STRUCTURE OF THE MURINE CYTOMEGALOVIRUS GENOME AND ITS EXPRESSION IN PRODUCTIVE AND NON-PRODUCTIVE INFECTIONS by VIKRAM MISRA B.Sc., Jodhpur University, Rajasthan (India), 1970 B.Sc, (Microbiology), University of British Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1977 © Vikram Misra, 1977 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes i s for f i nanc ia l gain sha l l not be allowed without my writ ten pe rm i ss i on. Depa rtmen t The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i . ABSTRACT The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study i t s expression during productive and non-productive infections caused by the virus. The kinetic complexity of MCMV DNA was not less than i t s molecular weight implying the absence of major reiterations. The restriction endonuclease EcoR^ cleaved this molecule into twenty-five fragments, which were present in the digest in equimolar amounts and ranged in molecular weights from 20 to 1 million. The sum of the molecular weights of the fragments was 136 million. The genomes of the 'K 181' and 'Smith' strains of MCMV appeared to share more than 99 percent of their sequences, although the DNAs exhibited slightly different fragmentation patterns when treated with EcoR^ and Hind III endonucleases. Control was exerted on the transcription of the MCMV genome at temporal, quantitative, and processing levels. During productive infections, approximately 25 percent of the genome was represented as stable transcripts in the c e l l at 6 hours post infection, i.e., before the onset of v i r a l DNA synthesis, whereas RNA transcribed from 35 to 40 percent of the DNA was present in the cells in the later stages of infection. RNA sequences corresponding to 6 h (early) transcripts would be detected in the c e l l throughout the infectious cycle. Both 'early' and 'late' RNA comprised two RNA classes differing about 7 to 10 fold in concentration. i i . V i r a l DNA synthesis in the host c e l l was required for the expression of 'late' genes since in the presence of inhibitors of protein and DNA synthesis only 'early' transcription occurred. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm of infected ce l l s . Although RNA extracted from the nuclei of infected cells arose from 25 (early) and 35 (late) percent of the v i r a l genome, transcripts from only 11 (early) and 15 (late) percent of the DNA were detected in the cytoplasm. Cells of mouse origin (3T3.cells), arrested in the G^ phase of the c e l l cycle, retained the v i r a l genome in a non-replicating state, but could be induced to enter the l y t i c cycle by serum activation. Trans-cripts from 19 percent of the genome were observed in G^ arrested, MCMV-infected ce l l s . V i r a l RNA in these cells comprised only one abundance class, which was similar to the scarce class in 'early' RNA from infected exponentially growing c e l l s . Some evidence was also obtained for the transmission of latent MCMV genomes from mother to progeny. Cells cultured from embryos of infected mice did not normally produce infectious virus. However, the presence of the virus, at least in some of these c e l l s , could be demonstrated by immunofluorescence, and by in-situ hybridization, using iodinated MCMV DNA as probe. i i i . TABLE OF CONTENTS Chapter Page I. INTRODUCTION 1 I. Herpes viruses: History and classification 1 II. Cytomegaloviruses 2 A. General 2 B. Structure of cytomegaloviruses 3 III. Properties of herpes virus genomes 4 A. Herpes Simplex type 1 4 B. Human cytomegalovirus 5 C. Murine cytomegalovirus 6 IV. Growth of murine cytomegalovirus in tissue culture 7 A. General 7 B. Adsorption, penetration and 'centrifugal enhancement' of infectivity 7 C. Replication 8 D. Transcription 9 ^ E. Effect of infection on host cells 9 F. Protein synthesis in infected cells 10 V. Transcription of the HSV-1 genome in l y t i c systems 11 (A prototype of herpes virus transcriptional control) A. Solution hybridization and i t s advantages over the two phase system 11 B. Temporal and abundance control of transcription 12 C. Post transcriptional modification of RNA 13 VI. Pathogenesis of cytomegaloviruses 14 A. Human cytomegalovirus 14 B. Murine cytomegalovirus 17 II. MATERIALS AND METHODS 19 I. Reagents 19 II. Solutions 20 ' III. Growth medium 22 IV. Cells 23 V. Cell culture conditions 23 iv. Chapter Page II. MATERIALS AND METHODS VI. Mouse embryo cells 24 VII. Arrest of 3T3 cells in G1 24 VIII. Cell transfer 24 IX. Viruses 25 X. Growth of murine cytomegalovirus 25 A. Standard inoculation 25 B. Centrifugal inoculation 26 XI. Plaque assays ' 26 XII. Labelling of MCMV with radioactivity 27 A. 3 2P labelling of MCMV 27 B. Labelling of MCMV with 3H-TdR and 3H-UR 27 C. Iodination 28 XIII. Purification of MCMV 28 XIV. Purification of DNA 29 A. V i r a l DNA 29 B. Cellular DNA 30 XV. Purification of RNA 31 XVI. Fractionation of cells 32 XVII. Fragmentation of DNA with restriction enzymes 32 A. EcoRi 32 B. Hind III 33 XVIII. Separation of DNA fragments by agarose gel 33 electrophoresis XIX. Staining and photography of gels 33 XX.Autoradiography of gels 33 XXI. Elution of DNA from agarose gels 34 XXII. Nuclei and hybridization 35 A. F i l t e r hybridization 35 B. Solution hybridization 36 XXIII. Melting profiles of renatured DNA 36 V. Chapter Page III. RESULTS 1 37 Structure of the murine cytomegalovirus genome I. Kinetic complexity of MCMV DNA 37 II. Absence of ribonucleotides linked to MCMV DNA 42 III. Fragmentation of MCMV DNA with EcoRi endonuclease 46 IV. Relative density of high MW fragments 50 V. Effect of high multiplicity passage on the restriction endonuclease pattern of MCMV DNA 51 VI. Comparison between MCMV (K 181) and MCMV (Smith) 56 IV. RESULTS 2 Transcription of the murine cytomegalovirus genome in l y t i c infections I. Experimental design and treatment of data 66 II. Characterization of Sj nuclease 71 III. Determination of the onset of v i r a l DNA synthesis in mouse embryo cells infected with MCMV 78 IV. Transcription of MCMV DNA in l y t i c a l l y infected cells 86 A. Abundance classes of RNA and the fraction of the genome transcribed 86 B. 'Early' RNA is a subset of 'late' RNA 91 C. Transcription of MCMV DNA in the presence of inhibitions of protein and DNA synthesis 96 D. Absence of symmetrical transcripts in MCMV infected cells 99 E. Nuclear and cytoplasmic RNA 104 V. RESULTS 3 Non-productive infections caused by MCMV I. ME-D cells 110 A. MCMV penetration 113, B. MCMV transcription in ME-D cells 113 II. Gi arrested 3T3 cells 120 A. Persistence of the MCMV genome in arrested 3T3 cells and the induction of the l y t i c cycle following serum activation 120 v i . Chapter Page V. RESULTS 3 II. G 1 arrested 3T3 cells B. Extent of transcription of the MCMV genome in G.^ arrested cells 123 VI. RESULTS 4 Vertical transmission of MCMV: An example of latency in vivo I. EM-2 cells 130 II. Pathogenesis of MCMV and vertical transmission 142 VII. DISCUSSION I. Structure of the MCMV genome 149 II. Expression of the MCMV genome 153 A. Onset of v i r a l DNA synthesis 153 B. Transcription in l y t i c a l l y infected cells 154 C. Non-productive infection in vitro 157 III. Vertical transmission of MCMV 158 v i i . LIST OF TABLES Table - Page I. Sensitivity of 3H-TdR and 3H-UR labelled DNA to ribonuclease, deoxyribonuclease and a l k a l i 45 II. MCMV DNA fragments generated by EcoRi 49 III. Effect.of ser i a l high multiplicity passage on the infectivity of MCMV ' 59 IV. Effect of RNA and NaCl concentration on activity of Si nuclease against single and double stranded DNA 75 V. Best f i t a and 3 values for RNA extracted from MCMV infected cells 92 VI. Relationship between 'early' and 'late' and 'early' and cyclohexamide arrested RNA 93 VII. Penetration of MCMV into mouse embryo fibroblasts and ME-D cells 114 VIII. DNA-DNA hybridization 118 IX. DNA-RNA hybridization 119 X. Transcription in 3T3 cells 128 XI. Protocol for studying pathogenesis of MCMV 143 XII. Infectious MCMV in salivary gland, kidney, spleen -and uterus of mice 146 LIST OF PLATES E f f e c t of high m u l t i p l i c i t y passage on the fragmentation pattern of MCMV DNA Immunofluorescence of uninfected and infe c t e d mouse c e l l s Immunofluorescence of EM-2 c e l l s H ybridization of infec t e d and uninfected ME c e l l s with iodinated MCMV DNA Hybridization of EM-2 c e l l s with iodinated MCMV DNA Immunofluorescence of c e l l s from embryos inf e c t e d with MCMV ix. LIST OF FIGURES Figure Page 1. HSV-1 DNA 4-5 2. Reassociation kinetics of 3 2P labelled MCMV and T7 DNA 39 3. 'Banding of 3H-TdR and 3H-UR labelled MCMV DNA in CsCl gradients 43 4a. MCMV (K 181) DNA fragments generated by ECORT^ endonuclease 48 4b. Comparison of the mobility of MCMV DNA fragments to that of known molecular weight markers 48 5. Analysis of MCMV (K 181) DNA fragments on CsCl gradients 53 6. Relative density of MCMV (K 181) DNA fragments 55 7a. Reassociation kinetics of 3 2P labelled MCMV (K 181) and (Smith) DNAs in the presence of excess homologous or heterologous unlabelled DNA 62 7b. Melting curves of homologous and heterologous hybrids _ 62 8. A comparison of the fragmentation patterns of MCMV (K 181) and (Smith) DNA 64 9. Correction of data for self-annealing of iodinated probe 73 10a. Effect of increasing concentrations of S^ on the susceptibility of DNA 77 10b. Effect of time of incubation on the susceptibility of DNA 77 11. Comparison of the melting curve of 3H-3T3 DNA as . monitored by a change in A 26 0 a n d by increased sensitivity to Sj nuclease 80 12. Reassociation of 1 2 5 I labelled MCMV-DNA in the presence of unlabelled v i r a l DNA 82 13. Vi r a l DNA synthesis in ME cells infected with MCMV 85 Figure Page 14. Transcriptional analysis of RNA extracted from MCMV infected ME-cells 6 h post infection 88 15. Transcriptional analysis of RNA extracted from ME cells 24 h post infection 90 16. Relationship between 'early' and 'late' RNA 95 17. Enumeration of MCMV genomes in infected untreated cells or infected cells treated with Ara-C 98 18. A transcriptional analysis of 'early' RNA and RNA extracted from Ara-C treated cells 101 19. A transcriptional analysis of 'early' RNA and RNA from cells treated with cyclohexamide 103 20. Analysis of 'early' and 'late' RNA for symmetric transcripts 106 21. Analysis of cytoplasmic and nuclear RNA 109 22. ME-D cells infected with MCMV 112 23. CsCl gradient analysis of DNA extracted from MCMV infected and uninfected ME-D and 3T3 cells 117 24. MCMV DNA and infectious virus associated with &i; arrested, serum activated, and exponentially-growing 3T3 cells 122 25. Extent of transcription of MCMV in exponentially growing cells 125 26. Extent of v i r a l transcription in arrested cells 127 27. MCMV in salivary glands and kidneys of mice 144 x i . ABBREVIATIONS C - Concentration of single stranded DNA at time = t. Co - I n i t i a l concentration of single stranded DNA. Cot - Product of the i n i t i a l concentration of single stranded DNA in molesdeoxyribonucleotides per l i t r e x length of hybridization in seconds. 1/2 Cot - Cot value at which 50 percent of a particular species of DNA reassociates = 1/K. Ci - Curie. (M) CMV - (Murine) cytomegalovirus. cpm - counts per minute. DNA - Deoxyribonucleic acid. DNase - Deoxyribonuclease (Pancreatic). EDTA - Ethylene diamine tetra acetic acid. Fig. - Figure. Xg .- X gravity. G - Guanine. A - Adenine. T - Thymine C - Cytosine. U - Uracil. h - hours HSV - Herpes Simplex virus. K - Reassociation or hybridization rate constant. ME-cells - Mouse embryo cell s . MEM - Minimal essential medium. x i i . m o i - Multiplicity of infection. PBS - Phosphate buffered saline, pfu - plaque forming unit. RNA - Ribonucleic acid. RNase - Ribonuclease. Ro - I n i t i a l concentration of unhybridized DNA i n mole nucleotides per l i t r e . S - Sedimentation coefficient. SDS - Sodium dodecyl sulphate. SSC - Standard sodium citrate. 3T3 - Mouse fibroblast line. Tm - Melting temperature of DNA. Tris - Tris (hydroxymethyl) aminomethane. TdR - Thymidine. UR - Uridine. UV - Ultraviolet. x i i i . ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my supervisor Dr. J.B. Hudson for his support and encouragement during the period of graduate study. Members of the advisory committee, Drs. R.A.J. Warren, D.E. Vance, D.M. McLean, as well as Drs. G.M. Tener and J.K. Chantler are gratefully acknowledged for their advice and assistance in the preparation of this report. I am particularly grateful to Ms. Jessica Suzuki for technical assistance and inexhaustable patience displayed in the face of unre-lenting assaults on her sense of order and neatness. I would also like to thank Ms. Mary Vorvis fdr typing this thesis. Above a l l I would like to give special thanks to my wife, Lyn for her encouragement and invaluable support. 1. CHAPTER I INTRODUCTION I. Herpes Viruses: History and Classification Although the term 'Herpes' has been used in medicine for at least the past twenty-five centuries, i t s meaning has changed considerably during that time. Since Hippocrates the term has been used to describe such disease conditions as eczema, cancer of the skin, shingles, faci a l herpes, ringworm, erythema multiforme and the lik e (Kaplan, 1973). The modern concept of Herpes was introduced by Cowdry in a series of papers,which he reviewed in 1934. He distinguished two types of intra-nuclear inclusions, A and B: "The type A inclusions are pleomorphic, resistant to organic solvents and contain l i t t l e or no thymonucleic acid or mineral. The nuclear involvement is total and proceeds to complete destruction and the inclusion is separated by a clear halo from the chromatin which eventually marginates." Two thirds of Cowdry's l i s t of situations in which 'Type A' intra-nuclear inclusions were found are recognizable as being caused by members of the Herpes group of viruses as we know them today. The introduction of electron microscopy and negative staining (Brenner and Home, 1959) made possible the characterization of virus particles with respect to size, shape, surface structure, and sometimes, symmetry. In 1961 and 1962 (Home and Wildy, 1961; Wildy, 1962) the use of morpho-logical c r i t e r i a for virus taxonomy were discussed for the f i r s t time. In 1962, Andrews put forward a classification of vertebrate viruses on the basis of: a) nucleic acid component, b) size and morphology, c) mode of replication and d) resistance to ether and b i l e salts. Among the NITA viruses (Nuclear Inclusion Type A Viruses, a term later changed to Herpes viruses), Andrews listed, in addition to the previously recognized herpes viruses, cytomegaloviruses of 4 species, 4 avian viruses, pig inclusion body r h i n i t i s , etc. Since 1962, other viruses have been added to the Herpes group using morphology as the principal criterion. In 1971, Melnick, in the semi-annually published chapter on the classification and nomenclature of animal viruses in "Progress in Medical Virology," described the members of the Herpes virus group as follows: "These medium sized viruses contain a core of DNA and possess a l i p i d containing envelope which surrounds the v i r a l capsid. These agents have 162 capsomeres arranged with 5:3:2 icosahedral symmetry. The enveloped virion is 180-250 nm in diameter, but the naked virus or nucleo-capsid has a diameter of about 110 nm. Herpes viruses are divided into two groups on the basis of their behaviour in tissue cultures. The viruses in group A (herpes simplex type 1 and 2 of man, the herpes viruses of monkeys and of several domestic mammals and fowls) are readily released from infected ce l l s , while those of a group B (varicella zoster, cytomegaloviruses, EB virus of man and a number of animal herpes viruses) are strongly c e l l associated and infectious virus is released with d i f f i c u l t y . " II. Cytomegaloviruses A. General. The cytomegaloviruses were originally known as salivary gland viruses because of their particular association with these glands. Their new name, which came into general use in recent years, reflects 3. their a b i l i t y to cause the cytoplasm of the infected host c e l l to swell (Plummer, 1967). Electron microscopy has shown them to possess the structure of Herpes viruses (Smith and Rasmussen, 1963; Wright, et a l . , 1964) although i t is unclear whether they form a distinct subgroup of Herpes viruses, or whether they merge in amongst them with no clear delineating features (Plummer, 1967). A number of agents have by convention become known as cytomegalo-viruses. They have been isolated from eight animal species: humans (Smith, 1956), African green monkey (Black, Hurtley, Rowe, 1963), mice (Smith, 1954), guinea pigs (Hartley, et a l . , 1957), horse (Plummer and Waterson, 1963), pig (L'Ecuyer, et a l . '1966), ground squirrel (Diosi, 1970), and rat (Rabson, et a l . , 1969). B. Structure of cytomegaloviruses. Examination in the electron microscope (Smith and Rasmussen, 1963; Wright, et a l . , 1964) of phosphotungstate negatively stained preparations of human cytomegaloviruses revealed a structure similar to that of herpes simplex virus (Wildy, et a l . , 1960): 162 capso-meres were arranged to form an icosahedral capsid. The capsid was about 100 mm in diameter and each of the hollow capsomeres measured 13.5 x 9.5 nm. Many of the particles had a loose outer envelope characteristic of the herpes group. In addition, the prevalent form of the virion, in tissue culture passaged preparations of the murine cytomegalovirus (MCMV) was the 'multicapsid virion', a single infectious unit, consisting of a collection of typical herpes-like nucleocapsids embedded in a dense staining matrix and surrounded by a single envelope (Hudson, Misra, Mosmann, 1976). The buoyant 4. density of murine CMV p a r t i c l e s i n CsCl has been determined to be 1.219 g/cm3 (Misra, unpublished observation). I l l . Properties of Herpes Virus Genomes A. Herpes simplex v i r u s type-1. The HSV-1 genome i s a l i n e a r double stranded DNA molecule (Becker, et a l . , 1968) with a molecular weight of approximately 1.0 x 10 8 (Graham, 1972; K i e f f , et a l . , 1971; Frenkel and Roizman, 1971; Mosmann and Hudson, 1973), and a k i n e t i c complexity of 99 x 10 6 (Frenkel and Roizman, 1971). The molecule contains terminal as well as i n t e r n a l redundancies (Sheldrick and Berthelot,-1974; Wadsworthand Roizman, 1975, 1976) with terminal redundancies amounting to approximately 2 percent of the genome. Digestion of HSV-1 DNA with the r e s t r i c t i o n enzymes EcoRi and Hind I II generates fragments that are present i n the digest either i n unimolar, h a l f molar, or quarter molar amounts, with the sum of the molecular weights of the i n d i v i d u a l fragments exceeding the molecular weight of the i n t a c t genome by as much as 50 percent (Skare, et a l . , 1975; Hayward, et a l . , 1975). These observations ind i c a t e the existence of four d i s t i n c t s t r u c t u r a l forms of HSV-1 DNA which d i f f e r i n the r e l a t i v e o r i e n t a t i o n of two subregions (designated 'L' and 'S'), each of which has inverted terminally redundant regions (Hayward, et a l . , 1975) ( F i g . 1). When HSV DNA i s denatured i n a l k a l i or released from v i r i o n s under a l k a l i n e conditions, s i n g l e strands with a molecular weight consistent with i n t a c t DNA are observed, along with fragments smaller Fig. 1 a b I 1 b a a c c a S • H S V - 1 D N A 5. than unit length (Kieff, et a l . , 1971; Wilkie, 1973). There is some disagreement among workers as to the location of these a l k a l i sensi-tive regions. Frenkel and Roizman (1972) contend that-their results indicate that the interruptions are mainly present on one strand of the duplex, whereas Wilkie (1973), and Wilkie, et a l . (1974), claim that the interruptions are randomly located on both strands. Ribonucleotides have been found associated with both HSV-1 DNA in virions (Hirsh and Vonka, 1974) and in newly synthesized DNA (Biswal, et a l . , 1974), and are thought to count at least partially, for the a l k a l i labile nature of HSV-1 DNA. Serial passage of HSV-1 in tissue culture at a high multiplicity of infection results in 84 to 98 percent loss of infectivity and the appearance of new DNA with a higher buoyant density in CsCl than DNA from HSV-1 passaged at a low multiplicity (Bronson, et a l . , 1973). Although the defective DNA co-sediments in sucrose gradients with wild-type non-defective HSV DNA, in contrast to non-defective DNA i t lacks cleavage sites for Hind III, and exhibits an altered frag-mentation pattern with EcoRi. Defective high m.o.i. DNA consists of tandem repetitions of sequences of limited complexity (Frenkel, et a l . , 1975), with the DNA sequences contained in the repeating unit originating from the 'S' region of the wild-type v i r a l DNA molecule (Frenkel, et a l . , 1976). B. Human cytomegalovirus (HCMV). The DNA of the human cytomegalovirus is a linear duplex with a molecular weight of 1.0 x 108. It has a buoyant density in CsCl equal to 1.716 g/cm3 (Huang, et a l . , 1973). 6. By examining the kinetics of the reassociation of radioactive labelled HCMV DNA in the presence of homologous or heterologous unlabelled DNA, Huang, et al . (1974) were unable to detect any sequence homology between HCMV DNA and DNA from non-human cytomegalo-viruses or HSV-1, HSV-2, and EBV. Kilpatrick, et a l . (1976) have examined the restriction endo-nuclease cleavage pattern of DNA from eleven human, one simian, and • one simian-related, isolates of CMV. Although DNA fragments of each isolate gave a distinctive electrophoretic profile, extensive comigration in agarose gels among human CMV isolates was observed. The digests contained both major and minor molar classes and the summed molecular weights of the major molar classes of several isolates closely approx-imated the molecular weight of the intact genome. Comparison of Hind III and EcoRi patterns showed that human isolates differed greatly from non-human isolates. C. Murine cytomegalovirus (MCMV). The genome of the murine cytomegalovirus i s a double stranded linear DNA molecule (Mosmann and Hudson, 1974). The molecular weight of this genome is 1.32 x 108, as determined by comparison of its sedimentation co-efficient with those of HSV-1 and T4 DNAs (Mosmann and Hudson, 1973). Like the DNA of other herpes viruses (MDV, Lee, et a l . , 1971; HSV-1, Kieff, et a l . , 1971; EBV, Nonoyama and Pagano, 1971), MCMV DNA contains a l k a l i sensitive regions (Mosmann and Hudson, 1973). Intact MCMV DNA bands in CsCl at a density of 1.718 g/cm3. The molecule i s , however, internally hetero-geneous in G + C content,since molecules sheared to quarter or smaller 7. sizes band at two discrete densities corresponding to 57.5 and 61.5% G + C. IV. Growth of Murine Cytomegalovirus in Tissue Culture A. General. MCMV replicates readily in growing ME fibroblasts and 3T3 cells, and to a lesser extent in monkey, hamster, and rabbit c e l l lines, and, after adaptation, in sheep cells (Kim and Carp, 1971). Although no growth of the virus in human cells has ever been recorded, human fibroblast c e l l lines - WI38 (Kim and Carp, 1972) and H.Ep-2 cells (Mosmann, 1973) undergo abortive infections leading to inclusion bodies, virus specific antigens, and c e l l death in the case of WI38 cells, and an altered c e l l morphology and growth rate in H.Ep-2 cells. Although attempts to detect v i r a l RNA in H.Ep-2 cells were inconclusive, an intact v i r a l genome was required for the effect of the virus, as UV-irradiated virus caused only a transient change in growth rate. B. Adsorption, penetration and 'centrifugal enhancement' of infectivity. MCMV adsorbs readily to mouse embryo fibroblasts, 60% being adsorbed after 30 min. (Henson, et a l . , 1966) under the conditions of assay. In common with at least one isolate of HCMV, the infectivity of suspensions of murine cytomegalovirus can be enhanced by applying a centrifugal f i e l d to the ce l l s . This phenomenon was f i r s t observed by Osborn and Walker (1968) and subsequently studied in our laboratory 8. (Hudson, Misra, Mosmann, 1976b). E s s e n t i a l l y , the a p p l i c a t i o n of vir u s to monolayer cultures of Swiss, Balb/C, or ICR mouse embryo c e l l s ; 3T3; and NRK c e l l s , r e s u l t s i n a 20 to 80 f o l d increase i n the number of in f e c t i o u s particles.. C e n t r i f ugation enhances the i n f e c t i v i t y of both si n g l e and 'multicapsid' v i r i o n s of MCMV (Hudson, Misra, Mosmann, 1976b). The phenomenon i s an inherent property of MCMV p a r t i c l e s and cannot be explained by increased penetration of the v i r u s or i t s DNA into the c e l l s or the i r n u c l e i , or by the presence of i n t e r f e r i n g organisms. There i s some evidence that i n the absence of a c e n t r i f u g a l f i e l d some MCMV p a r t i c l e s may have a tendency to enter into a non-replicating state i n mouse f i b r o b l a s t s . C. R e p l i c a t i o n of murine cytomegalovirus. The s i n g l e step growth cycle of MCMV takes approximately 28-32 h (Henson, et a l . , 1966; Tegtmeyer, et a l . , 1969; Osborn and Walker, 1968; Mosmann and Hudson, 1974). V i r a l DNA synthes'is i n infected c e l l s was demonstrated to begin between 10-12 h post i n f e c t i o n , using the 5-IUdR i n h i b i t i o n technique (Henson, et a l . , 1966), or 3H-TdR l a b e l l i n g followed by separation of v i r a l DNA on CsCl gradients (Moon, et a l . , 1976). However, both the techniques mentioned above r e l i e d on the presence of a thymidine kinase i n infected c e l l s for the phosphorylation of halogenated or t r i t i a t e d nucleosides before t h e i r incorporation into v i r a l DNA. Since MCMV does not induce a thymidine kinase, and the l e v e l of the c e l l u l a r enzyme i s depressed i n infected c e l l s (Muller and Hudson, 1977), the estimates for the onset of v i r a l DNA synthesis are u n r e l i a b l e . 9. Replication of the virus, at least in 3T3 cel l s , appears to be dependent on some event in the S phase of the c e l l cycle (Muller and Hudson, 1977). D. Transcription of the murine cytomegalovirus genome. The internal structural heterogeneity of the MCMV genome is extended to i t s expression in productive infections. Mosmann and Hudson (1974) separated the two density components of MCMV DNA on CsCl gradients, and used them in RNA-DNA hybridization reactions to test for virus specific RNA synthesized in mouse embryo cells at different times after infection. The results suggested that there was a specific control of synthesis or degradation of v i r a l RNA sequences - AT rich regions being preferentially transcribed (or retained) early in infection, whereas transcripts from GC rich regions of the DNA were present in a greater concentration late in infection. E. Effect of infection on host c e l l s . Inhibition of mitosis, and chromosomal aberrations, were observed from 18 h after infection with MCMV (Tegtmeyer, et a l . , 1969). On the basis of a decrease in incorporation of 3H-TdR into TCA insoluble materials in infected ce l l s , Moon et a l , (1976) con-cluded that infection with MCMV caused an inhibition of host DNA synthesis. Inhibition of host DNA did not require v i r a l protein synthesis, since i t was accomplished by UV irradiated virus and to a lesser extent by heat inactivated MCMV. These interpretations also rely on the presence of a thymidine kinase i n infected cells for the phosphorylation of 3H-TdR before incorporation into DNA, and since, as mentioned earlier, infected cells have depressed levels of this enzyme, the above results are open to question. F. Protein synthesis i n infected ce l l s . Unlike HSV-1 (Roizman and Furlong, 1974) and pseudorabies virus (Ben Porat and Kaplan, 1971), MCMV does not inhibit host translation (Chantler and Hudson, 1977), and host protein syn-thesis continues unabated u n t i l 24 h after infection. Virus induced protein synthesis in infected cells occurs in three phases. Synthesis of three non-structural virus induced proteins i s observed almost immediately following infection. The synthesis of these proteins stops by 4 h and is followed by a period of 4-8 h when a low level of synthesis of several v i r a l proteins may occur, but which cannot be detected over the background of continued host protein synthesis. The third phase begins after the onset of v i r a l DNA synthesis at 8-10 h, and lasts until the end of the infectious cycle. Late in infection 55 virus induced polypeptides can be detected. Thirty-four of these are precipitable by virus specific antisera and include most of the virus structural proteins as well as 8 non-structural proteins (Chantler and Hudson, 1977). A l l the proteins in i n -fected c e l l s , which have been tentatively identified as v i r a l proteins, account for not more than 50% of the coding capacity of the MCMV genome. Transcription of the HSV-1 Genome in Productive Systems (A protojtyjpe for herpes virus transcriptional control) A. Solution hybridization and i t s advantages over the two-phase system. Differentiation between v i r a l and cellular RNA is based solely on sequence recognition in hybridization tests with v i r a l DNA. I n i t i a l l y a l l hybridization tests involving herpes viruses were done in a two phase system consisting of labelled RNA in solution exposed to v i r a l DNA on f i l t e r s . This technique suffered from several shortcomings and at best provided a qualitative answer. The problems were as follows: 1. Bonds holding DNA to f i l t e r s were unstable and DNA completely saturated with RNA tended to f a l l off the f i l t e r (Haas, et a l . , 1972). 2. Analysis of the rates of hybridization did not permit detection of two or more classes of RNA complementary to different regions of DNA and differing in molar concentrations. 3. Quantitative comparisons of two or more preparations of RNA labelled at different times in the productive cycle necessitated corrections for changes in nucleotide pools during labelling intervals. 4. Analysis of RNA labelled late in infection was complicated by the possible presence of RNA, sharing the same sequences but made before the label was added. To overcome these problems, Frenkel and Roizman (1972) developed a more sensitive one phase hybridization technique, which relied upon the hybridization of a large amount of unlabelled RNA, extracted from infected cells, with a minute quantity of v i r a l DNA labelled with radioactivity to a high specific activity. In this system DNA, since i t is present in a relatively small concentration, does not reassociate to any appreciable extent. RNA-DNA hybrids are identified on hydroxyapatite or by virtue of their resistance to single strand specific nucleases. The results are readily amenable to analysis which is not complicated by any of the shortcomings associated with the two phase system. By means of this analysis i t is possible to determine the fraction of the genome represented as transcripts in a particular RNA preparation, as well as to identify classes of RNA differing in molar concentration. However, i t should be kept in mind that the technique only measures stable RNA species which arise by asymmetrical transcription. It also does not directly reveal the time of synthesis of the various species or their turnover rates. B. Temporal and abundance control of transcription. Synthesis of v i r a l RNA i n infected cells is divided into two phases. The term 'early' is assigned to v i r a l RNA present in HSV-1 infected cells 2 h post infection, i.e., before the onset of v i r a l DNA synthesis. 'Late' RNA is present in infected cells 8 h post infection, i.e., after v i r a l DNA replication starts. The RNA present i n infected cells 2 h after infection with HSV-1 is transcribed from 44% of the v i r a l genome, whereas late in infection RNA in infected cells i s homologous to 48-50% of the v i r a l DNA. Both 'early' and 'late' v i r a l RNA consist of two species 13. differing in molar concentration by 133 and 40 fold respectively. 'Early' RNA is a subset of 'late' RNA. According to these results temporal control of transcription in HSV-1 infected cells is exerted over only 4 to 6% of the genome. It is possible that due to the short growth cycle of HSV-1, RNA from cells harvested 2 h after infection may represent 'early' RNA as well as some 'late' transcripts. Only 'early' RNA (44% of the genome) is transcribed in cells in which protein synthesis is arrested by cycloheximide (Frenkel et a l . , 1973). This implies that early RNA is transcribed by either a host polymerase or a polymerase brought into the c e l l by the virus. In view of the a b i l i t y of f u l l y deproteinated HSV-1 DNA to i n i t i a t e a l y t i c infection (Sheldrick, et a l . , 1973), i t is plausible that HSV-1 is able to u t i l i z e a host enzyme for the transcription of 'early' genes. C. Post transcriptional modification of RNA. 1. Cleavage: Nuclear RNA species range in size from 10s to 60s. Polyribosomal RNA, on the other hand, ranges in size from 10s to a maximum of 35s. In hybridization tests unlabelled polyribosomal RNA competed with labelled nuclear RNA fractionated into size classes - 10-28s, 28-50s as well as 50-60s, indicating that at least a fraction of v i r a l RNA is made as high molecular weight precursor before appearing in polyribosomes (Wagner and Roizman, 1969; Roizman, et a l . , 1970). 2. Adenylation: Polyadenylated sequences comprise 15-20% of total HSV-1 mRNA present in polyribosomes at 4.5 h.p.i. 14. and consist of chains ranging up to 160 nucleotides in length (Bachenheimer and Roizman, 1972). Adenylation is a post transcriptional event (Roizman, 1974) and takes place i n the nucleus. 3. Selective transport of RNA from nucleus to cytoplasm: There appears to be some degree of selectivity exerted by the c e l l i n the transport of transcripts from the nucleus to the cytoplasm, and i t appears that only those transcripts that are candidates for translation are allowed to pass from the nucleus to the cytoplasm. Although transcripts that accumulate in the nucleus of infected cells 'late' in infection are homologous to almost 50% of the genome, only those homologous to 35% of the genome are products of asymmetric transcription. These asymmetric transcripts are transported to the cytoplasm and translated in polyribosomes. The remainder, which are capable of annealing to themselves, never appear in the cyto-plasm (Kozak and Roizman, 1975). Similarly, although in the absence of protein synthesis, transcripts from 44% of the genome accumulate in the nucleus (early RNA), RNA from only 10% of the genome is transported to the cytoplasm. On release of the cells from inhibition, i t is these transcripts which are translated into a or 'immediate early' proteins. VI. Pathogenesis of Cytomegaloviruses A. Human cytomegalovirus. Human cytomegalovirus (HCMV) is ubiquitous, with at least 80% of the population being infected by the age of 35 to 40 years. In the majority of healthy i n d i v i d u a l s , infected l a t e i n l i f e , the viru s probably causes a mild asymptomatic i n f e c t i o n . However, i n infants and adults where acute viremia occurs, i n f e c t i o n can lead to a myriad of conditions and symptoms ranging from mental retardation (Marx, 1975), neurological damage (Weller, et a l . , 1957, 1962), heterophile negative mononucleosis (Kaariainen, et a l . , 1966), l i v e r diseases (Hanshaw, 1965) and immunological disorders (Kantor, 1970). Certain i n d i v i d u a l s appear to be predisposed towards acute cytomegaloviruria: a) pregnant women, acute HCMV i n f e c t i o n i n which almost i n v a r i a b l y r e s u l t s i n congenital i n f e c t i o n of the c h i l d ; b) persons recovering from organ transplants and/or immunosuppressive therapy; c) persons receiving blood transfusions and d) i n d i v i d u a l s s u f f e r i n g from malignancies. The high incidence of acute HCMV i n f e c t i o n i n these i n d i v i d u a l s may be the r e s u l t of primary i n f e c t i o n , or more l i k e l y due to r e a c t i v a t i o n of lat e n t v i r u s , acquired by the patient at an e a r l i e r date and/or, i n the case of persons r e c e i v i n g transfusions and transplants, from the donor tissue (Lang, 1972; Weller, 1971). Lang has suggested a possible mechanism for the r e a c t i v a t i o n of latent HCMV. He points out that a l l conditions that have been associated with HCMV r e a c t i v a t i o n - namely pregnancy, organ trans-plants, transfusions and malignancies, share two common features. These are: 1) varying degrees of immunosuppression and 2) the presence of foreign antigens. The presence of fore i g n antigens stimulate the lymphoid c e l l s to divide. If HCMV i s harbored i n lymphoid c e l l s as some investigators have suggested (Lang, 1972; Stulberg, et a l . , 1966; Diosi, et a l . , 1969) and i f the virus replicates preferentially in dividing cells, this stimulation could reactivate the virus. The activated virus in an immuno-suppressed individual would then spread unhindered to other susceptible tissues and cause acute viremia. Oncogenic potential of HCMV: Certain human viruses of the herpes group, such as EB virus (Epstein, 1964; Henle, 1967), HSV (Naib, et a l . , 1966, 1969; Rawls, et a l . , 1969, Nahmias, 1970) have been associated to a variable degree with the induction of cancer. Although there is no clear-cut evidence to implicate HCMV as a causative agent of cancer, certain observations imply that the virus may possess at least the potential for acquiring this dubious distinction. HCMV infected cells have been shown to possess the capacity to grow in abnormal environments, as do transformed cell s . Introduction of a million HCMV infected cells into hamster cheek pouches produced a nodule that did not enlarge but regressed more slowly than a control implant (Kissling, et a l . , 1964). Lang et a l . (1970; 1974) grew HCMV infected cells for several generations in agarose, however, the altered clones of human fibroblasts eventually underwent lysis and permanent transformation could not be demonstrated. Albrecht and Rapp (1973) were able to transform hamster embryo fibroblasts, albeit at a very low frequency, with UV irradiated HCMV. The presence of the virus could be demonstrated in these cells by immunofluorescence and one out of 30 weanling hamsters injected with 10 million of these 'transformed' cells developed a tumor 10 weeks after injection. Whether this tumor was benign or malignant is not known. B. Murine cytomegalovirus. Infe c t i o n of mice in-utero or at b i r t h with MCMV r e s u l t s i n 3 p o s s i b i l i t i e s : 1) death within a month of i n j e c t i o n , with the f a t a l i t y rate depending on the s t r a i n of mice used (Henson and Neopolitan, 1970; Olding, et a l . , 1976), or growth retardation (Schwartz, et a l . , 1975) and vi r u s induced tissue damage i n heart, kidneys, l i v e r and s a l i v a r y glands; 2) c h r o n i c a l l y infected mice which shed v i r u s i n urine and s a l i v a with no apparent i l l e f f e c t s (Olding, 1976); 3) l a t e n t l y infected mice, i n the s a l i v a r y glands and spleens of which the presence of the v i r u s can only be demon-strated by either c o - c u l t i v a t i o n with allogeneic f i b r o b l a s t s , or by DNA-DNA h y b r i d i z a t i o n (Olding, et a l . , 1976). E f f e c t of maternal MCMV i n f e c t i o n : Infection of female mice at the time of ovulation and implantation (Neighbour, 1976), or during pregnancy (Manini and Medearis, 1961; Medearis, 1964; Johnson, 1969), causes pregnancy wasteage manifested by decreased l i t t e r s i z e , abortion or de l i v e r y of macerated s t i l l - b o r n f o e t i . Although f o e t i and the newborn from i n f ected mothers are susceptible to super-infection with MCMV (Johnson, 1969; Neighbour, 1976), attempts to i s o l a t e i n f e c t i o u s v i r u s from these animals have f a i l e d . On the basis of these observations, Neighbour (1976) has suggested that damage to f o e t i i n infected mothers may be due to an a l t e r a t i o n i n the placental environment rather than congenital i n f e c t i o n of the embryos. If t h i s i s true, then i t would represent an important d i s t i n c t i o n between the pathology of murine and human cytomegaloviruses, as acute maternal infections in the latter almost invariably result in the congenital infection of the offspring. The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study i t s expression during productive and non-productive infections caused by the virus. CHAPTER MATERIALS AND I. Reagents: Description 1. Agarose 2. Cesium chloride 3. Cordycepin 4. Cycloheximide 5. Cytosine arabinoside 6. Diethylpyrocarbonate 7. Deoxyribonuclease (electrophoretically purified) 8. Gentamicin 9. Hydroxyapatite 10. Mycostatin 11. NP-40 12. 3 2P orthophosphoric acid (NEX053) 13. Penicillin-streptomycin-Fungizone 14. Pronase (Streptomyces griseus) 15. Proteinase K 16. Restriction endonuclease EcoRi 17. Restriction endonuclease Hind III 18. Ribonuclease A 19. Si nuclease 20. Spectrafluor 19. II METHODS Source Seakem, distributed by Bausch and Lomb Schwarz-Mann Co. or Sigma Chemical Co. Sigma Chemical Co. GIBCO Sigma Chemical Co. Aldrich Worthington Biochemical Co. Sigma Chemical Co. BIORAD Laboratories DIFCO Shell New England Nuclear Co. GIBCO Sigma Chemical Co. Beckman Miles Laboratories Inc. Miles Laboratories Inc. Sigma Chemical Co. Sigma Chemical Co. Amersham Searle Co. 20. Description Source 21. 125j s o d i u m iodide (low pH NEZ 033L) 22. T h a l l i c (III) chloride 23. (Methyl- 3H) thymidine 41 Ci/mmole or 48.4 Ci/mmole 24. (2-lkC) thymidine 61.5 mCi/mmole or 62 mCi/mmole 25. Trypsin 26. T r i t o n X-100 27. (5,6- 3H)-uridine 41 Ci/mmole New England Nuclear Co. K and K Laboratories Amersham Searle Co. Amersham Searle Co. DIFCO BDH Chemical Co. Amersham Searle Co. II. A l l other reagents and chemicals were obtained from Fisher Chemical Co. Solutions Buffer A: (Kozak and Roizman, 1975) Tris-HCl Potassium ch l o r i d e Magnesium chloride EDTA 0.01 M 0.05 M 2.5 x 10" 3 M 0.2 x 10~ 3 M pH 7.5 Acetate buffer: Sodium acetate Acetic acid 0.25 M 0.1 M pH of the buffer was 5.0 and needed no further adjustment. 21. Phosphate buffered saline (PBS): Sodium chloride 0.13 M Potassium chloride 2.7 x 10" •3 M Sodium phosphate (Na 2 HPOi t ) 8.1 x 10" •3 M Potassium phosphate (KH2PO4) 1.5 x 10" •3 M Calcium chloride 1.0 x 10" •3 M Magnesium chloride 0.5 x 10" •3 M Gel electrophoresis buffer: Tris-acetate 0.4 M Pi Sodium acetate 0.2 M Sodium chloride 0.018 M EDTA 2 x 10"3 M Gel electrophoresis buffer was made up to 20x and diluted as required. Phosphate buffer (pH 6.8): Stock IM solution was prepared by mixing equal volumes of IM solutions of monobasic and dibasic sodium phosphate. RNA buffer: (Hudson, et a l . , 1970) Sodium chloride 0.1 M Sodium acetate 0.01 M Magnesium chloride 1 x 10 3 M adjusted to pH 5.2 Sci n t i l l a t i o n f l u i d : Spectrafluor 42 ml per l i t r e of toluene 22. Standard Saline C i t r a t e (SSC) ( G i l l e s p i e and Spiegelman, 1965) Sodium c h l o r i d e 0.15 M Sodium c i t r a t e 0.015 M Stock s o l u t i o n was made up to 24x and d i l u t e d as required. S x Buffer: Sodium chloride 0.3 M Sodium acetate 0.03 M Zinc sulphate 3 x 1 0 - 3 M adjusted to pH 4.5 Buffered T h a l l i c (III) chloride: T h a l l i c chloride 3 x 1 0 - 3 M i n sodium acetate (pH 5.0) TNE buffer: Tris-HCl 0.01 M pH 7.5 Sodium chloride 0.15 M EDTA 1 x 10" 3 M TNM buffer: Tris-HCl 0.01 M pH 7.5 Sodium chl o r i d e 0.15 M Magnesium chloride 1 mM I I I . Growth Medium Dulbecco's modified Eagle's medium was obtained from Grand Island B i o l o g i c a l Company (GIBCO) i n powdered form and dissolved and f i l t e r -23. sterilized before use. In addition, MEM-A contained 3.7 g/1 NaHCOg, and MEM-B contained 1.5 g/1 NaHCO^. After ste r i l i z a t i o n , gentamicin was added to 50 ug/ml. Foetal calf serum was added just before use to a concentration of five or ten per cent. Phosphate-deficient medium was made by adding 24.8 mg/1 of sodium phosphate to phosphate-free medium obtained from GIBCO. IV. Cells Mouse embryo c e l l cultures were prepared from randomly bred Swiss white mice, purchased from the Faculty of Medicine Animal Unit, University of B.C. Mouse 3T3 cells (Swiss 3T3) were obtained from Flow Laboratories. ME-D cells were a continuous line of mouse cells, developed in our laboratory by serial passage from mouse embryo cells. V. Cell Culture Conditions Cells were grown in MEM-A at 37°C i n a well-humidified atmosphere supplemented with 5% CO2 in Falcon plastic tissue culture dishes (35, 50, or 90 mm diameter). For experiments requiring large numbers of cells, the cells were grown i n MEM-B in stoppered glass bottles at 37°C. Bottles were rotated at a speed of 0.25 revolution per minute on a Bellco c e l l production roller apparatus. The surface areas of the various containers used for c e l l culture were a) 35 mm dishes - 8 cm2; b) 50 mm dishes - 20 cm2; c) 90 mm dishes - 64 cm2 and d) roll e r bottles - 1400 cm2. 24. VI. Mouse Embryo Cells Embryos were removed from pregnant mice in their second week of gestation, rinsed i n Hank's balanced salt solution, and cut into small fragments using forceps and scissors. The fragments were washed once with Hanks' and transferred to a bottle containing 50 ml. of 0.25% trypsin in Hanks'. After having been stirred vigorously for 20 minutes, the supernatant was drawn off and added to 1 ml of foetal calf serum in a 40 ml centrifugation tube. Cells were pelleted by centrifugation at 2,000 rpm in an International CS centrifuge for 10 min. The remaining tissue fragments were stirred for another 20 min. with fresh trypsin solution and the cells again collected from the supernatant. This procedure was repeated until a l l available cells had been extracted. Usually four cycles of trypsinization were sufficient. The c e l l pellets were pooled, suspended in MEM-A supplemented with 10% serum, dispensed into 90 mm Falcon tissue culture dishes and incubated at 37°C. VII. Arrest of 3T3 Cells in Gx Confluent monolayers of 3T3 cells were s p l i t 1 in 4 and seeded in Falcon tissue culture dishes in MEM-A containing 5% foetal calf serum. Twenty-four hours later, the growth medium was replaced with MEM-A containing 2% serum. Three days later, when no mitotic cells were observed and incorporation of 3H-TdR into acid insoluble material by the cells had dropped to less than 0.5% of control cultures, the cells were assumed to be arrested i n G\. VIII. Cell Transfer Medium on the monolayers was replaced with half i t s volume of 0.25% trypsin in Hank's balanced salt solution. Following incubation for 5 to 10 min at 37°C, the trypsin was discarded and cells resuspended in an appropriate volume of MEM-A or B. Gentle syringing was sometimes necessary to break up c e l l clumps. Foetal calf serum was then added to the c e l l suspension, which was then dispensed into fresh vessels. IX. Viruses The Smith strain of MCMV was obtained from the American Type Culture Collection and the K181 strain from Dr. J. Osborn. Both strains were passaged in.to mouse embryo cells at an input multiplicity not exceeding 0.1 plaque forming units per c e l l , or in 3 week old mice by intraperitoneal injection. Virus could be obtained from the salivary glands of the mice one or two weeks after infection. 3 2P labelled T7 was obtained from Dr. R.C. Miller and X and XSpc-1 DNA from Dr. P.P. Dennis. X. Growth of Murine Cytomegalovirus Murine cytomegalovirus was grown in secondary or tertiary mouse embryo cells. Cells were infected by either the standard or centrifugal technique. A. Standard inoculation. Medium was replaced with a minimum volume of MEM-B or Hank's containing the virus and 5% foetal calf serum. Adsorption was carried out for 30-60 min at 37°C. B. Cent r i f u g a l i n o c u l a t i o n (Osborn and Walker, 1968; Hudson, Misra, Mosmann, 1976). Medium was drained from c e l l monolayers i n 35 mm or 50 mm tissue culture dishes and replaced with 1 ml (for 35 mm dishes) or 2 ml (for 50 mm dishes) of MEM-B containing v i r u s and 5% serum. The dishes were stacked up to 6 (35 mm dishes) or 5 (50 mm dishes) per bucket i n the 242 rotor of the IEC-CS centrifuge, and c e n t r i -fuged f o r 30 min at 2,000 rpm. After i n f e c t i o n by either method, MEM-A containing 5% serum was added to the cultures, and incubation continued u n t i l most of the c e l l s i n the monolayer exhibited cytopathic e f f e c t . For large scale production of v i r u s , r o l l e r b o t t l e cultures of ME f i b r o b l a s t s were infected at an input m u l t i p l i c i t y of 0.1 plaque forming unit per c e l l , and harvested when a majority of c e l l s showed cytopathic e f f e c t . Virus was p u r i f i e d from the supernatant medium. XI. Plaque Assays Virus samples were s u i t a b l y d i l u t e d i n MEM-B supplemented with 5% serum. Inf e c t i o n was car r i e d out by the standard or c e n t r i f u g a l methods of ino c u l a t i o n as described above. Aft e r adsorption the inoculum was removed and the infected monolayers o v e r l a i d with MEM-A containing 0.5% agarose and 5% serum. After 4-8 days of incubation 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 . 27. XII. L a b e l l i n g of MCMV with R a d i o a c t i v i t y A. 3 2 P l a b e l l i n g of MCMV. Secondary or t e r t i a r y mouse embryo f i b r o b l a s t s were seeded 3-into glass r o l l i n g b o t t l e s i n PO^ - d e f i c i e n t MEM-B supplemented „ 3-with 10% f o e t a l c a l f serum and 4 mCi of ^ PO^ . (It was necessary 3- 3-to use PO^ - d e f i c i e n t medium rather than PO^ -fre e medium as v i r u s 3" y i e l d s i n medium containing l e s s than 24.8 mg/L of P01+ were d r a s t i c a l l y reduced). Twelve hours l a t e r MCMV was added at an input m u l t i p l i c i t y of 0.1 p f u / c e l l . At 48 h post i n f e c t i o n medium from the b o t t l e was removed and v i r u s p u r i f i e d from i t 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 above. B. L a b e l l i n g of MCMV with 3H-TdR or 3H-UR. Medium was drained from confluent monolayers of secondary or t e r t i a r y mouse embryo f i b r o b l a s t s i n 50 mm diameter Falcon p l a s t i c t i s s u e culture dishes. Virus i n 1 ml of MEM-B supplemented with 5% f o e t a l c a l f serum was added at an input m u l t i p l i c i t y of 10 p f u / c e l l . A f t e r subjecting the c e l l s to a c e n t r i f u g a l force of 900 x g for 30', the inoculum was replaced with 5 ml of MEM-A containing 10% f o e t a l c a l f serum and the c e l l s incubated at 37°C i n a i r + 5% CO2. Eight hours post i n f e c t i o n (when, under the above conditions, c e l l DNA synthesis i s a l l but completely shut o f f ; unpublished observations), the medium was replaced by 3 ml of MEM-A containing 10% f o e t a l c a l f serum and 25 yCi of 3H-TdR or 3H-UR. Virus was p u r i f i e d from the medium and harvested 24-30 hours post i n f e c t i o n 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. C. Iodination MCMV DNA was iodinated by the method of Commerford (1971) modified to minimize damage or alteration of labelled DNA. Purified and denatured MCMV DNA in 5 y l of d i s t i l l e d water was heated to 65°C for 20 min with 10 yl acetate buffer, 5 yl of buffered 3 x 10"3 M Thallic chloride solution, and 2.5 yl Na 1 2 5I contained in a stoppered plastic v i a l . After diluting with 200 yl of 0.05 M phosphate buffer the iodination mixture" was applied to a 0.5 cm x 1 cm hydroxyapatite column. The column was then washed extensively with 0.05 M phosphate buffer until less than 40,000 cpm of unreacted 1 2 5 I was eluted per ml of buffer. Iodinated DNA was then eluted i n 0.40 M phosphate buffer. Fractions containing DNA, in 0.40 M phosphate buffer, were reheated at 65°C for 1 hour to convert the intermediate 5-iodo-6-hydroxydihydrocytosine to 5-iodocytosine. Iodinated DNA was dialyzed extensively against 2 x SSC and stored at -20°C in 50% glycerol. XIII. Purification of MCMV Virus was purified from the medium after the majority of cells in the infected monolayer exhibited cytopathic effect. After removal of c e l l debris by centrifugation at 2,000 rpm, in an IEC-CS centrifuge, virus was pelleted from the medium by centrifugation in a Sorvall RC-2B centrifuge for 3 hours at 24,000 x g (maximum). In order to remove traces of cellular DNA, the virus pellet was resuspended in TNM and treated with 50 yg/ml of pancreatic deoxyribonuclease for 15 min at 37°C. The suspension was then diluted with twice i t s volume of TNE buffer and, after low speed centrifugation, the virus was pelleted again by high speed centrifugation. XIV. Purification of DNA A. Viral DNA. Virus, purified by differential centrifugation, was resus-pended in TNE buffer. Solid NaCl was added to increase the Na + concentration to IM. SDS and pre-digested pronase were added to 1% and 1 mg/ml respectively, followed by incubation of 37°C for 30'. An equal volume of water-saturated phenol was added and the mixture rolled at 65°C for 2 min. The phenol phase was then removed and the DNA solution was rolled in the chloroform until the aqueous phase cleared. At times centrifugation at 3,000 rpm for 10' in an IEC-CS centrifuge was necessary to clear the aqueous phase. DNA was spooled out in cold 95% ethanol, dissolved in TNE, and reprecipitated with 95% ethanol. The DNA was purified further by banding in CsCl gradients. The DNA solution was made up to a density of 1.72 (calculated from refractive index determinations) with CsCl and centrifuged for 72 hour at 30,000 rpm in the SW 50.1 rotor of a Beckman L 265B centrifuge. Ten-drop fractions were collected from the bottom of the tube and the refractive index of every 10th fraction measured on an ABBE-3-L Bausch and Lomb refractometer. After the addit ion of 0.5 ml of TNE to each fraction, the DNA band was 30. localized by the measurement of absorbance at 260 nM in a Gilford Spectrophotometer. Fractions containing DNA at the density of v i r a l DNA (1.718 g/cm3) were pooled, and DNA precipitated from them in 95% ethanol. B. Cellular DNA. Method 1): Cells were lysed in TNE by the addition of SDS to 1%. After incubation for 60' at 37°C with 1 mg/ml of pronase, phenol was added to the tube and the mixture agitated. Phenol and aqueous phases were separated by centrifugation at 3,000 rpm in an IEC-CS centrifuge and the lower phase removed without disturbing the interphase. An equal volume of chloroform was then added, the mixture was agitated, and the lower phase removed after centrifugation. Chloroform extractions were repeated until the interphase disappeared. The aqueous phase was then drawn off and DNA precipitated from i t by spooling in 95% ethanol. The DNA precipitate was dissolved in TNE and treated with 50 yg/ml of RNase for 60' at 37°C (RNase had been previously heated in boiling water for 10' to inactivate possible traces of DNase activity). Pronase was added to 1 mg/ml and incubation continued for an addition 30'. Phenol and chloroform extractions and ethanol precipitations were repeated as described above, and the DNA dissolved in the appropriate buffer. Method 2): (Meinke, et a l . , 1974). Hydroxyapatite, suspended in PBS, was poured into a 4 cm diameter column to a depth of approximately 0.5 cm. The column was then washed thoroughly with 0.24 M phosphate buffer and the c e l l lysate, in 8 M urea, 0.24 M phosphate, and 1% SDS was applied to the column. The column was 31. then washed with 100 ml of 0.14 M phosphate buffer and the DNA eluted with 0.48 M buffer. After dialyzing overnight against TNE, DNA was precipitated with 95% ethanol. XV. RNA Purification Cells were lysed in 10 mis of RNA buffer containing 1% SDS. Pro-teinase K was added to 100 yg/ml and the lysate heated at 65°C for 1 hr. Seven mis of water-saturated phenol were then added and the mixture was vortexed. Seven mis of chloroform were then added and the mixture was shaken again. After centrifugation at 3,000 rpm in an IEC-CS centrifuge to separate the phases, the lower phase was removed without upsetting the interphase. Fifteen ml of chloroform were then added and the mixture shaken and centrifuged as before. The lower phase was then removed and the chloroform extraction repeated un t i l the interphase disappeared or no longer changed. The aqueous phase was then withdrawn, leaving any interphase behind, and nucleic acids were precipitated from this phase by standing overnight in two volumes of ethanol at -20°C. The nucleic acid precipitate was collected by centrifugation and dissolved in 5 mis of TNM buffer. After digesting with 25 yg/ml pan-creatic DNase at 37°C, the phenol:chloroform extraction was repeated and RNA precipitated with ethanol. The precipitate was dissolved and reprecipitated. The f i n a l precipitate was dissolved in 1 or 2 ml of 2 x SSC. A l l glassware used for RNA purification was washed with st e r i l e d i s t i l l e d water containing 50 y l per 100 ml of diethyl pyrocarbonate (DEP), and then autoclaved. A l l solutions were also treated with DEP to inactivate any contaminating RNase activity. The solutions were heated in a boiling water bath for 10 minutes to remove DEP before using. XVI. Fractionation of Cells (Kozak and Roizman, 1975) Cells were collected by centrifugation, washed twice with PBS, and resuspended in five ml of buffer-A. Cells were lysed by the addition of an equal volume of buffer-A containing 2% NP-40 and swirling at 4°C for 15 minutes. After pelleting the nuclei at 2,000 rpm in an IEC-CS centrifuge, the cytoplasmic fraction was removed, and, after recentri-fuging to remove c e l l debris, dialysed overnight against RNA buffer prior to RNA purification. Nuclei were washed with buffer-A containing 1% NP-40, pelleted and resuspended in 10 mis of RNA buffer. Fractionation was monitored by phase contrast microscopy. XVII. Fragmentation of DNA by Restriction Enzymes A. EcoR^ endonuclease. The reaction mixture, in 50 Ul, contained 0.09 M Tris-HCl, pH 7.4; 0.01 M MgCl2, 0.05 M NaCl, 1 to 2.5 yg of DNA and 70 units of EcoR^. Following incubation at 37°C for 1.5 hr. the reaction was terminated by the addition of an equal volume of a solution containing 30% sucrose, 50 mM EDTA, 0.005% bromophenol blue, and heating for 10 min. at 65°C. 33. B. Hind III endonuclease. The reaction mixture, in 50 y l , contained 0.09 M Tris HCl pH 7.4, 0.01 M MgCl2, 0.05 M NaCl, 1-2.5 yg of DNA, 20 yg of bovine serum albumin and 75 units of Hind III. Conditions of incubation and termination of the reaction were the same as those used for EcoR^. XVIII. Separation of DNA Fragments by Agarose Gel Electrophoresis Restriction endonuclease digests were electrophoresed on horizontal 0.5 or 1.0% agarose slabs. Molten agarose in 4x concentrated gel buffer was poured onto a clean glass plate placed on a level surface. A well template was placed at one end of the slab and the agarose allowed to solidify. The gel slab, with the exception of 3 cm strips at each end, was coated with vaseline and the well template removed. Contact was established between the gel and the electrophoresis buffer (4x gel buffer) with 'J-cloth' strips. The potential difference between the two ends of the gel was adjusted to 35V. Samples were placed in the wells and electrophoresed for 24 hrs. XIX. Staining and Photography of Gels Bands of DNA were stained by immersing the gel in lx gel buffer containing 4 yg/ml ethidium bromide for 1 hr, and visualized under UV light. The bands were photographed through a KODAK No. 23A f i l t e r using Polaroid type 55 P/N film. An exposure of 20 minutes was usually necessary. XX. Autoradiography of Gels 32 P-labelled DNA was electrophoresed under conditions outlined above. The gels were then dehydrated by press drying. The gel was placed on a sheet of HAWP 304C5 ( M i l l i p o r e ) f i l t e r and covered with a sheet of mylar. The sandwich was placed on three layers of Whatman #1 f i l t e r paper and weighed down with a glass plate. Twenty minutes l a t e r the f i l t e r paper was replaced and the glass plate weighed down with 300 gms. F i l t e r paper changes were repeated at 45 min. i n t e r v a l s . Each time the weight on the glass plate was increased by 300 gms. Dried gels were placed against Kodak X-OMAT-R X-ray f i l m and auto-radiographed for three days to two weeks. The autoradiograms were scanned and the peaks integrated on a Helena Quick Scan Jr. densitometer equipped with an integrator.-. XXI. E l u t i o n of DNA from Agarose Gels DNA i n preparative agarose gels was v i s u a l i z e d under UV l i g h t a f t e r s t a i n i n g with ethidium bromide. Fluorescent bands were cut out with a razor and squirted through an 18 g needle into 5 volumes of a saturated potassium iodide s o l u t i o n i n 0.01 M Tris-HCl pH 8.5, 0.001 M EDTA (saturated s o l u t i o n of KI contained 30 g of KI i n 21 ml of b u f f e r ) . A g i t a t i o n at 37°C for 20 minutes was s u f f i c i e n t to disso l v e the agarose. DNA was removed from s o l u t i o n with 0.1 g of hydroxyapatite, which was then washed with two ml of 0.01 M potassium phosphate buffer followed by a wash with 0.1 M potassium phosphate. Hydroxapatite containing DNA was then poured into a 1 ml syringe stoppered with a scintered d i s c and the DNA eluted i n 0.48 M potassium phosphate buffer. The DNA s o l u t i o n was extracted once with phenol to remove any traces of agarose i f present, dialysed overnight against TNE, and p r e c i p i t a t e d with ethanol. 35. 90-95% of DNA in the agarose disc could be recovered by this technique. XXII. Nucleic Acid Hybridization A. F i l t e r Hybridization. A modification (Hudson, 1971) of the membrane f i l t e r technique of Gillespie and Spiegelman (1965) was used for both DNA-DNA and DNA-RNA hybridization reactions. B. Solution Hybridization. DNA-DNA: Radioactive v i r a l DNA was mixed with an appropriate amount of the unlabelled DNA to be analysed in 2 X SSC. Calf thymus DNA was added to a f i n a l concentration of 500 yg/ml. The mixture was sonicated for 3 minutes with the microprobe of a Biosonik sonicator. This procedure sheared DNA into fragments that co-sedimented in 5-20% sucrose gradient with 1 2 5 I labelled Drosophila tRNA. The DNA was then sealed into 100 y l corning micropipettes, denatured in a boiling water bath for 20 mins, and transferred to a water bath equilibrated at 67°C. Before denaturation, immediately after, and at various times during the incubation process, sealed tubes were removed, frozen by immersion into ethanol at -20°C and the contents added to 1 ml of cold Si buffer. Single stranded DNA was degraded by incubating at 37°C with 60 units of Si nuclease for 1.5 hr, and double stranded DNA was precipitated with cold 10% TCA. TCA precipitates were trapped on glass fibre f i l t e r s , washed with ethanol, and counted in an ISOCap 300 liquid s c i n t i l l a t i o n counter. Under the conditions used, nuclease degraded double stranded DNA to less than 5% and single stranded DNA to 98%. After subtraction of background, Si resistant cpm for each time point were expressed as a percentage of Si resistant cpm in that sample before denaturation. DNA-RNA: 5 ng iodinated MCMV DNA was mixed with 500-1000 yg of the RNA to be tested in. 1.0 ml.of 2 x SSC. After shearing the nucleic acids by sonication, the hybridization mixture was sealed into 50 or 100 y l micropipettes. Conditions of hybridization and treatment of samples were similar to those outlined for DNA-DNA hybridization in solution, except that Si buffer contained 10 yg/ml denatured calf thymus DNA. XXIII. Melting Profiles of Renatured DNA DNA that had been allowed to reassociate in 2 x SSC was dialysed extensively against 0.1 x SSC and sealed in 100 y l micropipettes. The sealed pipettes were placed in a Haake circulating water bath equilibrated at 25°C. The temperature of the water bath was then gradually increased to 100°C. After permitting the waterbath to equilibrate for 5 minutes at each sampling temperature,one micropipette was removed, quenched in ethanol and dry ice, and i t s contents trans-ferred to 1 ml of Si buffer. Single stranded DNA was degraded as described earlier and Si resistant material precipitated with cold 10% TCA. CHAPTER III RESULTS 1 Structure of the Murine Cytomegalovirus Genome The genome of the murine cytomegalovirus is a double stranded linear DNA molecule, which, with a molecular weight of 1.32 x 10 8, is significantly larger than the genome of any other herpes virus studied to date (Mosmann and Hudson, 1973). The molecule is internally heterogeneous, consisting of segments that band in CsCl at densities corresponding to 57.5 and 61.5% G + C (Plummer, 1966; Mosmann and Hudson, 1973). Intact MCMV DNA molecules band at a density corres-ponding to 59% G + C. The structure of this molecule was examined with respect to: a) kinetic complexity; b) unusual features, such as the presence of ribonucleotides; c) characterization of fragments generated by restriction endonucleases; d) the effect of high multi-p l i c i t y passage on the endonuclease cleavage pattern of the DNA, and e) comparison of the properties of the genomes of the 'Smith' and 'K181' strains of MCMV. I. Kinetic Complexity of MCMV DNA The complexity of MCMV DNA was determined by comparing the kinetics of i t s reassociation with that of bacteriophage T7 DNA. The kinetics of DNA-DNA reassociation as described by Britten and Kohne follow the second order equation: C 7T" = 1 + K.C t C o where: C is the i n i t i a l concentration of single stranded DNA, C is the concentration of single stranded DNA at time t, K is the reassociation rate constant and equals the reciprocal of C t, or the C t value at o i o which half the molecule reassociates. The C ti of a DNA molecule is inversely proportional to it s kinetic complexity. Thus the complexity of a double stranded DNA molecule can be determined by comparing i t s C t x to that of a molecule of known complexity and similar G + C content, 0 2 sheared to the same average size, and permitted to reassociate under identical conditions of temperature and salt concentration. The kinetics of reassociation of sheared and denatured 3 2P-labelled MCMV DNA were compared with that of T7 DNA in 2 X SSC at 67°C (Fig. 2). The C ti of the two molecules, determined from their reassociation rate o t constants, were 0.09 and O.0314mole nucleotides.sec.litre _ 1 respectively. T7 DNA is non permuted molecule with terminal reiterations accounting for only 0.7% of i t s entire length (Ritchie, et a l . , 1967). The G + C content of the molecule has been estimated to be 48% (Wyatt and Cohen, 1953) and it s molecular weight to be equal to 2.6 x 10 7 (Studier, 1965). Using these parameters and adjusting the C t i values for the difference 0 2 in the G + C content of MCMV and T7 DNA ( G i l l i s , et a l . , 1970), the complexity of MCMV DNA was calculated to be 1.83 x 10 8. Fig. 2.—Reassociation kinetics of 3 2P labelled MCMV- and T7-DNA. 2A. 1.13 and 0.56 yg of 3 2P labelled T7 DNA and 3.1 and 5.8 yg of 3 2P labelled MCMV DNA,were each dissolved in one ml of 2 x SSC. Each reaction mixture was made up to 500 yg DNA/ml with calf thymus DNA. Fig. 2B. According to the straight line equation: C TT" = 1 + K.C T C o The slope of each of the curves intercepting the Y axis at 1 are equal to K, the reassociation constant of that DNA. The specific activity of the DNAs were: T7 DNA = 1 x lOVpm/yg 0 MCMV DNA = 4 x 10Vpm/yg • o . 0 7 5 C o T 15 M C M V APPENDIX I: C a l c u l a t i o n of the K i n e t i c Complexity of MCMV DNA. DNA K MCMV 11.11 T7 88 There i s a 1.1% decrease i n the value of K for each percent increase i n G + C content ( G i l l i s , et a l . , 1970). The dif f e r e n c e i n G + C content of MCMV and T7 i s = 59-48 = 11%. The experimentally determined K value of MCMV DNA i s therefore 12.1% lower than i t would be i f i t s G +C content was the same as that of T7 DNA. .*. Adjusted K for MCMV DNA = 11.11 + 1.34 = 12.45 .". C tx of MCMV DNA = 1 = .0803 12745 C t x MCMV DNA .0803 0 2 = = 7 04 T7 DNA - ° 1 1 4 MW of T7 DNA (and therefore i t s complexity i n view of the lack of r e s t r i c t i o n s ) = 2.6 x 10 7. .*. Complexity of MCMV DNA = 2.6 x 10 7 x 7.04 = 1.83 x 10 8 I I . Absence of Ribonucleotides Linked to MCMV DNA DNAs from various sources, including bacteriophages T2, T4 (Speyer, et a l . , 1972), T5 (Rosenkranz, 1973), Colicinogenic factor E x ( B l a i r , et al.,1972 )» and herpes simplex type 1 (Hirsch and Vonka, 1974; Biswal, et a l . , 1974), have been found to contain ribonucleo-tides. I t has been suggested that i n the case of HSV-1, these ribonucleotides are responsible for fragmentation of the genome i n al k a l i n e sucrose gradients (Biswal, et a l . , 1974). As MCMV DNA also exhibits a heterogeneous pattern of peaks when analyzed on a l k a l i n e sucrose gradients (Mosmann and Hudson, 1973), DNA p u r i f i e d from MCMV was examined for the presence of ribonucleotides. MCMV DNA was l a b e l l e d with either 3H-TdR or 3H-UR as outlined i n the Materials and Methods Section. I t was anticipated that 3H-TdR would be incorporated into DNA as thymidine residues, whereas 3H-UR would be incorporated into RNA as uri d i n e residues or into RNA or DNA as c y t i d i n e or deoxycytidine residues (Hirsh and Vonka, 1974; Biswal, et a l . , 1974). P u r i f i e d DNAs l a b e l l e d with either nucleotide were analyzed on isopycnic CsCl gradients. Both DNAs banded at a density of 1.718 (F i g . 3). Fractions containing r a d i o a c t i v i t y were pooled, the DNA pr e c i p i t a t e d , dissolved i n TNM buffer, and incubated with RNase ;or' DNase, or heated i n 0.5 N KOH (Table I ) . Treatment with RNase caused a decrease of 14 to 23% i n the TCA p r e c i p i t a b l e r a d i o -a c t i v i t y associated with 3H-UR l a b e l l e d DNA. This can probably be attr i b u t e d to traces of DNase i n the ribonuclease preparation as a si m i l a r decrease was also observed when 3H-TdR l a b e l l e d DNA was treated with RNase. None of the r a d i o a c t i v i t y associated with either DNA was Fig. 3.—Banding of 3H-UR and 3H-TdR labelled MCMV DNA in CsCl gradients MCMV DNA labelled with either 3H-UR (A) or 3H-TdR (B) in TNE buffer were made up to an average density of 1.72 gm/ml with solid CsCl and centrifuged in the buckets of a SW 50.1 Berkman rotor for 60 hours at 35 x 10 3 rpm. 20 drop fractions were collected from the bottom of the tubes, and the retractive index of every 4th fraction measured 0.5 ml of TNE was added to each fraction and the radioactivity in 25 yl measured. radioactivity - ( A ) in cpm x 10~3 density of CsCl - ( • ) in gm/ml 44. Fig. 3.—Banding of 3H-UR and 3H-TdR labelled MCMV DNA-^l'nSCsd^radients. •H (ff fl Q F r a c t i o n No. TABLE 1.—Sensitivity of 3H-TdR and 3H-UR labelled DNA to Ribonuclease, Deoxyribonuclease and a l k a l i . Sample Test no Control RNase (50 yg) DNase (50 yg) 100°C Control KOH 0.5 N 100°C 3H-UR MCV DNA 1 5,103 4,426 (0.86) 533 (0.1) 4,941 4,687 (0.94) 2 4,760 3,700 (0.77) 409 (0.08) 4,632 5,097 (1.1) 3H-TdR MCV DNA 1 10,234 8,475 (0.82) 1,297 (0.12) 9,665 11,125 (1.15) 2 11,352 8,745 (0.77) 700 (0.06) 10,290 10,240 (0.99) 3H-UR 3T3 RNA 1 11,958 170 (0.014) - 12,900 352 (0.027) 2 5,463 243 (0.044) - 7,332 404 (0.05) 125 j MCV DNA 37,045 RNase (400 yg/ml) 36,448 (0.98) DNase (20 yg/ml) 6,263 (0.16) s e n s i t i v e to heating i n 0.5 N KOH. 3H-UR l a b e l l e d 3T3-RNA was almost completely degraded when treated with RNase and KOH under s i m i l a r condi-tions. MCMV DNA l a b e l l e d i n cytosine residues with 1 2 5 I , when treated with RNase that had been heated i n b o i l i n g water for 10 mins. was sol u b l i z e d to less than 2%. II I . Fragmentation of MCMV DNA with EcoRi Endonuclease 3 2 P l a b e l l e d or unlabelled MCMV (K 181) DNA was treated with the r e s t r i c t i o n endonuclease EcoR^, and the digest electrophoresed on 1% or 0.5% agarose gels. F i g . 4A shows a densitometer tracing of an autoradiogram of a 1% gel. 19 bands were v i s i b l e . The bands B-C, D and E could be resolved further into t h e i r constituent bands on 0.5% gels. Band B-C comprised 5 bands; B,B^, B"", C,.C"; band D, two bands, Di and D2; and band E, two bands, Ej and E 2 . Although 0.5% gels provided better r e s o l u t i o n , the bands were more d i f f u s e than those resolved on 1% gels. The molecular weights of the fragments were determined by co-electrophoresing MCMV DNA digests with bacteriophage A DNA and X spc-1 DNA fragmented with EcoR^ (Fig. 4B). The molecular weight estimates of of the A-DNA fragments have been documented by Fiandt, et a l . (1976). MCMV DNA fragments ranged i n s i z e from 20 to 1 x 10 6 (Table IT) and the sum of t h e i r molecular weights was equal to 1.33 to 1.36 x 10 8. This f i g u r e agrees c l o s e l y with the molecular weight of MCMV DNA as determined by Mosmann and Hudson (1973). The fragments described above were generated by incubating MCMV DNA with EcoRi at a concentration of 35 units of enzyme per microgram of DNA for 1 hr at 37°C. Incubation for longer periods of time, or with higher concentrations of enzyme, did not r e s u l t i n any change i n Fig. 4A—-MCMV (K 181) DNA fragments generated by EcoRi endonuclease: electrophoresis on 1% agarose slab gel. DNA digests were electrophoresed on a 1% agarose slab gel for 24 h at 35V in 4x gel buffer. After electrophoresis the DNA bands were stained with 4 yg Ethidium bromide/ml of lx gel buffer for 1 h. The bands were visualized under UV light, photographed and the negative analysed on a densitometer. Fig. 4B.—Comparison of the mobility of MCMV DNA fragments with fragments of known molecular weight derived from X and X spc-1 DNA, on a 0.5% agarose gel. X fragments - • X spc-1 fragments - # \ MCMV fragments - A -+ S 48. TABLE II.—Murine cytomegalovirus DNA fragments generated by EcoR^. Fragments MW(x 106) % of total MW % of total radioactivity Ratio: % radioactivity/% MW A 18-20 14 18 1.28 B 9.6 7.2 B' = 9.6 7.2 W" 9.0 6.8 - 25 28 1.12 C 8.3 6.2 <r = 8.3 6.2 D D' 6.9 = 6.9 5 ' 2 - 10.4 5.2 10.4 1.0 El E 2 ' 6.0 5.7 4 , 5 - 88 4.3 12 1.3 F 4.8 3.6 3.8 1.05 G 4.6 3.4 3.2 0.94 H 4.2 3.1 2.7 1.0 I 3.6 2.7 2.7 1.0 J 3.48 2.6 2.7 1.03 K 3.3 2.5 2.1 0.84 L 2.9 x 2 2.19, 4.3 2.04 M 2.7 2.0 1.6 0.8 N = 2.7 2.0 1.6 0.8 0 2.2 1.6 1.6 1.0 P 2.0 x 2 1.5 2.2 1.46 Q 1.6 1.2 1.09 0.9 R 1.3 0.98 1.09 1.11 S 1.1 0.83 1.0 1.25 T 1.0 135 + 137 the electrophoretic pattern of the digests. However when MCMV DNA was digested for less than 20 minutes at 37°C numerous additional frag-ments, probably the result of partial digestion, were vi s i b l e . Autoradiograms of gels containing 3 2P labelled DNA were scanned on a Helena Quick Scan Jr. densitometer fitted with an integrator. The area of each peak was taken as a measure of the radioactivity associated with that fragment, and was expressed (Tablell) as a per-centage of the sum of the areas of the peaks on the gel. Column 5 in Table 2 shows the ratio of the percentage of radioactivity associated with each band to i t s size expressed as a percentage of the total molecular weight of the genome. In a l l cases, except for the low molecular weight bands L and P (which probably comprise two fragments each) the ratio is close to one, implying that the fragments are present in the digest in unimolar quantities. Tracings of autoradiograms exposed for two different time intervals gave similar results. IV. Relative Density of High Molecular Weight Fragments MCMV DNA appears to be internally heterogeneous in base composition. Intact DNA bands in CsCl as a homogeneous collection of molecules of density corresponding to 59% G + C. Molecules sheared to quarter size or smaller, however, band at two discrete densities corresponding to 57.5 and 61.5% G + C (Mosmann and Hudson, 1973). To determine whether this heterogeneity was also reflected in fragments generated by EcoRj, the following experiment was conducted. 3 2P MCMV DNA was digested with EcoRn, purified, and centrifuged in CsCl i n a type 50 rotor at low speed to obtain maximum density separation. Most of the radioactivity banded at the density of MCMV DNA (1.718), but prominent shoulders were observed at densities 1.723 and 1.716 (Fig. 5). Radioactive fractions were combined into three pools: Fractions 14-16 into the 'heavy' pool, fractions 17-20 into the 'medium' pool and fractions 21-24 into the 'light' pool. The three pools and the unfrac-tionated digest were each electrophoresed on a 1% agarose gel, auto-radiographed and analyzed on a densitometer. It was expected that fragments that diverged from the density of intact MCMV DNA would be present in a graded manner in the three pools, i.e. greater amounts of the denser fragments would be present in the 'heavy' pool than in the 'medium' and 'light' pools and vice-versa. The results are presented as histograms in Fig. 6. A descending histogram denotes a 'heavy' fragment whereas an ascending histogram denotes a 'light' fragment. Severity of ascent or descent indicates divergence from average density. Fragment A, which makes up approximately 15% of the genome,as well as fragments F and H,are denser than the other fragments, whereas frag-ments B, B', B", C, 0,' (which together make up 25% of the genome, and appear to have a similar density) and fragment G,are lighter than the other fragments. V. Effect of High Multiplicity Passage on the Restriction Endonuclease Pattern of MCMV DNA When HSV-1 is serially passaged in tissue cultures at a high input multiplicity, a decrease in the yield of infectious virus produced by the cells is observed. These virus populations contain DNA that differs in buoyant density and restriction endonuclease patterns from DNA Fig. 5.—Analysis of MCMV (K 181) DNA fragments on a CsCl gradient. 3 2P MCMV DNA digested with EcoR^, was deproteinated and centri-fuged in CsCl in a Type 50 Beckman rotor at 30 x 10 3 rpm for 72 h. 10 drop fractions were collected from the bottom of the tube and the refractive index of every f i f t h fraction determined. 0.1 ml of TNE was added to each fraction and radioactivity in 10 yl measured. Radio-active fractions were combined into three pools: fractions 14 -»- 16 into the 'heavy' pool, fractions 17 -»- 20 into the medium pool, and fractions 21 -> 24 into the 'light' pool. Arrow marks density of intact MCMV DNA (1.718 g/ml). Fig. 5.—Analysis of MCMV (K 181) DNA fragments ona CsCl gradient. F r a c t i o n No. Fig. 6.—Relative density of MCMV (K 181) DNA fragments. 'Heavy', 'medium', and 'light' pools (Fig. 5)^ and the unfrac-tionated digest^were dialysed overnight against 4x gel buffer and electrophoresed on a 1% agarose slab gel. The gel was then dried and autoradiographed for two weeks. Autoradiograms of the restriction patterns were scanned on a densitometer and the radioactivity associated with each peak (or the area of each peak) determined on the integrator. Fig. 6A. The relative areas of the bands A, B-C, D, E, and E 2 for the three pools are expressed as a fraction of the total area of the bands. e.g. Area of band A in 'heavy' pool ' etc. Total area of bands A to E 2 in 'heavy' pool 'heavy* pool |||||| 'medium' pool jjjjjj 'light' pool Fig. 6B. The relative areas of bands F -»- L in the 'medium' and 'light' pools were calculated as a fraction of the sum of the areas of bands F to L. It was necessary to consider only the 'medium' and the 'light' pools for fragments F -»• L because in the 'heavy' pool (which contained the least amount of radioactivity) these bands were barely visible. 'medium' pool 'light' pool |H 55. Fig. 6.—Relative density of MCMV (K 181) DNA fragments. extracted from virus passaged at a low m.o.i. (Bronson, et a l . , 1973; Frenkel, et a l . , 1975; Frenkel, et a l . , 1976). Freshly isolated salivary gland virus was passed once in mouse embryo fibroblasts at a low m i l t i p l i c i t y of infection and subsequently used to infect centrifugally mouse embryo cells at a multiplicity of 200 plaque forming units per c e l l . This was continued for several passages. V i r a l DNAs from passages 1, 2, 3, 5 and 7 were treated with EcoRx and the digests analyzed on 1% agarose gels (Plate 1). Serial high m.o.i. passage appeared to have no effect on the number or size of the fragments generated by EcoR^. The enzyme generated 25 fragments from DNA extracted from MCMV passages 1 through 7. There also appeared to be no change in the relative molar concentrations of the fragments as the staining intensity of the bands remained unchanged. Over seven serial passages the t i t r e of infectious virus produced by ME cells remained in the range of 1.3 to 1.65 x 10 8 plaque forming units per million cells (Table III). VI. Comparison between MCMV (K 181) and MCMV (Smith) Our laboratory possesses two strains of the murine cytomegalovirus. The 'Smith' strain obtained from the American Type Culture Collection, and the 'K 181' strain obtained from Dr. June Osborn. Viruses of both strains appear to be identical in negatively stained preparations viewed by electron microscopy, exhibiting typical herpes virus like nucleocapsids 85 nm in diameter and a preponderance of multicapsid virions (Hudson, Misra, Mosmann, 1976a). Both strains exhibit similar cytopathic effects in mouse embryo fibroblasts and 3T3 cells, and intact DNAs from the two PLATE 1.—Effect of high multiplicity passage on the restriction enzyme fragmentation pattern of MCMV (K 181) DNA. DNA from virus obtained by high multiplicity passages 1, 2, 3, 5, and 7 were digested with EcoRj and the digests analysed on a 1% agarose slab gel. DNA bands were stained with ethidium bromide, illuminated with UV light and photographed. 58. 59.' TABLE I I I . — E f f e c t of serial 'high-multiplicity'.passage on the infectivity of murine cytomegalovirus. Passage # pfu/10 6 cells (x 10 8) 1 1.60 2 1.41 3 1.50 5 1.30 6 1.64 7 1.55 strains band at a density of 1.718 in CsCl gradients. The extent of homology between the DNAs of the two strains was examined by reassoci-ation kinetics and by comparison of fragments generated by the restriction enzymes EcoR^ and Hind III. 3 2P labelled DNA from the Smith and K 181 strains of MCMV were reassociated in 2 x SSC at 67°C, in the presence of a 10 fold excess of DNA from the same or heterologous strain. Labelled DNA from either strain reassociated with identical kinetics in the presence of homologous or heterologous DNA with a C t x of 9.0 0 2 x 10"2 (Fig. 7). To determine whether there was any degree of base mismatch between the heterologous DNAs, the reassociated molecules were dialyzed against 0.1 x SSC, sealed into 100 yl corning micropipettes, placed in a Haake circulating water bath, and the temperature slowly increased from 25°C to 100°C. Melting of double stranded DNA was monitored by i t s sensitivity to Si nuclease. Fig. 7B depicts the melting profiles of 3 2P labelled K 181 DNA reassociated with homologous or heterologous DNA. The profiles are identical, indicating less than 1.4% base mismatch between hetero-logous DNAs (Laird, et a l . , 1969 ) . DNA from the two strains were digested with EcoRi or Hind III endonucleases, electrophoresed on 1% agarose gels, stained with ethidium bromide and photographed. Fig. 8 depicts the densitometer tracings of the restriction patterns. The EcoRi patterns are similar with the exception of the 4.6 x 106 fragment G, which in Smith DNA is present in considerably less than unimolar quantities. An increase in the Fig. 7A.—Reassociation kinetics of 3 2P labelled MCMV (K 181) and (Smith) DNAs in the presence of excess homologous or heterologous unlabelled DNA. 3 2P labelled MCMV (K 181) and (Smith) DNA (1.23 and 1.30 x 10 5 cpm/yg) were mixed with a ten fold excess of unlabelled homologous or heterologous DNA. A l l samples were made up to 500 yg/ml with calf thymus DNA in 2 x SSC, sonicated, sealed in 100 yl micropipettes, denatured and allowed to reassociate at 67°C. Reassociation was monitored as a decrease in sensitivity of 3 2P labelled DNAs to Sj nuclease. 3 2P K 181 DNA + unlabelled K 181 DNA • 3 2P K 181 DNA + unlabelled Smith DNA S 3 2P Smith DNA + unlabelled Smith DNA # 3 2P Smith DNA + unlabelled K 181 DNA © Fig. 7B.—Melting curves of heterologous and homologous hybrids. Homologous and heterologous reaction mixtures which had been permitted to reassociate i n 2 x SSC to a Log^o C^T of 1.5, were dialysed overnight against 0.1 x SSC and sealed into 100 y l micropipettes. Hybrids were melted by gradually increasing the temp, of a Haake water bath iii which the micropipettes were submerged. Melting of the molecules was measured by an increase in their sensitivity to Sjnuclease. 3 2P labelled MCMV (K 181) DNA a) homologous A b) heterologous H Fig. 7A.—Reassociation kinetics of 3 2P labelled MCMV (K 181) and (Smith) DNAs in the presence of excess homologous or heterologous unlabelled DNA. Fig. 7B.—Melting curves of heterologous and homologous hybrids. 90 60 30 80 T e m p . ( C ° ) Fig. 8.—A comparison of the restriction enzyme fragmentation patterns of MCMV (K 181) and (Smith) DNA. K 181 and Smith DNA samples were treated with either EcoR^ or Hind III and the digests analysed on a 1% agarose gel. The figure depicts densitometer tracings of the restriction patterns. 64. .—A comparison of the restriction enzymes fragmentation patterns of MCMV (K 181) and (Smith) DNA.. molecular weight of any of the other fragments by 4.6 x 105, as might be expected i f Smith DNA lacked one EcoRi site responsible for fragment G, was not observed. K 181 DNA appears to lack the Hind III generated fragments F (9.7 x 106) and K (1.4 x 106) that are present in Smith. Again the lack of a DNA fragment with a molecular weight of 10 x 10 6 was apparently not compensated for by any of the other K 181 fragments. CHAPTER IV RESULTS 2. Transcription of the Murine Cytomegalovirus Genome in Lytic Infections The genome of murine cytomegalovirus i s a double stranded DNA molecule 1.32 x 10 8 in size. Experiments described in this chapter were designed to elucidate the manner in which the expression of this molecule is controlled at the transcriptional level during l y t i c infections caused by the virus. The techniques used were similar to those described by Frenkel and Roizman (1972), and were based on the kinetics of conversion of single stranded DNA fragments, in the presence of a large excess of infected c e l l RNA, to DNA-RNA hybrids. The technique is a one phase hybridization reaction and i t s primary advantage over the two phase, filter-hybridization technique is that i t does not depend upon isotope incorporation, and therefore upon constant levels of precursor pools. As w i l l be described later, v i r a l DNA synthesis in infected cells begins about 6-8 hours after infection with MCMV. By convention, RNA in infected cells at 6 hours post infection, i.e. before the onset of v i r a l DNA synthesis, was termed 'early' RNA, whereas RNA present in cells 24 h.p.i., i.e. after the onset of v i r a l DNA synthesis, was termed 'late' RNA. I. Experimental Design and Treatment of Data The kinetics of hybridization of 1 2 5 I MCMV DNA with a large excess of unlabelled whole c e l l RNA were studied. DNA-RNA hybrids were identified by virtue of their insensitivity to the single strand specific nuclease, S x. The results obtained by this technique were readily amenable to analytical treatment. The analysis identified the fraction of the genome represented as transcripts in a particular RNA preparation, as well as classes of RNA differing in molar concentration. However, i t could only be used to measure stable RNA species which arose by asym-metrical transcription, and did not reveal the time of synthesis of the various species, nor their turnover rates. The reaction of single stranded DNA fragments with homologous RNA under conditions where the reassociation of the DNA is negligible, due to i t s low concentration, can be described by the equation: (1) - 4r = K.R.C. dt Where t is the length of incubation, C i s the concentration of single stranded DNA at time t, R is the molar concentration of single stranded RNA, and K is the hybridization rate constant. Equation (1) is a modification of the second order equation, ^ = - KC2 dt K C derived by Britten and Kohne (1965) to describe reassociation kinetics of DNA. Under conditions of RNA excess,- where the concentration of RNA in hybrid i s small compared to that of single stranded RNA remaining, R can be assumed to equal to R q , the input R N A concentration. Equation (1) upon integration yields: (2) C - K . R . t - = e o where C q is the i n i t i a l concentration of single stranded D N A . Equation (2) assumes that a l l the D N A is transcribed to yield R N A with a single abundance, R q . This equation can be applied to a more general case with n classes of R N A , each appearing in molar concentration, R ^ . Such a reaction can be actually visualized as the sum total of the independent reactions of each R N A class with the D N A homologous to i t . For each such class the fraction of D N A remaining single stranded i s : ( 3 ) C n - K . R . t However, ( C )n equals C .a , where a is the fraction of the total o o n n v i r a l D N A serving as a template for this R N A class, and C q is the total input D N A . It follows therefore, that: <4> Cn - K . R .t = e n n C .a o n The observed fraction of single stranded D N A remaining at time t would be: /c.\ C Cj C o o o Where l-(an + .... a ) is the fraction of DNA that is not transcribed n and w i l l therefore remain single stranded throughout the hybridization. Therefore: (6) ^ - K i . R i . t + a .e Kn"^n" t + l - ( a i + a ) — = a i . e 1 1 • • n 1 n 0 The absolute numerical value of the DNA-RNA hybridization rate constant K i s required for calculations of the absolute values of RNA concentrations i n the c e l l . It i s not required, however, for the calculation of the relative concentration of the RNA species, or for determination of the size of template from which they are transcribed. Assuming the rate constants for a l l species of v i r a l RNA to be the same, equation (6) can be written as: (?) c - P i t _ i _ -e t , . — = a i . e 1 + a .e n + l-(oti + a ) C n 1 n o Where Bi . . . . B are the products of the molar concentrations of the x n ; RNA species (Rx R ) and the rate constant, K. Comparison of the values of B for the different RNA species is then an indication of their relative concentrations in the infected ce l l s . Q — for each time point, t, was determined experimentally. a and 0 C B values were calculated from a — vs t plot fitted to equation (7) on L i 0 an IBM 370, model 168 computer, by non-linear, least-squares optimization of the parameters through and B i through B n , for n = 1, 2, etc. The actual curves were generated with the aid of a Hewlett Packard 9810A calculator fi t t e d with a 9862A plotter. Curves that best f i t equation (7) were obtained using a non-linear optimization routine called FLETCH. This program was written by R. Fletcher and Associates at the Atomic Energy Research Establishment in Harwell, England and is documented in UBC-NLP, 1975. FLETCH minimizes the function value, F, or the sum of the squares, defined as: data points Ci , - S i t i , -3 t i , . . — - (.oii.e x + a .e n + 1-ai .... a ) C n 1 n o i=l C l Where — is the experimentally determined dependent variable of the 0 fraction of DNA remaining single stranded at time, t. It follows, therefore, that the closer the fi n a l function value is to zero, the better the f i t . The best f i t values for a and 3 were evaluated by giving FLETCH an i n i t i a l parameter estimate (or estimates) for 6 (n = 1) or 3i and 3 2 (n = 2). When the optimal values for a and 3 had been obtained, the programme was repeated with several different starting parameters (i.e. 3 estimates) to determine whether a l l runs converged to the same values of a and 3 • This procedure insured that the optima were 'real' or 'global' and not just a function of the i n i t i a l parameters specified. The concentration of 1 2 5 I - l a b e l l e d MCMV DNA in the hybridization mixtures was rigorously controlled to minimize self-annealing. Although reassociation of the iodinated probe in no case exceeded 5%, i t could seriously affect calculations for determining the various parameters, particularly in cases where a was less than 10%. DNA self-annealing was therefore monitored by incubating the iodinated probe with heterologous RNA under conditions identical to those utilized for test RNA. A (8) F = c 1 - ~ vs t plot of the DNA probe in the presence of heterologous RNA gave a straight line with slope = 1.3 x 10 ^ and intercept at 0. For a l l hybridization reactions each — value was then corrected for self 0 c annealing of the probe by adding to i t the corresponding 1 - — value, o calculated from the equation (Fig. 9). (9) 1 - = 0 + 1.3 x 10 _ t +.t o II. Characterization of Nuclease The crux of the one phase hybridization technique is the a b i l i t y to quantitate accurately the formation of RNA-DNA hybrids. This can be achieved either by separation of hybrids from unhybridized DNA on hydro-xyapatite, or by the selective degradation of unhybridized DNA by single strand-specific nucleases. Although both techniques were used with similar results, digestion of unhybridized DNA with the single strand specific nuclease was the favoured method, as i t was less time-consuming and simpler than the use of hydroxyapatite. A l l hybridization results documented in this chapter were generated with the Si nuclease technique. Sj i s a nuclease purified from Aspergillus oryzae. It is relatively inexpensive and readily available commercially. S^ possesses exo- as well as endo-nucleolytic activity against single stranded nucleic acids from a wide variety of sources. Its activity is stimulated by 10 - l t M Zn 2 + and inhibited by large concentrations of RNA (Ando, 1966; Sutton, 1971). The enzyme is unique i n that i t requires at least 1 yg of substrate per 10 units of enzyme for maximum activity. This 'plateau-effect' is thought to be due to a competing reaction which modifies single stranded Fig. 9.—Correction of data for self annealing of iodinated probe. DNA self-annealing was monitored by incubating the iodinated probe with heterologous (yeast) RNA under conditions identical to those used for test RNA. Q a) A 1 - — vs t plot of the DNA probe in the presence of yeast RNA o gave a straight line with slope = 1.3 x 10-1+ and intercept at 0. b) For a l l hybridization reactions each — value was corrected for 0 C self annealing of the probe by adding to i t the corresponding 1 - — 0 value calculated from the straight line equation. 1 _ | j _ = o + 1.3 x 10~k.t o Experimental slope Corrected slope 73. Fig. 9.—Correction of data for self annealing of iodinated probe. k Experimental i tzitzi H O U R S i s e a DNA i n a manner such as to render i t resistant to further hydrolysis by the enzyme (Ando, 1966; Sutton, 1971). In view of this property, denatured calf thymus DNA was added to a concentration of 10 yg/ml to every digestion mixture. Before using S^ to analyze hybrids i t was necessary to confirm i t s a b i l i t y to degrade single stranded rather than double stranded nucleic acid molecules, under the conditions of salt and RNA concentration used in the hybridization reactions, and to establish optimal conditions of enzyme concentration and time of incubation. The data in Table IV demonstrate the effect of NaCl and RNA concen-tration on the sensitivity of native and denatured 3H-labelled 3T3 DNA to Sj. In 0.1 M NaCl the enzyme was active against single stranded DNA, and also, albeit to a very small extent, against native DNA. This marginal activity against native DNA could be decreased further by increasing the NaCl concentration in the digestion mixture to 0.3 M. RNA had a slightly inhibitory effect on the activity of the enzyme. However, since this inhibitory effect was minimal, and since a l l hybridi-zation mixtures would contain the same amount of RNA, i t would not be expected to affect the results to any significant extent. Fig. 10A and 10B il l u s t r a t e the effect of increasing concentrations of enzyme and different periods of incubation on the susceptibility of denatured and native DNA to Sj nuclease. The data are self-explanatory and on this basis, a l l subsequent samples were digested for 1.5 hours with 60 units/ml of enzyme. 75. TABLE IV.—Effect of RNA and NaCl concentration on activity of Sx nuclease against single (SS)- and double-stranded (DS) DNA. Sample DNA Si (units) NaCl : RNA TCA ppt cpm % sensitivity 1. DS - .IM - 5.73 x 10 5 0.0 SS - .IM - 1 T 2. DS 60 u/ml .IM • - 534,480 6.8 SS 2,855 99.51 3. DS i i .IM 250 yg/ml 533,417 7 SS .IM 11 12,785 97.8 4. SS .IM 500 yg/ml 17,759 96.9 5. DS .3M 250 yg/ml 550,340 4 SS .3M i i 17,142 97 Concentration of DNA in digestion mixture was approximately 15 yg/ml. 76. Figure 10A.—Effect of increasing concentrations of on the suscepti-b i l i t y of 3H labelled single and double stranded DNA to the enzyme. 3H-labelled heat denatured or native 3T3 DNA was treated with i n -creasing amounts of Sj nuclease for 1.5 hr at 37°C. Each reaction mixture contained approximately 10 yg of DNA/ml. Undigested material was precipitated with cold 10% TCA, trapped on f i l t e r s and counted. Results are depicted as TCA insoluble cpm expressed as percentage of TCA insol. cpm without treatment with Si." Native DNA • Denatured DNA • Fig.lOB.—Effect of time of incubation on the susceptibility of denatured and native 3H-labelled 3T3 DNA to Sj nuclease. Native or denatured DNA in Si buffer was heated with 50 units/ml of Si nuclease for different periods. Insoluble material was then precipitated and radioactivity counted. 77. Fig. 11 is a comparison of the melting curve of 3T3 DNA in 0.1 x SSC with i t s sensitivity to Sj nuclease. 3H-labelled 3T3 DNA was heated in 0.1 x SSC from 20°C to 100°C. The melting curve of 3T3 DNA correlates well with i t s susceptibility to SI nuclease, further substantiating the single strand specific nature of the enzyme. III. Determination of the Onset of Viral DNA synthesis in Mouse Embryo Cells Infected with MCMV In order to select a time after infection when cells contain only RNA transcribed before the onset of v i r a l DNA replication, i t was imperative to determine accurately when v i r a l DNA Synthesis began in infected cells. Onset of v i r a l DNA synthesis in MCMV infected cells has been estimated at 12 h.p.i. These estimates were based on the time of appearance of radioactivity in v i r a l DNA in infected cells labelled with 3H-TdR (Moon, et a l . , 1976) or on the a b i l i t y of IUdR to inhibit the synthesis of infectious virus (Henson, et a l . , 1966). The above techniques relied upon the continued presence of thymidine kinase in infected cells for the phosphorylation, of. radioactive or halogenated nucleotides before their incorporation into v i r a l DNA. However, this requirement is not met in MCMV infected cells, since the cellular enzyme activity decreases after infection and the virus does not code for a new thymidine kinase (Muller and Hudson, 1977). In view of this, MCMV DNA synthesis was reassessed by a technique that did not depend on radioisotope incorporation or a constant level of precursor pools. The technique was based upon the principle that the rate of reassociation of 1 2 5 I - l a b e l l e d MCMV DNA is accelerated by Fig. 11.—Comparison of the melting curve of 3H-3T3 DNA as monitored by a change in A26O a n d bY increased sensitivity to Si nuclease. 3H-labelled 3T3 DNA was heated in 0.1 x SSC from 20°C to 100°C. At various temperatures, duplicate samples were removed, quenched in ice water, and their absorbance at 260 nm measured. (Results are expressed as percentage of total increase in absorbance). Samples were then diluted in Si buffer and heated with 60 u/ml Si nuclease for 1.5 hr., precipitated with cold TCA, and radioactivity measured. The results ( # ) are expressed as percentage of total increase i n sensitivity to Si'. 80. Fig. 11.—Comparison of the melting curve of 3H-3T3 DNA as monitored by a change in A 2 6 o a n d b v increased sensitivity to Sj nuclease. unlabelled MCMV DNA, the extent of this acceleration being proportional to the mass of the unlabelled DNA. The kinetics of DNA-DNA reassociation as described by Britten Q and Kohne (1965) follow the equation o _ 1 A plot of C C o Tj - vs t results i n a straight line with slope directly proportional to the v i r a l DNA concentration, i f the reaction follows second order kinetics. C Fig. 12 is a plot of vs t for 1 2 5 I - l a b e l l e d MCMV DNA in the presence of different quantities of unlabelled MCMV DNA. It shows that the reassociation of 125I-MCMV DNA follows second order kinetics and C that the slope of — vs t plot is proportional to the concentration of unlabelled v i r a l DNA. This relationship was exploited to determine the concentration of v i r a l DNA in DNA extracted from mouse cell s . C Fig. 13A shows a —— vs t plot for the DNA samples extracted from infected and uninfected mouse c e l l s . The determination of absolute quantities of v i r a l DNA in infected cells and the calculations of v i r a l genome equivalents (Fig. 13B) were based on the following data: A. The MCMV genome has a molecular weight of 132 x 10 8 (Mosmann and Hudson, 1973) and a kinetic complexity equal to i t s molecular weight. B. The molecular weight of diploid mammalian DNA is 3.92 x 10 1 2 (Gelb, et a l . , 1971). ~ There was an increase i n infectious virus between 8 and 13 h.p.i. (Fig. 13B) which was in agreement with results published by other workers for mouse embryo cells infected at an input multiplicity of 10 pfu/cell. Fig. 12.—Reassociation of i Z b I labelled MCMV-DNA in the presence of unlabelled MCMV DNA. 4 ng/ml of 1 2 5 I - l a b e l l e d MCMV DNA (10 5 cpm) were reassociated in 2 x SSC at 67°C in the presence of:- (i) 0.00 yg ( + ), ( i i ) 0.13 yg/ml (• ); ( i i i ) 0.26 yg/ml (• ); or (iv) 1.31 yg/ml ( A ) unlabelled MCMV DNA. The concentration of unlabelled DNA i n each reaction mixture was adjusted to 500 yg/ml with calf thymus DNA. The slopes were calcu-lated by means of a Hewlett—Packard HP9810 calculator fit t e d with a HP9862A calculator plotter, and were, for (i) 0.0002; ( i i ) 0.010; ( i i i ) 0.019; (iv) 0.100. Fig. 13.—Viral DNA synthesis i n ME fibroblasts infected with MCMV. A. Reassociation kinetics. B. Vi r a l genome equivalents and plaque forming units per c e l l . A. 4 ng/ml of 1 2 5 I - l a b e l l e d MCMV DNA (105cpm) were reassociated in the presence of (i) 93 yg/ml of uninfected c e l l DNA (•); ( i i ) 64 yg/ml DNA from cells 2 h.p.i. (•); ( i i i ) 88 yg/ml of DNA from cells 6 h.p.i. (<Q); (iv) 94 yg/ml DNA from cells 8 h.p.i. (•); (v) 101 yg/ml DNA from cells 13 h.p.i. (•); (vi) 95 yg/ml DNA from cells 15 h.p.i. ( V ) ; (vii) 114 yg/ml DNA from cells 22 h.p.i. ( A ) . Total unlabelled DNA in each reaction mixture was adjusted to 500 yg/ml with calf thymus DNA. Slopes calculated for each sample were as follows: (i) 0.0003; ( i i ) 0.0048; ( i i i ) 0.0036; (iv) 0.0076; (v) 0.041; (vi) 0.136; (vii) 0.252. B. The concentration of v i r a l DNA in each of the above samples was calculated from the data in Fig. 12 and were expressed as genome equivalents: 1 genome equivalent - 1 yg of v i r a l DNA/2.96 x IO1* yg c e l l DNA. • - log^Q genome equivalents per c e l l # - logio P f u (cells + supernatants) per c e l l 85. 86. Vir a l DNA was found associated with infected cells as early as 2 h.p.i. This probably represents input parental DNA and at 28 genome equivalents, agrees reasonably well with the input multiplicity of 10 pfu/cell. The v i r a l DNA content of the cells decreased slightly between 2 and 6 h.p.i. followed by an almost two fold increase between 6 and 8 hours, when v i r a l DNA synthesis probably commenced. Viral DNA content of the cells continued to increase u n t i l 22 h.p.i. (the last time point examined) when each c e l l contained almost 900 genomes of v i r a l DNA. Similar results were obtained for MCMV-infected exponen-t i a l l y growing 3T3 cells. IV. Transcription of MCMV DNA in Lytically Infected Cells A. Abundance classes of RNA and the fraction of the genome transcribed. Unlabelled RNA extracted from cells 6 h (early) and 24 h (late) after infection was hybridized to sheared, denatured, 125I-MCMV C DNA. Experimentally determined residual single strand DNA(—)values for various time points were fitt e d to equation (7) for n = 1, 2 etc. Fig.l4A illustrates the data from the hybridization reaction using 'early' RNA f i t t e d to n = 1, and in Fig. 14B to n = 2. When more than two parameters were used (i.e. n = 3 or more) the results were meaningless. As observed from the function values of the two curves, the curve for n = 2 f i t s the data slightly better than the curve for n = 1. Figures 15A and 15B depict a similar treatment of 'late' RNA. Fig. 14.—Transcriptional analysis of RNA extracted from MCMV infected ME cells 6 hr. post infection. (Early) 5 ng of iodinated MCMV DNA (5 x 10 7 cpm/ug) were added to 500 yg of 6 hr. RNA in 1 ml of 2 x SSC. Hybridization was carried out as outlined in the text. Figures are computer hard copies of: A. Data f i t t e d to equation (7) for 1 class of RNA. (n = 1) B. Data fi t t e d for 2 classes of RNA. (n = 2) Fig.-14.—Transcriptional analysis of RNA extracted from MCMV 88. ISI infected ME cells 6 h post infection. (Early) ® RSriZ.IZIIZIIZIlZIIZIIZI B 2 = IZI.IZIIZ!IZIIZIIZJ[ZI . 3 FINRL FUNCTION VRL-UETslZI.IZIEaZSIZIEa IZI I 1ZIIZI H O U R S I S Z I . 3 ® R I =C3.lZI5:3S:t2Il2I B I = —IZI. I IZI 5T IZI IZI IZI R 2 e l Z J . I W t Z U Z J I Z I B 2 = -IZI.IZI I H S T 12IZI FtNBL FUNCTION VHLUEs £3.01321211313 IZI I LZItZI H O U R ! LZI 89. Fig. 15.—Transcriptional analysis of RNA extracted from MCMV infected ME cells 24 hr. post infection. (Late) Details are similar to those outlined in Fig. 14 and the text. Computer hard copy of: A. Data fitt e d for 1 class of RNA (n = 1). B. Data f i t t e d for 2 classes of RNA (n = 2). 90. Fig.15.—Transcriptional analysis of RNA extracted from MCMV infected ME cells 24 h post infection. (Late) .E3 .7 R I s E L I B S E I E I E I E I B I = — E l . E l B 3 " 7 0 0 R 2 = 0 . E I E I E ! E I E I E ] B 2 = E1.E1EIEIEJE]E1 r i N R L FUNCTION VBLUCsB. iaBSHBei s r a i iz i ia i s i a H O U R S FINHL- FUNCTIDN VHL-LJEc 0.002HEB 1 > i i SIZI I IZII2] H O U R S The r e s u l t s of these experiments, summarized i n Table V, indic a t e the following: i ) 6 hours a f t e r i n f e c t i o n , c e l l s contained RNA transcribed from 23% (17.7 + 5.3) of the v i r a l genome, or assuming asymmetric t r a n s c r i p t i o n , from 46% of i t s coding capacity; i i ) 24 hours a f t e r i n f e c t i o n , genes from 37% (18.7 + 18.7) of MCMV DNA, i . e . 74% of the coding capacity of the genome, were represented i n the c e l l as stable t r a n s c r i p t s ; i i i ) both 'early' and ' l a t e ' RNA comprised two classes of RNA d i f f e r i n g i n molar concentration. In 'early' RNA, the abundant cl a s s , which was present i n a 7 f o l d excess over the scarce cl a s s , was transcribed from 5% of the genome, whereas the scarce class arose from 18% of the genome. In ' l a t e ' RNA each of the two classes arose from 18% of the genome. The abundant class was, however, present i n an 8 to 10 f o l d excess. B. 'Early' RNA i n a subset of ' l a t e ' RNA. The next question to be answered was whether 'early' and 'late' RNA were transcribed from mutually exclusive regions of the genome, or whether ' l a t e ' RNA comprised 'early' RNA as w e l l as sequences transcribed only ' l a t e ' i n i n f e c t i o n . To answer t h i s question a summation test was performed. Such an analysis i s designed to determine whether any sample of RNA contains the same nucleotide sequence as any other sample of RNA, and involves the h y b r i d i z a t i o n of a mixture of equal amounts of the two RNA samples ( i n t h i s case, 'early' and ' l a t e ' RNA). The sum of the a values of the i n d i v i d u a l RNA samples by themselves, indicates p a r t i a l s i m i l a r i t y , t o t a l s i m i l a r i t y or d i s s i m i l a r i t y between the two RNA samples (Table VI). The r e s u l t s depicted i n Table VI and F i g . 16 suggest that 'early' RNA was a subset of ' l a t e ' RNA. TABLE V.—Best f i t a and 3 values for RNA extracted from murine cytomegalovirus infected ce l l s . Sample Best f i t n = 1 or 2 •<*1 a2 ai + a2 6 i 3 2 Bi / e 2 (e) 6 h RNA 2 .054 .18 .23 .105 .014 7.2 (1) 24 h RNA 2 .18 .18 .37 .16 .018 8.7 6 h RNA + 24 h RNA (summation) (E+L) 1 .36 - .36 .03 -'C hex RNA' 2 .078 .19 .27 .152 .0101 14 'C hex RNA' + 6 h RNA (E) 2 .052 .19 .25 .105 .018 5.77 'Ara C RNA' 2 .053 .18 .23 .166 .026 6.36 TABLE VI.—Relationship between 'early' and 'late' and 'early' and 'cyclohexamide-arrested' RNA. Summation analysis Sample a values for individual samples Expected a values Observed a value i f mutually exclus ive i f subset i f partial subset i f asymmetric 'early' RNA .23 .60 .37 between .37 and < -37 .36 'late' RNA .37 . 60 'early' RNA 'C hex RNA* .23 .26 .49 .26 - ,< .26 .25 Fig. 16.—Relationship between 'early' and 'late' RNA: A summation analysis. a) 500 yg/ml of 'early', b) 500 yg of 'late', and c) a mixture of 250 yg each of 'early' and 'late' RNA, were analysed in a hybridization reaction with iodinated MCMV DNA. early RNA A. late RNA % early & late RNA . . - . p . . . : . Fig. 16.—Relationship between 'early' and 'late' RNA: A summation analysis. H D U R 5 C. Transcription of MCMV DNA in the presence of inhibitors of protein and DNA synthesis. In order to confirm that RNA in infected cells at 6 h.p.i. did i n fact represent functions expressed before the onset of v i r a l DNA synthesis, RNA from cells in which v i r a l DNA synthesis had been a r t i f i c i a l l y arrested was analysed, and compared with 'early' RNA. Two approaches were used: DNA synthesis in infected cells was inhibited either directly, by treating cells with cytosine arabinoside, or indirectly, by inhibiting protein synthesis in the cells with cycloheximide. The latter alternative was based on the observation that v i r a l DNA synthesis, at least in other herpes virus infected cells, requires prior protein synthesis and is not initiated in cells treated with inhibitors of protein synthesis from the time of exposure of the cells to the virus (Roizman, 1969). Cytosine arabinoside-treated, MCMV-infected mouse embryo cells acquired a swollen appearance 6-8 h.p.i. in a manner similar to untreated infected cel l s . Infectious virus was not produced however (J. Chantler, personal communications). To ascertain that v i r a l DNA synthesis in cytosine arabinoside treated cells was indeed inhibited, the following experiment was performed. As demonstrated in Fig. 17, the v i r a l DNA content of the treated cells continued to decrease after infection, whereas untreated cells exhibited an exponential increase in v i r a l DNA content from 6-8 h.p.i. These data confirm the inhibitory effect of cytosine arabinoside Fig. 17.—Enumeration of MCMV genomes in infected untreated mouse cells or infected cells treated with cytosine arabinoside. (Ara-C) Mouse embryo monolayers in 60 mm Falcon tissue culture dishes were infected with MCMV at a multiplicity of approximately 10 pfu/cell. After infection half the dishes were overlaid with MEM + 10% serum containing 50 yg/ml of cytosine arabinoside. The remainder were overlaid with MEM + 10% serum without cytosine arabinoside. At various times after infection cells of untreated or Ara-C treated cells were harvested and DNA extracted from them analysed for v i r a l genomes as outlined in the text and in Fig. 13. untreated cells: _ Q Ara-C treated cells: C.D». 98. Fig. 17.—Enumeration of MCMV genomes in infected untreated mouse cells or in infected cells treated with cytosine arabinoside. (Ara-C) and also indicate that v i r a l DNA synthesis is not required for the manifestation in the c e l l of MCMV-induced cytopathic effect (swelling). Fig. 18 and Table V depict a transcriptional analysis of RNA extracted from cytosine arabinoside-treated, MCMV-infected cells 24 h.p.i. (Ara-C RNA). Like 'early' RNA, the 'Ara-C RNA' comprised two abundance classes coded for by 5 and 17% of the MCMV genome. The abundant class was present in a seven fold excess over the scarce class. Mouse embryo cells were infected as before with MCMV in the presence of cycloheximide. After infection the cells were main-tained in cycloheximide and RNA harvested at 6 h.p.i. (C hex RNA). An analysis of this RNA generated results similar to those obtained for 'early' RNA and 'Ara-C RNA' (Fig. 19, Table V). Summation experiments performed with 'early' RNA and 'C hex RNA' indicated that both RNA samples comprised the same nucleotide sequences (Table VI). D. Absence of symmetrical transcription in MCMV infected c e l l s . The equation derived by Frenkel and Roizman (1972) (equation ) for DNA-RNA hybridization assumed that transcription i n herpes virus infected cells was asymmetric and that RNA from these cells lacked the a b i l i t y to form double-stranded RNA structures. Kozak and Roizman (1975) subsequently discovered that 'late' during infection with HSV-1, RNA transcribed from as much as 15% of the DNA was the Fig. 18.—A transcriptional analysis of 'early' RNA and RNA extracted from cytosine arabinoside treated cells 24 hr. post infection. 700 yg/ml each of 'early' RNA (•) and RNA extracted from Ara-C treated cells (©) 24 hr. post infection were analysed in a hybridi-zation reaction with iodinated MCMV DNA. Early (6 hr) RNA H Ara-C RNA © 101. Fig. 18.—A transcriptional analysis of 'early' RNA and RNA extracted from cytosine arabinoside treated cells 24 hr. post infection. • B 1 .-7 1 , E 3 1 i izuzi H O U R S i B : tzi 102. Fig. 19.—Transcriptional analysis of 'early' RNA, RNA from cells treated with cycloheximide, and a mixture of the two RNAs. a) 700 yg/ml 'early* (6 hr) RNA -> b) 700 yg/ml RNA from cycloheximide treated cells -> c) 350 yg/ml 'early' RNA + 350 yg/ml cycloheximide RNA -> 103. Fig. 19.—Transcriptional analysis of 'early' RNA, RNA from cells treated with cycloheximide, and a mixture of the two RNAs. i tz itzi HIZ ILJF5S t s e a 104. product of symmetrical transcription and could be rendered unavail-able for hybridization i f the RNA sample were preincubated. To determine whether stable MCMV RNA was the product of asymmetrical transcription or was partially symmetrical in nature, 'early' as well as 'late' RNA samples were preincubated at 67°C in 2 x SSC to a R Qt of 2,500. RNA samples were then rapidly cooled, mixed with denatured 125I-MCMV DNA, sheared, and sealed into 50 y l micropipettes. Half the micropipettes for each RNA sample were denatured in boiling water before incubating at 67°C, whereas the remainder were incubated without denaturation of the RNA. If symmetric transcripts were present to any appreciable extent, in either of the RNA samples the preincubated RNA would drive less 125I-MCMV DNA into DNA-RNA hybrids than i f the same RNA were denatured before incubation, the difference in a values representing the fraction of DNA giving rise to symmetric transcripts. If on the other hand, transcription was asymmetric the hybridization kinetics of preincubated and denatured RNA would be similar. The data depicted in Fig. 20A and 20B indicate that transcription of both 'early' and 'late' RNA was largely asymmetric. The possibility that small amounts of symmetric transcripts, randomly distributed in the RNA samples, were present, cannot however be ruled out. E. Nuclear and Cytoplasmic RNA. A transcriptional analysis of whole infected-cell RNA revealed that early in infection 23-27%, and late in infection 38-40%, of the v i r a l genome was represented as stable, asymmetric transcripts in the infected c e l l (Figs. 14, 15). Fig.20.—Analysis of 'early' and 'late' RNA for symmetrical transcripts. 'Early' RNA (A) and 'late' RNA (B) were preannealed i n 2 x SSC at 67°C to a R Q T of 2,500 (10 mg/ml' for 24 h) before mixing with denatured 125I-MCMV DNA and sealing into tubes. Before hybridizing at 67°C half the tubes in each batch were heated in boiling water to denature any double stranded RNA formed. The remaining tubes were directly incubated at 67°C. preincubated RNA • preincubated, denatured RNA • Fig. 20.—Analysis of 'early' and 'late' RNA for s y i m e t r i c a C l t f a n s K r i p . t s . s: tzi i LZIIZI i s tzi MIZ1LJF3S To determine what fraction of this RNA represented transcripts present in the cytoplasm of infected cells, RNA extracted from nuclear and cytoplasmic fractions of the cells 6 and 24 hr. after infection were analysed. Although early (6 hr.) in infection the nucleus contained RNA derived from 23-25% of the MCMV genome, only 11% of the DNA was represented as stable transcripts in the cytoplasm (Fig. 21A). Similarly, 24 hr. post infection, although the nucleus contained RNA from 37% of the genome, only 15% of the v i r a l genome was represented as stable transcripts in the cytoplasm (Fig. 21B). Early nuclear and cytoplasmic RNA, and late cytoplasmic RNA, were composed entirely of asymmetric transcripts. Late nuclear RNA, however, contained symmetric transcripts derived from 5% of the genome. These transcripts were capable of forming double stranded RNA and could be removed from the hybridization mixture by preincubating the RNA sample. Fig. 21A.—Analysis of cytoplasmic and nuclear RNA extracted from cells 6 hr. after infection. 'Early' cytoplasmic RNA: preincubated. g] preincubated and then denatured. • 'Early' nuclear RNA: preincubated. © preincubated and then denatured. £ Fig.21B.—Analysis of cytoplasmic and nuclear RNA extracted from cells 24 hr. after infection. 'Late' cytoplasmic RNA: preincubated. [T] preincubated and then denatured. • 'Late' nuclear RNA: preincubated. © preincubated and then denatured. # 110. CHAPTER V RESULTS 3. Non-Productive Infection Caused by MCMV Murine cytomegalovirus, like other members of the herpes group of viruses, i s known to cause latent as well as productive infections (Henson, et a l , 1972; Gardner, et a l . , 1974; Misra and Hudson, 1974; Hudson, et a l . , 1976; Muller and Hudson, 1977). Two situations in which the virus caused non-replicating infections i n mouse fibroblasts were examined. The f i r s t involved ME-D cells , a c e l l line derived from mouse embyro fibroblasts by continued passage. Unlike the parent cel l s , growing ME-D cells were unable to replicate MCMV. Unfortunately, experiments with ME-D cells could not be pursued beyond the preliminary stages, due to loos of the c e l l line. The second situation involved 3T3 cells arrested in the G^ phase of the cell-cycle. Replication of MCMV in 3T3 cells appears to be dependent upon some event in the S-phase of the cell-cycle (Muller and Hudson, 1977). G.^ arrested 3T3 cells failed to replicate the virus. I. ME-D Cells Murine cytomegalovirus normally replicates i n fibroblast cultures derived from mouse embyros, viz . in early passage mouse embryo fibro-blasts, 3T3 and 3T6 ce l l s . Optimum yields of >100 pfu/cell can be obtained and the process i s accompanied by c e l l death. In contrast, titres of virus associated with freshly seeded ME-D cel l s , infected with MCMV at four different input multiplicities, decreased exponentially over a period of 4 days (Fig. 22). The cells continued to grow unabated. Fig. 22.—ME-D cells infected with MCMV. Exponentially growing ME-D cells were infected with a) .01, b) .1, c) 1, d) 10 pfu of MCMV/cell. After infection cells were washed once with MEM and overlaid with MEM containing 5% FCS. Cells and supernatant were harvested at various times after infection and assayed for infectious virus. Fig. 22.—ME-D cells infected with MCMV. 1 2 3 4 Days post Inf. 113. A. MCMV penetration. The resistance of ME-D c e l l s to MCMV r e p l i c a t i o n could be due to a block i n an early stage of the i n f e c t i o n cycle, such as adsorption or penetration. This was examined by using v i r u s l a b e l l e d i n i t s DNA with 3H-TdR. Cultures of ME-D c e l l s and permissive f i b r o b l a s t s (MEF or 3T3) were infected simultaneously with the radioactive v i r u s , and at various times fractionated into nuclear and cytoplasmic compartments. Results of one such experi-ment are shown i n Table VII,from which i t i s evident that the v i r u s penetrated equally well into the n u c l e i of both c e l l types. In th i s and other experiments, 50-90% of the radioactive v i r u s was taken up by the c e l l s during the 30 min. adsorption period, and about two-thirds of t h i s penetrated the n u c l e i . By 24 h.p.i. much of the nuclear MCMV l a b e l had been l o s t i n the MEF c e l l s , presumably due to degradation accompanying v i r u s r e p l i c a t i o n , whereas about 70% of the nuclear l a b e l was retained i n the ME-D c e l l s . In other experiments, ME-D c e l l s were compared with 3T3 c e l l s , with r e s u l t s s i m i l a r to those presented i n Table VII. Therefore i t can be concluded that the block i n MCMV r e p l i c a t i o n i n ME-D c e l l s was not due to i n e f f i c i e n t uptake or penetration into the c e l l s or the i r n u c l e i , nor was i t due to rapid degradation of the input v i r a l genome. B. MCMV t r a n s c r i p t i o n i n ME-D c e l l s . In order to as c e r t a i n the nature of the block i n v i r u s r e p l i -c a tion i n ME-D c e l l s , MCMV DNA synthesis was examined by CsCl gradient TABLE V I I . — P e n e t r a t i o n of MCMV into mouse embryo f i b r o b l a s t s (MEF) and ME-D c e l l s . 30 mins. p . i . 24 h.p.i. C e l l f r a c t i o n MEF ME-D MEF ME-D Nucleus 32,690a 31,674 9,990 22,040 Cytoplasm 15,360 16,720 5,144 6,070 TCA-insoluble cpm per aliquo t of c e l l f r a c t i o n . 115. c e n t r i f u g a t i o n and by DNA-DNA hy b r i d i z a t i o n . Figure 23 shows a t y p i c a l CsCl gradient analysis of 3H-TdR l a b e l l e d DNA extracted from uninfected and infected ME-D c e l l s (23a and 23b), and from uninfected and infected 3T3 c e l l s (23c and 23d). The l a t t e r p r o f i l e s show the dominant s h i f t of 3H l a b e l from c e l l u l a r to v i r a l DNA, whereas no DNA of v i r a l density was evident i n the infected ME-D c e l l s . This r e s u l t was confirmed by DNA-DNA hy b r i d i z a t i o n t e s t s , an example of which i s shown i n Table VIII. The values obtained for the ME-D samples approximated background l e v e l s , and at the l e v e l of s e n s i t i v i t y of the test represented at most 1.6% of the corres-ponding l e v e l i n l a t e i n f e c t e d 3T3 c e l l s , i n terms of % input cpm hybridized. V i r a l s p e c i f i c RNA synthesis was determined by DNA-RNA hy b r i -d i z a t i o n t e s t s . An example of such an analysis i s shown i n Table IX. V i r a l RNA synthesis occurred both before and af t e r DNA r e p l i c a t i o n i n permissive f i b r o b l a s t s , as noted previously, and also i n s i g n i -f i c a n t amounts l a t e i n infec t e d ME-D c e l l s , although the quantity was much less than i n the permissive MEF c e l l s . In other experiments, v i r a l RNA synthesis was also detected l a t e i n ME-D c e l l s , although we cannot be sure about i t s synthesis at early times, since these values were too close to the background l e v e l . Nevertheless i t i s evident that the v i r u s can transcribe to a l i m i t e d extent i n ME-D c e l l s , which suggests that the input v i r a l genome i s probably uncoated normally. Figure 23.—CsCl gradient analysis of DNA extracted from MCMV-infected (10 pfu / c e l l ) , and uninfected ME-D c e l l s (a and b) and 3T3 c e l l s (c and d). Cultures were incubated between 18 and 24 h.p.i. with 25 uCi of 3H-TdR per 6.5 cm dish. The extracted DNA was mixed with p u r i f i e d li+C-TdR l a b e l l e d mouse DNA (from 3T3 c e l l s ) and centrifuged for 72 h at 35,000 rpm i n the Beckman SW 50.1 rotor. Fractions were c o l l e c t e d dropwise from the bottoms of the tubes and 3H and 1 I +C cpm counted. a) uninfected ME-D c e l l s b) infected ME-D c e l l s c) uninfected 3T3 c e l l s d) infected 3T3 c e l l s 118. TABLE V I I I . — DNA-DNA hybridization. Sample cpm hybridized to MCMV DNA (0.2 yg) ME-D uninfected 0 (<0.02)a ME-D infected 0-8 h.p.i. 0 (<0.03) ME-D infected 20-28 h.p.i. 20 ( 0.015) 3T3 uninfected 0 (<0.036) 3T3 infected 0-8 h.p.i. 0 (<0.043) 3T3 infected 20-28 h.p.i. 380 ( 0.92) igures in parentheses represent percentage of input cpm hybridized. Cultures of ME-D and 3T3 cells were infected with MCMV (10 pfu/cell), or mock infected, and at the indicated times incubated with 10 yCi of 3H-TdR per 5 cm dish. Early and late mock-infected samples were combined. Hybridizations were carried out with duplicate f i l t e r s containing 0.2 yg MCMV-DNA, or no DNA (blanks). The blank values, ranging from 39 to 82 cpm, have been subtracted. TABLE IX.—DNA-RNA h y b r i d i z a t i o n . Sample cpm hybridized to MCMV DNA (0.2 yg) ME-D uninfected 32 (0.013) a ME-D infected 0-8 h.p.i. 40 (0.019) ME-D infec t e d 20-28 h.p.i. 82 (0.035) 3T3 uninfected 21 (0.010) 3T3 infected 0-8 h.p.i. 117 (0.065) 3T3 infected 20-28 h.p.i. 642 (1.23) Figures i n parentheses r e f e r to percentages of input cpm hybridized. Experimental d e t a i l s were s i m i l a r to those described i n the legend to Table VIII, except that the isotope used was 3H-UR at 20 yCi per 5 cm dish. Values on blank f i l t e r s (15-20 cpm) have been subtracted. 120. II. G^ Arrested 3T3 Cells A. Persistence of the MCMV genome in G^ arrested 3T3 cells and the induction of replication following serum activation. 3T3 c e l l s , arrested in the G-^ phase of the cell-cycle as outlined in 'materials and methods', were infected with MCMV at an input multiplicity of 25 pfu/cell. After permitting the virus to adsorb for 1.5 h at 37°C the inoculum was replaced with MEM lacking serum. At 6, 24, and 48 h following infection the cells were examined for v i r a l DNA and infectious virus. 24 h after infection parallel cultures of G^ arrested, infected cells were serum activated, and examined for v i r a l DNA and infectious virus at various times after serum activation. As depicted in Fig. 24, G 1 arrested 3T3 cells failed to replicate MCMV. The v i r a l DNA content of these cells decreased over the 24 h period following infection and then remained constant at approximately 50 v i r a l genomes per c e l l , u n t i l 48 h post infection. In contrast, v i r a l DNA synthesis in exponentially growing 3T3 cells began at about 10 h post infection. 24 h after infection these cells contained almost 1000 v i r a l genomes/cell. Infected G^ arrested cells did not exhibit MCMV-induced cytopathic effect. Serum activation of infected, G± arrested c e l l s , 24 h post infection, caused an increase in their v i r a l DNA content, until at 48 h post infection, each c e l l contained on average, 300 v i r a l genomes. Serum activation was also followed by an increase i n infectious virus associated with the c e l l s . Thus, G1 arrested 3T3 cells did not replicate MCMV although 121. Fig. 24.—MCMV DNA and infectious virus associated with Gx arrested, serum activated, and exponentially growing 3T3 cell s . V i r a l genome equivalents in MCMV infected expo cells • Vir a l genome equivalents in MCMV infected Gx cells A Vir a l genome equivalents in G^ cells following serum activation "•••"" ^ ""'" L°g10 pfu/culture in serum activated cells Arrow indicates time of serum activation. 123. although v i r a l genomes were retained in these cells in a non replicating state. These non-replicating v i r a l genomes could be induced to undergo a l y t i c cycle, i f the host cells were activated with fresh serum. B. Extent of transcription of the MCMV genome in Gi arrested cell s . A transcriptional analysis of MCMV DNA in exponentially growing 3T3 cells (Fig. 25) revealed a pattern similar to that observed in ME cells ( Fig. 14, 15). At'6(Early) and 24 (Late) h post infection cells contained v i r a l RNA transcribed from 26% and 38% of the genome, respectively. Both 'early' and 'late' RNA comprised two classes differing in abundance. To determine the extent and nature of transcription of the v i r a l genome in Gi arrested c e l l s , RNA extracted from these cel l s , 6 and 24 h post infection was analysed by solution hybridization with iodinated MCMV DNA. RNA transcribed from 16% and 19% of the genome,respectivelyiand comprising only one abundance class, was found in these cells (Fig. 26a and b; Table X). A summation analysis, in which mixtures of Gi 6 h RNA and expo 6 h RNA,or Gi 24 h RNA and expo 6 h RNA,were hybridized to MCMV DNA, revealed that v i r a l RNA in Gi arrested cells was a subset of 6 h (early) RNA from exponentially growing 3T3 cell s . Both mixtures of Gj RNA and expo 6 h RNA comprised only one abundance class. This may have been brought about by an enrichment, 124. Fig. 25.—Extent of transcription of MCMV in exponential 3T3 cells. 6 h (expo early) RNA # 24 h (exp late) RNA • 125. Fig. 25.—Extent of transcription of MCMV i n exponential 3T3 c e l l s . S E 3 I I SZCZI I — I D U R S Fig. 26.—Extent of transcription of Best f i t curves for RNA from i) early, iv) early + expo early, v) a) i) G]_ early RNA i i i ) expo early RNA iv) Gi early + expo early RNA b) i i ) Gi late RNA i i i ) expo early RNA v) Gi late + expo early RNA 126. MCMV DNA in Gi arrested 3T3 cells. Gi early, i i ) Gi late, i i i ) expo Gi late + expo early ce l l s . 1 E3EIIZI © • V J ^ . — -. E3 . ."7 . E3 . t S E J 1 izica 1 s r z i HnURB S : E J 1 tziizj 1 SZIZI HDUR5 TABLE X. Sample n a - l a2 Sum of as 3 2 B 1 / B 2 3T3, Expo 6 h (Early) 2 .05 .21 .26 .18 .018 9.9 3T3, Gi 6 h 1 - .16 .16 - .010 -3T3, Gi 24 h 1 - .19 .19 - .015 -Summation Analysis Gi 6 h RNA + Expo 6 h RNA 1 .27 - .27 .014 - -Gi 24 h RNA + Expo 6 h RNA 1 .27 .27 .016 i n the mixture of the scarce class, detected in expo 6 h RNA (which was transcribed from approximately 21 percent of the genome), to the level of the abundant class. This would be expected to occur i f only the scarce transcripts were present in arrested cel l s . CHAPTER VI RESULTS 4. V e r t i c a l Transmission of Murine Cytomegalovirus: An Example of Latency Inf e c t i o n of female mice at the time of ovulation and implantation (Neighbour, 1976), or during pregnancy (Mannini and Medearis, 1961; Medearis, 1964; Johnson, 1969), causes pregnancy wastage manifested by decreased l i t t e r s i z e , abortion,or de l i v e r y of macerated s t i l l - b o r n f o e t i . Pregnancy wastage has been observed i n c h r o n i c a l l y infected mice almost a year a f t e r i n f e c t i o n with MCMV (personal observation). Although f o e t i and the newborn from infected mothers are susceptible to superinfection with MCMV (Johnson, 1969; Neighbour, 1976) attempts to i s o l a t e i n f e c t i o u s v i r u s from these animals have f a i l e d . F a i l u r e to i s o l a t e i n f e c t i o u s v i r u s from the f o e t i of infected mothers, however, does not conclusively r u l e out the presence of late n t MCMV genomes sequestered i n the c e l l s of these embryos. The experiments described i n t h i s chapter were performed to detect the presence of murine cytomegalovirus, whether i n f e c t i o u s or i n a latent form, i n c e l l s obtained from embryos of mice infected with MCMV. I. EM-2 C e l l s Three pregnant mice,examined a year a f t e r i n t r a - p e r i t o n e a l (IP) i n f e c t i o n with MCMV,were found to contain abnormal u t e r i : two of the mice contained u t e r i bearing two embryos each, as opposed to the normal complement of 7 to 14 embryos. The t h i r d contained a si n g l e , rather Plate 2a and 2b. Acetone fixed mouse embryo cells incubated with rabbit anti MCMV-infected c e l l serum and fluorescent goat anti-rabbit serum. Rabbit anti-MCMV serum was absorbed with uninfected ME.cells before use. 2a. uninfected mouse embryo cells. 2b. mouse embryo cells 24 h after infection with MCMV. 132. PLATE: 2,a PLATE: 2,b Plates 3a and 3b. EM-2 cells treated as in Plates 2a and 2b. 134. PLATE: 3,a Plates 4a and 4b. Acetone fixed ME cells hybridized with iodinated MCMV DNA. 4a. - uninfected ME cell s . 4b. - ME cells 24 h after infection with MCMV. 136. PLATE: 4 , a PLATE: 4,b Plates 5a and'5b. EM-2 cells treated as in Plates 4a and Plates 6a and 6b. Acetone fixed ME-cells from mothers infected with MCMV, incubated with anti-MCMV infected c e l l serum and fluorescent goat anti rabbit serum. 140. large, dead, foetus. Embryo c e l l s from the three mice were cultured as outlined i n materials and methods and designated, EM-1, EM-2, and EM-3. The si n g l e large foetus, EM-1 f a i l e d to y i e l d v i a b l e c e l l s . EM-2 and EM-3 c e l l s were c u l t i v a t e d over several passages. At passage four EM-3 c e l l s developed c h a r a c t e r i s t i c MCMV induced cytopathic e f f e c t , leading to the l y s i s of the c e l l s and release of v i r i o n s that morpho-l o g i c a l l y resembled MCMV. There was no in f e c t i o u s v i r u s associated with EM-2 c e l l s from passage one to passage seven (a f t e r which I was unable to c u l t i v a t e them). Virus l i k e p a r t i c l e s were not observed i n the medium, or i n c e l l lysates of EM-2 c e l l s . The c e l l s were examined for the presence of the v i r a l DNA by so l u t i o n - h y b r i d i z a t i o n and by in=-situ h y b r i d i z a t i o n , using iodinated MCMV DNA as probe and for the presence of v i r a l antigens by i n d i r e c t immunofluoresence, using rab b i t antisera,.; directed against MCMV infected mouse c e l l s . In s i t u h y b r i d i z a t i o n and immunofluorescence were performed by Dr. J.K. Chantler. No v i r a l DNA was detected i n EM-2 c e l l s by s o l u t i o n h y b r i d i z a t i o n . The technique (on t h i s occasion) was only s e n s i t i v e enough to detect more than 10 v i r a l genome equivalents. Therefore, the presence of small amounts of v i r a l DNA i n these c e l l s could not be ruled out. In s i t u h y b r i d i z a t i o n of acetone fixed EM-2 c e l l s revealed one c e l l i n 40 to contain c l u s t e r s of grains (Plates 5 a,b). A s i m i l a r proportion of c e l l s exhibited granular fluorescence when incubated with fluorescent a n t i -r a b b i t antibodies a f t e r p r i o r incubation with rabbit serum directed against MCMV infected mouse c e l l s (Plates 3a and b). 142. I I . Pathogenesis of MCMV and V e r t i c a l Transmission Experiments were conducted to obtain evidence for the v e r t i c a l transmission of MCMV, as outlined i n Table XI. E s s e n t i a l l y , two or three week-old mice were injected by the IP route with either t i s s u e culture passaged MCMV or vir u s f r e s h l y i s o l a t e d from s a l i v a r y glands of mice. Salivary gland passage v i r u s was used i n view of the findings of Osborn and Walker (1971) that MCMV could be ra p i d l y attenuated by passage through mouse embryo c e l l s . This attenuation was manifested by a loss of capacity to multiply i n some organs of infected mice. Two weeks a f t e r i n f e c t i o n the mice were divided into groups such that either males or females, or both, i n a group were infected. At various times a f t e r i n f e c t i o n s a l i v a r y glands, kidneys, and spleens were removed from the mice, weighed and assayed for i n f e c t i o u s v i r u s . When mice became pregnant, one from each group was s a c r i f i c e d , the embryos removed, and the target organs from the mother assayed for i n f e c t i o u s v i r u s . C e l l s from these embryos were assayed for i n f e c t i o u s v i r u s and infectious-centers (IC) on mouse-embryo monolayers, or by IP i n j e c t i o n i n weanling mice. The c e l l s were also examined for v i r a l DNA by s o l u t i o n h y b r i d i z a t i o n and for v i r a l antigens by immunofluorescence. The l a t t e r was performed by Dr. J.K. Chantler. High t i t r e s of v i r u s were detectable i n the s a l i v a r y glands of the mice injected with either t i s s u e culture passaged or s a l i v a r y gland v i r u s , two weeks a f t e r i n f e c t i o n . The t i t r e s then decreased s t e a d i l y u n t i l 90 days af t e r i n f e c t i o n , when mice from most groups exhibited undetectable l e v e l s of vi r u s ( F i g . 27a; Table XII). The v i r u s , however, appeared to 1 4 3 . TABLE XI. Protocol for studying pathogenesis of MCMV DAY 0 2 to 3 week old mice injected IP with either tissue culture passaged MCMV or virus freshly isolated from salivary glands. 12 Mice segregated according to sex. 14 Divided into groups: SG virus infected mice a) Inf. 8 + £ Inf. b) Uninfected $ + % Inf. c) Inf. (3 + Uninf. <j) d) Uninf. 6* + Uninf. C7 d) Infected (5 TC passaged virus infected mice as above Target organs (salivary glands, kidneys, spleen) weighed and assayed for infectious virus. 23 Target organs assayed for infectious virus 35 to 1st pregnancy 37 Target organs from mothers assayed for virus. Mouse embryos were a) Cultivated b) Assayed for infectious virus c) Assayed for infectious centers on ME monolayers. d) Injected into newborn mice, the salivary glands from which were assayed for MCMV 2 weeks p. i . e) Examined for MCMV DNA by solution hybridi-zation. f) Examined for v i r a l proteins by immuno-fluoresence. 53 Target organs assayed. 83 [Second Pregnancy] Mothers and embryos assayed as before. 93 Target organs assayed. Fig. 27.—MCMV in the salivary glands (A) and kidneys (B) of mice infected with tissue culture passaged virus, or virus isolated from salivary glands. 2 to 3 week-old mice were injected intraperitoneally with either 10 6 pfu of tissue culture passaged MCMV or 4 x IO 4 pfu of virus isolated from salivary glands of mice previously infected with MCMV. Infected mice were divided into groups such that either the males, or the females, or both the males and females in the group were infected. At various times after infection salivary glands and kidneys from mice were removed, weighed, homogenised and assayed for infectious virus. Tissue culture passaged virus infected ® • infected cT + infected Q ^ uninfected + infected Y infected + uninfected f- T Salivary gland virus inf ec ted $ © infected O + infected 9 E uninfected + infected ? A infected + uninfected ¥ v 145. Fig.27.—MCMV in the salivary glands (A) and kidneys (B) of mice infected with tissue culture passaged virus, or virus isolated from salivary glands. 0J3 O Hi 50 75 Days post Inf. 146. TABLE XII. Infectious v i r u s associated with organs of infected mice. Day Aft e r I n f e c t i o n Group Infectious v i r u s per gram of tis s u e . Saliv.gland Kidneys Spleen Uterus 1 2 14 TC infected 2.85 x 10 7 8.2 x 10 1 x 10 ND SG infected 6.0 x 10 6 3.82 x 10 3 < 100 ND 23 TC 1.8 x 10 6 3.6 x 10 1 < 100 ND SG 1.52 x 10 5 4.3 x 10 2 < 80 ND 1st Preg. 35 to 37 TC 1 ^ Icf 1.30 x 10 5 < 40 < 100 ND TC I <J> U & 9.5 x 10 2 < 40 < 80 ND TC U $? I d 3.34 x 10 5 < 40 < 100 ND SG I f I ^ 1.35 x IO 4 1.5 x 10 2 < 160 ND SG i f U § 7.5 x IO 4 6 x 10 1 < 160 ND SG U ? I <B < 80 < 40 < 100 ND Uninfected mice < 80 < 40 < 100 ND 53 TC 6.0 x 10 3 < 40 < 100 ND SG 1.2 x 10 3 < 40 < 100 ND 83 2nd Preg. TC I<p> IC^ 3.5 x 10 3 < 40 < 100 < 30 1C l o U ^ < 80 < 40 < 100 < 30 TC I @ < 80 < 40 < 100 < 30 SG I ? I 51 2 x 10 3 8 x 10 2 < 100 < 30 SG I % U cf 6.6 x 10 3 < 40 < 100 < 30 SG u ? i ef < 80 < 40 < 100 < 30 TABLE XII. Continued. Day After Group Infectious virus per gram of tissue. Infection Saliv.gland Kidneys Spleen Uterus Uninfected mice < 80- < 40 < 100 ND 92 TC < 80 < 40 < 100 ND SG < 80 < 40 < 100 ND 1 4 8 . p e r s i s t i n the s a l i v a r y glands of mice from the groups i n which both males and females were i n i t i a l l y infected. Considerably higher l e v e l s of v i r u s were observed i n the kidneys of mice injected with s a l i v a r y gland v i r u s than those injected with t i s s u e culture passaged v i r u s ( F i g . 27b;Table XII). This was i n accordance with r e s u l t s obtained by Osborn and Walker (1971), who found f r e s h l y i s o l a t e d v i r u s to be more v i r u l e n t and to have the a b i l i t y to i n f e c t a largerrange of murine tissues than tis s u e culture passaged v i r u s , which could only multiply to high t i t r e s i n the s a l i v a r y glands. Virus was detected i n the spleen of only one mouse. Mouse embryo c e l l s from infected mothers grew at a slower rate than embryo c e l l s from uninfected mothers. In addition the c e l l s could not be c u l t i v a t e d beyond the 4th or 5th passage. None of the c e l l cultures, derived from the 1st or the 2nd pregnancy, contained detectable l e v e l s of v i r a l DNA (to l e s s than 0.5 genome equivalents per c e l l ) . The presence of the v i r u s i n these c e l l s could not be demonstrated by means other than immunofluorescence. The c e l l s derived from the embryos of infected mothers exhibited fluorescence with sera directed against MCMV infected c e l l s (Plate 6 ). C e l l s from uninfected mothers did not fluoresce when treated i n a s i m i l a r manner. » 149. CHAPTER VII DISCUSSION I. The Structure of the MCMV Genome The murine cytomegalovirus genome is a double stranded linear DNA molecule which, with a molecular weight of 1.2 to 1.3 x 10 8, i s larger than the genome of any other herpes virus studied to date. The molecule was further characterized with respect to i t s kinetic complexity, the presence of ribonucleotides, and fragments generated by restriction endonucleases. The G + C content of T7 DNA differs from that of MCMV DNA by 11%. This discrepancy precludes the accurate determination of the complexity of MCMV DNA by direct comparison of the reassociation kinetics of the two molecules. However, by adjusting the C t x values of the two DNAs, O ^ 2 using the estimates of G i l l i s , Deley, Decleen (1970) for discrepant G + C contents, a value of 1.8 x 10 8 was obtained for the complexity of MCMV DNA. This i s greater than the value of 1.32 x 10 8 for the molecular weight of the DNA as determined in this laboratory (Mosmann and Hudson, 1973). Since the kinetic complexity of the genome is not less than i t s molecular weight, i t i s suggested that the molecule i s largely unique. However, although repetitive sequences were not detected by the technique used, the presence of minor reiterations cannot be ruled out. Further confirmation of the large size of the MCMV genome is provided by the analysis of fragments generated by EcoR^. The enzyme cleaves MCMV DNA into 25 fragments which are present in the digest in equi-molar amounts. The sum of the molecular weights of the fragments adds up to approximately 1.36 x 10 8, which again i s in agreement with the molecular weight of the unfragmented genome as determined by sediment-ation velocity. The replication of MCMV was dependent on the physiological state of the host cells (Muller and Hudson, 1977). Also at no time during the infectious cycle were proteins (Chantler and Hudson, personal communication) or transcripts that were coded for by more than 40% of the genome, detected in the infected c e l l . In view of these observations the question remains: What is the function of this large genome? Since infections with MCMV, in vivo, as well as in vitro, have been shown to result in consequences other than l y t i c infections (Henson, et a l . , Gardner, et a l . , 1972; Misra and Hudson, 1974; Hudson, Misra, Mosmann, 1976; Muller and Hudson, 1977) the possibility exists that a major portion of the genome is devoted to functions necessary for the main-tenance of non-productive states, functions which may not be required during productive infections. Another possibility i s that a major portion of the genome i s not expressed, but i t s presence is in some manner v i t a l for maintaining the structural configuration of the molecule necessary for i t s replication and/or expression. Like HSV-1, MCMV DNA exhibits marked fragmentation in alkaline sucrose gradients. In the case of HSV-1 i t has been speculated that ribonucleotides found linked to this molecule may at least in part be responsible for i t s a l k a l i sensitive nature (Biswal, et a l . , 1974). 151. Ribonucleotides associated with MCMV DNA would explain i t s behavior i n al k a l i n e sucrose gradients and also i t s heterogeneous nature as observed i n isopycnic gradients (Mosmann and Hudson, 1974). Although r e s u l t s obtained by digesting 3H-UR l a b e l l e d MCMV DNA with ribonuclease were inconclusive, b o i l i n g i n 0.5 N KOH f a i l e d to s o l u b l i s e any of the radio-a c t i v i t y associated with l a b e l l e d DNA, implying that ribonucleotides, i f at a l l associated with MCMV DNA, were present i n very small amounts. Fragments generated from MCMV DNA by EcoRi were present i n the digest i n equimolar amounts and the sum of t h e i r molecular weights added up to approximately equal to the molecular weight of the unfragmented genome. This was i n contrast with r e s u l t s obtained for HSV-1 (Skare, et a l . , 1975, Hayward, et a l . , 1975) and EBV(Hayward, et a l . , 1976), r e s t r i c t i o n enzyme fragments from which f a l l into two molar classes: Fragments which are present i n unimolar amounts, and fragments which are present i n the digest i n . quarter or half molar amounts. The sum of the molecular weights of the fragments i n these cases i s considerably greater than the mol. wt. of the genomes. For HSV-1, t h i s behaviour has been a t t r i b u t e d to the existence of four alternate sequence arrangements i n the DNA (Hayward, et a l . , 1975). Caution must however be exercised i n i n t e r -preting the lack of hypomolar fragments i n MCMV/EcoRi digests to mean that MCMV DNA exis t s i n only one sequence arrangement, as t h i s could merely be a r e s u l t of the absence of EcoR^ s i t e s i n regions of sequence inversion. Hayward, et a l . (1976) observed that, although the sum of the mol. wts. of fragments generated from EBV (B-95) DNA by EcoRi endonuclease equalled the mol. wts. of the EBV genome, the sum of the mol. wts. of the 152. fragments generated by the r e s t r i c t i o n enzymes Kpn and Sal exceeded the molecular weight of the EBV genome by almost 30 and 50 percent respec-t i v e l y . Comparison of the r e a s s o c i a t i o n k i n e t i c s of 3 2 P l a b e l l e d DNA from two s t r a i n s of MCMV i n the presence of an excess of homologous or hetero-logous unlabelled DNA revealed almost complete homology. Differences were however, observed i n the r e s t r i c t i o n enzyme patterns of the DNAs from the two s t r a i n s . Although incomplete separation of a group of high molecular weight fragments generated by Hind III precludes any comment on t h i s phenomenon, differences i n the fragmentation of the two DNAs by EC0R4 brings f o r t h an i n t e r e s t i n g and puzzling observation: The 4.6 x 10 6 molecular weight fragment 'G', which was present i n uni-molar amounts i n digests of MCMV (K 181) DNA was a l l but absent from MCMV (Smith) digests. If t h i s elimination was due to the absence of one EcoRi cleavage s i t e from (Smith) DNA due to a point mutation, i t should lead to a decrease i n the m o b i l i t y of some other fragment, corresponding to an increase i n i t s molecular weight by 4.6 x 10 6. Such a change i n mobility was not observed. A delet i o n i n fragment 'G' i n (Smith) DNA would be expected to lead to the generation of a smaller fragment. Such a fragment would either appear as a new low MW band or comigrate with some other fragment thereby increasing the amount of DNA associated with that band by a factor of 2. Neither of these e f f e c t s was observed i n the Smith digest, although the second p o s s i b i l i t y cannot be ruled out because of the inaccuracies inherent i n determining the r e l a t i v e amount of DNA associated with the smaller fragments. A th i r d p o s s i b i l i t y i s that DNA corresponding to the e n t i r e fragment 'G' 153. was absent from a majority of Smith DNA molecules. Such a deletion, although unlikely, might not be detected by conventional techniques for determining molecular weights of large DNA molecules, since the missing piece would account for not more than 3.4 percent of the entire genome. II. Expression of the MCMV Genome A. Onset of v i r a l DNA synthesis in productively infected mouse ce l l s . In view of the recent findings of Muller and Hudson (1977) that MCMV does not induce i t s own thymidine kinase, and that MCMV infected cells exhibit low levels of this enzyme, v i r a l DNA syn-thesis was measured by the technique of DNA-DNA reassociation kinetics. The technique measured absolute amounts of unlabelled v i r a l DNA in infected cells and was therefore not affected by the problems inherent in conventional techniques used for this purpose that u t i l i z e radioactive or halogenated precursors (Henson, et a l . , 1966; Misra and Hudson, 1974; Moon, et a l . , 1976). An increase in the v i r a l DNA content of the cells was observed 6 to 8 hours after infection. The DNA content continued to increase in an exponential manner for the next 10 hours at which time i t appeared to level off. It must be emphasized that the curve in Fig. 13b depicts the v i r a l DNA content of the infected cells and does not take into account DNA in virions excreted into the supernatant. The levelling of the curve between 18 and 24 hours may therefore be attributed to an increase in excreted virus late in infection rather than cessation of v i r a l DNA synthesis. 154. B. Transcription in productively infected cel l s . V i r a l transcription in infected cells was analysed by a technique similar to that developed by Frenkel and Roizman (1972). The analysis permitted the determination of the fraction of the genome represented as transcripts in a particular RNA preparation, as well as i t s consituent RNA classes that differed in molar concentration. The technique, however, measured only stable RNA species that arose by asymmetric transcription and revealed neither the time of synthesis of the various species nor their turnover rates. Three basic forms of control have been postulated to exist in the expression of DNA v i r a l genomes at the level of transcription (Frenkel and Roizman, 1972; Roizman, 1974; Brandner and Mueller, 1974; F l i n t and Sharp, 1976). These include: i) Temporal or on-off controls, which involve the turning 'on' or ' o f f of the transcription of regions of the genome i n relation to the various stages of the infectious cycle. Thus genes transcribed before the onset of v i r a l DNA synthesis are termed 'early' whereas transcripts present in the c e l l during the later stages of infection are said to be coded for by 'late' genes. There is a requirement for the i n i t i a t i o n of v i r a l DNA synthesis in the c e l l for the tran-sition from 'early' to 'late' transcription, although the mechanism of this transition i s as yet unclear. i i ) Abundant and scarce controls: These have probably evolved as a means of coping with the unequal requirement in the c e l l for d i f -ferent v i r a l gene products. Thus, i t has been suggested that 155. transcripts from certain regions of the genome (e.g. "structural" genes) are present in higher concentrations than transcripts for gene-products that are required in small amounts. i i i ) Processing controls: This term encompasses various controls including the selective transport of transcripts from the nucleus to the cytoplasm and the post-transcriptional modification of RNA. The data indicated that a l l three forms of control were exerted on the expression of the MCMV genome during l y t i c infections. Approximately 25 percent of the genome was represented as stable transcripts in the c e l l at 6 h post infection, whereas RNA transcribed from 35 to 40 percent of the MCMV DNA was present in the cells in the later stages of infection. Assuming asymmetric transcription, these figures represented 50 and 70 to 80 percent of the coding capacity of the DNA molecule respectively. 'Early' transcripts were retained in the c e l l throughout the infectious cycle and formed a subset of 'late' RNA. Temporal control was therefore exerted over about 10 percent of the genome as transcripts from this region were only present 'late' in infection. Transcripts from the remaining 10 to 15 percent of the genome (50-40 or 35) were never observed in infected c e l l s . This region (or regions) was either not transcribed, or RNA coded from i t was present in undetectable amounts or was preferentially degraded soon after i t s synthesis. Both 'early' and 'late' RNA comprised two RNA species differing about 7 to 10 fold in concentration. 'Early' in infection the abundant and scarce classes were coded for by approximately 5 and 19 percent of the genome respectively, whereas 'late' in infection each of the two classes was transcribed from 18 percent of the genome. As observed with other DNA viruses (Swanstrom and Wagner, 1974 ; Frenkel, et a l . 1973; Brandner and Mueller, 1974), v i r a l DNA synthesis in the host c e l l was required for the expression of 'late' genes, as in the presence of cytosine arabinoside or cycloheximide only 'early' transcription occurred. Virus induced cytopathic effect, as manifested by the rounding up of the host ce l l s , was observed in the cytosine arabinoside-treated cells, implying that i t was an 'early' function. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm (or the degradation of RNA from certain regions of the genome was enhanced once i t entered the cytoplasm). Although RNA extracted from the nuclei was similar to whole c e l l RNA in that i t arose from 25 percent (early) and 35 percent (late) of the genome, transcripts from only 11 percent (early) and 15 percent (late) of the v i r a l DNA were detected in the cytoplasm. These data were in accordance with the observations of Chantler and Hudson (1977) who, 'late' in infection, were able to detect v i r a l proteins that accounted for only 30 percent of the coding capacity of the genome (15 percent of the total DNA). i Although symmetric v i r a l transcripts were not detected in RNA 157 _ extracted from whole c e l l s , ' l a t e ' nuclear RNA contained RNA transcribed from 5 percent of the genome that was capable of annealing to i t s e l f . These symmetric t r a n s c r i p t s were probably present i n small quantities as they were not detected when whole c e l l RNA was examined. No symmetric t r a n s c r i p t s were detected i n either 'early' nuclear or cytoplasmic, or i n ' l a t e ' cytoplasmic RNA. C. Non-productive i n f e c t i o n s i n - v i t r o . The r e p l i c a t i o n of MCMV i n mouse c e l l s appears to be dependent upon some event In the 'S' phase of the c e l l - c y c l e (Muller and Hudson, 1977). Gi arrested c e l l s retained the v i r a l genome i n a non-replicating state and could be induced to enter the l y t i c c ycle by serum a c t i -vation. RNA transcribed from approximately 19 percent of the genome, and comprising only one abundance c l a s s , was observed i n infected Gi arrested c e l l s . Gi RNA was a subset of RNA from infected, expo-nentially-growing c e l l s 6 h post i n f e c t i o n . Expo. 6 h RNA comprised 2 classes transcribed from 5 and 20 percent of the genome, yet only one class could be detected i n a mixture of GiRNA and expo.6 h RNA. Although conclusive evidence was lacking, i t was tempting to i n t e r -pret t h i s r e s u l t to imply that only those regions of the genome that represented the scarce class i n expo. 6 h RNA were transcribed i n Gi c e l l s . In a mixture of the two RNA samples, the scarce class was probably enriched such that i t was in d i s t i n g u i s h a b l e from the abundant c l a s s . 158 . Thus, within the l i m i t a t i o n s of the data, the t r a n s c r i p t i o n of MCMV DNA can be postulated to comprise three d i f f e r e n t compartments c o n t r o l l e d by 'on-off mechanisms. The f i r s t represents the region of genome that g i v e s r i s e to RNA i n infected G x c e l l s , and the scarce class i n expo, 'early' RNA. This region of the genome probably codes for gene products necessary for the maintenance of latency. The second compartment involves genes that are not transcribed i n G^ c e l l s but are expressed as the abundant RNA clas s i n infected expo, c e l l s and i n infected c e l l s treated with i n h i b i t o r s of DNA synthesis. The t r a n s c r i p t i o n of these genes probably requires some factor or factors present i n exponentially growing c e l l s but not i n G^ arrested c e l l s . Trans-c r i p t i o n of genes i n the t h i r d compartment, comprising 10 to 15 percent of the genome, required the i n i t i a t i o n of v i r a l DNA synthesis, and probably codes f o r gene products required i n the terminal stages of the l y t i c cycle. I I I . V e r t i c a l Transmission of MCMV Infec t i o n of female mice leads to pregnancy wastage. A number of workers (Medearis, 1964; Johnson, 1969; Neighbour, 1976) have f a i l e d to i s o l a t e i n f e c t i o u s v i r u s from the f o e t i or newborns from infected mothers, and have suggested that damage to f o e t i i n infected mothers i s due to an a l t e r a t i o n of the placental environment rather than to congenital i n f e c t i o n of the embryos. I f t h i s i s the case i t would represent an important d i s t i n c t i o n between the pathology of murine and human CMV, as acute maternal i n f e c t i o n of the l a t t e r almost i n v a r i a b l y r e s u l t s i n the congenital i n f e c t i o n of the o f f s p r i n g (Lang 1970, 1974). 159. However, as pointed out earlier, failure to detect infectious virus in the cells of newborns does not conclusively rule out the presence of MCMV present in a non-infectious, latent form. Although detectable levels of v i r a l DNA were not observed i n cells of foeti or offspring born of infected mothers, antigens normally present in MCMV infected cells could be demonstrated in these cel l s , but not in embryo cells derived from uninfected mothers. In addition, after two weeks of culture, one batch of cells (EM-3) underwent productive infection and produced virus that morphologically resembled MCMV. 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Vikram MISRA: PUBLICATIONS Misra, V., J.B. Hudson. The i n t e r a c t i o n of murine cytomegalovirus with mouse c e l l s . Proc. Canadian Fed. of B i o l . Soc. 17_: 127. 1974. Hudson, J.B., V. Misra, T.R. Mosmann. Properties of the multicapsid v i r i o n s of murine cytomegalovirus. Virology _72: 224-234. 1976. Hudson, J.B., V. Misra, T.R. Mosmann. Cytomegalovirus i n f e c t i v i t y : Analysis of the phenomenon of c e n t r i f u g a l enhancement of i n f e c t i v i t y . Virology _7_2: 235-243. 1976. Misra, Vikram, J.B. Hudson. Structure and function of the murine cytomegalovirus genome. Presented as a poster-session at the ICN-UCLA Symposium of Moi. and C e l l u l a r B i o l . , Squaw Valley. 1976. Misra, Vikram, J.B. Hudson. Structure of the murine cytomegalovirus genome and i t s expression i n l y t i c and l a t e n t i n f e c t i o n s . Canadian Fed. of B i o l . Soc. 20: 90. 1977. Misra, Vikram, M.T. Muller, J.B. Hudson. The enumeration of v i r a l genomes i n murine cytomegalovirus infected c e l l s . Virology ( i n press). 1977. Misra, Vikram, J.B. Hudson. Murine cytomegalovirus i n f e c t i o n i n a non-permissive l i n e of mouse f i b r o b l a s t s . Arch. V i r o l , ( i n press). 1977. Hudson, J.B., L. Loh, V. Misra, B. Judd, J . Suzuki. Mu l t i p l e i n t e r -actions between murine cytomegalovirus and lymphoid c e l l s i n v i t r o . Journal of General Virology, ( i n press). 1977.
UBC Theses and Dissertations
Structure of the murine cytomegalovirus genome and its expression in productive and non-productive infections Misra, Vikram 1977
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