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UBC Theses and Dissertations

Genome properties and non-productive infections of herpesviruses Mosmann, Timothy Richard 1973

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J 7 Z J Z > d GENOME PROPERTIES AND NON-PRODUCTIVE INFECTIONS OF HERPESVIRUSES by TIMOTHY R. MOSMANN B.Sc. U n i v e r s i t y of N a t a l (Durban) 1968 B.Sc. (Hons.), Rhodes U n i v e r s i t y 1969 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEC-REE OF PHILOSOPHY In the Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced, degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of h ^ C ^ Q 9 JOLO The University of British Columbia Vancouver 8, Canada i ABSTRACT Herpesvirus infections have been studied with the purpose of establishing conditions under which control of latent infection could be investigated. Several relevant properties of the viruses were also studied. Murine cytomegalovirus (MCV) preparations grown in vitro in mouse embryo cells contained large numbers of multiple capsid virions. These multiples were apparently infectious, but were not the main cause of the unusual enhancement of infectivity of MCV caused by application of centrifugal force during adsorption. The DNA of three herpesviruses was studied. Herpes simplex (HSV) DNA did not contain a significant number of cross-links. By comparison with bacteriophage T^ -DNA (molecular weight 110 x 10^ ) after neutral sucrose gradient sedimentation, the molecular weights of MCV and HSV-DNA's were calculated as 132 x 10^ and 85 x 10^ respectively. Both these viral DNA's contained alkali sensitive single strand interruptions, and in the case of MCV-DNA i t was found that the number of interruptions was dependent on the purification procedure, and preparations were obtained in which some molecules were free, of alkali sensitive regions in both strands. Human cytomegalovirus preparations contained a DNA g component with an approximate molecular weight of 3 x 10 . MCV-DNA was analyzed by centrifugation to equilibrium in cesium chloride solution, and evidence obtained for a heterogeneous distribu-i i t i o n of d e n s i t y along the molecule. I n t a c t molecules banded at a s i n g l e d e n s i t y corresponding to a G + C content of 59$>. whereas fragmented molecules of 18 x 10^ molecular weight banded as two components, at d e n s i t i e s corresponding to 5 7 - 5 and 6 1 . 5 % G + C. This heterogeneity was confirmed by a n a l y s i s of UV-absorbance/ temperature p r o f i l e s of MCV-DNA, which provided evidence f o r two or p o s s i b l y three components present i n unequal amounts. Evidence was obtained from DNA-RNA h y b r i d i z a t i o n s t u d i e s to suggest that there was d i f f e r e n t i a l t r a n s c r i p t i o n or degradation of the v i r a l RNA synthesized during i n f e c t i o n , from the two DNA components separated on cesium c h l o r i d e g r a d i e n t s . A f t e r i n f e c t i o n of a continuous l i n e of human c e l l s w i t h MCV, the t i t r e of i n f e c t i o u s v i r u s r a p i d l y d e c l i n e d t o undetectable l e v e l s . The growth r a t e of the i n f e c t e d c e l l s decreased, and the morphology of the c e l l s was a l t e r e d . Both these changes l a s t e d f o r a few weeks, a f t e r which the c u l t u r e s r e v e r t e d t o the normal growth r a t e and morphology. DNA-DNA annealing was used i n an attempt to detect the presence and synthesis of v i r a l DNA i n these c e l l s . I n most cases, n e i t h e r the presence nor absence of MCV-DNA at the l e v e l of one genome per c e l l genome, could be e s t a b l i s h e d , but i n one experiment, synthesis of v i r a l DNA apparently occurred i n MCV i n f e c t e d c e l l s l U days a f t e r i n f e c t i o n . V i r a l RNA synthesis i n 5 these c e l l s was undetectable, i . e . l e s s than two p a r t s i n 10 of the t o t a l RNA syn t h e s i s . i i i TABLE OF CONTENTS Page CHAPTER 1: INTRODUCTION A. General 1 B. H i s t o r i c a l 3 1. Herpes simplex v i r u s 3 2. Human and murine cytomegaloviruses l± C. B i o p h y i s i c a l and Biochemical P r o p e r t i e s . . . . 5 1. Herpes simplex v i r u s 5 2. Human and murine cytomegaloviruses 6 D. Pathogenesis 7 1. Herpes simplex v i r u s 7 2. Cytomegaloviruses 11 E. Growth i n Tissue C u l t u r e 12 1. Herpes simplex v i r u s 12 2. Cytomegaloviruses 13 F. V i r u s Growth Cycle Ik 1. Herpes simplex v i r u s Ik A d s o r p t i o n ih E n t r y and uncoating 15 RNA synthesis and t r a n s p o r t 16 P r o t e i n synthesis 17 Assembly and r e l e a s e 18 Enzymes induced by herpes simplex v i r u s . . 19 E f f e c t of i n f e c t i o n oh host c e l l s . . . . 19 2. Cytomegaloviruses 20 A d s o r p t i o n and p e n e t r a t i o n 20 R e p l i c a t i o n 20 E f f e c t s of i n f e c t i o n on host c e l l s . . . 21 iv TABLE OF CONTENTS Page G. Herpes Simplex V i r u s Types 1 and 2 22 H. Latency and Oncogenesis i n Herpesvirus I n f e c t i o n s . . 23 CHAPTER 2: MATERIALS AND METHODS A. C e l l s 25 B. V i r u s e s 25 C. Growth Medium . 26 D. Reagents 26 E. S o l u t i o n s 27 F. C e l l C u l t u r e Conditions 29 G. Mouse Embryo C e l l s 30 H. C e l l T r ansfer ' 30 I. Growth o f Vir u s e s : 31 Murine cytomegalovirus 31 1. Standard adsorption 31 2. C e n t r i f u g a l adsorption 32 Herpes simplex v i r u s 33 Human cytomegalovirus 33 J. Plaque Assays 33 Murine cytomegalovirus 33 Herpes simplex v i r u s ( s t r a i n P) 3I4. K. E l e c t r o n Microscopy 3^ L. P u r i f i c a t i o n of Herpesviruses 35 D i f f e r e n t i a l c e n t r i f u g a t i o n 35 Deoxyribonuclease treatment 35 Sucrose d e n s i t y gradient v e l o c i t y sedimentation . 35 E q u i l i b r i u m gradient c e n t r i f u g a t i o n 36 V TABLE OF CONTENTS Page M. P u r i f i c a t i o n of DNA 36 V i r a l DNA 36 1. Large sca l e p u r i f i c a t i o n . 36 2. High molecular weight v i r a l DNA 39 C e l l u l a r DNA 39 N. A n a l y s i s o f V i r a l DNA 0^ Hydroxyapatite (HA) chromatography kO Stepwise e l u t i o n h2 Gradient e l u t i o n . h3 N e u t r a l sucrose g r a d i e n t v e l o c i t y sedimentation A l k a l i n e sucrose gradient v e l o c i t y sedimentation Cesium c h l o r i d e 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 5^ 1. P r e p a r a t i v e . . . . . . . . 5^ 2. A n a l y t i c a l 6^ U l t r a v i o l e t absorbance/temperature p r o f i l e s . . 7^ 0 . L a b e l l i n g and P u r i f i c a t i o n of RNA ^8 P. N u c l e i c A c i d H y b r i d i z a t i o n F i x a t i o n of DNA 50 DNA-DNA annealing 50 DNA-RNA h y b r i d i z a t i o n . 51 Q. R a d i o a c t i v i t y Measurement 52 R. C e l l Counting 52 CHAPTER 3: RESULTS I: Growth P r o p e r t i e s A. Plaque assays 5^  1. C o n d i t i o n of monolayers . 5^  2. G e l l i n g agent 55 v i TABLE OF CONTENTS Page A. 3- Serum con c e n t r a t i o n 55 k. V i s u a l i z a t i o n of plaques 55 5. Adsorption time 56 B. C e n t r i f u g a l Assay 56 C. Morphology 56 D. Growth Curves 65 E. S t a b i l i t y 65 F. P u r i f i c a t i o n 70 1. D i f f e r e n t i a l c e n t r i f u g a t i o n 70 2. Deoxyribonuclease and resedimentation . . . . 73 3. Sucrose g r a d i e n t v e l o c i t y sedimentation . . . 77 k. 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 78 CHAPTER k: RESULTS I I : P r o p e r t i e s of Herpesvirus Genomes A. Radi o a c t i v e L a b e l l i n g of V i r a l DNA 79 B. P u r i f i c a t i o n of V i r a l DNA 80 C. Hydroxyapatite (HA) Chromatography 83 D. Sucrose Density Gradient Sedimentation of DNA . . . 90 N e u t r a l pH. Murine cytomegalovirus . . . . 9° N e u t r a l pH. Herpes simplex v i r u s 95 N e u t r a l pH. Human cytomegalovirus 98 A l k a l i n e pH. Murine cytomegalovirus . . . . 102 A l k a l i n e pH. Herpes simplex v i r u s 108 A l k a l i n e pH. Human cytomegalovirus 108 E. Cesium C h l o r i d e Gradient 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 . I l l F. U l t r a v i o l e t Absorbance/Temperature P r o f i l e . . . . 120 G. T r a n s c r i p t i o n of MCV-DNA 123 v i i TABLE OF CONTENTS Page CHAPTER 5: RESULTS I I I : Non-Productive I n f e c t i o n s A. Herpes Simplex V i r u s . A r g i n i n e d e p r i v a t i o n . . . . 130 B. Host Range 131 C. Non-Productive I n f e c t i o n of H.Ep.2 C e l l s 132 1. Growth r a t e 133 2. Morphology 133 D. Attempts t o Detect MCV-DNA i n I n f e c t e d H.Ep.2 C e l l s . 137 CHAPTER 6: DISCUSSION A. Morphology of MCV 1^ 3 B. C e n t r i f u g a l Enhancement of I n f e c t i v i t y ihk C. Hydroxyapatite Chromatography ihh D. Sedimentation of V i r a l DNA1 s 1^ 5 1. Murine cytomegalovirus ll+5 2. Herpes simplex v i r u s 150 3. Human cytomegalovirus 153 E. Heterogeneity of MCV-DNA 15k F. T r a n s c r i p t i o n of MCV-DNA Components 157 G. Non-Productive I n f e c t i o n s 159 REFERENCES 162 " v i i i LIST OF TABLES Table Page 1. Loss of i n f e c t i v i t y of MCV and HSV. 69 2. P u r i f i c a t i o n of herpesviruses. 74 3. D e n s i t i e s of fragmented MCV-DNA. 119 1+. Annealing o f 3 H - c e l l DNA and RNA t o ll+o MCV-DNA i x LIST OF PLATES Plate Page 1. Morphology of herpesviruses 60 2 . Morphology of H.Ep.2 cells 1 3 6 X LIST OF FIGURES Fi g u r e Page 1. Hydroxyapatite column kl 2. C e n t r i f u g a l adsorption of MCV. 58 3. I n f e c t i v i t y of d i f f e r e n t v i r u s populations. 63 k. Growth curves of MCV and HSV. 67 5. Loss of i n f e c t i v i t y and degradation of HSV. 72 6. Sucrose gradient sedimentation of HSV. 77 7. C s C l 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 HSV and 8l H.Ep.2 DNA's. 8. V e l o c i t y sedimentation of DNA from sucrose- 85 g r a d i e n t p u r i f i e d v i r i o n s . 9. Rate of denaturation of HSV-DNA. 89 10. E l u t i o n of denatured HSV-DNA from hydroxy- 92 a p a t i t e . 11. V e l o c i t y sedimentation of MCV-DNA. 9I+ 12. V e l o c i t y sedimentation of HSV-P and MCV-DNA's. 97 13. V e l o c i t y sedimentation of HCV-DNA. 99 LIST OF FIGURES Fi g u r e ik. V e l o c i t y sedimentation of sheared HCV-DNA. 15. A l k a l i n e v e l o c i t y sedimentation of MCV-DNA. 16. A l k a l i n e v e l o c i t y sedimentation of HSV-DNA. 17. P r e p a r a t i v e C s C l gradient c e n t r i f u g a t i o n of MCV-DNA. 18. A n a l y t i c a l C s C l c e n t r i f u g a t i o n . 19. Sedimentation a n a l y s i s of sheared MCV-DNA. 20. M e l t i n g p r o f i l e of MCV and HSV-DNA. 21. Separation of the two d e n s i t y components of MCV-DNA. 22. T r a n s c r i p t i o n of the two d e n s i t y components of MCV-DNA. 23. E f f e c t s of i n f e c t i o n of H.Ep.2 c e l l s w i t h MCV. x i i ABBREVIATIONS ADB - Alkaline DNA buffer Ci - Curie CMV - Cytomegalovirus cpm - Counts per minute DNA - Deoxyribonucleic acid DNase - Pancreatic deoxyribonuclease dThd - Thymidine EDTA - Ethylenediaminetetraacetic acid fig. - Figure x g - x gravity (force) G + C - Guanine + cytosine HA - Hydroxyapatite HCV - Human cytomegalovirus H.Ep.2 - Human epidermoid carcinoma #2 HSV-P - Herpes simplex virus, strain P HSV-F - Herpes simplex virus, strain F IgA - Immunoglobulin A L 9 2 9 - Mouse fibroblast cells M - Molecular weight MCV - Murine cytomegalovirus ME - Mouse embryo (cells) ABBREVIATIONS MEM - Minimal e s s e n t i a l medium MKSA - SV-UO transformed mouse kidney (ce mRNA - Messenger RNA NDB - N e u t r a l DNA b u f f e r PBS - Phosphate b u f f e r e d s a l i n e pfu - Plaque forming u n i t J - Density (g/cc) RNA - R i b o n u c l e i c a c i d RNase - P a n c r e a t i c ribonuclease s - Sedimentation c o e f f i c i e n t SDS - Sodium dodecyl sulphate SSC - Standard s a l i n e c i t r a t e SY-kO - Simian v i r u s kO 3T3 - Mouse e p i t h e l i a l c e l l s Tm - M e l t i n g temperature (of DNA) T r i s - T r i s (hydroxymethyl) aminomethane Ur - U r i d i n e UV - U l t r a v i o l e t ( r a d i a t i o n ) v/v - volume/volume WI38 - Human d i p l o i d f i b r o b l a s t ( c e l l s ) w/w - weight/weight ACKNOWLEDGEMENT S The encouragement and advice of Dr. J. B. Hudson were g r e a t l y appreciated throughout t h i s study. Thanks are a l s o due to Dr. J.J.R. Campbell, who provided t h i s o p p o r t u n i t y f o r research, and t o my other committee members f o r t h e i r i n t e r e s t and suggestions. I am g r a t e f u l to Dr. R.C. M i l l e r f o r u s e f u l d i s c u s s i o n s and s e v e r a l g i f t s of Tk bacteriophage. The t e c h n i c a l a s s i s t a n c e of L. McGrath, G. Steele, L. W a t e r f i e l d , B. Bryant and T. Walters was appreciated during t h i s work. F i n a l l y , I wish t o thank R. Morgan, who p a t i e n t l y typed t h i s manuscript. INTRODUCTION A. General Herpesviruses are f o r m a l l y defined as l a r g e , enveloped v i r u s e s c o n t a i n i n g double stranded DNA enclosed i n an i s o c o h e d r a l c a p s i d w i t h 162 capsomers (Roizman, 1969)• A lar g e number of herpesviruses i s known, and most animal species appear to act as host f o r at l e a s t one h e r p e s v i r u s . The h o s t - v i r u s r e l a t i o n s h i p i n the n a t u r a l . h o s t i s o f t e n well-balanced and does not l e a d to death of the host. Long-term, p e r s i s t e n t i n f e c t i o n s are common, w i t h p e r i o d i c increased a c t i v i t y of the v i r u s . S e v e r a l herpesviruses are known to be oncogenic, and se v e r a l others are suspected of having a causative or 'helper' r o l e i n oncogenesis. The d i f f e r e n c e s between cytomegaloviruses and other herpes-v i r u s e s are not profound. In general, the cytomegaloviruses induce cytomegalia i n v i v o , form i n t r a n u c l e a r and o f t e n cytoplasmic i n c l u s i o n bodies, cause f o c a l c y t o p a t h o g e n i c i t y i n t i s s u e c u l t u r e because of the c e l l - a s s o c i a t e d nature of i n f e c t i o u s v i r u s , and are markedly h o s t - s p e c i f i c (Weller, 1971). However, a n t i g e n i c c r o s s - r e a c t i o n s suggest t h a t cytomegaloviruses are f a i r l y c l o s e l y r e l a t e d to some other herpesviruses (Wentworth and French, 1970). A study of the DNA d e n s i t y of s e v e r a l cytomegaloviruses and herpesviruses (Plummer, et a l , 1969) d i d not support the s e p a r a t i o n of the two groups. 2 The herpesviruses used i n t h i s study were:- Herpes simplex v i r u s , murine cytomegalovirus, and human cytomegalovirus. 3 B. Historical 1. Herpes simplex virus Herpetic, or 'creeping' eruptions were recognized over 2,000 years ago (Roizman, 1969). Although the term 'herpes' was ini t i a l l y used to refer to a wide variety of spreading, ulcerative lesions of the skin, later use of the word was restricted to describing certain vesicular eruptions. Several different herpetic diseases were recognized by the beginning of the 20th century, including herpes zoster, herpes febrilis, herpes facialis and herpes genitalis (Nahmias and Dowdle, 1968). The fi r s t definite successful transmission of a causative agent was reported by Gruter (192U) who transmitted a human herpetic ocular infection to the rabbit cornea during 1911-191^. The infectious agent was shown to be filterable (Luger and Lauda, 1921), and Loewenstein (±919) showed that material from other human herpetic infections (of the skin and mucous membranes) could yield a similar infection on rabbit corneas. Since apparently the same causative agent could be isolated from several different clinical diseases, the general name herpes simplex was given to the diseases herpes genitalis, herpes facialis and herpes febrilis. Lipschutz (1921, 1932) considered that herpes genitalis and herpes febrilis should be considered separate diseases, but only recently has i t been established that there are at least two subtypes of herpes simplex virus (reviewed in Nahmias and Dowdle, 1968). E a r l y c h a r a c t e r i z a t i o n of the agent e s t a b l i s h e d t h a t i t was approximately 100-150 nm i n diameter ( E l f o r d et a l , 1933). I n f e c t i v i t y was s e n s i t i v e to detergents (Burnet and Lush, 19 -^0). Morgan et a l (195*0 used the e l e c t r o n microscope to observe v i r u s p a r t i c l e s c o n s i s t i n g of a dense core surrounded by one or two membranes, having a diameter of .120-130 nm. R u s s e l l (1962) and BenPorat and Kaplan (1962) showed t h a t the v i r a l p a r t i c l e s c o n t a i n deoxyribonucleic a c i d , and l i t t l e , i f any, r i b o n u c l e i c a c i d . 2. Human and Murine Cytomegaloviruses Enlarged c e l l s w i t h nuclear i n c l u s i o n s , c h a r a c t e r i s t i c of cytomegalic i n c l u s i o n disease, were f i r s t r e p o r t e d i n man by Jesionek and Kiolemenoglou (190*0. S i m i l a r cytomegaly was seen i n s e v e r a l other animal species, but the enlarged c e l l s were o f t e n considered t o be a protozoan p a r a s i t e (Smith and Weidman, 1910-1911). Cole and Kuttner (1926), r e p o r t e d the f i r s t s u c c e s s f u l t r a n s m i s s i o n of a f i l t e r a b l e agent causing cytomegalic i n c l u s i o n disease i n guinea p i g s , e s t a b l i s h i n g the v i r a l nature of the disease. Smith (195*0 i s o l a t e d and propagated the murine cytomegalovirus i n mouse embryo f i b r o -b l a s t explant c u l t u r e s , and human cytomegalovirus was i s o l a t e d by Smith ( I 9 5 6 ) and Rowe et a l (1956). 5 C. B i o p h y s i c a l and Biochemical P r o p e r t i e s 1. Herpes Simplex V i r u s Herpes simplex v i r u s i s a la r g e ( 1 5 O - I 6 0 nm (Roizman and Spear, 1971)), enveloped (Morgan et a l , 195*+), DNA v i r u s (BenPorat and Kaplan, 1962) which r e p l i c a t e s i n the n u c l e i of i n f e c t e d c e l l s (Gray and Scott, 195*+; Morgan et a l , 195*+). The v i r i o n c o n s i s t s of a c e n t r a l c y l i n d r i c a l core, w i t h the DNA present i n a torus surrounding the equator of the core (Furlong et a l , 1972). This i s surrounded by a p r o t e i n c a p s i d w i t h 162 e x t e r i o r capsomers i n i c o s o h e d r a l symmetry (Caspar and Klug, 1962). The ca p s i d i s surrounded by two membranes, probably c o n t a i n i n g p r o t e i n and l i p i d (reviewed by Roizman, 1969). The e x t e r n a l membrane i s probably h o s t - d e r i v e d , and contains both host and v i r a l antigens (Watson and Wildy, 1963). The p a r t i c l e : i n f e c t i v i t y r a t i o i s approximately 10:1 at best (Watson et a l , 196I+), and may be 100:1 i n common preparations (Roizman, I969). I n f e c t i v i t y i s r a p i d l y l o s t i n a v a r i e t y of s o l u t i o n s at 37°C and *+°C, but i n a c t i v a t i o n can be retarded by use of d i s t i l l e d water or serum s o l u t i o n s as d i l u e n t s ( W a l l i s and Melnick, 1965)• I n f e c t i v i t y i s a l s o s e n s i t i v e to l i p i d s o l v e n t s (e.g. e t h y l ether, (Andrewes and Horstmann, 19*+9)» 1$ sodium dodecyl sulphate s o l u t i o n , (Kaplan, I969)), t r y p s i n (Gresser and Enders, 1961), and l i g h t ( W a l l i s and Melnick, 1969). 6 The chemical composition of p u r i f i e d v i r i o n s i s : -6. % DNA 70$ P r o t e i n 1.6$ Carbohydrate 22$ P h o s p h o l i p i d ( R u s s e l l et a l , 1963). The DNA of the herpes simplex v i r i o n i s a s i n g l e l i n e a r duplex DNA molecule of approximately 100 x 10^ molecular weight (Becker e t a l , 1968; Graham et a l , 1972; K i e f f et a l , 1971). I n both p u r i f i e d v i r i o n s and v i r a l DNA i n i n f e c t e d c e l l s , s i n g l e - s t r a n d i n t e r r u p t i o n s are present i n some or a l l DNA molecules ( F r e n k e l and Roizman, 1972a), and these i n t e r r u p t i o n s may be sequence-specific. The genetic complexity of herpes simplex DNA i s equ i v a l e n t to 95 x 10^ daltons of DNA (F r e n k e l and Roizman, 1971). The guanine + cyt o s i n e content of the DNA i s 68$ (type l ) or 70$ (type 2) (Goodheart et a l , 1968). Methylated or unusual bases are not known to be present (Roizman and Spear, 1971; Low et a l , 1969). 2. Human and Murine CMV MCV and HCV are l a r g e , enveloped (Smith, 1959)? DNA v i r u s e s (Henson et a l , ±966; Plummer et a l , 1969), w i t h i c o s o h e d r a l nucleo-7 capsids of l62 capsomers (Hanshaw, 1968). The DNA of murine c y t o -megalovirus i s double stranded, and apparently contains two components of d e n s i t i e s i n C s C l s o l u t i o n corresponding to guanine + cyt o s i n e contents of 59 and 6% (Plummer et a l , 1969). The DNA of human cytomegalovirus has a d e n s i t y i n CsCl corresponding to a G + C content of 57$ (Plummer et a l , 1969) and a molecular weight not l e s s than 32 x 106 (Crawford and Lee, 196U). D. Pathogenesis 1. Herpes Simplex V i r u s I n the n a t u r a l host, man, primary i n f e c t i o n by herpes simplex v i r u s normally r e s u l t s i n a m i l d , systemic, s e l f - l i m i t i n g i n f e c t i o n which i s o f t e n s u b c l i n i c a l ( S c o t t , 1957) but o c c a s i o n a l l y more serious (Kaplan, 19^9)• A l a r g e percentage of the adult p o p u l a t i o n c a r r i e s antibody to herpes simplex v i r u s , i n d i c a t i n g p r i o r i n f e c t i o n , This percentage v a r i e s from approximately 60% to 100$ (Kaplan 1969) and i s i n f l u e n c e d by the socio-economic status of the group surveyed (Becker, 1966). This v i r u s i s unusual i n that secondary i n f e c t i o n s i n the presence of c i r c u l a t i n g antibody occur very f r e q u e n t l y . These i n f e c t i o n s are c h a r a c t e r i s t i c a l l y l o c a l i z e d , v e s i c u l a r eruptions which tend to recur at the same s i t e i n subsequent eruptions i n the 8 same p a t i e n t (Kaplan, 1969). They occur i n the presence of c i r c u -l a t i n g antibody against herpes simplex v i r u s , and can be induced by a wide v a r i e t y of agents: e.g. i l l n e s s ; a r t i f i c i a l l y induced f e v e r (Carpenter et a l , 19 -^0); menstruation ( S c o t t , 1957); an Arthus r e a c t i o n (Anderson et a l , 1961); s u n l i g h t (Kaplan, 1969); i n j e c t i o n of adrenaline (Schmidt and Rasmussen, i960); treatment w i t h c o r t i -c o s t e r o i d s (Leopold and Levy, 1963), and emotional s t r e s s (Blank and Brody, 1950). The recurrence of eruptions i n the same s i t e , p r e d i c t -a b l y f o l l o w i n g v a r i o u s p h y s i c a l s t i m u l i , suggests t h a t secondary i n f e c t i o n s are normally due to r e a c t i v a t i o n of inapparent v i r u s present i n the host at a l l times. The nature of t h i s 'dormant' v i r u s has not yet been e s t a b l i s h e d . Roizman (1965), has suggested two p o s s i b i l i t i e s to e x p l a i n t h i s p e r s i s t e n c e : -1. The v i r u s m u l t i p l i e s at a very low l e v e l i n some t i s s u e without causing overt p a t h o l o g i c a l e f f e c t s . 2 . The reproductive c y c l e of the v i r u s i s i n h i b i t e d soon a f t e r i n f e c t i o n and before the synthesis of i n f e c t i o u s v i r u s . These two p o s s i b i l i t i e s have not yet been s a t i s f a c t o r i l y i n v e s t i g a t e d i n the n a t u r a l host, man, but more in f o r m a t i o n i s a v a i l a b l e regarding i n f e c t i o n i n mice and r a b b i t s . Johnson (196^ -) showed t h a t a f t e r p e r i p h e r a l i n o c u l a t i o n of herpes simplex v i r u s i n t o mice, v i r u s could reach the nervous system v i a the blood or by ascending i n f e c t i o n of n e u r a l c e l l s of the nervous system. Stevens et al (1972), have shown that the virus can be recovered from the trigeminal ganglia of rabbits with recurrent herpes simplex eye infection, and from the spinal ganglia of mice that had recovered from posterior paralysis caused by herpes simplex virus infection (Stevens and Cook, 1972). No virus could be detected in ganglia on direct assay. However, several weeks after culturing spinal ganglia associated with sciatic nerves (in mice) or trigeminal nerve ganglia (in rabbits), infectious herpes simplex virus was detected in the supernatant fluid of the tissue cultures, and virions were seen in thin sections of the cultured cells. This suggests that the virus existed in these ganglia in a latent, non-infectious form. In man, trigeminal ganglia have been implicated as the reservoir of virus between recurrences (reviewed by Chang, 1971)> and i t is possible that the mechanism of latency in man is similar to that in rabbits. However, there is also evidence that virus can be excreted between recurrences in apparently normal rabbits (Ashe and Rizzo, 19^7), and humans (Kaplan, 1969), so that i t is s t i l l possible that dorm-ancy of the virus could be due to a chronic, inapparent infection. Recurrent herpes simplex virus infections are unusual in that circulating antibody against herpes simplex virus is present before and after the eruption, and does not change during the infection (Chang, 1971). Spread of the virus through a localized eruption could possibly involve direct cell-to-cell transmission of the virus (Kaplan, 1969) which is known to occur in cell culture 1 0 ( R u s s e l l , 1962). This could e x p l a i n why the e r u p t i o n remains l o c a l i z e d , but does not e x p l a i n why the r e c u r r e n t l e s i o n i s s e l f -l i m i t i n g and hea l s i n a few days, since i n f e c t i o n of human c e l l s i n t i s s u e c u l t u r e normally proceeds t o completion. Two p o s s i b l e mechanisms f o r r e p r e s s i o n / a c t i v a t i o n i n v i v o have r e c e n t l y been revealed: -1. Although t o t a l c i r c u l a t i n g antibody i s not s i g n i f i c a n t l y d i f f e r e n t between p a t i e n t s w i t h r e c u r r e n t and l a t e n t i n f e c t i o n s , there i s a s i g n i f i c a n t l y g r e a t e r amount of IgA i n the blood of p a t i e n t s w i t h l a t e n t i n f e c t i o n than i n the blo o d of p a t i e n t s w i t h a c t i v e r e c u r r e n t i n f e c t i o n (Greenberg and Brightman, 1971). Although these p r e l i m i n a r y r e s u l t s are not as s t a t i s t i c a l l y s i g n i f i c a n t .as claimed (Bodmer, 1971)? c o n f i r m a t i o n w i t h a l a r g e r number of p a t i e n t s would provide a p o s s i b l e e x p l a n a t i o n f o r recurrence and l a t e n c y . 2. C e l l u l a r immunity, a c t i v e against v i r u s - i n f e c t e d c e l l s but not against f r e e v i r i o n s , has r e c e n t l y been t e s t e d i n p a t i e n t s w i t h and without a h i s t o r y of r e c u r r e n t herpes simplex i n f e c t i o n ( W i l t o n et a l , 1972). I n p a t i e n t s w i t h r e c u r r e n t i n f e c t i o n , s i g n i f i c a n t l y lower lymphocyte c y t o t o x i c i t y and macrophage migra-t i o n i n h i b i t i o n were observed. These e f f e c t s were s p e c i f i c f o r herpes v i r u s antigens. I t i s p o s s i b l e t h a t a complex i n t e r a c t i o n of host f a c t o r s may serve t o i n h i b i t or a c t i v a t e the v i r u s . I n a d d i t i o n to c i r c u l a t i n g IgA antibody and c e l l u l a r immunity, temperature (Waddell and S i g e l , 1966), i n t e r f e r o n (reviewed i n Kaplan, 1969), and t h y r o i d and p a r a t h y r o i d hormones (Roizman, 1962) can a f f e c t the r e p l i c a t i o n of herpes simplex v i r u s . 2. Cytomegaloviruses Murine cytomegalovirus i s f a t a l when i n j e c t e d i n l a r g e doses i n t o mice (Henson and Neapolitan, 1970). At lower doses, a chronic i n f e c t i o n i s e s t a b l i s h e d , w i t h v i r u s p e r s i s t i n g i n the s a l i v a r y glands, lymphoid t i s s u e s , and kidneys (Henson and Neapolitan, 1970; Henson and Stranoy 1972; Henson et a l , 1972). The r a t e of d i s -appearance of the v i r u s v a r i e s according to the s t r a i n of mouse, and i n some cases, i n f e c t i o n can p e r s i s t f o r l i f e (Brodsky and Rowe, 1958). Disappearance of v i r u s from the su b m a x i l l a r y s a l i v a r y glands i s a s s o c i a t e d w i t h inflammation of the i n f e c t e d t i s s u e s , and degeneration of i n f e c t e d and neighbouring u n i n f e c t e d a c i n a r c e l l s (Henson'.and Strano, 1972). I t has been suggested (Henson and Neapolitan, 1970) t h a t the presence of i n t a c t i n c l u s i o n - b e a r i n g c e l l s was necessary f o r the p e r s i s t e n c e of i n f e c t i o n and t h a t the a b i l i t y of inflammatory c e l l s to reach i n f e c t e d c e l l s was a major f a c t o r i n determining the r a t e of disappearance of v i r u s . Recovery of v i r u s from lymphoid t i s s u e (Henson et al,1972) suggests a p o s s i b l e mechanism of dissemination. Human cytomegalovirus i n f e c t i o n i s very common, and by a d u l t -hood a m a j o r i t y of persons have experienced contact w i t h the v i r u s , as shown by antibody responses. However, i n f e c t i o n s a f t e r b i r t h are o f t e n asymptomatic. Cytomegalovirus mononucleosis ( K a a r i a i n e n et a l , 1966) e s p e c i a l l y f o l l o w i n g blood t r a n s f u s i o n , c e r t a i n types of l i v e r disease (Hanshaw et a l , 1965), and p o s s i b l y other diseases can be caused by human cytomegalovirus. F e t a l i n f e c t i o n appears t o be c o r r e l a t e d w i t h m i l d or severe damage t o the c e n t r a l nervous system, i n approximately 10$ of i n f e c t e d c h i l d r e n (Hanshaw, 1971; Weller, 1971)• V i r u s i s excreted by i n f e c t e d persons i n the s a l i v a and u r i n e (Rowe et a l , 1958), and cytomegalic c e l l s can be found i n the kidneys (Fetterman, 1968). There i s some evidence suggesting t h a t human cytomegalovirus p e r s i s t s i n the c i r c u l a t i n g lymphocytes i n the b l o o d , ' i n the presence of c i r c u l a t i n g antibody (reviewed i n Weller, 1971)• Some immunological a b e r r a t i o n s may be r e l a t e d to cytomegalovirus i n f e c t i o n (Weller, 1971). "E. Growth i n Tissue C u l t u r e 1. Herpes Simplex V i r u s Herpes simplex v i r u s m u l t i p l i e s i n t i s s u e c u l t u r e i n c e l l s d e r ived from a v a r i e t y of animals. The v i r u s r e p l i c a t e s i n s e v e r a l species of mammalian c e l l s (Kaplan, 1969), t o r t o i s e kidney c e l l s (Faueonnier, 1963), and chick embryo c e l l s (Lowry et a l , 1971). High t i t r e s can be obtained (100-200 plaque forming u n i t s per c e l l ) and the v i r u s can be r e a d i l y separated from gross c e l l u l a r d e b r i s . Cytopathic e f f e c t s v a r y according t o the s t r a i n of v i r u s ( E j e r c i t o et a l , 1968), and can i n c l u d e rounding and clumping of c e l l s , l y s i s , or polykaryocyte formation. A c h a r a c t e r i s t i c f e a t u r e of i n v i v o and i n v i t r o i n f e c t e d c e l l s i s the presence of l a r g e i n t r a n u c l e a r i n c l u s i o n bodies which have been i m p l i c a t e d i n the s y n t h e s i s of v i r a l DNA (Crouse et a l , 1950; N i i et a l , 1961). 2 . Cytomegaloviruses Murine cytomegalovirus grows r e a d i l y i n mouse embryo f i b r o -b l a s t c e l l c u l t u r e s , and a l s o grows to a much l e s s e r extent i n monkey, r a b b i t and hamster c e l l l i n e s , and a f t e r adaptation, i n sheep c e l l s (Kim and Carp, 1971). No growth i n human c e l l s has been recorded, but human d i p l o i d f i b r o b l a s t s (W138) undergo an ab o r t i v e i n f e c t i o n (Kim and Carp, 1972). I n c l u s i o n bodies, v i r a l -s p e c i f i c antigens, and c e l l death are caused, but s i g n i f i c a n t v i r a l r e p l i c a t i o n does not occur. This behaviour has not yet been reported f o r other known cytomegaloviruses, which are more s p e c i e s - s p e c i f i c , and normally m u l t i p l y o n l y i n homologous c e l l s (Plummer, 1967). However, f o r both human and murine cytomegalo-v i r u s e s , c y t o p a t h i c e f f e c t s i n v i t r o are seen i n f i b r o b l a s t but not epithelial cells, in contrast to the in vivo involvement of mainly epithelial cells (Smith, 1959; Weller, 1971). Murine cyto-megalovirus infection of mouse embryo fibroblasts causes cell rounding and release of some infectious virus, although the majority of the infectious virus remains cell-associated at the end of the replicative cycle. Similarly, infection of human fibroblasts with a laboratory-adapted strain of human cytomegalovirus led to cell rounding and readily detectable extracellular virus, but infection with a fresh isolate resulted in spread of infection and cell lysis, but virtually no release of infectious virus (Kanich and Craighead, 1972a). Growth of the viruses in tissue culture results in the production of limited amounts of interferon (Henson et al, 1966), but the viruses are relatively insensitive to inhibition by inter-feron (Osborn and Medearis, 19^7; Glasgow et al, 1967)• In addition, human cytomegalovirus depresses the interferon response of cells infected with other viruses (Glasgow et al, 1967). F. Virus Growth Cycle. 1. Herpes Simplex virus Adsorption: Herpes simplex virus adsorbs rapidly to H.Ep.2 cells at temperatures from k°C to 37°C, suggesting that a d s o r p t i o n does not r e q u i r e energy (Roizman, 1969). P a r a t h y r o i d hormone i n h i b i t s adsorption, and t h y r o i d hormone s t i m u l a t e s adsorp-t i o n (Roizman, 1962). E n t r y and Uncoating: P e n e t r a t i o n of the v i r u s i n t o the c e l l i s temperature dependent, and at 37°C, 90% of adsorbed v i r u s penetrates .in ten minutes (Huang and Wagner, 196U), as measured by i n f e c t i v i t y . This i s not n e c e s s a r i l y a v a l i d measure of adsorb-t i o n . F u r t h e r i n f o r m a t i o n on the e n t r y and uncoating of the v i r u s i s mainly d e r i v e d from e l e c t r o n microscope s t u d i e s , and to a l e s s e r extent from bi o c h e m i c a l s t u d i e s . U n f o r t u n a t e l y , h i g h m u l t i p l i c i t i e s o f i n f e c t i o n (e.g. 1000 p a r t i c l e s per c e l l ) must be used t o v i s u a l -i z e s u f f i c i e n t p a r t i c l e s i n the e l e c t r o n microscope. A l s o , the p a r t i c l e : plaque forming u n i t r a t i o f o r herpes simplex v i r u s i s 10:1 or more, so t h a t both e l e c t r o n microscopic and b i o c h e m i c a l observa-t i o n s are r e s t r i c t e d by the p o s s i b i l i t y t h a t over 90$ of the v i r i o n s may not be going through an i n f e c t i o u s process during e n t r y and uncoating. Information on uncoating of v i r a l p a r t i c l e s i s at present confusing and of d o u b t f u l s i g n i f i c a n c e (reviewed i n Roizman, 1969). However, v i r a l DNA, but not p r o t e i n , accummulates i n the nucleus of i n f e c t e d c e l l s from l e s s than 60 minutes a f t e r i n f e c t i o n (Hochberg and Becker, 1968). By autoradiography, p a r e n t a l v i r a l 16 DNA -without capsids can be seen i n the nucleus from 30 minutes post i n f e c t i o n (Hummeler et a l , 1969)• E l e c t r o n microscope evidence a l s o supports the suggestion t h a t the v i r a l DNA i s uncoated before or during e n t r y i n t o the nucleus (Morgan et a l , 1968). RNA Synthesis and Transport: V i r u s - s p e c i f i c RNA can be ex t r a c t e d from the n u c l e i of i n f e c t e d c e l l s at three hours post i n f e c t i o n , and reaches a maximum at about 8 hours post i n f e c t i o n (Flanagan, 1967). Nuclear v i r a l RNA i s heterogeneous i n s i z e and contains molecules w i t h sedimentation c o e f f i c i e n t s greater than 80s (Roizman et a l , 1970). V i r a l RNA i s t r a n s p o r t e d t o the cytoplasm a f t e r a .10 t o 15 minute l a g , and the s i z e range of cytoplasmic RNA i s s h i f t e d to lower molecular weights than nuclear RNA. Since the cytoplasmic RNA contains at l e a s t 80$ of the sequences present i n hi g h molecular weight nuclear RNA, i t i s probable t h a t the c y t o -plasmic RNA i s derived from t h i s nuclear RNA by cleavage before or during t r a n s p o r t (Wagner and Roizman, 1969a,b). H o s t - s p e c i f i c polyribosomes disaggregate a f t e r i n f e c t i o n , and v i r u s - s p e c i f i c polyribosomes are formed ( S y d i s k i s and Roizman, 1967, 1968). RNA synthesized e a r l y during i n f e c t i o n contains sequences not synthesized i n s i g n i f i c a n t q u a n t i t i e s l a t e during i n f e c t i o n , and v i c e versa, suggesting c o n t r o l of herpes simplex v i r u s genome expression at the t r a n s c r i p t i o n a l l e v e l (Wagner, 1972). 17 P r o t e i n Synthesis: As f a r as i s known, a l l v i r a l p r o t e i n synthesis occurs i n the cytoplasm of i n f e c t e d c e l l s ( S y d i s k i s and Roizman, 1966). V i r a l p r o t e i n synthesis reaches a maximum r a t e at fou r to s i x hours post i n f e c t i o n , but there i s some evidence t h a t s t r u c t u r a l p r o t e i n synthesis i s asynchronous (Spear and Roizman, 1968). Transport of p r o t e i n s to the nucleus i s slow (Olshevsky et a l , 1967; Spear and Roizman, 1968), and s e l e c t i v e (Spear and Roizman, 1970). A f t e r disaggregation of host polyribosomes and reaggregation of v i r a l polyribosomes ( c o n t a i n i n g mainly v i r a l m RNA, (Wagner and Roizman, 1969)), at l e a s t 9 - H polypeptides are synthesized and incorporated i n t o v i r i o n s (Olshevsky and Becker, 1970; Roizman and Spear, 1971). However, some of these polypeptides might be a r t i f a c t s of the e x t r a c t i o n procedure, and i t i s not yet known i f a l l these polypeptides are coded f o r by the v i r a l genome (Roizman and Spear, 1971). An a n a l y s i s of a l l the p r o t e i n s made a f t e r i n f e c t i o n r evealed at l e a s t 25 d i f f e r e n t polypeptides (Spear and Roizman, 1968). At l e a s t three of these appeared t o be r e s t r i c t e d t o the cytoplasm. Of the p r o t e i n s synthesized a f t e r i n f e c t i o n and inc o r p o r a t e d i n t o v i r i o n s , at l e a s t s i x are g l y c o s y l a t e d (Roizman and Spear, ±971). These g l y c o p r o t e i n s are ass o c i a t e d w i t h the c e l l membranes and the envelope of v i r i o n s (Olshevsky and Becker, 1970), and are d i f f e r e n t from the g l y c o p r o t e i n s synthesized before i n f e c t i o n (Spear et a l , 1970). The most probable s i t e of g l y c o s y l a t i o n i s the c e l l membrane 18 (Spear and Roizman, 1970). The gl y c o p r o t e i n s synthesized i n c e l l s i n f e c t e d w i t h d i f f e r e n t s t r a i n s of v i r u s d i f f e r i n s e v e r a l c h a r a c t e r -i s t i c s ( K e l l e r et a l , 1970). Assembly and Release: V i r a l nucleocapsids are assembled i n the n u c l e i of i n f e c t e d c e l l s (Roizman et a l , 1969). A smaller, core p a r t i c l e , c o n t a i n i n g p r o t e i n and DNA, has been i m p l i c a t e d as a pre-cursor of complete nucleocapsids of pseudorabies v i r u s ( S y d i s k i s , 1969), and s i m i l a r p a r t i c l e s have been seen i n herpes simplex v i r u s i n f e c t e d c e l l s (Spring et a l , 1968). There i s some evidence t h a t the inner l i p i d envelope of the v i r i o n i s a l s o added i n the nucleus, from e l e c t r o n microscopic (Roizman and Spear, 1971), b i o p h y s i c a l (Spring and Roizman, 1968), and b i o l o g i c a l data (Roizman, et a l , 1969). The outer envelope of the v i r i o n i s derived from the inner l a m e l l a of the nuclear membrane by a budding process ( N i i et a l , 1968; Schwartz and Roizman, 1969)- Both s i n g l y enveloped and doubly enveloped v i r i o n s are i n f e c t i v e (Roizman and Spear, 1971). V i r i o n s are r e l e a s e d i n t o the e x t r a c e l l u l a r space without l y s i s of the host c e l l (Roizman, 1962a). Released v i r i o n s are predominantly enveloped ( N i i et a l , .1968; Spring et a l , 1968). Late i n i n f e c t i o n , the cytoplasm of i n f e c t e d c e l l s contains a network of tubules, and v i r i o n s are seen i n the tubules (Schwartz and Roizman, 1969b). I t has been suggested t h a t these tubules may be i n v o l v e d w i t h egress of v i r i o n s from the c e l l (Roizman, 1969)• Enzymes Induced by Herpes Simplex "Virus: Although s e v e r a l enzymes change i n a c t i v i t y and pr o p e r t i e s a f t e r i n f e c t i o n w i t h the v i r u s , none of these enzymes has yet been c o n c l u s i v e l y shown to be coded f o r by the v i r a l genome. A c t i v i t y of thymidine kinase i n i n f e c t e d BHK-21 c e l l s increases t w e n t y f o l d a f t e r i n f e c t i o n , and the new a c t i v i t y has s e v e r a l p r o p e r t i e s t h a t are d i f f e r e n t from the a c t i v i t y i n u n i n f e c t e d c e l l s (Klemperer et a l , 1967)• Host and v i r u s mutants ( K i t and Dubbs, 1963a,b), and d i f f e r e n t herpesviruses (Buchan and Watson, 1969) have provided f u r t h e r evidence f o r the vi r u s - i n d u c e d nature of the enzyme. Thymidine monophosphate kinase (Hamada et a l , 1966; Nohara and Kaplan, 1963), DNA polymerase ( K e i r , 1968), and a l k a l i n e DNA exonuclease a c t i v i t i e s (Morrison and K e i r , 1967, 1968) a l l increase i n a c t i v i t y and change i n p r o p e r t i e s i n herpes simplex v i r u s -i n f e c t e d c e l l s . E f f e c t of I n f e c t i o n on Host C e l l s : M i t o s i s can be prevented or aborted by herpes simplex v i r u s i n f e c t i o n (Vantis and Wildy, I962). A m i t o t i c nuclear d i v i s i o n ( N i i and Kamehara, 1963) and chromosome breakage (Waubke et a l , 1968) have a l s o been reported a f t e r i n f e c t i o n . Host DNA synthesis i s i n h i b i t e d (Roizman, 1969)• Host RNA synthesis i s i n h i b i t e d a f t e r i n f e c t i o n (Roizman et a l , I965) and processing of ribosomal RNA i s independently i n h i b i t e d (Roizman et a l , 1970). Host polyribosomes are disaggregated a f t e r infection (Sydiskis and Roizman, 1966, 1967) which may account for the inhibition of host protein synthesis observed after infection (Roizman et al, 19^5; Spear and Roizman, 19^3; Roizman and Spear, 1971)-2. Cytomegalovirus e s Adsorption and Penetration: Murine cytomegalovirus absorbs to mouse embryo fibroblasts fairly rapidly, with 60$ being adsorbed after 30 minutes (Henson et al, 1966) under the conditions of assay. However, Osborn and Walker (1968) have reported that the titre of several preparations could be enhanced 10 to 100 fold by inoculation under a centrifugal force of 1,900 g. It was suggested that there were two populations of virions differing in their ease of adsorption. Human cytomegalovirus adsorption is not temperature -dependent (Vonka and Benyesh-Melnick, 1966) and proceeds at about the same rate as murine cytomegalovirus adsorption. Human CMV penetration is temperature-dependent ("Vonka and Benyesh-Melnick, 1966). Replication: The single-step growth cycle of murine cyto-megalovirus takes approximately 28 - 32 hours (Henson et al, 1966; Tegtmeyer et al, 19^9; Osborn and Walker, 1968). Using 5-iodo-deoxyuridine, Henson et al (1966) showed that DNA synthesis starts at about 12 hours a f t e r i n f e c t i o n . E x t r a c e l l u l a r v i r u s can be detected from 20 hours a f t e r i n f e c t i o n (Tegtmeyer et a l , 1969; Osborn and Walker, 1968). Human cytomegalovirus i n f e c t i o n shows a l a t e n t p e r i o d of 55 hours (Plummer et a l , 1969) and the amount of v i r u s r e l e a s e d i n t o the medium depends on the s t r a i n of v i r u s used. A l a b o r a t o r y adapted s t r a i n produced h i g h y i e l d s of i n f e c t i o u s v i r u s , whereas a ' w i l d ' f r e s h l y i s o l a t e d s t r a i n produced strong c y t o p a t h i c e f f e c t s but l i t t l e or no i n f e c t i o u s v i r u s (Kanich and Craighead, 1972a). During i n f e c t i o n of human d i p l o i d , f i b r o b l a s t c e l l s , both s t r a i n s showed assembly of nucleocapsids i n the nucleus (Kanich and C r a i g -head, 1972b). Some m a t e r i a l was added t o the nucleocapsid by budding through the nuclear membrane, but the outer envelope of the v i r i o n was probably acquired by budding i n t o cytoplasmic tubules. E f f e c t s of I n f e c t i o n on Host C e l l : M i t o s i s i n h i b i t i o n and chromosomal a b e r r a t i o n s were seen during murine cytomegalovirus i n f e c t i o n , from 18 hours a f t e r i n f e c t i o n (Tegtmeyer et a l , 1969). P a t h o l o g i c a l mitoses have a l s o been seen i n human embryo f i b r o b l a s t s i n f e c t e d w i t h human cytomegalovirus (Blyumkin et a l , 1969). Sur-v i v a l of human d i p l o i d c e l l s t r a n s p l a n t e d i n t o hamster cheek pouches was enhanced by p r i o r i n f e c t i o n of the human c e l l s w i t h human cytomegalovirus ( K i s s l i n g and Addison, 196^-), and such i n f e c t i o n a l s o conferred a t r a n s i e n t a b i l i t y f o r growth i n s o f t agarose (Lang and Montagnier, 1970). G. Herpes Simplex V i r u s Types 1. and 2 Two groups of s t r a i n s of herpes simplex v i r u s are now recognized (reviewed i n Nahmias and Dowdle, 1968). The f i r s t major d i f f e r e n c e to be c l e a r l y e s t a b l i s h e d was a n t i g e n i c v a r i a t i o n . Using a micro-n e u t r a l i z a t i o n t e s t (Dowdle et a l , 1967), n e u t r a l i z a t i o n k i n e t i c s (Plummer et a l , 1970) or simultaneous n e u t r a l i z a t i o n w i t h a known intermediate s t r a i n ( T e r n i and Roizman, 1970), two groups of s t r a i n s can r e a d i l y be defined. Various other d i f f e r e n c e s can a l s o be found between the two groups, although s e r o l o g i c a l d i f f e r e n c e s are the most c l e a r c u t . Type 2 s t r a i n s are almost i n v a r i a b l y recovered from g e n i t a l i n f e c t i o n s , w h i l e type 1 s t r a i n s are normally recovered from o r a l , f a c i a l or s k i n i n f e c t i o n s (Dowdle et a l , 1967; Plummer et a l , 1970). A n t i b o d i e s i n the serum of p a t i e n t s w i t h g e n i t a l or other h e r p e t i c i n f e c t i o n s were most e f f e c t i v e against type 2 and type 1 s t r a i n s r e s p e c t i v e l y (Rawls et a l , 1970). Type 2 s t r a i n s grow to lower t i t r e than type 1 s t r a i n s i n s e v e r a l c e l l l i n e s (Rawls et a l , 1968; Plummer et a l , 1968). Type 2 s t r a i n s have a higher p a r t i c l e : p l a q u e forming u n i t r a t i o than type .1 s t r a i n s , and are more t h e r m o l a b i l e (Figueroa and Rawls, 1969)• cyto p a t h i c e f f e c t of the two s t r a i n s i n t i s s u e c u l t u r e can be d i f f e r e n t i a t e d ( E j e r c i t o et a l , 1968). Type 2 s t r a i n s are more neurotropic i n mice (Plummer et a l , 1968). The guanine + cyt o s i n e contents of v i r a l DNA i s 68$ f o r type 1 and 70$ f o r type 2 s t r a i n s (Goodheart et a l , 1968), and there i s about 50$ sequence homology between the DNA's of type 1 and type 2 s t r a i n s ( K i e f f et a l , 1972). D i f f e r e n c e s can be detected i n the thermal s t a b i l i t y and immunological s p e c i f i c i t y of thymidine kinases induced i n c e l l s i n f e c t e d w i t h type 1 or type 2 s t r a i n s (Thouless and Skinner, 1971). H . Latency and Oncogenesis i n Herpesvirus I n f e c t i o n s Many herpesviruses enter i n t o inapparent, longterm, p e r s i s t e n t i n f e c t i o n s , which can be r e a c t i v a t e d under c e r t a i n circumstances. The mechanism of t h i s p e r s i s t e n c e i s not yet understood, but there are two broad p o s s i b i l i t i e s , as pointed out by Eoizman (1965) f o r herpes simplex v i r u s : -.1. Low l e v e l p e r s i s t e n t m u l t i p l i c a t i o n 2. Latency, i . e . p e r s i s t e n c e of the v i r u s i n a no n - i n f e c t i o u s i n t r a c e l l u l a r form. Although these could both apply to inapparent herpes simplex i n f e c t i o n s , as discussed above, more evidence:is a v a i l a b l e f o r some other herpesviruses. EB vi r u s - p r o d u c i n g c e l l l i n e s show o n l y a few per cent of c e l l s producing v i r a l antigens, and i n some cases no i n f e c t i o u s v i r u s can be detected, yet a l l c e l l s i n the c u l t u r e apparently c o n t a i n the v i r a l genome and have the a b i l i t y to synthesize v i r a l antigens (Zajac and Kohn, 1970). A l s o , Lucke adenocarcinoma tumours i n fr o g s produce v i r u s when l e f t at low temperature, but at higher temperatures, i n c l u s i o n bodies, v i r u s p a r t i c l e s and tumor-i g e n i c i t y are a l l absent (Granoff, 1972). Both human and murine cytomegaloviruses can apparently p e r s i s t i n c i r c u l a t i n g lymphocytes w i t h l i t t l e , i f any, r e p l i c a t i o n (Henson et a l , 1972; Weller, 1971). S e v e r a l herpesviruses are known to cause or suspected of causing cancer i n v a r i o u s hosts. These v i r u s e s i n c l u d e Marek's disease v i r u s i n chickens (Purchase, 1972); Herpesviruses s a i m i r i and a t e l e s i n monkeys (Melendez et a l , 1972); a v i r u s causing Lucke adenocarcinoma i n f r o g s (Granoff, 1972); a herpesvirus causing lymphomas i n r a b b i t s (Hinze and Chipman, 1972); and p o s s i b l y EB v i r u s (Henle, 1972), and herpes simplex type 2 i n humans ( A u r e l i a n , 1972; Rapp and Duff, 1972). In most cases, i n f e c t i o u s v i r u s can be i s o l a t e d from tumor c e l l s . Human cytomegalovirus has not yet been l i n k e d w i t h a p a r t i c u l a r type of cancer, but the behaviour i n t i s s u e c u l t u r e shows s e v e r a l s i m i l a r i t i e s t o tha t ,of EB v i r u s and other p o s s i b l y oncogenic v i r u s e s . HCV-infected c e l l s show p a t h o l o g i c a l mitoses (Blyumkin et a l , 1969)5 increased s u r v i v a l i n the cheek pouches of hamsters ( K i s s l i n g and Addison, 196^), and growth i n s o f t agarose (Lang and Montagnier, 1970). Growth of lymphoblastoid c e l l l i n e s i n v i t r o (Klemola et a l , ±969)5 i s p o s s i b l y due to HCV or EBV i n f e c t i o n . MATERIALS AND METHODS A. C e l l s Mouse embryo c e l l c u l t u r e s were prepared from randomly bred, Swiss white mice, purchased from the F a c u l t y of Medicine Animal U n i t , U n i v e r s i t y of B r i t i s h Columbia. Human Epidermoid Carcinoma #2 (H.Ep.2) c e l l s were obtained from Dr. D. M. McLean. A continuous l i n e of SV-40-transformed mouse kidney c e l l s (MKSA) were obtained from Dr. Saul K i t . Human d i p l o i d f i b r o b l a s t (WI38) c e l l s and the continuous mouse l i n e s L-929 and 3T3, were purchased from Flow La b o r a t o r i e s . B. Viruses The herpes simplex v i r u s s t r a i n , designated HSV-P, used f o r most of t h i s study was obtained from Dr. D. M. McLean, who i s o l a t e d i t from a l a b i a l l e s i o n . The F s t r a i n of HSV was obtained from Dr. B. Roizman. The Smith s t r a i n of murine cytomegalovirus (MCV) and the ADI69 s t r a i n of human cytomegalovirus (HCV) were obtained from the American 32 Type C u l t u r e C o l l e c t i o n . Bacteriophage ih, l a b e l l e d w i t h P at a s p e c i f i c a c t i v i t y of 1 mCi/mg phosphorus, was obtained from Dr. R. C. M i l l e r . C. Growth Medium Dulbecco's modified Eagle's medium was obtained from Grand I s l a n d B i o l o g i c a l Company (GIBCO), catalogue number Hl6, i n powdered form, and d i s s o l v e d and f i l t e r - s t e r i l i z e d before use. I n a d d i t i o n , MEM-A contained 3-7 g / l NaHCOg, and MEM-B contained 1.5 g / l NaHCO^. A f t e r s t e r i l i z a t i o n , gentamycin ( B i o c u l t L a b o r a t o r i e s ) was added to a f i n a l c o n c e n t r a t i o n of 50 ug/ml. F e t a l c a l f serum (Flow L a b o r a t o r i e s ) was added j u s t before use, at a conce n t r a t i o n of two, f i v e or ten per cent. D. Reagents D e s c r i p t i o n Source Agarose Seakem, Bausch and Lomb Cesium C h l o r i d e Schwartz-Mann Co. Deoxyribonuclease ( E l e c t r o p h o r e t i c a l l y p u r i f i e d ) Pronase (Fungal protease) Worthington Biochemical Co. Sigma Chemical Company Sigma Chemical Company Geigy Ribonuclease A Sar k o s y l NL30 S p e c t r a f l u o r Thymidine-methyl- H (k8.k uCi/mmole) Thymidine -2- l i +C (62 mCi/mmole) T r i t o n X-100 (Detergent) T r y p s i n U r i d i n e - 3 H (k2 Ci/mmole) Amersham Searle Company New England Nuclear Co. Amersham Searle Company Rohm and Haas DIFCO New England Nuclear Co. A l l other reagents were obtained from F i s h e r Chemical Company. S o l u t i o n s A l k a l i n e DNA B u f f e r (ADB) ( K i e f f et a l , 1971) NaCI 0.8 M NaOH 0.3 M EDTA 0.001 M Hank's Balanced S a l t S o l u t i o n (modified) Glucose 0.0055 M NaCI O.lkjO M KC1 0.0053 M KHgPO^ 0.0041+ M Na 2 HP0^ 0.0150 M Phenol red 0.0006 M S t e r i l i z e d by a u t o c l a v i n g at 15 l b s steam pressure f o r 20 minutes. NaCI-EDTA NaCI 0.15 M Ethy l e n e d i a m i n e - t e t r a - a c e t i c a c i d (EDTA) 0.001 M . Adjusted t o pH 7. 5 N e u t r a l DNA Buf f e r (NDB) ( K i e f f et a l , 1971) NaCI 1 M EDTA 0.001 M Tris(hydroxymethyl)aminomethane(Tris) 0.05 M Adjusted t o pH 7.5 28 Phosphate B u f f e r Stock s o l u t i o n : NagHPO^ 0.25 M NaH 2P0^ 0.25 M pH 6.8 D i l u t e d to s p e c i f i e d m o l a r i t y Phosphate B u f f e r e d S a l i n e (PBS) 0.13 M 0.0027 M 0.0081 M 0.0015 M 0.001 M 0.0005 M RNA B u f f e r (Hudson et a l , 1970) NaCl 0.1 M CH^COONa 0.01 M MgClg 0.001 M Adjusted to pH 5-2. S c i n t i l l a t i o n F l u i d A Per l i t e r of tolu e n e : -NaCl K C 1 Na 2HP0 ; KH 2P0 U C a C l 2 MgCl k g 2 ,5 5-Diphenyloxazole 50 mg 1, 4-Bis - (2 - (5-phenyloxazolyl))-Benzene S c i n t i l l a t i o n F l u i d B Toluene:Triton X-100:water:methanol i n the proporti o n s 5: 3:1:1, c o n t a i n i n g per l i t e r : -h g 2 ,5 ,Diphenyloxazole 50 mg 1 ,4-Bis - (2 - (5-phenyloxazolyl))-benzene Standard S a l i n e C i t r a t e (SSC) ( G i l l e s p i e and Spiegelman, 1965) NaCl 0.15 M Na^ c i t r a t e 0.015 M Stock s o l u t i o n was made up at 2h x t h i s c oncentration, and d i l u t e d as re q u i r e d . Tris-EDTA T r i s 0.01 M EDTA 0.001 M Adjusted to pH 7-5 F. C e l l C u l t u r e Conditions C e l l s were grown i n MEM-A under a 5$ COg, w e l l - h u m i d i f i e d atmos-phere at 37°C i n Falco n p l a s t i c p e t r i dishes (353 50 or 90 mm diameter) or i n MEM-B i n stoppered g l a s s b o t t l e s at 37°C. B o t t l e s were e i t h e r s t a t i o n a r y (Brockway b o t t l e s ) or r o l l i n g on a B e l l c o c e l l p r o duction r o l l e r apparatus. G. Mouse Embryo C e l l s Pregnant mice i n t h e i r second week of g e s t a t i o n were used. Embryos were removed, r i n s e d i n Hank's s o l u t i o n , and cut i n t o s m a l l fragments u s i n g forceps and s c i s s o r s . The fragments were s t i r r e d f o r f i v e minutes i n Hank's s o l u t i o n w i t h a magnetic s t i r r i n g bar, and then allowed to s e t t l e . The supernatant was discarded, and Hank's s o l u t i o n c o n t a i n i n g 0.25$ t r y p s i n was added. A f t e r s t i r r i n g v i g o r o u s l y f o r 15 minutes, the l a r g e r fragments were allowed to s e t t l e , and the supernatant was drawn o f f and added to 1 ml serum i n a ho ml c e n t r i f u g e tube. The c e l l s were c o l l e c t e d by c e n t r i -f u g a t i o n at 2,000 rpm f o r 10 minutes i n an I n t e r n a t i o n a l Equipment Corporation CS c e n t r i f u g e . The remaining t i s s u e fragments were s t i r r e d f o r another 15 minutes w i t h f r e s h t r y p s i n s o l u t i o n , and the c e l l s again c o l l e c t e d from the supernatant. This procedure was repeated u n t i l a l l a v a i l a b l e c e l l s had been e x t r a c t e d . The c e l l p e l l e t s were pooled, suspended i n MEM-B supplemented w i t h 10$ serum, dispensed i n t o b o t t l e s and incubated at 37°C. H. C e l l T r ansfer A l l c e l l t r a n s f e r s onto new surfaces were c a r r i e d out by t r y p s i n i z a t i o n . The growth medium was discarded from confluent monolayers of the c e l l s to be t r a n s f e r r e d , and a minimal volume of 0. 25$ t r y p s i n i n Hank's s o l u t i o n was added. A f t e r r o t a t i n g to ensure thorough bathing of a l l c e l l s , the s o l u t i o n was decanted. A f t e r f i v e minutes, a s m a l l q u a n t i t y of growth medium was added, and the t r y p s i n -i z e d c e l l s suspended by p i p e t t i n g . The c e l l suspension was then d i l u t e d t o the r e q u i r e d d e n s i t y and dispensed i n t o f r e s h v e s s e l s . Immediately p r i o r to p l a c i n g the v e s s e l s i n the incubator, the p l a t e s or b o t t l e s were s w i r l e d thoroughly to ensure t h a t the c e l l s were u n i -f o r mly i n suspension, and then l e f t untouched i n the incubator f o r at l e a s t an hour to all o w the c e l l s to s e t t l e and attach. This was a necessary p r e c a u t i o n to avoid uneven c e l l d i s t r i b u t i o n on the p l a t e , p a r t i c u l a r l y i n the case of H.Ep.2 c e l l s . 1 . Growth of Viru s e s : murine cytomegalovirus Murine cytomegalovirus (MCV) was grown i n monolayers of secondary t e r t i a r y or quaternary mouse embryo c e l l s . A d sorption was c a r r i e d out by e i t h e r of two methods: 1. Standard adsorption Medium was drained from the c e l l monolayers, and a minimal volume of MEM-B c o n t a i n i n g the v i r u s and 2$ serum was added. Adsorption was c a r r i e d out at 37°C f o r 30 minutes, during which time the inoculum was spread by r o c k i n g the dishes at ten-minute i n t e r v a l s . 2. C e n t r i f u g a l adsorption Medium was drained from c e l l monolayers i n 35 mm or 50 mm p e t r i dishes, and 0.5 ml ( f o r 35 mm dishes) or 1.5 ml ( f o r 50 nim dishes) MEM-B co n t a i n i n g the v i r u s and 2$ serum was added. The dishes were stacked up to s i x (35 mm dishes) or three (50 mm dishes) per basket i n the buckets f o r the 2^ 2 r o t o r of the IEC-CS c e n t r i -fuge and c e n t r i f u g e d at 2,000 rpm f o r 30 minutes. A f t e r adsorption by e i t h e r method, MEM co n t a i n i n g 2% serum was added to the c u l t u r e s , and in c u b a t i o n continued f o r 36 hours at 37°C. I f r a d i o a c t i v e v i r u s was req u i r e d , the medium was repla c e d at 12, l6 or 20 hours a f t e r i n f e c t i o n w i t h 5 mis MEM-A co n t a i n i n g 2aj0 c a l f serum and the r e q u i r e d amount of thymidine-methyl- H (^dThd) or thymidine -2- 1^C ( l i +CdThd). At 36 hours a f t e r i n f e c t i o n , the medium was c a r e f u l l y removed, and a minimal volume of 1PJ0 serum i n d i s t i l l e d water was added. A f t e r t e n minutes at room temperature, the c e l l s were suspended by p i p e t t i n g and f r o z e n at -70°C. For l a r g e - s c a l e p roduction of v i r u s , r o l l e r - b o t t l e c u l t u r e s of ME c e l l s were i n f e c t e d at low m u l t i p l i c i t y (e.g. 0.01 plaque forming u n i t s per c e l l ) and harvested when the m a j o r i t y of the c e l l s showed severe c y t o p a t h i c e f f e c t . The supernatant medium was a l s o r e t a i n e d as a source of v i r u s i n these cases. Herpes simplex v i r u s Growth of HSV i n H.Ep.2 c e l l s was c a r r i e d out as described above f o r MCV.in ME c e l l s , except f o r the f o l l o w i n g changes:- MEM without serum was added at the end of the adsorption period. For r a d i o a c t i v e v i r u s , the medium was replaced at k hours 3 a f t e r i n f e c t i o n w i t h MEM co n t a i n i n g the r e q u i r e d amount of HdThd Ik or CdThd. I n f e c t e d c e l l s were harvested at 20 hours a f t e r i n f e c t i o n . Human cytomegalovirus Growth of HCV i n WI38 c e l l s was c a r r i e d out as described above f o r MCV i n ME c e l l s , except t h a t c e l l s were harvested when the m a j o r i t y of c e l l s showed strong c y t o p a t h i c e f f e c t s . L a b e l l i n g c o n d i t i o n s are described i n i n d i v i d u a l experiments. J . Plaque Assays Murine cytomegalovirus V i r u s samples were s u i t a b l y d i l u t e d i n MEM-B c o n t a i n i n g 2% serum. I n f e c t i o n was c a r r i e d out by e i t h e r standard or c e n t r i f u g a l a d s o r p t i o n as described above. A f t e r adsorption, the inoculum was removed, and 2 ml o v e r l a y medium were added t o each d i s h . Overlay medium c o n s i s t e d of 0.5$ agarose, and 5$ f e t a l c a l f serum i n MEM-A. A f t e r 4-8 days i n c u b a t i o n at 37°C, plaques were c l e a r l y v i s i b l e without s t a i n i n g , and could be most conveni e n t l y counted i f i l l u m i n a t i o n was oblique and the plaques were v i s u a l i z e d by sca t t e r e d , r a t h e r than t r a n s m i t t e d l i g h t . Herpes simplex v i r u s ( s t r a i n P) Plaque assays were c a r r i e d out as described above f o r MCV, except that f r e s h l y confluent monolayers of H.Ep.2 c e l l s were used, and the o v e r l a y medium contained no c a l f serum. Plaques were v i s i b l e , without s t a i n i n g , a f t e r 2-4 days at 37°C. K. E l e c t r o n Microscopy V i r u s preparations were examined by negative s t a i n i n g . A formvar-coated g r i d was i n v e r t e d onto a d r o p l e t of v i r u s suspension f o r 60 seconds, then onto a drop of f r e s h l y prepared 2$ phospho-t u n g s t i c a c i d , pH 6.4, f o r 60 seconds, and b l o t t e d dry. Specimens were immediately examined i n a P h i l i p s EM 300 e l e c t r o n microscope. 35 L. P u r i f i c a t i o n of Herpesviruses D i f f e r e n t i a l c e n t r i f u g a t i o n Frozen i n f e c t e d c e l l suspensions were thawed and c e n t r i f u g e d at 3,000 rpm i n the 2*4-0 r o t o r of the I.E.C.-CS c e n t r i f u g e f o r 10 minutes to remove large c e l l u l a r d e b r i s . The supernatants were decanted and ce n t r i f u g e d f o r 60 minutes at *4°C, e i t h e r at 15,000 rpm i n the SW 50.1 r o t o r , or 18,000 rpm i n the SW 27 r o t o r i n a Spinco L 2 - 6 5 B u l t r a -c e n t r i f u g e . The supernatants were discarded, and the p e l l e t s r e s u s -pended i n a minimal volume of 1$ serum i n d i s t i l l e d water. Deoxyribonuclease treatment To remove t r a c e s of host DNA from v i r u s p r e p a r a t i o n s , the h i g h -s p e e d - p e l l e t t e d v i r u s was resuspended i n PBS and t r e a t e d w i t h p a n c r e a t i c DNase (50 ng/ml) f o r 15 minutes at 37°C. The suspension was d i l u t e d to 5 mis w i t h d i s t i l l e d water, c l a r i f i e d by low speed c e n t r i f u g a t i o n , and the v i r u s p e l l e t t e d by hi g h speed c e n t r i f u g a t i o n as above. Sucrose d e n s i t y gradient v e l o c i t y sedimentation From 0.2 to 0.5 ml v i r u s s o l u t i o n was l a y e r e d on top of a l i n e a r 5 ml gradient of 10-30$ (w/w) sucrose i n phosphate b u f f e r e d s a l i n e . Gradients were c e n t r i f u g e d f o r 30 minutes at k C and 15,000 rpm i n the S W 5 0 . 1 r o t o r i n a Spinco L2-65B u l t r a c e n t r i f u g e . F r a c t i o n s were c o l l e c t e d dropwise from the bottom and assayed f o r plaque forming u n i t s (pfu) and/or r a d i o a c t i v i t y . E q u i l i b r i u m gradient c e n t r i f u g a t i o n A v i r u s s o l u t i o n was made up to 0 . 1 $ bovine serum albumin and s o l i d C s C l was added to give a d e n s i t y of 1.26 g/cc, as c a l c u l a t e d from r e f r a c t i v e index determinations. Ei g h t mis of the s o l u t i o n were added t o a c e l l u l o s e n i t r a t e tube f o r the Spinco type 50 r o t o r , and the tube topped up wi t h m i n e r a l o i l , C e n t r i f u g a t i o n was c a r r i e d out i n a Spinco L2-65B u l t r a c e n t r i f u g e at ho,000 rpm and k°C f o r 24-hour s. F r a c t i o n s were c o l l e c t e d dropwise from the bottom and assayed f o r plaque forming u n i t s and/or r a d i o a c t i v i t y . M. P u r i f i c a t i o n of DNA V i r a l DNA 1. Large sca l e p u r i f i c a t i o n of DNA f o r h y b r i d i z a t i o n or m e l t i n g p r o f i l e purposes. V i r u s p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n was suspended i n NaCI-EDTA, fjronase added t o 1 mg/ml and 1 0 $ SDS s o l u t i o n added to 37 1$ (The Pronase had p r e v i o u s l y been s e l f - d i g e s t e d at 37 C f o r 2 hours to remove p o s s i b l e t r a c e s of nuclease a c t i v i t y ) . The e x t r a c t was incubated at 37° C f o r 2 hours, or at room temperature overnight. An equal volume of r e d i s t i l l e d phenol, s a t u r a t e d w i t h NaCl-EDTA was added and the two phases mixed thoroughly and then c e n t r i f u g e d i n a r e f r i g e r a t e d S o r v a l l RC-2B c e n t r i f u g e i n a SS3*+ head at 10,000 rpm f o r 10 minutes. The lower phenol phase was removed w i t h a Pasteur p i p e t t e , an equal volume of f r e s h phenol added, and the e x t r a c t i o n repeated. The supernatant from the second e x t r a c t i o n was t r a n s f e r r e d to a f r e s h tube, and two volumes of 95$ ethanol added s l o w l y down the side of the tube. Gentle s w i r l i n g caused mixing of the two l a y e r s , and p r e c i p i t a t i o n of the DNA. I f the s w i r l i n g was c a r r i e d out g e n t l y , the DNA could a l l be p r e c i p i t a t e d as a s i n g l e loose b a l l of threads. The l i q u i d phase could then be c a r e f u l l y removed, and s u c c e s s i v e l y r e p l a c e d by more ethanol, u n t i l the DNA was suspended i n 95$ ethanol. This procedure was devised to remove an appreciable amount of RNA and polysaccharide which d i d not c o p r e c i p i t a t e w i t h the DNA threads. This method i s analogous t o " s p o o l i n g " of DNA at a water-ethanol i n t e r f a c e , but r e s u l t s i n a much l o o s e r p r e c i p i t a t e which d i s s o l v e s very r a p i d l y i n a s u i t a b l e aqueous b u f f e r . A f t e r a l l o w i n g to stand i n ethanol f o r s e v e r a l minutes t o remove tra c e s of phenol, the p r e c i p i t a t e was drained, r i n s e d again w i t h ethanol, drained, and d i s s o l v e d i n Tris-EDTA at a con c e n t r a t i o n of 50-100 pg/ml. This DNA was r e l a t i v e l y f r e e of RNA, host DNA, 38 p r o t e i n and polysaccharide, as determined by c e n t r i f u g a t i o n to e q u i l i -brium i n p r e p a r a t i v e C s C l gr a d i e n t s . S o l i d CsCl was added to the DNA s o l u t i o n to give a d e n s i t y of 1.71 g/cc, c a l c u l a t e d from r e f r a c t i v e index determinations. S i x mis of the s o l u t i o n were added to a polyallomer tube f o r the Spinco type 50 r o t o r , and the r e s t of the tube f i l l e d w i t h m i n e r a l o i l . The s o l u t i o n was c e n t r i f u g e d at 4-0,000 rpm i n a type 50 r o t o r f o r hd hours at 20°C i n a Spinco L 2 - 6 5 B u l t r a c e n t r i f u g e . Twenty-drop f r a c t i o n s were c o l l e c t e d from the bottom of the tube. 0.5 ml T r i s -EDTA was added to each f r a c t i o n , and the o p t i c a l d e n s i t y at 260 nm wavelength was measured on a Beckman DB-G spectrophotometer. U s u a l l y , the sharp v i r a l DNA band was the o n l y UV-absorbing m a t e r i a l i n the gradient. DNA-containing f r a c t i o n s were pooled, and the DNA pre-c i p i t a t e d w i t h ethanol and d i s s o l v e d i n 0.1 x SSC. This method was modified s l i g h t l y f o r s m a l l q u a n t i t i e s of r a d i o a c t i v e l y l a b e l l e d DNA. Although as l i t t l e as k. ug/ml DNA could be p r e c i p i t a t e d q u a n t i t a t i v e l y w i t h ethanol, the p r e c i p i t a t e i n such cases was not v i s i b l e , and was p e l l e t t e d at 15,000 rpm f o r 20 minutes i n a r e f r i g e r a t e d S o r v a l l RC - 2 B c e n t r i f u g e . R adioactive DNA could be q u a n t i t a t i v e l y recovered i n the i n v i s i b l e p e l l e t . DNA could a l s o be recovered by adding yeast RNA to 100 ug/ml, and c o - p r e c i p i t a t i n g RNA and DNA w i t h ethanol. A l t e r n a t i v e l y , the ethanol p r e c i p i t a t i o n step could be omitted, and the phenol removed by s e v e r a l e x t r a c t i o n s w i t h chloroform-isoamyl a l c o h o l (2h:l, v / v ) . The p o s i t i o n of the 39 DM pgak on CsCl gradients was determined by measuring r a d i o a c t i v i t y i n a sm a l l a l i q u o t of each f r a c t i o n . 2. High molecular weight v i r a l DNA. The method of K i e f f et a l (1971) was used to prepare i n t a c t DNA f o r v e l o c i t y sedimentation and e q u i l i b r i u m gradient c e n t r i f u g a t i o n . Appropriate mixtures of p u r i f i e d v i r i o n s were t r e a t e d w i t h 0.5% sodium dodecyl sulphate and 2$ S a r k o s y l NL30 i n NDB and r o l l e d w i t h r e d i s t i l l e d phenol ( s a t u r a t e d w i t h NDB) at 6o°C f o r two minutes. The phenol phase was removed a f t e r c o o l i n g t o k°C, and the DNA s o l u t i o n r o l l e d w i t h isoamyl a l c o h o l (U$ v/v) i n chloroform u n t i l the aqueous phase was c l e a r . C e l l u l a r DNA Method (.1) f o r v i r a l DNA, described above, was a l s o used to p u r i f y c e l l u l a r DNA, up to the ethanol p r e c i p i t a t i o n step. The p r e c i p i t a t e was then d i s s o l v e d i n Tris-EDTA, made up to approximately 0.2 M i n NaCl by the a d d i t i o n of concentrated NaCl s o l u t i o n , and the ethanol p r e c i p i t a t i o n repeated. The second p r e c i p i t a t e was d i s s o l v e d i n Tris-EDTA, and made up to 0.15 M NaCl and 50 ug/ml RNase. (RNase had been p r e v i o u s l y heated t o 6o°C t o i n a c t i v a t e p o s s i b l e t r a c e s of DNase a c t i v i t y ) . A f t e r i n c u b a t i o n at 37°C f o r 15 minutes, pronase was added to 1 mg/ml, and i n c u b a t i o n at 37 C continued f o r 30 iminutes Phenol e x t r a c t i o n and ethanol p r e c i p i t a t i o n were then repeated as described above, and a f t e r the second ethanol p r e c i p i t a t i o n , the DNA was c e n t r i f u g e d t o e q u i l i b r i u m i n CsCl gradients as described f o r v i r a l DNA. Concentrations of DNA were measured by absorbance at 260 nm wave len g t h , assuming t h a t an o p t i c a l d e n s i t y of 1.0 corresponded to a DNA conc e n t r a t i o n o f 45 ug/ml. P u r i f i e d DNA preparations were s t o r e d at -20° C i n 0.1 x SSC. Sheared DNA f o r h y b r i d i z a t i o n purposes was obtained by passing p u r i f i e d DNA through a French pressure c e l l under a pressure of 12,000 p s i , or, f o r sm a l l volumes, by s q u i r t i n g f o r c e -f u l l y t e n times through a 26-gauge needle. Lower degrees of shear were obtained u s i n g 18, 20, 21, and 22 gauge needles. N. A n a l y s i s of V i r a l DNA Hydroxyapatite (HA) chromatography Hydroxyapatite was prepared e s s e n t i a l l y according to Miyazawa and Thomas (1965). About 100 mis water were poured i n t o a 2 1 g l a s s beaker. 500 mis each of 0.5 M Na^HPO^ and 0. 5 M C a C l 2 were added dropwise through two pasteur p i p e t t e s at a r a t e of 120 drops/minute. The contents of the beaker were mixed g e n t l y w i t h a magnetic s t i r r i n g bar. The f i n a l p r e c i p i t a t e was allowed to s e t t l e , and the supernatant decanted. The p r e c i p i t a t e was washed f o u r times w i t h d i s t i l l e d water by decantation, and suspended i n 1 l i t e r of water. 25 mis of h-0% w/w NaOH were added, and the mixture b o i l e d w i t h s t i r r i n g f o r 1 hour.- The supernatant was decanted and the p r e c i p i t a t e washed w i t h 1 l i t e r of water f o u r times. The p r e c i p i t a t e was resuspended i n 1 l i t e r of 0.01 M phosphate b u f f e r , and brought j u s t to b o i l i n g . The p r e c i p i t a t e was c o l l e c t e d by decantation and then b o i l e d i n the same s o l u t i o n f o r 5 minutes. The p r e c i p i t a t e was again c o l l e c t e d and b o i l e d f o r 15 minutes i n 0.01 M phosphate b u f f e r . A l l b o i l i n g treatments were accompanied by s t i r r i n g . The f i n a l p r e p a r a t i o n was s t o r e d at room temperature i n 0.01 M phosphate b u f f e r w i t h a s m a l l amount of chloroform. A s m a l l volume ( u s u a l l y 0.5-1.0 ml packed volume) of hyd r o x y a p a t i t (HA) suspension was added t o a column c o n s i s t i n g of a g l a s s tube w i t h a transverse s i n t e r e d g l a s s septum, enclosed by a water j a c k e t which was maintained at 60°C (See f i g . 1 f o r column d e t a i l s ) . The DNA s o l u t i o n was added t o the top of the column i n 0.05 M or l e s s , phosphat b u f f e r , and allowed to f l o w s l o w l y through the HA by g r a v i t y . Stepwise e l u t i o n The column was then washed w i t h 0.01 M phosphate, then 0.12 M phosphate, and f i n a l l y 0.5 M phosphate. F i v e to 10 mis of each b u f f e r 43 were used. F r a c t i o n s were c o l l e c t e d dropwise from the bottom of the column, and 100 u l a l i q u o t s i n d u p l i c a t e were taken from each f r a c t i o n , d r i e d onto f i l t e r paper d i s c s , and r a d i o a c t i v i t y measured. Gradient e l u t i o n A f t e r adsorption of the DNA, the top of the column was connected to an ISCO Di a l a g r a d gradient apparatus, and the a i r vent closed. The D i a l a g r a d was used t o generate a l i n e a r gradient of 0.03 M to 0.3 M phosphate b u f f e r according to the i n s t r u c t i o n s s u p p l i e d w i t h the machine. However, the normal dead volume between the pumps and the column (approximately 5 mis) was considered unacceptable, and so narrow t u b i n g was used, and an a u x i l i a r y mixing chamber, c o n t a i n i n g a magnetic s t i r r i n g bar, was s u b s t i t u t e d f o r the standard v i b r a t i n g mixer. This reduced the dead volume to approximately 0.'5 ml. The gradient was pumped through the column at a r a t e s l i g h t l y slower than g r a v i t y f l o w would proceed on the same column, t o avoid excessive compacting of hydroxyapatite. The f l o w r a t e was u s u a l l y 20 mis/hour. F r a c t i o n s were c o l l e c t e d dropwise d i r e c t l y i n t o s c i n t i l l a t i o n v i a l s . Phosphate concentrations were measured by r e f r a c t i v e index determinations and reference t o a c a l i b r a t i o n curve. Ten volumes of s c i n t i l l a t i o n f l u i d B were added, the v i a l s shaken, and r a d i o a c t i v i t y measured. kk N e u t r a l sucrose gradient v e l o c i t y sedimentation F r e s h l y prepared r a d i o a c t i v e l y l a b e l l e d v i r i o n s were used f o r the a n a l y s i s of DNA on sucrose g r a d i e n t s . F a i r l y low s p e c i f i c a c t i v i t i e s were used (See Resul t s ) and DNA was normally analyzed w i t h i n one day of h a r v e s t i n g the v i r u s . These precautions were expected t o reduce r a d i a t i o n - i n d u c e d breakage to n e g l i g i b l e amounts (Rosenthal and Fox, 1970). DNA was e x t r a c t e d from a mixture of two r a d i o a c t i v e l y l a b e l l e d v i r i o n p reparations as above, Method 2 , and c a r e f u l l y p i p e t t e d onto an 1 8 ml l i n e a r 10-30$ (w/w) sucrose gradient which had been prepared i n n e u t r a l DNA b u f f e r i n a s i l i c o n i z e d c e l l u l o s e n i t r a t e tube, u s i n g a Buchler twin-cone g r a d i e n t apparatus and p e r i s t a l t i c pump. Gradients were c e n t r i f u g e d at 2 6 , 0 0 0 rpm f o r 9 hours at 20°C i n the S W 2 7 . 1 r o t o r i n a Spinco L 2 - 6 5 B u l t r a c e n t r i f u g e . Tubes were then p i e r c e d w i t h a needle and 2 0-drop f r a c t i o n s c o l l e c t e d from the bottom, d i r e c t l y i n t o s c i n t i l l a t i o n v i a l s . Three mis s c i n t i l l a t i o n f l u i d B per v i a l were added, and the v i a l s counted at l4-°C i n a Nuclear Chicago U n i l u x S c i n t i l l a t i o n Counter. A l k a l i n e sucrose g r a d i e n t v e l o c i t y sedimentation DNA s o l u t i o n s prepared as above were denatured by adding 0 . 5 volume of 1 M NaOH. A f t e r being allowed t o stand at room temperature f o r 10 minutes, the samples were c a r e f u l l y p i p e t t e d onto 18 ml l i n e a r 10-30$ (w/w) sucrose gradients prepared i n a l k a l i n e DNA b u f f e r i n s i l i c o n i z e d c e l l u l o s e n i t r a t e tubes. C e n t r i f u g a t i o n and c o l l e c t i o n were c a r r i e d out as described above f o r n e u t r a l sucrose g r a d i e n t s . Cesium c h l o r i d e 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 1. P r e p a r a t i v e : DNA was e x t r a c t e d from a mixture of l a b e l l e d v i r i o n s and d i l u t e d w i t h d i s t i l l e d water. Cesium c h l o r i d e was added t o give a d e n s i t y of approximately 1.710 g/ml, and 1 ml of the s o l u t i o n was added t o a s i l i c o n i z e d c e l l u l o s e n i t r a t e tube, Spinco #303369. A l l operations were c a r r i e d out u s i n g precautions t o avoid shear breakage. For d e l i b e r a t e l y sheared DNA pre p a r a t i o n s , p a r t of the DNA s o l u t i o n was s q u i r t e d f o r c e f u l l y through a 22-gauge needle f i v e times before C s C l was added. D e n s i t i e s were c a l c u l a t e d from r e f r a c t i v e index determinations. The tubes were topped up w i t h m i n e r a l o i l and c e n t r i f u g e d w i t h D e l r i n adapters i n the Spinco type 50 r o t o r , f o r kQ hours at kO, 000 rpm, or f o r 80 hours at 30,000 rpm, and 20°C i n a Spinco L 2 - 6 5 B u l t r a c e n t r i f u g e . The tubes were then punctured w i t h a needle and three-drop f r a c t i o n s c o l l e c t e d from the bottom, i n t o s c i n t i l l a t i o n v i a l s . One ml of s c i n t i l l a t i o n f l u i d B per v i a l was added, and the v i a l s shaken and r a d i o a c t i v i t y measured. k6 2. A n a l y t i c a l : MCV l a b e l l e d w i t h CdThd was p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n and DNase treatment as described p r e v i o u s l y . I n t a c t DNA was ex t r a c t e d from the v i r i o n s as described above, and d i l u t e d to 0.5 ml w i t h d i s t i l l e d water. 0.25 ml of the s o l u t i o n was then passed f i v e times f o r c e f u l l y - t h r o u g h a 21, 20 or 18 gauge needle, depending on the d e s i r e d degree of fragmentation. P u r i f i e d i n t a c t ^ ^P-TU-D N A and u n l a b e l l e d HSV-P-DNA were then added t o both sheared and unsheared a l i q u o t s of MCV-DNA, and samples taken from each f o r s e d i -mentation a n a l y s i s on n e u t r a l sucrose gradients t o determine fragment s i z e s . S o l i d C s C l was added t o the remainder of each s o l u t i o n , t o give d e n s i t i e s of 1.71 g/cc and the f i n a l s o l u t i o n s were c a r e f u l l y p i p e t t e d d i r e c t l y i n t o the centrepiece c a v i t y of Beckman Model E u l t r a c e n t r i f u g e c e l l s , a v o i d i n g shear breakage (The c e l l s had been assembled i n the normal manner, and the top windows and holders then removed to all o w the DNA s o l u t i o n s t o be added). The c e l l s were then closed, and a d d i t i o n a l C s C l s o l u t i o n , d e n s i t y 1.71 g/cc, added through the normal f i l l i n g h ole to f i l l the c e l l . The s o l u t i o n s were c e n t r i f u g e d t o e q u i l i b r i u m at 25°C and kk,000 or 30,000 rpm i n the An-D r o t o r of a Beckman Model E u l t r a c e n t r i f u g e . DNA bands were then photographed u s i n g the U.V. o p t i c a l system, the i n i t i a l images were enlarged onto a second f i l m , and the f i n a l images were scanned on a Joyce-Loebl scanning densitometer. D e n s i t i e s were c a l c u l a t e d according to Mandel et a l (1968) from the p o s i t i o n of known marker DNA bands i n the same gradi e n t , u s i n g the equation: -*j = J0 + 4.2 2 ( r 2 - r Q 2 ) x 1 0 - 1 0 g/ml where ^ = d e n s i t y of unknown DNA, Ja = d e n s i t y of marker DNA, O J = speed of r o t a t i o n i n r a d i a n s / s e c , r = distance from unknown band t o centre of r o t a t i o n , and r Q = d i s t a n c e from marker band to centre of r o t a t i o n . G + C contents were c a l c u l a t e d from the equation:-(G+C) = •/- 1.660 g/ml 0.098 U l t r a v i o l e t absorbance/temperature p r o f i l e s . Mandel and Marmur (1968). Samples of DNA were p u r i f i e d as described p r e v i o u s l y , Method 1 and d i a l y z e d against 0.1 x SSC. Three semimicro quartz cuvettes were used f o r each determination: - two c o n t a i n i n g d i f f e r e n t DNA samples, and one c o n t a i n i n g 0.1 x SSC as a blank. The cuvettes were stoppered and pl a c e d i n a G i l f o r d r e c o r d i n g spectrophotometer f i t t e d w i t h a sample chamber temperature monitor and connected t o a Haake c i r c u l a t i n g waterbath. The temperature c o n t r o l of the water bath was connected to a v a r i a b l e - s p e e d motor to provide a slow and r e g u l a r temperature increase. O p t i c a l d e n s i t y at 260 nm was measured at 25 C, and the temperature r a p i d l y r a i s e d t o 50°C. The cuvettes were tapped t o remove a i r bubbles, and the temperature increased to 65°C at the r a t e of 0.5°C per minute. The r a t e of increase was then slowed down to 0.1°C per minute or l e s s , and h e a t i n g continued u n t i l a temperature of 90° C had been reached. O p t i c a l d e n s i t y was measured from the chart at 0 .5° i n t e r v a l s , and p l o t t e d as the increment per degree Centigrade versus temperature, t o a l l o w minor i r r e g u l a r i t i e s i n the m e l t i n g p r o f i l e to be seen more r e a d i l y . 0. L a b e l l i n g and P u r i f i c a t i o n of RNA C e l l c u l t u r e s were incubated w i t h a s m a l l volume ( l m l per 35 mm p e t r i dish) of MEM-A c o n t a i n i n g the d e s i r e d q u a n t i t y of t r i t i a t e d u r i d i n e . The times of l a b e l l i n g are given i n i n d i v i d u a l experimental d e t a i l s . At the end of the l a b e l l i n g p e r i o d , the monolayers were washed once w i t h RNA b u f f e r , and 1 ml per 35 mm d i s h of 1$ SDS i n RNA b u f f e r was added. A f t e r f i v e minutes, the c e l l l y s a t e was f r e e d from attachment to the p l a t e by s w i r l i n g , decanted i n t o a Corex tube, and immediately f r o z e n at -20°C u n t i l r e q u i r e d f o r p u r i f i c a t i o n . The l y s a t e was thawed and mixed by v o r t e x i n g at room temperature w i t h an equal volume of chloroform,-phenol s o l u t i o n ( 1 : 1 ) . The mixture was then c e n t r i f u g e d at 10,000 rpm f o r 10 minutes i n a S o r v a l l R C 2 B c e n t r i f u g e , and the lower phase removed w i t h a pasteur p i p e t t e . This e x t r a c t i o n was repeated u n t i l the denatured p r o t e i n at the i n t e r f a c e was h a r d l y v i s i b l e , and the RNA was then p r e c i p i t a t e d by the a d d i t i o n of NaCl s o l u t i o n t o 0.2 M, and two volumes of 95$ ethanol. The samples were cooled t o -20°C t o complete p r e c i p i t a t i o n , and h e l d at -20°C f o r s e v e r a l hours i f the amount of p r e c i p i t a t e was very small. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n at 15,000 rpm f o r 20 minutes i n the SS3*+ r o t o r i n a S o r v a l l RC-2B c e n t r i f u g e . The p r e c i p i -t a t e was d i s s o l v e d i n RNA b u f f e r , and the p r e c i p i t a t i o n step repeated. The second p e l l e t was d i s s o l v e d i n RNA b u f f e r , pH 7.1, and t r e a t e d w i t h 20 pg/ml DNase f o r 20 minutes at 37°C. Pronase was added to a f i n a l c o n c e n t r a t i o n of 1 mg/ml, and in c u b a t i o n continued at 37°C f o r 30 minutes. SDS was added to a con c e n t r a t i o n of 1$, and phenol-chloro-form e x t r a c t i o n c a r r i e d out as above, u n t i l no i n t e r f a c e m a t e r i a l was seen. The f i n a l aqueous phase was t r a n s f e r r e d t o a cl e a n tube, and the RNA p r e c i p i t a t e d twice w i t h ethanol as described above. The f i n a l p r e c i p i t a t e was d i s s o l v e d i n 10~^ M EDTA, pH 7-5, and stored at -20°C. P. N u c l e i c A c i d H y b r i d i z a t i o n Method 1 above was used to p u r i f y DNA f o r h y b r i d i z a t i o n r e a c t i o n s . A m o d i f i c a t i o n (Hudson, 1971) of the membrane-filter technique of G i l l e s p i e and Spiegelman (1965) was used f o r both DNA-DNA and DNA-RNA h y b r i d i z a t i o n r e a c t i o n s . A l l glassware i n v o l v e d i n h y b r i d i z a t i o n r e a c t i o n s was washed w i t h chromic a c i d . 5 0 F i x a t i o n of DMA Denaturation of DNA was c a r r i e d out by h e a t i n g i n 0 . 1 x SSC i n a b o i l i n g waterbath f o r 1 0 minutes, f o l l o w e d by r a p i d c o o l i n g under c o l d running water. The DNA s o l u t i o n was then made up to 5 mis or 2 0 mis of 6 x SSC, and f i l t e r e d by g r a v i t y through 1 . 7 cm or 3 . 5 cm M i l l i p o r e HAWP, 0 . ^ 5 urn pore s i z e n i t r o c e l l u l o s e f i l t e r s , which had been soaked i n b o i l i n g 6 x SSC and washed by s u c t i o n w i t h 6 x SSC. A f t e r f i l t r a t i o n of DNA, the f i l t e r s were washed, by f i l t r a t i o n w i t h three a l i q u o t s of 6 x SSC, and allowed to a i r dry. The DNA was f i x e d t o the f i l t e r s by h e a t i n g at 80°C f o r fo u r hours. Small d i s c s , 6.h mm i n diameter, were then cut from the l a r g e f i l t e r d i s c s u s i n g a punch. These d i s c s were l i g h t l y l a b e l l e d w i t h a p e n c i l f o r i d e n t i f i c a t i o n . DNA-DNA annealing The r a d i o a c t i v e sheared DNA to be t e s t e d i n s o l u t i o n was denatured by h e a t i n g i n a b o i l i n g water bath f o r t e n minutes, and cooled r a p i d l y under c o l d running water. The s o l u t i o n was made up to 6 x SSC, 0 . 1 $ SDS, and 1 . 0 ml placed i n t o a flat-bottomed s c i n t i l l a t i o n v i a l . The r e q u i r e d DNA-containing f i l t e r d i s c s were presoaked i n a separate 6 x SSC s o l u t i o n , and the wet f i l t e r s added to the t e s t DNA s o l u t i o n . Up to e i g h t s m a l l d i s c s were pla c e d i n one v i a l (Under these c o n d i t i o n s , d u p l i c a t e s were found t o agree c l o s e l y ) . The v i a l s were t i g h t l y capped and placed i n a 65 C water bath. The r e a c t i o n was allowed to continue f o r 1-k days, depending on the experiment, and the v i a l s were s w i r l e d approximately twice a day, care being taken t h a t overlap of f i l t e r s was minimal. At the end of the r e a c t i o n , the v i a l s were cooled, and the r e a c t i o n s o l u t i o n decanted. The f i l t e r s were washed twice by decantation w i t h 3 x 10 M T r i s , pH 9-3, and t r a n s f e r r e d to clea n v i a l s . The d i s c s were washed twice more by decantation, and then shaken at room temperature f o r two periods of 20 minutes each w i t h _3 f r e s h 3 x 10 M T r i s . The f i l t e r s were then washed twice more, b l o t t e d dry, heated at 80°C u n t i l completely dry, and r a d i o a c t i v i t y measured i n s c i n t i l l a t i o n f l u i d A. DNA-RNA h y b r i d i z a t i o n The r e a c t i o n was c a r r i e d out as described above, except that the r a d i o a c t i v e RNA s o l u t i o n was not heated p r i o r to h y b r i d i z a t i o n . At the end of the r e a c t i o n , the v i a l s were cooled, and the f i l t e r s washed twice by decantation w i t h 2 x SSC. The d i s c s were then t r a n s f e r r e d t o clea n v i a l s , washed twice more w i t h 2 x SSC, then shaken at 37°C f o r two periods of 20 minutes each w i t h two a l i q u o t s of 2 x SSC c o n t a i n i n g 20 ug/ml RNase. The f i l t e r s were then washed twice more, b l o t t e d dry, heated at 80°C u n t i l completely dry, and r a d i o a c t i v i t y measured i n s c i n t i l l a t i o n f l u i d A. Q. R a d i o a c t i v i t y Measurement Seve r a l methods of pre p a r i n g samples f o r s c i n t i l l a t i o n counting were i n v e s t i g a t e d , and the f o l l o w i n g two were found to be most u s e f u l . 1 . Samples were d r i e d onto Whatman GF/A gl a s s f i b r e f i l t e r s , and 5 mis s c i n t i l l a t i o n f l u i d A were added to each f i l t e r i n a v i a l . 2 . Aqueous samples c o n t a i n i n g up to 3 0 $ sucrose, 1 M NaCI or 0 . 5 M phosphate were counted i n s c i n t i l l a t i o n f l u i d B. Ten volumes of s c i n t i l l a t i o n f l u i d were added to the samples, and the mixture was cooled to k°C and shaken u n t i l a c l e a r s o l u t i o n was obtained. Samples were counted i n a Nuclear Chicago U n i l u x s c i n t i l l a t i o n counter at k°C. Machine backgrounds (e.g. 1 5 counts per minute f o r t r i t i u m ) were subtracted from a l l values. R. C e l l Counting T r y p s i n i z e d c e l l suspensions were counted i n a haemacytometer. C e l l s i n monolayers were counted by means of a g r i d i n the eyepiece of a W i l d i n v e r t e d phase c o n t r a s t microscope. The r a t i o between the areas of the g r i d image and the p l a t e was determined by c a l i b r a t i n g ' the eyepiece g r i d image area w i t h a haemacytometer g r i d w i t h l a r g e squares 1 mm across, and measuring the diameter of the p l a t e . A l l counts were converted t o numbers/plate. F i v e f i e l d s were counted on each p l a t e f o r each determination, i n areas s e l e c t e d to cover a l l regions of the p l a t e . The exact area to be counted was decided without observing the c e l l s through the microscope, to prevent b i a s towards dense or sparse regions. CHAPTER 3: RESULTS I Growth P r o p e r t i e s Growth p r o p e r t i e s , assay techniques and p u r i f i c a t i o n of HSV have been reported (e.g. Kaplan, 1969), but i n s e v e r a l respects these were not s u i t a b l e f o r the s t r a i n of HSV used i n t h i s study. Techniques f o r assaying and p u r i f y i n g MCV were a l s o r e q u i r e d , and so s u i t a b l e methods were developed f o r both v i r u s e s . A. Plaque Assays Herpes simplex v i r u s was assayed on confluent monolayers of H.Ep.2 c e l l s or o c c a s i o n a l l y on ME c e l l s , and MCV on confluent monolayers of ME c e l l s . S everal parameters were v a r i e d i n order to maximize both the plaque count and the convenience of measurement. 1. C o n d i t i o n of monolayers: For the assay of HSV, young, f r e s h l y confluent monolayers of H.Ep.2 c e l l s r e s u l t e d i n higher t i t r e s and more d i s t i n c t plaques than o l d e r , densely confluent monolayers, e.g. i n one experiment, the plaque count was 8-fold higher i n c e l l s seeded 2\ days p r i o r to i n f e c t i o n than i n c e l l s seeded h\ days before i n f e c t i o n . The younger c e l l s a l s o produced sharper, more d e f i n i t e white plaques. For MCV a l s o , the plaque appearance was most d i s t i n c t when young, f r e s h l y confluent monolayers were used. A r e d u c t i o n i n plaque count w i t h o l d e r c e l l s was not c o n s i s t e n t l y observed. 2. G e l l i n g agent: Agar and agarose were both t e s t e d as g e l l i n g agents f o r the o v e r l a y medium, but o n l y agarose allowed the p r o d u c t i o n of d i s t i n c t plaques. The concentration of agarose was important, and f o r both MCV and HSV, the o p t i m a l c o n c e n t r a t i o n was 0 . 5 $ . Concentrations below t h i s d i d not give a s u f f i c i e n t l y f i r m g e l , and concentrations above 0 . 5 $ r e s u l t e d i n i n d i s t i n c t plaques. 3. Serum concentration: Plaque appearance was a f f e c t e d c o n s i d e r a b l y by the c o n c e n t r a t i o n of serum i n the o v e r l a y medium. The most c l e a r c u t r e s u l t s f o r HSV plaques were obtained w i t h 0 $ serum, wh i l e 5$ serum was found t o be o p t i m a l f o r the MCV assay. k. V i s u a l i z a t i o n of plaques: Both MCV and HSV-P are non-l y t i c , so t h a t plaques produced by e i t h e r v i r u s c o n s i s t of f o c i of rounded, r e f r a c t i l e c e l l s . Although n e u t r a l red s t a i n i n g i s necessary f o r the v i s u a l i z a t i o n of l y t i c plaques, i t was found t h a t H.Ep.2 c e l l s d i d not take up the s t a i n i n a r e p r o d u c i b l e manner, and t h a t unstained plaques of both v i r u s e s could be adequately observed when viewed by s c a t t e r e d r a t h e r than t r a n s m i t t e d l i g h t . The appearance of unstained plaques under these c o n d i t i o n s was d i s t i n c t i v e and con-v e n i e n t f o r counting. 5. Adsorption time: The r a t e of adsorption of HSV i n f e c t i v i t y t o H.Ep.2 monolayers was measured by a l l o w i n g v i r u s to adsorb f o r d i f f e r e n t times, removing the i n o c u l a , washing, and adding o v e r l a y medium. A f t e r 30 minutes adsorption, the number of plaques obtained was 93$ of the number obtained a f t e r 60 minutes adsorption. B. C e n t r i f u g a l Assay Osborn and Walker (1968) reported t h a t the i n f e c t i v i t y of mouse -passaged MCV can be enhanced approximately 30-fold by i n f e c t i n g c e l l s under a c e n t r i f u g a l f i e l d . I n the present study, t h i s f i n d i n g was confirmed f o r t i s s u e culture-passaged v i r u s , and some parameters a f f e c t i n g the c e n t r i f u g a l adsorption of i n f e c t i v i t y were measured. F i g . 2 shows the e f f e c t of v a r y i n g the time and speed of c e n t r i f u g a -t i o n , and the volume of the inoculum. I n f e c t i v i t y o f HSV was enhanced h.5-fold by c e n t r i f u g a l i n o c u l a -t i o n onto H.Ep.2 c e l l s , and 7 -7-fold i n mouse embryo c e l l s . C. Morphology N e g a t i v e l y s t a i n e d preparations of p a r t i a l l y p u r i f i e d HSV, MCV and HCV were examined under the e l e c t r o n microscope. HSV showed t y p i c a l herpesvirus morphology ( p l a t e l a ) as reported elsewhere (Roizman and Spear, 1971). Murine cytomegalovirus preparations F i g . 2 ; C e n t r i f u g a l a d s o r p t i o n of MCV Murine cytomegalovirus, p u r i f i e d o n ly by low-speed c e n t r i f u g a t i o n , was d i l u t e d t o concentrations of approximately 200 and 2,000 c e n t r i f u g a l pfu per ml. T e r t i a r y mouse embryo c e l l s were i n f e c t e d by c e n t r i f u g a l a d s o r p t i o n as described i n the M a t e r i a l s and Methods chapter, except f o r the changes described below. a) C e n t r i f u g a t i o n was c a r r i e d out f o r 10 and 20 minutes, and d i f f e r e n t volumes of i n o c u l a were used. R e s u l t s from the two adsorption times were averaged, and used to c a l c u l a t e the t i t r e of the o r i g i n a l p r e p a r a t i o n , so that the f i g u r e shows the r e l a t i v e f r a c t i o n s of v i r u s adsorbed from each inoculum volume. b) C e n t r i f u g a t i o n was c a r r i e d out f o r 10 and 20 minutes at v a r i o u s speeds. The standard speed of 2,000 rpm c o r r e s -ponds to a c e n t r i f u g a l f o r c e of 1,900 g. c) Standard adsorption was c a r r i e d out as described i n the M a t e r i a l s and Methods chapter, except t h a t 0.5 ml inoculum was used. C e n t r i f u g a l adsorption was c a r r i e d out f o r the times shown. 58 Fig. 2: VOLUME OF INOCULUM (mis) 0.2 0.4 RELATIVE CENTRIFUGAL FORCE (xg) 1,000 2,000 I L 30 40 50 60 TIME OF CENTRIFUGATION (MINUTES) P l a t e 1 : a. Herpes simplex v i r u s b. Murine cytomegalovirus, m u l t i c a p s i d form c. Murine cytomegalovirus, s i n g l e c a p s i d form d. Human cytomegalovirus The bars i n d i c a t e 1 0 0 nm 6 2 showed a l a r g e number of m u l t i p l e forms, c o n s i s t i n g of s e v e r a l capsids enclosed by an apparently s i n g l e envelope ( p l a t e l b ) . S i n g l e v i r i o n s were a l s o present ( p l a t e l c ) many of them unenveloped, but i t was estimated t h a t approximately h a l f of the capsids seen were i n m u l t i p l e forms. Human cytomegalovirus preparations contained mainly s i n g l e , enveloped v i r i o n s ( p l a t e l d ) w i t h some naked capsids and o c c a s i o n a l aggregates of capsids without an envelope. No enveloped m u l t i p l e -capsid forms were seen. Since the l a r g e m u l t i p l e s seen i n MCV preparations would be expected to sediment r a p i d l y i n a c e n t r i f u g a l f i e l d , the r e l a t i o n s h i p of r a p i d l y - s e d i m e n t i n g v i r u s to c e n t r i f u g a l enhancement of i n f e c t i v i t y was i n v e s t i g a t e d . Preparations of MCV and HSV were p e l l e t t e d by c e n t r i f u g a t i o n at v a r i o u s speeds, and i n f e c t i v i t y measured by standard and c e n t r i f u g a l adsorption. The m a j o r i t y of the MCV i n f e c t -i v i t y was p e l l e t t e d at lower speeds than HSV i n f e c t i v i t y , but the r a t i o of c e n t r i f u g a l : s t a n d a r d i n f e c t i v i t y was greater than 30 f o r a l l f o u r populations of MCV ( f i g . 3). E l e c t r o n microscopic examination showed t h a t a l l f o u r HSV populations comprised predominantly s i n g l e , enveloped v i r i o n s . Populations of MCV p e l l e t t e d at lower speeds comprised mainly m u l t i p l e v i r i o n s , and the populations p e l l e t t e d at higher speeds contained r e l a t i v e l y l e s s m u l t i p l e s and more s i n g l e , 40H 0 15 30 45 60 CENTRIFUGAL FORCE x TIME (xg min x 10"^ ) F i g . 3: I n f e c t i v i t y of d i f f e r e n t v i r u s populations Preparations of MCV and HSV were p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . The resuspended p e l l e t s were c e n t r i f u g e d at 5,000 rpm i n the SW 50.1 r o t o r at k°C f o r 15 minutes i n a Spinco L2-65B u l t r a c e n t r i f u g e . The supernatants were then t r a n s f e r r e d to clean tubes, and c e n t r i f u g e d at 8,000 rpm f o r 15 minutes. The supernatants were again removed and c e n t r i f u g e d at 11, 500 rpm f o r 15 minutes, and f i n a l l y at 13,000 rpm f o r 30 minutes. A l l p e l l e t s were r e -suspended i n MEM-B co n t a i n i n g 2% serum, and assayed on mouse embryo c e l l s by both standard and c e n t r i f u g a l a dsorption methods. Results are shown as the i n f e c t i v i t y (pfu) i n each f r a c t i o n expressed as a percentage of the t o t a l i n f e c t i v i t y recovered, p l o t t e d against the product of the time and the c e n t r i f u g a l f o r c e used to p e l l e t each population. enveloped v i r i o n s . Separation of the m u l t i p l e and s i n g l e v i r i o n p opulations was not complete. However t h i s r e s u l t demonstrated t h a t both types of MCV v i r i o n s were subject to c e n t r i f u g a l enhancement of i n f e c t i v i t y . D. Growth Curves The growth c y c l e s of MCV and HSV are shown i n f i g . k. I n agreement w i t h Kaplan (1969) and Henson et a l (1966), the len g t h of the s i n g l e step growth curve under our co n d i t i o n s was about 20 hours f o r HSV and 28 hours f o r MCV. I n f e c t i v i t y was mainly c e l l - a s s o c i a t e d f o r both v i r u s e s . E. S t a b i l i t y Loss of i n f e c t i v i t y was measured under a v a r i e t y of c o n d i t i o n s f o r MCV and HSV. I n p a r t i c u l a r , s u i t a b l e storage c o n d i t i o n s were re q u i r e d , under which v i r u s preparations could be maintained w i t h a n e g l i g i b l e l o s s of i n f e c t i v i t y . The r e s u l t s are summarized i n Table 1. Since f r e e z i n g and thawing caused a s l i g h t l o s s of i n f e c t i v i t y , v i r u s preparations were stored at *+°C i n 1% s e r u m - d i s t i l l e d water f o r short periods (a few days) and at -70°C i n the same d i l u e n t f o r longer periods. Loss of p a r t i c l e i n t e g r i t y was a l s o measured f o r HSV, by Growth curves of MCV and HSV H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of 0 . 8 p f u per c e l l , and the supernatant and c e l l s harvested s e p a r a t e l y at v a r i o u s times a f t e r i n f e c t i o n . Mouse embryo c e l l s were i n f e c t e d w i t h MCV at a m u l t i -p l i c i t y of 0 . 0 5 p f u per c e l l . A l l t i t r e s r e f e r .to standard adsorption. C e l l - a s s o c i a t e d i n f e c t i v i t y Supernatant i n f e c t i v i t y A. r- — r — i — -10 20 30 HOURS AFTER INFECTION B . 10 20 30 40 50 HOURS AFTER INFECTION Table 1: Loss of i n f e c t i v i t y of MCV and HSV I n f e c t i v i t y was measured by plaque assay as described i n the M a t e r i a l s and Methods chapter. Adsorption was c a r r i e d out by standard i n o c u l a t i o n f o r HSV and by c e n t r i -f u g a l adsorption f o r MCV. The time r e q u i r e d f o r h a l f of the i n f e c t i v i t y t o be l o s t , was read d i r e c t l y from a p l o t of i n f e c t i v i t y versus time, or c a l c u l a t e d by e x t r a p o l a t i o n of the l a s t value, assuming t h a t the l o g a r i t h m of the i n f e c t i v i t y v a r i e d l i n e a r l y w i t h time. Table 1: Loss of i n f e c t i v i t y of MCV and HSV V i r u s D i l u e n t Temperature °C Time r e q u i r e d f o r l o s s of h a l f of Lqg3_o l o s s per i n f e c t i v i t y freeze-thaw-cycle (hours) MCV H 20 + 2$ serum > 200 0.03 MCV H 20 MCV MEM-B + 2% serum k k 9-5 35 l.k 0.025 MCV MEM-B HSV H 20 + 1$ serum k k 10.3 13k 1.5 0.05 HSV H20 HSV H 20 HSV PBS + 10$ serum k 37 37 2.7 1.6 0.5 HSV PBS 37 0.6 70 measuring the q u a n t i t y of r a d i o a c t i v e v i r u s sedimenting i n sucrose gradients at the r a t e c h a r a c t e r i s t i c of i n t a c t v i r u s p a r t i c l e s . F i g . 5 shows the l o s s of i n f e c t i v i t y compared to the degradation of v i r i o n s to smaller components. I n f e c t i v i t y was l o s t f a s t e r than p a r t i c l e i n t e g r i t y . F. P u r i f i c a t i o n The r e q u i r e d degree of p u r i f i c a t i o n v a r i e d according t o the purpose f o r which the v i r u s p r e p a r a t i o n was to be used. The f o l l o w i n g methods were i n v e s t i g a t e d (See Methods f o r d e t a i l s ) . Table 2 shows the recovery of input p f u from v a r i o u s procedures. 1. D i f f e r e n t i a l c e n t r i f u g a t i o n : C l a r i f i c a t i o n by low speed c e n t r i f u g a t i o n was used to remove l a r g e c e l l u l a r d e b r i s , and the v i r u s was then p e l l e t t e d at h i g h speed and resuspended i n the d e s i r e d volume of d i l u e n t . This method was u s e f u l f o r a l l three of the v i r u s e s used. Routine p u r i f i c a t i o n and c o n c e n t r a t i o n of v i r u s p reparations was c a r r i e d out by d i f f e r e n t i a l c e n t r i f u g a t i o n , because of the speed and convenience of t h i s method. A l s o , the DNA i n the h i g h speed p e l l e t was greater than 95$ v i r a l i n nature. This was determined by e x t r a c t i o n of DNA from h i g h speed p e l l e t s and 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 t h i s DNA i n CsCl gr a d i e n t s . The DNA present at the d e n s i t y of host DNA, measured by r a d i o a c t i v i t y , was l e s s than 5$ of the t o t a l f o r HSV-P and F i g . 5: Loss of i n f e c t i v i t y and degradation of HSV H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of approximately 10 pfu per c e l l , and incubated from 5 to 19 hours a f t e r i n f e c t i o n w i t h h uCi/ml HdThd. The v i r u s p r e p a r a t i o n was then p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n and suspended i n Hank's s o l u t i o n c o n t a i n i n g 1$ serum. A f t e r i n c u b a t i n g at 37°C f o r the times shown i n the f i g u r e , a l i q u o t s were t r e a t e d w i t h DNase (20 ug/ml, 10 mM Mg + +) f o r a f u r t h e r 15 minutes at 37°C. The samples were thera. cooled to k°C and assayed f o r i n f e c t i v i t y , by plaque assay, and f o r v i r i o n i n t e g r i t y , by measuring the t o t a l amount of r a d i o a c t i v i t y sedimenting at the v e l o c i t y of i n t a c t v i r i o n s on sucrose g r a d i e n t s . I n f e c t i v i t y R a d i o a c t i v i t y i n v i r u s band F i g . 5: 1 2 HOURS AT 37°C MCV. This allowed h i g h - s p e e d - p e l l e t t e d v i r u s to he used f o r some DNA sedimentation analyses, since the t r a c e of contaminating host DNA was of low molecular weight and d i d not i n t e r f e r e w i t h the i n t e r p r e t a t i o n of the r e s u l t s . 2 . Deoxyribonuclease and resedimentation: To remove f i n a l t r a c e s of host DNA, the h i g h speed p e l l e t was resuspended, t r e a t e d w i t h DNase, and subjected t o another c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n . This a d d i t i o n a l step was r e q u i r e d when t r a c e s of host DNA were unaccept-able, as i n the case of a n a l y t i c a l C s C l c e n t r i f u g a t i o n experiments. 3. . Sucrose gradient v e l o c i t y sedimentation: Herpes simplex v i r u s was p u r i f i e d on sucrose gradients when v i r u s of higher p u r i t y was requ i r e d . The v i r u s band, i f s u f f i c i e n t l y concentrated, was v i s i b l e as a t u r b i d l a y e r i n the grad i e n t , and the peak of r a d i o a c t i v e v i r u s was w e l l separated from low molecular weight r a d i o a c t i v e m a t e r i a l and l a r g e c e l l u l a r d ebris ( f i g . 6 ) . No r a d i o a c t i v i t y appeared i n the f e c t e d c e l l p r e p a r a t i o n was analyzed i n t h i s way. Sucrose gradient a n a l y s i s of HSV was used as a measure of the amount of r a d i o a c t i v i t y i n c o r p o r a t e d i n t o v i r i o n s , since r a d i o a c t i v i t y i n the appropriate r e g i o n of the gradient was s p e c i f i c f o r v i r i o n s . This method was not u s e f u l f o r MCV because of the heterogeneous s i z e of v i r a l p a r t i c l e s . r e g i o n of the v i r a l band when Table 2: P u r i f i c a t i o n of herpesviruses Treatment of v i r u s Percentage recovery-p r e p a r a t i o n (pfu) HSV MCV Low-speed c e n t r i f u g a t i o n 89 88 High-speed c e n t r i f u g a t i o n 85 126 Cesium c h l o r i d e g r a d i e n t 37 Sucrose gradient 70 DNase treatment 77 Table 2: P u r i f i c a t i o n of herpesviruses I n f e c t i v i t y was measured by plaque assays, u s i n g standard adsorption f o r HSV and c e n t r i f u g a l adsorption f o r MCV. P u r i f i c a t i o n was c a r r i e d out as described i n the M a t e r i a l s and Methods chapter, and the amount of i n f e c t i v i t y remaining a f t e r each procedure was expressed as a percentage of the i n f e c t i v i t y of the p r e p a r a t i o n immediately p r i o r t o t h a t procedure. 6: Sucrose g r a d i e n t sedimentation of HSV H.Ep.2 c e l l s were i n f e c t e d w i t h HSV and incubated from 9 to 2 0 hours a f t e r i n f e c t i o n w i t h k pCi/ml "HdThd. V i r u s was harvested and p u r i f i e d by low-speed c e n t r i f u g a t i o n only, and then analyzed on a sucrose gradient as described i n the M a t e r i a l s and Methods chapter, except t h a t 0 . 5 n i l 6 o $ (w/w) sucrose was added to the bottom of the gradient. An a l i q u o t was taken from each gradient f r a c t i o n f o r r a d i o a c t i v i t y measurement, and f r a c t i o n s were pooled as shown i n the f i g u r e , and assayed f o r i n f e c t i v i t y by plaque assay. Standard i n o c u l a t i o n was used. The j o i n e d c i r c l e s show r a d i o a c t i v i t y and the histogram represents i n f e c t i v i t y . D i r e c t i o n of sedimentation i s t o the l e f t . 77 Fig. 6 : 10(H 5CH FRACTION NUMBER k. 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 : Herpes simplex v i r u s was a l s o p u r i f i e d on C s C l and sodium potassium t a r t r a t e d e n s i t y g r a d i e n t s ; but n e i t h e r method proved u s e f u l . V i r a l bands were not w e l l r e s o l v e d from c e l l u l a r d ebris under the c o n d i t i o n s used. C e n t r i f u g a t i o n of HSV i n CsCl r e s u l t e d i n 63$ l o s s of i n f e c t i v i t y . CHAPTER h: RESULTS I I P r o p e r t i e s of Herpesvirus Genomes The purpose of t h i s study was to i n v e s t i g a t e p o s s i b l e s t a t e s of l a t e n t i n f e c t i o n w i t h herpesviruses. I f a l a t e n t i n f e c t i o n could be e s t a b l i s h e d , the l o c a t i o n and s t a t e of the v i r a l genome i n such circumstances would be of considerable importance. Hence an i n v e s t i g a t i o n was made of the p r o p e r t i e s of the genomes of HSV, MCV and HCV as they e x i s t w i t h i n the v i r i o n s . A. Rad i o a c t i v e L a b e l l i n g of V i r a l DCTA 3 Ill-V i r a l DNA was l a b e l l e d w i t h "HdThd or ' CdThd by adding the appropriate precursor t o the growth medium a f t e r i n f e c t i o n . The op t i m a l time f o r a d d i t i o n of l a b e l was determined by measuring the r e l a t i v e amounts of l a b e l i n c o r p o r a t e d i n t o the p u r i f i e d v i r u s preparations when l a b e l was added at d i f f e r e n t times a f t e r i n f e c t i o n . HSV was p u r i f i e d by sucrose g r a d i e n t v e l o c i t y s e d i -mentation, and MCV by d i f f e r e n t i a l c e n t r i f u g a t i o n . The o p t i m a l l a b e l l i n g times were:- fo u r hours a f t e r i n f e c t i o n of H.Ep.2 c e l l s w i t h HSV at high m u l t i p l i c i t y , and 12-20 hours a f t e r i n f e c t i o n of ME c e l l s w i t h MCV. B. P u r i f i c a t i o n of V i r a l DNA A convenient a n a l y t i c a l method was a v a i l a b l e f o r assay of HSV and MCV DNA, i n t h a t v i r a l and host DNA's have markedly d i f f e r e n t G + C contents, and hence can be r e a d i l y separated by CsCl 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 . F i g . 7 i l l u s t r a t e s the se p a r a t i o n obtained between t r i t i u m - l a b e l l e d HSV-DNA and carbon - lU l a b e l l e d H.Ep.2 c e l l DNA. This method was used f o r assessing the p u r i t y of v i r a l DNA a f t e r v a r i o u s p u r i f i c a t i o n procedures. Although CsCl gradients could be used t o separate t o t a l host and v i r a l DNA, t h i s method was not used to prepare v i r a l DNA i n t h i s study. Contamination by host DNA segments of h i g h G + C content (e.g. ribosomal gene t r a c t s ) could have s e r i o u s l y a f f e c t e d the r e s u l t s of some h y b r i d i z a t i o n experiments. Therefore, DNA was e x t r a c t e d from p u r i f i e d v i r u s p r e p a r a t i o n s , which contained o n l y 10$ of the t o t a l v i r a l DNA i n the case of HSV-P. A s i n g l e c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n was s u f f i c i e n t to remove a l l detectable t r a c e s of host DNA from most HSV-P prepara-t i o n s , measured e i t h e r by r a d i o a c t i v i t y of incorp o r a t e d t r i t i a t e d thymidine, or by o p t i c a l d e n s i t y at 260 nm. However, preparations obtained from l a r g e r o l l e r b o t t l e s r e q u i r e d DNase treatment and r e p e l l e t t i n g . One c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n of a MCV p r e p a r a t i o n y i e l d e d DNA c o n t a i n i n g l e s s than 5$ host DNA, and the l a t t e r could be removed by DNase treatment and r e p e l l e t t i n g (e.g. 81 Fig. 7: FRACTION NUMBER 7: C s C l 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 HSV and H.Ep.2 DNA's H.Ep.2 c e l l s were incubated f o r ih hours w i t h 0.05 / ih uCi/ml CdThd. The growth medium was then r e p l a c e d by medium l a c k i n g r a d i o a c t i v e thymidine, and i n c u b a t i o n continued f o r 9 hours. The c e l l s were then i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of approximately 10 p f u per c e l l , and incubated from 5 to 19 hours a f t e r i n f e c t i o n w i t h h uCi/ml 3HdThd. At 19 hours a f t e r i n f e c t i o n , DNA was e x t r a c t e d from the i n f e c t e d c e l l s , p u r i f i e d , and analyzed on cesium c h l o r i d e e q u i l i b r i u m gradients as described i n the M a t e r i a l s and Methods chapter. The bottom of the gradient i s to the l e f t . see F i g . 1 8 , i n which host DNA would appear between the MCV and T*+-DNA peaks). Sucrose gradient v e l o c i t y sedimentation was found to be an e f f e c t i v e method f o r p u r i f i c a t i o n of s m a l l q u a n t i t i e s of HSV f o r DNA e x t r a c t i o n . This method was a l s o attempted f o r MCV, but i n repeated experiments the DNA e x t r a c t e d from such v i r i o n s was found to be heterogeneous i n molecular weight when analyzed by sucrose d e n s i t y gradient v e l o c i t y c e n t r i f u g a t i o n ( F i g . 8 a ) , i n c o n t r a s t t o the s i n g l e d i s c r e t e band obtained w i t h DNA from sucrose gradient p u r i f i e d HSV-P ( F i g . 8 b ) . The reason f o r the breakage of MCV-DNA under these c o n d i t i o n s i s not known. Since DNA from v i r i o n s p u r i f i e d by d i f f e r -e n t i a l c e n t r i f u g a t i o n d i d not show t h i s breakage, o n l y d i f f e r e n t i a l c e n t r i f u g a t i o n was used to p u r i f y MCV f o r DNA e x t r a c t i o n . Complete d e t a i l s of the methods f i n a l l y adopted are described i n the M a t e r i a l s and Methods chapter. C. Hydroxyapatite (HA) Chromatography I n i t i a l s t u d i e s concerned the use of HA chromatography f o r a n a l y s i s of DNA-DNA and DNA-RNA h y b r i d i z a t i o n . However, unexpected p r o p e r t i e s of HSV-DNA were discovered, and t h i s l e d to a more d e t a i l e d i n v e s t i g a t i o n of parameters a f f e c t i n g e l u t i o n of HSV-DNA from hydroxy-a p a t i t e . The HSV-DNA was denatured by h e a t i n g i n a b o i l i n g water bath F i g . 8: V e l o c i t y sedimentation of DNA from sucrose gradient p u r i f i e d v i r i o n s ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one plaque forming u n i t (pfu) per c e l l , and incubated from 16 to 36 hours a f t e r i n f e c t i o n w i t h k uCi/ml ^HdThd. H.Ep.2 c e l l s were i n f e c t e d w i t h HSV-P at a m u l t i p l i c i t y of 4-5 p f u per c e l l and incubated from 4 to 20 hours a f t e r i n f e c t i o n w i t h 20 u-Ci/ml 3HdThd. V i r i o n s were p u r i f i e d by sucrose d e n s i t y gradient v e l o c i t y s e d i -mentation as desc r i b e d i n Methods. DNA was e x t r a c t e d from the v i r a l band as described i n Methods, l a y e r e d onto 5 ml l i n e a r 10-30$ (w/'w)fp sucrose gradients prepared i n n e u t r a l DNA b u f f e r , and c e n t r i f u g e d i n the SW 50-1 r o t o r i n a Spinco L2-65-B u l t r a c e n t r i f u g e f o r 2.5 hours at 20°C and 42,000 rpm. 15-drop f r a c t i o n s were c o l l e c t e d from the bottom, and r a d i o a c t i v i t y measured a f t e r the a d d i t i o n of 3 mis s c i n t i l l a t i o n f l u i d . D i r e c t i o n of sedimentation i s to the l e f t . a) MCV-DNA HSV-P-DNA f o r 10 minutes i n 0.05 M phosphate b u f f e r (Na con c e n t r a t i o n 0.075 M) i n which the Tm of HSV-DNA should be 90°C (Mandel and Marmur, 1968). The denatured m a t e r i a l was adsorbed to HA and e l u t e d w i t h 0.12 M phosphate b u f f e r , as described i n Methods, t o e l u t e s i n g l e stranded DNA (Miyazawa and Thomas, 19&5; B r i t t e n and Kohne, 1968; Bendich and McCarthy, 1970; L a i r d , 1971), and then w i t h 0.5 M phosphate b u f f e r to e l u t e double stranded DNA. I n repeated experiments, approximately 25$ of the DNA e l u t e d i n the 0.5 M phosphate f r a c t i o n s . The f r a c t i o n of s t a r t i n g m a t e r i a l remaining apparently double-stranded was inde-pendent of the con c e n t r a t i o n of DNA over a UO-fold range, but v a r i e d from one batch of DNA t o another. This 1 double-stranded' m a t e r i a l was d i l u t e d t o 0.05 M phosphate, heated i n a b o i l i n g water bath f o r 10 minutes, and analyzed again on HA. *+7$ of t h i s m a t e r i a l e l u t e d i n the 0. 5 M f r a c t i o n s . Shearing the DNA t o low molecular weight before denaturation allowed e s s e n t i a l l y complete denaturation upon h e a t i n g (96$ of m a t e r i a l i n 0.12 M f r a c t i o n s ) . This behaviour suggested t h a t c r o s s l i n k i n g of DNA strands such as occurs i n V a c c i n i a v i r u s DNA (Berns and Silverman, 1970), could have been causing r a p i d reannealing of a p o r t i o n of the HSV-DNA. This was i n v e s t i g a t e d f u r t h e r u s i n g phosphate b u f f e r g r a d i e n t e l u t i o n , i n s t e a d of stepwise e l u t i o n . I t was found t h a t the double-stranded f r a c t i o n was an a r t e f a c t of the previous denaturation and chromatography c o n d i t i o n s , and that i n r e a l i t y no heat or a l k a l i -s t a b l e c r o s s l i n k i n g was present i n HSV-DNA (e.g. F i g . 10). From a number of experiments the f o l l o w i n g f a c t o r s were found t o a f f e c t the phosphate e l u t i o n behaviour of the DNA. 1. The (G + C) content of s i n g l e - s t r a n d e d DNA. Thus DNA w i t h a h i g h G + C content (e.g. HSV-DNA, 68$ (G + C)) e l u t e d at a higher phosphate m o l a r i t y than low (G + C) DNA (e.g. T4-DNA). 2. The molecular weight of s i n g l e stranded DNA. Unsheared denatured HSV-DNA e l u t e d at a m o l a r i t y of approximately 0.13 M phosphate whereas sheared s i n g l e - s t r a n d e d DNA e l u t e d at about 0.09 M. The e f f e c t of molecular weight on e l u t i o n of double stranded DNA was l e s s ( i n agreement w i t h Be r n a r d i (1971)). Because of these f a c t o r s , the m o l a r i t y at which s i n g l e - s t r a n d e d HSV-DNA e l u t e s can be greater than 0.12, and so stepwise e l u t i o n u s i n g 0.12 M and 0.5 M phosphate buffer.produced anomalous r e s u l t s . 3. Conditions of denaturation. A peak of apparently double-stranded m a t e r i a l was als o seen i n e a r l y p r o f i l e u s i n g gradient e l u t i o n of denatured HSV-DNA. I t was then shown th a t heat and a l k a l i d enaturation i n e a r l y experiments was inadequate ( f i g . 9)« Denatura-t i o n at 100°C i n 0.075 M Na + proceeded at a r e l a t i v e l y slow r a t e , and was 99$ complete a f t e r 25 minutes, but o n l y 87$ complete a f t e r 5 minutes, f o r the p r e p a r a t i o n of DNA t e s t e d . Heating at 100°C f o r 10 minutes i n 0.03 M Na + r e s u l t e d i n complete denaturation of i n t a c t F i g . 9: Rate of denaturation of HSV-DNA H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of 10 p f u per c e l l and incubated from 5 to 20 hours a f t e r i n f e c t i o n w i t h 50 p C i / ml ^HdThd. V i r u s was p u r i f i e d by sucrose d e n s i t y g r a d i e n t v e l o c i t y sedimentation, and DNA e x t r a c t e d from the v i r u s band, u s i n g method 1 as described i n the M a t e r i a l s and Methods chapter, up t o the phenol e x t r a c t i o n step. The s o l u t i o n was then e x t r a c t e d w i t h c h l o r o f o r m - i s o -amyl a l c o h o l (2h:l, v/v) and made up t o 0 .03 M phosphate, 0.075 M Na +. The s o l u t i o n was immersed i n a b o i l i n g waterbath, and 0 . 5 ml a l i q u o t s withdrawn and cooled at 5 minute i n t e r v a l s . The samples were made up to 10 mM Mg + +, and Neurospora c r a s s a endonuclease (obtained from Dr. J. B. Hudson) added. A f t e r i n c u b a t i o n at 37°C f o r 2.5 hours, the samples were loaded on hydroxyapatite as described i n the M a t e r i a l s and Methods chapter. E l u t i o n was c a r r i e d out by a l i n e a r g r a d i e n t of phosphate b u f f e r , f o l l o w e d by a wash w i t h 0 . 5 M phosphate. The amount of DNA e l u t i n g above 0.15 M phosphate was expressed as a percentage of the t o t a l DNA recovered. F i g . 9a shows the amount of 'double-stranded' DNA ( e l u t i n g above 0.15 M phosphate) a f t e r i n c r e a s i n g times of h e a t i n g . F i g . 9"° shows the gradient p r o f i l e of the e l u t i o n of the 5 minute sample from hydroxyapatite. The m a j o r i t y of DNA e l u t e d at v e r y low phosphate m o l a r i t y a f t e r degradation to nuc l e o t i d e s or short o l i g o n u c l e o t i d e s by N. cr a s s a endonuclease. 8 9 Fig. 9: DURATION OF HEATING (MINUTES) 10 20 , 1 1 , FRACTION NUMBER molecules. No peak of double stranded m a t e r i a l could then be seen on gradient e l u t i o n p r o f i l e s of such m a t e r i a l (e.g. F i g . 10) . D. Sucrose Density Gradient V e l o c i t y Sedimentation of DNA N e u t r a l pH. Murine cytomegalovirus I n t a c t DNA was e x t r a c t e d from a mixture of r a d i o a c t i v e l y l a b e l l e d MCV and ih v i r i o n s as described i n the M a t e r i a l s and Methods Section. The DNA from each v i r u s y e i e l d e d a s i n g l e , sharp peak when analyzed by sucrose d e n s i t y gradient v e l o c i t y sedimentation ( f i g . 11a). However, overload i n g the gradients w i t h more than approximately 0 .5 Mg t o t a l DNA per gradient r e s u l t e d i n d i s t o r t i o n of the peaks (e.g. f i g . l i b ) and v a r i a t i o n i n the apparent r e l a t i v e sedimentation c o - e f f i c i e n t s . Using t o t a l amounts of DNA ranging from 0 . 5 Mg to 0 .09 Mg> "the r e l a t i v e r a t e s of sedimentation of MCV and T^-DNA's were compared, u s i n g the r e l a t i o n s h i p of Bur g i and Hershey (1963) as mo d i f i e d by F r e i f e l d e r (1970). a 1 = s x = ( MX ) 0 .38 d~ 5" [ M" ] where d = distance sedimented, s = sedimentation c o e f f i c i e n t , and M = molecular weight of the DNA molecule. 1 0 : E l u t i o n of denatured HSV-DNA from hydroxyapatite H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of approximately 1 0 pfu per c e l l and incubated from h.5 to 2 6 hours a f t e r i n f e c t i o n w i t h KO uCi/ml HdThd. V i r u s was p u r i f i e d and DNA e x t r a c t e d as described i n the legend to f i g u r e 9? except t h a t precautions were taken to avoid shear breakage. The DNA s o l u t i o n was then d i l u t e d t o 0 . 0 3 M Na + and heated i n a b o i l i n g water bath f o r 1 0 minutes. The de-natured DNA was adsorbed to hydroxyapatite and e l u t e d as described i n the legend to f i g u r e 9> 92 Fig. 10: FRACTION NUMBER 93 F i g . 11: V e l o c i t y sedimentation of MCV-DNA ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one p f u per c e l l and incubated from 20 to 36 hours a f t e r i n f e c t i o n w i t h 50 uCi/ml ^HdThd (a) or w i t h 1.6 uCi/ml l [ |CdThd (b). V i r i o n s were p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n , 32 DNA was e x t r a c t e d from mixtures of MCV and P - l a b e l l e d T*+ v i r i o n s , and analyzed on n e u t r a l sucrose gradients as described i n Methods, except t h a t i n the case of (b) c e n t r i f u g a t i o n was f o r 8 hours at 25,000 rpm and 36-drop f r a c t i o n s were c o l l e c t e d . D i r e c t i o n of sedimentation i s to the l e f t . (a) JH - MCV DNA 1.2 x 10 cpm/pg DNA 3 2 P - Th DNA - MCV DNA 72,000 cpm/ng DNA - Th DNA FRACTION NUMBER C a l c u l a t i o n s were made from seventeen determinations, u s i n g t e n d i f f e r e n t p r eparations of MCV l a b e l l e d w i t h e i t h e r CdThd or ~HdThd and an average sedimentation c o e f f i c i e n t of 6 l . 5 t 1 .Is obtained f o r MCV DNA, r e l a t i v e t o T4-DNA as 57.5s ( L e v i n and Hutchinson, 1973). I f the genome of MCV i s a l i n e a r , duplex DNA molecule, t h i s c o r r e s -ponds to a molecular weight of 132 t 5 x 10^, r e l a t i v e to T4-DNA as 110 x 10°" ( F r e i f e l d e r , 1970). Recovery of input DNA from n e u t r a l or a l k a l i n e g r a d i e n t s , where checked, was grea t e r than 90$. N e u t r a l pH. Herpes simplex v i r u s The DNA from HSV-P was als o cosedimented w i t h MCV-DNA, and from the r e l a t i v e sedimentation c o e f f i c i e n t s obtained from s i x determina-M M C V t i o n s (e.g. f i g . 12) , the r a t i o - was found to be 1.58 t O.Oh, ^SV-P u s i n g the r e l a t i o n s h i p of F r e i f e l d e r as above. R e v e r s a l of the l a b e l s ("^ C and 3H) d i d not a l t e r the r e s u l t s . MT4 Cosedimentation of HSV-P-DNA w i t h T4-DNA showed the r a t i o 6 £ s v t o be 1.25. Assuming molecular weights of 110 x 10 and 132 x 10° f o r Tk and MCV-DNA's r e s p e c t i v e l y , a value of 85 t 2. 5 x 10°" was c a l c u l a t e d f o r the molecular weight of HSV-P-DNA. This value i s i n good agree-ment w i t h the value of 88 t 13.5 x 10^, determined by 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 of f o u r s t r a i n s of HSV (Graham et a l , 1972; Ludwig, 1972), but does not agree w i t h the value of 99 — 5 x 10^, determined by cosedimentation of HSV-F and HSV-G-DNA w i t h T4-DNA ( K i e f f et a l , Fig. 12: 12: V e l o c i t y sedimentation of HSV-P and MCV-DNA ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one pfu per c e l l , and incubated from 2k to 36 hours a f t e r / Ill-i n f e c t i o n w i t h 2 pCi/ml CdThd. H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of lUcrpfu per c e l l and incubated from k t o 20 hours a f t e r i n f e c t i o n w i t h 20 uCi/ml ^dThd. DNA was e x t r a c t e d from a mixture of the p u r i f i e d v i r i o n s and analyzed on sucrose gradients as described i n Methods. D i r e c t i o n of sedimentation i s to the l e f t . MCV - DNA s p e c i f i c a c t i v i t y 33,000 cpm/ug HSV - DNA s p e c i f i c a c t i v i t y 39,000 cpm/ug 98 1971). To check the p o s s i b i l i t y of molecular weight d i f f e r e n c e s between s t r a i n s , the F s t r a i n of HSV was obtained from Dr. B. Roizman. Under c o n d i t i o n s of low DNA conce n t r a t i o n ( 0 . 0 2 to 0 . 3 Mg t o t a l DNA per gradient) we determined, by co-sedimentation of HSV-P wi t h ik-DNA's and HSV-F w i t h MCV-DNA's, a value of 87 t 1.5 x 10 (3 d e t e r -minations) f o r the molecular weight of HSV-F-DNA. N e u t r a l pH. Human cytomegalovirus Although d i f f i c u l t i e s were encountered i n growing and l a b e l l i n g s u f f i c i e n t q u a n t i t i e s of HCV, sucrose gradient analyses were c a r r i e d out on s e v e r a l preparations. The q u a n t i t i e s of HCV-DNA added t o the gradients were not known, but the shape of the ik-DNA peak was used as an i n d i c a t i o n of p o s s i b l e h i g h concentrations of HCV-DNA. The r e s u l t i n g g r a d i e n t p r o f i l e s were compared w i t h the r e s u l t s of Le v i n and Hutchinson (1973a), who cosedimented T2-DNA and very l a r g e B a c i l l u s s u b t i l i s DNA on n e u t r a l sucrose g r a d i e n t s . The d i s t o r t i o n seen i n the HCV + T4-DNA p r o f i l e was l e s s than t h a t seen by Le v i n and Hutchinson (1973a) u s i n g very l a r g e DNA at a con c e n t r a t i o n of 0 . 2 ug/ml. Since HCV-DNA. sedimented more s l o w l y than the DNA i n Le v i n and Hutchinson's experiments, i t i s probable t h a t l e s s than 0 . 2 ug/ml ( 0 . 0 4 ug per gradient) HCV-DNA was present on the gradients i n t h i s study. A l l g radients contained a v a r i a b l e amount of l a b e l l e d m a t e r i a l Fig. 13: FRACTION NUMBER F i g . 13: V e l o c i t y Sedimentation of HCV-DNA WI38 c e l l s were i n f e c t e d w i t h HCV, and 50 uCi/ml ^HdThd was added to the c u l t u r e s at 2k and kO hours a f t e r i n f e c t i o n . Some c u l t u r e s were harvested at 3 days a f t e r i n f e c t i o n , and the r e s t at 7 days a f t e r i n f e c t i o n . C e l l s i n d i s t i l l e d water, and the supernatant medium, were f r o z e n , thawed, and p u r i f i e d by one c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n , DNase treatment, and a second c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n as described i n the M a t e r i a l s and Methods chapter. An a l i q u o t of the f i n a l p r e p a r a t i o n , c o r r e s -ponding to the v i r u s from approximately 2 x 10°" c e l l s , was mixed 32 w i t h P - l a b e l l e d Tk, and the DNA e x t r a c t e d and analyzed on a n e u t r a l sucrose gradient as described i n the M a t e r i a l s and Methods chapter, except that c e n t r i f u g a t i o n was c a r r i e d out at 22,500 rpm. D i r e c t i o n of sedimentation i s to the l e f t . remaining in the top few fractions of the gradient, and a band of HCV-DNA sedimenting considerably faster than T*+-DNA (e.g. figure 13). Some gradients also contained material sedimenting in a very broad band centred at approximately the position of T*+-DNA. The fast-sedimenting band had a sedimentation coefficient of 73-3 "t 0.8s (six determinations) i f centrifugation was carried out at 26,000 rpm or 27,000 rpm. However, i f the DNA was centrifuged at 22,500 rpm, the fastest-sedimenting band had a sedimentation coefficient of 79-1 t 1.3s (three determinations). Sedimentation coefficients were calculated relative to cosedimented TU-DNA as 57.5s (Levin and Hutchinson, 1973). Although these results could not be satisfactorily interpreted at the time, the recent results of Levin and Hutchinson (1973a) show that the speed of centrifugation has a marked effect on the sedimentation coefficients of very large DNA molecules. The equat-ions derived to explain these data are fitted to the conditions of centrifugation through 5 to 20$ sucrose gradients in a SW 50.1 rotor. However, the parameters involved have similar values during centrifugation through 10 to 30$ sucrose in the SW 27-1 rotor, and so the equations should hold reasonably well for the results described here. Using these equations and the two sets of sediment-ation coefficients, the molecular weight of HCV-DNA was calculated as 3lh x 10 6 . In an attempt to determine whether the HCV-DNA molecule was extremely large, or had an unusually compact (e.g. circular) structure, a mixture of intact HCV-DNA and T4-DNA was passed through an 18 gauge needle five times, which was expected to shear most but not a l l of the TU-DNA molecules. Results of sedimentation analysis of this material are shown in figure ik. Before shearing, this DNA preparation showed single peaks for both HCV and T^ -DNA's with an apparent sedimentation coefficient of 73-7 for HCV-DNA. After shearing, most of the T4-DNA sedimented at the rate expected for half molecules (the centre of the peak corresponded to a molecular weight of 56 x 10 )^, and a small amount sedimented at the velocity characteristic of intact molecules. The sheared HCV-DNA sedimented as a broad peak in the region of the sheared T4-DNA molecules, the centre of the peak corres-ponding to a molecular weight of 53 x 10 .^ Alkaline pH. Murine cytomegalovirus Alkali-denatured MCV and TU-DNA's were cosedimented on alkaline sucrose gradients (Fig. 15). In contrast to the single peak seen on neutral gradients, denatured MCV-DNA gave a heterogeneous pattern of peaks, suggesting that our preparations of MCV-DNA contained single-strand breaks. Similar patterns have also been seen with three other herpesviruses, Marek's disease virus (Lee et al, 1971), HSV (Kieff et al, 1971) and EB virus (Nonoyama and Pagano, 1971). This pattern was F i g . lh: F i g . Ik: V e l o c i t y Sedimentation of Sheared HCV-DNA WI38 c e l l s were i n f e c t e d w i t h HCV, and 20 uCi/ml HdThd was added t o the c u l t u r e s every 2k hours f o r 6 days a f t e r i n f e c t i o n . I n f e c t e d c e l l s and supernatant medium were harvested s e p a r a t e l y at 8 days a f t e r i n f e c t i o n , and p u r i f i e d by two c y c l e s of d i f f e r -e n t i a l c e n t r i f u g a t i o n . An a l i q u o t of the f i n a l p r e p a r a t i o n d e r i v e d from the supernatant o n l y of 1.5 x 10^ i n f e c t e d c e l l s , was mixed w i t h 3 2 P - l a b e l l e d ik, and the DNA ex t r a c t e d . H a l f of t h i s DNA was analyzed on a n e u t r a l sucrose gradient, and the other h a l f was passed f i v e times through an l8-gauge needle, and analyzed on a second n e u t r a l sucrose gradient as descibred i n the M a t e r i a l s and Methods chapter, except t h a t c e n t r i f u g a t i o n was f o r 6 . 5 hours. The p r o f i l e of the sheared DNA i s shown. D i r e c t i o n of sedimentation i s to the l e f t . Fig. 15: A l k a l i n e v e l o c i t y sedimentation of MCV-DNA The same p r e p a r a t i o n of MCV as i n F i g . 12 was used. lU DNA was e x t r a c t e d from a mixture of p u r i f i e d C-32 MCV and F-lk v i r i o n s , denatured and analyzed on a l k a l i n e sucrose gradients as described i n Methods. D i r e c t i o n of sedimentation i s to the l e f t . Ik / C-MCV - DNA s p e c i f i c a c t i v i t y 33,000 cpm/ug 3 2P-T4 - DNA 107 Q lU seen w i t h both H and C - l a b e l l e d DNA. Using the r e s u l t of Rosenthal and Fox (1970) i t was found by c a l c u l a t i o n that the amount of r a d i a t i o n -induced breakage would be f a r l e s s than the a c t u a l breakage seen. A l s o , the amount of breakage d i d not depend on the s p e c i f i c a c t i v i t y of the DNA. DNase treatment was not c a r r i e d out during the p u r i f i c a t i o n o f v i r u s preparations used i n t h i s s e c t i o n . The f a s t e s t - s e d i m e n t i n g component of denatured MCV-DNA was a l s o the major component, and by comparison w i t h cosedimented denatured TU-DNA, the s i n g l e - s t r a n d e d molecular weight was c a l c u l a t e d as 61.7 1 0.6 x 10^ (12 determinations), u s i n g the r e l a t i o n s h i p of Studier (1965): -S° = 0.0528 M 0 ^ 0 0 20.w and hence * 1 = f l = W °- k 0° d2 S2 (M2) When the i n t e g r a l of the MCV-DNA p r o f i l e i n f i g u r e 15 was p l o t t e d , i t was found t h a t approximately 56$ of the DNA was i n the major peak. On other gradients the f r a c t i o n of the DNA i n t h i s peak was o f t e n lower. To determine whether or not the amount of s i n g l e - s t r a n d breaks was dependent on the p u r i f i c a t i o n procedure, one p r e p a r a t i o n of MCV was prepared w i t h a d d i t i o n a l precautions to preserve v i r i o n i n t e g r i t y . The normal p u r i f i c a t i o n method was f o l l o w e d except t h a t a l l operations were performed at 0°C, and i n the presence of 10$ f e t a l c a l f serum. DNA was e x t r a c t e d from a mixture of MCV and ik v i r i o n s , and analyzed on an a l k a l i n e sucrose gradient as usual. 6 5 $ of the t o t a l MCV DNA was found i n the major peak, at a p o s i t i o n corresponding t o a molecular weight of 62 x .10^. A l k a l i n e pH. Herpes simplex v i r u s A l k a l i - d e n a t u r e d HSV and T^-DNA's were cosedimented on a l k a l i n e sucrose g r a d i e n t s . HSV-DNA showed a fragmented p r o f i l e ( f i g . .16) s i m i l a r t o th a t of denatured MCV-DNA. The fa s t e s t - s e d i m e n t i n g major component, compared t o cosedimented T4-DNA, had an apparent s i n g l e -stranded molecular weight of kk.8 ± 0 . 7 x 10°" (three determinations). Although the amount of fragmentation seen i n denatured HSV-P DNA was much l e s s than t h a t reported f o r HSV-F and HSV-G ( K i e f f et a l , 1 9 7 1 ) , the DNA i n the major peak d i d not exceed 50$ i n any of these three p r o f i l e s . A l k a l i n e pH. Human cytomegalovirus Denatured HCV-DNA sedimented on a l k a l i n e sucrose g r a d i e n t s i n a ver y heterogeneous p a t t e r n . From f o u r experiments, the sedimentation v e l o c i t y of the f a s t e s t - s e d i m e n t i n g component corresponded to a .6 s i n g l e - s t r a n d e d molecular weight of .158 t 5 x 10"', compared to 55 x 6 10 f o r cosedimented T4-DNA. The l e a d i n g component was d i s t i n c t but very s m a l l on a l l f o u r g r a d i e n t s , and the r e s t of the DNA was spread 16: A l k a l i n e v e l o c i t y sedimentation of HSV-DNA H.Ep.2 c e l l s were i n f e c t e d w i t h HSV and incubated from k.5 to 2 0 hours a f t e r i n f e c t i o n w i t h 5 pCi/ml 3HdThd. V i r i o n s were p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n , op mixed w i t h P-T*+ v i r i o n s , and DNA e x t r a c t e d as described i n M a t e r i a l s and Methods. The DNA s o l u t i o n was denatured and analyzed on an a l k a l i n e sucrose gradient. D i r e c t i o n of sedimentation i s to the l e f t . 3 H-HSV-DNA s p e c i f i c a c t i v i t y 6 , 0 0 0 cpm/ug 32P-T*+-DNA over the whole of the upper portion of the gradient, starting immediately behind the leading peak. Since the specific activity of the DNA was not known, radioactive disintegration cannot be ruled out as a cause of some or a l l fragmentation. The sedimentation co-efficient of the fastest-sedimenting component of denatured HCV-DNA was calculated as 111.1s, relative to T4-DNA as 72.7s (Studier, 1965)• E. Cesium Chloride Gradient Equilibrium Centrifugation Although Plummer et al (1969) reported that MCV-DNA showed two reproducible components on centrifugation to equilibrium in CsCl solution in an analytical ultracentrifuge, the present study showed that the DNA was homogeneous on velocity sedimentation. To determine whether the population of molecules contained two different classes of eq[ual size but different density, or whether the population con-sisted of identical molecules with internal heterogeneity, intact and artificially sheared radioactive MCV-DNA were centrifuged to equilibrium in preparative CsCl gradients. Some results are shown in figure 17. Intact MCV-DNA yielded a sharp single peak, while sheared MCV-DNA gave at least two, and occasionally more peaks close together. The pattern of sheared DNA peaks was not reproducible from one experiment to another. HSV-P or TU-DNA present in the same gradients as markers, showed single, sharp peaks for intact DNA, and 112 F i g . 17: P r e p a r a t i v e CsCl gradient c e n t r i f u g a t i o n of MCV-DNA (a) ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one p f u per c e l l and incubated from 20 to 36 hours a f t e r i n f e c t i o n w i t h 1.6 (iCi/ml "^CdThd. H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of Ik pfu per c e l l and incubated from h to 20 hours a f t e r i n f e c t i o n w i t h 10 uCi/ml ^HdThd. I n t a c t DNA was e x t r a c t e d from a mixture of the two p u r i f i e d v i r i o n p r e p a r a t i o n s , and analyzed on a 1 ml p r e p a r a t i v e C s C l gradient as described i n M a t e r i a l s and Methods. The bottom of the tube i s to the l e f t . l l +C - MCV - DNA s p e c i f i c a c t i v i t y 118,000 cpm/ug 3H - HSV - DNA s p e c i f i c a c t i v i t y 82,000 cpm/ug (b) ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one p f u per c e l l and incubated from 2h to 36 hours a f t e r i n f e c t i o n w i t h 2 uCi/ml CdThd. H.Ep.2 c e l l s were i n f e c t e d w i t h HSV at a m u l t i p l i c i t y of 15 p f u per c e l l and incubated from h to 20 hours a f t e r i n f e c t i o n w i t h 20 uCi/ml 3HdThd. I n t a c t DNA was e x t r a c t e d from a mixture of the two p u r i f i e d v i r i o n p r e p a r a t i o n s , sheared by passing f i v e times through a 22-gauge needle, and analyzed on a 1 ml p r e p a r a t i v e C s C l gradient as described i n M a t e r i a l s and Methods. The bottom of the tube i s to the l e f t . DNA s p e c i f i c a c t i v i t y 33,000 cpm/ug DNA s p e c i f i c a c t i v i t y 39,000 cpm/ug ih C - MCV JE - HSV F i g . 17: Ilk s i n g l e , broader peaks f o r sheared DNA. The width of the marker DNA peak was always l e s s than the t o t a l width of the MCV-DNA peaks i n the case of sheared DNA. Since the separation of the MCV-DNA peaks (approximately 0.00*+ g/cc) was clos e to the l i m i t of r e s o l u t i o n of p r e p a r a t i v e c e n t r i f u g a t i o n under our c o n d i t i o n s , t h i s was i n v e s t i g a t e d f u r t h e r u s i n g 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 . The a d d i t i o n a l steps of DNase treatment and r e - p e l l e t t i n g were used t o prepare MCV preparations that d i d not co n t a i n detectable contamination w i t h host DNA. The u l t r a v i o l e t absorbance p r o f i l e s of i n t a c t and sheared MCV-DNA are shown i n f i g u r e 18, and the sedimentation p r o f i l e s of the sheared DNA samples, on n e u t r a l sucrose g r a d i e n t s , are shown i n f i g u r e 19. The d e n s i t i e s and s i z e s of fragments of MCV-DNA are summarized i n Table 3- D e n s i t i e s were c a l c u l a t e d according to Mandel et a l (1968), from the d e n s i t y of T*+-DNA as 1.7005 g/cc ( S z y b a l s k i , I968) and HSV-DNA as 1.726 g/cc ( K i e f f et a l , 1971). HSV-P and HSV-F DNA have been c e n t r i f u g e d to e q u i l i b r i u m i n the same CsCl gradients i n our l a b o r a t o r y and found to d i f f e r i n d e n s i t y by l e s s than 0.001 g/cc. I n t a c t MCV-DNA banded at a d e n s i t y of 1.7178 (three determinations), corresponding to a G + C content of 59$- "Half-molecules" apparently banded at t h i s same d e n s i t y whereas "quarter molecules" or smaller fragments banded i n two regions of d e n s i t i e s I.7165 and 1.7200, corresponding t o G + C contents of 57-5$ and 6l. 5$ r e s p e c t i v e l y . F i g . 18: A n a l y t i c a l C s C l c e n t r i f u g a t i o n ME c e l l s were i n f e c t e d w i t h MCV at a m u l t i p l i c i t y of one pfu per c e l l , and incubated from l 6 to 36 hours a f t e r i n f e c t i o n w i t h / li+ 0 . 0 2 uCi/ml CdThd. H.Ep.2 c e l l s were i n f e c t e d w i t h HSV-P at a m u l t i p l i c i t y of two pfu per c e l l and harvested at 2h hours a f t e r i n f e c t i o n . HSV-P and MCV v i r i o n s were p u r i f i e d as described i n M a t e r i a l s and Methods f o r a n a l y t i c a l CsCl c e n t r i f u g a t i o n experiments. I n t a c t DNA was e x t r a c t e d from MCV, HSV-P and ik v i r i o n s , sheared as d e t a i l e d below, and c e n t r i f u g e d to e q u i l i b r i u m i n CsCl g r a d i e n t s . U.V. absorbance p r o f i l e s are shown. The meniscus i s to the r i g h t i n a l l p r o f i l e s . a) I n t a c t HSV-P, MCV and T^-DNA. 3 0 , 0 0 0 rpm b) Sheared HSV-P, MCV and TU-DNA. 18 gauge needle, kk,000 rpm c) I n t a c t T*+-DNA, sheared MCV and HSV-P DNA. 20 gauge needle. 3 0 , 0 0 0 rpm d) I n t a c t T*4- and HSV-P DNA, sheared MCV-DNA. 21 gauge needle. 3 0 , 0 0 0 rpm 118 F i g . 19: Sedimentation a n a l y s i s of sheared MCV-DNA Each sheared DNA p r e p a r a t i o n used to o b t a i n the r e s u l t s shown i n f i g u r e 18 was a l s o analyzed f o r fragment s i z e . Samples were c e n t r i f u g e d through 5 ml 10-30$ (w/w) sucrose gradients i n n e u t r a l DNA b u f f e r , i n a SW 50.1 r o t o r at k2,000 rpm and 20°C f o r 2 .5 hours, i n a Beckman L2-65 B u l t r a c e n t r i -fuge. F i f t e e n drop f r a c t i o n s were c o l l e c t e d from the bottom. D i r e c t i o n of sedimentation i s to the l e f t . a) Sheared, 18 G needle, corresponds to f i g . l8b b) Sheared, 20 G needle, corresponds to f i g . l8c c) Sheared, 21 G needle, corresponds to f i g . l8d The v e r t i c a l arrow i n d i c a t e s the p o s i t i o n of the marker 32 i n t a c t P-T4-DNA sedimented on the same grad i e n t s . Table 3: D e n s i t i e s of Fragmented MCV-DMA Treatment of D e n s i t i e s of bands Predominant s i z e c l a s s i n t a c t DNA on CsCl gradient (approx. m.w.) None 1 . 7 1 7 8 1 3 . 2 x 1 0 7 1 . 7 1 8 0 8 x 1 0 7 •7 1 . 7 1 6 7 h x 1 0 ' Sheared 18 G- needle Sheared 20 G needle 1 . 7 1 8 5 1 . 7 2 0 3 Sheared 1 . 7 1 6 3 2 x 1 0 7 2 1 G needle 1 . 7 2 0 0 Although the p a t t e r n of bands seen at kk,000 rpm or 30,000 rpm was the same, the greater separation at 30,000 rpm allowed greater r e s o l u t i o n of the bands by the scanning densitometer. Bands obtained w i t h HSV-P-DNA or T^-DNA were always s i n g l e , whether the DNA was i n t a c t or sheared (e.g. see f i g u r e l 8 c ) . F. U l t r a v i o l e t Absorbance/Temperature P r o f i l e s Although the experiments described above showed t h a t the MCV-DNA molecule was heterogeneous i n dens i t y , t h i s could conceivably have been due to reasons other than heterogeneity i n base composition. The m e l t i n g p r o f i l e of MCV-DNA was obtained to serve as an independent measure of the degree of base-composition heterogeneity. M e l t i n g p r o f i l e s were obtained as described i n the M a t e r i a l s and Methods chapter f o r MCV and HSV-DNA's. Representative p r o f i l e s are shown i n f i g u r e 20. Herpes simplex v i r u s DNA melted over a r e l a t i v e l y short range. However, MCV-DNA showed marked heterogeneity, i n the p r o f i l e , and at l e a s t two and p o s s i b l y three components could be d i s t i n g u i s h e d i n f o u r separate p r o f i l e s obtained. By comparison w i t h the m e l t i n g temperature of HSV-DNA, the estimated m e l t i n g temperatures of the MCV-DNA components were c a l c u l a t e d to correspond to approximate G + C contents of 63$, •57$ and k6%, from the equation:-% GC = (Tm - 53-9)2.kk (Mandel and Marmur, 1968b), Fig. 20: TEMPERATURE (°C) 2 0 : M e l t i n g p r o f i l e of MCV and HSV-DNA P u r i f i e d MCV and HSV-DNA's were prepared by procedure 1 as described i n the M a t e r i a l s and Methods s e c t i o n . The f i n a l preparations were d i s s o l v e d i n 0 . 1 x SSC, and both d i a l y z e d against 0 . 1 x SSC i n the same beaker f o r two days. M e l t i n g p r o f i l e s were obtained f o r the two DNA samples, and absorbances were c o r r e c t e d f o r the thermal expansion of the solvent. Results are p l o t t e d as the t o t a l (a) or incremental (b) percentage of absorbance increase, compared to the absorbance at 25°C as 1 0 0 $ . MCV-DNA HSV-DNA and assuming t h a t a maximum i n the incremental p l o t ( f i g u r e 20b) corresponded t o the mid-point of an absorbance r i s e . I n the case of HSV-DNA, t h i s was c o r r e c t . The average m e l t i n g temperature of MCV-DNA was obtained from the equation: T = <T. A. average 1 1 ^ t o t a l where T i s the average m e l t i n g temperature, A. i s the increment average - D ' 1 of absorbance increase per °C at a temperature T., and Aj_ , -, i s the 1' t o t a l t o t a l increase i n absorbance. The value obtained was c a l c u l a t e d to correspond t o a G + C content of 5 9 $ 5 i n agreement w i t h the average G + C content ( 5 9 $ ) c a l c u l a t e d from the d e n s i t y of i n t a c t MCV-DNA i n CsCl g r a d i e n t s . G. T r a n s c r i p t i o n of MCV-DNA The se p a r a t i o n of MCV-DNA i n t o two components on cesium c h l o r i d e gradients provided a u s e f u l t o o l w i t h which to i n v e s t i g a t e the t r a n s -c r i p t i o n of MCV-DNA. Herpes simplex v i r u s ( F r e n k e l and Roizman, 1972b; Wagner, 1 9 7 2 ) , and p o s s i b l y equine a b o r t i o n v i r u s (Huang et a l , 1971) show c o n t r o l of v i r a l t r a n s c r i p t i o n during i n f e c t i o n , and so the t r a n s c r i p t i o n of the two components of MCV was i n v e s t i g a t e d f o r two purposes: 1. To determine whether or not both d e n s i t y components were t r a n s c r i b e d during i n f e c t i o n . 2. To i n v e s t i g a t e the r e l a t i v e amounts of RNA made from each component at d i f f e r e n t times a f t e r i n f e c t i o n . Ik A p r e p a r a t i o n of G - l a b e l l e d MCV-DNA was p u r i f i e d , sheared, and banded at e q u i l i b r i u m on a p r e p a r a t i v e cesium c h l o r i d e gradient ( f i g . 2 1 ) . The average s i z e of the fragments i n t h i s DNA p r e p a r a t i o n was ±8 x 10°" daltons, as determined by sucrose gradient v e l o c i t y sediment-a t i o n . F r a c t i o n s from two cesium c h l o r i d e gradients were combined i n t o two pools of 'dense' and ' l i g h t ' DNA, u s i n g the mid-point between the two peaks as the sep a r a t i o n point. Assuming t h a t the p r o f i l e was made up of two symmetrical overlapping peaks, the best f i t t h a t could be obtained e m p i r i c a l l y , suggested t h a t contamination of each component by the other was 5 to 1 5 $ . The two DNA components were t e s t e d i n h y b r i d i z a t i o n r e a c t i o n s , w i t h RNA from MCV-infected c e l l s l a b e l l e d w i t h H u r i d i n e at d i f f e r e n t times a f t e r i n f e c t i o n . F i g u r e 22 shows the r e s u l t s of one h y b r i d -i z a t i o n experiment. E s s e n t i a l l y the same r e s u l t s were obtained when the h y b r i d i z a t i o n t e s t s were repeated on f u r t h e r a l i q u o t s of the same n u c l e i c a c i d preparations. From the r e l a t i v e amounts of RNA b i n d i n g t o each DNA component, i t appears that:some sequences of both DNA components are t r a n s c r i b e d at l e a s t at some stage during i n f e c t i o n , 21: Separation of the two d e n s i t y components of MCV-DM Mouse embryo c e l l s were i n f e c t e d w i t h MCV at a m u l t i -p l i c i t y of k pfu per c e l l and incubated from 12 to 36 hours a f t e r i n f e c t i o n w i t h 0 . 0 1 uCi/ml "^CdThd. The v i r u s p r e p a r a t i o n was p u r i f i e d by three c y c l e s of d i f f e r e n t i a l c e n t r i f u g a t i o n , and i n t a c t DM was e x t r a c t e d as described i n the M a t e r i a l s and Methods chapter, method 2 . The DM s o l u t i o n was passed f i v e times through a 2 1 G needle, and c e n t r i f u g e d to e q u i l i b r i u m i n a 1 .6 ml e'esium c h l o r i d e g r a d i e n t , u s i n g the D e l r i n adaptors f o r the Spinco type 50 r o t o r . C e n t r i f u g a t i o n was c a r r i e d out at 20 C and 3 0 , 0 0 0 rpm f o r &k hours. Three-drop f r a c t i o n s were c o l l e c t e d from the bottom, 25 u l Tris-EDTA b u f f e r were added t o each f r a c t i o n , and a 5 M-1 a l i q u o t of each f r a c t i o n was taken f o r r a d i o a c t i v i t y measurement i n s c i n t i l l a t i o n f l u i d B. g. 21: loon 5 OH F R A C T I O N N U M B E R Fig. 22: 12 24 TIME A F T E R INFECTION (hours) F i g . 2 2 : T r a n s c r i p t i o n of the two d e n s i t y components of MCV-DNA The C - l a b e l l e d DNA f r a c t i o n s shown i n f i g . 2 1 were combined i n t o two pools of ' l i g h t ' and 'dense' DNA. A f t e r p r e c i p i t a t i o n from the cesium c h l o r i d e s o l u t i o n w i t h ethanol, the two f r a c t i o n s of DNA were d i s s o l v e d i n 0 . 1 x SSC, and the absorbance at 2 6 0 nm measured. A l i q u o t s of each DNA f r a c t i o n were denatured and bound to M i l l i p o r e f i l t e r s as described i n the M a t e r i a l s and Methods chapter, such t h a t each s m a l l 6 . 5 mm d i s c contained 0 . 0 3 M-g DNA. C o n t r o l f i l t e r d i s c s were prepared, each c o n t a i n i n g 0 . 0 3 M-g HSV-DNA. S p e c i f i c a c t i v i t i e s of the MCV-DNA components were 2 , 3 0 0 and 2 , 2 0 0 cpm/ug DNA f o r dense and l i g h t components r e s p e c t i v e l y . Mouse embryo c e l l s were i n f e c t e d c e n t r i f u g a l l y w i t h MCV at a m u l t i p l i c i t y of 5 p f u per c e l l . At d i f f e r e n t times a f t e r i n f e c t i o n , 100 pCi 3 n-uridine/ml was added to one sm a l l p e t r i d i s h . Four hours a f t e r the a d d i t i o n of the l a b e l , the medium was discarded, and RNA ex t r a c t e d from the c e l l s . At the same time, the i n f e c t e d c e l l s from another p e t r i d i s h were harvested f o r i n f e c t i v i t y t i t r a t i o n s . Con-t r o l RNA preparations were obtained from mock i n f e c t e d mouse embryo c e l l s l a b e l l e d as above. The RNA preparations were p u r i f i e d and t e s t e d i n DNA-RNA h y b r i d i z a t i o n r e a c t i o n s w i t h the X ^ C - l a b e l l e d DNA f r a c t i o n s described above Each r e a c t i o n v i a l contained: -One RNA sample ( 2 0 ng) Two d i s c s c o n t a i n i n g 'dense' MCV-DNA Two d i s c s c o n t a i n i n g ' l i g h t ' MCV-DNA Two d i s c s c o n t a i n i n g HSV-DNA The r a d i o a c t i v i t y bound to the HSV-DNA f i l t e r s ( l e s s than 5 cpm above machine background) was subtracted from the counts obtained from the other f i l t e r s i n the same v i a l , and the r e s u l t s expressed as the r a t i o of RNA: DNA present on the f i l t e r , i n terms of r a d i o -a c t i v i t y . (Both 3fj and l i +C were measured). The times shown f o r the RNA preparations r e f e r to the time of ha r v e s t i n g . The zero-time sample was obtained from mock-infected c e l l s . I n f e c t i v i t y t i t r a t i o n s were c a r r i e d out i n mouse embryo c e l l s , u s i n g c e n t r i f u g a l i n o c u l a t i o n . a) 'dense' DNA (A) ,' l i g h t ' DNA (B) b) RNA bound to A RNA bound t o B l o g i n P e r c u l t u r e (approximately 10^ c e l l s ) F i g . 22: (continued) Background counts from HSV-DNA f i l t e r s were l e s s than 10 cpm ( i n c l u d i n g machine background). H y b r i d i z a t i o n values ranged from 30 to 130 cpm per f i l t e r d i s c . The v e r t i c a l bars show the se p a r a t i o n of the two d u p l i c a t e s . The times shown f o r the RNA preparations r e f e r t o the time of h a r v e s t i n g . The zero-time sample was obtained from mock-infected c e l l s . I n f e c t i v i t y t i t r a t i o n s were c a r r i e d out i n mouse embryo c e l l s , u s i n g c e n t r i f u g a l i n o c u l a t i o n . a) 'dense' DNA (A) ' l i g h t ' DNA (B) b) RNA bound to A RNA bound t o B • ' • l 0 § - i n ^ u £ e r c u l " t u r e (approximately 1 0 ° " c e l l s ) and the s l i g h t v a r i a t i o n i n the r a t i o of the amounts of RNA bound to each DNA component during i n f e c t i o n , suggests t h a t there may be some c o n t r o l over the synthesis or breakdown of s p e c i f i c v i r a l RNA sequences. CHAPTER 5: RESULTS I I I Non-Productive I n f e c t i o n s During the l a t e n t i n f e c t i o n s c h a r a c t e r i s t i c of s e v e r a l herpes-v i r u s e s i n t h e i r n a t u r a l hosts, the nature of the bl o c k i n v i r a l r e p l i c a t i o n , and the causes of a c t i v a t i o n or r e p r e s s i o n , are not known. A s u i t a b l e model system of l a t e n t i n f e c t i o n i n t i s s u e c u l t u r e would be extremely u s e f u l i n studying l a t e n t i n f e c t i o n s , and so i n t h i s study s e v e r a l attempts were made t o e s t a b l i s h a l a t e n t i n f e c t i o n i . e . p e r s i s t e n c e of v i r a l g e netic i n f o r m a t i o n without c e l l death or p roduction of i n f e c t i o u s v i r u s . A. Herpes Simplex V i r u s . A r g i n i n e D e p r i v a t i o n D e p r i v a t i o n of a r g i n i n e has been reporte d t o i n h i b i t the r e p l i c a t i o n of HSV i n continuous l i n e c e l l s ( i n g l i s , 1968). This was confirmed f o r HSV-P i n H.Ep.2 c e l l s . HSV-P was grown i n H.Ep.2 c e l l s i n normal growth medium, and i n H.Ep.2 c e l l s which were deprived of a r g i n i n e f o r 30 hours before i n f e c t i o n and during i n f e c t i o n . A l l c e l l s were harvested at 2k hours a f t e r i n f e c t i o n , and the v i r u s t i t r e of the a r g i n i n e - d e p r i v e d p r e p a r a t i o n was k.6 l o g lower than t h a t o f the normal l y s a t e . However, H.Ep.2 c e l l s maintained i n the absence of a r g i n i n e stopped growing, changed i n morphology, and e v e n t u a l l y died. I n c r e a s -i n g amounts of serum were added t o the a r g i n i n e - d e f i c i e n t medium i n an attempt t o f i n d c o n d i t i o n s i n which HSV would not r e p l i c a t e , but i n f e c t e d c e l l s would s u r v i v e . These could not be obtained. High serum concentrations l e d t o c h a r a c t e r i s t i c v i r a l c y t o p a t h i c e f f e c t and c e l l death, and low serum concentrations caused c e l l death due to a r g i n i n e d e p r i v a t i o n . Since the a r r e s t e d i n f e c t i o n of dying c e l l s c o uld not be considered as a model system f o r i n v i v o l a t e n c y , t h i s was not i n v e s t i g a t e d f u r t h e r . B. Host Range Seve r a l c e l l types were i n f e c t e d w i t h HSV and MCV i n an attempt to f i n d a c e l l type i n which the v i r u s would p e r s i s t without r e p l i -c a t i o n or c e l l death. HSV was l e t h a l f o r a l l c e l l s t e s t e d . S e v e r a l mouse c e l l types were i n f e c t e d w i t h MCV, since a non-productive i n f e c t i o n of MCV i n c e l l s d e rived from the n a t u r a l host would be an i d e a l model system. Mouse embyro c e l l s were i n f e c t e d at low m u l t i p l i c i t y , and i n f e c t i o n allowed to spread through the mono-l a y e r , w i t h repeated medium changes. C e l l death proceeded to completion ( l e s s than one i n 10 c e l l s s u r v i v e d ) . Two continuous l i n e s of mouse c e l l s , 3T3 and MKSA (SVUO-transformed mouse kidney) were al s o i n f e c t e d w i t h MCV, r e s u l t i n g i n complete spread of i n f e c t i o n and c e l l death. Mouse f i b r o b l a s t (L.929) c e l l s , i n f e c t e d at high m u l t i p l i c i t y , showed an i n i t i a l p e r i o d of c e l l death, f o l l o w e d by r e -growth of l a r g e r , more e p i t h e l i a l - l i k e c e l l s , and a second c y c l e of c e l l death e i g h t days a f t e r i n f e c t i o n . I n f e c t i o u s MCV was detected i n the supernatant of the c u l t u r e at a l l times a f t e r i n f e c t i o n , and the t i t r e rose during the c e l l death phase. This behaviour i s s i m i l a r to the c y c l i c v i r u s r e p l i c a t i o n and c e l l regrowth seen a f t e r i n f e c t i o n of L - c e l l s w i t h polyoma v i r u s (Henle et a l , 1963) or HSV ( N i i , 1970). I n f e c t i o n of H.Ep.2 c e l l s w i t h MCV at low m u l t i p l i c i t y had ho obvious e f f e c t on the c e l l s . The uptake and r e t e n t i o n of r a d i o a c t i v e v i r u s were measured, u s i n g MCV l a b e l l e d w i t h " CdThd or "HdThd. A f t e r i n f e c t i o n , 80-90$ of the r a d i o a c t i v i t y remained a s s o c i a t e d w i t h the c e l l s , and t h i s was l o s t s l o w l y , w i t h 18$ s t i l l a s s o c i a t e d w i t h the c e l l s at s i x days. I n another experiment, 23$ of the input r a d i o -a c t i v i t y was a s s o c i a t e d w i t h the c e l l s a f t e r h days, and 76$ of t h i s was a s s o c i a t e d w i t h i s o l a t e d n u c l e i . The l a b e l l e d DNA e x t r a c t e d from these n u c l e i annealed t o MCV-DNA at 8$ of the e f f i c i e n c y of the p a r e n t a l r a d i o a c t i v e DNA. This l a t t e r system was t h e r e f o r e chosen f o r more d e t a i l e d i n v e s t i g a t i o n . C. Non-Productive I n f e c t i o n of H.Ep.2 C e l l s A f t e r i n f e c t i o n w i t h MCV at high m u l t i p l i c i t y , H.Ep.2 c e l l s were observed f o r s e v e r a l weeks a f t e r i n f e c t i o n . I n f e c t i o u s v i r u s i n the c u l t u r e s d e c l i n e d r a p i d l y , and was undetectable f i v e days a f t e r i n f e c t i o n ( l e s s than 2 pfu per ml of medium, and l e s s than 10 p f u per 10^ c e l l s ) . However, two e f f e c t s were r e p r o d u c i b l y seen a f t e r i n f e c t i o n . 1 . Growth rat e : I n f e c t e d c e l l s grew at a slower r a t e than mock-infected c o n t r o l c e l l s , grown under the same c o n d i t i o n s . This r e d u c t i o n p e r s i s t e d f o r a v a r i a b l e length of time, from ten up to twenty days a f t e r i n f e c t i o n . During t h i s time, the c e l l s grew ten to one hundred f o l d ( F i g . 2 3 ) . I n f e c t i o n w i t h the same q u a n t i t y of UV- l i g h t i r r a d i a t e d v i r u s , caused a t r a n s i e n t r e d u c t i o n i n growth r a t e f o r approximately two days, a f t e r which the c e l l s grew at the same r a t e as the mock-infected c o n t r o l c e l l s . 2. Morphology: The appearance of MCV-infected H.Ep.2 c e l l s c ould r e a d i l y be d i s t i n g u i s h e d from t h a t of u n i n f e c t e d c e l l s . I n f e c t e d c e l l s were more pleomorphic, and i n p a r t i c u l a r showed a la r g e number of 'streaky' c e l l s ( p l a t e 2 ) . S l i g h t overlap of i n f e c t e d c e l l s was observed, i n co n t r a s t to the appearance of u n i n f e c t e d c e l l s . Before confluency was reached, i n f e c t e d c e l l s were l a r g e r . C e l l s i n f e c t e d w i t h U V - i r r a d i a t e d v i r u s showed an intermediate morphology between th a t of i n f e c t e d and u n i n f e c t e d c e l l s . Although ob s e r v a t i o n of these changes was n e c e s s a r i l y s u b j e c t i v e , Fig. 2 3 : 135 F i g . 23: E f f e c t s of i n f e c t i o n of H.Ep.2 c e l l s w i t h MCV Approximately 10°" H.Ep.2 c e l l s each were i n o c u l a t e d by c e n t r i f u g a l adsorption w i t h MCV, U V - i r r a d i a t e d MCV,- or an u n i n f e c t e d mouse c e l l l y s a t e . A l l three i n o c u l a were p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . UV i r r a d i a t i o n was c a r r i e d out at 5 -5 cms from a Syl v a n i a G15T8 UV l i g h t f o r 2 0 minutes i n 0 . 5 mis MEM-B i n a 35 mm p e t r i d i s h . D i l u t i o n s of the v i r u s preparations were simultaneously i n o c u l a t e d onto mouse embryo c e l l s to determine the m u l t i p l i c i t y of i n f e c t i o n , which i n t h i s experiment was 180 pfu per c e l l . A f t e r i n f e c t i o n , the number of c e l l s i n each c u l t u r e was counted, u s i n g a c a l i b r a t e d g r i d i n the microscope eyepiece. The c e l l s were maintained i n MEM-A co n t a i n i n g 5$ serum, and t r a n s f e r r e d by t r y p s i n i z a -t i o n every f o u r days. The medium was changed two days a f t e r each sub-c u l t u r e . The c e l l s i n each c u l t u r e were counted immediately p r i o r t o each t r a n s f e r , and the same number of c e l l s from each c u l t u r e ( 1 . 0 x 10 per 50 mm dish) added t o new dishes, t o ensure s i m i l a r growth c o n d i t i o n s . The morphology was evaluated by p l a c i n g the dishes on the microscope stage without reading the l a b e l s , and then a s s i g n i n g them to the three c a t e g o r i e s of i n f e c t e d , u n i n f e c t e d and UV-MCV i n f e c t e d , on the b a s i s of the microscopic morphology. The f i g u r e shows the accuracy w i t h which the c u l t u r e s could be recognized. I f no d e f i n i t e d e c i s i o n could be made, the accuracy was taken as zero. N Q number of c e l l s present at the time of i n f e c t i o n N t o t a l number of c e l l s present H.Ep.2,mock i n f e c t e d H.Ep .2, UV-MCV i n f e c t e d H.Ep .2, MCV i n f e c t e d $> accuracy i n r e c o g n i z i n g morphology Plate 2: Morphology of H.Ep.2 cells Photographs were taken of the three H.Ep.2 cell cultures described in figure 23, at Ik days after infection. The photographs show living cells, as seen by phase contrast microscopy. a. H.Ep.2 cells, mock infected b. H.Ep.2 cells, MCV infected c. H.Ep.2 cells, UV-MCV infected !3<o O S 1 3 6 t 137 there were obvious d i f f e r e n c e s between the c e l l morphologies since the i n d i v i d u a l c e l l c u l t u r e s could be e a s i l y i d e n t i f i e d by independent observers. F i g u r e 2 3 a l s o shows the accuracy w i t h which the c u l t u r e could be i d e n t i f i e d by the author as i n f e c t e d , u n i n f e c t e d or i n f e c t e d w i t h U V - i r r a d i a t e d v i r u s . A l s o , photomicrographs were taken of random f i e l d s from each c e l l c u l t u r e at ih days a f t e r i n f e c t i o n , and twelve people were asked t o score the three sets of photographs i n terms of the a l t e r a t i o n s described above. Eleven out of twelve were able to ass i g n the sets of photographs c o r r e c t l y . I n t h i s experiment, a l l the e r r o r s made i n i d e n t i f y i n g the c u l t u r e s up t o 2 0 days a f t e r i n f e c t i o n r e s u l t e d from confusion of UV-MCV i n f e c t e d c u l t u r e s w i t h e i t h e r of the other two c u l t u r e s . Up to 2 0 days, MCV-infected and mock-infected c u l t u r e s were never confused w i t h one another. The change i n morphology a l s o l a s t e d f o r o n l y a l i m i t e d time (see • f i g . 2 3 ) , a f t e r which the c e l l s could no longer be d i s t i n g u i s h e d . D. Attempts to Detect MCV-DNA i n I n f e c t e d H.Ep .2 C e l l s The r e s u l t s described above demonstrate t h a t MCV i n f e c t i o n of H.Ep .2 c e l l s d i d not r e s u l t i n the production of i n f e c t i o u s v i r u s , but d i d cause two r e l a t i v e l y l o n g - l a s t i n g e f f e c t s on the c e l l s . The presence or absence of v i r a l DNA i n such c e l l s could not be i n f e r r e d from these data, and was i n v e s t i g a t e d u s i n g DNA-DNA annealing. 138 U n l a b e l l e d DNA was e x t r a c t e d from i n f e c t e d and u n i n f e c t e d H.Ep.2 c e l l s , p u r i f i e d , denatured, and immobilized on n i t r o c e l l u l o s e f i l t e r s . These were then incubated w i t h h i g h l y r a d i o a c t i v e denatured MCV-DNA i n s o l u t i o n , t o determine the amount of MCV-DNA on the f i l t e r s . The f i r s t experiments c a r r i e d out appeared to i n d i c a t e t h a t MCV-DNA was present i n i n f e c t e d H.Ep.2 c e l l s , at a conce n t r a t i o n of about 50 genome equiva-l e n t s of MCV-DNA per c e l l genome (assuming molecular weights of 1.3 x 8 12 10 and 7 x 10 f o r the two genomes). However, l a t e r experiments d i d not support t h i s . Ten batches of DNA from MCV-infected H.Ep.2 c e l l s were analyzed, w i t h appropriate c o n t r o l s , and v a r y i n g the amounts of r a d i o a c t i v e MCV-DNA i n s o l u t i o n , the time of in c u b a t i o n (from one to three days) and the presence or absence of bovine serum albumin and u n l a b e l l e d H.Ep.2 c e l l DNA i n s o l u t i o n . DNA from MCV-infected c e l l s was e x t r a c t e d at times ranging from 15 to 4-7 days a f t e r i n f e c t i o n at m u l t i p l i c i t i e s v a r y i n g from 53 to 500 pfu per c e l l . Considering a l l the r e s u l t s , i t became apparent t h a t the amount of r a d i o a c t i v i t y bound to one batch of DNA was c o n s i s t e n t , but t h a t t h i s v a r i e d from one batch of DNA t o another, even of u n i n f e c t e d H.Ep.2 c e l l DNA. This 'background' r a d i o a c t i v i t y bound t o the f i l t e r s was p r o p o r t i o n a l to time and the amount of r a d i o a c t i v e DNA i n s o l u t i o n , and depended upon the batch of DNA on the f i l t e r , but not on the p r e p a r a t i o n of r a d i o -a c t i v e DNA i n s o l u t i o n . The 'background' r a d i o a c t i v i t y bound, was r e l a t i v e l y h i g h i n s p i t e of the presence of SDS during the h y b r i d i z a t i o n r e a c t i o n . From the v a r i a b i l i t y i n 'background' b i n d i n g seen i n s e v e r a l experiments, i t was c l e a r t h a t the r a d i o a c t i v i t y b i n d i n g to i n f e c t e d c e l l DNA i n excess of t h a t b i n d i n g t o u n i n f e c t e d c e l l DNA, could not be taken as an accurate measure of MCV-DNA present i n the c e l l . I n no experiment was i t p o s s i b l e t o detect MCV-DNA, i f present, i n the c e l l DNA at a con c e n t r a t i o n of one genome equivalent per c e l l genome. However, i n some experiments, i t could be concluded t h a t l e s s than 10 genome equiv a l e n t s per c e l l were present. The a d d i t i o n of MCV-DNA t o H.Ep.2 c e l l DNA on a f i l t e r always r e s u l t e d i n a p r e d i c t a b l e increase i n the amount of r a d i o a c t i v i t y bound. Provided the f i l t e r s were a l l present i n the same r e a c t i o n s o l u t i o n , the increase i n r a d i o a c t i v i t y bound was d i r e c t l y p r o p o r t i o n a l to the amount of MCV-DNA on the f i l t e r , so t h a t o n l y the v a r i a b i l i t y i n background b i n d i n g prevented q u a n t i -t a t i v e conclusions from being obtained. This v a r i a b i l i t y i n background would not a f f e c t the r e s u l t s i f the c o n d i t i o n s of h y b r i d i z a t i o n were reversed, and a s i n g l e batch of u n l a b e l l e d , f i l t e r - B o u n d MCV-DNA was annealed w i t h r a d i o a c t i v e c e l l DNA preparations i n s o l u t i o n . This procedure n e c e s s i t a t e d the use of ver y h i g h inputs of r a d i o a c t i v i t y , and backgrounds were i n i t i a l l y high. These could be reduced i f u n l a b e l l e d H.Ep.2 DNA was added t o the s o l u t i o n , w i t h the purpose of competing w i t h r a d i o a c t i v e DNA f o r 'non-s p e c i f i c 1 b i n d i n g s i t e s . The r e s u l t s of such an experiment (Table k) appear to i n d i c a t e the presence of r a d i o a c t i v e MCV-DNA i n DNA ex t r a c t e d from MCV-infected c e l l s 1*+ days a f t e r i n f e c t i o n , but not i n DNA from u n i n f e c t e d c e l l s or c e l l s i n f e c t e d w i t h U V - i r r a d i a t e d MCV. However, Table 4: Annealing of ^ H - c e l l DMA and RNA t o MCV-DNA Source of H-DNA Input c.p.m. bound c.p.m. bound "A" - "B" i n s o l u t i o n c.p.m. to MCV-DNA to HSV-DNA II A " " 1 3 " H.Ep.2, 5.0 x i o 6 1+96 9^ 3 - 300.5 mock i n f e c t e d 993 1146 H.Ep.2, 5.8 x i o 6 554 1202 - 73.0 UV-MCV i n f e c t e d 1088 588 H.Ep.2, 5-3 x i o 6 4U58 910 2339 MCV i n f e c t e d 1. 1695 565 H.Ep.2, 5.1 x i o 6 5337 884 2017 MCV i n f e c t e d 2. 2772 1191 MCV-DNA 1.3 x 10* 786.6 28.6 . 853.2 979.8 31.4 Source of 3H-RNA Input c.p.m. bound c.p.m. bound "A" - "B" i n s o l u t i o n c.p. m. to MCV-DNA to HSV-DNA "A" "B" H. Ep.2, 1.6 x 107 118.7 81.3 36.7 mock i n f e c t e d 115.7 79.7 H.Ep.2, 1.1 X 107 118.5 77.1 45.3 UV-MCV i n f e c t e d 112.0 62.9 H.Ep.2, 1.2 x 107 115.1 76.1 41.4 MCV i n f e c t e d 1. 118.9 75.0 H.Ep.2, 1.4 x 107 146.7 86.7 52.8 MCV i n f e c t e d 2. 135.2 89.6 Table k: Annealing of " H - c e l l DNA and RNA t o MCV-DNA Four c u l t u r e s of H.Ep.2 c e l l s (approximately 1 x 10 c e l l s each) were i n f e c t e d by. c e n t r i f u g a l adsorption w i t h MCV at 1020 p f u per c e l l , 20k pfu per c e l l , UV-MCV (equ i v a l e n t t o 1020 pfu per c e l l ) and an un i n f e c t e d mouse c e l l l y s a t e . A l l f o u r i n o c u l a were p u r i f i e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . U V - i r r a d i a t i o n , v i r u s assay and growth of the c u l t u r e s were c a r r i e d out as described i n the legend to f i g u r e 2 3 . Fourteen days a f t e r i n f e c t i o n , 1 ml MEM-A co n t a i n i n g 250 uCi ^ H dThd was added t o one d i s h each of the fou r c u l t u r e s , and 1 ml MEM-A 3 c o n t a i n i n g 250 pCi H Ur was added t o each d i s h of a second set of four c u l t u r e s . A f t e r k hours, t h i s was repeated, and a f t e r a t o t a l l a b e l l i n g p e r i o d of 2k hours ( 3HdThd l a b e l ) or 10 hours (3E Ur l a b e l ) the c u l t u r e s were harvested f o r DNA and RNA r e s p e c t i v e l y , as described i n the M a t e r i a l s and Methods s e c t i o n . P u r i f i c a t i o n was c a r r i e d out as described i n M a t e r i a l s and Methods except t h a t the CsCl gr a d i e n t step was omitted f o r the DNA p u r i f i c a t i o n . U n l a b e l l e d H.Ep.2 c e l l DNA was added to each DNA pr e p a r a t i o n , and each then sheared by passing f i v e times through a 26-gauge needle. Each n u c l e i c a c i d p r e p a r a t i o n was t e s t e d i n s o l u t i o n against f i l t e r d i s c s c o n t a i n i n g 0 . 5 M-g MCV-DNA or HSV-DNA as described i n the M a t e r i a l s and Methods chapter. Each r e a c t i o n v i a l contained one n u c l e i c a c i d p r e p a r a t i o n , two f i l t e r s c o n t a i n i n g MCV-DNA, and 2 f i l t e r s c o n t a i n i n g HSV-DNA. Machine backgrounds (approximately 20 cpm) have not been sub-t r a c t e d from the values shown. the d u p l i c a t e s show poor agreement, and u n t i l the nature of the v a r i a b l e background b i n d i n g , described above, i s understood, these r e s u l t s must be regarded w i t h caution. V i r a l RNA synthesis was a l s o t e s t e d i n MCV-infected H.Ep.2 c e l l s , and the r e s u l t s (Table k) i n d i c a t e that no detectable v i r a l RNA synth e s i s occurred i n these c e l l s at Ik days a f t e r i n f e c t i o n . CHAPTER 6: DISCUSSION A. Morphology of MCV The l a r g e m u l t i p l e - c a p s i d v i r i o n s seen i n MCV preparations appear to be p e c u l i a r t o t h i s v i r u s , since n e i t h e r HSV nor HCV showed such forms under s i m i l a r c o n d i t i o n s of growth and p u r i f i c a t i o n . These m u l t i p l e s are found before or a f t e r h i g h speed c e n t r i f u g a t i o n , i n e i t h e r the supernatant or c e l l s of i n f e c t e d c u l t u r e s . S i m i l a r forms have al s o been seen i n t h i n s e c t i o n s of i n f e c t e d c e l l s ( j . B . Hudson and V. M i s r a , p e r s o n a l communications), and so i t does not appear l i k e l y t h a t m u l t i p l e s are a r t i f a c t s of the e x t r a c t i o n or the p u r i f i c a t i o n procedure. The p o s s i b l e s i g n i f i c a n c e of these m u l t i p l e v i r i o n s i s not yet known. However, i t i s l i k e l y that both m u l t i p l e and s i n g l e - c a p s i d forms are i n f e c t i o u s , since MCV preparations c o n t a i n a r e l a t i v e l y l a r g e p r o p o r t i o n of r a p i d l y sedimenting i n f e c t i v i t y , and the prepar-a t i o n s can be enriched f o r m u l t i p l e s by c o l l e c t i n g o n l y r a p i d l y -sedimenting p a r t i c l e s . The r e s u l t s shown i n f i g u r e 3 f o r HSV are approximately c o n s i s t e n t w i t h the presence of on l y one type of p a r t i c l e , i n agreement w i t h the r e s u l t s from e l e c t r o n microscopic examination. I n the same experiment, MCV showed a d i s t r i b u t i o n of i n f e c t i v i t y t h a t could not be f i t t e d to the r e s u l t s expected f o r a s i n g l e type of i n f e c t i o u s p a r t i c l e . B. C e n t r i f u g a l Enhancement of I n f e c t i v i t y This study confirmed the o r i g i n a l o b s e r v a t i o n by Osborn and Walker ( 1 9 6 8 ) ; t h a t MCV i n f e c t i v i t y can be enhanced up t o 1 0 0 - f o l d by i n o c u l a t i o n under a c e n t r i f u g a l f i e l d . Since r a p i d l y sedimenting i n f e c t i o u s p a r t i c l e s , as found i n t h i s study, might be expected to show more c e n t r i f u g a l enhancement of i n f e c t i v i t y (Osborn and Walker, I968), t h i s enhancement was measured f o r the d i f f e r e n t MCV and HSV populations p a r t i a l l y separated by d i f f e r e n t i a l c e n t r i f u g a t i o n . F i g ure 3 shows t h a t a l l populations of MCV d i s p l a y e d strong enhance-ment (gr e a t e r than 3 0 - f o l d ) w h i l e a l l populations of HSV showed o n l y 8 - 1 0 f o l d enhancement. This suggests t h a t the r a p i d sedimentation of some i n f e c t i o u s MCV p a r t i c l e s i s not the major cause of c e n t r i f u g a l enhancement. C. Hydroxyapatite Chromatography Although i n i t i a l s t u d i e s appeared to i n d i c a t e t h a t HSV-DNA contained unusual s t r u c t u r e s , t h i s was l a t e r shown t o be an a r t i f a c t of the denaturation and chromatography procedures. Conditions were e s t a b l i s h e d i n which HSV-DNA could be completely denatured by a l k a l i or heat treatment, and no double-stranded DNA was formed, immediately a f t e r n e u t r a l i z i n g or c o o l i n g . This showed t h a t HSV-DNA d i d not con t a i n a s i g n i f i c a n t number of heat or a l k a l i - s t a b l e c r o s s l i n k s , i n agreement w i t h the r e s u l t s of sucrose gradient v e l o c i t y sediment-a t i o n work. This c o n t r a s t s w i t h the s i t u a t i o n f o r v a c c i n i a v i r u s DNA, i n which c r o s s l i n k i n g of the DNA strands i s present i n most v i r a l DNA molecules (Berns and Silverman, 1970). D. Sedimentation of V i r a l DNA's 1. Murine cytomegalovirus The sedimentation c o e f f i c i e n t o f the DNA genome of MCV was determined by co-sedimentation w i t h T4-DNA i n n e u t r a l sucrose d e n s i t y g r a d i e n t s . Reproducible r e s u l t s , and u n d i s t o r t e d peaks, could o n l y be obtained when the t o t a l amount of DNA on the 18 ml gradient was less' than 0.5 Mg, f o r clo s e l y - s e d i m e n t i n g DNA's such as MCV and Th-DNA's. This e f f e c t was l e s s n o t i c e a b l e i f the d i f f e r e n c e between the sedimentation r a t e s of the two DNA's was greater , as i n the case of MCV and HSV-DNA's. This r e s u l t would be expected i f the d i s t o r t i o n i s due t o the v i s c o s i t y of the DNA bands, since closer-sedimenting DNA's would sediment together f o r a grea t e r distance before separating i n t o two bands. From these r e s u l t s , i t appears t h a t sedimentation p r o f i l e s showing overlapping peaks (e.g. Berns and Silverman, 1970; K i e f f et a l , 1971), should be i n t e r p r e t e d w i t h caution. In a d d i t i o n to the apparent i n t e r f e r e n c e between the two DNA species at higher concentrations, as seen i n t h i s study, the sedimentation r a t e of DNA has been shown to be concentration-dependent (Rosenbloom and Schumaker 1963). This e f f e c t i s al s o n e g l i g i b l e at concentrations below 0.5 M-g DNA/gradient. Only gradient p r o f i l e s which showed undisturbed peaks, at DNA concentrations l e s s than 0.5 M-g/gradient, were used f o r c a l c u l a t i o n s . A f i v e - f o l d d i f f e r e n c e i n c o n c e n t r a t i o n below t h i s value d i d not s i g n i f i c a n t l y a l t e r the r e l a t i v e sedimentation r a t e s , and a value of 132 t 5 x 10^ was c a l c u l a t e d f o r the molecular weight of the MCV genome. I t should be emphasized t h a t t h i s value depends upon f o u r assumptions: 1. The molecular weight of TU-DNA i s 110 x 10^ ( F r e i f e l d e r , 1970). 2. The genome of MCV i s l i n e a r (see below) 3- MCV- and TU-DNA's have eq u i v a l e n t c o n f i g u r a t i o n s under the con-d i t i o n s used. The presence of a l k a l i - s e n s i t i v e regions i n the MCV-DNA molecule could a l t e r the c o n f i g u r a t i o n . However, i f these regions c o n s i s t o n l y of s i n g l e n i c k s , the sedimentation c o e f f i c i e n t would probably not be a f f e c t e d (Hays and Zimm, 1970). k. The Burgi-Hershey formula, as mo d i f i e d by F r e i f e l d e r (1970) i s 0 38 v a l i d as s = KM . This equation was designed t o f i t recent data on absolute molecular weights of coliphage DNA ( F r e i f e l d e r , 1970). The o r i g i n a l equation of B u r g i and Hershey (1963), s = KM , had been confirmed by Leighton and Rubenstein (1969), u s i n g whole and half-molecules of T7-DNA, so that absolute molecular weights do not a f f e c t the exponent obtained. However, L e v i n and Hutchinson (1973a) have c o r r e c t e d the c a l c u l a t i o n s of the l a t t e r two determinations, r e s u l t i n g i n exponents of 0.38 and 0.36. I f an exponent of 0.35 i s used, a c a l c u l a t e d value of 134 x 10°" i s obtained f o r MCV-DNA. Leighton and Rubenstein (1969) a l s o showed t h a t r e l a t i v e sedimentation c o e f f i c i e n t s determined on sucrose gradients are not the same as those determined by 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 . F r e i f e l d e r (1970) has suggested t h a t t h i s may be due to w a l l e f f e c t s because of the non-sector shape of the tube used f o r sucrose gradient sedimentation. I f t h i s i s the case, i t might be expected t h a t the magnitude of the w a l l e f f e c t would be dependent on the diameter of the tube used, and the exponent of the Burgi-Hershey r e l a t i o n s h i p would d i f f e r from one tube to another. Thus i t i s p o s s i b l e t h a t the l a r g e r diameter tubes used i n our work would produce s l i g h t l y d i f f e r e n t r e l a t i v e sedimentation c o e f f i c i e n t s . However, from s t u d i e s on the sedimentation of sheared h a l f -molecules of TU and MCV-DNA ( r e s u l t s not shown) t h i s e f f e c t , i f present, i s s l i g h t . A recent f i n d i n g by Le v i n and Hutchinson (1973a) may a f f e c t the molecular weight determination of MCV-DNA. These workers showed a speed dependence of sedimentation v e l o c i t y of la r g e DNA molecules Although t h e i r - equations are derived f o r 5 to 20$ sucrose gradients i n an SW 50.1 r o t o r , approximate e v a l u a t i o n of the r e l e v a n t para-meters i n d i c a t e s that they probably a p p l i e d f a i r l y c l o s e l y to the sedimentation c o n d i t i o n s used i n t h i s study. C o r r e c t i n g f o r the e f f e c t of r o t o r speed, a value of 137 x 10^ was obtained f o r the molecular weight of MCV-DNA. Co-sedimentation of denatured MCV and ik-DNA i n a l k a l i n e sucrose gradients showed t h a t s i n g l e - s t r a n d e d MCV-DNA c o n s i s t e d of fragments of v a r i o u s s i z e s . The f a s t e s t sedimenting and major component was c a l c u l a t e d t o have a molecular weight of 6l.7 t 0.6 x 10 ,^ c l o s e to h a l f of the double-stranded molecular weight. I n repeated e x p e r i -ments, t h i s value was c o n s i s t e n t , but the p a t t e r n of lower molecular weight fragments v a r i e d c o n s i d e r a b l y from one p r e p a r a t i o n t o another, both' i n amount and d i s t r i b u t i o n . S i m i l a r patterns of fragments have been shown f o r denatured DNA of HSV, Marek's disease v i r u s , and EB v i r u s ( K i e f f et a l , 1971; Lee et a l , 1971; F r e n k e l and Roizman, 1972; Nonoyama and Pagano, 1971), and these were a l s o found t o be v a r i a b l e . The fragments are thought to a r i s e because of s i n g l e - s t r a n d i n t e r r u p t i o n s i n the DNA. I t i s not c e r t a i n whether these i n t e r r u p -t i o n s were present i n i n t a c t v i r i o n s or whether they were a r t i f a c t s of the p u r i f i c a t i o n and DNA e x t r a c t i o n procedures. Since the p a t t e r n of fragments was v a r i a b l e , and the q u a n t i t y of DNA i n the f a s t e s t sedimenting peak could exceed 50$ of the t o t a l i n the experiments i n t h i s study, some molecules i n the MCV-DNA preparations were i n t a c t , w i t h no i n t e r r u p t i o n s i n e i t h e r strand. Since the more c a r e f u l l y prepared v i r i o n s showed l e s s s i n g l e - s t r a n d i n t e r r u p t i o n s , i t appears l i k e l y t h a t at l e a s t some of these were a r t i f a c t s introduced during p u r i f i c a t i o n . This i s i n con t r a s t to the s i t u a t i o n w i t h HSV-F (F r e n k e l and Roizman, 1972a) where the amount of DNA i n i n t a c t strands i s l e s s than 50$ of the t o t a l and the breaks do not appear to occur during p u r i f i c a t i o n . There was a s l i g h t discrepancy between the c a l c u l a t e d molecular weight of the l a r g e s t s i n g l e stranded species ( 6 l . 7 1 0.7 x 10^) and the expected molecular weight of an i n t a c t s i n g l e strand (66 + 2.5 x 10^). There are s e v e r a l p o s s i b l e explanations: a) The equations used f o r these c a l c u l a t i o n s are e m p i r i c a l . The r e l a t i o n s h i p used f o r the a l k a l i n e sedimentation a n a l y s i s , S = KM^* was der i v e d from a n a l y t i c a l c e n t r i f u g a t i o n experiments ( S t u d i e r , 1965), or from p r e p a r a t i v e c e n t r i f u g a t i o n analyses u s i n g r a d i a t i o n damage to va r y the molecular weight of DNA (Levin and Hutchinson, 1973b). However, t h i s l a t t e r determination may not be very accurate, and an e a r l i e r determination of the c o e f f i c i e n t as O.38 (Abelson and Thomas, 1966) may be more n e a r l y c o r r e c t . In any case, n e i t h e r n e u t r a l nor a l k a l i n e sedimentation equation was designed to f i t the sedimentation c o n d i t i o n s used i n t h i s study, and so p e r f e c t agreement i s not n e c e s s a r i l y to be expected. b) The sedimentation c o e f f i c i e n t of DNA molecules may va r y s l i g h t l y according t o the G + C content, the degree of g l y c o s y l a t i o n , and the presence of unusual bases (e.g. hydroxymethylcytosine). Although these parameters do not appear to a f f e c t sedimentation c o e f f i c i e n t s on n e u t r a l sucrose gradients ( F r e i f e l d e r , 1970) i t i s p o s s i b l e that they could be s i g n i f i c a n t f a c t o r s i n a l k a l i n e sedimentation. c) I t has been assumed f o r these c a l c u l a t i o n s t h a t the genome of MCV i s l i n e a r , not c i r c u l a r . Although c i r c u l a r i t y would l e a d to an apparent discrepancy between n e u t r a l and a l k a l i n e sedimentation r a t e s , q u a n t i t a t i v e c a l c u l a t i o n s (according to Gray et a l , 1967, and Vinograd et a l , 1965) show th a t the magnitude of t h i s discrepancy would be much l a r g e r than t h a t seen i n t h i s work. A l s o , the sep a r a t i o n of the two d i f f e r e n t d e n s i t y components of sheared MCV-DNA was d i s c r e t e . Unless s p e c i f i c s h e a r - s e n s i t i v e regions are po s t u l a t e d , shear breakage of a c i r c u l a r heterogeneous DNA molecule should r e s u l t i n fragments w i t h a complete spectrum of d e n s i t i e s between the two extremes. F i n a l l y , the p a t t e r n of fragmentation of MCV-DNA on a l k a l i n e sucrose gradients i s not con-s i s t e n t w i t h a c i r c u l a r model. 2. Herpes simplex v i r u s HSV-P DNA was cosedimented w i t h MCV- and TU-DNA's through n e u t r a l sucrose g r a d i e n t s , and a molecular weight of 85 t 2.5 x 10* was c a l c u l a t e d f o r the HSV-P genome. This value agrees w i t h i n experimental e r r o r w i t h the value of 88 t 13. 5 x 10°" determined by Graham et a l (1972), by v e l o c i t y sedimentation i n the 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 , but not w i t h the value of 99 - 5 x 10°" obtained by K i e f f et a l (1971) by cosedimentation of HSV-P-DNA w i t h T4-DNA i n n e u t r a l sucrose d e n s i t y g r a d i e n t s . This discrepancy was als o seen when HSV-F-DNA was cosedimented w i t h T4-DNA or MCV-DNA i n t h i s study, and a value of 87 t !•7 x w a s obtained f o r HSV-F-DNA at low DNA concentration. K i e f f et a l (1971) d i d not quote s p e c i f i c a c t i v i t i e s , or amounts of DNA added to t h e i r g r a d i e n t s , and i t i s not p o s s i b l e to t e l l from t h e i r f i g u r e s whether or not the peaks were d i s t o r t e d . However, overloa d i n g the gradients would tend to decrease the sep a r a t i o n of the peaks, and hence a hi g h value f o r the sedimentation c o e f f i c i e n t of HSV-F-DNA could be obtained. A l s o , the high speeds used i n the experiments of K i e f f et a l (1971) would have caused serious speed e f f e c t s s i m i l a r to those seen by Le v i n and Hutchinson (1973), r e s u l t i n g i n slower sediment-a t i o n of both DNA's, but p a r t i c u l a r l y of T4-DNA. This would r e s u l t i n the c a l c u l a t i o n of a high molecular weight f o r HSV-F-DNA. The p a t t e r n of sedimentation of denatured HSV-P DNA was s i m i l a r to that obtained f o r MCV, except t h a t no preparations were obtained w i t h more than 50$ of the DNA i n the l e a d i n g band. I t i s p o s s i b l e t h a t each HSV-DNA molecule contains at l e a s t one s i n g l e -s t r a n d i n t e r r u p t i o n . The major component was c a l c u l a t e d to have a molecular weight of kk.Q t 0.7 x 10 ,^ s l i g h t l y g reater than h a l f the double-stranded molecular weight. Although the discrepancy between a l k a l i n e and n e u t r a l sedimentation behaviour was not as s i g n i f i c a n t as was the case f o r MCV-DNA, the d i r e c t i o n of t h i s s l i g h t e r r o r suggests t h a t the cause of the discrepancy was the accuracy of the equations as discussed under a) above f o r MCV-DNA. The nature of the s i n g l e strand i n t e r r u p t i o n s i n herpesvirus DNA molecules i s not known. Since the i n t e r r u p t i o n s are found a f t e r a l k a l i treatment, i t i s p o s s i b l e that they could be s i n g l e - s t r a n d breaks, or short lengths of p o l y r i b o n u c l e o t i d e s . However, the appearance of an increased number of i n t e r r u p t i o n s during p u r i f i c a t i o n and storage suggests t h a t at l e a s t some i n t e r r u p t i o n s are present as s i n g l e strand breaks before a l k a l i treatment. Since both MCV and HSV are r e s i s t a n t t o p a n c r e a t i c DNase treatment of i n t a c t v i r i o n s , the i n t r o d u c t i o n of breaks i n the DNA during h a n d l i n g of the v i r u s would appear to be due to an unusual l a b i l i t y of the DNA as i t e x i s t s w i t h i n the nucleocapsid, or t o an endonuclease present i n the v i r i o n . F r e n k e l and Roizman (1972a) have reported t h a t the i n t e r r u p t i o n s i n HSV-F DNA are not introduced during p u r i f i c a t i o n , and t h a t seven s p e c i f i c fragments are produced. However, n e i t h e r HSV-P nor MCV-DNA showed a re p r o d u c i b l e p a t t e r n of fragments i n t h i s study, and the t o t a l amount of fragmentation was f a r l e s s than t h a t reported by Fr e n k e l and Roizman (1972a). I t i s unclear at t h i s stage whether or not the s i n g l e s t r a n d i n t e r r u p t i o n s have any s i g n i f i c a n c e during the r e p l i c a t i v e c y c l e of the v i r u s . 3 . . Human cytomegalovirus An u n u s u a l l y f a s t - s e d i m e n t i n g DNA component was repeat e d l y found i n HCV-DNA preparations. The sedimentation c o e f f i c i e n t of t h i s component was not re p r o d u c i b l e , but recent s t u d i e s by Le v i n and Hutchinson ( 1 9 7 3 a ) have shown th a t the sedimentation c o e f f i c i e n t of lar g e DNA molecules v a r i e d according to the speed of c e n t r i f u g a t i o n . A p p lying t h e i r equations to the r e s u l t s f o r HCV-DNA, a molecular weight of 3 l 4 x 10°" was c a l c u l a t e d . This corresponds c l o s e l y to the s i n g l e - s t r a n d molecular weight of 1 5 8 x 1 0 c a l c u l a t e d f o r the f a s t e s t sedimenting component on a l k a l i n e sucrose g r a d i e n t s . This c l o s e agreement i s probably f o r t u i t o u s , because of the assumptions t h a t were made, and the s e n s i t i v i t y of the c a l c u l a t i o n t o small v a r i a t i o n s i n sedimentation v e l o c i t y . However, i t appears f a i r l y c l e a r t h a t a l a r g e Q DNA component, of approximately 3 x 1 0 molecular weight, was present i n HCV-DNA preparations. This m a t e r i a l was very s e n s i t i v e to shear f o r c e s , which supported the c o n c l u s i o n t h a t i t was very l a r g e . The l a r g e s i z e of t h i s DNA r a i s e s the p o s s i b i l i t y t h a t i t could be d e r i v e d from a b a c t e r i a l or mycoplasma contaminant of the i n f e c t e d c e l l s . Although t h i s p o s s i b i l i t y cannot be excluded, i t i s u n l i k e l y f o r the f o l l o w i n g reasons: a) The la r g e component was present i n a l l DNA preparations, derived from v i r u s p u r i f i e d from e i t h e r i n f e c t e d c e l l s or the super-natant medium. b) The supernatant from uninfected, l a b e l l e d c e l l s yielded less than 3$ of the r a d i o a c t i v i t y yielded by an infected c e l l super-natant after p u r i f i c a t i o n . E. Heterogeneity of MCV-DMA Plummer et a l (1969) reported that DNA, extracted from MCV infected mouse c e l l s , showed two extra components on CsCl equilibrium gradients that were not present i n uninfected mouse c e l l DNA. Since the two components were also seen with a second s t r a i n of MCV, thi s was apparently not due to contamination with another virus. The present studies on the size of MCV-DNA indicated that only one very large component, homogeneous i n size, was present, and so the p o s s i b i l i t y was investigated that the genome was i n t e r n a l l y hetero-geneous i n base composition. Normal DNA p u r i f i c a t i o n and loading of a Spinco Model E ultracentrifuge c e l l , v i a a 26-gauge needle, would cause shear breakage of a large DNA molecule such as MCV-DNA, so that the results of Plummer et a l (1969) were probably obtained with sub-genomic fragments. I f the average G + C content of various fragments was different, multiple bands would then appear on centrifugation i n a CsCl gradient. To test f o r i n t e r n a l heterogeneity, radioactively l a b e l l e d MCV-DNA was centrifuged to equilibrium i n preparative CsCl gradients, with added HSV-P or T4-DNA as marker. In agreement with the hypothes 155 i n t a c t MCV-DNA banded as a s i n g l e species, w h i l e fragmented DNA banded i n a m u l t i p l e p a t t e r n . HSV or T4-DNA banded as s i n g l e components whether i n t a c t or sheared, but gave broader bands a f t e r shearing. The t o t a l width of the m u l t i p l e MCV-DNA band was greater than the width of the marker DNA band i n the same gradient. These r e s u l t s were confirmed by a n a l y t i c a l CsCl 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 , and p r e c i s e measurements obtained f o r the number and d e n s i t i e s of the d i f f e r e n t components. The d e n s i t y of the s i n g l e band of i n t a c t MCV-DNA was found to correspond to a G + C content of 59-0$> and t h i s separated i n t o two components of 57-5$ and 6l.5$ G + C a f t e r shearing. From the p r o f i l e obtained on both v e l o c i t y sedimentation and CsCl e q u i l i b r i u m gradients w i t h DNA sheared through d i f f e r e n t s i z e needles, i t appears t h a t whole and approximately h a l f -genome molecules have a s i n g l e d e n s i t y corresponding t o 59$ G + C, while smaller fragments f a l l i n t o two c l a s s e s of 57-5 and 6l. 5$ G + C content. Heterogeneity i n G + C content was confirmed by examination of the m e l t i n g p r o f i l e of MCV-DNA. Two and p o s s i b l y three components were r e p r o d u c i b l y seen, corresponding to G + C contents of 63, 57 and h6°lo. The average G + C content c a l c u l a t e d from the m e l t i n g p r o f i l e agreed w i t h the G + C content c a l c u l a t e d from the d e n s i t y i n cesium c h l o r i d e s o l u t i o n , suggesting t h a t few or no unusual modified bases or other compounds (e.g. polysaccharide) were present. The sepa r a t i o n of the DNA i n t o two components of equal q u a n t i t y on e q u i l i b r i u m g r a d i e n t s , and i n t o three unequal components during melting, i s probably due to the nature of the two methods: even the sma l l e s t DNA fragments analyzed on cesium c h l o r i d e gradients had a molecular weight of 1 8 x 1 0 ^ , and would be expected t o band at the average d e n s i t y of a l l regions of the fragment. However, the increase i n UV-absorbance during h e a t i n g i s due t o strand s e p a r a t i o n over r e l a t i v e l y short d i s t a n c e s , without s i g n i f i c a n t i n t e r f e r e n c e by neighbouring s t r e t c h e s of d i f f e r e n t G + C contents. The me l t i n g p r o f i l e should thus correspond to a p r o f i l e of v e r y s m a l l fragments banded at e q u i l i b r i u m i n cesium c h l o r i d e s o l u t i o n . The o r i g i n of regions of d i f f e r e n t G + C content i s not known. I t i s p o s s i b l e t h a t MCV could have acquired regions of d i f f e r e n t G + C content by recombination w i t h DNA from host c e l l s or other v i r u s e s . P r e l i m i n a r y annealing experiments ( r e s u l t s not shown) have i n d i c a t e d t h a t DNA homology between MCV and mouse DNA i s a maximum of 0.3 genome equivalents of MCV-DNA per mouse genome and probably much l e s s , suggesting t h a t recombination has not occurred w i t h mouse DNA i n amounts s u f f i c i e n t to account f o r the l i g h t (k6°lo GC) component of MCV-DNA. However, a l t e r a t i o n of acquired sequences by mutation could remove homology as detected i n anneal-i n g experiments without a p p r e c i a b l y a l t e r i n g the base composition. F. T r a n s c r i p t i o n of MCV-DMA Components The t r a n s c r i p t i o n of the two d e n s i t y components of MCV-DNA was i n v e s t i g a t e d , and evidence obtained f o r d i f f e r e n t i a l RNA synthesis or degradation. At e a r l y times a f t e r i n f e c t i o n (before 8 hours) s l i g h t l y more of the l a b e l l e d RNA bound t o the M i g h t ' component of DNA. RNA l a b e l l e d from 8 hours a f t e r i n f e c t i o n onwards, bound p r e f e r e n t i a l l y to the 'dense' component, and from 12 hours a f t e r i n f e c t i o n , there was a marked increase i n the amount of l a b e l l e d RNA b i n d i n g t o both components. This sudden increase i n the amount of r a d i o a c t i v e RNA bi n d i n g to DNA occurs immediately a f t e r the onset of DNA r e p l i c a t i o n at 12 hours a f t e r i n f e c t i o n ( j . B. Hudson, pers o n a l communication, Henson et a l , 1 9 6 6 ) . Although these r e s u l t s show a d i f f e r e n c e i n the r e l a t i v e amounts of RNA der i v e d from the dense and l i g h t DNA components, q u a n t i t a t i v e conclusions cannot be obtained, f o r s e v e r a l reasons. a) The sepa r a t i o n of the two d e n s i t y components of the DNA was not complete, and each component probably contained 5 to 15$ of the other component. I f the r e s u l t s are co r r e c t e d f o r t h i s overlap, the d i f f e r e n c e between the r e l a t i v e amounts of a p a r t i c u l a r RNA pr e p a r a t i o n b i n d i n g to each component would increase. b) The se p a r a t i o n of the two DNA components i s due on l y to the d i f f e r e n c e i n d e n s i t y of fragments of a c e r t a i n s i z e . This s e p a r a t i o n c o n v e n i e n t l y provides two components i n equal amounts, but does not separate the molecules i n t o the regions shown by the m e l t i n g p r o f i l e of MCV-DNA, which would be more l i k e l y t o correspond t o f u n c t i o n a l u n i t s i n the DNA. So i t i s u n l i k e l y t h a t the regions of DNA t r a n s -c r i b e d at any p a r t i c u l a r time would f a l l w h o l l y w i t h i n one d e n s i t y component. c) The complexity of t r a n s c r i p t i o n a l c o n t r o l i s l i k e l y to be c o n s i d e r a b l y more complicated than a simple two-component system. d) S a t u r a t i o n of a DNA species w i t h RNA during the h y b r i d i z -a t i o n process would a l s o l e a d t o underestimation of the a c t u a l t r a n s c r i p t i o n a l heterogeneity. Although the a c t u a l experiments shown were not c a r r i e d out under c o n d i t i o n s which would a l l o w s a t u r -a t i o n , t h i s e f f e c t could a l s o occur to a l e s s e r extent at sub-s a t u r a t i n g concentrations of RNA. These f o u r f a c t o r s would a l l tend to minimize the d i f f e r e n c e between the amounts of RNA bound by each DNA component. So t h i s d i f f e r e n c e i n the amounts of d i f f e r e n t RNA sequences i s probably more marked than i s apparent from these experiments. The r e s u l t s a l s o show t h a t at l e a s t some sequences of each d e n s i t y component are t r a n s c r i b e d during i n f e c t i o n , since each DNA component binds a m a j o r i t y of RNA l a b e l l e d at some'stage a f t e r i n f e c t i o n . The v a r i a t i o n i n the r e l a t i v e amounts of s p e c i f i c RNA sequences could he due e i t h e r t o d i f f e r e n t i a l t r a n s c r i p t i o n , or to s e l e c t i v e degradation of d i f f e r e n t RNA species. G. Non-Productive I n f e c t i o n s I n f e c t i o n of H.Ep.2 c e l l s w i t h MCV was i n v e s t i g a t e d by s e v e r a l techniques. Uptake and r e t e n t i o n of the v i r u s , as measured by r a d i o a c t i v i t y , was f a i r l y e f f i c i e n t , but some of t h i s uptake apparently l e d to degradation of v i r a l DNA, since l a b e l l e d DNA ex t r a c t e d from the n u c l e i f o u r days a f t e r i n f e c t i o n was o n l y 8$ v i r a l i n nature. This suggests t h a t most of the v i r a l DNA had been degraded, and the r a d i o a c t i v e n u c l e o t i d e s r e - i n c o r p o r a t e d i n t o host c e l l DNA. However, 8$ of the p a r e n t a l DNA was apparently undegraded. Although i n f e c t i o u s v i r u s d e c l i n e d r a p i d l y to undetectable l e v e l s , a r e d u c t i o n i n growth r a t e could be c o n s i s t e n t l y observed a f t e r i n f e c t i o n . This e f f e c t p e r s i s t e d f o r s e v e r a l generations i f normal v i r u s was used, but f o r o n l y two to f o u r generations when the same q u a n t i t y of UV - i n a c t i v a t e d v i r u s was used. This suggests t h a t some UV- s e n s i t i v e component of the v i r i o n caused the e f f e c t . The most p l a u s i b l e explanation i s th a t some component of the v i r i o n caused the i n h i b i t i o n , t h a t the v i r a l DNA coded f o r more of t h i s component a f t e r i n f e c t i o n , and t h a t the damaged DNA of U V - i r r a d i a t e d v i r u s could not code f o r f u r t h e r synthesis of the component. However, i t is also possible that some other UV-sensitive component of the virion caused the reduction in growth rate. The changed morphology after infection also reverted to normal after several generations of growth, but in this case the effect persisted for about the same time for both normal and UV-irradiated virus infections, during which time the cells grew several hundred-fold. Since the heavily-irradiated virus would not be expected to be capable of coding for meaningful virus products, this suggests that some viral component in the inoculum was responsible for the change, but was not required to maintain the cells in the altered state for several generations. The presence or absence of MCV-DNA in infected H.Ep.2 cells was investigated, using DNA-DNA annealing between filter-bound DNA and radioactive DNA in solution. Although early results appeared positive, the results of a series of annealing experiments indicated that the technique had severe limitations under the conditions used in this study. Background radioactivity bound to filters during DNA-DNA annealing was 0 . 0 2 $ of input at best, in contrast to the backgrounds of 0 . 0 0 0 5 $ obtained in DNA-RNA hybridization in this study. However, high inputs of radioactivity were required to detect a possible quantity of DNA corresponding to one MCV genome per cell k genome, i.e. a ratio of 1:5 x 10 . This meant that annealing values were expected to be near the background values. Since i t became apparent after a series of experiments that backgrounds were variable from one batch of f i l t e r bound DNA t o another, but c o n s i s t e n t w i t h i n one batch, s m a l l d i f f e r e n c e s i n the b i n d i n g of MCV-DNA to c e l l DNA were not s i g n i f i c a n t . A d d i t i o n of MCV-DNA t o c e l l DNA on the f i l t e r r e s u l t e d i n a re p r o d u c i b l e increase i n the a b i l i t y of th a t DNA t o b i n d r a d i o a c t i v e MCV-DNA, but such experiments showed t h a t the q u a n t i t y of MCV-DNA bound t o c e l l DNA c o n t a i n i n g e.g. 1 genome of MCV-DNA per c e l l genome, would not be d i s t i n g u i s h a b l e from the v a r i a t i o n i n background b i n d i n g . The r e s u l t s of the reverse annealing, u s i n g v a r i o u s r a d i o a c t i v e c e l l DNA's i n s o l u t i o n , would not have been a f f e c t e d by v a r i a b i l i t y between backgrounds on d i f f e r e n t DNA preparations. 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