n t r a t i o n o f l i q u i d t o 90% a c e t o n e . I f M i l l i p o r e f i l t r a t i o n s had been done, the f i l t e r p l u s a t t a c h e d a l g a l c e l l s were immersed d i r e c t l y i n t o 90% a c e t o n e . The acetone and c e l l s were t h o r o u g h l y mixed and p l a c e d i n a dark -19-TABLE ,111. B e i j e r i n c k ' s mineral medium (Stein 1973) For 1000 ml of medium Stock I — Add to 500 ml of 100 ml d i s t i l l e d water: NH 4N0 3 0.75 g K 2 H P 0 4 0.10 g MgS0 4'7H 20 0.10 g C a C l 2 . 2 H 2 0 0.05 g I I — Add to 500 ml of K H 2 P 0 4 d i s t i l l e d water: 4.53 g Stock I I I — Add to 500 ml of d i s t i l l e d water: K 2 H P 0 4 5.30 g Stock IV -- Trace elements, see below 6 0 ml 40 ml 1 ml Add d i s t i l l e d water to 1000 ml, and adjust pH to 6.5. Trace elements -- Add to 70 ml warmed d i s t i l l e d water: H3B03 1. 00 g C u S 0 4 . 5 H 2 0 0. 15 g Na 2EDTA a 5. 00 g Z n S 0 4 . 7 H 2 0 2. 20 g MnCl 2. 4H"20 0. 50 g F e S 0 4 . 7 H 2 0 0. 50 g C o C l 2 . 6 H 2 0 0. 15 g (NH.) Mo_0„,.4H„0 4 6 7 ^4 2. 0. 10 g Add s a l t s i n the order given. Allow each a d d i t i o n to d i s s o l v e completely, and adjust the pH to 5.0'with KOH p e l l e t s before adding the next s a l t . F i n a l volume should be 100 ml. A f t e r 2 to 3 days at 4 C, the pH should have r i s e n to 6.5 and the s o l u t i o n i s ready f o r use. Discard i f i r o n has p r e c i p i t a t e d . a) disodium ethylene diaminetetraacetate -20-r e f r i g e r a t o r f o r approximately 18 hours. Acetone blanks were included when M i l l i p o r e f i l t e r s were used. A f t e r the e x t r a c t i o n the samples were warmed to room temperature and c e n t r i f u g e d at 3,000 g f o r 10 minutes to remove d e b r i s . Absorbance at 660 nm was read f o r each sample i n a Bausch & Lomb Spectronic 2 0 c o l o r i m e t e r . C h l o r o p h y l l a_ concentrations of the o r i g i n a l volume of c u l t u r e medium were c a l c u l a t e d by the equation below: ... , . . . 0.95 (A,,.. ) (11.6) ( t o t a l volume of solvent used) ( c h l o r o p h y l l a.) = 6 60 nm  o r i g i n a l volume of medium i n l i t e r s where 11.6 i s the e x t i n c t i o n c o e f f i c i e n t of c h l o r o p h y l l a_. This equation approximates the true absorbance due to c h l o r o p h y l l a when measured i n p l a n t e x t r a c t s which a l s o c o n t a i n c h l o r o p h y l l s  b and c. (Hansmaan 1973) . V i r u s p u r i f i c a t i o n I. Uronema gigas VLP (UGV) I n i t i a l attempts to p u r i f y the VLP from U. gigas were hampered by three f a c t o r s : the extremely low c o n c e n t r a t i o n of VLP's i n l a r g e volumes of c u l t u r e medium? the presence of small dense spheres, about one t h i r d the diameter of the UGV p a r t i c l e , i n the c u l t u r e medium, which co-sedimented w i t h UGV i n a l l c e n t r i f u g a t i o n steps; and the tendency of the UGV p a r t i c l e s to aggregate i r r e v e r s i b l y at pH 5.5 or below. The p u r i f i c a t i o n scheme d e t a i l e d below was designed to circumvent these f a c t o r s . The dense bodies were p u r i f i e d by two c y c l e s of sucrose d e n s i t y gradient c e n t r i f u g a t i o n (conditions described below) and then i d e n t i f i e d by e l e c t r o n microprobe a n a l y s i s i n -21-the Geology Department of U.B.C. The dense bodies are a mineral p r e c i p i t a t e c o n t a i n i n g mainly i r o n w i t h magnesium and calcium; they seem to accumulate as the pH of the medium f a l l s below 5.5. A l g a l c u l t u r e s were f i r s t f i l t e r e d through 35 u b o l t i n g s i l k to remove long f i l a m e n t s . The f i l t r a t e was then passed through Whatman #1 f i l t e r paper which removed most germlings and zoospores. In some cases 1.20 y m M i l l i p o r e membrane f i l t e r s were used f o r t h i s step, w i t h i d e n t i c a l r e s u l t s . The a l g a l c e l l s were c o l l e c t e d from the f i l t e r s and s i l k and pooled f o r c h l o r o p h y l l a_ determinations. The c l e a r straw-colored f i l t r a t e was c e n t r i f u g e d at approximately 10,000 g f o r one hour i n a S o r v a l l GSA r o t o r . The r e s u l t i n g supernatant was dis c a r d e d ; the p e l l e t was. resuspended i n a small volume of a b u f f e r c o n t a i n i n g 0.05 M sodium c i t r a t e , 0.01 M disodium ethylenediamine t e t r a a c e t a t e (Na 2EDTA), and 0.1% T r i t o n X-100, t i t r a t e d to pH 6.0 w i t h 1 M c i t r i c a c i d . The VLP suspension was d i a l y z e d against 2 l i t e r s of t h i s b u f f e r overnight. C i t r a t e and Na2EDTA w i l l c h elate i r o n at pH 1s between 5.0 and 7.0; t h i s step removed the dense bodies. Sometimes complete removal of dense bodies r e q u i r e d s e v e r a l days of d i a l y s i s w i t h b u f f e r changes. Removal of dense bodies can be judged f a i r l y a c c u r a t e l y judged by eye, as the i n t a c t dense bodies give a yellow t i n g e to the VLP suspension. The d i a l y z e d VLP suspension was then r e - c e n t r i f u g e d at 10,000 g f o r one hour i n the GSA r o t o r ; the supernatant was dis c a r d e d , and the p e l l e t resuspended i n 0.05 M 2-amino-2 (hydroxymethyl)-1,3-propanediol ( T r i s ) - H C l b u f f e r at pH 8.0 to prevent p a r t i c l e aggregation. -22-Further p u r i f i c a t i o n was accomplished by centrifugation through 10-40% sucrose gradients i n 0.05 M Tris-HCl pH 8.0. Small volumes of concentrated VLP's (0.2 to 4.0 ml) were layered on 12 or 36 ml gradients i n SW 41 or SW 27 and centrifuged at 10,000 g i n a Beckman L5-75 ultracentrifuge for 30 minutes. The resolved gradients were scanned at 254 nm i n an ISCO Model UA-5 absorbance monitor; absorbance peaks were coll e c t e d by hand. Sucrose from the gradient was removed by d i a l y s i s and the p u r i f i e d VLP's were stored at 4C i n 0.05 M Tris-HCl pH 8.0 with sodium azide or chlorbutanol added as an a n t i b a c t e r i a l preservative. I I . P u r i f i c a t i o n of Tipula i r i d e s c e n t virus (TIV) Tipula iridescent virus was p u r i f i e d following the method of Kalmakoff and Tremaine (1968). Infected Tipula  paludosa larvae were kindly donated by Mr. Fred Wilkinson of the Canada Agriculture Research Station, Vancouver; they were o r i g i n a l l y obtained from Dr. Claus Karl of the Institute of B i o l o g i c a l Control, Delemont, Switzerland. The larvae were cut into small pieces with a scissors and the r e s u l t i n g homogenate was magnetically s t i r r e d i n d i s t i l l e d water at 4C overnight. The r e s u l t i n g suspension was centrifuged at 1,000 rpm for 10 minutes i n a Sorvall SS-34 rotor. The p e l l e t was discarded. The supernatant was then centrifuged i n a type 30 rotor using the Beckman L5-75 ultracentrifuge at 20,000 rpm for 30 minutes. The p e l l e t s were suspended i n a 0.01 M potassium phosphate buffer, pH 7.4. Further p u r i f i c a t i o n of TIV was achieved by centrifugation through li n e a r gradients of 5 to 40% sucrose i n phosphate b u f f e r . V i r u s suspensions (0.2 ml) were l a y e r e d onto g r a d i e n t s and c e n t r i f u g e d at 15,000 rpm i n the SW 41 r o t o r f o r 20 minutes. Gradi e n t s were scanned i n the ISCO Model UA-5 absorbance monitor and f r a c t i o n s were c o l l e c t e d as described, f o r UGV. V i r u s f r a c t i o n s were d i a l y z e d a g a i n s t 0.01 M phosphate b u f f e r pH 7.4, and s t o r e d i n t h i s b u f f e r at 4 C with added sodium a z i d e . I l l , P u r i f i c a t i o n of c a u l i f l o w e r mosaic v i r u s (CaMV) F r e e z e - d r i e d Chinese cabbage ( B r a s s i c a p e k i n e n s i s ) leaves i n f e c t e d w i t h c a u l i f l o w e r mosaic v i r u s (CaMV) were obtained from Dr. P.. Stace-Smith, of the Canada A g r i c u l t u r e Research S t a t i o n , Vancouver. T h i s CaMv s t r a i n was o r i g i n a l l y i s o l a t e d at the Rothamsted Experimental S t a t i o n , and was donated by Dr. A.A. Brunt e t a l . (1975). The leavesifere homogenized w i t h a mortar and p e s t l e i n 0.01 M potassium phosphate b u f f e r , pH 7.2, and C e l i t e added, and the homogenate was rubbed onto fo u r Chinese cabbage p l a n t s . A f t e r 2h to 3% weeks, the p l a n t s developed n o t i c e a b l e s y m p t o m s — c h l o r o t i c m o t t l i n g and v e i n c l e a r i n g on young leaves --and were t h e n c e f o r t h used as the v i r u s source f o r f u r t h e r i n o c u l a t i o n s . I n f e c t e d l e a v e s were s t o r e d f r o z e n up t o fo u r months without l o s i n g i n f e c t i v i t y . The v i r u s was p u r i f i e d by the method of H u l l , Shepherd, and Harvey (1976), E i g h t Chinese cabbage or Tendergreen mustard ( B r a s s i c a compestris) p l a n t s were i n o c u l a t e d w i t h CaMV and harvested approximately 2 5 days l a t e r . I n f e c t e d leaves were c h i l l e d and homogenized i n a Waring blendor with 0.5 M potassium phosphate b u f f e r , 0.75% sodium s u l f i t e , pH 7.2, u s i n g 1 ml buffer per gram of leaves. The slurry was f i l t e r e d through cheesecloth and the volume noted. Triton X-100 and urea were added to 2.5% and 1 M, respectively; the mixture was then s t i r r e d overnight at 4 C. A low speed centrifugation followed (6,000 g, 10 min), and the re s u l t i n g p e l l e t was discarded. The supernatant f l u i d was resuspended i n one tenth the o r i g i n a l volume of 0.0 M potassium phosphate buffer pH 7.2 made to 2.5% Trito n X-100 and 1 M urea, and again s t i r r e d overnight at 4 C. This suspension was centrifuged at 27,000 rpm for 1.5 hours i n the Spinco Type 30 rotor. The r e s u l t i n g supernatant f l u i d was discarded and the p e l l e t s were slowly dispersed i n d i s t i l l e d water overnight. A f i n a l low speed centrifugation was performed (10,000 g for 10 minutes), and the supernatant was c a r e f u l l y c o l l e c t e d for sucrose density gradient centrifugation. The gradients used were 10-40% sucrose i n 0.01 M potassium phosphate pH 7.2 i n Beckman SW 41 tubes. Conditions of centrifugation were two hours at 23,000 rpm i n the Beckman L5-75 ultr a c e n t r i f u g e . Resolved gradients were scanned at 254 nm using an ISCO Model Ua-5 absorbance monitor. Fractions containing virus were collected manually, diluted 1:1 with d i s t i l l e d water, and pelleted by centrifugation at 45,000 rpm for one hour i n the Type 65 rotor. The p e l l e t was resuspended i n d i s t i l l e d water for storage. Virus y i e l d , determined using an extinction c o e f f i c i e n t 0 1% uncorrected for l i g h t scatter (E *_° = 7, Shepherd 1970), ^ 26 0 nm ' - ' averaged about 1 mg/100 g fresh weight leaves. Electron microscopy Viruses were prepared for electron microscopy by negative staining or by f i x a t i o n , embedding, and sectioning. Two negative s t a i n s were used: 2% phosphotungstic a c i d (PTA) i n water, pH 7.2, and 2% aqueous ur a n y l acetate, pH 5,0. U s u a l l y 2% PTA was the s t a i n of choice, since u r a n y l acetate overstained the UGV p a r t i c l e and caused aggregation of v i r u s p a r t i c l e s due to the low pH. One drop of a VLP-containing suspension was placed on a 200 mesh, carbon/collodion-coated copper g r i d f o r 10 minutes at room temperature. Excess suspension was then washed o f f using 20 drops of s t a i n . The f i n a l drop of s t a i n was held on the g r i d f o r 15 seconds before b l o t t i n g w i t h f i l t e r paper. Conditions of grid-making and s t a i n i n g were standardized-as much as p o s s i b l e to allo w use of p a r t i c l e - c o u n t i n g as a measure of v i r u s c o n c e n t r a t i o n . Negatively s t a i n e d p a r t i c l e s were viewed w i t h a P h i l i p s EM300 e l e c t r o n microscope at a m a g n i f i c a t i o n of 14,000 times; counts of ten l i n e a r t r a n s e c t s of g r i d squares were averaged f o r an a r t i f i c i a l index of v i r u s c o n c e n t r a t i o n , l a t e r c o r r e c t e d to a 100X concen t r a t i o n of o r i g i n a l c u l t u r e medium. Counts were found t o be inaccurate i f detergent ( T r i t o n X-100 or SDS) was present during i n c u b a t i o n on the g r i d . Very few VLP's adhered to the g r i d i n the presence of detergent; extensive d i a l y s i s t o remove the detergent r e s u l t e d i n much higher estimations of VLP index. One procedure was used f o r f i x a t i o n and embedding of a l g a l c e l l s and p u r i f i e d v i r u s e s . Clumps of a l g a l t i s s u e or p e l l e t e d v i r u s were f i x e d f o r one hour w i t h 5% glutaraldehyde i n 0.1 M potassium phosphate b u f f e r , pH 7.2, r i n s e d twice i n 15 minute changes of the b u f f e r , and p o s t - f i x e d i n 1% osmium t e t r o x i d e i n Palade's b u f f e r (Palade 1952) f o r at l e a s t one hour. Sometimes the a l g a l t i s s u e was mixed i n 2% Noble agar a f t e r osmium f i x a t i o n to allow e a s i e r handling of the small c e l l s . ( B i s a l p u t r a e_t a l . 1973) . The f i x e d m a t e r i a l was dehydrated through ten minute steps of 30%, 50%, 70%, and 95 % eth a n o l , and f i n a l l y three times i n 100% eth a n o l , followed by two-f i f t e e n minute changes of propylene oxide; then i t was i n f i l t r a t e d overnight w i t h a 1:1 mixture of propylene oxide and Epon (1 p a r t Epon A to 1 part Expon B plus 1.5% DMP-30 ac c e l e r a t o r ) (Luft 1961). The next day the samples were embedded i n pure Epon and hardened at 60 C f o r 36 hours. Sections were cut using g l a s s knives on a Reichert OMU-2 ultramicrotome, t r a n s f e r r e d to 100 mesh copper g r i d s , and d r i e d . They were f i r s t s t a i n e d w i t h 5% u r a n y l acetate i n 50% methanol f o r 20 minutes, thoroughly r i n s e d , and then s t a i n e d f o r ten minutes w i t h Reynold's lead c i t r a t e (1961) d i l u t e d 1:4 wi t h 0.01 N NaOH. Stained s e c t i o n s were examined using the P h i l i p s EM200 or EM3 00 e l e c t r o n microscopes operated at 6 0 KV. Photographs were taken on 3 5 mm f i l m . An a l t e r n a t e f i x a t i o n and embedding method was used f o r enzyme d i g e s t i o n s t u d i e s . Samples were f i x e d i n a 1:1 mixture of 5% a c r o l e i n and 5% f o r m a l i n , i n 0.1 M phosphate b u f f e r , pH 7.2, f o r 90 minutes, except f o r those to be DNAase-digested, which were f i x e d i n 5% f o r m a l i n alone (Weintraub et a l . 1969). In l a t e r experiments, the usual glutaraldehyde/ osmium t e t r o x i d e double f i x a t i o n was used w i t h s i m i l a r r e s u l t s (Kazama and Schornstein 1972). Fixed t i s s u e was embedded i n g l y c o l methacrylate (GMA), f o l l o w i n g the procedure of Leduc and Bernard (1967). The embedding procedure was c a r r i e d out at 4 C; a UV l i g h t source (315 nm) was used f o r p o l y m e r i z a t i o n of the GMA -27-i n the cold room over 3 to 7 days. The enzyme solutions used for digestion of sections on grids were: 1. Ribonuclease A--bovine pancreas Type 12A--(Sigma) 0.5 mg/ml i n d i s t i l l e d water, adjusted to pH 6.8 with 0.01 N NaOH; 2. Deoxyribonuclease I—bovine pancreas—(Sigma)—0.5 mg/ml in 0.05 M Tris-HCl, 0.005 M MgCl 2, pH 6.8; 3. Trypsin--bovine pancreas Type 1--(Sigma) at 0,6 mg/ml i n 0.05 M Tris-HCl pH 8.0; 4. Pronase—Type 6—(Sigma) at 0.2 mg/ml i n 0.05 M Tris-HCl pH 7.4; and 5. Lipase 448 (Nutritional Biochemical Company) at 0.5 mg/ml i n 0.05 M Tris-HCl pH 7.8. Grids were floated on drops of the enzyme solutions i n wells of a porcelain spot plate at 3 7 C; the incubation time varied from 10 minutes to overnight. Control grids were floated on drops of the appropriate buffer. The spot plate was wrapped i n aluminum f o i l to prevent evaporation. After the appropriate time, the sections were rinsed, and then stained with 2% aqueous uranyl acetate for one hour at 6 0 C, followed by Reynold's lead c i t r a t e for 10 minutes. Scanning electron microscopy Young Tj_. gigas germlings were prepared for scanning electron microscopy by growth on a d i a l y s i s membrane. Ten ml of newly released zoospores were added to a beaker containing 50 ml of fresh medium, with a s t e r i l e d i a l y s i s membrance adhering to the inner circumference of the beaker. Many zoospores se t t l e d -28-i n a t h i n l i n e on the membrane at the surface of the medium. Pieces of the membrane wit h adhering l i n e s of germlings were c o l l e c t e d a f t e r two days of growth, f i x e d i n 5% glutaraldehyde f o r one hour, r i n s e d i n 0.01 M phosphate b u f f e r pH 7.2 t w i c e , and dehydrated i n the normal ethanol steps. The samples were then loaded i n t o small baskets and d r i e d by the c r i t i c a l p o i n t d r y i n g method (Cohen 1975). Small pieces of d i a l y s i s membrane wi t h attached zoospores were mounted on aluminum stubs and observed without coating i n a H i t a c h i S500 scanning e l e c t r o n microscope w i t h a Tracor-Northern NS 880 x-ray m i c r o a n a l y s i s u n i t attached. Pounding w i t h a small hammer p a r t i a l l y r e l e a s e d v i r u s - l i k e bodies from the d r i e d a l g a l c e l l s . Photographs were taken using 35 mm f i l m or P o l a r o i d 52 f i l m . Samples were analyzed using the x-ray microanalyzer f o r 100 second counting periods at a voltage of 2 0 KV. The sm a l l e s t o b j e c t i v e aperture was used, w i t h the detector 3 cm away from the specimen. Phosphorus counts were c o l l e c t e d at 2.02 KeV. L i g h t s c a t t e r i n g p r o p e r t i e s of UGV The methods of Noordam (197 3) were used to measure the l i g h t s c a t t e r i n g c h a r a c t e r i s t i c s of s e v e r a l UGV pr e p a r a t i o n s . L i g h t s c a t t e r i s dependent on p a r t i c l e s i z e and p a r t i c l e aggregation, so i t can vary between preparations or under d i f f e r e n t c o n d i t i o n s of storage of v i r u s e s . The Beckman DK-2A recording spectrophotometer was used to determine absorbance of VLP suspensions from 230 nm to 600 nm. Curves were drawn representing true absorbance of the VLP's, wi t h absorbance due to l i g h t s c a t t e r subtracted, using Noordam's graphing techniques and c a l i b r a t i n g overlays. In l a t e r work d i r e c t measurements of absorbance at 2 6 0 nm could be used for an approximation of true VLP concentration, using the correction factor determined from these curves. Sedimentation c o e f f i c i e n t of UGV The sedimentation c o e f f i c i e n t of in t a c t UGV p a r t i c l e s was determined using the Beckman Model E. a n a l y t i c a l u l t r a c e n t r i f u g e . Sedimentation runs were at 4 0 5 9 or 6 1 6 6 rpm; photographs were taken at 2 or 4 minute inter v a l s using the schlieren and u l t r a v i o l e t optics. The temperature for a l l runs was maintained at 2 0 C. Determinations were made on preparations containing the highest concentrations of UGV available, usually about 3 0 0 p a r t i c l e s per transect by electron microscopy. virus suspensions had to appear b l u i s h -white by eye to obtain a r e s u l t with the Model E. Suspensions of TIV at 0.2 mg/ml were always co-sedimented i n a second quartz c e l l . Centrifugations were performed at pH 8.0 and pH 5 . 5 . Schlieren images were photographed on Professional f i l m , with 1 5 second exposure times. Calculations of S 2 Q / W were done by the method of Markham ( 1 9 6 7 ) . Measurements of UGV dimensions Dimensions of virus p a r t i c l e s were determined by measurements from electron micrographs of negatively stained preparations. Mixtures of TIV at 0 . 1 5 mg/ml and UGV at a VLP index of 6 0 p a r t i c l e s / t r a n s e c t were stained using the standard procedure i n 2% phosphotungstic acid, pH 7 . 2 . The VLP's were photographed and then printed at a magnification of 3 2 , 0 0 0 times. -30-P a r t i c l e dimensions were determined using a map measuring device (Dietzgen). In a second experiment samples of TIV and UGV were mixed with uniformly sized latex beads (790 nm diameter, Sigma). Measurements were made at 20,000 times. Dimensions of glutaraldehyde-fixed UGV, and UGV stained with uranyl acetate, pH 5.5, had been previously determined by Dr. J. A l l a n Dodds. Acridine orange staining The method of Bradley (1965) as described by Spendlove (1967) was followed for acridine orange staining of virus smears. Suspensions of bromegrass mosaic virus (BMV), tobacco mosaic virus (TMV), Tipula iridescent virus (TIV), and cauliflower mosaic virus (CaMV) were prepared for comparison with UGV suspensions. Drops of virus suspensions were applied to clean glass s l i d e s with micropipets and allowed to dry. The virus smears were fixed by immersion i n Carnoy's solution ( g l a c i a l acetic acid/absolute ethanol/reagent grade chloroform, 1:6:3) for 5 to 10 minutes. The smears were then stained for 5 minutes with a modified Mcllvaine's buffer containing 6 ml of 0.1 M c i t r i c acid, 4 ml of 0.15 M Na2HPC>4, and 0.1 ml of 1% acridine orange, pH 3.8. The s l i d e s were thoroughly rinsed i n the same buffer without acridine orange, and transferred to 0.15 M Na2HPO^ for 15 minutes. After shaking to remove excess l i q u i d , the s l i d e s were observed i n a darkened room using a Zeiss photomicroscope with an u l t r a v i o l e t l i g h t source; the use of exciter f i l t e r No. 1 and b a r r i e r f i l t e r s Nos. 44 and 53 i n the l i g h t beam resulted i n an emitting shortwave u l t r a v i o l e t l i g h t -31-of approximately 257 nm. Under these conditions a si n g l e -stranded nucleic acid w i l l fluoresce red and double-stranded nucleic acids w i l l fluoresce green. If a red fluorescence i s seen, a test can be performed to di s t i n g u i s h RNA from DNA by exposing the smears to 0.1 M c i t r i c acid for 1 to 5 minutes. A single-stranded DNA w i l l show fading of the red fluorescence, possibly to green; a single-stranded RNA remains red-fluorescing (Bradley 1965). Even with very successful staining, the amount of l i g h t available for photography i s extremely limited using t h i s technique. The microscope i r i s must be completely open, and the d i f f u s i o n disk f i l t e r at the back of the microscope must be removed from the l i g h t beam. Exposure times of up to two minutes were common, using Kodak Ektachrome ASA 160. Diphenylamine and o r c i n o l tests The diphenylamine and o r c i n o l t e s t s , which di s t i n g u i s h ribonucleic acid from deoxyribonucleic acid (Pederson 1969), were used to determine the type of nucleic acid of UGV, following the methods of Shatkin (1969). For either t e s t , samples of whole virus were brought to 5% t r i c h l o r o a c e t i c acid (TCA), incubated at 90 C for 20 minutes, and centrifuged at 3,000 g for 10 minutes. Otherwise tests were performed exactly as described. Eventually i t become apparent that samples of UGV and TIV prepared i n t h i s manner gave misleading r e s u l t s , perhaps due to the presence of sugar residues other than those present i n the nucleic acids. Diphenylamine and o r c i n o l tests were found to be more r e l i a b l e when p u r i f i e d v i r a l nucleic -32-acids from the v i r u s e s were t e s t e d . The RNA's of tobacco mosaic and bromegrass mosaic v i r u s e s were used as RNA standards; DNA from TIV and CaMV were used as DNA standards. N u c l e i c a c i d p u r i f i c a t i o n methods are described i n a separate s e c t i o n of M a t e r i a l s and Methods. P r o t e i n e l e c t r o p h o r e s i s V i r u s e s were d i s s o c i a t e d f o r a n a l y s i s of p r o t e i n components by a m o d i f i c a t i o n of a method of M a i z e l (1971). V i r u s samples (1 mg/ml) were mixed w i t h a d i s s o c i a t i o n b u f f e r (24% urea, 1% SDS, and 1% B-mercaptoethanol i n 0.1 M NaH 2P0 4, f r e s h l y prepared) i n a r a t i o of 3:1 (w/v) . The mixture was b o i l e d f o r 90 seconds. A l t e r n a t i v e l y , a small volume of VLP's was made 1 N i n HC1, allowed to stand at room temperature over n i g h t , and c e n t r i f u g e d at 6,000 rpm f o r 15 minutes; the p e l l e t was then d i s s o l v e d i n d i s s o c i a t i o n b u f f e r (J.H. Tremaine, personal communication). D i s s o c i a t e d p r o t e i n s were electrophoresced i n gels c o n t a i n i n g 1% SDS and 5, 7.5 or 10% polyacrylamide. Stock s o l u t i o n s f o r p r o t e i n e l e c t r o p h o r e s i s i n c l u d e d : 1) 10E: 1.0 M Na 2HP0 4/NaH 2P0 4 b u f f e r , pH 7.2, 0.1% SDS, w i t h 0.1 ml B-mercaptoethanol added per l i t e r . This s o l u t i o n was used f o r the e l e c t r o p h o r e s i s b u f f e r (IE) at a 1/10 d i l u t i o n w i t h d i s t i l l e d water; i t was used at 3/10 (3E) f o r the g e l b u f f e r , w i t h 3 ml of 10% SDS added to each 97 ml of d i l u t e d stock b u f f e r (Agrawal and Tremaine 1972). 2) Acrylamide (Eastman Kodak), 30%, i n d i s t i l l e d water, f i l t e r e d -33-a f t e r d i s s o l v i n g , and w i t h 0.75% N,N 1-methylene-bis-acrylamide (Eastman Kodak) added. 3) TEMED (N,N,N',N'-tetramethyladiamine, Eastman Kodak), 1% i n d i s t i l l e d water. 4) Ammonium p e r s u l f a t e , 10% i n d i s t i l l e d water, made f r e s h every tv/o weeks (Fraenckel-Conrat and Rueckert 1967 , Maurer 1971) . Twelve gels were cast using a t o t a l volume of 36 ml of g e l mixture. The proportions of the mixture f o r 10% gels were 12 ml acrylamide stock, 12 ml 3E b u f f e r , 7 ml d i s t i l l e d water, 0.36 ml 10% SDS, 3.6 ml TEMED, and 0.18 ml 10% ammonium p e r s u l f a t e , added l a s t w i t h mixing. P l a s t i c g e l tubes were each q u i c k l y f i l l e d w i t h 3 ml of the mixture, and d i s t i l l e d water was c a r e f u l l y layered over the g e l mixture to ensure a l e v e l g e l surfac e . A minimum of one hour was allowed f o r p o l y m e r i z a t i o n . When the gels had set the tubes were i n s e r t e d i n t o the upper trough of the e l e c t r o p h o r e s i s chamber, which was then f i l l e d w i t h 800 ml of IE b u f f e r . This assembly was lowered i n t o the lower trough c o n t a i n i n g 1200 ml of IE b u f f e r . P r o t e i n samples (50 t o 200 u l ) i n d i s s o c i a t i o n b u f f e r were made more dense w i t h the a d d i t i o n of 10% g l y c e r o l c o n t a i n i n g 1% bromophenol blue as a marker, and layered on the g e l surface using a micropipet under the b u f f e r surface. The gels were electrophoresed at 10 mA/gel f o r 4.5 hours. Gels were removed from g e l tubes by gentle blowing or by f o r c i n g water through a syringe needle between the i n t e r i o r of the g e l and the inner surface of the p l a s t i c tube, and were immediately immersed i n s t a i n . The s t a i n used was 0.1% Coomassie b r i l l i a n t blue R-50 i n 15% acetic acid, 50% ethanol. The gels were stained overnight at 37 C and then destained i n 7% acetic acid, 50% ethanol at 37 C overnight. The gels were re-swollen and stored i n 7% acetic acid. Protein molecular weights were determined by preparing a standard plo t for each electrophoresis run, showing distance migrated i n the gels versus the logarithms of the molecular weights of protein standards, as described by Loening (1969). Protein standards used were carbonic anhydrase, myoglobin, ovalbumin, bovine serum albumin, and alcohol dehydrogenase (molecular weights of 29,000, 17,500, 43,000, 66,000 and 37,000 daltons, respectively—Sigma Biochemical Company). In one instance a slab gel was used instead of c y l i n d r i c a l gels. A 10% polyacrylamide separating gel i n a slab holder was capped with an 8% gel after protein samples had been added to i n d i v i d u a l wells. Buffers were the same as for c y l i n d r i c a l gels. Voltage was supplied from a Buchler power supply, with a slow increase i n voltage from 10 V to 40 V over the f i r s t hour of electrophoresis; 40 to 50 V or approximately 15 0 mA were maintained over 3 to 4 hours u n t i l the dye front had almost reached the gel end. Estimates of molecular weights determined using t h i s system corresponded well with values determined using c y l i n d r i c a l gels; protein band resolution was better using t h i s system. Lipids Preparations of VLP's were treated with ether or .chloroform/methanol (2:1) i n order to investigate the presence or absence of l i p i d s i n these p a r t i c l e s . In the f i r s t experiment small aliquots of VLP's i n storage buffer were gently shaken for 20 minutes at room temperature with 3 times t h e i r volume of each solvent. The extracted VLP's were pelleted at 10,000 g for one hour, and then fixed, dehydrated, and embedded for electron microscopy using the standard methods. Sections were cut, stained, and examined for a l t e r a t i o n s of virion structure after the solvent treatments. Control grids were sections of VLP's pelleted and fixed without a solvent extraction. In a second experiment, solvent extraction of VLP's was followed by slab gel protein electrophoresis. The methods of Tas et a l . (1977) and Amako and Yasunaka (1977) were followed with s l i g h t modifications. A small volume (0.6 ml) of concentrated VLP's was shaken with either chloroform/methanol or ether, using 30 volumes of solvent for every volume of v i r u s . The chloroform/methanol sample was shaken vigorously for 5 minutes and then mixed slowly for two hours on a rotary shaker. One t h i r d of the t o t a l sample of methanol was then added and the mixture was centrifuged at 3,000 g for 15 minutes. The p e l l e t was taken up i n 0.9 ml of protein d i s s o c i a t i o n buffer for electrophoresis. The chloroform/methanol supernatant was concentrated to near dryness using a rotary evaporator and the concentrate was taken up i n 0.9 ml of d i s s o c i a t i o n buffer. The ether-treated sample was mixed for 2 0 minutes at room temperature, the ether removed by pipette, and 0.3 ml of d i s s o c i a t i o n buffer was added to the remaining aqueous phase. The solvent treated samples were analyzed as described i n the section on protein -36-electrophoresis; samples were co-electrophoresed with dissociated virus that had not been treated with organic solvents. DNA i s o l a t i o n and p u r i f i c a t i o n DNA was is o l a t e d from the VLP's of JJ. gigas by the method of Shepherd et al.. (197 0) . The concentration of virus i n each sample was determined, by absorbance at 26 0 nm, corrected for l i g h t scatter, and brought to 0.25% (w/v) sodium dodecyl sulfate (SDS). Pronase (1 mg/ml) was added to a f i n a l concentration of 5 0 ug/mg of v i r u s . The mixture was incubated at 37 C for 2 hours, then more SDS was added to a f i n a l concentration of 1% SDS. The incubation was continued for one hour or u n t i l opalescence had disappeared. The DNA was further p u r i f i e d by two extractions with saline-sodium-citrate (SSC)-saturated phenol (SSC: 0.15 M Na c i t r a t e , 0.015 M NaCl, pH 7.0). An equal volume of SSC-saturated phenol was added to each virus sample, and the mixtures were gently mixed on a rotary shaker at low speed for 15 minutes. The r e s u l t i n g emulsion was centrifuged at 6,000 rpm for 10 minutes; the lower phenol layer and some denatured protein at the interface were removed by pipette. The upper aqueous layer was re-extracted with phenol. After a second centrifugation, the aqueous layer was shaken gently with ether to remove traces of phenol. Care was taken during a l l manipulations of the DNA to minimize shearing. F i n a l l y , the DNA was dialyzed against 2 l i t e r s of SSC, overnight at 4 C. P u r i f i e d DNA was stored frozen i n small aliquots. When DNA of low concentrations was handled, a l l glassware was p r e - t r e a t e d w i t h " S i l i c l a d , " a commercial co a t i n g compound, f o l l o w i n g d i r e c t i o n s on the package. Untreated glassware binds DNA: S i l i c l a d c o ating forms a hydrophobic surface and prevents b i n d i n g . This method of DNA p u r i f i c a t i o n worked w e l l f o r p u r i f i c a t i o n of DNA from '..CaMV and TIV as w e l l as from UGV. Concentration of DNA was determined by absorbance 2 at 260 nm, using an e x t i n c t i o n c o e f f i c i e n t of 20 cm /mg (Shepherd et a l . 1968). Gentler methods of o b t a i n i n g DNA i n v o l v i n g only SDS or mixtures of SDS and m i l d p r o t e i n denaturants ( B e l l e t t and Inman 1967 , K e l l y and Avery 1974, Garwes ejb a l . 1975) d i d not d i s r u p t the v i r u s . A milder d i g e s t i o n using the fungal enzyme proteinase K (Hansen 1974, G r o s s - B e l l a r d et a l . 1973) was not severe enough to rel e a s e t h i s DNA, even wi t h overnight i n c u b a t i o n s . In one instance DNA was prepared f o r e l e c t r o n microscopy by b r i n g i n g a small a l i q u o t of VLP's to 0.1 M NaOH, pH 13.0. A f t e r 2 to 6 hours at room temperature e s s e n t i a l l y a l l i n t a c t VLP's had disappeared; the l i b e r a t e d DNA was used without f u r t h e r p u r i f i c a t i o n . Buoyant d e n s i t y determinations Determinations of buoyant d e n s i t y were made by c e n t r i f u g a t i o n of UGV-DNA i n cesium c h l o r i d e (CsCl) g r a d i e n t s . P u r i f i e d DNA at approximately 5 to 10 ug/ml i n Tris-EDTA b u f f e r (0.1 M T r i s , 0.01 M Na2EDTA at pH 8.5) was added to 0.951 g of o p t i c a l l y pure CsCl to b r i n g to 1 ml t o t a l volume. Marker DNA was added (5 to 10 u l of 1 to 2 mg/ml of DNA from E s c h e r i c h i a -38-c o l i . Micrococcus l y s o d e i k t i c u s or TIV). The r e s u l t i n g d e n s i t y of CsCl was measured at 25 C using an Abbe refractometer w i t h a sodium lamp. A d d i t i o n a l CsCl was added, i f necessary to b r i n g the d e n s i t y of the s o l u t i o n to approximately 1.700 g/ml. When cesium s u l f a t e gradients were used, the i n i t i a l d e n s i t y was approximately 1.40 g/ml. C e n t r i f u g a t i o n s were run on a Beckman Model E a n a l y t i c a l u l t r a c e n t r i f u g e at 44,000 rpm, 20 C, f o r 20 to 22 hours. P i c t u r e s were taken using the u l t r a v i o l e t l i g h t source, at zero time, a f t e r 4 to 8 hours, and at the end of each run, using Kodak P r o f e s s i o n a l Pan f i l m #4155. Exposure s e r i e s of 10 seconds to 2 minutes were u s e f u l . The negatives were scanned using a Joyce-Loebel microdensitometer w i t h the 0 to 2.0 O.D. u n i t wedge. Two methods of c a l c u l a t i o n of buoyant d e n s i t y were used: the reference DNA method (Sueoka 1961, Mandel e_t a l . 196 8) , and the absolute method, r e q u i r i n g no marker DNA (Chervenka 1973). The molar f r a c t i o n of guanine plus c y t o s i n e was c a l c u l a t e d using the equation of S c h i l d k r a u t et a l . (1962) . In order to prove that the i s o l a t e d n u c l e i c a c i d was DNA, the banded n u c l e i c a c i d was hydrolyzed w i t h DNAse and then re-run i n a CsCl g r a d i e n t . A f t e r a s u c c e s s f u l Model E, run the CsCl was removed from the DNA s o l u t i o n by d i a l y s i s versus SSC overnight. The DNA s o l u t i o n was brought to 100 ug/ml DNAse (Worthington) and 0.01 M MgCl 2, and digested f o r two hours. The d i g e s t was r e - d i a l y z e d versus SSC w i t h added EDTA (0.01 M), CsCl was re-added w i t h marker DNA and the s o l u t i o n was c e n t r i f u g e d again to e q u i l i b r i u m . -39-E l e c t r o n microscopy of DNA P u r i f i e d DNA was prepared f o r e l e c t r o n microscopy by the technique of Kl e i n s c h m i d t (1968) with some m o d i f i c a t i o n s Davis .et a l . 1971, Younghusband and Lee 1974, McClements and Kaesberg 1 9 7 7 ) . Tabbed copper g r i d s ( P e l c o — 2 2 mesh) were coated w i t h a t h i n f i l m prepared from 3.5% P a r l o d i o n i n iso-amyl a c e t a t e . The P a r l o d i o n was baked 24 hours a t 60 C bef o r e use: the d i s s o l v e d P a r l o d i o n was s t o r e d d e s s i c a t e d i n a brown b o t t l e . One drop of P a r l o d i o n prepared i n t h i s way was a p p l i e d t o a water f i l l e d Buchner f u n n e l c o n t a i n i n g a s m a l l number of c l e a n g r i d s r e s t i n g on Whatman #1 f i l t e r paper. The water was slo w l y d r a i n e d from the f u n n e l , a l l o w i n g the P a r l o d i o n f i l m t o s e t t l e on the g r i d s . The f u n n e l was s e c u r e l y covered and the g r i d s were l e f t i n p l a c e to dry f o r a t l e a s t 2 days. G r i d s made i n t h i s way g i v e high r e s o l u t i o n and are very c l e a n , but must be used w i t h i n f i v e days, as the f i l m becomes u n s t a b l e . A l l glassware used was coated with " S i l i c l a d . " The f o l l o w i n g stock s o l u t i o n s were prepared f o r the K l e i n s c h m i d t technique: 1) hyperphase s t o c k — 1 0 mg cytochrome 'c' i n 10 ml of 5 M ammonium ace t a t e pH 8.5; 2) hypophase s t o c k — 0 . 2 5 M ammonium a c e t a t e pH 8.5; 3) T r i s - E D T A— 0 . 1 M T r i s , 0.01 M Na 2EDTA pH 8.5. Samples of DNA (1 to 45 u l ) at 10 to 50 ug/ml were combined wi t h 5 u l of hyperphase sto c k , and the Tris-EDTA b u f f e r was added to b r i n g the t o t a l volume t o 50 u l . A 90 mm square p l a s t i c p e t r i d i s h was f i l l e d w ith hypophase to form a high meniscus. A g l a s s rod was run over the p e t r i d i s h to c l e a n the hypophase s u r f a c e . A thoroughly cleaned g l a s s s l i d e r e s t i n g on a smal l block of p l a s t i c formed a ramp into the hypophase approximately 45° to the hypophase. A glass c a p i l l a r y pipette was used to transfer the hypophase to the top of the ramp; the hyperphase was allowed to run down the ramp to the hypophase surface. Grids were touched to the hyperphase surface, held i n stai n 30 seconds, and then transferred to isopentane for 10 seconds, and f i n a l l y dried by touching to f i l t e r paper. The stain was made as follows: 10 u l of concentrated HC1 were added to 250 mg of uranyl acetate, af t e r which 10 ml of d i s t i l l e d water were added (UA stock). For use, 10 u l of UA stock were dil u t e d with 10 ml of 70% ethanol. The di l u t e d s t a i n i s stable for one hour only. Grids may be observed without shadowing or may be shadowed with platinum at an angle of 5 to 7°. Shadowing increases contrast and gri d s t a b i l i t y (from one day to one week). Grids were observed and photographed i n a P h i l i p s EM300 electron microscope at magnifications of 10,000 to 40,000 times. At each photography session a c a l i b r a t i o n g r i d (E.F. Fullam, 2160 l i n e s / mm) was photographed at each magnification used. DNA lengths were measured on photographic enlargements using a map measuring device (Dietzgen) . Radioisotope l a b e l l i n g of UGV-nucleic acid JJ. gigas cultures were grown for two weeks i n Beijerinck's medium with t r i t i u m - l a b e l l e d u r a c i l or thymidine added i n order to produce lab e l l e d VLP nucleic acid. One 10 ml seeder test tube was added to 100 ml of fresh unlabelled medium, shaken, and then 10 ml was dispensed to each of 11 te s t tubes. The new cultures were heat-shocked at 38 C i n the dark for 6 hours. Then 0.2 ml of H-uracil (1 mCi/ml, s p e c i f i c a c t i v i t y of 20 Ci/mmol, Amersham/Searle; f i n a l concentration i n medium of 20 uCi/ml) was added to each of two tubes with 0.1 ml of 10 micromolar unlabelled u r a c i l . To two other 3 tubes 0.10. ml of H-thymidine (0.5 mCi/ml, sp. act. 23 Ci/mmol, Amersham/Searle; f i n a l concentration i n medium of 5 uCi/ml) was added with 0.1 ml of unlabelled 10 y-M thymidine. S t e r i l e technique was used throughout. Two samples of 0.1 ml each were withdrawn from each tube immediately after the la b e l was added and every other day for the fourteen days following. The 0.1 samples were each added to 3 ml of ice cold 5% t r i c h l o r o a c e t i c acid (TCA). Bovine serum albumin (BSA, 1 mg/ml, 0.1 ml) was added, and the mixture was l e f t on ice for 5 to 10 minutes. The acid-insoluble material was col l e c t e d by f i l t r a t i o n through a 0.45 y M i l l i p o r e membrane, which was then washed 3 times with cold 5% TCA, twice with cold 95% ethanol, and then dried at 70 C. The f i l t e r was then put i n a s c i n t i l l a t i o n v i a l containing 10 ml of Aquasol (New England Nuclear). A Picker Nuclear Liquimat s c i n t i l l a t i o n counter was used; samples were equilibrated at 4 C i n the dark overnight before counting. The counting e f f i c i e n c y for the t r i t i u m - l a b e l l e d nucleosides was determined to be 74% for the u r a c i l and 56% for the thymidine by adding known volumes of pure la b e l d i r e c t l y to s c i n t i l l a n t and comparing actual counts to counts expected from the s p e c i f i c a c t i v i t i e s . At the end of the growth period, 0.1 ml "of the remaining culture medium was plated onto nutrient agar to test for b a c t e r i a l contamination; the remaining medium (about 8 ml) was passed through a 0.45 u M i l l i p o r e f i l t e r . The f i l t e r e d culture medium was centrifuged at 9,000 rpm for one hour with 0.2 ml of unlabelled VLP's and loaded onto an SW 41 gradient at pH 8.0 (10 to 40% sucrose i n 0.05 M T r i s Buffer pH 8.0) which was then centrifuged at 10,000 rpm for 30 minutes i n the Beckman L5-75 ultra c e n t r i f u g e . Resolved gradients were scanned at 254 nm i n an ISCO Model UA 5 absorbance monitor;, ten-1.2 ml fractions were coll e c t e d from each gradient. Each f r a c t i o n was dil u t e d with 2 ml of ice cold 10% TCA, incubated at 4 C for 30 minutes, and f i l t e r e d through Whatman 3 MM paper previously soaked i n 5% sodium pyrophosphate and dried. The f i l t e r s were batch rinsed i n 5% TCA for 30 minutes; the r i n s i n g was repeated twice. The f i l t e r s were then rinsed 2 to 3 times with cold ethanol and f i n a l l y with one change of ether and a i r dried. The experiment was repeated with both the thymidine and u r a c i l labels supplied at 10 uCi/ml and harvest of a l g a l c e l l s after one week of growth. Amicon 0.45 u f i l t e r s substituted for both the M i l l i p o r e f i l t e r s and the Whatman 3 MM paper after comparisons revealed no differences i n retention c h a r a c t e r i s t i c s . Heat shock experiments A growth experiment was performed twice to test for heat-induced release of VLP's. In the f i r s t experiment one-10 ml seeder test tube of mature U. gigas filaments was added to each of twelve-125 ml flasks containing 50 ml of Beijerinck's medium. Immediately aft e r seeding, eight of the flasks were held at 39 C for 6 hours with normal lighting? - four were maintained at the normal temperature of approximately 22 C. After heat treatment the flasks were transferred to a rotary shaker at low speed for two weeks of growth. Then the contents of each flask were i n d i v i d u a l l y f i l t e r e d through Whatman #1 f i l t e r paper; the culture medium was centrifuged at 9,0 00 rpm for one hour, and the r e s u l t i n g p e l l e t was taken up i n 2 ml of 0.05 M Tris-HCl pH 7.25. Numbers of VLP's released i n each f l a s k was determined by the electron microscopical counting procedure previously described. In the second experiment, zoospores were produced the night before seeding by adding ten-10 ml seeder tubes to 400 ml of fresh medium; i n the morning 10 ml of zoospore-containing medium was added to each of 24-125 ml f l a s k s . Twelve flasks were then heat^-shocked at 38 C for seven hours i n the dark, during what would normally be the culture's photoperiod. The twelve controls had the normal day-time temperature and l i g h t regime. Assay methods for VLP's at the end of two weeks were i d e n t i c a l to experiment 1. Alg a l c e l l s were collected by f i l t r a t i o n and pooled i n two groups, heat-shocked and controls, for assay of chlorophyll a. content. In l a t e r culturing of JJ. gigas, the heat shock at 3 8 C i n the dark, for 6 to 8 hours of the photoperiod, was routinely used to promote release of VLP's. -44-RESULTS I. Interaction of UGV with Uronema gigas A. Morphology of UGV and i t s host alga  Uronema gigas, Indiana University Culture C o l l e c t i o n number 17 4, was obtained i n 197 4 by Dr. J. Allan Dodds. Electron microscopic examination of sub-cultured c e l l s confirmed the presence of v i r u s - l i k e p a r t i c l e s (VLP's) i n the cytoplasm of a few c e l l s , as described by Mattox, Stewart, and Floyd (1972). Negative staining of aliquots of the medium in which the alga was grown revealed that similar VLP's had been released from the a l g a l c e l l s . Figures l a and lb show a healthy filament i n longitudinal and cross sections. A d i s t i n c t nucleus with nucleolus and i n t a c t nuclear membrane can be seen; mitochondria, ribosomes, and the large g i r d l i n g chloroplast containing a pyrenoid are also present. The c e l l s are bound by a unit membrane and a r i g i d c e l l wall; plasmodesmata between neighboring c e l l s can be seen i n Figure l a . Figures l c , d, and e show three c e l l s at r e l a t i v e l y late stages of i n f e c t i o n when c e l l u l a r membrane systems have become completely disorganized. VLP's (also referred to as UGV, for convenience) are scattered throughout the cytoplasm; no nuclear membrane can be detected, and degeneration of cytoplasmic and chloroplast membrane systems i s far advanced. The VLP's are unusual i n morphology and s i z e . As shown i n Figure 2b, approximately 10% of the released p a r t i c l e s have t a i l s which vary i n length to a maximum of approximately one -45-Figure 1. Thin sections of healthy and infected c e l l s of Uronema gigas. Tissue was fixed i n glutaraldehyde and osmium tetroxide, embedded i n Epon, and stained with uranyl acetate and lead c i t r a t e . a) Healthy filament showing several i n t a c t c e l l s . b) Cross-section of healthy c e l l . c) Cross-section of c e l l containing VLP's. d) Two-celled germling with VLP's. . e) Cross-section of c e l l containing VLP's. -46-F i g u r e 2. P u r i f i e d p a r t i c l e s of UGV i n negative s t a i n (5% phosphotungstic a c i d pH 7,2) a) VLP's with dense bo d i e s . b) VLP's a f t e r p u r i f i c a t i o n to s o l u b i l i z e dense bod i e s . c) VLP's wi t h p a r t i c l e s of TIV and TMV f o r s i z e comparisons. -47-Figure 3. a) High magnification photograph of a VLP within an a l g a l c e l l . Note membrane-like coat, non-homogeneous core. b) High magnification photograph of a p u r i f i e d VLP, without t a i l , i n negative s t a i n . c) Tailed VLP's within degenerating c e l l s of JJ. gigas. d) Higher magnification of (c). e) An early stage of i n f e c t i o n of VLP's. Note in t a c t chloroplast and angularity of VLP coats -'4 8-micron, and which o f t e n e x h i b i t a knob or round protruberance at about h a l f t h e i r length. Seen i n c r o s s - s e c t i o n s w i t h i n the c e l l s , the UGV head or capsid appears to be i c o s a h e d r a l ; pentagonal and hexagonal forms are both seen i n the VLP's shown i n Figure 1, suggesting the various planes of c u t t i n g an icosahedron, as described by Home (1974). The VLP's vary i n the f u l l n e s s of the c a p s i d ; o f t e n a capsid appears to have a c e n t r a l empty space, w i t h a s t r o n g l y o s m i o p h i l i c area next to the VLP coat and d e l i c a t e f i b e r s f i b e r s r a d i a t i n g throughout the c e n t r a l area. The outer s h e l l of the UGV p a r t i c l e i s a l s o of i n t e r e s t : i t appears to c o n s i s t of a membranous l a y e r , somewhat resembling a c e l l u l a r plasmalemma, however i t a l s o shows a c e r t a i n r i g i d i t y i n shaping i n t o the hexagonal forms seen i n s e c t i o n s (e.g. Figure 3a). F i v e or s i x rounded v e r t i c e s , s t r o n g l y n e g a t i v e l y s t a i n i n g , were o c c a s i o n a l l y seen, embedded i n or attached to the outer s h e l l (Figure 3b). The diameter of the VLP's was approximately 390 nm when p a r t i c l e s were f i x e d i n glutaraldehyde before negative s t a i n i n g or embedding; VLP diameter ranged up to 590 nm under v a r y i n g c o n d i t i o n s of pH and s t a i n (see Table I X ) . B. Cytopatholoqy V i r u s - l i k e p a r t i c l e s were u s u a l l y found i n c e l l s at advanced stages of degeneration, w i t h l i t t l e o r g a n i z a t i o n of membranes l e f t . An i n t a c t , normal nucleus, as seen i n Figures l a and l b , was u s u a l l y present i n s e c t i o n s of healthy a l g a l c e l l s , but was never observed i n an i n f e c t e d c e l l , even at e a r l y stages of VLP maturation. The r o l e of the nucleus i n e a r l y "49-events of UGV r e p l i c a t i o n i s not clear however, since the breakdown of the nuclear membrane i s followed by di s i n t e g r a t i o n of a l l the major c e l l u l a r membrane systems, including the lamellae of the chloroplast, at l a t e r stages of UGV i n f e c t i o n . A very early stage of i n f e c t i o n i s shown i n Figure 3e. The photosynthetic lamellae appear normal. There i s no clear demarcation of the nucleoplasm. Outer s h e l l s of the UGV p a r t i c l e can be seen forming i n pieces with the angularity of completed polyhedra, enclosing some ground substance which appears almost i d e n t i c a l to the surrounding material. A h a l o - l i k e , clear area i s sometimes apparent around the outer edge of the developing p a r t i c l e s . T a i l s were occasionally observed on developing UGV p a r t i c l e s with JJ. gigas c e l l s (Figures 3c and d) . The sequence of capsid assembly, packing, and t a i l attachment i s not clear from the few c e l l s observed i n section at intermediate stages of UGV development. V i r u s - l i k e p a r t i c l e s were observed i n a l g a l germling c e l l s only; 'none of the zoospores or expanded c e l l s of filaments which were sectioned ever showed signs of i n f e c t i o n . It i s possible that the s e t t l i n g germling i s the most susceptible stage i n the l i f e cycle of JJ. gigas for VLP release and that i n d i v i d u a l germlings which become infected at t h i s stage f a i l to develop further. Examination of t h i n sections of heat-shocked populations of germlings revealed c e l l s containing VLP's at about f i v e times the frequency of scans of mixtures of c e l l s at a l l stages i n the l i f e cycle of the alga, prepared without -50-heat shock. About one out of twenty heat-shocked germlings that were sectioned contained VLP's at some stage of assembly. C. Scanning electron microscopy of germlings Newly released zoospores were allowed to s e t t l e on d i a l y s i s membranes at the surface of the culture medium i n small beakers. After one to two days of growth, the membranes were co l l e c t e d , fixed, and then c r i t i c a l - p o i n t - d r i e d for examination by scanning electron microscopy. No metal coating or treatments with heavy metal stains was done i n order to allow use of the x-ray microanalysis unit for characterization of the composition of the a l g a l c e l l s . A representative f i e l d of the young germlings i s shown i n Figure 4a. L i t t l e structure i s revealed beyond the general outline of the c e l l s ; one type of f i x a t i o n caused collapse of the chloroplast into an elongated cup-like form. At higher magnifications, about 5% of the germlings appeared to contain multiple, opaque spheres of roughly 500 nm diameter which could be seen through the c e l l wall and after crushing to p a r t i a l l y release the p a r t i c l e (Figure 4c), could be analysed separately. Elemental x-ray microanalyses consistently revealed the presence of more phosphorus i n the spheres as compared to a l g a l cytoplasm; potassium was somewhat increased as well. Figure 4d shows representative analysis of cytoplasm versus the dense spheres; the large peak occurs at 2.02 KeV, the energy l e v e l of phosphorus secondary electrons. The diameter of the spheres and t h e i r elemental phosphorus content suggest that they may be UGV p a r t i c l e s , fixed in. s i t u within infected c e l l s . Potassium and sodium occur with nucleic acids as counter-ions; the f i x a t i o n i n potassium glutaraldehyde would replace any sodium ions with potassium. An attempt was made to analyse p u r i f i e d VLP's by x-ray. P u r i f i e d VLP's, dried down d i r e c t l y onto a scanning electron microscope stub, could not be seen--the structure was lost--but x-ray analysis indicated the presence of phosphorus, s u l f u r , sodium, and calcium, when many applications of a solution of VLP's were applied to a carbon stub. Sulfur and calcium may be d i f f i c u l t to detect i n the minute quantities present i n a single p a r t i c l e , fixed i n s i t u i n the al g a l c e l l . - 5 2 -F i g u r e 4 . Scanning e l e c t r o n microscopy and x-ray m i c r o a n a l y s i s of JJ. gigas germlings. a) A l g a l germlings on d i a l y s i s membranes. b) Close-up of germling t i p c o n t a i n i n g opaque spheres. c) Crushed germling w i t h sphere made a c c e s s i b l e f o r x-ray m i c r o a n a l y s i s . d) Elemental a n a l y s i s of dense sphere (lower curve) compared to a l g a l cytoplasm (upper c u r v e ) . Large peak on l e f t i s at the energy l e v e l of phosphorus; the s m a l l e r peak on the r i g h t r e p r e s e n t s potassium. S 9.0U -53-5n Figure 5. Growth of U. g igas and re lease of VLP's in two c u l t u r e media: B e i j e r i n c k ' s medium (BJ), and Br i s t o l ' s med ium (BR). -54-I I . I s o l a t i o n and p u r i f i c a t i o n o f UGV A. G r o w t h o f t h e h o s t a l g a J J . g i g a s was gr o w n i n l a r g e v o l u m e s o f B e i j e r i n c k ' s m i n e r a l medium f o r p u r i f i c a t i o n o f l i b e r a t e d V L P ' s . U n d e r t h e e n v i r o n m e n t a l c o n d i t i o n s a v a i l a b l e - - n o n - s h a k i n g c u l t u r e s , e x p o s e d t o a p p r o x i m a t e l y 5000 l u x d u r i n g t h e p h o t o p e r i o d , a t 20 t o 23 C, m a x i m a l v i r u s r e l e a s e was o b s e r v e d a f t e r a b o u t two months o f g r o w t h , w i t h o u t a d d i t i o n o f f r e s h medium ( r e s u l t s o f Dr . J.A. D o d d s ) . C o m p a r i s o n o f B e i j e r i n c k ' s medium w i t h a r i c h e r g r o w t h medium, ( B r i s t o l ' s medium, S t e i n 1 9 7 3 ) , r e v e a l e d t h a t i n t h e two month g r o w t h p e r i o d , b o t h a l g a l g r o w t h a n d VLP r e l e a s e w e r e g r e a t e r i n B e i j e r i n c k ' s medium. R e s u l t s a r e shown i n F i g u r e 5. When l a r g e v o l u m e s o f c u l t u r e d U. g i g a s w e r e n o t e s s e n t i a l , t h e a l g a was grown i n s m a l l f l a s k s on a r o t a r y s h a k e r , w h i c h s h o r t e n e d t h e g r o w t h p e r i o d t o a p p r o x i m a t e l y one month. W i t h o u t an i n f e c t i v i t y a s s a y , t h e e l e c t r o n m i c r o s c o p e c o u n t i n g p r o c e d u r e ( d e t e r m i n a t i o n o f a "VLP i n d e x " ) h a d t o be r e l i e d upon f o r a c c u r a t e e s t i m a t i o n o f VLP c o n c e n t r a t i o n p e r c u l t u r e and o v e r t h e c o u r s e o f v a r i o u s p u r i f i c a t i o n p r o c e d u r e s . The c o u n t i n g m e thod u s e d was r e l a t i v e l y c r u d e c o m p a r e d t o s p r a y d r o p l e t t e c h n i q u e s f o r a c c u r a t e e l e c t r o n m i c r o s c o p i c a l d e t e r m i n a t i o n s o f p a r t i c l e s p e r u n i t v o l u m e ( H a s c h e m e y e r and M e y e r s 1 9 7 2 , S h a r p 1 9 7 5 ) . However, t h e number o f a s s a y s o f VLP c o n c e n t r a t i o n t o be d o n e , e s p e c i a l l y t h r o u g h t h e s t e p s o f a p u r i f i c a t i o n , d i c t a t e d t h e u s e o f a s i m p l e r m e t h o d . Some -55-v e r i f i c a t i o n o f t h e a c c u r a c y o f o u r t e c h n i q u e was o b t a i n e d by c o u n t i n g a d i l u t i o n s e r i e s o f T i p u l a i r i d e s c e n t v i r u s as w e l l as t h e a l g a l V L P ' s . R e s u l t s a r e p r e s e n t e d i n T a b l e I V . One f u r t h e r d r a w b a c k o f t h i s m e t hod o f d e t e r m i n i n g VLP c o n c e n t r a t i o n was t h e p o o r b i n d i n g o f V L P ' s t o a g r i d s u r f a c e i n t h e p r e s e n c e o f d e t e r g e n t s . P u r i f i c a t i o n s t e p s i n v o l v i n g u s e o f d e t e r g e n t c o u l d n o t by a s s a y e d a c c u r a t e l y f o r VLP c o n c e n t r a t i o n . A c o n s i s t e n t o b s e r v a t i o n f r o m r e p e a t e d h a r v e s t s o f U_. g i g a s was t h a t VLP s y n t h e s i s and r e l e a s e was h i g h l y v a r i a b l e i n t e r m s o f a c t u a l numbers o f p a r t i c l e s l i b e r a t e d and i n t e r m s o f VLP i n d e x p e r u n i t o f a l g a l g r o w t h , e x p r e s s e d as m i l l i g r a m o f c h l o r o p h y l l a p e r m i l l i l i t r e o f c u l t u r e medium ( T a b l e V ) . Numbers o f V L P ' s r e l e a s e d c o u l d n o t be a b s o l u t e l y c o r r e l a t e d w i t h c u l t u r e a g e , and o n l y i n a b r o a d s e n s e w i t h a d e c l i n e i n c h l o r o p h y l l a c o n t e n t . D r . Dodds i s o l a t e d 49 s u b c u l t u r e s by s i n g l e z o o s p o r e t r a n s f e r ; w h i l e none o f t h e i s o l a t e s had l o s t t h e c a p a c i t y t o r e l e a s e V L P ' s , t h e r e was a w i d e r a n g e o f r a t e s o f r e l e a s e . H i g h v i r u s - y i e l d i n g s u b c u l t u r e s w e r e s e l e c t e d f o r a l g a l g r o w t h f o r v i r u s p u r i f i c a t i o n . E x t e n s i v e t e s t i n g t o e n s u r e t h a t t h e r a t e o f VLP l i b e r a t i o n was a s t a b l e c h a r a c t e r i s t i c o f e a c h i s o l a t e was n o t p e r f o r m e d . B. P u r i f i c a t i o n o f t h e V L P ' s I n i t i a l a t t e m p t s t o p u r i f y t h e VLP f r o m J J . g i g a s w e r e h a m p e r e d by f o u r f a c t o r s : 1. t h e p r e s e n c e i n t h e g r o w t h medium o f s p h e r e s a p p r o x i m a t e l y one f i f t h t h e d i a m e t e r o f t h e V L P , b u t i d e n t i c a l t o t h e VLP i n d e n s i t y (shown i n F i g u r e 2a) ; "56" TA'BLE IV -- Electron microscope counting technique for assay of VLP concentration Concentration (ug/ml) of d i l u t i o n of preparation P a r t i c l e s per tr a n s e c t 3 P a r t i c l e d i l u t i o n TIV 6.9 102 102 3.5 43 86 1.8 25 100 UGV 1:5 70 350 1:10 39 390 1:50 6.6 330 1:100 3.2 320 Average of p a r t i c l e s counted i n 10 gr i d square transects. TABLE V — Release of UGV p a r t i c l e s i n r e l a t i o n to a l g a l growth, measured by chlorophyll a. content per ml of culture medium.3 Age of culture (weeks) Chlorophyll a content (mg/ml) VLP index VLP index chi a_ (mg 9 2.7 1100 407 8 3.2 392 123 8 2.8 244 87 10 1.1 70 64 8 3.2 200 63 10 2.8 150 54 7 2.1 112 53 9 3 . 2 165 52 8 3.3 70 21 8 1.9 40 21 6 5.5 37 7 8 3.7 16 4 6 4.6 8 2 Data from thirteen representative harvests. -57-2. the low concentrations of VLP's i n large volumes of culture medium; 3. the tendency of the VLP's to aggregate i r r e v e r s i b l y at pH 5.5 or below, when present i n high concentrations during a p u r i f i c a t i o n ; and 4. the presence of a l g a l c e l l u l a r debris i n the medium (even though no homogenization of a l g a l c e l l s had been done). Methods assayed to circumvent these problems are detailed i n Table VI. This table describes r e s u l t s of various p u r i f i c a t i o n s i n terms of VLP qu a l i t y rather than quantity since, as previously mentioned, harvests of U. gigas varied widely and unpredictably i n numbers of VLP's released. Direct comparisons of y i e l d s of VLP's from harvest to harvest would not be meaningful as a measure of the e f f i c i e n c y of a p a r t i c u l a r p u r i f i c a t i o n procedure. The f i n a l procedure developed i s presented i n Figure 6 with a representative example of the r e l a t i v e amounts of VLP's at each stage of p u r i f i c a t i o n . A l g a l filaments were f i r s t removed from the culture medium by f i l t r a t i o n through bolting s i l k , followed by membrane or paper f i l t r a t i o n to remove zoospores. C e l l s were reserved for determinations of chlorophyll aj the f i l t r a t e was centrifuged at 9,000 rpm for one hour to p e l l e t VLP's, dense bodies, and some debris. The dense bodies were p u r i f i e d by two cycles of d i f f e r e n t i a l centrifugation of a harvest which contained very few VLP's. Energy-dispersive spectroscopy of the dense bodies, kindly performed by Mr. George J. Georgakopoulos of the Department of Geology, U.B.C., demonstrated that iron , calcium, -58-TABLE VI -- Comparison of concentration and p u r i f i c a t i o n methods f o r UGV as determined by e l e c t r o n microscopy of ' prep a r a t i o n s . Method assayed F i l t r a t i o n , then 10,000 x g f o r one hour Dense bodies Aggregated p a r t i c l e s C e l l u l a r d e b r i s + Loss of p a r t i c l e s + Sucrose cushion f o r + concentr a t i o n and f u r t h e r p u r i f i c a t i o n 45-90% sucrose ± de n s i t y g radient c e n t r i f u g a t i o n Polyethylene g l y c o l (PEG) p r e c i p i t a t i o n df VLP 1s from medium 4% PEG and 0.25 M NaCl 4% PEG, no NaCl + + + No VLP 1s were p r e c i p i t a t e d , w i t h or without NaCl. PEG/sucrose i n v e r s e g r a d i e n t s : 10% sucrose, 10% PEG to 40% sucrose, 2.5% PEG + Na acetate b u f f e r , pH ± 5.5 to resuspend VLP p e l l e t a f t e r c e n t r i f u g a t i o n To reverse aggregation of VLP's: added 0.01 M EDTA added 0.03 M MgCl s o n i c a t i o n added Igepon T-73, a detergent 0.01 M EDTA added to resuspension b u f f e r , pH 5.5 Sodium c i t r a t e or Sodium acetate b u f f e r , pH 6.0 f o r resuspension of VLP' s N/Ac N/A N/A N/A No change No change No change + + + + N/A N/A N/A N/A (made soluble) (made soluble) - 5 9 * .Method assayed Dense bodies Aggregated p a r t i c l e s 0.05 M sodium c i t r a t e / 0.01 M EDTA pH 6.0 (made soluble) f o r resuspension of VLP's Ammonium s u l f a t e + p r e c i p i t a t i o n of VLP's (assayed 15 to 55%) Potassium t a r t r a t e / g l y c e r o l gradient c e n t r i f u g a t i o n ( O b i j e s k i et a l . 1974) T r i t o n X-100 det e r - -gent added at 0.5% with citrate/EDTA b u f f e r + C e l l u l a r d e b r i s + + Loss of p a r t i c l e s Very few l o s t . not a p p l i c a b l e . e l e c t r o n microscope assay was u n r e l i a b l e i n the presence of detergent. -60-PURIFICATIQN OF UGV Relative yields: 100 % | Filtr ation F cells chlorophyll a assay f iltVatc I Centrifuga T 10,000 g ; 1  t ion hour JE pellet + resuspend in Citrate! EDTA! Triton buffer I dialysis ^ Centrifugation r supernatant (discard) 7 4 % supernatant (discard) 1 pellet J resuspend in Tris-HCI buffer i 56% dialysis I Sucrose density gradient centrifugation 10 000 g ; 30 minutes I collect band of VLP's dialysis 4 2 % • store in Tris-HCI Figure 6. F ina l p u r i f i c a t i o n procedure fo r UGV p a r t i c l e s . For d e t a i l s see Mater ia l s and Methods. potassium, manganese, and copper were present. The technique i s not q u a n t i t a t i v e . I t proved p o s s i b l e to remove the dense bodies by c h e l a t i o n w i t h EDTA at a pH below 6.0; however, extensive aggregation of VLP's r e s u l t e d i f the c h e l a t i o n step was prolonged, e.g. over 48 hours. A l g a l d e b r i s was removed during the c h e l a t i o n step by the a d d i t i o n of 0.5% T r i t o n X-100 detergent to the EDTA-containing b u f f e r . A f t e r a second c e n t r i f u g a t i o n , the VLP's were resuspended i n a 0.05 M T r i s - H C l b u f f e r at pH 8.0 to l i m i t aggregation of the VLP's. Loss of VLP's over the course of a p u r i f i c a t i o n was due mainly to aggregation and consequent p e l l e t i n g i n c e n t r i f u g a t i o n steps. When adequate amounts of VLP's were a v a i l a b l e a f t e r the d i a l y s i s step of p u r i f i c a t i o n , the p a r t i c l e s were f u r t h e r p u r i f i e d by d e n s i t y gradient c e n t r i f u g a t i o n on 10-40% or 20-50% sucrose gradients at pH 8.0. A r e p r e s e n t a t i v e scan of resolved gradients i s shown i n Figure 7a. Suspensions of TIV were always c e n t r i f u g e d i n one gradient of each run as a standard. Comparing the two curves, i t i s immediately apparent t h a t the VLP from JJ. gigas i s much l e s s homogeneous i n the p r o p e r t i e s which a f f e c t sedimentation than i s TIV. Bands from the gradients of UGV were always broad, o c c a s i o n a l l y r e s o l v i n g i n t o two separate areas, as shown i n Figure 7b. By e l e c t r o n microscopy, the upper band appeared to c o n t a i n p a r t i a l l y empty VLP cap s i d s , and some p a r t i a l l y degraded p a r t i c l e s ; the lower band contained complete p a r t i c l e s which looked homogeneous i n morphology. T a i l e d p a r t i c l e s were d i s t r i b u t e d i n both bands. In some runs a f a s t e r sedimenting band c o n t a i n i n g aggregates of the VLP's were detected. The r e l a t i o n s h i p between sedimentation depth, -62-A. 0.01 sodium acetate pH5.5 12,000 g x 26 min B. 0.05 M Tris-HCI 10,000 g x I. empty VLP's II. incomplete VLP's , tails III-intact VLP's , tails Bottom F i g u r e 7- C e n t r i f u g a t i o n o f UGtf p a r t i c l e s i n 10-40% s u c r o s e g r a d i e n t s . -6 3-UV absorbance, and p a r t i c l e counts of UGV i s shown i n Figure 7a. The area of the gradient with' the highest absorbance at 254 nm also contained the greatest number of p a r t i c l e s by electron microscopy. Alga l c e l l disruption was attempted by various methods described i n Table VII to l i b e r a t e increased amounts of VLP's. Alga l c e l l s were d i f f i c u l t to homogenize; most methods were only p a r t i a l l y e f f e c t i v e . The only procedure which resulted i n disrupted c e l l s , sonication followed by freezing and thawing, showed no increased y i e l d of VLP's. Because of t h i s r e s u l t , no further attempts were made at homogenization. C. Heat shock e f f e c t s on VLP y i e l d The re s u l t s of the two heat shock experiments are shown i n Table VIII. In the f i r s t experiment, i n which heat shock was applied during the normal photoperiod, no s i g n i f i c a n t increase i n the number of released VLP's was observed between heat-shocked and control f l a s k s , although the number of VLP's released per heat-shocked flask had a higher mean. The wide v a r i a b i l i t y of numbers of VLP's released among each treatment could-have been due to the differences i n maturity of the seeding c e l l s supplied; i n th i s experiment a mixture of mature filaments, young germlings, and zoospores was used to begin the culture. In the second experiment the heat shock was administered i n darkness during what would have been a normal photoperiod. There were approximately six times as many VLP's released from heat-shocked flasks' as from the controls, comparing the means. Control flasks had an average chlorophyll e -64-TABLE V I I — C e l l d i s r u p t i o n methods assayed f o r U. gigas Grinding w i t h sand Grinding w i t h l i q u i d n i t r o g e n Homogenization w i t h Waring blendor S o n i c a t i o n of c e l l s S o n i c a t i o n followed by f r e e z i n g and thawing + TABLE V I I I — Heat-shock e f f e c t s on UGV r e l e a s e Method C e l l u l a r d i s r u p t i o n Release of VLP's Contro l s Experiment 1 L i g h t at 38 C 4 1 ± 5 0 a ( 4 ) b Dark at 38 C 36±5 (12) Experiment 2 Chi a content 3.2 mg/ml Heat-shocked 98193 (8) 2 2 8 1 8 0 ( 1 2 ) ° 2.5 mg/ml a F i g u r e s are given i n terms of "VLP index": a c t u a l numbers of VLP's counted converted to a 100 X concentr a t i o n of the o r i g i n a l volume of c u l t u r e medium. The means of each group, plus or minus one standard d e v i a t i o n , are given, Figures i n parentheses r e f e r to numbers of f l a s k s assayed. Heat shock was administered i n the dark during what would be a normal photoperiod. -6 5-content of 3.2 mg/ml; heat-shocked flasks averaged 2.5 mg/ml of chlorophyll a_. Both the controls and the heat-shocked cultures showed less v a r i a b i l i t y i n VLP numbers released than i n experiment one, perhaps because these cultures were begun with a uniform suspension of freshly released zoospores only. In l a t e r culturing of JJ_. gigas for virus p u r i f i c a t i o n , a six to eight hour heat shock i n darkness was included after zoospore release, based on the increase i n y i e l d of VLP's observed i n the second experiment. -66-I I I . P a r t i a l characterization of UGV and i t s components A. Dimensions of UGV Data for measurements of UGV are presented i n Table IX. P a r t i c l e s were measured in. s i t u i n infected c e l l s and i n  v i t r o after p u r i f i c a t i o n . Measurements were made under a variety of conditions of stain and pH, Aside from the data shown, i n which uranyl acetate (UA) and phosphotungstic acid (PTA) stains were used, uranyl formate, potassium molybdate, and potassium permanganate stains were also tried, at various pH's. Effects on VLP dimensions were similar and resolution of the structure of the VLP's was not as good i n most cases. V i r u s - l i k e p a r t i c l e s which were fixed with glutaralde-hyde (and often osmium tetroxide as well) before negative staining or embedding and sectioning showed an edge-to-edge diameter of about 390 nm. Fixation and sectioning without post-staining showed a diameter of about 350 nm, suggesting that an outer layer of the coat of the p a r t i c l e was not v i s u a l i z e d by t h i s method. Dimensions of p u r i f i e d VLP's i n negative stai n seemed to be highly dependent on pH conditions and type of s t a i n . Two percent UA at pH 6,0 revealed a VLP diameter of 390 nm, i d e n t i c a l to the fixed diameter. Two percent PTA at pH 6.0 to pH 7.0 showed p a r t i c l e s of about 480 nm diameter. UGV p a r t i c l e s which had been stored i n buffer at pH 8.0 and then stained with pH 7.0 PTA enlarged even further to about 550 to 5 90 nm i n diameter; TIV p a r t i c l e s showed a similar enlargement from 13 9 to 17 2 nm after storage at pH 8.0. Other authors (Bellett 196 8) have also reported enlargement of -67-TABLE IX — UGV Measurements Mean Length (nm) ± Dimension measured Fixation Stain and pH Storage buffer and pH Number measured standard deviation UGV diameter i n v i t r o g l u t a none 0.05 M T r i s -HCI, pH 8.0 42 355 ± 39 UGV diameter i n s i t u g l u t / 0s0 4 UA/Pb — 29 389 ± 37 UGV diameter i n vitro* 3 glut UA,pH 6 . 0 0.01 M Na acetate pH 6.0 390 UGV diameter i n v i t r o D glut PTA, pH 7.2 0.01 M Na acetate pH 6.0 390 UGV diameter i n v i t r o none PTA pH 5.5 0.01 M KPO buffer, pH 6.0 9 475 ± 57 TIV diameter none II II II 10 139° ! ± 15 UGV diameter'0 none PTA, pH 7.2 0.01 M KPO. pH 7.0 4 490 UGV diameter TIV diameter none none PTA pH 7 . 2 0.05 M T r i s -HCI pH 8.0 50 40 595 171 ± 66 ± 17 UGV diameter TIV diameter Latex beads^ none none none PTA pH 7.2 0.05 M T r i s -HCI pH 8.0 17 5 20 550 173 775 ± 49 ± 20 ± 62 Stains and f i x a t i v e s abbreviated as follows: PTA i s 2% phosphotungstic acid, used as a negative stai n ; UA i s 2% uranyl acetate, also a negative stai n ; glut i s 5% glutaraldehyde, used as a f i x a t i v e with or without OsO., 2% osmium tetroxide; and UA/Pb i s uranyl acetate followed by Reynold's lead c i t r a t e s t a i n , used on thin sectioned material. Measurements of June 1975 done by Dr. J.A. Dodds. GPublished TIV diameter i s 130 nm (Kalmakoff and Tremaine 1968). ^Latex beads (Sigma) were of average diameter 790 nm according to manufacturer's description. T 6 8 - , TIV p a r t i c l e s from 130 nm t o 170 nm i n n e g a t i v e l y s t a i n e d p r e p a r a t i o n s , w i t h o u t p r i o r f i x a t i o n . I n d i v i d u a l s i d e s o f th e UGV h e x a g o n a l p a r t i c l e a l s o e x h i b i t e d s w e l l i n g : from 2 20 nm i n a s t o r a g e b u f f e r a t pH 7.0 t o 27 0 nm i n pH 8.0 b u f f e r . Lengths o f s i d e s o f h i g h l y s w o l l e n p a r t i c l e s were d i f f i c u l t t o measure a c c u r a t e l y . T a i l l e n g t h s and d i a m e t e r s p r o b a b l y do n o t i n c r e a s e as much p r o p o r t i o n a t e l y as t h e VLP heads. T a i l l e n g t h s were e x t r e m e l y v a r i a b l e , most l i k e l y due t o breakage d u r i n g p u r i f i c a t i o n , w i t h maximum l e n g t h s c l o s e t o one m i c r o n ; the t a i l w i d t h i s a p p r o x i m a t e l y 45 nm. The "knob" was a p p a r e n t on a l l l o n g e r t a i l s , b u t many t a i l s seemed t o have brok e n between t h e knob and t h e head, l e a v i n g o n l y a s h o r t stump. A s h o r t e n e d , w i d e r t a i l s u g g e s t i v e o f c o n t r a c t i l i t y was never o b s e r v e d . Measurements were c a l i b r a t e d by the use o f TIV p a r t i c l e s (130 nm i n d i a m e t e r ) , t o b a c c o mosaic v i r u s p a r t i c l e s (300 nm), and l a t e x beads (790 nm d i a m e t e r ) ( F i g u r e 2 c ) . Measurement o f t h e d i a m e t e r s o f 20 l a t e x beads gave an average o f 775 nm, a d i f f e r e n c e o f o n l y 2%, Tobacco mosaic v i r u s l e n g t h s were t o o v a r i a b l e , due t o breakage and a g g r e g a t i o n , t o be u s e f u l as s i z e m a r k e r s . The d i a m e t e r o f 390 nm f o r UGV f i x e d by g l u t a r a l d e h y d e i n s i t u was c o n s i d e r e d t o be most u s e f u l f o r d e s c r i p t i o n o f t h i s VLP, g i v e n t h e s w e l l i n g o b s e r v e d under v a r i o u s c o n d i t i o n s o f s t a i n and pH. B. E l e c t r o n m i c r o s c o p y and enzyme d i g e s t i o n o f p e l l e t e d VLP's  P u r i f i e d VLP's from JJ. g i g a s were f i x e d and embedded f o r e l e c t r o n m i c r o s c o p y and the n examined i n t h i n s e c t i o n . A -69-r e p r e s e n t a t i v e group of p a r t i c l e s i s shown i n Figure 8a. As i n i n f e c t e d c e l l s , 5- and 6- sided p r o f i l e s of the membrane-like coat were observed w i t h a v a r y i n g f u l l n e s s i n s i d e the coat. The diameter corresponded to the 390 nm diameter of i n t r a c e l l u l a r VLP's, or VLP's f i x e d before negative s t a i n i n g . No t a i l e d p a r t i c l e s were observed by t h i s method, perhaps due to the repeated c e n t r i f u g a t i o n s needed to r e - e s t a b l i s h a p e l l e t i n the course of f i x a t i o n and dehydration. I n i t i a l l y , p e l l e t e d VLP's were examined to v e r i f y t h a t the p a r t i c l e s which were being p u r i f i e d were i d e n t i c a l to the o r i g i n a l p a r t i c l e s observed by Mattox et a l . (1972). Both s i z e measurements and u l t r a s t r u c t u r a l d e t a i l s suggested t h a t the VLP's p u r i f i e d were the same as those described i n the e a r l i e r paper. The d i s t r i b u t i o n of the major biochemical components of the UGV p a r t i c l e was determined by s p e c i f i c enzyme d i g e s t i o n i n t h i n s e c t i o n s of p u r i f i e d p e l l e t s of VLP's. P e l l e t s of VLP's were f i x e d i n f o r m a l i n alone, f o r m a l i n and a c r o l e i n , or glutaraldehyde and were embedded i n the h y d r o p h i l i c medium, g l y c o l methacrylate (GMA, Leduc and Bernard 1967). The a l t e r e d f i x a t i o n and embedding had a dual purpose; to r e t a i n molecular s t r u c t u r e during f i x a t i o n i n order to maintain the c o n f i g u r a t i o n s recognized by s p e c i f i c enzymes, and to enclose the t i s s u e to be s t u d i e d i n a h y d r o p h i l i c medium to allow f o r exposure to the enzymes, which were prepared i n aqueous s o l u t i o n s . U n f o r t u n a t e l y , g e n t l e r f i x a t i o n s and the use of GMA reduced the r e s o l u t i o n and c o n t r a s t of s t r u c t u r e s viewed by e l e c t r o n microscopy. P u r i f i e d VLP's prepared using these methods are shown i n Figure 8b. -70-Figure 8. Enzyme treatments of embedded p u r i f i e d VLP's. a) P e l l e t e d VLP's, f i x e d i n glutaraldehyde and osmium t e t r o x i d e and embedded i n Epon; stained w i t h u r a n y l acetate and lead c i t r a t e . b) P e l l e t e d VLP's, f i x e d i n f o r m a l i n / a c r o l e i n and embedded i n g l y c o l methacrylate. Normal s t a i n i n g . c) As i n (b), incubated 10 minutes w i t h pronase. d) As i n (b), incubated two hours i n pronase. e) As i n (b), incubated w i t h RNAse f o r two hours. f) As i n (b), incubated w i t h DNAse f o r two hours. - 7 1 -Enzyme t r e a t m e n t s - a r e shown i n F i g u r e s 8 c , d , e, and f . P l a t e s 8c and d show t h e r e s u l t s o f 1 0 - m i n u t e and 2 - h o u r d i g e s t i o n w i t h p r o n a s e . C l e a r l y , t h e d e n s e , h e a v i l y s t a i n i n g a r e a i n t h e c e n t e r o f t h e p a r t i c l e c o n t a i n e d p r o t e i n ; a f t e r l o n g d i g e s t i o n s t h e c e n t r a l c o n t e n t s o f t h e V L P ' s w e r e c o m p l e t e l y r e m o v e d and t h e c o a t l o o k e d c o n s i d e r a b l y t h i n n e r . The r e s u l t s o f d i g e s t i o n s u s i n g t h e n u c l e a s e s a r e shown i n 8e (RNase) and 8 f ( D N a s e ) . (Some d i g e s t i o n o f t h e c o n t e n t s o f t h e V L P ' s was s e e n a f t e r DNAase t r e a t m e n t , as i n d i c a t e d by a s l i g h t l y c l e a r e d a r e a ; no d i g e s t i o n was o b s e r v e d w i t h RNAase t r e a t m e n t . D i g e s t i o n p e r i o d s r a n g e d f r o m 10 m i n u t e s t o 12 h o u r s ; f o r DNAase t h e m o s t m a r k e d e f f e c t was o b s e r v e d a f t e r p r e - s o a k i n g t h e s e c t i o n s i n b u f f e r f o l l o w e d by d i g e s t i o n f o r 2 h o u r s a t 37 C. The e f f e c t s o f one l i p i d - d i g e s t i n g enzyme, l i p a s e 4 48, w e r e a l s o a s s e s s e d . No d i g e s t i o n was o b s e r v e d o f any component o f t h e V L P . Two o t h e r i n d i r e c t t e c h n i q u e s w e re u s e d i n a t t e m p t s t o d e t e c t t h e p r e s e n c e o f l i p i d u s i n g e l e c t r o n m i c r o s c o p y o f p e l l e t e d V L P ' s . I n t h e f i r s t m e t h o d , r u t h e n i u m r e d s t a i n i n g f o r l i p i d s a n d c a r b o h y d r a t e s was t r i e d ( H a y a t 1 9 7 0 , and K o b a y a s h i and A s b o e - H a n s e n 1 9 7 1 ) , b o t h as a f i x a t i o n t e c h n i q u e , and f o r s t a i n i n g o f t h i n s e c t i o n s . R e s u l t s w e r e i n c o n c l u s i v e . A l l p o r t i o n s o f t h e V L P ' s w e r e s t a i n e d , b u t i n d i s t i n c t l y and w i t h a g r e a t d e a l o f b a c k g r o u n d d i r t . I n t h e s e c o n d m e t h o d , VLP p r e p a r a t i o n s w e r e t r e a t e d w i t h two d i f f e r e n t l i p i d s o l v e n t s p r i o r t o f i x a t i o n and e m b e d d i n g i n E p o n . P r e p a r a t i o n s o f V L P ' s w e r e s h a k e n w i t h e t h e r o r s t i r r e d o v e r n i g h t w i t h c h l o r o f o r m : m e n t h a n o l (2:1) b e f o r e p e l l e t i n g and f i x a t i o n . The r e s u l t s o f t h e s e p r o c e d u r e s a r e shown i n F i g u r e 9 a , b , and c . E t h e r -7 2-Figure 9. Sectioned p e l l e t s of UGV, pre-treated with l i p i d solvents. a) No treatment. Usual f i x a t i o n , embedding, and s t a i n . b) Pre-treated with ether. c) Pre-treated with chloroform/methanol ( 2 : 1 ) . treatment (9b) had no marked e f f e c t . Chloroform/methanol treatment (9c) caused extensive disruption of normal VLP structures, and aggregations of disorganized p a r t i c l e s were always seen, with long extensions of material resembling the membranous VLP coat. One other attempt was made to determine the presence of l i p i d , and i s described i n the section on the electrophoresis of proteins. C. Light scattering correction Optical absorbance of the U_. gigas VLP's from 240 to 310 nm, corrected for l i g h t scattering absorbance due to the size of the p a r t i c l e , was determined by the method of Noordam (1973). By t h i s method a l l absorbance above 310 nm i s considered to be due to l i g h t scatter. A l i n e i s extrapolated back below 310 nm using the slope of the absorbance above 310 nm to determine the portion of the absorbance i n the lower wavelength region which i s due to l i g h t scatter. Results of t h i s correction are shown i n Figure 10. Light scattering properties vary from preparation to preparation, dependent on the degree of aggregation of the p a r t i c l e s ; absorbance due to l i g h t scatter varied from 74% to 87% of t o t a l absorbance i n d i f f e r e n t preparations. Corrected A„,„ /Aor>_ ^ ^ 26 0nm 2 80nm rat i o s were i n the range of 1.20 to 1.29, showing l i t t l e increase over the uncorrected values of 1.14 to 1.21. When a rough estimate of VLP concentration based on absorbance was needed, 2 0% of t o t a l absorbance was considered to be due to true absorbance by the p a r t i c l e s . A corresponding figure of 54% was used for true absorbance by TIV (Kalmakoff and Tremaine -74-N M F i g u r e 10. Absorbance o f one UGV s u s p e n s i o n b e f o r e and a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g a b s o r b a n c e . 1968) . -75-D. S e d i m e n t a t i o n c o e f f i c i e n t The s e d i m e n t a t i o n c o e f f i c i e n t (s„_ ) o f t h e VLP was 20 ,w det e r m i n e d i n f i v e s e p a r a t e c e n t r i f u g a t i o n s . R e s u l t s a r e summarized i n T a b l e X. TIV was u s u a l l y c e n t r i f u g e d as a ' s t a n d a r d i n t h e second c e l l o f t h e a n a l y t i c a l u l t r a c e n t r i f u g e . An average v a l u e o f 6350 S was o b t a i n e d f o r UGV; TIV averaged 2450 S. The r e p o r t e d s e d i m e n t a t i o n c o e f f i c i e n t o f TIV i s 2200 S ( B e l l e t t 1968); our h i g h e r v a l u e may r e f l e c t d i f f e r i n g c o n d i t i o n s o f c e n t r i f u g a t i o n . The s v a l u e f o r UGV was measured a t pH 5.5 i n 0.01 M a c e t a t e b u f f e r and a t pH 8.0 i n 0.05 M T r i s - H C I b u f f e r w i t h o u t s u b s t a n t i a l d i f f e r e n c e s b e i n g o b s e r v e d . I n s e v e r a l o t h e r r u n s , however, t h e s e d i m e n t a t i o n c o e f f i c i e n t c o u l d n o t be measured a t pH's lower than 6.0 due t o p a r t i c l e a g g r e g a t i o n , w h i c h r e s u l t e d i n e x t r e m e l y r a p i d p e l l e t i n g . A p p a r e n t l y , f r e e VLP's a t pH 5.5 o r pH 8.0 a r e s i m i l a r i n t h e p r o p e r t i e s which a f f e c t s e d i m e n t a t i o n , E. D e n s i t y i n s u c r o s e o f UGV The c o n c e n t r a t i o n o f cesium c h l o r i d e n e c e s s a r y f o r e q u i l i b r i u m g r a d i e n t c e n t r i f u g a t i o n was found t o aggregate and d i s r u p t UGV p a r t i c l e s . I n the o n l y cesium c h l o r i d e e q u i l i b r i u m r u n w h i c h was done, the c a l c u l a t e d d e n s i t y f o r TIV was 1.32 g/ml, i n agreement w i t h r e p o r t s i n t h e l i t e r a t u r e ( G l i t z e t a l . 196 8). The d e n s i t i e s o f UGV, TIV, and BSMV ( b a r l e y s t r i p e mosaic v i r u s ) were a l s o measured i n 45 t o 90% s u c r o s e g r a d i e n t s which had e q u i l i b r a t e d f o r 24 h o u r s , a l e s s a c c u r a t e d e t e r m i n a t i o n t h a i i n cesium c h l o r i d e . R e s u l t s a re shown i n F i g u r e 11. A f t e r 20 hours - 7 6 -10 15 20 25 Fraction number F i g u r e 11. E q u i l i b r i u m c e n t r i f u g a t i o n o f i n 45-90% s u c r o s e g r a d i e n t s , d e t e r m i n e d by r e f r a c t o m e t r y . UGV, T I V , and BSMV S u c r o s e c o n c e n t r a t i o n s -77-TABLE X — Sedimentation c o e f f i c i e n t of UGV S20. w Buffer conditions rpm TIV UGV 0.01 M acetate pH 5. 5 4059 2300 6400 0.01 M acetate pH 5. 5 4059 2340 6200 0.01 M acetate pH 5. 5 4059 not done 6000 0.05 M Tris-HCl pH 8 .0 4059 2700 6650 0.05 M Tris-HCl pH 8 .0 6166 2450 6450 Average S20 values: ,w 2450 6340 TABLE XI — UGV polypeptides detected by SDS-polyacrylamide electrophoresis Observation of protein bands proteih band Molecular weiaht Tube crels Per cent 5 % 7.5% Slab ael polyaerylamide 10% 10% l 26,000 daltons + + - + 2 30,000 - - + 3 32,000 - - - + 4 35,000 + + - + 5 37,000 - + + + 6 40,000 + + - + 7 42,000 - - - + 8 45,000 + + + + 9 53,000 + + + 10 64,000 + - + + - 7 8 -o f c e n t r i f u g a t i o n , T I V o c c u r r e d as a b a n d a t 1.2 8 g / m l , BSMV a t 1.265 g / m l , and UGV a t a p p r o x i m a t e l y 1,30 g/ml ( d e n s i t i e s d e t e r m i n e d by r e f r a c t o m e t r y ) . The l o w e r e d d e n s i t y o f T I V i n s u c r o s e may be due t o i o n i c e f f e c t s i n c r e a s i n g t h e d e n s i t y i n C s C l , o r may be a r t i f a c t u a l , i f t h e s e g r a d i e n t s h ad n o t c e n t r i f u g e d t o e q u i l i b r i u m . P a r t i c l e s o f UGV a p p e a r t o be somewhat d e n s e r t h a n T I V p a r t i c l e s . F. UGV p r o t e i n s P r o t e i n s o f UGV p a r t i c l e s w e r e a n a l y z e d by SDS-p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n c y l i n d r i c a l g e l s o f 5, 7.5, and 10% p o l y a c r y l a m i d e and on a 10% p o l y a c r y l a m i d e s l a b g e l . The V L P ' s w e r e d i s s o c i a t e d by b o i l i n g f o r 90 s e c o n d s i n a b u f f e r c o n t a i n i n g u r e a , SDS, and m e r c a p t o e t h a n o l , o r by i n c u b a t i o n o v e r n i g h t a t room t e m p e r a t u r e w i t h 1 N H C l . Ten p o l y p e p t i d e b a n d s w e r e d e t e c t e d by e l e c t r o p h o r e s i s i n t h e s l a b g e l ; many o f t h e b a n d s p r e s e n t i n t h i s g e l w e r e a l s o r e s o l v e d i n t h e c y l i n d r i c a l g e l s . A p r e d o m i n a n t p o l y p e p t i d e s p e c i e s o f 45,000 d a l t o n s was n o t e d i n a l l g e l r u n s . R e s u l t s a r e s u m m a r i z e d i n T a b l e X I and a r e p r e s e n t a t i v e g e l i s shown i n F i g u r e 12. A m i c r o d e n s i t o m e t e r s c a n o f a p h o t o g r a p h i c n e g a t i v e o f t h e s l a b g e l i s shown i n F i g u r e 13. As i n a l l o t h e r b i o c h e m i c a l w o r k w i t h UGV, amounts o f m a t e r i a l a v a i l a b l e f o r a n a l y s i s w e r e m i n i m a l . Two p r o b l e m s w e r e e n c o u n t e r e d i n p r o t e i n a n a l y s i s : i n c o m p l e t e d i s r u p t i o n o f p o l y p e p t i d e s f r o m UGV, p r o d u c i n g a h i g h m o l e c u l a r w e i g h t c o mponent w h i c h w o u l d n o t p e n e t r a t e t h e g e l s , and f a i n t b a n d s due t o t h e l i m i t e d amount o f s t a r t i n g m a t e r i a l . Many o f t h e - 7 9 -Figure 12. Polyacrylamide g e l e l e c t r o p h o r e s i s of UGV p o l y p e p t i d e s , i n . a 10% polyacrylamide s l a b g e l . S l o t 1: D i s s o c i a t e d VLP's, no pretreatment. S l o t 2: VLP's p r e t r e a t e d w i t h chloroform/ methanol before d i s s o c i a t i o n . S l o t 3: Concentrated chloroform/methanol e x t r a c t . 7 1 a 45,000 d I Figure 13. Microdensi tometer scan of UGV p o l y p e p t i d e s , a f t e r e l e c t r o p h o r e s i s in SDS-polyacrylamide in F igure 12. sta ined with Coomassie blue s t a i n s lab gel (10%). Scan of s l o t (1) -81-bands noted could not be seen i n gel photographs or by scanning negatives, but only by d i r e c t observation of the g e l . A l l measurements of band position for calculations of molecular weights were made from measurements of the gels themselves; photographs were not used for t h i s purpose. The slab gel electrophoresis was used for d i r e c t comparison of the polypeptide components of VLP samples before and af t e r various treatments with organic solvents, to test for the presence of l i p i d s or proteins associated with l i p i d s (TaS et a l . 1977). No polypeptide bands disappeared when the i n t a c t VLP was treated with . ether or with chloroform/methanol p r i o r to d i s s o c i a t i o n . A f a i n t l y s taining, d i f f u s e region which electrophoresed with the buffer front was noted i n the concentrat-ed chloroform/methanol solvent. This region may contain l i p i d , or g l y c o l i p i d (Tas et a l . 1977). Results are included i n Figure 12. G. Nucleic acid characterization Two methods of characterizing the UGV nucleic acid were used on i n t a c t VLP's, i n addition to s p e c i f i c enzyme digestion: acridine orange staining and the standard diphenylamine and o r c i n o l t e s t s . The biochemical tests did not give consistent r e s u l t s for UGV and TIV; however, some assays of UGV p a r t i c l e s were weakly po s i t i v e i n the diphenylamine te s t and a l l o r c i n o l tests of UGV were negative. Results of the two kinds of assay are summarized i n Table XII. The acridine orange staining r e s u l t s suggest that UGV contains a double-stranded nucleic acid. The sta i n does not d i s t i n g u i s h between double-TABLE XII -- Nucleic acid colorimetric tests Acridine orange fluorescence Nucleic acid Diphenylamine Orcinbl color Virus standard type re s u l t s r e s u l t s Green Red UGV not known + - + -TIV ds DNAa + + CaMVb ds DNA +. - + -TMV ss RNA° - + - + BMV ss RNA ± + - + Double-stranded DNA. Cauliflower mosaic v i r u s . Single-stranded RNA. -83-stranded RNA and double-stranded DNA. H. Nucleic acid p u r i f i c a t i o n Nucleic acid was successfully p u r i f i e d from UGV preparations at 15 to 30 ug/ml of undissociated p a r t i c l e s , corrected for l i g h t scatter (A„,_ times 0.2). By analogy with ^ 260nm 1 t)-TIV, about 15% of the non-light scattering absorbance i s due to the nucleic acid of the p a r t i c l e , so a nucleic acid p u r i f i c a t i o n from the samples above, i f no nucleic acid were l o s t and the f i n a l volume of nucleic acid was i d e n t i c a l to the sta r t i n g volume of VLP's, would have a concentration of 2.3 to 4.5 ug/ml. Since this was normally the maximum amount of virus available, the nucleic acid often required concentration, and again, characterizations of the nucleic acid were done at the l i m i t s of s e n s i t i v i t i e s of the tests and instruments used. Rather vigorous digestion with pronase, i n the presence of 1% SDS, was necessary for disruption of VLP's and l i b e r a t i o n of the nucleic acid. A milder p r o t e o l y t i c enzyme, proteinase K, was not e f f e c t i v e i n digesting the VLP coat. Relative a b i l i t i e s to degrade the p a r t i c l e s could be judged by loss of opalescence. The s t a b i l i t y of the UGV p a r t i c l e was also seen i n i t s resistance to disruption for protein analysis; b o i l i n g i n SDS/urea/mercaptoethanol for 90 seconds was not completely e f f e c t i v e . Orcinol and diphenylamine tests were repeated using p u r i f i e d nucleic acid. The nucleic acid from UGV gave a clear p o s i t i v e reaction i n the diphenylamine t e s t , and a negative reaction with o r c i n o l , Controls were p u r i f i e d nucleic acids from TIV and CaMV (DNA's) and TMV and BMV (RNA 1s). No anomalous results were noted: a l l the DNA's gave strong blue reactions i n the diphenylamine t e s t and no color change with o r c i n o l ; and RNA1s gave a strong green color i n the or c i n o l tests. Another technique was used to lib e r a t e DNA for the Kleinschmidt technique: a pH 13.0 treatment required 1 to 2 hours for UGV disruption, i n contrast to a 10 minute exposure for CaMV. I. Buoyant density of UGV-DNA Cesium chloride buoyant density determinations were made using two Beckman Model E ultracentrifuges, one at the Agriculture Canada Research Station, and the other at the Depart-ment of Microbiology, U.B.C., Vancouver. Results are presented in Table XIII. In two runs at the Agriculture Canada Research st a t i o n , UGV-DNA was run alone and with added TIV-DNA as a marker. Using two ca l c u l a t i o n methods (reference DNA vs no reference), and taking TIV-DNA as 1.690 g/ml (Be l l e t t and Inman 1967), a buoyant density of 1.714 g/ml was estimated for UGV-DNA. Several measurements of buoyant density using the Model E at the Microbiology Department gave a density of 1.719 g/ml, with M. lysodeikticus DNA as the marker (1.731 g/ml, Szybalski 1968). Diffuse and f a i n t minor bands were observed i n a l l runs; one which was seen regularly had a density i n the region of 1.693*0.003 g/ml. Scans of resolved equilibrium grandients, before and after DNAase digestion, are shown i n Figure 14. The major band (band 1, F i g . 14) at 1.719 g/ml disappeared following DNAase digestion; the minor bands did not. In an equilibrium centrifugation using -85-F i g u r e 14. E q u i l i b r i u m c e n t r i f u g a t i o n of UGV-DNA: densitometer scans of pho t o g r a p h i c n e g a t i v e s . 1. UGV-DNA; 2. DNA from Micrococcus l v s o d e i k t i c u s ; 3, DNA from E.. c o l i . a. Buoyant d e n s i t y i n cesium c h l o r i d e . b. As i n (a) , a f t e r DNAse d i g e s t i o n . M.. l v s o -d e i k t i c u s marker DNA re-added. c. Buoyant d e n s i t y i n cesium s u l f a t e . -86-TABLE XIII — Buoyant density of UGV-DNA Marker DNA Main band Minor band Run # added Gradient Model E density density 1 none CsCl Agr. Can. 1.713 g/ml 1.699 g/ml 2 TIV-DNA CsCl Agr. Can. 1.714 3 E. c o l i Cs 2S0 4 Agr. Can. 1.436 1 .404 4 _M, lvso-deikticus dei CsCl Microbiol. 1.719 1.692 5 M. lvso. CsCl Microbiol. 1.719 1.698 6. M. lyso. C s C l a Microbiol. no band 1.694 DNAase-digested, marker DNA re-added. TABLE XIV — Guanine plus cytosine molar f r a c t i o n of UGV-DNA (G + C) = a - 1.660 g/ml 0.098 = 1.714 - ,1-fifin - 55.1% (Agr. Can.) = 1 , 7 1 9 - 1.660 = 60.2% (UBC Microbiol.) 0 . 098 The symbol a represents the buoyant density of the DNA i n question, (Schildkraut .et a l . 1962) . - 8 7 -cesium su l f a t e , two bands were observed: a major band at 1 . 4 3 6 g/ml and a minor one at 1 , 4 04 g/ml. A buoyant density of 1 . 7 1 9 g/ml, by the equation of Schildkraut et a l . ( 1 9 6 2 ) , corresponds to a molar f r a c t i o n of guanine plus cytosine of 6 0 , 2 % (Table XIII). Using the graphical estimation method of Erikson & Szybalski ( 1 9 6 4 ) , a cesium sulfate density of 1 . 4 3 6 g/ml corresponds to a (G + C) molar f r a c t i o n of 70% i f the DNA i s not at a l l glucosylated; i t corresponds to approximately 60% (G + C) i f the DNA i s 10% glucosylated. J. Nucleic acid v i s u a l i z a t i o n by Kleinschmidt technique  Nucleic acid molecules i s o l a t e d from p u r i f i e d VLP's were examined by electron microscopy using the Kleinschmidt technique of spreading nucleic acid i n thin films of protein. Standards included for comparison were CaMV-DNA, a 2 . 3 to 2 . 5 micron c i r c l e (Shepherd and Wakeman 1 9 7 1 ) ; and TIV-DNA, t h e o r e t i c a l l y a 6 5 micron l i n e a r molecule (Bellett and Inman 1 9 6 7 , Kelly and Avery 1 7 4 ) . Both TIV-DNA and CaMV-DNA are double-stranded. CaMV c i r c u l a r molecules and long TIV molecules were observed; both were of a width t y p i c a l of double-stranded molecules (Figure I5). UGV-DNA was i d e n t i c a l to the other DNA's in width, and i s presumably double-stranded as well. TIV-DNA was always seen i n a large tangled form, usually with , free ends v i s i b l e . CaMv preparations showed c i r c u l a r forms and shorter l i n e a r molecules, with the proportion of li n e a r molecules increasing with the storage time of p u r i f i e d CaMV-DNA or in t a c t v i r i o n s , as reported by Shepherd et a l . ( 1 9 6 8 , 1 9 7 0 ) . -8 8-The UGV n u c l e i c a c i d molecules observed by t h i s technique were long and l i n e a r (Figure 15 a ) . Many were well-extended by spreading and could be measured using a map measuring device on photographic enlargements at about 40,000 times m a g n i f i c a t i o n . The e r r o r f a c t o r due to t h i s measuring procedure was estimated by repeated measurements of one molecule, and was found to be approximately ± 10%. One hundred and three molecules of UGV-DNA were measured, i n c l u d i n g e i g h t y - s i x prepared by SDS/pronase and seventeen by pH 13.0 treatment. A histogram of the d i s t r i b u t i o n of lengths i n microns i s included i n Figure 16. No d e f i n i t e peak rep r e s e n t i n g a modal length of UGV-DNA was found. Lengths of DNA molecules v a r i e d from 4 microns to 36 microns, corresponding to DNA's of molecular weights 8 x 10^ to 72 x 10^ daltons (using an approximation of 2 x 10^ daltons molecular weight per micron of DNA len g t h , Lang 1970). S i m i l a r d i s t r i b u t i o n patterns were obtained from both methods of DNA p u r i f i c a t i o n , although fewer short molecules were observed w i t h the pH treatment, suggesting t h a t the d i s t r i b u t i o n was not an a r t i f a c t of pr e p a r a t i o n methods. The length d i s t r i b u t i o n of UGV-DNA's was p l o t t e d on p r o b a b i l i t y paper (Harding 1949) to t e s t f o r n o r m a l i t y . Using t h i s paper, normal d i s t r i b u t i o n s w i l l r e s u l t i n a s t r a i g h t l i n e when p l o t t e d by frequency c l a s s e s . Measurements of CaMV-DNA were i n c l u d e d , to a r t i f i c i a l l y introduce a second, known d i s t r i b u t i o n i n t o one area of the range of values f o r UGV-DNA, to t e s t t h a t a d e v i a t i o n from normality would be detected using t h i s graphing procedure (Figure 16). With the exception of the -89-Figure 15. Molecules of DNA v i s u a l i z e d by the Kleinschmidt technique, a) UGV-DNA; approximately 15 microns. b) CaMV-DNA c i r c l e . c) TIV-DNA being released from TIV p a r t i c l e s , a f t e r spreading on 4 M urea. - 3 0 -M5 Mo h5 LENGTH (MICRONS) 16. D i s t r i b u t i o n o f l e n g t h s o f m o l e c u l e s measured o f UGV-DNA and CaMV-DNA (118 t o t a l ) . H i s t o g r a m ( d o t t e d a r e a ) and c u m u l a t i v e f r e q u e n c i e s p l o t t e d on p r o -b a b i l i t y paper ( l i n e ) . area of the curve i n c l u d i n g CaMV-DNA lengths (roughly a l l values below 3.0 microns), the set of values of UGV-DNA lengths appear to be normally d i s t r i b u t e d , i . e . they l i e along a reasonably s t r a i g h t l i n e on the p r o b a b i l i t y graph. There i s no suggestion of a d e v i a t i o n from normality over the range of 6 microns to 25 microns, which would argue against the presence of a s i n g l e modal l e n t h . However, the sample s i z e may have been too sm a l l ; there are c l u s t e r s of length values around 15 u and 22 u. A l s o , three molecules were seen which had lengths of approximately 32 u, were well-spread and easy to measure. N u c l e i c a c i d from UGV prepared by the a l t e r n a t i v e technique of in c u b a t i o n i n 0.3 N NaOH showed a s i m i l a r v a r i a b i l i t y i n lengths, w i t h a maximum of about 32 u. A l l RNA would have been hydrolyzed under these c o n d i t i o n s . The strands of UGV n u c l e i c a c i d completely disappeared when pre-incubated w i t h p a n c r e a t i c deoxyribonuclease f o r 10 minutes before spreading CaMV and TIV-DNA's were a l s o d i g e s t e d . An attempt to show emergence of more than one strand of DNA from p a r t i c l e s of UGV by spreading of i n t a c t VLP's on 4 M urea (Vasquez and Kleinschmidt 1968) revealed no e x t r u s i o n of n u c l e i c a c i d ; TIV t r e a t e d i n the same way immediately began to lose DNA i n long, c o i l e d loops (Figure 15b). Both UGV and CaMV spread on 4 M urea appeared to lose coat s t r u c t u r e but n u c l e i c a c i d was not seen i s s u i n g out of these p a r t i c l e s . K. Radioisotope l a b e l l i n g of UGV-DNA Because f u r t h e r c h a r a c t e r i z a t i o n of UGV-DNA i n sucrose or CsCl gradients or by r e s t r i c t i o n enzyme cleavage and g e l electrophoresis required more DNA than was ever available, an attempt was made to produce t r i t i u m - l a b e l l e d nucleic acid. For further evidence that UGV p a r t i c l e s contain DNA rather than RNA, t r i t i a t e d u r a c i l as well as t r i t i a t e d thymidine were supplied as precursors of both nucleic acids, i n separate culture tubes. U r a c i l , the base form, was supplied at 20 uCi/ml of culture medium. Thymidine, the nucleoside, was added to the culture medium at 5 uCi/ml. The choices-of the form of the precursor and the concentration to be used were made on the basis of previous l a b e l l i n g studies involving other algae (Swinton and Hanawalt 1972, Dr. L i n Kemp, personal communication). In the f i r s t experiment uptake of the l a b e l l e d precursors was followed over a two week time period. Two 10 ml test tube cultures, seeded with freshly released zoospores, were used to assay uptake of each prectirsor. Total concentration of either u r a c i l or thymidine was 0.1 u mole/ml culture medium. Beginning immediately after the addition of l a b e l , the tubes were sampled every two days (2 x 100 u l from each tube) for two weeks. The uptake of the lab e l was determined by the following c a l c u l a t i o n : Percent la b e l absorbed = maximum counts/100 u l x 10 concentration of lab e l (uCi/ml) x e f f i c i e n c y x constant = cpm/ml  (uCi/ml) x (cpm/dpm) x 2,2 x 10^ (dpm/uCi) Results are shown i n Figure 17a. Uptake of u r a c i l or thymidine by U. gigas was i n e f f i c i e n t : only 0.6% of the t o t a l u r a c i l counts per ml and 0.7% of the t o t a l thymidine counts per ml were taken up by the a l g a l c e l l s . The amount of lab e l i n a l g a l c e l l s did not increase appreciably after one week of growth. The c e l l s were harvested by f i l t r a t i o n after two weeks growth. The culture f i l t r a t e , when examined by electron microscopy, contained bacteria as well as UGV p a r t i c l e s . Aliquots of 100 u l plated onto nutrient agar showed extensive b a c t e r i a l growth after 48 hours. The f i l t r a t e s were subjected to sucrose density gradient centrifugation, with the addition of unlabelled UGV as c a r r i e r , and fractions were assayed by s c i n t i l l a t i o n counting to determine i f any UGV p a r t i c l e s had been lab e l l e d under these conditions. Results were i d e n t i c a l to those of the second experiment (in which b a c t e r i a l contamination was reduced), which are presented i n Figure 17 b. Apparently the labe l l e d precursors were not u t i l i z e d i n VLP nucleic acid synthesis to any detectable extent. In the second experiment c e l l s were grown i n 10 uCi/ml of u r a c i l or thymidine, and were harvested after one week, with no. p r i o r sampling, to r e s t r i c t b a c t e r i a l contamination. Algal c e l l s c o l l e c t e d on f i l t e r s were treated with 5% TCA and counted; t o t a l uptake of each la b e l c l o s e l y p a r a l l e l e d the results of experiment one. The f i l t r a t e from each tube was pelleted, and the p e l l e t re-suspended with added unlabelled UGV, and the sample analysed on sucrose gradients. Gradient fractions were examined by electron microscopy for the presence of VLP's and were assayed for r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. Results are shown i n Figure 17 b. Most counts were detected near the top and the bottom of the gradients; UGV p a r t i c l e s were located near the gradient centers, i n a region of e s s e n t i a l l y no counts, for either u r a c i l or thymidine. This d i s t r i b u t i o n of VLP's and lab e l -94-Uptake by alga 2.-1 i o « H Q . O T 4 8 Days after inoculation A -3 H-urac i l • - ^ H - t h y m i d i n e B. Label in UGV Fraction number F i g u r e 17- A t t e m p t e d l a b e l l i n g o f t h e n u c l e i c a c i d o f UGV by f e e d i n g w i t h 3 H - u r a c i l and H - t h y m i d i n e . was seen after sucrose gradient analysis of eight test cultures Production of labe l l e d UGV-DNA was not apparent using t h i s method of supplying l a b e l l e d precursors. No difference i n u t i l i z a t i o n of u r a c i l or thymidine were noted. -96-DISCUSSION I. I n t r o d u c t i o n The i s o l a t i o n and c h a r a c t e r i z a t i o n of v i r u s e s i n f e c t i n g e u c a r y o t i c algae has proceeded very slowly i n comparison with work on the v i r u s e s of p r o c a r y o t i c , blue-green algae. With the e x c e p t i o n of the r e s e a r c h d e s c r i b i n g the v i r u s of Chara  c o r a l l i n a (Gibbs e_t a l . 1975, S k o t n i c k i e t a l . 1976), a l l p u b l i s h e d work on these algae as v i r a l hosts has been r e s t r i c t e d to u l t r a s t r u c t u r a l o b s e r v a t i o n . Often the host a l g a was c o l l e c t e d i n the f i e l d f o r e l e c t r o n m i c r o s c o p i c a l examination o n l y , so by the time VLP's in s i t u had been seen the host was no longer a v a i l a b l e f o r c u l t u r i n g . S e v e r a l authors have s t a t e d t h a t f u r t h e r c h a r a c t e r i z a t i o n of a VLP they had observed was being attempted, but no r e p o r t s have as y e t appeared, perhaps due to d i f f i c u l t i e s i n p u r i f y i n g the necessary q u a n t i t y of p a r t i c l e s as w e l l as i n c u l t u r i n g the host a l g a . T h i s t h e s i s has been concerned w i t h the f i r s t p a r t i a l c h a r a c t e r i z a t i o n of a p o l y h e d r a l v i r u s -l i k e - p a r t i c l e from a e u c a r y o t i c a l g a l host. I I . Morphology of the p a r t i c l e The VLP's from JJ. gigas showed str o n g s i m i l a r i t i e s i n s t r u c t u r e t o many of the p o l y h e d r a l p a r t i c l e s observed i n other s p e c i e s of algae, p a r t i c u l a r l y to the VLP's over 100 nm i n diameter. The 400 nm, t a i l e d UGV p a r t i c l e s appeared to be i c o s a h e d r a l , with a membrane-like outer coat and a densely-packed but non-homogeneous c e n t r a l core, as noted i n other a l g a l VLP's (Pickett-Heaps 1972, Toth and Wilce 1972, Swale and B e l c h e r 1973, Clitheroe and Evans 1974, Markey 1974, Moestrup and Thomsen 1974, Hoffman and Stanker 1976). The p u r i f i e d VLP's when pelleted and sectioned were similar i n morphology to those seen i n thin sections of infected c e l l s . A recurrent problem i n culture and i s o l a t i o n of VLP's from fungi, protozoans, and eucaryotic algae has been the maintenance of axenic culture conditions, to ensure that the VLP's isol a t e d represent true i n t r a c e l l u l a r agents of the host organism being studied, and not phages of procaryotic contaminants or symbionts. Because of the large size and d i s t i n c t i v e morphology of the UGV p a r t i c l e , b a c t e r i a l contamination was not as severe a problem i n t h i s study. P a r t i c l e s of UGV are much larger than any reported bacteriophages, approaching bacteria i n s i z e , and no i n t r a c e l l u l a r symbionts of _U. gigas were ever observed i n thin section. Also the p u r i f i e d p a r t i c l e s matched those within U_. gigas c e l l s , described by Mattox et al.. (1972) and seen i n our lab, i n both size and morphology. P a r t i c l e s of UGV seemed to vary i n f u l l n e s s of the capsid, and i n capsid constituents, including dense, s o l i d -looking material and a f i b r i l l a r substance. Two aspects of the preparation methods could account for seemingly empty space within the capsid: f i x a t i o n and embedding procedures could shrink or d i s t o r t the contents of the VLP, as i n the case of bacteriophage ft6 (Gonzalez e_t al_. 1977) ; or varying planes of sectioning might include VLP constituents of d i f f e r i n g staining reactions (Hayat 1970) , i f t h e i r d i s t r i b u t i o n v/as not uniform within the capsid. The enzyme digestion experiments suggest that the extremely dense material inside the capsid i s composed of -98-protein. Cores of other complex viruses are known to contain nucleoprotein, a nucleic acid and protein complex; some double-stranded DNA viruses, e.g. SV40, have true histones complexed with the DNA, l i k e c e l l u l a r chromosomes. For UGV, the protein portion of a possible nucleoprotein complex may be more accessible during enzyme digestion, or may be present surrounding and bound to the DNA, so that p r o t e o l y t i c enzyme digestion would remove the nucleic acid as well; digestion by the s p e c i f i c nuclease alone might be harder to detect v i s u a l l y . Deoxyribonuclease digestion resulted i n only p a r t i a l clearing of the core area of the VLP's. The p o s s i b i l i t y that the UGV p a r t i c l e may include a l i p i d component has not been eliminated. Results from three types of i n d i r e c t experiments which tested for the presence of l i p i d s were ambiguous, and the VLP's were not available i n s u f f i c i e n t quantity for a d i r e c t assay of l i p i d s using chromatographic methods. Digestion of p e l l e t e d VLP's with a lipase (with no a c t i v i t y s p e c i f i c for phospho- or g l y c o - l i p i d s ) af t e r embedding for electron microscopy did not r e s u l t i n a noticeable change of u l t r a s t r u c t u r e . Other workers have reported d i f f i c u l t i e s using lipases (Hayat 1970). Treatment of VLP's with chloroform/methanol caused extensive disruption of t h e i r structure. Organic solvents are routinely used to test for the presence of l i p i d s ; many animal viruses are rendered non-infectious following the disruption of the v i r a l envelope by such solvents. However, s t r u c t u r a l alterations caused by solvent treatments can be seen by electron microscopy even for -99-some viruses known to be free of l i p i d components (Amako and Yasunaka 197 7) . Chlorof orm/methanol treatment can apparently-disrupt protein configurations as well as remove l i p i d . Enveloped viruses also lose i n f e c t i v i t y after treatment with ether, but ether treatment caused no noticeable u l t r a s t r u c t u r a l changes i n UGV. Certainly UGV i s not as susceptible to organic solvents as the true enveloped viruses. But UGV p a r t i c l e s may contain some l i p i d : chloroform/methanol, aft e r being used for extraction of VLP's, produced a d i f f u s e band i n polyacrylamide gels, i n the region where l i p i d s would run. This band was not seen i n gels of ether concentrates, or i n gels of untreated VLP's. Kelly and Vance (1973) demonstrated the presence of l i p i d i n two i r i d e s c e n t viruses, after a ten year controversy concerning e a r l i e r reports of l i p i d i n these viruses. Other workers had thought that the l i p i d detected was due to contamination of v i r i o n s with host c e l l membranes during p u r i f i c a t i o n . Kelly and Vance provided evidence that there was enough l i p i d for a single b i l a y e r inside the capsid, and that the l i p i d s present i n virus extracts were d i f f e r e n t from those of the host c e l l s . Stoltz (1971, 1973) has proposed a structure for a l l ICDV's (icosahedral cytoplasmic deoxyriboviruses) which includes an i n t e r n a l layer of l i p i d . He postulated that the l i p i d layer encloses the inner capsid core, and that the angular s h e l l of ICDV's i s composed of protein alone. Using t h i s model, only solvents which disturbed the external protein structure would gain access to the i n t e r n a l membrane layer. The extremely t h i n , single layer of stained material l e f t i n t a c t at the periphery -100-of UGV p a r t i c l e s after lengthy p r o t e o l y t i c digestion (Figure 8c,d) could represent t h i s l i p i d layer. In Stoltz's model, the l i p i d layer does not provide the r i g i d angularity seen i n the i n t a c t capsid; the protease-digested VLP's in t h i s study are c i r c u l a r i n outline, i n contrast to the hexagonal p r o f i l e of i n t a c t p a r t i c l e s . The diameter of the UGV p a r t i c l e (400 nm) i s the largest observed among the eucaryotic a l g a l viruses or the ICDV's. To date the largest reported a l g a l virus was from Oedogonium (diameter 240 nm) (Rickett-Heaps 1972), and the largest ICDV's were certain variants of the lymphocystis virus of f i s h (Kelly and Robertson 1973). The diameters of the ICDV's range from 130 nm for many of the insect iridescent viruses, frog virus 3, and the octopus ICDV to a maximum of 26 0 nm, with intermediate diameters from other insect ICDV's, African swine fever v i r u s , and other amphibian viruses. Wide v a r i a t i o n i n diameter--from 13 0 to 26 0 nm--has been reported among known strains of lymphocystis v i r u s . The significance of t h i s v a r i a b i l i t y i s unclear (Howse and Christmas 1971) and a t y p i c a l of most known viruses. Several workers have suggested that a virus group such as the ICDV's which includes such d i v e r s i t y of sizes must be a r t i f i c i a l , e s p e c i a l l y since the l i t t l e amount of s e r o l o g i c a l data available suggests that s i g n i f i c a n t differences e x i s t among the viruses included i n t h i s group (Kelly and Robertson 197 3), Structural s i m i l a r i t i e s , an i n t e r n a l l i p i d membrane, multiple proteins, a double-stranded DNA genome of approximately 130 to 160 x 10^ daltons, and accumulation of v i r i o n s within the cytoplasm of infected c e l l s have been designated as the -101-c h a r a c t e r i s t i c s unifying t h i s diverse group (Fenner 1976). The UGV p a r t i c l e can apparently change dimensions under various conditions of s t a i n and pH: swelling was observed from 390 nm (in. s i t u f after f i x a t i o n , or i n uranyl acetate stain) to a maximum of 590 nm (in phosphotungstic acid s t a i n and after storage at pH 8.0). If other seemingly 'related VLP's and viruses have similar capacities to enlarge comparisons of size would be meaningful only where conditions of s t a i n and pH were s p e c i f i e d . Only one other a l g a l virus has been reported to be t a i l e d (Aulacomonas VLP—Swale and Belcher 1973); the t a i l s are quite broad and are less than one capsid diameter i n length. Swale and Belcher have not reported on the appearance of the e x t r a c e l l u l a r VLP i n negative stain; the t a i l has only been seen i n sections of c e l l s . No other t a i l e d VLP's or viruses have been observed i n eucaryotic c e l l s . Tailed p a r t i c l e s are completely unknown among the ICDV's. Bacteria and blue-green algae are hosts to a number of t a i l e d p a r t i c l e s . T a i l s of viruses of blue-green algae can be c o n t r a c t i l e or non-c o n t r a c t i l e (Padan and Shilo 1973). The c o n t r a c t i l e forms, found on N-l and AS-1 viruses, are about twice as long as the capsid diameter and are quite thin (about 20 nm wide). The non-c o n t r a c t i l e t a i l s of the LPP virus group are r e l a t i v e l y short i n comparison to capsid diameter (only 20 nm long). The r e l a t i v e sizes of capsid and t a i l of UGV—400 nm capsid and 1000 nm t a i l — are closer i n proportions to the c o n t r a c t i l e - t a i l e d blue-green a l g a l viruses. However, no evidence of c o n t r a c t i l i t y was seen for UGV, and the UGV diameter i s 4 to 8 times larger than the diameters of AS-1 and N-l cyanophages. By u l t r a s t r u c t u r a l -102-appearance, UGV p a r t i c l e s have a capsid that shares many-properties with ICDV's, and a t a i l , of unknown function, and varying i n appearance from the t a i l s of phages. II I . I n t r a c e l l u l a r development Mattox, Stewart, and Floyd (1972) reported that UGV p a r t i c l e s develop i n the nucleoplasm of infected U. gigas c e l l s . They never observed VLP's within the chloroplast, and l y s i s of the nucleus was the f i r s t c y t o l o g i c a l a l t e r a t i o n associated with i n c i p i e n t UGV development. My examination of UGV-infected c e l l s was i n agreement with these r e s u l t s . C e l l s at the e a r l i e s t stages of i n f e c t i o n observed (Figure 3e) had no nuclear membranes. As UGV assembly progressed, more of the c e l l u l a r membrane systems degenerated, u n t i l f i n a l l y even the chloroplast became disorganized. Observations that UGV development begins i n the area of the nucleus are i n accord with reports on the development of other eucaryotic a l g a l viruses of t h i s s t r u c t u r a l type (Lee 1971, Swale and Belcher 1973, Pearson and Norriss 1974, Moestrup and Thomsen 1974, Clitheroe and Evans 1974, and Hoffman and Stanker 1976). These researchers and others working on i n t r a c e l l u l a r development of the ICDV's agree that some type of nuclear role i s implicated i n the VLP r e p l i c a t i o n process, and that nuclear degeneration occurs as a r e s u l t of a l l these i n f e c t i o n s , whether virus assembly takes place i n the nucleus or cytoplasm. Pearson and Norris (197 4) suggested that the diameter of the nuclear pore (60 nm) might be the factor which allows for the establishment of a viroplasm i n the nucleus; p a r t i c l e s larger than 6 0 nm would -103-be assembled i n a viroplasm i n the cytoplasm. However, Pienaar (1976) has observed 100-130 nm p a r t i c l e s i n the nuclei of phytoplankton; the theory of entrance by nuclear pores may be too s i m p l i s t i c . Two viruses which are assembled i n cytoplasmic viroplasms have recently been shown to require the presence of a nucleus: frog virus 3(FV3—Goorha et a l . ,1978), and African swine fever virus (AFSV—Ortin and Vinuela 1977). Enucleated or UV-irradiated host c e l l s w i l l not support virus r e p l i c a t i o n of AFSV or FV3; pulse-chase autoradiographic studies of FV3 r e p l i c a t i o n suggest that a low molecular weight v i r a l DNA i s made i n the nucleus early i n the i n f e c t i o n cycle, and then transported to the cytoplasm. Since these viruses contain double-stranded DNA, as does UGV, nuclear involvement may consist of an enzyme requirement for DNA r e p l i c a t i o n , or simply the breakdown of host DNA for a source of deoxyribonucleotides. Investigations of i n t r a c e l l u l a r development of several ICDV's (FV3, lymphocystis, TIV) and several viruses of eucaryotic algae have included observations of nascent angular forms of incomplete capsids i n c e l l s at early stages of in f e c t i o n (Yule and Lee 1973). Usually c e l l s contain many of these incomplete hexagonal or pentagonal forms, so the p o s s i b i l i t y that the forms are a sectioning a r t i f a c t i s s l i g h t . These were seen i n germlings of U. gigas infected by UGV as well (Figure 3e). U gigas and other algae seemed to be most l i k e l y to contain VLP's during the zoospore and germling stages of th e i r l i f e cycles (see Table I ) . Pickett-Heaps (1972) suggested that -104-transmission of these viruses might occur only during the zoospore stage, when the host alga i s not enclosed i n a thick c e l l wall. The zoospore without i t s wall may also be more susceptible to environmental stress; several c e l l d i v i s i o n s and a development sequence into a germling must occur i n a short period of time subsequent to zoospore release. Cvlindrocapsa  geminella, another Ulotrichalean alga which has been shown to contain VLP's (Hoffman and Stanker 1976), seems to be most susceptible to VLP i n f e c t i o n — o r most l i k e l y to express a latent infection--during the germling stage, e s p e c i a l l y i f stress-ed by heat shock. A heat shock treatment of U. gigas at the zoospore and germling stages produced a s i x - f o l d increase i n the number of VLP's released. The increase may be due to a greater percentage of a l g a l c e l l s becoming infected; a l t e r n a t i v e l y more c e l l s could have been induced to show active i n f e c t i o n after the heat shock. My r e s u l t s , though preliminary, indicate that the e f f e c t of heat shock i s augmented i n the dark. Lack of l i g h t during a normal photoperiod would increase the stress on a heat-shocked photosynthetic organism, by forcing u t i l i z a t i o n of c e l l u l a r reserves. In future experiments, heat shock treatments could be done i n conjunction with a time course to test the timing of active i n f e c t i o n s . Cultures of zoospores only, s e t t l i n g zoospores, or germlings, could be assayed at various times aft e r a heat shock to determine at which stage heat shock has the greatest e f f e c t . Uronema gigas VLP's were seen only i n sectioned germling c e l l s , or c e l l s of very young filaments, never i n the expanded c e l l s of mature filaments. Infected c e l l s may be more -105-f r a g i l e than healthy c e l l s , i . e . they may have l o s t portions of the plasmalemma as v/ell as other c e l l u l a r membranes, and be destroyed during the f i x a t i o n and embedding processes, p a r t i c u l a r l y i f attached at both ends to the sturdy c e l l s of the healthy filament. At t h i s point there i s no evidence that mature c e l l s of the filament show i n f e c t i o n with UGV. The p o s s i b i l i t y that mature c e l l s contain the information for VLP synthesis i n some form - a s i t u a t i o n analogous to lysogeny , i n bacteria - w i l l be considered separately. The evidence from scanning electron microscopy of germlings i s also suggestive of increased s u s c e p t i b i l i t y at t h i s stage of the l i f e cycle. Dense spheres of a diameter similar to that of the UGV p a r t i c l e were observed through the c e l l walls of hog; germlings. By x-ray microanalysis the spheres were found to contain more phosphorus than the surrounding a l g a l cytoplasm; they may thus represent UGV p a r t i c l e s , with increased phosphorus due to DNA content. Since p u r i f i e d UGV p a r t i c l e s l o s t a l l structure when dried on stubs for scanning electron microscopy, i t i s unclear whether these spheres would match reference UGV i n appearance. Scanning of mature filaments was not done, but the absence of VLP's i n t h i n sections of such filaments would suggest that they would not have been detected by t h i s method. The spheres were seen i n approximately 5% of the hundreds of c e l l s which were scanned. JJ_. gigas c e l l s at a l l stages of the l i f e cycle, examined by transmission electron microscopy, included about 1% obviously infected c e l l s . However, many more c e l l s were examined by scanning than by transmission microscopy, so comparisons of frequencies may be inaccurate. -106-The dense spheres seen by scanning microscopy may i n f a c t be completely d i f f e r e n t e n t i t i e s , the s o - c a l l e d polyphosphate granules, reported by Harold (1966) to be found i n b a c t e r i a l c e l l s , some f u n g i , and i n a few algae t h a t were examined ( C h l o r e l l a , Scenedesmus, and Chlamydomonas). Polyphosphate i s b e l i e v e d to act as a storage form of phosphate, accumulating under c o n d i t i o n s unfavorable f o r growth. Harold (1966) describes c y t o l o g i c a l i d e n t i f i c a t i o n of the granules by use of b a s i c dyes, a r a t h e r n o n - s p e c i f i c t e s t , or by the use of lead s t a i n s . By e l e c t r o n microscopy, the granules s t a i n e d w i t h lead are extremely opaque and may v o l a t i l i z e under the e l e c t r o n beam, l e a v i n g c h a r a c t e r i s t i c h oles. Lead s t a i n i n g of i n f e c t e d JJ. gigas c e l l s was r o u t i n e l y used, and d a r k l y s t a i n i n g pyrenoids were seen w i t h i n c h l o r o p l a s t s (Figures l a and l b ) , but no v o l a t i l i z a t i o n ever occurred. I f polyphosphate granules mainly accumulate under favorable growth c o n d i t i o n s , one would not expect t h e i r e x i s t e n c e , i n q u a n t i t y , i n a f r e s h young c u l t u r e of mainly 2- to 4 - c e l l e d f i l a m e n t s . IV. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n C h a r a c t e r i z a t i o n of the UGV p a r t i c l e depended on adequate amounts of p u r i f i e d p a r t i c l e s f o r the various methods of a n a l y s i s . Various methods of i n c r e a s i n g y i e l d of VLP's from an a l g a l harvest were assessed i n the course of developing a p u r i f i c a t i o n procedure, but they could doubtless be improved. The r o u t i n e heat-shocking of freshly-seeded f l a s k s should increase y i e l d of VLP's. A d d i t i o n a l s t r e s s , perhaps i n the form of a l e s s r i c h medium, lengthened heat shock, or prolonged dark treatment might increase the y i e l d of VLP's. An attempt to -107-increase the number of VLP's released by UV-light treatment of the culture resulted i n extensive fungal contamination. S u f f i c i e n t testing of c e l l u l a r disruption methods, to release more VLP's from a l g a l t i s s u e , was not done; i n p a r t i c u l a r a gentler but more e f f i c i e n t method than those t r i e d , such as the use of a Braun homogenizer, might r e s u l t i n the increased l i b e r a t i o n of VLP's. The simple p u r i f i c a t i o n used at present seems adequate for i s o l a t i o n of VLP's from the culture medium; a c l a r i f i c a t i o n step would probably be needed i f the a l g a l c e l l s were homogenized. The f i n a l steps of p u r i f i c a t i o n with VLP's kept at pH 8.0 i n 0.05 M. Tris-HCl, might be more e f f e c t i v e at a lower pH (pH 7.0 to 7.5), i n order to l i m i t the swelling of the p a r t i c l e s . P a r t i c l e s may be more unstable duri centrifugation steps when they are highly swollen. Properties of the in t a c t UGV p a r t i c l e which were successfully assessed were l i g h t scattering, sedimentation c o e f f i c i e n t , and density i n sucrose. The extent of absorbance due to l i g h t scattering, about 80% of t o t a l absorbance at 260 nm, and the sedimentation c o e f f i c i e n t of approximately 6300 S could be expected on the basis of the very large size of t h i s VLP. The density i n sucrose should be re-tested, as the f i r s t run was probably not centrifuged to equilibrium; however, an estimate of 1.32 g/ml, very close to the density of TIV, was obtained, implying a similar d i s t r i b u t i o n of protein, nucleic acid, and possibly l i p i d i n the p a r t i c l e . A comparison of the properties of UGV and TIV i s included i n Table XVI. The analysis of the l i p i d content of UGV was most obviously affected by lack of material. A d e f i n i t i v e statement -108-about the presence of l i p i d i n UGV cannot be made on the basis of the evidence obtained, much less a comparison of VLP l i p i d s to those of host o r i g i n . Similar d i f f i c u l t i e s were involved i n the analysis of the polypeptide components of UGV: although ten species of polypeptides were detected, a l l but three were vi s u a l i z e d i n barely resolved bands. Based on the size of the capsid, and by analogy with such viruses as TIV, which codes for 20 polypeptides (Kelly and Robertson 1973), one would expect to have detected more bands. Cyanophages of t h i s s t r u c t u r a l complexity, with t a i l s , contain more than ten polypeptides. There very l i k e l y was too l i t t l e material applied to the gels to resolve other bands. In addition, the UGV p a r t i c l e was d i f f i c u l t to d i s s o c i a t e , and a residual undissociated f r a c t i o n was always seen at the tops of the gels, i n several d i f f e r e n t methods of d i s s o c i a t i o n . With more UGV available and a more vigorous d i s s o c i a t i o n , one would expect an increase i n i n t e n s i t y of a l l the bands noted here, and more bands would probably be resolved. The nucleic acid of the UGV p a r t i c l e s was shown to be double-stranded DNA. The evidence on which th i s conclusion i s based i s summarized i n Table XV. The DNA appears to have a buoyant density of 1.719 g/ml i n cesium chloride, corresponding to a molar f r a c t i o n of cytosine plus guanine (G + C) of 60 percent. Two d i f f e r e n t estimates of buoyant density were found on the two Model E a n a l y t i c a l ultracentrifuges used (1.714 g/ml and 1.719 g/ml). The density value of the major DNA band from the centrifuge i n the U.B.C. Department of Microbiology was estimated by more r e l i a b l e techniques (the use of M_. -109-TABLE XV — Characterization of UGV nucleic acid Test or assay Enzyme digestion of sectioned VLP's a) RNAse b) DNAse Diphenylamine Orcinol Acridine orange staining Buoyant density i n CsCl i n Cs 2S0 4 added DNAse Result Type of N.A. Indicated no digestion digestion of cores posi t i v e negative green fluoresence 1.719 g/ml 1.436 g/ml main band disappears DNA DNA DNA or RNA DNA DNA DNA Strandedness double-stranded Kleinschmidt appearance wide lin e a r bands DNA or RNA double-stranded -110-lysodeikticus DNA as a marker and the reference method of ca l c u l a t i o n ) , and t h i s value was consistent from run to run, so a buoyant density of 1.719 g/ml was considered to be the more accurate of the two values. The discrepancy between the two instruments may be due to the d i f f e r e n t marker DNA's used. A buoyant density value of 1.436 g/ml i n cesium sulfate was determined for the major band seen, corresponding f a i r l y well with a cesium chloride density of 1.719 g/ml, and possibly i n d i c a t i n g up to 10% glucosylation of the DNA (Erikson and Szybalski 1964, Szybalski 1968). A density of 1.719 g/ml i n CsCl or 1.436 i n Cs2SO^ i s i n the range c h a r a c t e r i s t i c of DNA: RNA molecules are considerably denser and would be pelleted i n th i s cesium chloride density range. Several minor bands were usually also observed. One which was present i n every run had a density of approximately 1.695 g/ml i n cesium chloride, inconsistent with i t s density of 1.404 g/ml i n cesium s u l f a t e . The major band i n cesium chloride disappeared after treatment with DNAse; t h i s minor band did not. Because of i t s anomalous behavior i n density gradients and i t s resistance to DNAse, t h i s band does not appear to represent DNA. Anomalous bands of polysaccharides and glycogen have been reported by other authors (Mandel e_t a l . 1968); the DNA p u r i f i c a t i o n used did not involve any treatment to r i d the samples of these compounds. The v i s u a l i z a t i o n of the nucleic acid from UGV by the Kleinschmidt technique reinforced e a r l i e r evidence that the p a r t i c l e contains double-stranded DNA. The width of the molecule was i d e n t i c a l to CaMV-DNA and TIV-DNA, the two double-- I l l -stranded DNA markers used. No c i r c u l a r UGV-DNA molecules were seen, although c i r c l e s of CaMV-DNA were e a s i l y found; TIV-DNA occurred as large tangled masses, but was c l e a r l y a l i n e a r molecule, w i t h f r e e ends. Strands of UGV-DNA prepared by the Kleinschmidt technique disappeared a f t e r i n c u b a t i o n on drops of DNAse but not on RNAse. Use of a high pH treatment (pH 13.0) f o r r e l e a s e of UGV-DNA r e s u l t e d i n the same d i s t r i b u t i o n of lengths as tha t seen a f t e r DNA re l e a s e by SDS/pronase treatment. A large RNA molecule, or a DNA i n c l u d i n g r i b o n u c l e o t i d e r e s i d u e s , would be degraded by t h i s procedure. C a u l i f l o w e r mosaic v i r u s DNA, which contains r i b o n u c l e o t i d e s ( H u l l and Shepherd 1977, V o l o v i t c h et al.. 1977), disappeared when released by using high pH. Esti m a t i o n of the molecular weight of the UGV-DNA by the Kleinschmidt technique was not p o s s i b l e . A range of lengths from approximately 4 microns to 3 2 microns was obtained, corresponding to DNA's of molecular weights of approximately 8 to 64 x 10 6 d a l t o n s . As shown i n Figure 16, none of the intermediate lengths occurred as an obvious peak i n a histogram, suggesting a modal length w i t h extensive v a r i a t i o n . The d i s t r i b u t i o n seen could represent random breakage of a long molecule. Perhaps the three long DNA molecules (about 32 microns) are the na t i v e DNA and a l l others represent fragments produced during DNA p u r i f i c a t i o n . Care was taken through p u r i f i c a t i o n to l i m i t breakage, however, and long, seemingly i n t a c t TIV-DNA molecules were seen a f t e r p u r i f i c a t i o n using the same method. An accurate length measurement of 2.3 microns was obtained f o r c i r c u l a r CaMV-DNA molecules, i n -112-agreement wi t h the published value (Shepherd and TVakeman 1971). The long UGV-DNA molecules could represent end-to-end aggregates of a s h o r t e r , n a t i v e molecule of approximately 16 microns, but t h i s type of aggregation occurs r e l a t i v e l y r a r e l y under the co n d i t i o n s used (Davis et. a l . 1971) , A t h i r d e x planation f o r the d i s t r i b u t i o n of DNA lengths i s tha t the v i r u s does i n f a c t c o n tain DNA's of va r y i n g lengths. That the m u l t i p l e lengths may not be a r t i f a c t u a l i s im p l i e d by o b t a i n i n g s i m i l a r length d i s t r i b u t i o n s using two d i f f e r e n t DNA p u r i f i c a t i o n procedures. (The pH 13.0 procedure d i d not i n v o l v e p i p e t t i n g , phenol, ether e x t r a c t i o n , or overnight d i a l y s i s , which were features of the other method; the DNA was released and immediately used f o r e l e c t r o n microscopy). No w e l l - c h a r a c t e r i z e d DNA v i r u s has been reported to c o n t a i n v a r i o u s lengths of DNA, or m u l t i p l e pieces of DNA, although the r e o v i r u s group i s c h a r a c t e r i z e d by c o n t a i n i n g m u l t i p l e pieces of double-stranded RNA. The presence of more than one DNA molecule could i n d i c a t e e i t h e r a divided',, genome of UGV or some type of d e f e c t i v e packaging process, where by various lengths of VLP-DNA, and p o s s i b l y pieces of host DNA, are encapsidated. Sectioned UGV p a r t i c l e s always showed some c e n t r a l "empty space", not a feature of most i c o s a h e d r a l v i r u s e s , and perhaps due to a r t i f a c t u a l e f f e c t s of dehydration and f i x a t i o n . On the other hand, space w i t h i n a p a r t i c l e could i n d i c a t e incomplete packing w i t h DNA. The UGV p a r t i c l e d i d not r e a c t to spreading on 4 M urea as d i d the TIV p a r t i c l e s which released l a r g e whorls of n u c l e i c a c i d , suggesting t h a t the TIV-DNA pushed out under pressure as the p r o t e i n coat was denatured. The UGV -113-p a r t i c l e s may c o n t a i n l e s s DNA, and l e s s t i g h t l y - p a c k e d DNA, tha;n TIV and most other DNA v i r u s e s . There i s no evidence t h a t , f o r i t s three times l a r g e r diameter, there i s a p r o p o r t i o n a t e l y greater amount of DNA i n UGV than i n TIV. Another observation t h a t may support the hypothesis of heterogeneity of DNA contents w i t h i n UGV p a r t i c l e s was the anomalous sedimentation p r o f i l e of the p a r t i c l e s i n sucrose d e n s i t y gradients (Figure 7a). A h i g h l y heterogeneous pop u l a t i o n of p a r t i c l e s i s i m p l i e d by the broad band obtained a f t e r c e n t r i f u g a t i o n , e s p e c i a l l y when compared to TIV c e n t r i f u g e d under the same c o n d i t i o n s . A range of p a r t i c l e d e n s i t i e s i s suggested by t h i s p r o f i l e . Varying amounts of the most dense component of the VLP, the DNA, could lead to v a r y i n g p a r t i c l e d e n s i t i e s . Heterogeneity of DNA contents i n UGV p a r t i c l e s could be t e s t e d by c o l l e c t i n g p a r t i c l e s from the uppermost ( l e a s t dense) and lowermost (most dense) regions of a sucrose d e n s i t y g r a d i e n t , and analysing the DNA components of each f r a c t i o n by the Kleinschmidt technique., The attempts to produce l a b e l l e d VLP-DNA by a d d i t i o n of t r i t i a t e d thymidine or u r a c i l to the c u l t u r e medium were unsuc c e s s f u l : very l i t t l e l a b e l was absorbed by the host a l g a , and e s s e n t i a l l y no l a b e l was incorporated i n t o VLP-DNA. This r e s u l t i s i n agreement w i t h r e s u l t s of other l a b e l l i n g experiments f o r a l g a l n u c l e i c a c i d (Steffensen and Sheridan 1965, Chapman et a l . 1966, Swinton and Hanawalt 1972), a l l of which re p o r t t h a t thymidine was found to be incorporated i n t o c h l o r o p l a s t DNA only. Several authors have speculated t h a t the a l g a l cytoplasm may lack thymidine k i n a s e , the necessary enzyme -114-f o r i n c o p o r a t i o n of thymidine i n t o DNA, and that i t i s encoded only i n the c h l o r o p l a s t genes (Swinton and Hanawalt 1972, Steffensen and Sheridan 1965) . La b e l l e d DNA could be more completely c h a r a c t e r i z e d i n the small amounts a v a i l a b l e from UGV than has been p o s s i b l e w i t h u n l a b e l l e d DNA. One approach to produce l a b e l l e d UGV-DNA 32 would be to use P - l a b e l l e d phosphate s a l t s i n the c u l t u r e medium. However, the l a b e l l i n g p e r i o d r e q u i r e d by t h i s host 32 organism i s r e l a t i v e l y long, and incorporated P could cause a u t o l y s i s , or a r t i f a c t u a l breaks, i n the VLP-DNA by the time the VLP's were p u r i f i e d . P u r i f i e d r a d i o a c t i v e DNA could be studied by two methods: d e n s i t y gradient a n a l y s i s f o r m u l t i p l e s i z e s , and d i g e s t i o n using r e s t r i c t i o n endonucleases followed by polyacrylamide g e l e l e c t r o p h o r e s i s , to estimate the t o t a l molecular weight of UGV-DNA. C l e a r l y , a r e l i a b l e estimate of the molecular weight of UGV-DNA i s e s s e n t i a l f o r c l a s s i f i c a t i o n of t h i s VLP. The ICDV's are q u i t e homogeneous i n t h e i r content of DNA, wi t h DNA molecular weights of 130 to 160 x 10^ d a l t o n s . Blue-green a l g a l v i r u s DNA's are of molecular weights between 27 and 62 x 10 dal t o n s . M u l t i p l e pieces of DNA would e s t a b l i s h UGV as the f i r s t member of a new v i r u s group; a u n i t length of 3 2 microns (or 6 4 megadaltons) would suggest s i m i l a r i t i e s w i t h cyanophages; a l a r g e r DNA might imply a r e l a t i o n s h i p w i t h the ICDV's. I f f u r t h e r work v e r i f i e s the present evidence that UGV p a r t i c l e s c o n t a i n v a r y i n g lengths of DNA, i n v e s t i g a t i o n of the o r i g i n of these DNA fragments would be of i n t e r e s t . In p a r t i c u l a r , h y b r i d i z a t i o n of UGV-DNA to JJ.. gigas (host) DNA -115-should be done to test for d i r e c t packaging of host DNA without true VLP-DNA synthesis. A defective phage of B a c i l l u s  s u b t i l i s , PBSX, lysogenizes i t s host c e l l s . Upon induction with mitomycin C the host c e l l s lyse and phage p a r t i c l e s are produced, even when DNA synthesis i s completely i n h i b i t e d (Okamoto et a l . 1968b). By DNA/DNA hybridization, buoyant density determinations i n cesium chloride, and transformation studies on the bacterium using 'phage DNA, the DNA i n the phage p a r t i c l e s was determined to be b a c t e r i a l DNA, cut to a uniform length and packaged i n phage coat protein (Okamoto et, a l , 1968a) . This phage i s considered to be defective i n the synthesis of i t s own DNA. If UGV were s i m i l a r l y defective, and less s p e c i f i c i n i t s cutting of host DNA, a range of DNA lengths packaged into UGV p a r t i c l e s might r e s u l t . PBSX i s not infect i o u s since i t contains only host DNA; the analogous case for UGV might explain d i f f i c u l t i e s i n demonstrating transmission and i n obtaining "healthy" cultures. The molar f r a c t i o n of G + C does not aid i n c l a s s i f i c a t i o n of these viruses. The ICDV's are very heterogeneous i n t h i s property, ranging from 30 to' 60% (G +C); the cyanophages also show great v a r i a t i o n , from 37 to 67%. This parameter cannot be used as a measure of relatedness of these viruses, as has been done for higher organisms. The DNA of UGV, with a (G + C) f r a c t i o n of 60%, i s not outside the range of either of the other two virus groups. On the evidence available now, summarized i n Table XVI, the UGV p a r t i c l e appears to be d i s t i n c t i v e , bearing no strong s t r u c t u r a l or biochemical rel a t i o n s h i p to the ICDV's or the -116-TABLE XVI — Comparison of the properties of ICDV's, cyanophages, and UGV Property Size (nm) T a i l Envelope S20,w TIV 130 2300 CsCl density 1.32 (g/ml) Ether sensitive Polypeptides: number L i p i d component DNA: mol. wt. (x 106 daltons) (G + C) % nature 20 M.W. of major species 65,000 + 130-160 30% li n e a r d s c FV3 130 1.32 17 49,000 + 130-160 56? Other ICDVa 130-260 4450 (MIV) 30-60% Cyanophage 55-90 + UGV 390 (nonswollen) + 550, approx. 6 35 0 1.48 10 38,000 & 14 ,000 27-62 37-67% li n e a r l i n e a r l i n e a r ds ds ds 1.32 10 detected 45 ,000 60% lin e a r ds Data from Kelly and Robertson 1973. Data from Padan and Shilo 1973. 'Double-stranded. -117-cyanophages. On the basis of morphology alone, the capsid appears to be similar to those of the ICDV's, only substantially larger than most; but i t also has a t a i l , uncharacteristic of the ICDV's and suggesting a r e l a t i o n s h i p with phages. As there i s d i v e r s i t y within the ICDV's and among cyanophage types (Kelly and Robertson 1973, Padan and Shilo 1973), the e x i s t i n g c l a s s i f i c a t i o n s may be subject to revisions as new information becomes available. On the other hand, the VLP from U. gigas may represent the f i r s t characterized member of a new group of viruses. V. B i o l o g i c a l considerations In further study of the r e l a t i o n s h i p of t h i s VLP to i t s a l g a l host, a primary concern would be establishing the i n f e c t i v i t y of t h i s p a r t i c l e . At the moment i t i s unclear whether any c e l l s of U. gigas can be shown to be healthy, or free of VLP's under any environmental conditions imposed. There i s no d e f i n i t i v e evidence to show whether i n f e c t i o n proceeds from outside the c e l l , by absorption of f r e e - f l o a t i n g p a r t i c l e s to a l g a l zoospores (or possibly other c e l l types) or whether the information for VLP synthesis i s i n some way carried by a l l c e l l s of U_. gigas, with varying extents of expression. A l l i s o l a t e s derived by single c e l l transfers have been shown to release VLP's. The p o s s i b i l i t y that some kind of latency phenomenon i s involved cannot be ruled out. The occurrence of latent infections of VLP's i n other algae has been suggested previously. Hoffman and Stanker (1976) working on the Cylindrocapsa VLP, also found that heat shock could induce VLP production i n young, apparently healthy colonies and -118-repeated transfers gave r i s e to seemingly healthy filaments which could always be induced to produce VLP's. Markey (197 4) also postulated a dormant i n f e c t i o n from a study of the brown alga P y l a i e l l a , i n which VLP's are seen only i n sporangial c e l l s . Synthesis of VLP's i s synchronous from c e l l to c e l l i n a sporangium, and thus unlike l y to be caused by external i n f e c t i o n . Markey suggests that i n f e c t i o n of c e l l s without thick w a l l s -gametes or zoospores—may occur, but induction of VLP production only happens i n the mature plant. Similar timing of i n f e c t i o n was found i n Ectocarpus, and Clitheroe and Evans (1974) f e e l that the VLP i n f e c t i o n i s probably systemic, with production of VLP's limited to one c e l l type only, the sporangia. Lemke (1976) points out that most of the double-stranded RNA viruses of fungi are mainly latent, transmitted by cytoplasmic exchange, and have multi-component genomes packaged i n multiple capsids. The lower eucaryotes may have evdv ed less dramatic forms of v i r a l i n f e c t i o n than are seen i n the diseases of plants and animals., No author has speculated on the mechanism of latency: whether some phenomenon l i k e lysogeny i n phages i s involved, or i f the VLP p a r t i c l e s remain i n t a c t i n some form i n the cytoplasm of the a l g a l c e l l s . Further characterization of the i n f e c t i v i t y of UGV would require healthy a l g a l cultures as t e s t organisms. Since no authenticated healthy JJ_, gigas i s available from culture c o l l e c t i o n s , e f f o r t s would have to be made to either derive a healthy i s o l a t e or to demonstrate i n f e c t i v i t y i n another host. Several methods could be t r i e d for derivation of a healthy culture, such as selection for the most vigorous zoospores -119-(perhaps by greater m o t i l i t y i n moving toward a l i g h t source; U. gigas zoospores are p o s i t i v e l y phototactic); growth of i s o l a t e d germlings on agar to look for altered morphological c h a r a c t e r i s t i c s which might correlate with i n f e c t i o n ; repeated s i n g l e - c e l l transfer from repeatedly heat-shocked cultures; and possibly curing by a n t i b i o t i c treatments, e.g., mitomycin C, as i s routinely done with lysogenic bacteriophages. One d i f f i c u l t y i n assessing i n f e c t i o n i n i s o l a t e s i s the absolute reliance at present on electron microscopical methods for detection of VLP production. Negative staining of aliquots of culture medium may not be r e l i a b l e i f very low levels of VLP synthesis and release are involved (or i f release i s defective). Examination of a l g a l c e l l s i n thin section i s probably more inaccurate as a measure of how many c e l l s are infected, since only very few c e l l s can be scanned. Preparation of an antiserum to p u r i f i e d UGV would simplify these tests immeasurably; to date p u r i f i e d UGV has not been available i n the quantity needed for antiserum production. If future experiments suggest that latent infections of UGV do occur i n U. gigas, systematic investigation of the conditions which trigger active infections would be of great i n t e r e s t . Those conditions which might occur as cues i n nature for a cycle of i n f e c t i o n of the host alga would have implications for control of eucaryotic a l g a l blooms. Cowlishaw and Mrsa (1975) found that a stable equilibrium of the blue-green alga Plectonema boryanum and i t s v i r u s , LPP-1, developed and that most host c e l l s were re s i s t a n t to severe infections (leading to l y s i s ) . A similar adaptation of host alga and virus may have -120-developed between U. gigas and UGV, and possibly other eucaryotic algae and t h e i r VLP's. Lemke (1976) points out that a demonstration of true i n f e c t i v i t y — n e e d e d to confirm UGV as a true v i r u s — r e s t s on s a t i s f y i n g Koch's postulates, which may not be possible i n cases of latent i n f e c t i o n or obligate symbiosis. Given the p o s s i b i l i t y of latency as a widespread phenomenon among eucaryotic microorganisms, the VLP's observed may never be amenable to designation as viruses, i n the usual sense of the term. If latency does occur i n U. gigas, a question arises concerning the taxonomic status of the alga: does the presence of a latent UGV i n f e c t i o n a f f e c t the host's morphological c h a r a c t e r i s t i c s ? V i r u s - l i k e p a r t i c l e s i n other eucaryotic microorganisms have been shown to a l t e r host phenotype, e.g., the k i l l e r systems i n Paramecium and yeast (Lemke 1976), and hypovirulence and sometimes colony color i n the chestnut b l i g h t fungus and the t a k e - a l l fungus of wheat (Moffitt and L i s t e r 1975). Ramanathan (1964) emphasized that the c h a r a c t e r i s t i c s separating Uronema species from Ulothrix species were r e l a t i v e l y minor and variable according to environmental conditions. Mitra (1974) regarded fragmentation of long filaments due to continual zoospore production as a feature of many Uronemas. If mature c e l l s of the a l g a l filament can become infected with VLP's and can lyse, releasing VLP's, fragmentation could also be due to t h i s process. Certainly, species of Ulothrix should be tested at a l l stages of t h e i r l i f e cycle for s u s c e p t i b i l i t y to UGV or to changes i n culture c h a r a c t e r i s t i c s after exposure to UGV. -121-To summarize, a VLP r e l e a s e d by c e l l s o f Uronema g i g a s , a f r e s h w a t e r e u c a r y o t i c a l g a , has been p u r i f i e d and p a r t i a l l y c h a r a c t e r i z e d . W h i l e i t s h a r e s some s t r u c t u r a l p r o p e r t i e s w i t h the i c o s a h e d r a l c y t o p l a s m i c d e o x y r i b o v i r u s e s o f e u c a r y o t e s and some w i t h t h e cyanophages o f p r o c a r y o t i c a l g a e , t h e s i z e and th e p r e s e n c e o f a t a i l a r e unique among v i r u s e s o f e u c a r y o t e s . The VLP always o c c u r s i n c e l l s w i t h d i s r u p t e d membrane systems, • and i t was ob s e r v e d i n young g e r m l i n g s o n l y , never i n c e l l s o f the mature f i l a m e n t . The c y t o p a t h o l o g i c a l e f f e c t s i n c e l l s c o n t a i n i n g VLP's suggest t h a t the VLP i s p a t h o g e n i c , but i n f e c t i v i t y o f t h e VLP's cannot be demonstrated w i t h o u t a h e a l t h y JJ_. g i g a s c u l t u r e . Methods have been worked out f o r growth of the a l g a t o maximize VLP r e l e a s e , and a p r o c e d u r e f o r p u r i f i c a t i o n o f UGV was d e v e l o p e d . T h i s system now shows p o t e n t i a l f o r f u r t h e r c h a r a c t e r i z a t i o n o f t h e p a r t i c l e i t s e l f , and o f t h e i n t e r a c t i o n o f the p a r t i c l e w i t h i t s h o s t a l g a . I n s p i t e o f t h e r e l a t i v e o b s c u r i t y o f the h o s t and i t s l i m i t e d d i s t r i b u t i o n , f u r t h e r d e s c r i p t i o n o f the UGV p a r t i c l e and i t s r e l a t i o n t o i t s h o s t may p r o v i d e a model system f o r s t u d y o f o t h e r VLP's o f e u c a r y o t i c m i c r o o r g a n i s m s . -122-LITERATURE CITED Adolph, K.W. and R.H. Haselkorn. 1971. Iso l a t i o n and chara-c t e r i z a t i o n of a virus i n f e c t i n g the blue-green alga Nostoc muscorum. Virology 46: 200-208. Agrawal, H.W. and J.H.Tremaine. 1972. 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