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Cell cycle dependent replication of the murine cytomegalovirus Muller, Mark T. 1977

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CELL CYCLE DEPENDENT REPLICATION OF THE MURINE CYTOMEGALOVIRUS by MARK T. MULLER B.S., Texas A & M University, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE, 1977 Mark T. Huller, 1977 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Mir.rohinl ngy The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date July 22, 1977 ABSTRACT The interaction between Murine Cytomegalovirus (MCMV) and the c e l l cycle has been investigated in synchronized murine c e l l s . Based on the following evidence i t was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of v i r a l growth in expo-nentially growing 3T3 cells (12 h). was protracted un t i l the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support v i r a l replication; (3) entry of the virus was equally efficient in G-l, S and exponential c e l l s ; (4) in expo-nential 3T3 cells v i r a l DNA synthesis began at 10 h post infection, and in synchronized cells i t began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of v i r a l DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in i t s a b i l i t y to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the a b i l i t y of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a v i r a l thymidine kinase, and thymidine kinase was not essential for normal v i r a l replication. When G-l arrested 3T3 cells were infected with MCMV, v i r a l DNA syn-thesis did not i n i t i a t e and the l y t i c cycle was reversibly blocked. The non-replicating v i r a l genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the c e l l cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was f i r s t studied in G-l, S phase, and exponential 3T3 c e l l s . Under the appropriate conditions, nuclei from infected cells synthesized v i r a l DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized c e l l DNA only and not v i r a l DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative v i r a l DNA polymerase was inhibited by antiserum prepared against infected c e l l proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a v i r a l gene product. The second approach involved a comparison of v i r a l transcription in permissive and non-permissive 3T3 c e l l s . The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of v i r a l RNA classes and the fraction of the v i r a l genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before v i r a l DNA synthesis), two classes of v i r a l transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the v i r a l DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the v i r a l DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the v i r a l DNA. (4) i n non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the v i r a l DNA. Summation hybridization experiments indicated that in non-permissive (G-l) c e l l s , only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe c e l l cycle dependent r e p l i -cation of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the i n i t i a t i o n of v i r a l DNA synthesis directly, or for the transcription of v i r a l DNA sequences. I V TABLE OF CONTENTS Page CHAPTER 1: INTRODUCTION A. The Herpesvirus Group 1 B. Herpesvirus Replication 2 1. Morphology, chemical and physical properties of the herpesvirion 2 2. Viral growth kinetics 5 3. Adsorption penetration and uncoating 5 4. Transcription 6 5. Protein synthesis 8 6. Virion assembly and release 10 7. Alterations in host metabolism and oncogenic potential 10 C. Latency 12 D. The Cell Cycle 14 1. G± and G Q 14 2. S phase 16 3. G2 and mitosis 20 E. Interaction. Between Animal DNA Viruses and the Cell Cycle 21 1. Importance of v i r a l and c e l l cycle studies 21 2. General survey of the DNA containing viruses and the c e l l 21 CHAPTER 2: MATERIALS AND METHODS 27 A. Reagents 27 B.1 Solutions and buffers 29 V TABLE OF CONTENTS Page C. Routine tissue culture techniques 32 1. Cells 32 2. Growth medium 32 3. Culture conditions . . . 33 4. Cell transfer 33 5. Cell counting 34 6. Freezing of cells 34 D. Viruses 35 1. Routine propagation of herpesviruses 35 E. Purification of Virus 35 F. Infection of Cells for Experimental Purposes 35 1. MCMV 36 2. HSV-1 and HSV-B2006 37 G. Plaque Assays and Infectious Center Determinations . . 37 H. Synchronization of Cells • 38 1. G^  arrest 38 2. Synchronization by serum split 38 3. Double TdR treatment 39 4. Mitotic harvests 39 I. Analysis of the Degree of Synchrony 40 1. DNA synthesis 40 2. Mitotic index 40 3. Cell numbers 41 J. Nucleic Acid Purification 41 1. Vi r a l DNA 41 2. Cellular DNA 41 3. Total cellular RNA 42 K. Hydroxyapatite Column Chromatography . . . 43 L. Sucrose Gradient Velocity Sedimentation . . . . . . . . 44 1. Neutral pH 44 2. Alkaline pH 44 v i TABLE OF CONTENTS Page M. Preparative Cesium Chloride Equilibrium Centrifugation . 45 N. Radioiodination of MCMV DNA 46 1. Preparation of the hydroxyapatite column and MCMV DNA 46 2. Assembly of the reaction mixture 47 3. The f i r s t heating step 47 125 4. Removal of unreacted ~~ Iodine 47 5. The second heating step 48 0. Fi l t e r Hybridization (DNA-DNA) 48 P. Solution Hybridizations 49 1. DNA-DNA and DNA-RNA 49 2. Hybrid detection . . . 50 3. Presentation of results 50 Q. In" Vitro DNA Synthesis 51 R. Thymidine Kinase Assay 52 S. Determination of Nucleotide Pools 53 32 1. P labelling, sampling and extraction of nucleotides 53 2. Spotting and development 54 3. Quantitation 55 T. Anti-Infected Cell Protein Serum (Anti-ICP Serum). . . . 56 1. Preparation of antiserum 56 2. Obtaining the IgG fraction from serum 56 3. Adsorption of antibody to cellular proteins . . . . 57 4. Experimental treatment of nuclear monolayers with anti-ICP and preimmune serum 58 U. Processing of TCA precipitates for radioactivity measurements 58 v i i TABLE OF CONTENTS Page CHAPTER 3: RESULTS SECTION I - Characterization of the Lytic System ' 59 A. Growth Cycle of MCMV in 3T3 Cells 59 B. Time of Onset of Vi r a l DNA Synthesis 59 C. DNA and RNA Synthesis in Infected 3T3 Cells 65 SECTION II - Synchronized of 3T3 Cells 70 A. Synchronization of 3T3 Cells 70 B. MCMV Replication of 3T3 Cells Synchronized by the Serum-Split Method 74 C. Inhibition of Vir a l Replication in Cell Cycle Blocked 3T3 Cells 80 D. Correlation Between Cell Cycle Traverse and Vir a l Replication i 80 E. Initiation of Vir a l DNA Synthesis in G-l Infected Synchronized Cells 84 F. Replication of HSV-1 in Serum-Split 3T3 Cells 88 G. Replication of MCMV and HSV-B2006 in Synchronized ME cells , 90 H. Thymydine Kinase in 3T3 Cells Infected with MCMV . . . . 93 I. Nucleoside Triphosphate Pools in Synchronized 3T3 Cells Infected with MCMV 99 1. Uninfected synchronized cells 100 2. MCMV infected synchronized cells 100 SECTION III - Replication of MCMV DNA In Vitro 103 A. Optimal Conditions for DNA Synthesis in Nuclear Monolayers 103 B. The Nature of DNA Synthesized in Permissive and Non-Permissive Nuclei 105 v i i i TABLE OF CONTENTS i Page C. Induction of a New DNA Polymerase Activity in Permissive and Non-Permissive Cells 110 D. Characterization of MCMV DNA Synthesized In Vitro . . . 113 SECTION IV - The Interaction Between G-l 3T3 Cells and MCMV . . 123 A. Persistence of MCMV in G-l Phase 3T3 Cells 123 B. Extent of Viral DNA Synthesis in G-l Cells 125 C. Transcriptional Analyses 125 1. Theory, analytical treatment, and data reduction . 127 2. Analysis of v i r a l RNA present in infected permissive (exponential) 3T3 cells 131 3. Analysis of v i r a l RNA present in infected non-permissive (G-l) 3T3 cells 135 4. Summation hybridization analyses 138 CHAPTER 4: DISCUSSION A. Summary of the Data 143 1. Vi r a l growth 143 2. MCMV DNA synthesis 146 3. Ribonucleoside triphosphate pools 149 4. Viral specific enzymes 149 5. Transcription analyses 151 B. The Proposed Model to Describe Cell Cycle Dependent Replication of MCMV 158 BIBLIOGRAPHY 162 J ix LIST OF TABLES CHAPTER 1 - Introduction Table Page I Biochemistry of the phase in cells traversing the c e l l cycle towards phase 17 II The biochemistry of S phase 18 III Vertebrate DNA polymerases 19 IV The interaction between animal DNA viruses and the c e l l cycle 23 CHAPTER 3 - Results Table Page 125 I Reconstruction experiment of I-MCMV reassociation with varying amounts of MCMV DNA 63 3 II Uptake of H-TdR labelled MCMV into G-l, S and asynchronous 3T3 cells 79 III Inhibition of MCMV replication by excess thymidine 81 IV DNA-DNA annealing to determine the onset of v i r a l DNA synthesis in synchronized 3T3 cells 86 V Initiation of v i r a l DNA replication in synchronized 3T3 cells infected in early G-l, as determined by reassociation kinetics 87 VI Reaction requirements for DNA synthesis in MCMV infected nuclear monolayers 104 VII Hybridization of in vitro synthesized DNA 109 VIII DNA-DNA annealing of in vitro synthesized MCMV DNA 118 IX Summary of transcription patterns in permissive and non-permissive 3T3 cells 137 CHAPTER 4 - Discussion Table LIST OF TABLES Page I Comparison of the l y t i c , synchronized and latent 3T3 c e l l virus systems 144 x i LIST OF FIGURES CHAPTER 1 - Introduction Figure Page 1 The c e l l cycle 15 CHAPTERS 3 AND 4 - Results and Discussion Figure Page 1 The growth cycle of MCMV in exponential 3T3 cells 60 2 Reconstruction experiment for the determination of the concentration of v i r a l DNA 62 3 Rate of reassociation of iodinated MCMV DNA with infected c e l l DNA harvested at various times after infection 64 4 - The time of onset of v i r a l DNA synthesis in expo-nential 3T3 cells 66 3 3 5 Incorporation of H-TdR and H-UR into infected and Mock infected 3T3 cells 67 6 Rates of RNA synthesis in MCMV infected 3T3 cells 69 7 Synchronization of 3T3 cells by double TdR treatment 71 8 Kinetics of G-l arrest in 3T3 cells 73 9 3T3 cells synchronized by the serum-split method 75 10 MCMV growth kinetics in synchronous and asynchronous 3T3 cells 76 11 Viral growth curves in synchronized 3T3 cells infected in early G-l (2 h. post serum-split) and late G-l (11 h. post serum-split) 78 12 Infection of G-l arrested 3T3 cells 82 13 Correlation between c e l l cycle traverse and v i r a l replication 83 LIST OF FIGURES DNA synthesis in infected and mock infected synchronized 3T3 cells Replication of HSV-1 in synchronized and non-synchronized exponential 3T3 cells Replication of MCMV in synchronized ME cells Replication of HSV-B2006 in synchronous and asynchronous ME cells Analysis of TK reaction products Changes in TK activity after infection 3 Incorporation of H-TdR into synchronized uninfected 3T3 and 3T3-4E cells and infected 3T3-4E cells Dose response of MCMV, HSV-1 and HSV-B2006 to BUdR Ribonucleoside triphosphate pools in synchronized uninfected and infected 3T3 cells Kinetics of DNA synthesis in nuclear monolayers CsCl equilibrium centrifugation of the DNA synthesized in vitro in infected G-l and exponential nuclei 3 Effect of ammonium sulfate concentration on H-dTMP incorporation 3 Effect of ammonium sulfate concentration on H-dTMP incorporation in G-l arrested nuclei Effect of antisera on DNA synthesis in nuclear monolayers Preparative CsCl gradient analysis of in vitro synthesized DNA Sedimentation analysis of DNA synthesized in vitro in the presence and absence of unlabelled dTTP Sedimentation analysis of MCMV DNA synthesized in vitro LIST OF FIGURES Alkaline sedimentation velocity of v i r a l DNA synthesized in vitro Persistence of MCMV in G-l 3T3 cells Detection of MCMV DNA by reassociation kinetics Self-annealing of iodinated MCMV DNA in the presence of heterologous RNA Hybridization of MCMV DNA with excess exponential RNA at 24 h post infection (permissive late RNA) Hybridization of MCMV DNA with excess exponential RNA at 6 h. post infection (permissive early RNA) Hybridization of MCMV DNA with excess G-l RNA at 6 and 24 h. post infection Summation analysis of permissive early and permissive late RNA Summation analysis of permissive early and non-permissive early RNA Summation analysis of permissive early and non-permissive late RNA A hypothetical model of MCMV transcription in permissive and non-permissive 3T3 cells The proposed model to describe c e l l cycle dependent replication of MCMV ABBREVIATIONS A N R N (A_ O A) Absorbance at 260 nm (or 280) zoO zoO a Fraction of the v i r a l DNA transcribed ATP Adenosine 5'-triphosphate 3 Relative v i r a l RNA concentration BUdR 5-bromodeoxyuridine C Concentration of single strand DNA at time t. C Q I n i t i a l concentration of single strand DNA C t DNA concentration times time o Ci Curie CPE Cytopathic effect CPM Counts per minute CTP Cytidine 5'triphosphate DPM Disintegrations per minute DNA Deoxyribonucleic acid DNase Pancreatic deoxyribonuclease dATP Deoxadenosine 5'triphosphate dCTP Deoxycytidine 5'triphosphate dGTP Deoxyguanosine 5'triphosphate dTMP Deoxythymidine 5'monophosphate dTDP Deoxythymidine 5'diphosphate dTMP Deoxythymidide 5'triphosphate dXTP Deoxynucleoside 5'triphosphate EAV Equine abortion virus EDTA Ethylenediaminetetraacetic acid Fletch Optimization algorithm by Quasi-Newton methods G + C Guanine + cytosine X V ABBREVIATIONS (Cont'd) GTP Guanosine 5'triphosphate G-l The f i r s t 'gap' in the c e l l cycle (after M phase) G-2 The second 'gap' (before M phase) h. Hours Hank's BSS Hank's balanced salt solution HCMV Human cytomegalovirus H.Ep.-2 Human epidermoid carcinoma #2 HnRNA Heterogeneous nuclear RNA HSV Herpes simplex virus IBR Infectious bovine rhinotracheitis virus IgG Immunoglobulin G K Rate constant of hybridizations M Mitotic phase of the c e l l cycle MCMV Murine cytomegalovirus ME Mouse embryo MEM Minimal essential medium mRNA Messenger RNA N Number of classes of transcripts differing in relative concentration NP-40 Nonidet-P 40 nonionic detergent PB Phosphate buffer PBS Phosphate buffered saline Pei Polyethyleneimine PFU Plaque forming units p . i . Post infection PRV Pseudorabies virus xvi ABBREVIATIONS (Cont'd) P.S.S. Post serum s p l i t RNA Ribonucleic acid RNase Pancreatic ribonuclease rRNA Ribosomal RNA R t RNA concentration times time o rXTP Ribonucleoside 5'triphosphate S DNA synthetic phase of the c e l l cycle s Sedimentation coefficient SDS Sodium dodecyl sulfate SSC Standard saline citrate SV-40 Simian virus 40 t time TCA Trichloroacetic acid TdR Thymidine 3T3 Mouse embryo fibroblast derived c e l l line TK Thymidine kinase TNE TRIS, NaCl, EDTA buffer TNM TRIS, NaCl, MgC± 2 buffer TRIS Tris(hydroxymethyl)aminomethane UR Uridine UTP Uridine 5'triphosphate UV Ultraviolet w/w Weight/weight ACKNOWLEDGEMENTS I would like to thank Dr. James B. Hudson for capable services rendered as my research supervisor. Acknowledgement is also due to my committee members, Dr. R.C. Miller, Dr. R. Reeves and Dr. G. Weeks for continued support and constructive commentary. Particular thanks are due to Dr. Miller and Dr. J. Benbasat for gifts of T-4 and T-7 bacterio-phage. I am grateful to Dr. B. Roizman and co-workers for helpful discussions and sending the details of their computer program. Special thanks are due to my Father, Col. M.T. Muller and the University of Texas Computational Center for supplying several alternate algorithms. Finally I would like to thank my wife, Annette, for her assistance in preparation of this thesis, and Ms. R. Morgan for her s k i l f u l l typing. CHAPTER 1 1 INTRODUCTION A. The Herpesvirus Group; The origin of the word 'herpes' can be traced back twenty-five centuries as a medical description of cutaneous lesions of varied etiology. It i s derived from the verb epTTEiv (= to creep) (Beswick, 1962). Herpesviruses are classically defined as large enveloped virions having double stranded DNA as their genetic material and an icosahedral capsid containing 162 capsomers (Roisman, 1969). According to Roizman and Furlong (197.4) the most frequent identification of a putative herpes-virus i s based upon the observation of particles (~ 100 mm in diameter) budding through the inner nuclear membrane. Accordingly, more than seventy different herpesviruses have been identified to date. Each herpesvirus i s named after the family to which i t s primary, natural host belongs (Roizman and Furlong, 1974) and arbitrarily assigned an arabic numeral (e.g. human herpesvirus 1 or murid herpesvirus 1). Latinized binomials (which indicate phylogenetic relationships) have been avoided since a l l the herpesviruses from a given species are not necessarily closely related to one another. L i t t l e , i f any, DNA-DNA homology exists among the different herpesviruses (save HSV-1 and 2), although antigenic relationships are more common. The G + C percentages for the herpesvirus group show a considerable range from 33% (canine herpesvirus 1) to 72% (porcine herpesvirus 1) (Plummer et a l , 1969). Clearly, some additional c r i t e r i a are needed to establish phylogenetic and biological relationships. Throughout the rest of the thesis, I shall use only the 'common names' instead of 'provisional labels' (Roizman and Furlong, 1974). 2 B. Herpesvirus Replication: 1. Morphology, chemical and physical properties of the herpes- virion The mature, enveloped Herpes simplex virion is generally agreed to be 150-170 nm in diameter (Roizman et ' a l , 1969). A single enveloped virion of MCMV is approximately the same size as HSV. However, MCMV displays a unique morphology not shared by other herpesviruses namely, multicapsid virions (Hudson et a l , 1976 ). The predominant form of the virion i s a 'cluster' of herpes-like capsids surrounded by a common envelope. These multicapsid virions may be as large as 500 nm in dia-meter. The innermost region of the herpesvirion is the core, which houses the v i r a l genome coiled around a central protein component (in HSV), and forms a 'toroid' structure (Furlong et a l , 1972). The core is enclosed by a capsid with a diameter between 85 and 110 nm (Roizman and Furlong, 1974). Each capsid i s composed of 162 morphologically distinguishable subunits termed capsomers (Wildy et a l , 1960). The capsid displays the geometry of an icosadeltahedron. The majority of the capsomers are viewed as hexameric structures 12.5 nm long with a diameter of 8-9 nm and a canal (4 nm in diameter) through the long axis (Wildy et a l , 1960; Roizman and Furlong, 1974). Located between the capsid and the envelope is the tegument, or inner membrane, which appears as a fibrous membrane-like structure in negatively stained virions (Roizman and Furlong, 1974). The structure and composition of the tegument are not known, although i t is anti-genically distinct from the outer membrane (Roizman et a l , 1969). 3 The outer layer of the herpesvirion i s the envelope, consisting of a trilaminar membrane with spikes projecting from i t s outer surface (Wildy et aJ., 1960). The phospholipid composition of the virion is similar to the host inner nuclear membrane (Ben-Porat and Kaplan, 1971) and contains host as well as v i r a l antigens (Watson and Wildy, 1963). The integrity of the envelope i s destroyed by phospholipase C as is infectivity (Spring and Roizman, 1968). The particle-to-PFU ratio may be, at best, 10 for HSV (Roizman and Furlong, 1974) but is usually 100 or more. The particle-to-PFU ratio for MCMV, although not known, may be significantly different than for other herpesviruses i n view of the multicapsid nature of MCMV. In addition, within each multicapsid virion, defective capsids were observed (Hudson et a l , 1976), a finding which complicates the inter-pretation of particle-to-PFU counts. The multicapsid nature of MCMV may result in lowered infectivity (e.g. compared to HSV-1) since a single envelope may enclose as many as twenty nucleocapsids, which cannot give rise to more than one infected c e l l (Hudson et: a l , 1976 ). The size and base composition of herpesvirus DNA's have been reviewed and catalogued by Roizman and Furlong (1974). HSV-1 DNA has been estimated to range from 85 x 10 (Mosmann and Hudson, 1973) to 100 x 10 (Kieff et a l , 1971) with a base composition of 67% G + C. The kinetic complexity of HSV-1 is close to the calculated molecular weight, indicating that the sum of the unique sequences i s equal to the molecular weight of the virion DNA (Frenkel and Roizman, 1971). Terminal re-petitions have been conclusively identified with HSV-1 (Grafstrom e^ t a l , 1974). In addition, the HSV genome has been shown to contain two palindromes (Sheldrick and Berthelot, 1974). The f i r s t , at the l e f t end of the duplex, contains A-B-L-B-A, and the second (continuing l e f t to right) consists of A-C-S-C-A. The l e f t end A-B is inverted and complementary to the internal region B-A. Likewise, the C-A region shows the same relationship to the internal A-C region. The L region comprises 71% of the molecule, and the S region, 9%. A common feature of herpesvirus DNA's i s their fragmentation upon denaturing with a l k a l i (Lee et a l , 1971; Kieff et^ a l , 1971; Nonoyama and Pagano, 1972; Mosmann and Hudson, 1973). Recently i t was reported that in HSV-1 the a l k a l i sensitive regions were located at random (Wilkie et_ a l , 1974; Hyman et a l , 1977) in contrast to a previous report (Frenkel and Roizman, 1972), which suggested the sites were unique. When HSV-DNA was pretreated with DNA polymerase and DNA ligase, alkaline-sensitivity was significantly decreased, which led Hyman et a l (1977) to conclude that pre-existing gaps (or nicks) were present, although the presence of ribonucleotides could not be ruled out entirely The molecular weight and base composition of MCMV DNA was reported to be 132 x 10 daltons and 59% G + C respectively (Mosmann and Hudson, 1973) . Electron microscopy of MCMV-DNA revealed linear duplex molecules and no obvious single strand regions (Mosmann and Hudson, 1974). Although MCMV DNA gives rise to a heterogeneous population of single strand fragments after alkaline denaturation, i t appears that these breaks were artifacts introduced during purification (Mosmann and Hudson 1974) . An interesting and unique feature of MCMV is the internal hetero-geneity in the G 4- C content of the genome (Mosmann and Hudson, 1973, 1974). By controlled shearing of the intact genome, two species of DNA were resolved by CsCl equilibrium centrifugation, corresponding to 57.5% and 61.5% G + C content (Mosmann and Hudson, 1973). 5 MCMV contains predominantly unique sequences as determined by DNA reassociation kinetics, using phage T-7 DNA as a basis for com-parison (V. Misra, personal communication). 2. Vi r a l growth kinetics Certain members of the herpesvirus group have relatively short growth cycles (15-20h. from the time of infection to the end of the exponential phase). The fast growing herpesviruses include HSV-1, HSV-2, PRV, EAV, IBR, and the B virus. The latent period i s usually less than 6 hours with these viruses (Darlington and Granoff, 1973). Cytomegaloviruses are classically considered to be slow growing herpes-viruses (Plummer, 1973). MCMV replicates in exponentially growing mouse cells with a latent period of 10 to 12 hours (Mosmann and Hudson, 1974). The appearance of the f i r s t progeny virions in HCMV may take up to 48 hrs. under optimal conditions (McAllister et a l , 1963), which distinguishes this herpesvirus as one of the slowest growing members of the group. 3. Adsorption, penetration and uncoating The attachment of a herpesvirus to a susceptible c e l l appears to be a random-hit process, independent of temperature, but dependent upon the presence of cations and the volume of inoculum (Farnham and Newton, 1959). The actual rate of adsorption varies depending on the virus, host c e l l , and physiological conditions. Direct observations in the electron microscope demonstrated that 90% of the input HSV was attached within 45 minutes, and enveloped particles adsorbed more 6 frequently than nucleocapsids (Holmes and Watson, 1963). The involve-ment of herpesvirus receptor sites on the plasma membrane has not been clearly resolved (Roizman, 1969). Unlike bacteriophages, where the virion 'coat' remains outside the host and only the nucleic acid penetrates (Hershey and Chase, 1952), herpesvirions enter the host c e l l by two proposed mechanisms: 1) they are taken into the c e l l by engulfment into phagocytic vesicles and subsequently uncoated (Dales and Silverberg, 1968); 2) they undergo a preliminary fusion with the plasma membrane prior to release of the nucleocapsid into the cytoplasm (Morgan et a l , 1968). Both interpretations depend primarily upon electron microscope evidence and are, therefore, subject to the usual shortcomings, such as artifacts introduced by high multiplicities and high particle-to-PFU ratios as well as trying to deduce from random photographs a totally dynamic process. Penetration requires an expenditure of energy, and enzymes may be involved (Kaplan, 1969). The fate of the nucleocapsid in the cytoplasm remains unresolved. Uncoating of v i r a l DNA occurs mainly in the cytoplasm by pre-existing enzymes (Hochberg and Becker, 1968). V i r a l DNA, devoid of capsid proteins, migrates to the nucleus where transcription begins (Roizman and Furlong, 1974). Details of the uncoating and transporting of v i r a l DNA within the cytoplasm remain to be elucidated. 4. Transcription The most detailed information on herpesvirus transcription has been reported by Roizman and co-workers with Herpes simplex. They concluded that, based upon the analysis of hybridization in solution 7 (Frenkel and Roizman, 1972), 48-50% of the HSV-1 genome was expressed at 8 h.p.i. ('late' RNA, i.e. after the i n i t i a t i o n of v i r a l DNA syn-thesis at 3 h. (Roizman and Furlong, 1974)) compared to 44% at 2 h.p.i. (Frenkel et a l , 1973). A computer aided analysis of the kinetics of hybridization of v i r a l DNA resolved the existence of two classes differing 140 fold in molar concentration at 2 h.p.i. and 40 fold at 8 h.p.i. (Frenkel et a l , 1973). Therefore, in both pre- and post-DNA replicative transcription, 'abundant' and 'scarce' classes were observed. Thus, there are two proposed forms of transcriptional control with HSV. The f i r s t i s an on-off type whereby certain sequences are transcribed 'early' and others, 'late'. The second i s an abundant-scarce type of control which reflects the number of copies of each transcript made. The synthesis of v i r a l RNA takes place in the nucleus of the infected host (Roizman et a l , 1970). Early transcription (prior to DNA synthesis) appears to be mediated by a host DNA dependent RNA polymerase, since cycloheximide does not affect early transcription (Frenkel ejt a l , 1973). In addition, HSV DNA was shown to be infectious (Sheldrick et a l , T973; Stow and Wilkie, 1976), eliminating the possibility that early sequences were being transcribed by a virion-bound RNA polymerase. The solution hybridization technique w i l l detect only stable tran-scripts which arise by asymmetrical transcription. However, i t was demonstrated that symmetrical transcripts arising from 30% of the v i r a l DNA accumulate in large amounts in the nucleus of the infected c e l l (Jacquemont and Roizman, 1975). Symmetrical, nuclear RNA i s capable of self-annealing and sediments faster than the corresponding RNA from polyribosomes (Jacquemont and Roizman, 1975). Apparently HSV nuclear RNA is processed through two post-transcription events. One i s cleavage of v i r a l RNA to yield lower molecular weight transcripts, 8 and the other i s the addition of poly A (Bachenheimer and Roizman, 1972; Silverstein et. a l , 1973). Although there is extensive evidence for multilevel regulation of transcription and transport of RNA into the cytoplasm, many details remain to be elucidated. For example, the nature of the mechanisms mediating abundance-scarce control of transcription i s unknown. Equally enigmatic i s the control of translocation of RNA from the nucleus to the cytoplasm. Furthermore, nothing definitive has emerged concerning the enzyme (probably host derived) involved in transcription of v i r a l DNA. Using the two-phase f i l t e r hybridization, Mosmann and Hudson (1974) investigated the transcriptional patterns of the two density components of MCMV DNA. Although the transcriptional patterns were similar for the 'dense' and 'light' sequences, a notable increase in transcription was observed late in infection, in accordance with an on-off control similar to HSV. F i l t e r hybridization, however, would not detect scarce RNA species (Roizman and Furlong, 1974). ..5. Protein synthesis In general, there is good evidence that v i r a l proteins are synthesized in the cytoplasm (Roizman and Furlong, 1974; Roizman and Kieff, 1975), and subsequently transported to the nucleus (Ben-Porat e_t a l , 1969), although a small minority remain cytoplasmic (Spear and Roizman, 1968). The need for arginine in the replication of herpesviruses has been well documented (Tankersley, 1964; Roizman, 1965; Becker et a l , 1967), and apparently, this amino acid is required for the transport of proteins into the nucleus (Courtney et al, 1971). Host polysomes disaggregate within 1-2 h.p.i., and faster sedimenting polysomes reappear containing v i r a l specific mRNA (Roizman et a l , 1970; Jacquemont and Roizman, 1975). HSV-1 protein synthesis is maximal at 4-6 h.p.i. (Honess and Roizman, 1973) , but there i s evidence that the majority of structural proteins are synthesized from early RNA (Frenkel and Roizman, 1972). However, this view is discrepant with two other herpesviruses, namely PRV (Rakusa-nova et a l , 1971) and EAV (Huang et a l , 1971), as well as an independent study on HSV-1 by Wagner (1972). Recent studies on enumeration of HSV-1 proteins may be summarized as follows: (1) 48 infected c e l l polypeptides are considered to be v i r a l specific (Honess and Roizman, 1974) of which 23 comigrated with structural proteins, 15 were non-structural and 9 unclassified (Roizman and Furlong, 1974) ; (2) 33 proteins have been identified in the purified virion (Heine et a l , 1974), approximately half of which are glycosylated and several phosphorylated after translation; (3) pulse-chase experiments indicate post-translational cleavage of some structural proteins (Honess and Roizman, 1973). Assuming a molecular weight for HSV-1 DNA of 10 / X. ' ,da7iEons'-, the polypeptides thus far enumerated may account for 87% of the information content of the virus (Roizman and Furlong, 1974). Based on the analysis of polypeptides made after the withdrawal of inhibitors of protein synthesis, Roizman has devised a model of co-ordinate regulation of HSV gene products (Honess and Roizman, 1974). The model predicts 3 classes of transcripts and corresponding proteins termed a, 3 and y. These proteins interact in a somewhat complex scheme to either turn 'on' their synthesis sequentially or turn ' o f f or reduce the rate of synthesis. These data suggest the existence of a complicated multilevel regulatory system operable at the level of trans-cription, transportation, and translation of HSV RNA. Kim jit al (1976) have i d e n t i f i e d 33 v i r i o n proteins i n MCMV, and at l e a s t 6 are glycosylated. Assuming asymmetric t r a n s c r i p t i o n of MCMV DNA, the s t r u c t u r a l proteins i d e n t i f i e d by Kim et a l (1976) account f o r 30% of the genome capacity of MCMV DNA. 6. V i r i o n assembly and release Most of the s t r u c t u r a l proteins migrate from the cytoplasm into the nucleus where morphogenesis of the core and capsid takes place (Roiz-man and Furlong, 1974). V i r a l DNA i s synthesized p r i o r to the assembly of nucleocapsids, (Ben-Porat and Kaplan, 1963). The condensation of v i r a l DNA into cores i s an i n e f f i c i e n t process since <20% of the DNA becomes associated with v i r i o n s (Ben-Porat and Kaplan, 1963). The de-t a i l s of events leading to capsid assembly are not well understood. Electron microscope evidence suggests that envelopment occurs, following nucleocapsid morphogenesis, v i a a 'budding' process through the inner lamella of the nuclear membrane (Roizman and Furlong, 1974). Movement of enveloped v i r i o n s through the cytoplasm to the extra-c e l l u l a r environment probably occurs v i a some membranous v e s i c l e such as the endoplasmic reticulum (Roizman and Furlong, 1974). V i r i o n s are li b e r a t e d into the e x t r a c e l l u l a r space without l y s i s of the host c e l l . 7. A l t e r a t i o n s i n host metabolism and oncogenic p o t e n t i a l A s i g n i f i c a n t i n h i b i t i o n of host DNA synthesis has been observed with several herpesviruses (Ben-Porat and Kaplan, 1965; O'Callaghan, 1968; Roizman, 1969; Nonoyama and Pagano, 1972; Moon et a l , 1976; Hudson et a l , manuscript i n preparation). In contrast, HCMV can cause 11 stimulation of c e l l DNA synthesis in arrested, permissive cells (St. Jeor et a l , 1974). Host protein synthesis declines rapidly after infection with HSV (Sydisilis and Roizman, 1966) and PRV (Kaplan et a l , 1970). Cessation of host protein synthesis after infection with MCMV was not observed (Chantler and Hudson, manuscript in preparation). An overall decrease i n the rate of RNA synthesis occurs in HSV-infected c e l l s , however, for certain RNA species i t i s more pronounced (Roizman and Kieff, 1975). Thus 45S ribosomal precursor RNA was made and methylated, but was not processed into 18S and 28S ribosomal RNA (Roizman et a l , 1970). Non-ribosomal RNA, synthesized post infection, did not form polyribosomes and was, therefore, incapable of directing host protein synthesis (Roizman et a l , 1970; Rakusanova et a l , 1972). An overwhelming amount of data suggests that in humans, EBV possesses a l l the attributes of an oncogenic virus. Based on similar evidence, an association between HSV-2 and cervical carcinoma has also been sug-gested, although the evidence i s more circumstantial than for EBV. These data have been recently reviewed by zur Hausen (1975) and Roizman and Kieff (1975). Obviously there i s no way to prove an etiological relationship between a given human herpesvirus and cancer. Other non-human herpesviruses have been strongly implicated as etiological agents in malignancies. These include certain non-human primate viruses (herpesvirus saimiri, herpesvirus ateles), Marek's disease virus i n chickens, and the Lucke tumor agent in frogs (zur Hausen, 1975). C. Latency: Latency i s quite characteristic of many herpes viruses and may be defined as a state in which the virus i s present in an individual, without showing overt signs of infection (Docherty and Chopan, 1974). Although extensively studied, the mechanism of latency i s poorly under-stood. HSV may be envisaged as existing in either a 'static state' or 'dynamic state' (Roizman, 1965). The former maintains that the v i r a l l y t i c cycle i s reversibly blocked in cells which harbour the quiescent HSV or HSV genome. The latter proposes that the latent virus is replicating at a very low level. HSV-1 is considered to be a neurotropic virus (Stevens, 1975), specifically housed within the trigeminal nerve system (Docherty and Chopan, 1974; Roizman and Kieff, 1975). Neurons, being highly d i f -ferentiated c e l l s , do not divide in the adult animal and have a long l i f e span. Thus, the quiescent nerve c e l l may lack specific 'factors' necessary for v i r a l replication (Roizman and Kieff, 1975). This suggests an involvement of the host c e l l cycle in mediating latency (Goodheart, 1970), although the host immune defenses probably come into play. Indeed, the activation of some latent viruses has been shown to be c e l l cycle dependent. Activation of SV-40 was maximal during S phase (Kaplan et al, 1975). Likewise, activation of the repressed EBV genome was shown to be S phase dependent (Hampar et^ a l , 1974, 1976). MCMV, like most herpesviruses, may cause latent or persistent infections in the natural host. MCMV was recovered from leukocytes of chronically infected mice by co-cultivation with mouse fibroblasts in tissue culture (Henson et a l , 1972). Similarly, HCMV has been re-covered by co-cultivation with susceptible human ce l l s , from peripheral blood leukocytes (Weller, 1971). With both HCMV and MCMV, activation of the latent virus is triggered by immunosuppression (Weller, 1971; Gardner et a l , 1974). Due to the r i g i d host specificity of cytomegalo-viruses, i t has not been possible to adapt the human virus to other • animals. However, animal models, in particular MCMV in mice, have been most useful in the study of latency among the cytomegalovirus group. Furthermore, the similarity between HCMV and MCMV makes the murine model extremely relevant to the study of HCMV induced birth defects (micro-cephaly, mental and motor retardation, congenital deafness) (Weller, 1971), and pathogenesis. Attempts at establishing a model latent system with MCMV in vitro have been of limited success, possibly due to the l y t i c nature of the virus in c e l l culture. However, Olding et al (1975) demonstrated that the in vitro activation of latent MCMV in B c e l l lymphocytes from in -fected mice, was enhanced by immunologic reactions to foreign antigens. A subsequent report showed that virus was readily recovered from spleen cells of infected mice by co-cultivation with allogeneic, but not syn-geneic, fibroblast feeder layers (Olding et a l , 1976). Therefore, rapidly dividing cells may provide a suitable environment for MCMV activation, a situation also observed with EBV (Hampar et a l , 1974, 1976) and SV-40 (Kaplan et a l , 1975). Cheung and Lang (1977) recently reported that from MCMV infected mice, salivary and prostatic tissue cultivated in vitro would eventually produce infectious virus within weeks (or months) after explantation. In this case, the authors speculated that in vitro activation was a consequence of enhanced cellular proliferation associated with explantation of tissue into c e l l culture. D. The Cell Cycle: The demonstration of a separate DNA synthetic period with inter-vening 'gaps' between mitotic events established the kinetics of the c e l l cycle (Howard and Pelc, 1953). The c e l l cycle consists of four distinct phases: (1) phase or the f i r s t 'gap', followed by (2) S phase or the DNA synthetic period, (3) G^ or the second 'gap' and (4) M phase or mitosis, followed by a subsequent G^ . A number of investi-gators recognize a G^ phase as an alternative to G^  phase (reviewed by Baserga, 1976). Each of the phases w i l l be described briefly below with emphasis on the biochemical events of S phase. A diagramatic view of the c e l l cycle is shown in Fig. 1. 1. G^ and G Q The most significant aspect of the G^  period concerns growth arrest. Mammalian cells in culture, upon depletion of growth factors, are 'restricted' (Pardee, 1974) in G 1 (Nilhausen and Green, 1965; Holley and Kiernan, 1968). The mechanism of G^  'restriction' (or G^  arrest) is not understood, but even less is known about the mechanism controlling the flow of cells from G^  into S. In order to discriminate between G^  arrested cells and cells traversing normally through G^  and into S, the concept of GQ has been introduced (Lajtha, 1963). G^  cells are defined as those which have been withdrawn from the c e l l cycle (Fig. 1) either transiently or permanently (e.g. confluent, stationary cells in culture are in G n). Fig. 1; 15 T H E C E L L C Y C L E The phase represents the f i r s t major biosynthetic period for G^ cells after growth stimulus and post-mitotic cells. The main biochemical events associated with G^  to S transition are summarized in Table I. 2. S phase The replication of cellular DNA proceeds in a distinct period of the c e l l cycle, representing 35-45% of the total c e l l cycle. The r e p l i -cation of eukaryotic DNA is characterized by: (1) a large number of replicating units (replicons) per genome (Prescott, 1976), (2) b i -directional mode of replication (Huberman and Riggs, 1968), and (3) discontinuous replication (Huberman and Horwitz, 1973). Besides DNA synthesis, other events in S phase include the synthesis of histones, non-histone chromosomal proteins, and RNA, a l l in a highly ordered manner (Mueller, 1971). The major biochemical milestones common to S phase are summarized in Table II. The majority of cellular enzymes are synthesized or expressed discontinuously during different phases of the c e l l cycle (reviewed by Mitchison, 1971). Only the S phase specific enzymes cognate to deoxynucleotide metabolism have been list e d in Table II. It i s now clear that several DNA polymerases are present in eu-karyotic cells (Romberg, 1974; Weissbach, 1975). A minimum of five DNA polymerases have thus far been recognized, in accord with: (1) their intracellular location, (2) their catalytic function performed on DNA (i.e. replication, repair), and (3) the appearance of v i r a l enzymes. Weissbach (1975) recently revised the pre-existing nomenclature on vertebrate DNA polymerases. The current scheme is summarized in Table III. Table I: Biochemistry of the phase in cells traversing the c e l l cycle toward S phase. Biochemical process Reference Rapid increase in synthesis of rRNA Increased uptake of uridine; decrease in cAMP levels Increased attachment of pre-existing, stable mRNA to free ribosomes Dramatic increase in protein synthesis and polysome reformation Synthesis of histone mRNA Increased phosphate transport Increase in deoxyribonucleotide pools Mauck and Green, 1973 Noonan and Burger, 1973 Rudland et a l , 1975 Levine e_t a l , 1965 Steward et a l , 1968 Borun ej: a l , 1967 Weber and Edlin, 1971 Tobey et a l , 1974 Table II: The biochemistry of S phase Molecular Event Reference Periodic Enzymes a. thymidine kinase b. dTMP kinase c. dCMP deaminase d. ribonucleotide reductase e. aspartate transcarbamylase f. DNA polymerase - a g. DNA polymerase - y h. DNA swivel enzyme Mitchison, 1969, 1971 L i t t l e f i e l d , 1966 Brent e_t a l , 1965 Mittermayer et^ a l , 1968 Turner et a l , 1968 Bostock £t a l , 1966 Spadari and Weissbach, 1974 Spadari and Weissbach, 1974 Rosenberg et a l , 1976 II. Histone biosynthesis and modification (phosphorylation, methylation, acetylation) Baserga, 1976 Tobey et a l , 1974 Mueller, 1971 III. Rapid expansion 'of deoxy-nucleotide pools Tobey et a l , 1974 Skoog et a l , 1973 IV. Increase in polyamines Sunkara et a l , 1977 Table III. Vertebrate DNA polymerases (Weissbach, 1975) DNA poly-merase % of total polymerase activity M.W. Location Distinguishing Features a 80-90 1.1-2.2 x 10 Nucleus and cytoplasm S phase limited sensitivity to sulfhydryl group inhibitors 3 5-15 4.5 x 10 4 Nucleus synthesized continuously throughout the c e l l cycle insensitive to sulfhydryl group inhibitors Y 1-2 1.1 x 10 5 Nucleus and cytoplasm S phase limited sensitive to sulfhydryl group inhibitors Mt - 1.5 x 10 5 Mitochondria copies circular duplex DNA V i r a l variable New DNA polymerases which appear after infection with a virus (Reverse transcriptase excluded from this cate-gory) 2. G„ and mitosis Upon completion of DNA replication, the c e l l progresses into G^ , a phase which may be viewed simply as a pre-staging area for mitosis. Thus, the G^  c e l l synthesizes RNA and protein necessary for the orderly progression into mitosis (Baserga, 1976). A class of proteins, limited to G^ , have been identified on acrylamide gels, but their function i s unknown (Kolodny and Gross, 1969). Actually, many events in G^ are obscure due to the problem of preparing adequate numbers of pure G^  cells (Tobey, 1971). Furthermore, the G^ phase i s not as well defined as the other c e l l cycle phases and may represent, at least in some ce l l s , an extended prophase (Prescott, 1976). Cell division, which i s limited to the M phase of the cycle, usually represents <5% of the total generation time and involves four stages: prophase, metaphase, anaphase and telophase. The morphological changes during M ensue rapidly and are very pronounced. Cells in metaphase are rounded and rather tenuously attached to the substratum, providing a convenient method of synchronization by 'selective wash-off of M cells (Terasima and Tolmach, 1963). Recently, evidence concerning the biochemical changes during M has emerged. It is generally accepted that the energy requirements for M are supplied by ATP-linked reactions (Tobey et a l , 1971). During M phase the rate of protein synthesis i s significantly decreased due to a reduction in the attachment of mRNA to ribosomes (Fan and Penman, 1970). The rate of transcription declines precipitously in late pro-phase, and RNA synthesis i s minimal just prior to metaphase (Prescott, 1976). The block in RNA synthesis has been ascribed to the condensation of chromosomes, in which case the DNA template would be inaccessible to 21 RNA polymerase. However, other results indicate that constituents of the chromosomal protein fraction are responsible for the inhibition of RNA synthesis during M phase (Farber ^ t a l , 1972). In conjunction with the inhibition of macromolecular synthesis, numerous other changes occur (summarized from a review by Prescott, 1976): (1) the nuclear envelope and nucleolus disappear; (2) nuclear proteins and -HnRNA are released into the cytoplasm; (3) the processing of precursor rRNA ceases. In late telophase, as the daughter c e l l nuclei reform, nuclear proteins and RNA return to the nucleus. E. Interaction Between Animal DNA Viruses and Synchronized Cells 1. Importance of v i r a l and c e l l cycle studies Synchronization allows one to infer the order of events in the replication of a virus in a highly quantitative manner, as well as exposing the intricacies of v i r a l induced changes upon the host c e l l cycle. The extra effort involved in establishing a synchronous system i s compensated for, since these studies are the closest approximation to the reciprocal influence of a single c e l l and single virus. The use of synchronous cells in virology is of major interest for two reasons. Fi r s t , the virus may be used as a simplified 'window' to study the c e l l cycle. Second, the details of how viruses act as gene regulators may be investigated (Lin and Munyon, 1974). 2. General survey of the DNA containing viruses and the c e l l cycle Table IV summarizes the available information on the interaction of DNA viruses with the c e l l cycle. • Several pertinent conclusions may be gleaned from this comparative survey: 1. The a b i l i t y of a virus to replicate independently of S phase appears to be unrelated to genetic complexity, at least for genomes greater than 25 x 10 daltons (adenovirus) in molecular weight. This aspect is particularly obvious in the herpesvirus group. 2. In general, the a b i l i t y of a virus to induce S phase enzymes (Table II) does not correlate with the a b i l i t y to replicate independently of S phase. The one possible exception is thymidine kinase and the herpesvirus group (see next point). 3. Within the herpesvirus group, the failure of EAV and MCMV to induce thymidine kinase correlates with S phase dependency. However, this association may easily be fortuitous and certainly does not hold for other virus groups. 4. Dependence upon the c e l l cycle actually means dependence upon S phase, since requirements for G^ , or M have never been reported. On the other hand, the possibility that a virus requires G^  or G^  limited functions exists, but would be d i f f i c u l t to prove. 5. Virtually a l l c e l l cycle dependent viruses require the host S phase for i n i t i a t i o n of v i r a l DNA synthesis. This is logical since the virus, li k e the host, must replicate DNA i n order to reproduce. 6. The observation that vaccinia replication is inhibited in M cells suggests that even c e l l cycle independent viruses are not entirely free of c e l l cycle constraints. Indeed, with herpesviruses and poxviruses v i r a l replication i s inhibitory for M and vice versa (Vantis and Wildy, 1962; Tegtmeyer, et a l , 1969; Groyon and Kniazeff, 1967). After infection with a c e l l cycle independent virus, the c e l l may enter a G n state in a similar manner to differentiated Table IV: The Interaction Between Animal DNA Viruses and the Cell Cycle Virus Group Genome S Phase Induction Character- Depend- of S Phase i s t i c s ency a Enzymes b Comments Primary Reference I. Poxvirus c m.w. = , No 1. Thymidine kinase 1. Initiation of v i r a l DNA Mantani and Kato, 160 x 10 2. DNA Polymerase synthesis i n G^  1970. A. vaccinia (D.S.) 2. Replication unimpeded by Groyon and Kniazeff, 2.5 mM TdR 1967 3. Vi r a l replication inhibited in M cells II. Herpesvirus A. HSV-1 m.w. = , No 1. Thymidine and 1. Initiation of v i r a l DNA Cohen et a l , 1971 100 x 10 dTMP kinase synthesis in G^  Cohen, 1972 (D.S.) 2. DNA Polymerase 2. Replication unimpeded by Cohen et a l , 1974 3. Ribonucleotide 2.0 mM TdR Roller and Cohen, reductase 3. Deoxynucleotide pools 1976 induced B. HSV-2 m.w. = , No 1. Thymidine kinase 1. Initiation of v i r a l DNA Roller and Cohen, 100 x 10 2. DNA Polymerase synthesis i n G^  1976 (D.S.) 3. Ribonucleotide 2. Vir a l replication sensitive Cohen £t a l , 1974 reductase to 2 mM TdR 3. Deoxynucleotide pools induced C. EAV m.w. = Yes 1. DNA Polymerase 1. Initiation of v i r a l DNA Lawrence, 1971 84 x 10 synthesis requires host (D.S.) S phase 2. Thymidine kinase not coded for Table IV: Cont'd. Virus Genome S Phase Induction Comments Primary Group Character- Depend- of S Phase Reference i s t i c s ency a Enzymes ^  D. PRV m.w. = 80 x 10 (D.S.) No 1. dTMP and thymidine kinase 2. DNA Polymerase 1. Replication in G^ cells 2. Replication unimpeded by 2 mM TdR Ben-Porat and Kaplan, 1973 E. EBV m.w. = , 105 x 10 (D.S.) Yes 1. DNA Polymerase Activation of the latent EBV genome occurs during S phase Hampar et a l , 1976 F. MCMV m.w. = ^ Yes 1. DNA Polymerase 1. Initiation of v i r a l DNA Muller and Hudson, 132 x 10 synthesis requires host 1977 (D.S.) S phase 2. Viral growth sensitive to 2.0 mM TdR 3. No v i r a l growth in G^ cells 4. Thymidine kinase not coded for G. HCMV m.w 1 x 10 (D.S.) 8 No 1. DNA Polymerase 2. Thymidine kinase 3. 4. HCMV may stimulate c e l l DNA synthesis Vi r a l DNA synthesis does not depend upon host S phase Replication in G^  cells Viral replication unimpeded when host c e l l cycle traverse blocked by TdR or Fu DeMarchi and Kaplan, 1975 Zavada et a l , 1976 K5 4S Table IV: Cont'd. Virus Genome S Phase Induction Comments Primary Group Character- Depend- of S Phase Reference i s t i c s ency a Enzymes b I I I . Adenovirus m.w.= 20 - No 1. DNA Polymerase 1. V i r a l DNA synthesis does Hodge and Scharff, 25 x 10^ 2. Thymidine kinase not depend upon host S 1969 (D.S.) phase S t r o h l , 1969 2. C e l l DNA synthesis stimulated a f t e r i n f e c t i o n of G^ c e l l s IV. Papovavirus m.w. = ^ Yes 1. DNA Polymerase 3 x 10 2. Thymidine kinase (D.S.) 3. dTMP kinase 1. I n i t i a t i o n of v i r a l DNA Pages jet a l , 1973 synthesis require host Thorn, 1972 a, 1972^ S phase 2. C e l l DNA synthesis stimulated a f t e r i n f e c t i o n of G, c e l l s V. Parvovirus m.w.= 1.2-1.8 x 10 6 (S.S.) Yes Unknown 1. V i r a l DNA synthesis requires l a t e S phase events 2. These viruses have a p r o c l i v i t y for r a p i d l y p r o l i f e r a t i n g t i s s u e i n vivo Tennant et a l , 1969 Hampton, 1970 Toolan, 1972 T a t t a r s a l l , 1972 Rhode, 1973 S i e g l and Gautschi 1973a, 1973 b a Dependency upon S phase correlated with productive i n f e c t i o n s only. D E i t h e r host or v i r a l coded enzymes. c Poxviruses r e p l i c a t e i n the cytoplasm, a l l others i n the nucleus. cells (e.g. neurons). This concept is not new since, according to Roizman (1974) " i t is heuristically profitable to consider as more correct the view that productive infection with viruses i s complex and that herpesvirus infection i s a form of extreme, i r -reversible differentiation resulting in the synthesis of one product, the virus, and in c e l l death". The purpose of this study was to investigate the interaction between a herpesvirus and the c e l l cycle at the molecular level. From Table IV, i t i s clear that a large number of viruses have been characterized as ' c e l l cycle dependent'. However, in no synchronous-virus c e l l system has any attempt been made to answer the question 'what i s the nature of the dependency upon S phase'. In this thesis an attempt is made to answer this query, and in so doing, a latent system has been characterized in c e l l culture which may serve as an implement to study: (1) the nature of the S-phase dependency; (2) v i r a l persistence; and (3) the l y t i c cycle of replication. CHAPTER 2 27 MATERIALS.AND METHODS Reagents A l l reagents, except those l i s t e d below, were purchased from Fisher Chemical Company. Reagent Agarose Aquasol 5-bromodeoxyuridine Cesium chloride Dimethyl sulfoxide Deoxyribonuclease (electrophoretically purified) Deoxyribonucleoside triphosphates Dithiothreitol Freund's Adjuvant Gentamycin H-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes) Hydroxyapatite (Bio Gel HTP) 2-Mercaptoethanol Mycostatin Nonidet P-40 Source Seakem, Bausch and Lomb New England Nuclear Sigma Chemical Co. Schwartz-Mann Co. or Sigma Chemical Co. Continental Biosystems Inc. Worthington Biochemical Co. or Sigma Chemical Co. Sigma Chemical Co. Miles Laboratories Inc. Dif co Sigma Chemical Co. Calbiochem. Bio-Rad Laboratories Eastman Organic Chemicals Gibco Shell Chemical Co. Ltd. Reagent Source Penicillin-streptomycin-fungizone Phosphonoacetic acid Pronase (Streptomyces griseus) Type VI. Proteinase K 32 32 P (as PO^)> carrier free Ribonucleoside triphosphates Ribonuclease A S-l nuclease 125 Sodium iodide I, carrier free low pH Spectrafluor Thallic chloride Thymidine 3 Thymidine-methyl- H (17 Ci/mmole or 44) Thymidine 5'-triphosphate-methyl-3 H tetrasodium salt (46 Ci/mmole) Trypsin Uridine (5,6,-3H) (44 Ci/mmole) Gibco Abbott Laboratories (a kind g i f t f Dr. A. 0. Geiszier). Sigma Chemical Co. Beckman New England Nuclear Sigma Chemical Co. Sigma Chemical Co. Sigma Chemical Co. New England Nuclear (NEZ-033L) Amersham Searle Co. K & K Laboratories Inc. Sigma Chemical Co. New England Nuclear (NET-027Z) New England Nuclear (NET-221X) Difco New England Nuclear 29 Solutions and Buffers: Hank's Balanced Salt Solution (lx) (Hank's BSS) Glucose 5.5 mM NaCl 147.0 mM KC1 5.3 mM KHoP0, 4.4 mM 2 4 Na„HP0. 15.0 mM 2 4 Phenol red 0.6 mM prepared as a lOx solution, stored at -20°C, diluted to lx and steri l i z e d by autoclaving (15 lbs steam pressure, for 20 minutes), Phosphate Buffer (stock solution) (PB) Na.HPO. 0.5 M 2 4 NaHoP0. 0.5 M I 4 pH 6.8 diluted to specified molarity Phosphate Buffered Saline (PBS) NaCl 130 mM KC1 2.7 mM NaoHP0. 8.1 mM 2 4 KH„P0, 1.5 mM 2 4 CaCl 2 1.0 mM MgCl 2 0.5 mM RNA Buffer (Hudson et a l , 1970) NaCl 100 mM CH3C00Na MgCl 2 10 mM 1 mM adjusted to pH 5.2 S-l Buffer (modified) NaCl 300 mM CH3C00Na ZNSO, 30 mM 1.8 mM adjusted to pH 4.5 with acetic acid. S c i n t i l l a t i o n Fluid per l i t e r of toluene: 4 g 2,5,-Diphenyloxazole 50 mg l,4-Bis-(2-(5-phenyloxazolyl))-benzene Sodium Acetate Buffer (Commerford, 1971) CH3C00Na 0.25 M CH„C00H 0.1 M 3 (pH 5.0) 31 Standard Saline Citrate (Gillespie and Spiegelman, 1965) (SSC) NaCl 0.15 M Na 3 citrate 0.015 M A stock solution was prepared at 12x this concentration, and diluted to the desired concentration. Thallic Chloride (Buffered) (Commerford, 1971) Thallic chloride 3 mM CH3C00Na 250 mM CH3C00H 100 mM pH 4.5 TNE TRIS 10 mM NaCl 100 mM EDTA 1.0 mM pH 7.5 TNM TRIS 10 mM NaCl 100 mM MgCl 2 1.0 mM pH 7.5 C. Routine Tissue Culture Techniques: 1. Cells ME cells were prepared by trypsinization and explantation of embryos from randomly bred Swiss white mice obtained from the Faculty of Medicine Animal Unit, University of British Columbia. Mouse 3T3 cells (Swiss-3T3) were obtained from Flow Laboratories. 3T3-4E (a TK~ mutant) and the wild type parental c e l l line, designated 3T3-H, were a kind g i f t of Dr. H. Green. H.Ep.2 cells were obtained from Microbiological Associates. 2. Growth medium For cultivation of a l l c e l l s , Eagle's minimal essential medium (MEM), Dulbecco's modification, was used. The MEM was obtained from Grand Island Biological Company in powdered form, rehydrated and f i l t e r s t e r i l i z e d (Bac-T-Kote, 0.2 uM pore size, Schleicher and Schuell Inc.). For open system cultivation (in petri dishes in a CO^ incubator), 3.7 g/1 NaHCO^ were added (MEM-A), and for closed systems (capped r o l l e r bottles) 1.5 g/1 were added (MEM-B). After s t e r i l i z a t i o n , antibiotics were routinely added (gentamicin, 20 yg/ml or p e n i c i l l i n 100 u/ml, streptomycin 100 u/ml, fungizone .25 yg/ml). Fetal bovine serum (Gibco Laboratories) was added at a concentration of 0.1, 1, 5, or 10%, just prior to use. 33 3. Culture conditions Cells were incubated in 35, 50, or 90 mm Falcon tissue culture dishes (Fisher Scientific Company) in a humidified atmosphere containing 5% C0 2 and 95% air at 37°C. Frequently, cells were incubated in stoppered glass r o l l e r bottles at 37°C on a Bellco c e l l production rol l e r apparatus. Two sizes of rol l e r bottles were used, the larger re-usable Bellco bottles and Bellco disposable units. In addition, 32 oz. prescription bottles (Brockway Bottles, Brown Bottle Company, Quebec) were used. These bottles were capped and incubated at 37°C on a f l a t surface. The surface areas of the various containers used for c e l l culture 2 2 are: (1) 90 mm plates - 64 cm ; (2) 50 mm plates - 20 cm ; (3) 35 mm 2 2 plates - 8 cm ; (4) Brockway bottles - 120 cm ; (5) Re-usable rol l e r 2 2 bottles - 1400 cm ; (6) Disposable r o l l e r bottles - 700 cm . 4. Cell transfer 3T3-4E and 3T3-H cells were subcultured when confluent, every 3 to 4 days. Swiss 3T3 cells were faster growing cells and had to be sub-cultured every 2 days as subconfluent monolayers to ensure exponential growth. ME cells were usually subcultured at confluence (every 2 to 5 days depending on passage number). The medium was decanted and the monolayers were gently rinsed once with pre-warmed MEM to remove debris. Trypsin (0.25% in Hank's BSS) was then added for 2 to 6 minutes and decanted. The cells were resuspended in 5-7 ml. of fresh MEM + serum by pipetting and were gently syringed twice (10 cc syringe with a 21 g needle) to disperse clumps. ME cells were not routinely syringed. The c e l l suspension was diluted to the appropriate density and dispensed into fresh culture vessels. Freshly seeded plates were rocked to-and-fro several times to ensure an even distribution of cells and placed in the incubator. For seeding r o l l e r bottles, 3T3 cells were syringed more vigorously and seeded at a lower density. These measures helped obviate clumping of the unattached . c e l l s . 5. Cell counting Cells were trypsinized and resuspended in a known volume of PBS or Hank's BSS. Accurate c e l l counts were performed using either a Levy-Hauser hemacytometer or a calibrated Coulter counter (50y probe, Model B, Coulter Electronics Inc.). Cell counting on plates was also performed with the aid of an 2 ocular grid (1 mm ) in a Wild inverted phase contrast microscope. The number of cells/grid was converted to cells/dish by interpolation from a standard curve, which was constructed for 35, 50 and 90 mm dishes. 6. Freezing of cells A l l 3T3 c e l l lines were prepared for freezing in the following way. Subconfluent exponential cells in 90 mm dishes were trypsinized in the usual manner (for c e l l transfer) and suspended in 1.8 ml MEM-A plus 20% fetal bovine serum. The c e l l suspensions were trans-ferred to vials on ice containing 0.2 ml dimethyl sulfoxide, mixed and placed at -70°C in a Revco freezer. 35 Approximately every 6 weeks or 20 passages, fresh cells were established from frozen stocks by simply thawing and re-plating in MEM-A + 20% serum. Immediately after the cells attached, fresh MEM con-taining 20% serum was added. D. Viruses: The herpesviruses used in this study were: (1) MCMV, the Smith strain; (2) HSV-1, P-Strain (Mosmann and Hudson, 1973); (3) HSV-B2006, a TK~ mutant of HSV-1, obtained from Dr. D.R. Dubbs. 1. Routine propagation of herpesviruses HSV-B2006 and MCMV were grown in secondary, tertiary or quaternary ME cells in roller-bottles. HSV-1 was routinely propagated in H.Ep.2 cells in roller-bottles. Medium was decanted from the r o l l e r , and 10 ml (for large roller) or 5 ml (for small roller) of virus in MEM-B + 10% serum added for a 2h. adsorption period. The input multiplicity was <0.01 pfu/cell. Usually, by 36 hours post infection, foci of infection were evident in the infected cultures. The medium was then decanted and *g volume of fresh MEM + 10% serum added to maintain exponential c e l l growth which significantly increased v i r a l yields. The supernatant was removed when CPE was >98%, and placed at -70°C in 2.0 ml aliquots. E. Purification of Virus Supernatant virus from ME c e l l roller-bottles was centrifuged at 1000 xg in the 240 rotor of the IEC-CS centrifuge for 10 minutes. The supernatants were pooled, added to 250 ml Nalgene centrifuge bottles, and centrifuged for 3 hrs. at 20,000 xg in the GSA rotor of an RC2-B Sorvall centrifuge. The supernatants were discarded; the v i r a l pellets resuspended by vigorous pipetting in 5 ml of TNM and treated with pan-creatic DNase (50 ug/ml) for 30 minutes at 37°C. The suspension was diluted with 3 to 4 volumes of TNE and c l a r i f i e d by a second 10 minute low speed spin in the 240 rotor of the IEC-CS centrifuge. The super-natant was then centrifuged at 20,000 xg in the SS-34 rotor in an RC2-B Sorvall for another 3 hrs. F. Infection of Cells for Experimental Purposes: 1. MCMV MCMV infections were performed by either of two methods: (1) standard adsorption: medium was drained from the cultures, and a minimal volume (1.0 ml for 35 mm plates, 2.0 ml for 50 mm plates and 4.0 ml for 90 mm plates) of either MEM-B plus 5% serum or Hank's BSS containing virus was added. The cells were exposed to the virus for 1 to 2 hours at 37°C on a rocker platform. After the adsorption period, the inocula were removed and the plates given pre-warmed medium and placed in a 37°C CO2 incubator. (2) centrifugal adsorption: centrifugal infections were essentially the same as above, except that the plates were stacked in the buckets of an IEC model CS centrifuge (35 mm and 50 mm plates) or a Damon/IEC model CU-5000 centrifuge (90 mm plates), and centrifuged at 600-800 xg for 30 minutes at room temperature. Infections were performed at an input m.o.i. of 10 to 20 pfu/cell unless stated other-wise. 37 2. HSV-1 and HSV-B2006 Infection of 3T3 cells by HSV-1 and HSV-B2006 was carried out by adsorption in MEM-B plus 5% serum, for 2 hours on a Rocker platform at 37°C. MEM-A plus 5% serum was added after decantation of the inocula. The input m.o.i. was adjusted to 20 pfu/cell unless stipulated otherwise. G. Plaque Assays and Infectious Center Determinations For plaque titrations, virus samples containing c e l l plus supernatant associated virus, were freeze-thawed three times and serially diluted in MEM-B plus 5% serum. MCMV infections were performed on 3T3 or ME cells by standard or centrifugal adsorption. HSV-1 and HSV-B2006 were assayed on either 3T3, ME or H.Ep.2 cells by standard adsorption. After adsorption, the inoculum was removed and the cells overlayed with MEM-A (5 to 10% serum) plus 0.5% agarose. Plates were l e f t to solidify at room temperature for 10 minutes and placed i n a 37°C CO^ incubator. Plaques were visible within 72 hours and were counted without staining. Infectious center determinations (MCMV only) were carried out in the following manner. Infected cells were rinsed ten times with Hank's BSS and trypsinized for 5-10 min. at room temperature. After resuspending the cells in MEM-A plus 5% serum by pipetting and gentle syringing, an aliquot was removed for c e l l number determination. The remaining cells were diluted s e r i a l l y in MEM-A plus 10% serum, and 1.0 ml of each dilution added to freshly plated, subconfluent 3T3 or ME cells , which were placed in the 37°C CO^ incubator un t i l the cells had attached (usually 2-4 hours). The medium was decanted and replaced with 0.5% agarose, MEM-A plus 10% serum. Infectious centers, which developed usually within 72 hours, were enumerated and the number of infectious centres per 10" cells calculated. H. Synchronization of Cells 1. G^  arrest 3T3 cells seeded in MEM-A + 10% serum were allowed to reach con-fluence (usually 2 days) and l e f t without medium change for a total of 4 to 10 days. At this time, the cells appeared crowded, but not stacked, and the cultures contained cells as indicated by the total 3 absence of mitotic cells and H-TdR labelled nuclei. The cultures could be maintained in G^  for varying lengths of time, up to two weeks, depending upon the i n i t i a l seeding density and serum concentration. The cells would eventually die and slough off, however, only those experiments u t i l i z i n g healthy, viable G^  cells were analyzed. Centrifugal or standard infections of G^  cells were carried out in MEM-B + 0.1% serum or Hank's BSS after adsorption, the inocula were decanted and G^  'depleted' medium re-added to maintain G^  arrest. 2. Synchronization by serum-split G^  arrested 3T3 cells were stimulated to enter S-phase by: (1) rinsing the c e l l sheet once with pre-warmed MEM-A; (2) trypsinizing for 1 to 2 min.; (3) suspending the cells by pipetting; (4) gently syringing the c e l l suspension with a 10 cc syringe (21 g needle); (5) replating i n a 4 to 10 fold larger volume of pre-warmed MEM-A plus 10% serum. This procedure is referred to as the serum-split method. 39 Quiescent cells could also be stimulated to enter S phase by a medium change, viz: fresh MEM-A plus 10-20% serum, without replating. However, the resulting degree of synchrony was not as good as the serum-s p l i t method. After the serum-split, the cells would rapidly attach to the plates (15 min.). Standard and centrifugal infections in early G^  were routinely carried out at 1 to 2 hours post serum-split. 3. Double TdR treatment A modification of the excess TdR method, reported by Bootsma et a l (1964) was used. 3T3 cel l s , seeded at a low density (1 x 10^ c e l l s / 50 mm plate), were treated with 2.0 mM TdR for 12 hours. The cells were then washed free of TdR and given fresh MEM-A + 10% serum for 8 hours, at which time 2 mM TdR was added for an additional 12 hours. The cells were infected by standard or centrifugal adsorption in TdR-free medium. After the adsorption period the inoculum was decanted, and fresh medium, with or without 2 mM TdR, was added. 4. Mitotic harvests Exponential 3T3 cells in Brockway bottles were used. One to three hours before the actual harvest, the bottles were agitated vigorously to detach debris and dead c e l l s . The growth medium from 10 bottles was decanted and 20 mX of pre-warmed MEM-B + 2% serum added to the f i r s t bottle in the series. After gently rocking the bottle 20 times from side to side for 60 seconds, the medium was transferred to the second bottle and the process repeated in sequence unt i l a l l bottles were harvested. The medium containing detached metaphase cells was emptied from the last bottle into a conical centrifuge tube on ice. Four to six harvests were routinely done (20 ml each). The cells were centrifuged (500 xg in an IEC-CS centrifuge) and resuspended in pre-warmed medium. An aliquot was removed and immediately processed for determination of mitotic fraction. I. Analysis of the Degree of Synchrony 1. DNA synthesis At various intervals after synchrony induction, the cells were 3 pulsed with 1 uCi H-TdR per culture for 30 minutes. Following the pulse, the radioactive medium was aspirated, cold TNE added, the cells scraped off and TCA added to 10%. The precipitates were kept at 4°C 3 for 1 hour and the level of H-TdR incorporation into acid precipitable material was determined as described below (Section U). The fraction of cells in S phase was also determined by auto-3 radiography. Cells in 35 mm plates were pulsed with H-TdR as described above, fixed for 30 minutes with methanol:acetic acid (3:1), washed for 30 minutes under cold running water and air dried. The plates were overlaid with Ilford liquid emulsion L4 (Ilford Ltd., Ilford, Essex, England), exposed for 1 week, developed with Kodak Microdol X and observed by phase contrast microscopy for counting. 2. Mitotic index The fraction of cells in mitosis was determined by swelling 41 detached cells in a hypotonic solution (1% sodium citrate) for 15 min. at 4°C. The suspension was then fixed by drop-wise addition of methanol: acetic acid (3:1), spread onto glass microscope slides, stained with crystal violet and counted in a phase contrast microscope. Between 500 and 1000 cells were scored for mitotic figures. 3. Cell numbers Cell numbers per plate were determined using a calibrated Coulter counter as described i n Section C-5. J. Nucleic Acid Purification 1. V i r a l DNA Virus, purified by differential centrifugation, was suspended in TNE + 1% SDS. Pronase (self-digested at 37°C for 2 hours) was added to 1 mg/ml and the protein digested for 3 hrs. at 37°C. Alternatively, proteinase K (100 ug/ml) was added and the protein digested overnight at 65°C. V i r a l DNA was then extracted as described by Mosmann and Hudson (1973). After dissolving the DNA in the suitable aqueous buffer the concentration was determined by absorption at 260 nm wavelength, assuming 1 A^gQ unit = 50 Ug/ml of DNA. 2. Cellular DNA Cellular DNA was purified by the SDS, pronase (or proteinase K), phenol-chloroform technique, as described above for v i r a l DNA. After the DNA was ethanol precipitated and washed, and then dissolved in TNE, 50 yg/ml RNase was added and the mixture incubated at 37°C for 30 minutes. The RNase had been previously boiled for 5-10 minutes to destroy trace amounts of DNase activity. SDS was added to 1%, pronase to 1 mg/ml or proteinase K to 100 yg/ml, and the mixture was incubated at 37°C for 1 hr. One phenol and one or more chloroform extractions were repeated as described above. The DNA was precipitated with 95% ethanol, rinsed in ethanol, and dissolved in either TNE or .01, 1 or 2x SSC. 3. Total cellular RNA A modification of the procedure described by Palmiter (1974) was used to purify RNA. The cells were scraped off the plates into the growth medium, deposited in 50 ml tubes, and centrifuged at 1000 xg i n the IEC-CS centrifuge for 10 minutes. The supernatant was discarded and the c e l l pellets resuspended in RNA buffer by vortexing, and then centrifuged again. After resuspending the cells in RNA buffer, SDS was added to 1% and the lysate stored at -20°C. At the end of the experiment, a l l lysates were thawed and proteinase K added to a f i n a l concentration of 100 ug/ml. After a 3 hr. incubation at 65°C, 0.5 volume of water saturated, r e d i s t i l l e d phenol was added and mixed by vortexing. Then, 0.5 volume of chloroform was added, the mixture vortexed again and centrifuged at 2000 xg for 10 minutes in the 240 rotor of the IEC-CS centrifuge. The lower phase was withdrawn, leaving the flocculent interface behind, and an equal volume of chloro-form added to repeat the extraction. Subsequent chloroform extractions were done unt i l the interface had either disappeared or no longer changed. Usually two or more extractions were required. The upper aqueous phase was removed leaving the interface material behind. RNA was precipitated with two to three volumes of 95% ethanol. For complete precipitation, the mixtures were stored overnight at -20°C. Then the RNA was collected by centrifugation at 22,000 xg for 30 minutes in a Sorvall RC2-B centrifuge. TNM was added and after complete dissolution of the precipitated RNA, DNAse (50 yg/ml) was added for 30 minutes at 37°C. SDS was added to 1%, proteinase K to a f i n a l concentration of 100 yg/ml, and incubation continued for.lhr. at 37°C. Phenol-chloroform extractions were repeated as described above, and the aqueous phase transferred to an acid washed 30 ml corex tube. The ethanol precipi-tation step was repeated twice as described, and the f i n a l precipitate dissolved i n 2 x SSC. K. Hydroxyapatite Column Chromatography A l l columns were run at room temperature by stepwise elution. A 5.0 ml volume of 0.05 M PB was added to a 2.5 cc disposable syringe f i t t e d with a porous polypropylene support disc. Hydroxyapatite was added to a depth of 5 mm and allowed to settle. The column was then washed with 5.0 ml of 0.05 M PB and drained completely. Radioactive DNA was added to the empty column bed in a 0.5 ml volume of .05 M PB, and the effluent collected. Then, 0.5 ml aliquots of 0.05 M PB were added, eluted and collected individually, to give 4 to 5 fractions. The process was repeated for 0.16 M and 0.48 M PB. Calf thymus carrier DNA (25 yg/fraction) was added and each fraction vortexed. An equal volume of .01 M pyrophosphate in 20% TCA was then added and the DNA precipitated at 4°C for lhr. The samples were processed for liquid s c i n t i l l a t i o n counting as described in Section U. Recovery of DNA from the column was greater than 95%. L. Sucrose Gradient Velocity Sedimentation 1. Neutral pH In vitro labelled DNA from MCMV infected, NP-40 treated nuclei, purified according to method 2, or marker DNAs, were gently layered onto a 5.0 ml linear 5-20% sucrose gradient containing a .25 ml, 60% sucrose cushion. Sucrose solutions at neutral pH were prepared in TNE on a w/w basis and the gradients formed in siliconized SW-50.1 poly-allomer tubes using a Buchler twin cone gradient generating device and p e r i s t a l t i c pump. The gradients were centrifuged for 2.5 to 4 hr. at 35,000 rpm in a preparative Beckman ultracentrifuge (L2-65B or L5-50). The tubes were fractionated from the bottom into 10 drop fractions, 25 ug of carrier DNA added to each tube and an equal volume of 0.01 M pyrophosphate in 20% TCA added. After lhr. at 4°C the precipitates were processed for liquid s c i n t i l l a t i o n counting as described below in Section U. 2. Alkaline pH Alkaline sucrose gradients were prepared and centrifuged exactly as above in TNE containing 0.2 M NaOH. The DNA was treated briefly i n 0.2 M NaOH at room temperature prior, to loading onto the gradients. After fractionation of the gradients, 0.2 ml of carrier DNA (100 yg/ml) in 0.5 M t r i s - C l (pH 7.0) plus 3 M NaCl was added to each fraction 45 with vortexing. An equal volume of .01 M pyrophosphate i n 20% TCA was added and the p r e c i p i t a t e s processed as above. M. Preparative Cesium Chloride Equilibrium Centrifugation Labelled or unlabelled DNA was d i l u t e d i n t o TNE and s o l i d CsCl added to give a density of 1.700 g/cc. Densities were determined by r e f r a c t i v e index measurements using a Bausch and Lomb Model ABBE 3-L refractometer. A l l centrifugations were performed at 20°C i n e i t h e r a Beckman L2-65B or L5-50 preparative u l t r a c e n t r i f u g e . At the end of the c e n t r i f u g a t i o n , the tubes were punctured from the bottom and fracti o n a t e d . Then, a f t e r determining the r e f r a c t i v e index of every f i f t h f r a c t i o n , 0.5 ml of TNE was added and the DNA bands l o c a l i z e d by: (1) A 0 £ . measurements i n a G i l f o r d recording spectrophotometer; or zoU (2) r a d i o a c t i v i t y measurements. Unlabelled MCMV DNA, p u r i f i e d by method 1 above, was loaded onto a preparative CsCl gradient and centrifuged at 30,000 rpm for 60 hr. i n a s i l i c o n i z e d c e l l u l o s e n i t r a t e tube, i n the SW50.1 rotor. U.V. absorbing material at the density of MCMV DNA (1.718 g/cc; Mosmann and Hudson, 1973) was p r e c i p i t a t e d with ethanol as described. The DNA was dissolved i n e i t h e r .01 SSC, TNE or .05 PB and concentration estimated by ^frQ measurement. Preparative CsCl gradients of l a b e l l e d DNA were centrifuged i n eit h e r the SW50.1 rotor at 30,000 rpm for 60hrs. or a type 40 rotor at 35,000 rpm for 80hrs. The DNA was l o c a l i z e d i n the gradient by spotting a 25 u l a l i q u o t of each f r a c t i o n onto glass f i b e r f i l t e r s , which were dried and counted by l i q u i d s c i n t i l l a t i o n spectrometry. MCMV DNA was separated from c e l l u l a r DNA according to t h e i r d i f f e r e n t 46 buoyant densities (1.718 and 1.699 g/cc respectively). The two species of DNA were adequately resolved in the SW50.1 and type 40 rotor; however, the resolution was slightly better in the type 40 rotor. N. Radioiodination of MCMV DNA The iodination reaction was carried out at room temperature in a well ventilated fume hood to ensure containment of volatile radioactivity. The space within the hood was divided into two areas. One-half of the hood was used as a working area, and the second half was used to separate iodinated waste materials behind a tabletop lead barrier shield. A l l operations were performed wearing a lead apron and two pairs of tru-touch examination gloves. The procedure of Commerford (1971) was used for iodination, with a few minor modifications. 1. Preparation of the hydroxyapatite column and MCMV DNA A 5.0 ml volume of .05 M PB was added to a disposable 10 cc syringe fi t t e d with a porous polypropylene support disc. Hydroxyapatite was added to a depth of 5 mm and the column washed with 5 to 10 ml of .05 M PB. Vir a l DNA, purified by method 1 above and preparative CsCl gradient centrifugation, was dissolved i n 0.1 ml of .05 M PB at a concentration of 500 ug/ml. The DNA was then heat-denatured (100°C for 10 minutes) and quenched in ice. 47 2. Assembly of the reaction mixture On a 2 by 2 cm piece of parafilm, 5 y l each of denatured DNA, buffered T l C l ^ and d i s t i l l e d water were mixed in a microdroplet To a Beckman microfuge tube containing 10 y l of acetate buffer, 2.5 to 5 125 y l of sodium Iodide (1 to 2 taCi) was added. Extreme care was necessary to prevent the two solutions from making contact by f i r s t dispensing the acetate on the bottom of the tube with an Oxford 10 y l pipette 125 and then gently adding the sodium Iodine as a microdroplet to the side of the tube with an Eppendorf 5 y l pipette. The denatured DNA solution was transferred from the parafilm to the side of the same tube, using a 25 y l Oxford pipette, again taking care not to mix the contents of the tube. 3. The f i r s t heating step The entire contents of the microfuge tube were mixed using an Oxford 50 y l pipette. The tube was capped and immediately placed in a 65°C waterbath for 20 minutes. 125 4. Removal of unreacted Iodine The reaction mixture was removed from the waterbath and allowed to cool to room temperature. A 200 y l volume of .05 M PB was carefully added to the tube and the contents drawn up and expelled several times with an Oxford 100 y1 pipette. The solution was then layered onto the hydroxyapatite column bed and the column allowed to run dry. The tube was washed three times with 1 ml aliquots of .05 PB, and the wash added to the column. A l l of the unreacted "^"""iodine was eluted by passing 75 to 100 ml of .05 PB through the column. Radioactivity in the effluent was monitored by collecting 3 drops into s c i n t i l l a t i o n vials, adding 10 ml of Aquasol and counting. The column was allowed to run dry, 0.5 ml aliquots of 0.4 M PB were added, and then collected. The process was repeated unt i l five 1.0 ml fractions were collected. A 10 ul aliquot from each fraction was counted in Aquasol, and two 'peak' fractions pooled. 5. The second heating step The pooled "^ ~*I-DNA in 0.4 M PB was heated a second time at 65°C 125 for 1 hr. and dialyzed against one to two l i t e r s of lx SSC. The I-DNA i n 1 x SSC was mixed with an equal volume of glycerol and 10 ul 125 TCA precipitated and counted on a glass fiber f i l t e r . The I-DNA in glycerol was stored at -20°C. Virtually a l l of the "*"^I was present in an acid precipitable form. The specific activity of the DNA was 7 8 between 10 and 10 cpm/ug. 0. F i l t e r Hybridization (DNA-DNA) A modification of the Gillespie and Spiegelman (1965) technique was used as described by Mosmann and Hudson (1973). The annealing reaction was carried out for 12-24h. at 65°C. P. Solution Hybridizations 1. DNA-DNA and DNA-RNA The technique of solution hybridization was established and characterized in this laboratory by Vikram Misra. Non-radioactive DNA purified from infected or mock-infected cells by method 2 was dissolved in 2 x SSC at a concentration of 500 to 850 yg/ml. Similarly, purified non:radioactive RNA was dissolved in 2 x SSC at a concentration of 900 yg/ml. Iodinated MCMV DNA, prepared as described above, was added in a volume of 35 to 50 y l (giving 2 to 4 x 10^ acid precipitable cpm) and the total volume made up to 2.0 ml. The mass ratio of non-radioactive DNA or RNA to iodinated DNA was estimated to be greater than 10^. The total reaction mixtures were sonicated as described above for the f i l t e r hybridization. Fragment size was estimated to be 5s by velocity sedimentation in neutral sucrose gradients (V. Misra, personal communication). The mixtures were then sealed in Corning disposable micropipettes (100 or 50 y l ) , placed in boiling water for 15 minutes and quickly immersed in 95% ethanol at -20°C. Several pipettes were withdrawn and individually dispensed into 1.0 ml aliquots of S-l buffer (4°C) containing 10 yg/ml denatured salmon sperm DNA. These tubes were stored at -20°C and processed for input and background cpm determinations. The remaining pipettes were placed in a 65°C waterbath to i n i t i a t e the hybridization reaction. Successive samples were then removed at pre-determined times, immediately frozen in -20°C 95% ethanol, dispensed into 1.0 ml aliquots of S-l buffer (4°C) containing 10 yg/ml single strand salmon sperm DNA, and stored at -20°C. 50 2. Hybrid detection After the last sample had been harvested, the solutions were thawed in a 37°C waterbath, and 60 units/sample of single strand specific nuclease S-l added to a l l but two tubes, which were used for determination of input cpm. Backgrounds were calculated as the number of acid precipit-able, S-l resistant cpm remaining in samples harvested immediately after denaturation. (Background levels were consistently less than 5% of input counts). Incubation was carried out for 1.5 hrs. at 37°C, followed by TCA precipitation and f i l t r a t i o n onto glass fiber discs. Radioactivity measurements were made as described below in Section U. This hybrid detection method separated DNA-RNA hybrids," DNA duplexes, 125 and renatured I-DNA probe from the bulk of single stranded material. Conditions were adjusted in the hybridization mixtures to minimize self-125 annealing of the I-DNA by using a vast excess of non-radioactive DNA 125 or RNA. Renaturation of I probe may introduce serious errors into quantitative analysis of hybridizations. However, the renaturation of 125 I-DNA was monitored for each DNA-RNA hybridization with heterologous yeast RNA (Sigma) or uninfected c e l l RNA. Similarly, calf thymus, salmon sperm, or 3T3 c e l l DNA were used in DNA-DNA reactions. 3. Presentation of results The DNA-RNA hybridization results were expressed as the percentage 125 of input I-DNA driven into hybrid form as a function of R Qt or the product of moles of ribonucleotides per l i t e r x seconds of incubation. The average molecular weight of a single ribonucleotide was taken to be 360. DNA-DNA reassociation experiments were expressed as /C as a 125 function of hours of incubation. C q is the total input cpm of I-DNA, and C i s the cpm present as single strand "^I-DNA. The plot of C°^C at various incubation times generated a straight line (Roizman and Frenkel 1973), the slope of which was directly proportional to the concentration of v i r a l DNA (from the original equation (Britten and Kohne, 1968) of C/ 1 Co = /(1 x K C Q t ) ) . The slopes were determined by computed-aided linear regression using a Hewlett-Packard 9810-A calculator containing a s t a t i s t i c s block and 9862-A plotter. Q. In Vitro DNA Synthesis The method of Bell (1974) was used to prepare nuclear monolayers for the endogenous assay of DNA synthesis jLn vitro-. At room temperature, the growth medium from infected or mock-infected cells i n 35 mm plates was decanted, and the monolayers were rinsed with 1.0 ml of 50 mM Hepes buffer (pH 7.6), containing 1 mM MgCl 2 > 0.5 mM CaCl 2 and 1 mM dithio-threitol. The monolayers were then treated with 1.0 ml of the same Hepes buffer containing 0.5% non-ionic detergent NP-40 for 2 minutes. The NP-40 was aspirated off the plates removing the cytoplasm and leaving the nuclei attached to the culture vessel as a nuclear monolayer. Sub-sequent washing of the nuclear monolayers was unnecessary and even un-desirable, since v i r a l infected nuclei were slightly more tenuously attached than uninfected nuclei. The basic incorporation mixture contained the following concentration of solutes in a f i n a l volume of 0.5 ml: 50 mM Hepes buffer (pH 7.6), 0.5 mM CaCl 2, 1 mM MgCl 2 > 1 mM dithiothreitol, 40 uM each of dATP, dGTP, 3 dCTP, 10 uCi (methyl- H) dTTP, 0.5 mM ATP and 80 uM each of GTP, CTP and UTp. The mixture was modified where indicated in the results to contain 1 to 4 M dTTP and/or 25 to 200 mM ammonium sulfate. The incorporation mixture was prepared fresh from stock solutions stored at -20°C, equilibrated at 37°C, and 0.5 ml gently added to plates containing nuclear monolayers. The plates were covered and incubated in a humidified 37°C NAPCO incubator. At the end of the incubation period (up to 60 minutes), nuclei were processed by either of two methods. For determination of TCA precipitable cpm, 0.5 ml of ice cold 1 x SSC, .01 M EDTA was added, the nuclei scraped off, trans-ferred to tubes containing 1.0 ml 20% TCA, .01 M pyrophosphate, and the samples processed as described in Section U. For extraction of DNA, an equal volume of 2 x TNE (.01 M EDTA) was added, the nuclei scraped off, transferred to a 15 ml corex tube and the DNA purified according to method 2. Variations in the above techniques are indicated in the results. R. Thymidine Kinase Assay Cell free extracts for TK assays were prepared as follows. Cell monolayers were washed twice with 3.0 ml buffer A (10 mM Tris-HCl pH 7.5; 5 mM 2-mercaptoethanol; 10 mM NaCl and 15 mM MgCl^), scraped off and centrifuged at 800 x g for 10 minutes. Cell pellets were resuspended in 3 to 5 volumes of buffer A and sonicated at 4°C for three 30 second intervals using a Bronwill Biosonik II probe sonicator set at 75% maximum intensity. The disrupted cells were then centrifuged at 15,000 rpm in a refrigerated RC2-B centrifuge (SS-34 rotor) for 20-25 minutes, and the supernatant retained for assay. The TK assay contained the following f i n a l concentrations of solutes 12.5 mM ATP, 12.5 mM MgCl 2, 2.5 mM Tris CI pH 7.5, 2.5 UCi methyl ( H) thymidine (NEN; specific activity 17 Ci/mmole) in a volume of 0.10 ml. The reaction was started by the addition of the c e l l free extract (0.1 ml, giving a f i n a l volume of 0.20 ml) and incubated at 35°C for 30 minutes. The assay was terminated by heat denaturation (5 minutes at 100°C) and the precipitated protein pelleted by centrifugation at 800 x g for 10 minutes. Fi f t y u l aliquots of the supernatant were spotted on DE-81 f i l t e r discs in t r i p l i c a t e . After the discs were washed 3 x in 0.1 mM ammonium formate, once in methanol and dried, the radioactivity was determined in a liquid s c i n t i l l a t i o n counter. The assay was linear with time (up to 45 minutes incubation). TK activity was proportional to protein concentration in a l l assays. It was shown that >95% of the radioactivity is retained on the f i l t e r s as 3 ( H)-dTMP, using thin layer chromatography on pel to resolve dTMP, dTDP and dTTP in a 0.5 M Li C l plus 5% sodium borate (pH 7.0) solvent system. S. Determination of Nucleotide Pools 32 1. P labelling, sampling and extraction of nucleotides 32 3T3 cells were pre-labelled with 20 UCi/35 mm plate of P-ortho-phosphate (carrier free) for 24 hours and infected with MCMV by centri-fugal adsorption. After the adsorption period, the inoculum was decanted 32 and fresh MEM-A + 10% serum added containing 20 UCi P. At various times post infection, the cells were acid extracted by the following method. The radioactive medium was decanted, the monolayer rinsed with 1.0 ml TNE and immediately overlayed with 0.5 ml of ice-cold 0.3 N formic acid. The plates were placed at 4°C for 1 hour, and the formic acid transferred to a Beckman microfuge tube. Detached cells were pelleted at 4°C for 5 minutes in a Beckman microfuge. Extracts were stored at 4°C and processed at the same time. 2. Spotting and development The method of Cashel et a l (1969) was used to separate the various nucleoside triphosphates on thin-layer, polyethyleneimene-cellulose chromatography plates. Polygram cel-300 pei/uv plates (20 x 20 cm), manufactured by Macherey-Nagel and Co., were used. The plates were washed by ascending chromatography with d i s t i l l e d water prior to use. For one dimensional development, origin spots were placed 2 cm from the bottom of the plate. As many as 18 samples could be analyzed on a single pei plate. For two dimensional development, a single origin spot was made 2 cm from the bottom and 2 cm from the side of the plate. Samples were applied in a volume of 10 to 25 y l directly onto the origin in a single application using a Corning micropipette. Nucleotide standards (usually 5 y l of a 10 mM solution) were co-chromatographed with the labelled nucleotides. Ribonucleoside triphosphates were separated by one dimensional ascending chromatography in 0.75 M KH^PO^ (pH 3.4). Development was terminated when the solvent front reached 17 cm above the origin. The plates were then water washed for 30 minutes, fan dried, washed in absolute methanol for 10 minutes, and air dried. Ribonucleoside triphosphate and deoxyribonucleoside triphosphate pools were analyzed by two dimensional chromatography. After developing the f i r s t dimension in 3.3 M ammonium formate, 4.2% boric acid (adjusted to pH 7.0 with ammonium hydroxide), the plates were washed in methanol for 15 minutes, dried, observed under short wavelength U.V., and the pH front (the second front below the solvent front) cut off. The plates were turned on their sides, and development in the second dimension carried out in 0.75 M KH^PO^ (pH 3.4). In both cases, the solvent front was allowed to migrate 15 to 17 cm above the origin. The plates were washed in water, then methanol and dried as in the one dimensional development. Chromatography was performed in glass tanks containing 100 ml of solution and fitt e d with air-tight l i d s . As many as 4 plates were developed in the same tank; however, care was taken to prevent the cellulose surfaces from touching. 3. Quantitation After development, the plates were marked with radioactive ink, and exposed to Kodak medical X-ray film overnight. The film was superimposed on the plate by aligning the ink marks. The exposed areas, which co-migrated with the standards were circumscribed by pricking holes through the film with a dissecting needle. These areas (plus a 'blank') were cut out, placed in s c i n t i l l a t i o n vials, and each disc treated for 15 minutes with 0.5 ml of 2N NH^ OH. One ml of water, and five ml of Aquasol were added, and the v i a l agitated prior to counting in a refrigerated Packard Tri-Carb liquid s c i n t i l l a t i o n spectrometer. Calculations of pool sizes were made according to the following formula: A x B x 1000 _ „ C x D x 3* 56 where A DPM in the spot B volume of acid used to extract the nucleotides C volume of extract spotted on the plate D 32 PdPM/y mole phosphate in the growth medium E = size of pool in nmoles of nucleotide/spot * A factor of 3 is used for triphosphate pools The f i n a l results were expressed as pmoles of nucleotide per 10 c e l l s . T. Anti-Infected Cell Protein Serum (Anti-ICP Serum) 1. Preparation of antiserum Anti-ICP serum (kindly provided by Dr. J.K. Chantler) was prepared by the following method. Infected M.E. cells were harvested at 30 h. post infection, suspended in PBS and sonicated for 40 seconds using a Bronwill Biosonik II probe sonieator at a setting of '40'. This material i s referred to as the infected c e l l protein or ICP. New Zealand, white rabbits were injected intravenously with 0.5 ml of ICP, which contained 2 mg/ml protein. An equal volume of Freund's adjuvant, emulsified with 0.5 ml of ICP was then injected intramuscularly. Four successive injections were done by the intravenous route at weekly intervals. The animals were bled from the ear vein 10 days after the last injection, and three additional bleedings were repeated at 2 month intervals. 2. Obtaining the IgG fraction from serum The clotted blood was centrifuged at low speed and the serum fraction transferred to a 10 ml beaker containing a 'flea' magnetic sti r r i n g bar. An equal volume of saturated ammonium sulfate was added drop-wise (1 drop/second) with vigorous s t i r r i n g . Stirring, at 4°C, was continued overnight, and the solution transferred to a 15 ml corex tube, followed by centrifugation at 10,000 rpm (10 minutes) in the SS-34 rotor of a refrigerated RC2-B Sorvall centrifuge. The pelleted precipitate was washed once with 50% saturated ammonium sulfate, and re-centrifuged. The precipitate, containing the globulin fraction, was taken up in PBS to give a total volume of about one quarter of the original serum volume. The re-dissolved precipitate was dialyzed against 1 to 2 l i t e r s of PBS at 4°C. Dialysis was continued until sulfate ions were no longer detected in an aliquot of the dialysate by the addition of a few drops of 10% barium chloride. Any insoluble material was removed by centrifugation as described. 3. Adsorption of antibody to cellular proteins Two adsorption steps were employed. Fi r s t , the gamma-globulin fraction was adsorbed overnight against PBS washed, exponential 3T3 cells at 4°C. The cells were removed by centrifugation and a second adsorption performed against 3T3 nuclear monolayers in mid-S phase (prepared at 20 h. post serum split) for 1 h. at 4°C. After another centrifugation,(10,000 rpm in the SS 34 rotor) the concentration of IgG was determined (assuming 1 A unit =1.5 mg/ml) and the anti-ICP serum stored at -20°C. Pre-immune serum was processed for the IgG fraction as described for the anti-ICP serum, omitting the adsorption step. 58 4. Experimental treatment of nuclear monolayers with anti- ICP and pre-immune serum Infected or mock infected nuclear monolayers were exposed to anti-ICP or pre-immune serum (diluted in Hepes buffer pH 7.6), at room temperature on a rocker platform for 30 minutes. The anti-sera were removed and the nuclei assayed for endogenous DNA synthesis as described. U. Processing of TCA Precipitates for Radioactivity Measurements Radioactive samples were precipitated in 10% TCA containing 0.01 M pyrophosphate for a minimum of 1 hour at 4°C, followed by f i l t r a t i o n onto glass fiber discs (Schleicher and Scheull, grade 29). The f i l t e r discs were washed twice with 5% TCA containing 0.01 M pyrophosphate and once with 95% ethanol. The discs were dried at 20°C for 1 hour, placed into glass vials and 3 ml of s c i n t i l l a t i o n f l u i d added per sample. The vials were tightly capped and counted at ambient temperature in an Isocap/300 liquid s c i n t i l l a t i o n spectrometer (Searle Analytical, Inc.). 58a CHAPTER 3 RESULTS SECTION I: CHARACTERIZATION OF THE LYTIC SYSTEM A. Growth Cycle of MCMV in 3T3 Cells Optimal conditions for v i r a l growth occurred in exponentially growing sparse monolayers of 3T3 cel l s . Supernatant virus yields were in the range of 10 to 100 pfu/cell. The lower yields (ca. 10 to 30) were ob-served i n confluent cultures, compared to 50 to 100 pfu/cell in sparse cultures. The single step growth of MCMV in 3T3 cells under optimal conditions takes 28 to 32 hours. MCMV replicated with a latent period of 12 hours in exponentially growing cells (Fig. 1). The kinetics of v i r a l growth were the same for both standard and centrifugal forms of inoculation... In a l l cases (at 30 h. post infection) the majority of progeny virus was found i n association with the c e l l s . B. Time of Onset of Vir a l DNA Synthesis 3 For reasons stated below (Section 2H), the use of H-TdR labelling to determine the time of i n i t i a t i o n of v i r a l DNA synthesis in the l y t i c system was avoided. An alternate technique was developed to assess exactly when DNA synthesis was initiated. It i s based on the observation by Britten and Kohne (1968), that the rate of reassociation of single strand DNA is directly proportional to the concentration according to the equation: 1 + kC t ( 1 ) o where C = total DNA concentration, C = concentration of single strand o DNA, t = time (seconds) and K i s a rate constant. If equation (1) i s 59a Fig. 1: The Growth Cycle of MCMV in Exponential 3T3 Cells 3T3 cells (1 x 10° cells/50 mm plate) were infected at an input multiplicity of 10 pfu/cell by standard inoculation, and the supernatants and cells harvested separately at various times post infection. Plaque assays were performed on freshly con-fluent ME cells by centrifugal inoculation. ( • ) Cell associated virus ( • ) Supernatant virus 60 1 0 2 0 H O U R S P . I . 61 inverted, the following equation is derived: C /C = C kt + 1 (2) o o Q In this equation _£ actually means 1/fraction remaining single stranded. C Q The resulting plot of a s a function of time generates a straight line C with a Y intercept of unity. The slope of the line is directly proportional to the concentration of v i r a l DNA, when the labelled 'probe' is MCMV DNA. Equation (2) was verified by a reconstruction experiment. Three concentrations of non-radioactive MCMV DNA were added to separate solutions of 2 x SSC containing 500 Ug/ml salmon sperm DNA (as a viscosity control) and 1 x 10^ cpm/ml of iodinated MCMV DNA. The solutions were sonicated and the annealing reaction carried out and analyzed as described in Materials and Methods. The results are shown in Fig. 2, and summarized in Table I. In a l l cases, the slopes were proportional to the concentration of v i r a l DNA. These data may therefore be used as standard curves to quantitate microgram levels of v i r a l DNA, as well as the number of v i r a l genomes per c e l l , for any annealing reaction performed under the same conditions of temperature, salt concentration, DNA fragment size and total DNA concentration. A time course experiment was performed to determine the onset of v i r a l DNA synthesis. The cells were infected, and at pre-determined times, c e l l DNA was extracted and purified. The kinetics of reassociation of infected c e l l DNA with iodinated MCMV DNA were analyzed under the same conditions described in the reconstruction experiment (Fig. 2). The results, illustrated in Fig. 3, demonstrate that the rate of reassociation did not increase un t i l 12 h. post infection. The concentration of v i r a l DNA almost doubled between 10 and 12 h. post infection. At later times, the concentrations of v i r a l DNA increased exponentially reaching a maximum Fig. 2: Reconstruction Experiment for the Determination of the Concentration of V i r a l DNA CsCl gradient purified, MCMV DNA was added to 2.0 ml 2 x SSC 125 containing 500 yg/ml purified salmon sperm DNA and 1-2 ng I-MCMV DNA. Each reaction mixture contained either: (1) No v i r a l DNA O (2) 0.18 yg/ml v i r a l DNA • (3) 0.36 yg/ml v i r a l DNA • (4) 1.8 yg/ml v i r a l DNA T The mixtures were sonicated, denatured, and incubated at 65°C. Samples were removed at intervals, and single- and double-stranded DNA separated. 62 4 8 1 2 H O U R S 63 125 Table I: Reconstruction Experiment of I-MCMV Reassociation  with Varying Amounts of MCMV DNA Non-radioactive Mathematical description ..Calculated v i r a l MCMV DNA Ug/ml of the best f i t line: genome equivalents (per c e l l ) : 0 Y = 0.0021 X + 1.0 0.18 Y = 0.0106 X + 1.0083 .10.6 0.36 Y = 0.0218 X + 1.0126 21.3 1.80 Y = 0.0897 X + 1.0083 106.5 " The best f i t was calculated by a computer aided linear regression. The equations are given in the form of Y = mx + B, where: Y = dependent variable (_£. ), x = independent variable C (time), m = slope (C Q.K), and b = the Y intercept, which should be unity, according to equation (2). k Calculated according to Gelb et a l . (1971) assuming the ratio 4 of molecular weights of c e l l : v i r a l DNA = 2.96 x 10 . 64 64a Fig. 3: Rate of Reassociation of Iodinated MCMV DNA with Infected Cell DNA Harvested at Various Times After Infection Exponential 3T3 cells were infected at an input multiplicity of 10 pfu/ c e l l by centrifugal infection. Total DNA was purified by method 2 from cells harvested at 6, 8, 10, 12, 14, 16, 18 and 24 h. post infection. In a f i n a l volume of 0.35 ml 2 x SSC the following were mixed: (1) 200 yg/ml infected c e l l DNA; (2) 300 yg/ml salmon sperm DNA, and (3) 10 y l 1 2 5 I DNA (6.3 x 10 4 cpm; 3 ng). The mixtures were sonicated, sealed i n 50 y l pipettes, and heat denatured. The annealing mixture was incubated and analyzed as in Fig. 2. Symbols: ( • ) 6, 8 and 10 h. post infection ( • ) 12 h. post infection ( A ) 14 h. post infection (• ) 16 h. post infection ( • ) 18 h.: post infection ( Y ) 24 h. post infection The slopes, as calculated by computer aided linear regression, were: (1) 6, 8, 10 h., 6.3 x 10~3; (2) 12 h., 1.13 x 10~2; (3) 14 h., 3.3 x 10"2; (4) 16 h., 9.35 x 10 _ 2; (5) 18 h., 0.191; (6) 24 h., 0.934. The s t a t i s t i c a l correlations for the best-fit curves were >0.95 in a l l cases. of 2.13 x 10 v i r a l genomes/cell, by 24 h. post infection. From the data in Figures 2 and 3, the number of genome equivalents were calculated and plotted on a l°g-^ Q scale shown in Fig. 4. Vir a l DNA synthesis began in the period between 10 and 12 h. post infection. However, v i r a l progeny were usually detected by 12 h. post infection. Therefore, i t was s t i l l possible that a slight, but significant, increase in the concentration of v i r a l DNA occurred between 6 and 8 or 8 and 10 h. post infection, which was below the degree of sensitivity of the assay. However, in another experiment, in which the concentration of infected c e l l DNA was increased 10 fold, thereby increasing the sensitivity, the same results were obtained. Control experiments indicated that the iodinated MCMV DNA probe reassociated to a very small extent (ca. 1% in 30 h.) in the presence of mock infected cellular DNA. Based on this observation, two points are significant: (1) iodinated MCMV DNA was not contaminated by detectable amounts of cellular DNA; (2) conditions i n the hybridization reaction 125 were established such that the self-annealing of single stranded I-DNA was minimal. C. DNA and RNA Synthesis in Infected 3T3 Cells Inhibition of host DNA synthesis in cells infected by herpesviruses has been well documented (Roizman and Furlong, 1974). An overall view of DNA synthesis in MCMV infected, exponentially growing, 3T3 cells was 3 obtained by 2 h. pulses with H-TdR at various times throughout the infection cycle. The results, shown in Fig. 5, are expressed as the ratio of infected c e l l to mock infected TCA precipitable cpm. During the f i r s t 10 h. post infection, incorporation decreased precipitously, to a Fig. 4: The Time of Onset of Vir a l DNA Synthesis in Exponential 3T3 Cells. From the reconstruction experiment (Fig. 2 and Table I), the number of MCMV genome equivalents was calculated for each curve i n Fig. 3. A representative v i r a l growth curve (input m.o.i. equal to 10 pfu/ cell) i s illustrated for comparison. Symbols: ( • ) ^-0&IQ g e n o m e equivalents ( • ) log^ pfu/ml supernatant plus c e l l associated virus Fig- 5: Incorporation of H-TdR and H-UR into Infected and Mock Infected 3T3 Cells Exponentially growing 3T3 cells were mock infected or infected at an input m.o.i. of 10 pfu/cell by standard inoculation. The cultures were pulsed with 2.0 uCi/35 mm dish 3H-TdR or 3H-UR for 2.0 h. at the indicated times. At the end of the pulse, the radioactive medium was decanted, the cells scraped off in a known volume of PBS, and 0.5 volume processed for TCA precipitable radioactivity. For deter-mination of total TCA soluble and insoluble radioactivity, the remaining 0.5 volume of cells was filt e r e d onto glass fiber discs, which were washed with PBS, dried, and counted as described. The results are expressed as 'v/c ratios' or the ratio of v i r a l infected to control TCA precipitable CPM. Symbols: ( • ) 3H-UR ( • ) 3H-TdR level of 20% of mock infected cultures. In particular, an increase in incorporation was not observed concurrently with the onset of v i r a l DNA synthesis (10 to 12 h. post infection). Therefore, the possibility must be considered that MCMV does not induce an enzyme capable of phosphory-lating TdR (see Section 2H). Moreover, this experiment (Fig. 5) may simply reflect an overall inhibition of host TK activity and not neces-sarily rates of DNA synthesis. MCMV, like other herpesviruses, probably does inhibit host DNA synthesis (Moon et a l , 1976); however, in the absence of detailed knowledge of v i r a l induced enzymes, studies using labelled deoxynucleosides to assess rates of DNA synthesis should be avoided. 3 Similar arguments may be levied against the use of H-UR to assess overall RNA synthesis in infected c e l l s . However, unlike the previous 3 situation, H-UR incorporation was stimulated in infected cells (Fig. 5). To determine i f this stimulation reflected an actual increase in RNA synthesis or a v i r a l induced change in the UTP pool, the infected c e l l data from the experiment shown in Fig. 5 were expressed as the percentage of total intracellular cpm (acid soluble plus insoluble) which were acid insoluble. This manipulation should compensate for changes in the pool size and more accurately reflect rates of RNA synthesis after infection. As illustrated in Fig. 6, a burst of RNA synthesis was observed at two time intervals in infected c e l l s , f i r s t at 3 h. post infection ('early') and another beginning at 12 h. and reaching a maximum at 24 h. post infection. 68a Fig. 6: Rates of RNA Synthesis in MCMV Infected 3T3 Cells The H-UR incorporation data from Fig. 5 (infected cells) were expressed as the percentage of total intracellular counts (TCA soluble and insoluble) which were TCA precipitable at various times after infection. SECTION II: SYNCHRONIZED CELL AND VIRUS STUDIES A. Synchronization of 3T3 Cells Three methods of synchronization were investigated: (1) the double TdR block (Bootsma et a l , 1964); (2) mitotic selection (Terasima and Tolmach, 1963), and (3) serum activation. The advantages and corresponding limitations of each one with respect to synchronous c e l l and virus studies, were evaluated before deciding which method was best.. The double TdR treatment traps cells at the Gl-S border, and upon release of the block, results i n an immediate synchronous wave of DNA synthesis. The method is technically easy to use and the yields of syn-chronized cells are high. Exponential 3T3 cells were synchronized by excess TdR as described and infected or mock infected. The resulting synchrony and v i r a l growth patterns are shown in Fig. 7. In mock infected c e l l s , two synchronous S phases occurred, 12 hours apart. An approximate doubling in c e l l numbers was observed in mock infected controls between 7 and 12 hours post-reversal (data not shown). The degree of synchrony was therefore acceptable; however, this method is vitiated by the failure . of MCMV to replicate in the doubly-TdR treated c e l l s . Although the extra-cellular nucleoside i s washed out at reversal, the endogenous dTTP pools may remain sufficiently expanded to preclude v i r a l replication. The replication of HSV-2 is inhibited by 2.0 mM TdR treatment in KB cells (Cohen et a l , 1974). 3 The H-TdR incorporation in infected synchronized 3T3 cells (Fig. 7) is significant for two reasons. F i r s t , these data indicate that the v i r a l infected cells are synchronized, and second, i t i s clear that the virus, although non-replicating, i s affecting the host S phase (altered 70a Fig. 7: Synchronization of 3T3 Cells by Double TdR Treatment Exponential 3T3 cells were synchronized by two TdR blocks (2 mM TdR), infected by standard inoculation at an input multiplicity of 5 pfu/cell in TdR free medium, and given fresh MEM and 10% serum without TdR. Control and infected cultures were pulsed 3 for 1.0 h. with 2 uCi/plate H-TdR and TCA insoluble material processed for radioactivity measurement. 3 Symbols: ( • ) H-TdR incorporation in MCMV infected cells ( • ) H-TdR incorporation in control cells ( A ) v i r a l titers per culture (supernatant virus) 71 dTTP pools and/or decreased rates of DNA synthesis). These aspects have not been further investigated, since the sensitivity of MCMV replication to 2.0 mM TdR suggests that synchronization by a nucleoside blockade is untenable for an analysis of synchronous c e l l and virus interactions. Therefore, other methods of synchronization were investigated. Selection synchrony was also attempted using the mitotic shake-off method described by Tferasima and Tolmach (1963). This method is based on the observation that mitotic cells adhere less firmly than interphase cells to glass or plastic surfaces. Numerous modifications of the original technique were tried, but with l i t t l e success. The principal problem is the fact that the attachment properties of 3T3 cells make them unsuitable for synchronization by this method. Mitotically harvested 3T3 cells were, in a l l cases, contaminated with a high percentage of interphase cel l s , resulting in a very low degree of synchrony. This problem has also been reported with other c e l l lines (Whitmore, 1971). The serum-split method of synchronization was developed in this laboratory as the ideal technique for several reasons. In addition to yielding large populations of synchronized cells required for detailed biochemical analyses, the serum-split method eschews the use of chemical inhibitors (i.e. TdR or hydroxyurea), which may conceivably destroy the very process being investigated (in this case, v i r a l replication). The procedure is based on the observation of Holley and Kiernan (1968) that 3T3 c e l l s , l e f t without medium change, eventually become density inhibited, forming a confluent monolayer of predominantly G—1 arrested c e l l s . Reversal of arrest is then accomplished by trypsinization, replacing and .replenish-ment with fresh MEM plus 10% serum. 3 The kinetics of growth arrest were followed by H-TdR incorporation (Fig. 8). At any time after day 4, the cells could be stimulated to enter Fig. 8: Kinetics of G-l Arrest in 3T3 Cells Exponential„3T3 cells were plated on day 0 at a density of 5 1 x 10 cells per 65 mm dish. Each succeeding day, several 3 dishes were incubated for 30 minutes with 1.0 yCi H-TdR and analyzed for TCA-precipitable radioactivity. 300 n Days After Plating 74 S phase in a synchronous fashion by serum-split. From Fig. 9, i t i s clear that the degree of synchrony i s acceptable. The main features of this synchrony are: (1) one synchronous S-phase, i n i t i a t i n g at 12 to 14 h. post-serum-split, with a 'peak' at 20 h.; (2) a second S phase 'peak' at 36 to 38 h. (followed by rapid desynchronization); (3) an approximate doubling of c e l l numbers between 22 and 28 h; (4) an increase in mitotic fraction at 26 h.; (5) greater than 85% labelled nuclei at 20 h. as deter-mined by autoradiography. The durations of S and M phases are 7 to 8 h. and 1 h. respectively, and the generation time of these cells is 17 to 18 h. B. MCMV Replication in 3T3 Cells Synchronized by the Serum-Split Method Figure 10 shows a comparison of v i r a l growth curves inasynchronous exponential 3T3 cells and synchronized 3T3 cells infected in the early G—1 period (1 to 2 h post serum-split). MCMV in randomly growing cells replicated with a latent period of 10 to 12 h. However, the latent period in the synchronized cells was protracted to 24 h. The f i n a l yield of virus per c e l l was approximately equal for synchronized and randomly growing c e l l s . The time course of v i r a l growth in G-l infected 3T3 cells was similar with both the standard and centrifugal modes of infection, and the protracted latent period could not be altered by increasing the input multiplicity. The trypsinization procedure used to induce synchrony was not responsible for the protracted latent period, since the same results were observed with cells synchronized without trypsinization, and trypsin-ization (2 h. prior to infection) did not protract the v i r a l growth curve in asynchronous c e l l s . Furthermore, i n both an early G-l infection (at 2h. post serum-split) and a late G-l infection (at 11 h. post serum-split) 74a Fig. 9: 3T3 Cells Synchronized by the Serum-Split Method Two 90 mm plates were l e f t without medium change for 9 days, and then serum s p l i t to a density of 3.5 x 10^ cells/plate. DNA synthesis, c e l l numbers/plate and mitotic fractions were determined as described in Section i of Materials and Methods Symbols: ( • ) DNA synthesis ( A ) c e l l numbers/plate ( O ) mitotic fractions 3 H CPM/ml x 10 - 3 Cn O Oi O O O _l I I 75a Fig. 10: MCMV Growth Kinetics in Synchronous and Asynchronous 3T3 Cells Top panel: Synchronous 3T3 cells ( • ) were 'serum-split' at a density of 1 x 10^ cells/plate and infected between 1 and 2 h. later at an m.o.i. of 20 pfu/ c e l l . Asynchronous cells ( O ) were present at a density of 8.2 x 10~* cells/plate and infected at the same m.o.i. (20 pfu/cell). Virus assays represent total intracellular plus extracellular virus. Bottom panel: Rate of DNA synthesis in mock infected synchronous 3T3 cells ( • ). 76 progeny virus appeared at the same time in the S-phase (Fig. 11). Thus a delay in the G-l infection by 9 hours resulted in a corresponding decrease in the latent period of v i r a l growth. Although the protracted latent period after infection in G-l was independent of the m.o.i., i t was necessary to ascertain i f there were quantitative differences in attachment or penetration of virus between G-l, S and asynchronous c e l l s . Since trypan blue exclusion assays on G-l arrested 3T3 cells (prior to serum-split) demonstrated that greater than 90% of the cells were viable, altered v i r a l growth could not be ascribed to a preponderance of moribund cells in the synchronous population. In addition the data'' in Table II argue against selective uptake of MCMV by G-l, S or exponential asynchronous 3T3 c e l l s . A similar proportion of radioactive virus was taken up by the three types of culture. Furthermore, infectious center assays from G-l phase cells and asynchronous cells (infected at the same m.o.i.) indicated >90% of the cells were susceptible to MCMV in both cases. These data are con-sistent with the notion that the protracted latent period i s a post-penetrational event. 77a Fig. 11; V i r a l Growth Curves in Synchronized 3T3 Cells Infected in Early G-l (2 h. Post Serum-Split) and Late G-l (11 h. Post Serum-Split) 3T3 cells were synchronized by the serum-split method and infected at an input m.o.i. of 20 pfu/cell at 2 h. post serum-split (early G-l) and 11 h. post serum-split (late G-l), indicated on the graph by arrows. Virus assays represent total supernatant and c e l l associated virus. DNA synthesis in uninfected cultures was 3 determined by 1 h. pulses with 1 uCi/plate H-TdR. Symbols: ( • ) early G-l infection growth curve ( • ) late G-l infection growth curve 3 ( • ) H-TdR incorporation 78 10 20 30 H P O S T S.S. 79 3 Table I I : Uptake of H-TdR Labelled MCMV into G-l, S and Asynchronous 3T3 C e l l s 3 Input cpm cpm adsorbed % of input adsorbed 2 x 10 5 c e l l s 2 x 10 5 c e l l s 2 x 10 5 c e l l s G-l phase c e l l s 2.5 x 10 3 1.85 x 10 3 74 S-phase c e l l s 1.76 x 10 3 .1.20 x 10 3 68 3 3 Asynchronous c e l l s 1.89 x 10 1.36 x 10 72 ~"H-TdR l a b e l l e d MCMV was prepared free of detectable soluble radio-a c t i v i t y 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. Radio-a c t i v e v i r u s was added to synchronous or asynchronous cultures and cpm (as TCA p r e c i p i t a b l e material within c e l l s ) determined. The m.o.i. was 20 p f u / c e l l f o r a l l three types of cult u r e . 80 G. Inhibition of V i r a l Replication in Cell Cycle Blocked 3T3 Cells Cell cycle traverse was stopped by two independent methods, with similar results. Table III shows the dose response relationship between MCMV replication and the concentration of TdR in the medium. V i r a l r e p l i -cation was inhibited under conditions which also inhibited cellular DNA synthesis and division, and this inhibition was dose dependent. Consistent with this observation, 3T3 cells maintained in G-l phase after infection did not support v i r a l replication (Fig. 12). These data strongly imply an S-phase dependency for MCMV. D. Correlation Between Cell Cycle Traverse and V i r a l Replication If MCMV requires host S-phase events for replication, there should be an exact and predictable correlation between c e l l cycle traverse and v i r a l growth. This is illustrated by the experiment shown in Figure 13. Confluent G-l arrested 3T3 cells were infected with MCMV and immediately after absorption, divided into 4 Aliquots (trypsinization and replating were not done in this experiment): (1) control, where the same depleted medium was added; (2) fresh serum only was added; (3) fresh MEM only was added; (4) fresh MEM and serum were added. In Aliquots 1, 2 and 3, c e l l cycle traverse was not observed, as indicated by residual H-TdR incorporation, and mitotic fractions (data for mitotic fractions not shown). In Aliquot 4, c e l l cycle traverse occurred concurrently with the production of infectious virus, after the usual protracted latent period. Thus, the failure of the virus to replicate in confluent G-l arrested c e l l s , was not due to a deficiency in the depleted medium (e.g. basic amino acids). Table I I I : I n h i b i t i o n of MCMV Replic a t i o n by Excess Thymidine Concentration of F i n a l y i e l d Percent of TdR (mM)a p f u / c e l l b maximum y i e l d 0 335 100 .002 59 18 .02 28 8 2.0 12 3 " 3T3 c e l l s were given a double TdR block as described i n Methods, infec t e d with MCMV c e n t r i f u g a l l y at an input m.o.i. of 10 p f u / c e l l i n TdR free medium and various amounts of TdR added back immediately a f t e r the adsorption period. k Expressed as c e l l associated plus supernatant v i r u s per c e l l . 81a Fig. 12: Infection of G-l Arrested 3T3 Cells 3T3 cells were l e f t without medium change for 1 week and infected centrifugally at an input m.o.i. of 5 pfu/cell in Hank's BSS plus 0.5% serum. Immediately after the adsorption period the G-l depleted medium was re-added. Supernatant virus was assayed at the indicated times. 82 Fig. 13; Correlation Between Cell Cycle Traverse and V i r a l Replication 3T3 c e l l s , l e f t without medium change for 9 days (3 x 10° cells/ 35 mm plate), were infected centrifugally at an input m.o.i. of 20 pfu/cell in Hank's BSS plus 0.5% serum. After the adsorption period the plates were divided into 4 different Aliquots: (a) control, G-l depleted medium re-added; (b) 10% fresh serum added in depleted medium; (c) fresh MEM only added (double strength diluted 1:1 into depleted MEM); (d) fresh serum (10%) plus fresh MEM. Assays represent total virus i n the c e l l s and supernatant. Mitotic cells were observed only in treatment (d). Symbols: (• •) log 1 n pfu/ml (culture) (• •) number of labelled nuclei/unit f i e l d Log PFU/ml o (ft I CO <t> c 3 ro o o o o J 1 1 1 I I 1.1 I I T 1—1 1—1 1 1 o o o o o o o o J i CO Cn J I I V KJ o 1—I—I u o o o I CO J L Cn o J I -I / C O "I—I—I KJ ^ O o o o •U Cn o -I 1 I / i—i—i IO ^ o o o o •• Number of Labelled Nuclei/Unit Field 00 LO 84 E. Initiation of Vir a l DNA Synthesis in G-l Infected Synchronized Cells. One logical explanation for delayed v i r a l growth in synchronized 3T3 cells i s that the replication of MCMV DNA requires S-phase events. Synchronized cells were infected in early G-l phase (0.5 to 1 h. post serum-split) and the appearance of v i r a l DNA assessed by several methods. To gain an overall view of DNA synthesis in infected, synchronized 3 ce l l s , incorporation of H-TdR was assayed (Fig. 14). In v i r a l infected ce l l s , DNA synthesis increased in concert with the mock infected 3 c e l l s , up to mid-S phase. The H-TdR incorporation was suppressed in the infected c e l l s , in agreement with previous results. After 18 h. however, an unscheduled increase occurred in infected c e l l s , which presumably re-presented v i r a l DNA synthesis. To verify this, DNA was extracted from 32 synchronized cel l s , continuously labelled with P, and examined for v i r a l DNA by f i l t e r hybridization (Table 4). Viral DNA was not detected by 16 h. either by annealing or CsCl gradient centrifugation (not shown). By 20 h. v i r a l DNA was detected, which suggests that v i r a l DNA synthesis began in the period between 16 and 20 h. post infection, in contrast to exponential c e l l s , which synthesized v i r a l DNA between 10 and 12 h. The onset of v i r a l DNA synthesis was more clearly defined using DNA-125 DNA annealing in solution with I MCMV probe (Table V). This method circumvents many of the problems associated with radioisotope incorporation, such as enzyme fluctuations or nucleotide pool changes after infection. The method is also more sensitive and more quantitative than f i l t e r hy-bridization. V i r a l DNA was found in association with G-l phase cells at 8 h. post infection. This DNA probably represents input, parental DNA which, at 20 genomes/cell (Table V), agrees reasonably well with an input multiplicity of 10 pfu/cell. Parenthetically, exponential 3T3 cells 84a F i g . 14: DNA Synthesis i n Infected and Mock Infected Synchronized 3T3 C e l l s 3T3 c e l l s were synchronized by the serum-split method and infec t e d c e n t r i f u g a l l y at an input m.o.i. of 10 p f u / c e l l or mock infected 3 within 1 h. a f t e r p l a t i n g . H-TdR incorporation (1 yCi/35 mm plate) was measured by 1 h. pulses, TCA p r e c i p i t a t i o n , and l i q u i d s c i n t i l l a t i o n counting. Symbols: ( a ) infec t e d 3T3 c e l l s ( • ) mock infec t e d 3T3 c e l l s Table IV: DNA-DNA Annealing to Determine the Onset of Vir a l DNA Synthesis in Synchronized 3T3 Cells Source of P labelled Input yg added CPM hybridized to 1.0 yg DNA in solution (CPM) DNA on f i l t e r s 3T3 calf MCV cells thymus Synchronized infected 65.7 „ 82 8 3 3T3 c e l l DNA harvested (3.55 x 10 ) at 16h post infection Synchronized infected 74.7 , 407 30 435 3T3 c e l l DNA harvested (2.85 x 10 ) at 20h post infection Synchronized infected 92.7 , 370 100 3333 3T3 c e l l DNA harvested (7.51 x 10 ) at 24h post infection Synchronized mock 67.0 . 483 51 5 infected 3T3 c e l l DNA (4.82 x 10 ) harvested at 16 h.p.i. Carrier free P was added to mock- or virus infected cells (250 yCi/culture) immediately after serum activation. DNA was extracted and purified at the indicated times and annealed to non-radioactive DNA on f i l t e r s . The input m.o.i. was adjusted to 10 pfu/cell. k Corrected for machine background of 20-30 cpm. 87 Table V: Initiation of Vi r a l DNA Replication in Synchronized 3T3 Cells Infected in Early G-l, as Determined by Reassociation Kinetics Hours post Mathematical description Genome Log^g genome infection: 3- of best f i t l i n e : b equivalents: 0 equivalents: 8 y = 0.00412 X + 1.005 20 1.3012 10 y = 0.00417 X + 1.005 20 1.3012 12 y = 0.0036 X .1.002 18 1.255 14 y = 0.00409 X + 1.002 20 1.3012 16 y = 0.00577 X + 1.012 28 1.451 18 y = 0.01607 X + 1.002 79 1.896 22.5 y = 0.03858 X + 1.009 188 2.275 32 y = 0.1197 X + 1.02 1363 3.134 " 3T3 cells were synchronized by serum-split and centrifugally infected in early G-l (0.5 to 1.0 h. post-serum-split) at an input m.o.i. of 10 pfu/cell. DNA was extracted from infected cells and annealed in 2 x SSC 125 Each annealing mixture contained 5 ng/ml I-MCMV DNA probe and 100 yg/ ml infected c e l l DNA (except the 32 h. sample which contained 50 yg/ml). Total unlabelled DNA in each mixture was adjusted to 500 yg/ml with salmon sperm DNA. Sampling times for each annealing reaction were 1, 2, 3, 6, 8, 10, 12.5, 24 and 27 hours. k See Table I for explanation. The s t a t i s t i c a l correlation for the best f i t curves varied from 0.80 to 0.98. Calculated according to Gelb et a l (1971) assuming the molecular weight of MCMV DNA = 1.32 x 10 (Mosmann and Hudson, 1973). 88 infected at the same moi, also contained 20 genomes/cell at 8 h. post infection. Assuming that a similar amount of virus enters exponential cells as in G-l phase cells (Table II), these data imply that the degree of intracellular degradation of the v i r a l genome is similar for the two. The v i r a l DNA content stayed relatively constant at 18 to 20 genomes/cell u n t i l 16 h., when this value increased to 28. Between 16 and 18 h. the number of genomes/cell increased 2.8 fold, and at later times increased to a maximum of 1363 genomes/cell. It i s not clear i f the 1.4 fold increase between 14 and 16 h. is substantial enough to localize the actual i n i t i a t i o n event in this period. However, the 2.8 fold increase between 16 and 18 h. strongly indicates the onset of MCMV DNA replication in this period. It is therefore concluded that v i r a l DNA synthesis depends upon a host event(s) which is manifest at 16 h. or approximately early S phase. F. Replication of HSV-1 in Serum-Split Synchronous 3T3 Cells In order to be certain that the protracted G-l period from 0 to 12 h post serum s p l i t was not artifactual, the a b i l i t y of a known c e l l cycle independent herpes virus, HSV-1, to replicate outside of S-phase was deter-mined in the serum-split synchronous system. Using doubly-TdR synchronized human KB cel l s , Cohen et a l (1971) demonstrated that HSV-1 could replicate without regard for host c e l l cycle events. Figure 15 shows the results with HSV-1 i n serum-split synchronized 3T3 cells infected at two points (early and late G-l) and for comparison, v i r a l growth in synchronous (exponential) 3T3 c e l l s . The kinetics of v i r a l growth did not vary significantly in any of the three situations suggesting that, in accord-ance with Cohen et a l (1971), HSV-1 does not have a proclivity for cells in any one particular phase of the c e l l cycle. Therefore, the results 88a Fig. 15: Replication of HSV-1 in Synchronized and Non-Synchronized  Exponential 3T3 Cells 3T3 cells were synchronized by the serum-split method and infected in early G-l (2 h. post serum split) or late G-l (8 h. post serum s p l i t ) . Non-synchronized exponential 3T3 cells were infected at the same time as the early G—1 synchronized cells (2 h.) for comparison. 3 The input m.o.i. was adjusted to 8 pfu/cell. The host S-phase ( H-TdR incorporation in uninfected synchronized cells) i s also shown. Total virus was assayed on 3T3 c e l l s . Symbols: (•- •) early G-l infection (0---0) late G-l infection •) rion-synchronized exponential infection 3 *) H-TdR incorporation 89 90 with MCMV are not vitiated by an aberrant synchrony induction procedure, and the serum-split method of synchronization is probably valid for the study of c e l l cycle and herpes virus interactions. G. Replication of MCMV and HSV-B2Q06 in Synchronized ME Cells The replication of MCMV in tertiary, serum-split, synchronous ME cell s was identical to the 3T3 c e l l system, viz : after an early G—1 infection progeny virus did not appear unt i l S phase (Fig. 16). Thus the long latent period observed in synchronous 3T3 cells after an early G-l infection is not unique to that c e l l line. The replication of a mutant of HSV-1 (HSV B2006; Kit and Dubbs, 1963), deficient i n i t s a b i l i t y to induce TK, was also investigated in synchronized ME c e l l s . This experiment was designed to test the relationship between TK induction and the a b i l i t y of a herpesvirus to replicate outside of S-phase; ME cells were synchronized by the serum-split method and infected with HSV-B2006 within 1 h after synchrony induction, and the kinetics of v i r a l growth determined throughout the c e l l cycle (Fig. 17). Growth of HSV-B2006 was protracted u n t i l early S-phase, compared to the exponential, asynchronous cells infected at the same time. Therefore, the mutant was replicating in a c e l l cycle (i.e. S-phase) dependent manner. These results are significant, since they indicate that the v i r a l TK may play a key role in the a b i l i t y of HSV-1 to replicate without c e l l cycle constraint. More-over, c e l l cycle dependency of HSV-B2006 is consistent with the observations of Jamieson et a l (1974) that the HSV-1 induced TK is essential for r e p l i -cation in G-l arrested cel l s , but not necessary in exponentially growing ce l l s . 90a Fig. 16: Replication of MCMV In Synchronized ME Cells ME cells were l e f t without medium change for 7 days and serum-s p l i t to induce synchrony as described for 3T3 cel l s . The synchronized cells were infected 1 h. later (arrow) at an input multiplicity of 20 pfu/cell by centrifugal inoculation. Virus in the supernatant and cells was assayed on ME ce l l s . 3 Incorporation of H-TdR was determined by 10 minute pulses (1 yCi/plate) i n mock infected cells, followed by TCA precipitation and counting. Symbols: ( O ) MCMV growth curve 3 ( • ) H-TdR incorporation. 91 91a Fig- 17: Replication of HSV-B2006 in Synchronous and Asynchronous ME Cells Serum-split synchronous and asynchronous, exponential ME cells were infected at an input m.o.i. of 10 pfu/cell with HSV-B2006 (TK~) at the time indicated by the arrow. Total virus was assayed at various times after infection. The host S-phase i s indicated (separate experiment). Symbols: ( • ) HSV-B2006 growth curve in synchronous ME cells infected in early G-l ( • ) HSV-B2006 growth in asynchronous ME cells H. Thymidine Kinase Activity in 3T3 Cells Infected with MCMV 93 The results from the preceding section demonstrate the importance of TK activity in the a b i l i t y of HSV to replicate in a c e l l cycle independent manner. As pointed out in the Introduction (Section E-2 and Table 4), another c e l l cycle dependent herpes virus, EAV, does not induce TK in infected cells (Jamieson et a l , 1974). Since a host S phase specific enzyme, such as TK, may be required for v i r a l DNA synthesis, i t was of interest to determine the abi l i t y of MCMV to induce a v i r a l TK activity. TK activity was assayed as described in the methods section. Although the DEAE disc method separates the labelled nucleoside substrate from the phosphorylated product, i t was demonstrated that subsequent phosphory-lation of the product (dTMP) was not occurring (Fig. 18). After a 20 minute incubation period, 95% of the labelled product was in the form of dTMP. Residual levels of labelled dTTP and dTDP represented radiochemical impurities which were present at 1.0% and 0.7% respectively of the total 3 H-TdR radioactivity obtained from the manufacturer (data not shown). 3 Therefore, significant conversion of H-dTMP to the d i - and tri-phosphate derivatives does not occur under these assay conditions. TK activity was. proportional to protein concentration in a l l assays. The assay was linear with time (up to 45 minutes), and the pH and temper-ature optima were identical in infected and mock infected c e l l extracts (viz: pH 7.5 and 35°C). The a b i l i t y of MCMV to stimulate TK was f i r s t investigated. The analysis simply compared changes in specific activity in uninfected and infected 3T3 cells at various times post infection. The data in Fig. 19 indicate that MCMV does not induce TK activity. The proliferating cultures of uninfected 3T3 cells showed a rapid increase 93a Fig. 18: Analysis of TK reaction products A c e l l free extract from MCMV infected 3T3 cells was prepared and assayed for TK activity. Incubation times varied from 0 to 20 minutes and 100 y l from each assay were mixed with 25 y l of marker nucleotides (approximately 0.5 mM each of dTTP, dTMP, dTDP) and spotted in one application on the origin. The chromato-grams were given two ascending rinses with water to mobilize the labelled TdR far above the nucleotides. Chromatography was then carried out in 0.5 M Li C l plus 5% sodium borate. The standards were localized under short wavelength UV light and the spots cut out for counting in the toluene based liquid s c i n t i l l a t i o n f l u i d . Symbols: ( • ) dTTP ( • ) dTDP ( • ) dTMP 94 MINUTET5 OF lNCUBBTlDN-> 94a Fig. 19: Changes in TK Activity After Infection Freshly confluent 3T3 cells were s p l i t into 50 mm dishes at a density of 5 x 10~* cells/plate and infected by centrifugal inoculation at an input multiplicity of 20 pfu/cell. Harvests for c e l l free extracts were made at various times, and these were assayed for TK activity (expressed as pMoles dTMP formed 2 x 10 /30'/mg protein). Symbols: ( • ) mock infected ( O ) MCMV infected TK Specific Activity VO i n TK by 12 h. post i n f e c t i o n . TK was considerably suppressed i n MCV inf e c t e d cultures to 2% of cont r o l (uninfected) cultures by 27h. A TK~ l i n e of 3T3 c e l l s (3T3-4E courtesy of Dr. H. Green (Matsuya and Green, 1969)) was used to determine the a b i l i t y of MCMV to u t i l i z e 3 exogenously added H-TdR. Uninfected wild-type 3T3 and 3T3-4E cultures were synchronized by the 'serum-split' method and analyzed f o r t h e i r a b i l i t y to incorporate H-TdR. Synchronized c e l l s were used i n t h i s case since a dis c r e t e S-phase q u a n t i t a t i v e l y l o c a l i z e s thymidine incorporation to a period between 12 and 24 h. post serum a c t i v a t i o n and may therefore amplify r e s i d u a l thymidine incorporation. As seen i n F i g . 20, the TK 3T3-4E incorporated only low l e v e l s of thymidine, compared to wild-type 3T3. The MCMV inf e c t e d 3T3-4E incorporated even l e s s thymidine than the uninfected 3T3-4E. The v i r a l growth curve i n synchronized 3T3-4E cultures, however, showed the c h a r a c t e r i s t i c protracted l a t e n t period. Uninfected wild-type 3T3 cultures were included as controls to demonstrate the normal 3 l e v e l of H-TdR incorporation. The data presented i n F i g . 21 show that the r e p l i c a t i o n of MCMV, i n 3T3-4E (TK~) c e l l s was r e s i s t a n t to l e v e l s of BUdR as high as 200 yg/ml. Wild-type HSV-1 y i e l d s were severely depressed at 5 yg/ml, while HSV-B2006 was not aff e c t e d . The i n h i b i t o r y e f f e c t s of BUdR. are contingent upon the conversion of BUdR to the nucleotide l e v e l by TK (Henderson and Paterson, 1973). Therefore, v i r a l y i e l d s should be unaffected by BUdR i n the absence of TK. This experiment demonstrates that neither MCMV nor HSV-B2006 induce s i g n i f i c a n t l e v e l s of TK a c t i v i t y , i n the 3T3-4E c e l l l i n e . To sum up, these r e s u l t s support the concept that MCMV induces neither a c e l l u l a r TK nor a v i r a l TK based on the following evidence. MCMV does not induce a detectable increase i n TK by 27 h. post i n f e c t i o n , and the 96a Fig. 20: Incorporation of H-TdR into SynchronizedUriinfected 3T3  and 3T3-4E Cells and Infected 3T3-4E Cells 3T3 and 3T3-4E cultures were synchronized by the serum-split method and the 3T3-4E cells mock infected or MCMV infected at an input m.o.i. 3 of 20 pfu/cell within 1 h. after re-plating. H-TdR incorporation was measured by means of 30' pulses with 1 uCi/culture. Acid insoluble radioactivity was determined as described in Materials and Methods. Virus assays represent total (supernatant plus c e l l associated) virus. 3 Symbols: ( • ) H cpm in uninfected wild-type 3T3 cultures 3 ( O ) H cpm in mock infected 3T3-4E cultures 3 ( • ) H cpm in MCMV infected 3T3-4E cultures ( A ) v i r a l growth curve in 3T3-4E cultures 97a Fig. 21; Dose Response of MCMV, HSV-1 and HSV-B2006 to BUdR Exponentially growing 3T3-4E cells (1.7 x 10J cells/35 mm plate) were infected at an input m.o.i. of 20 pfu/cell with MCMV, HSV-B2006, or wild-type HSV-1 in the presence of various levels of BUdR. After the adsorption period, the inocula were decanted and fresh medium plus 10% serum and BUdR added. The cultures were harvested when cpe was maximal, for assay of supernatant plus c e l l associated virus. Symbols: ( A ) ( • ) ( O ) MCMV infected 3T3 cells HSV-B2006 infected 3T3 cells HSV-1 infected 3T3 cells 98 jjg/ml BUdR virus does not stimulate a 3T3-4E TK host to incorporate exogenously 3 supplied H-TdR. Furthermore the virus i s resistant to the inhibitory effects of BUdR while replicating i n a TK host. However, the failure of MCMV to induce TK cannot solely account for the dependency on S-phase, since the virus replicates in a c e l l cycle dependent manner in syn-chronized 3T3-4E TK~ ce l l s . I. Nucleoside Triphosphate Pools in Synchronized 3T3 Cells Infected  with MCMV. The synthesis of v i r a l DNA at 16 to 18 h. post infection in syn-chronized cells is the event which is dependent upon the host S-phase. Thus, the failure of MCMV to replicate in G—l arrested 3T3 cells may be linked to the inabi l i t y of the virus to provide i t s own functional nucleo-tide pools. Consistent with this idea was the foregoing observation that MCMV did not stimulate TK after infection. Therefore, an analysis of nucleoside triphosphate pool levels in infected and uninfected 3T3 cells was carried out. In this experiment, G-l arrested 3T3 cells were pre-equilibrated 32 with P for 24 h., and then serum activated to induce synchrony at the time of infection. The acid soluble extracts, taken at various times, were analyzed on one dimensional chromatograms, as described by the Cashel method (Cashel et a l , 1969). Although the nucleoside triphosphates are separated from mono- and diphosphates, the procedure does not resolve dXTP's from the rXTP's. However the total dXTP pool amounts to about 1% of the rXTP pool (Hauschka, 1973). Therefore the one dimensional chromatograms resolve predominantly the rXTP pools. Due to the technical ease, sensitivity, and reproducibility, one dimensional development i s 100 ideal for survey purposes. Fig. 22 shows the general trend in the rXTP pool changes in infected and uninfected synchronized 3T3 ce l l s . 1. Uninfected synchronized cells During the f i r s t c e l l cycle after serum activation (up to 26 h.), the ATP, GTP and CTP pools did not fluctuate significantly, in agreement with other reports (Weber and Edlin, 1971). The one exception, the UTP pool, increased 3 to 4 fold as the cells approached mid S-phase. Mauck and Green (1973) reported a 3-4 fold increase in UTP incorporation in 3T3 cells after serum activation, due predominantly to accelerated ribosome synthesis during transition from G-l to S phase. 2. MCMV infected synchronized c e l l s . The virus had a profound effect on GTP and ATP pools. The infected c e l l purine pools were on the average 20 to 30% of the controls. Similar results were obtained from exponential infected and uninfected 3T3 ce l l s . A slight increase in the purine pools occurred during mid S phase ( ~20 h.). The infected, cellular pyrimidine ribonucleotide pools were, on the average, 70% of control (uninfected) c e l l s . The UTP pool increased more or less in concert with the control c e l l pool, and the infected c e l l CTP pool did not fluctuate significantly during the entire 28 h. Attempts to analyze the dXTP pools met with only limited success. Among the many d i f f i c u l t i e s encountered were: (1) extensive 'tailing' of the rXTP spots (particularly ATP and UTP) which often precluded localization of the much smaller deoxy-pools; (2) poor resolution of the dTTP spot 32 which migrated very near the inorganic P; and (3) poor reproducibility 100a Fig. 22: Ribonucleoside Triphosphate Pools in Synchronized Uninfected and Infected 3T3 Cells G-l arrested 3T3 cells were prelabelled for 24 h. with 20 uCi/35 mm 32 plate carrier free P, and infected at an input m.o.i. of 20 pfu/ c e l l by centrifugal inoculation. Immediately after the adsorption 32 period the inocula were removed and fresh P (20 uCi/plate) MEM, plus 10% serum added. The nucleotides were extracted at various times after serum activation, with 0.3 N formic acid. Cell numbers per plate were determined with a hemacytometer, enumerating between 500 and 1000 cells/time point for infected and uninfected parallel cultures. The c e l l density did not change in the infected cultures throughout the entire experiment. Cell density doubled in uninfected cells at 26 h. (see Fig. 9). The rXTP pool sizes were quantitated 32 by the one dimensional method. Counting efficiencies for P were approximately 100% and the concentration of total phosphate in the MEM was approximately 0.90 umoles/ml. Symbols: ( • ) uninfected serum activated cells ( • ) infected serum activated cells PICDMDLE5 UTP/10 G CELLS PICDMDLE5 ^^j-^j^ Tl 0 n c I 01 Tl r • in ^ in n c 3 I m ii r s P1C0M0LE5 CTP/10 G CELLS PICDMDLE5 HTP/I0 CELLS S TOT "of measurable dXTP pools. Two other procedures were tried, the two dimensional method described by Neuhard et a l (1965) and the one dimensional 'wick' method described by Yegian (1974). The data shown in Fig. 22 demonstrate that MCMV profoundly affects pools and probably host nucleotide metabolism in general. However, a conservative evaluation of the available data is that the rXTP pools, although suppressed in infected c e l l s , may s t i l l provide a source for the required dXTP's, for example by ribonucleoside diphosphate reductase (O'Donovan and Neuhard, 1970). Obviously, one cannot unequivocably eliminate the possibility that S phase dependency is a manifestation of dXTP pool requirements. 103 SECTION III: REPLICATION OF MCMV DNA IN VITRO The i n i t i a t i o n of v i r a l DNA synthesis appears to require information specified by the host genome. Therefore, an analysis of v i r a l DNA synthesis in vitro, under permissive and non-permissive conditions, was performed to help resolve the various components involved i n v i r a l DNA replication. A. Optimal Conditions for DNA Synthesis in Nuclear Monolayers When 3T3 cells are treated in situ with the non-ionic detergent, Nonidet-P 40 (NP-40), the c e l l membrane i s lysed which results in removal of the cytoplasm while the nuclei remain- attached to the substratum (Mauck and Green, 1973; Bell, 1974). A culture treated in this way i s referred to as a 'nuclear monolayer'. The nuclei, particularly infected nuclei, were rather tenuously attached, hence, subsequent washing procedures were avoided. Nuclear monolayers were prepared from exponential 3T3 cells at 14 to 16 h. post infection (when rates of v i r a l DNA synthesis were maximal in 3 vivo, see f i g . 4) and the conditions for optimal H-dTMP incorporation were determined (Table VI). From experiment 1, i t is clear that DNA synthesis i s dependent upon the addition of dXTP's, MgCl 2 and ATP. A 10 fold lower concentration of Mg resulted in a slight but reproducible increase in DNA synthesis. Experiment 1 and experiment 2 contained approximately equal numbers of nuclei and may be compared directly. The addition of 0.08 mM each of GTP, UTP and CTP increased DNA synthetic activity to 120% (Biswal and Murray, 1974) and were included in a l l subr: sequent reactions. Omission of unlabelled dTTP resulted in an increase in CPM and a higher specific activity (cpm/yg) DNA product. This 104 Table VI: Reaction Requirements for DNA Synthesis in MCMV  Infected Nuclear Monolayers3 Reaction Mixture Relative % H-TMP Incorporation 100 19 51 121 21 105 100 206 " Exponentially growing 3T3 cells were infected at an input m.o.i. of 20 pfu/cell by centrifugal inoculation. Nuclear monolayers were prepared at 16 h. post infection, and TCA precipitable cpm determined after a 60' incubation period at 37°C. b 'Complete' reaction: 50 mM HEPES (pH 7.6); 0.5 mM CaCl 2; 10 mM MgCl 2; 1 mM dithiothreitol; 0.04 mM each of dCTP, dATP, dGTP; 0.004 mM dTTP; 0.5 mM ATP. The 100% value corresponds to 16,801 TCA precipitable cpm. 'Complete' reaction: same as above except GTP, UTP, CTP added at 0.08 mM each and MgCl£ decreased to 1 mM. The 100% value corresponds to 31,737 cpm. Experiment 1: complete^ -dXTP's -ATP +GTP, UTP, CTP (.08 mM) -Mg"^ 1/10 Mg*4" Experiment 2: completec - unlabelled dTTP 105 aspect w i l l be discussed in more detail below (Section D). Unless stated otherwise, the reaction mixture in experiment 2 (Table VI) was used. The in vitro product was 95% solubilized by DNase and resistant to RNase (50 yg/ml and 100 yg/ml respectively, incubated for 1 h. at 37°C). The kinetics of DNA synthesis are shown in Fig. 23. A l l reactions contained equal numbers of c e l l s . Nuclei from G-l arrested mock infected cells synthesized less than 20% of the level of DNA synthesized in nuclei from infected G-l cells,, at 16 h. post infection. As expected, DNA syn-thesis in infected S phase nuclei was stimulated compared to mock infected S-phase nuclei. Infected G-l phase nuclei synthesized slightly more DNA than mock infected S-phase nuclei, although the i n i t i a l rates of synthesis were similar. After infection, total DNA synthesis was stimulated in permissive (S-phase) and non-permissive (G-l phase) nuclei. The rate of DNA synthesis was always linear during the f i r s t 30 minutes in infected G-l or S phase nuclei, and usually levelled out between 30 and 60 minutes. Addition of fresh substrate after 60 minutes restored DNA synthesis to 56% of the i n i t i a l rate. Addition of fresh nuclei at 60 minutes restored DNA synthesis to 25% of the i n i t i a l rate, suggesting that the plateau observed between 30 and 60 minutes was probably due to depletion (or destruction) of substrate and limiting factors in the nuclei. B. The Nature of DNA Synthesized in Permissive and Non-Permissive Nuclei The increased DNA synthetic activity of nuclei from MCMV infected cells (Fig. 23) suggested that v i r a l DNA was being synthesized. This was tested by CsCl gradient centrifugation and DNA annealing. Nuclear monolayers from infected, G-l arrested 3T3 cells and infected exponential cells were prepared and analyzed for DNA synthetic activity 105a Fig. 23: Kinetics of DNA Synthesis in Nuclear Monolayers 3T3 cells were l e f t without medium change for 5 days to effect G-l arrest. The cultures were mock infected or MCMV infected (input m.o.i. of 10 pfu/cell) by centrifugal inoculation in Hank's BSS plus 0.1% serum. After the adsorption period, the inocula were removed and half of the infected and mock infected cultures were given the same G—l depleted medium to maintain G-l arrest. The remaining infected or mock-infected cultures were fed fresh MEM plus 10% serum. Nuclear monolayers were prepared at 20 h. post infection except for the mock infected G-l cells which were assayed at 16 h. post infection. Since the assays were a l l done prior to 26 h. post serum activation (when the c e l l density doubles.in serum activated cultures, Fig. 9), a l l reactions contained equal numbers of nuclei. The 'standard' reaction mixture (experiment 2, Table VI) contained 0.004 mM dTTP. The reactions were incubated for _2 various periods of time, ice cold 1 x SSC plus 10 M EDTA added to stop the reaction, and TCA precipitable radioactivity was determined. Backgrounds were determined by the amount of TCA precipitable radioactivity in a time zero harvest. Symbols: (• ) S phase infected ( • ) S phase mock infected ( T ) G-l phase infected ( • ) G-l phase mock infected 106 107 at 14 to 16 h. post infection. The purified products were then centrifuged in CsCl gradients. The majority of the labelled DNA from the experimental nuclei, banded at the density of MCMV DNA (1-718 g/cc, Mosmann and Hudson, 1973), and the remainder at the cellular DNA density (Fig. 24A). Thus, under the appropriate conditions in vitro, nuclei from infected exponential cells detained theircapacity to replicate MCMV DNA. Infected nuclei from G-l arrested cel l s , on the other hand, did not synthesize detectable levels of v i r a l DNA (Fig. 24B). However, in view of the heterogeneity in base composition of MCMV DNA (Mosmann and Hudson, 1974), and the fact that v i r a l DNA synthesized in vitro may consist of several species of different buoyant densities (Bell, 1974), the results of this experiment were verified by DNA annealing. Infected, G-l arrested nuclei and mid-S phase nuclei were analyzed for in vitro DNA synthesis at 20 h. post infection. The DNA was purified and annealed to unlabelled DNA on nitrocellulose f i l t e r s (Table VII). The infected S phase nuclei synthesized DNA which was complementary to MCMV DNA as well as some cellular DNA. Approximately half of the total labelled DNA was v i r a l as determined by CsCl gradient centrifugation. Infected G-l nuclei (Table VII) did not synthesize detectable levels of MCMV DNA. The possibility that G—1 nuclei synthesized low levels of v i r a l DNA (i.e. below the range of sensitivity of the f i l t e r hybridization technique) cannot be entirely ruled out at present. However, the hybrid-ization data are in agreement with the CsCl analyses (Fig. 24). In addition, a control has been included in Table VII (hybridization #4) which demonstrates that the DNA banding at the buoyant density of MCMV DNA in CsCl is indeed v i r a l specific. A second approach was attempted to determine i f infected nuclei were synthesizing v i r a l DNA in vitro based on DNA reassociation kinetics. The 107a Fig. 24: CsCl Equilibrium Centrifugation of the DNA Synthesized  in vitro in Infected G—1 and Exponential Nuclei G-l arrested or exponentially growing 3T3 cells were infected at an input m.o.i. of 20 pfu/cell by centrifugal inoculation. In vitro DNA synthesis was performed at 14 to 16 h. post infection, and the purified DNA analyzed on 2.8 ml CsCl gradients. Centri-fugation was carried out in a Beckman SW-50.1 rotor for 42 h. (20°C) at 30,000 rpm. The bottom of the tube is to the l e f t . The arrow denotes DNA at the density of MCMV DNA (1.718 g/cc). 3 a. Exponential nuclei - 1.60 x 10 cpm/yg b. G-l arrested nuclei - 2.92 x 10 cpm/yg 109 Table VII: Hybridization of in vitro Synthesized DNA Source of3H-dTMP DNA labelled in v i t r o 3 Input cpm (yg) CPM Hybridized mouse cells calf thymus MCMV 1. nuclei from infected S phase cells 3.22 x 10 4 (17) 167 52 490 2. nuclei from infected G-l cells 1.178 x 10 4 (20) 67 21 27 3. nuclei from mock infected cells 1.47 x 10 4 (15) 95 22 24 3 4. H-MCMV synthesized in v i t r o b 2.56 x 10 4 (22) 72 34 1384 Infected 3T3 cells (G-l or S phase) or mock infected 3T3 cells were analyzed at 20 h. post infection for in vitro DNA synthetic activity. The DNA was purified and hybridized with f i l t e r s containing 2 yg of mouse c e l l , calf .thymus, or MCMV DNA for 24 h. at 65°C. H-MCMV DNA synthesized in vitro in exponential 3T3 nuclei was purified from a preparative CsCl gradient. ""H-TMP l a b e l l e d material from exponential,, i n f e c t e d n u c l e i was p u r i f i e d , and DNA banding at the buoyant density of MCMV DNA on CsCl was pooled, dialyzed against 2 x SSC, and the rate of reass o c i a t i o n determined i n the presence of 3.6 Ug/ml unlabelled v i r a l DNA, or 23 ug/ml unlabelled c e l l u l a r 3 DNA. The rate of reass o c i a t i o n of the H-TMP l a b e l l e d v i r a l DNA without any addition served as a co n t r o l . The rate of reassociation was accelerated 2 to 3 f o l d (compared to the control) by the ad d i t i o n of 3.6 ug/ml of MCMV DNA, and the rate was not affected by the addition of c e l l u l a r DNA. This approach was not amenable to infec t e d G-l DNA due to the low s p e c i f i c a c t i v i t y of the i n v i t r o product (see F i g . 24). C. Induction of a New DNA Polymerase A c t i v i t y i n Permissive and Non- Permissive C e l l s . From the foregoing data two conclusions may be drawn: (1) t o t a l i n v i t r o DNA synthesis i s stimulated a f t e r i n f e c t i o n of permissive and non-permissive c e l l s ; and (2) non-permissive c e l l s do not synthesize s i g n i f i c a n t l e v e l s of v i r a l DNA i n vivo or i n v i t r o . An obvious explanation i s that MCMV induces a v i r a l polymerase i n both permissive and non-permissive c e l l s , but the l a t t e r do not synthesize v i r a l DNA i n the absence of a fact o r (or factors) supplied by the host during S phase. When ammonium s u l f a t e was added to the i n v i t r o reaction mixture, i n the range of 25 to 200 mM, the DNA synthetic a c t i v i t y increased 200 to as much as 400% i n in f e c t e d exponential (permissive) 3T3 n u c l e i . The host c e l l DNA synthetic a c t i v i t y was i n h i b i t e d by 80 to 90% i n the presence of 200 mM ammonium s u l f a t e ( F i g . 25B). In exponential 3T3 c e l l s a s a l t dependent polymerase was beginning to appear at 6 h. post i n f e c t i o n . At 14 h. post i n f e c t i o n the stimulation by 100 mM ammonium s u l f a t e increased 110a Fig. 25: Effect of Ammonium Sulfate Concentration on H-dTMP  Incorporation A. Exponentially growing 3T3 cells were infected at an input m.o.i. of 20 pfu/cell and analyzed for in vitro DNA synthesis at 14 h. post infection. Assays were carried out (60 minute incubation period) using the standard reaction mixture (Table VI, experiment 2) in the absence of carrier dTTP. Ammonium sulfate was added to a f i n a l concentration of 0.1 to 0.2 M. Three 35 mm plates (1.54 x 10 nuclei/plate) were harvested at the end of the incu-bation period and the DNA purified. The specific activities and ammonium sulfate molarity were respectively: 3.391 x 10 cpm/yg, 0.0; 7.12 x 10 3 cpm/yg, 0.1; 2.841 x 10 3 cpm/yg, 0.15; 2.3 x 10 3 cpm/yg, 0.2. 30 yg from each DNA preparation were annealed to (1) MCMV DNA, 5 yg/ f i l t e r ; (2) MCMV DNA, 2yg/filter; (3) 3T3 c e l l DNA, 2 yg/fi l t e r ; (4) calf thymus DNA, 2 yg/filter; (5) blank f i l t e r . Each reaction v i a l contained duplicate f i l t e r s of each of the above. Backgrounds, estimated by hybridization to hetero-logous DNA, have been subtracted. The hybridization reaction was performed at 65°C for 24 h. Symbols: ( • ) 3T3 DNA (2 yg/filter) ( • ) MCMV DNA (5 yg/filter) ( • ) MCMV DNA (2 yg/filter) B. Exponentially growing 3T3 cells were mock infected or infected, and analyzed for i n vitro DNA synthesis, as described above, at 6 h., 14 h. and 48 h. post infection. After a 1 h. incubation period TCA precipitable radioactivity was determined for each salt concentration. Symbols: ( • ) MCMV infected (1 x 10 nuclei) 48 h. post infection ( • ) MCMV infected (1.32 x 10 6 nuclei) 14 h. post infection ( • ) MCMV infected (1.78 x 10 6 nuclei) 6 h. post infection ( O ) mock infected (1.42 x 10 nuclei) 14 h. post infection 112 to 325%. The degree of salt enhancement is probably directly proportional to the number of polymerase molecules present. Weisbach et a l (1973) reported that the purified HSV DNA polymerase was stimulated 25 to 50 fold by 150 mM ammonium sulfate, while host DNA polymeraseS/0, and 3 were inhibited by 90% at this salt concentration. The a b i l i t y of the salt dependent polymerase to replicate v i r a l and host DNA was investigated by f i l t e r hybridization. 3T3 cells in exponential growth were analyzed for their in vitro DNA synthetic activity in the presence of 0 to 200 mM ammonium sulfate. The annealing reactions were performed with a constant mass input of labelled DNA (Fig. 25^-). The amount of labelled v i r a l DNA increased to a maximum of 180% (100 mM ammonium sulfate). Therefore, the optimal salt concentration was established at 100 mM for the v i r a l polymerase. A rather unexpected finding was that the levels of cellular DNA increased to a maximum of 350% at 100 mM ammonium sulfate. Therefore the v i r a l polymerase was catalyzing some form of host DNA synthesis (e.g. repair) as well as v i r a l DNA synthesis. The labelled DNA's from the '0' and 0.1 M ammonium sulfate assays were also centrifuged in CsCl gradients The '0' salt assay contained 37% v i r a l DNA and 63% cellular DNA. The 0.1 M salt assay contained 56% v i r a l DNA and 44% cellular DNA (not shown). Comparing these data to Fig. 25A, i t i s clear that the 3.5 fold increase in cellular DNA at 0.1 M salt was not reflected i n the isopycnic analyses. Therefore the apparent increase in cellular DNA, as assessed by DNA f i l t e r annealing, possibly was a result of accelerated reassociation kinetics of the in vitro made, cellular DNA. For example, i f reiterated DNA was selectively synthesized in the presence of 100 mM salt, the rate of annealing would increase due to the lower Cot, value. This has not been tested directly. 113 The ammonium sulfate response of infected, G-l arrested, 3T3 nuclei was similar to the permissive system (Fig. 26). However, v i r a l DNA was not synthesized even when 100 mM ammonium sulfate was included in the reaction mixture, in agreement with previous data. Synthesis of total DNA by infected nuclei (in 100 mM ammonium sulfate) was inhibited by MCMV specific antiserum (anti-ICP). DNA synthesis in nuclei from infected exponential and G—l cells was inhibited by 80%, and greater than 40% respectively (Fig. 27). Pre-immune serum had no affect on the DNA synthetic activity of infected nuclei. Similarly, neither mock infected exponential nor G-l arrested nuclei were affected by the anti-ICP serum. Furthermore, the inhibitory action of the anti-ICP serum on infected c e l l nuclei was alleviated by a pre-adsorption to infected exponential nuclei (not shown). The data presented herein may be summarized as follows: (1) MCMV induces a 'new' DNA polymerase in infected exponential and G—l arrested nuclei with different reaction requirements than the host polymerases; (2) DNA synthesis by the salt dependent polymerase i s inhibited by antisera directed against total infected c e l l protein; (3) in infected exponential 3 nuclei, the salt dependent polymerase incorporates H-dTMP into cellular and v i r a l DNA sequences; (4) in infected, G—l arrested nuclei, the salt 3 dependent polymerase incorporates H-dTMP into cellular DNA only. The immunological data support the contention that the MCMV genome codes for a portion, and perhaps a l l of the 'new' DNA polymerase. D. Characterization of MCMV DNA Synthesized In Vitro As stated previously, the addition of unlabelled dTTP to the in vitro reaction mixture, was undesirable since i t led to a dilution of Fig. 26: Effect of Ammonium Sulfate Concentration on H-dTMP Incorporation in G-l Arrested Nuclei G-l arrested 3T3 cells were mock infected or infected at an input m.o.i. of 10 pfu/cell and maintained i n G-l after infection. At 20 h. post infection the nuclei were prepared and analyzed for DNA synthesis in the presence of ammonium sulfate (final concentration of 0.025 to 0.2 M). The reactions were incubated for 1 h. without added carrier dTTP. Each plate contained approximately 1.5 x 10^ 4 nuclei. The 100% value corresponds to 2 x 10 cpm.(mock) and 7.2 x 4 10 cpm (infected). Symbols: ( • ) MCMV infected ( O ) mock infected 114 114a Fig- 27: Effect of Antisera on DNA Synthesis in Nuclear Monolayers Nuclear monolayers were prepared from mock infected or infected exponential 3T3 cells (3.25 x 10"* cells/plate, input m.o.i. 20 pfu/ cell) at 14 h. post infection. Infected G—l cells (approximately 1 x 10^ cells/plate, same m.o.i.) were assayed at 20 h. post infection. The nuclei were pre-incubated for 20 minutes (at room temperature), with either pre-immune or anti-ICP serum in a volume of 0.5 ml. The antisera were diluted in Hepes buffer (pH 7.6) to the desired con-centration. The anti-ICP sera had been preadsorbed against uninfected intact 3T3 cells and mid-S phase nuclei as described in the Materials and Methods. To start the reaction, the antisera were removed and the standard reaction mixtures (minus carrier dTTP) added for 1 h. at 37°C. TCA precipitable radioactivity was then determined. A l l determinations were done in duplicate. Symbols: ( O ) anti-ICP + exponential mock infected nuclei ( • ) anti-ICP + G—l arrested mock infected nuclei ( A ) pre-immune control serum + exponential MCMV infected nuclei ( • ) anti-ICP + G-l arrested MCMV infected nuclei ( • ) anti-ICP + exponential MCMV infected nuclei The 100% values correspond to (1) exponential mock infected - 8.4 x 3 5 10 cpm; (2) exponential MCMV infected - 2.5 x 10 cpm; (3) G—1 arrested 4 mock infected - 1.2 x 10 cpm; (4) G-l arrested MCMV infected - 9.2 x i n 4 10 cpm. 115 -'H-dTTP and a subsequent lower extent of JH-dTMP incorporation. Therefore, experiments were carried out to assess the influence of unlabelled dTTP on the in vitro reaction. The buoyant density of v i r a l DNA made in vitro was 1.718 g/cc in the presence and absence of carrier dTTP (.004 mM). The gradient profile for DNA synthesized in the presence of 0.004 mM dTTP is shown in Fig. 28. Centrifugation in a fixed angle rotor (type 40) allowed good separation of the two DNA species. Fractions at the v i r a l and cellular DNA density were combined into two pools, and hybridized to MCMV DNA on nitrocellulose f i l t e r s (Table VIII). The pooled DNA at the cellular density contained 17 to 18% of the amount of v i r a l DNA contained i n the v i r a l DNA density pool. This is in agreement with the isopycnic analysis (Fig. 28) since the amount of overlap between the two peaks was estimated to be 18%. Analysis of total in vitro synthesized DNA from infected nuclei on hydroxyapatite indicated the majority of DNA (85-90%) was double stranded. The results were the same for DNA made in the presence or absence of .004 mM dTTP. A small but variable amount of labelled DNA (approximately 10 to 20% of the total) was released from nuclei into the supernatant. This pheno-menon was observed with or without added dTTP. However, in the absence of dTTP, 30% of the supernatant associated DNA was single-stranded compared to 10% in the presence of dTTP's as determined by hydroxyapatite column chromatography. The size of the in vitro labelled DNA was studied by sedimentation velocity in sucrose. Nuclear monolayers were prepared at 14 h. post infection from infected exponential 3T3 c e l l s . Sedimentation of the purified c e l l plus v i r a l DNAs was carried out i n 5-20% neutral sucrose gradients 32 (Fig. 29). By comparison with P labelled T-7 DNA in the same gradient, 116a Fig. 28: Preparative CsCl Gradient Analysis of in vitro Synthesized DNA Exponential 3T3 cells were infected at an input m.o.i. of 10 pfu/cell. Nuclear monolayers were prepared at 14 h. post infection and incubated 1 h. in the standard reaction mixture containing 100 mM (NH^^SO^ plus 4 yM dTTP. The DNA was purified and centrifuged to equilibrium in a 5.5 ml CsCl gradient using a type-40 rotor. Centrifugation was carried out at 35,000 rpm for 82 h. The tube was fractionated, refractive index determined for every f i f t h fraction and 0.5 ml TNE added. Small aliquots (25 yl) were withdrawn for radioactivity determinations and the A.260 °f t n e remaining material read. The arrow corresponds to 1.718 g/cc, the density of MCMV DNA. Symbols: ( ) cpm (* •> A260 117 118 Table VIII: DNA-DNA Annealing of in vitro Synthesized MCMV DNA a 3 Source of H-dTMP DNA labelled in vitro Input CPM: (ug) bCPM bound to f i l t e r s containing: calf thymus DNA (2 yg) MCMV DNA (5 yg) MCMV DNA (2 yg) 1. DNA at the buoyant density of MCMV DNA 25,680 (22.25) 34 2658 1380 2. DNA at the buoyant density of cellular DNA 5,112 (24) 42 498 237 125 3. I-MCMV DNA 37,800 (.002) 45 11696 4127 " The v i r a l or cellular fractions were pooled from the preparative CsCl gradient shown in Fig. 28, precipitated with ethanol, and dissolved in .01 SSC. The specific activities were: v i r a l DNA -3 2 1.154 x 10 cpm/yg; cellular DNA - 2.14 x 10 cpm/yg. Each reaction v i a l contained duplicate f i l t e r s . The annealing reaction was carried out for 24 h. b Machine backgrounds (20-30 cpm) have not been subtracted. Fig. 29: Sedimentation Analysis of DNA Synthesized in vitro in the Presence and Absence of Unlabelled dTTP Exponential 3T3 cells were infected at an input m.o.i. of 10 pfu/cell. Nuclear monolayers were prepared at 14 h. post infection and DNA syn-thesis was carried out in 100 mM (NH^^SO^, plus 1 yM dTTP or minus dTTP in the standard reaction mixture for a 1 h. incubation period. 2 The specific a c t i v i t i e s of the purified DNA's were: (1) 4 x 10 cpm/yg 2 (no dTTP); (2) 1.8 x 10 cpm/yg (1 yM dTTP). An aliquot of each was 32 mixed with P-labelled T-7 DNA and layered on 5.0 ml, 5-20% sucrose gradients containing a 0.25 ml cushion of 60% sucrose. Centrifugation was carried out for 4 h. at 35,000 rpm in the SW-50.1 rotor. The direction of sedimentation i s to the l e f t . A. DNA synthesized in the presence of dTTP B. DNA synthesized i n the absence of dTTP the majority of the in vitro product sedimented in the range of 24 to 32s, assuming the sedimentation coefficient of T-7 DNA is 32s (Studier, 1965) . The size distribution was similar for DNA synthesized in the presence or absence of unlabelled dTTP i n the reaction. Assuming these molecules were linear and double stranded, the molecular weight was calculated to be in the range of 12 to 23 million daltons (Freifelder, 1970). The size of in vitro synthesized v i r a l DNA, which was carefully separated from cellular DNA by preparative CsCl gradient centrifugation, was estimated to be approximately 17 million daltons (28s) relative to T-7 DNA (Pig. 30). Therefore, the major peak in Fig. 29, probably represented v i r a l DNA, since the high molecular weight (50 to 53s) and low molecular weight (8-10s) DNA species were not observed in gradients containing pure v i r a l DNA (Fig. 30). The size of nascent, in vitro made DNA was also determined by sedi-mentation in alkaline sucrose (Fig. 31). Denatured v i r a l DNA sedimented more slowly (14 to 19s relative to T-4 DNA at 57-.-5s (Levin and Hutchison, 1973)) than the native in vitro product (Fig. 30). These results suggested that nascent v i r a l DNAs made in vitro were either associated with a l k a l i -labile material or represented low molecular weight DNA non-covalently linked to larger structures. The former seems more lik e l y since heat denatured DNA, in neutral sucrose, was somewhat larger (18-28s) than a l k a l i denatured DNA. 120a Fig- 30: Sedimentation Analysis of MCMV DNA Synthesized in vitro Exponential 3T3 cells were infected at an input m.o.i. of 10 pfu/cell. At 14 h. post infection, nuclear monolayers were prepared and analyzed in the standard reaction mixture containing 100 mM (NH^^SO^. The purified DNA was centrifuged on a CsCl gradient and material at the density of v i r a l DNA pooled. The DNA (specific activity of 823 cpm/yg) hybridized exclusively to MCMV DNA on f i l t e r s and not to c e l l DNA. An 32 aliquot was mixed with P-MCMV DNA and centrifuged for 2.5 h. at 35,000 rpm i n 5.0 ml 5-20% neutral sucrose plus a 0.25 ml 60% cushion of sucrose. The sedimentation coefficient of T-7 DNA was determined in a parallel gradient, under exactly the same conditions. Sedimentation is to the l e f t . The arrows indicate the marker positions. 121 121a Fig. 31: Alkaline Sedimentation Velocity of V i r a l DNA Synthesized in vitro The same CsCl gradient purified v i r a l DNA preparation used in Fig. 30 32 was analyzed on alkaline sucrose (5-20%). In a parallel gradient, P labelled T-4 DNA from a l k a l i treated intact phage particles was analyzed. Centrifugation was carried out in the SW-50.1 rotor (at 20°C) for 3 h. at 35,000 rpm. Sedimentation is to the l e f t . 122 123 SECTION IV: THE INTERACTION BETWEEN G-l 3T3 CELLS AND MCMV The failure of MCMV to replicate in G-l 3T3 cells was shown to be due to the absence of a host S phase factor (or factors) required for v i r a l replication (Fig. 13). This, as well as other supporting data, indicates that the G-l latent system i s related to the phenomenon of c e l l cycle dependent replication of MCMV. Therefore, a detailed investigation of the G-l latent system should yield information concerning the nature of the S phase dependency. Moreover, the interaction between MCMV and G-l phase cells provides a useful model system to study v i r a l persistence in c e l l culture. A. Persistence of MCMV in G-l Phase 3T3 Cells The experiment illustrated in Fig. 32 shows that significant levels of MCMV replication did not occur in G-l 3T3 cells , when the latter were infected with MCMV and maintained in G—1. Over the entire 8 day experiment, 4 the level of infectious supernatant virus decreased by 10 . However, the virus could be activated by either of two methods: (1) by replating the infected G-l cells onto exponentially growing ME cells; or (2) by providing the appropriate stimuli (fresh MEM + 10% serum) to promote entry into S phase. The former method resulted in infectious centers, which decreased approximately one log over the 8 day period. Therefore, about 10% of the cells retained an inducible v i r a l genome. The actual percentage may be higher than 10% due to poor adherence of infected G-l cells to the ME c e l l monolayer. The second method of activation demonstrated that the progression of infected G-l cells into S phase was sufficient for induction of a l y t i c infection. 'Che G-l cells were stimulated to produce Fig. 32: Persistence of MCMV in G-l 3T3 Cells G-l arrested 3T3 cells were infected at an input m.o.i. of 10 pfu/ c e l l and maintained in G-l after infection. At 24 h. intervals the supernatant medium from 1 plate was removed for plaque ti t r a t i o n and the cells rinsed 5 times with MEM plus 10% serum, trypsinized for 5 minutes, and resuspended in 1.0 ml of medium. These cells were diluted serially and replated onto exponential ME c e l l mono-layers.for infectious center determination. Approximately 1 x 10 cells were recovered from each plate. On day 5, three plates were given fresh MEM plus 10% serum. Super-natant virus was then harvested at day 6 and day 8 for plaque titr a t i o n . Symbols: ( • ) infectious centers ( • ) supernatant virus, G-l c e l l s ( • ) supernatant virus, serum activated cells 125 infectious progeny virus on day five (Fig. 32). At any time up to two weeks post infection these cells could be activated to yield similar levels of progeny virus. A comparison of the number of infectious centers from G—1 3T3 cells and exponential (permissive) c e l l s , immediately after infection, demon-strated that in both cases greater than 90% of the cells were infected. In addition, at least 80% of radiolabelled virus was found in the nucleus of the infected G-l c e l l . Thus the i n a b i l i t y of the virus to replicate in the G-l c e l l cannot be ascribed to inefficient penetration of the c e l l or i t s nucleus. B. Extent of Vir a l DNA Replication in G-l Cells The extent of v i r a l DNA synthesis in G—l cells was determined using DNA reassociation kinetics as described in section IB. As shown in Fig. 33, the DNA from mock infected cells did not accelerate reassociation of the 125 I-DNA probe. Comparison of reassociation kinetics i n the presence of infected G-l or infected exponential c e l l DNA indicated that the MCMV DNA concentration in the former was only 3% of the latter. This value probably represented input parental v i r a l DNA, and not significant v i r a l DNA syn-thesis. C. Transcriptional Analyses The interaction between G—l arrested cells and MCMV i s characterized by three salient features: (1) v i r a l progeny are not produced; (2) v i r a l DNA synthesis is negative in vivo and in vitro; and (3) the virus i s maintained in a viable (i.e. inducible) state in at least some ce l l s . Fig. 33: Detection of MCMV DNA by Reassociation Kinetics Exponential 3T3 cells and G-l arrested cells were infected at an input m.o.i. of 10 pfu/cell. DNA was isolated and purified at 1 25 24 h. post infection and annealed with I MCMV DNA. Each reaction contained 800 ug unlabelled DNA plus 0.05 ml (ca. 1-2 ng) MCMV iodinated DNA probe in a total volume of 1.85 ml 2 x SSC. Duplicate samples were removed at the indicated times for S-l nuclease treatment. The solid lines represent best f i t s as determined by the 9810A minicomputer. The slopes were: (1) Expo-nential infected, 0.0808; (2) G-l arrested infected, 0.0028; (3) mock infected, 0.0007. Symbols: ( • ) DNA from infected exponential c e l l s ( G ) DNA from infected G-l arrested cells ( A ) DNA from mock infected cells 126 127 To gain further insight into the metabolic activity of the latent v i r a l genome, a detailed analysis of v i r a l RNA synthesis was performed. 1. Theory, analytical treatment, and data reduction The techniques are based upon the conversion of single strand DNA to DNA-RNA hybrids under rigorously controlled conditions of RNA con-centration, incubation time, DNA fragment size, self-annealing of probe, and temperature. The following data are pertinent to the design and interpretation of the solution hybridization experiments which follow: (i) the MCMV genome is a linear, double stranded molecule of 132 x 10^ molecular weight (Mosmann and Hudson, 1973), composed mainly, i f not entirely, of unique sequences (V. Misra personal communication); ( i i ) MCMV DNA synthesis, in exponentially growing 3T3 cells , begins at 10 to 12 h. post infection; ( i i i ) RNA extracted prior to the onset of DNA synthesis is termed 'early' RNA; (iv) RNA extracted after 12 h. is termed 'late' RNA. Essentially 3 important conclusions may be drawn" from a single solution hybridization experiment with RNA in excess: (1) the extent of transcription of v i r a l DNA; (2) the number of classes of v i r a l RNA present in infected cells differing in relative concentration; (3) the fraction of the total DNA which codes for the abundant, scarce or intermediate RNA classes. The principal advantages of solution hybridization with unlabelled RNA in excess are that the results are not biased by nucleotide pool fluctuations as a result of virus infection, and the analysis is sufficiently sensitive to detect scarce and abundant RNA species. The basis for the following series of equations was formulated by Britten and Kohne (1965) to describe the reassociation of complementary 128 DNA strands: dC/dt = -kC 2 (1) where C is the concentration of single strand DNA, t is time, and k i s the DNA-DNA reassociation rate constant. A similar relationship was used to describe the DNA-RNA hybridization reaction (Frenkel and Roizman, 1972): dC/dt = -kRC (2) where t is the time of hybridization, C is the molar concentration of single strand DNA at time t, R i s the molar concentration of total single strand RNA, and k is the DNA-RNA hybridization rate constant. Equation (2) assumes the reassociation of the DNA probe is either negligible or can be controlled throughout the hybridization reaction. The contribution of hybridized v i r a l RNA to total single strand RNA i s also negligible and R can be assumed constant, such that R = R q (i.e. input RNA concentration). Therefore, assuming R q and k do not depend upon t, equation (2) upon integration yields: ^ = e" k Ro t (3) o where is the concentration of single strand DNA at time t, and C q is the i n i t i a l concentration of single strand DNA. Equation (3) assumes the homologous RNA is contained in a single abundance class, Ro. However, equation (3) may be modified to pertain to N classes of RNA, each present in a molar concentration of R , such that the sum total of a l l classes n equals the total DNA transcribed. Hence, each class may be visualized as: ( Ct>n -kR t = e ° (4) (C ) o n 129 Since (C ) equals C • a (a being the fraction of total v i r a l DNA serving on o n n ° as template for this RNA class), i t follows that: (C ) -k R t t n n n / r. = e (5) C .a o n The observed fraction of single stranded DNA may therefore be visualized as: (6) ^ t = ( C t \ + + ^ _Vn + 1 - (ai + + a ) C C C n o o o Where 1 - (ai .... + a ) corresponds to that portion of the genome that n is not transcribed and w i l l remain single stranded throughout the hybrid-ization reaction. It follows, therefore, that: Q • t -kiRit , . -kT1RT,t , , / . \ (7) — = ai,.e 1 1 + + a e n " + l-(oti + + a ) C n n o (Frenkel and Roizman, 1972). For reasons stated below, equation (7) was modified to yield: Q ( 8 ) -r- = a 1 e " P l t + + a e " ^ + 1 - (ai + + a ) C n n o The 3 values correspond to the product of K'R^ . The K"Rn parameter was coded in this way since the K value for DNA-RNA hybridization i s an unknown, and may differ by 25% from the DNA-DNA constant (Galau et a l , 1977) . Although the K value i s required for absolute values of v i r a l molar RNA concentration, i t is not required for determination of relative concentrations of the RNA species or for the a values. In any event, n the R and K values introduce constraints into the optimizing routine n within the computer program and are therefore a constant source of problems. In a l l hybridizations, the total molar RNA concentration was kept constant 130 at 0.0025 moles nucleotlde/liter. To solve the problem of f i t t i n g the best curve to equation (8), a non-linear optimizing routine celled 'fletch' was used. This program was written by R. Fletcher at the Atomic Energy Research Establishment in Harwell, ..England, and is documented in UBC-NLP, 1975. The program actually minimizes the function value F, or sum of squares, defined as: -2-C i t - ( a ! e " e i t i + + a e " ^ ! + ± _ a _ ... a ) - — n n l_ o C t i Where — — i s the experimentally determined dependent variable of the o fraction of DNA remaining single stranded at time t. It follows therefore that the closer F i s to zero, the better the f i t . The best f i t curves were deduced on an interactive basis in the following way. The experimental observations were fed into fletch using an IBM 370 computer. The calculation of the number of classes is therefore based on f i t t i n g to the experimental data by non linear least squares optimization, the parameters through a and 3i through 3 for n equal n n to 1, 2, 3, 4 etc. The actual curves were then generated with the aid of a Hewlett-Packard 9810A Mini-computer and a 9862A plotter function. The program (called 'curv-plot') was written specifically to allow visual-ization of the best f i t data. The graphs shown below were generated by the curv-plot program on the 9810A. 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 $i (n = 1) or 3i and $2 (n = 2). After the optima for a and 3 were reached, the program was repeated from several different starting points (i.e. 3 estimates) to infer i f a l l runs converged to the same values for a and 3. This procedure 131 ensured that 'global', rather than 'local', optima were achieved and helped obviate premature or false convergance. The validity of fletch for deter-mination of the best f i t parameters was verified by curve-fits supplied by Dr. B. Roizman (University of Chicago). In a l l cases, the best f i t values determined by fletch were coincident with Dr. Roizman's parameters. The conditions of hybridization were manipulated to ensure minimal self-annealing of the iodinated probe DNA. In fact, self-annealing never exceeded 5% during a 140 h. incubation period. However, this could introduce a serious error into the calculation of a values of the various abundant-scarce classes, particularly when a was less than 10%. Therefore, DNA self-annealing was monitored by mixing the iodinated probe with heterologous RNA. A computer-aided linear least squares reduction analysis was carried out on these data (Fig. 34), and an equation derived to describe that line. Hence, for a l l subsequent hybridizations, each experimentally determined fc value at time t was corrected for DNA self-annealing, according to the Co following equation (from Fig. 33): Ct rr- = -0.000421 t + 0.996752 o where -0.000421 is the slope and 0.996752 is the Y intercept. 2. Analysis of v i r a l RNA present in infected permissive (exponential) 3T3 cells Total, unlabelled RNA was isolated from infected exponential 3T3 cells 125 at 6 h. and 24 h. after infection and hybridized with sheared I-MCMV DNA. Analyses of the kinetics of hybridization are shown in Fig. 35 (late) for n = 1 and n = 2, and Fig. 36 (early). When n was set equal to or 131a Fig. 34: Self-annealing of Iodinated MCMV DNA in the Presence  of Heterologous RNA Re-purified yeast RNA (Sigma) was mixed with 0.035 ul of ""'I MCMV DNA (2 x 10^ cpm or 5 ng/ml) in 2 x SSC. The concentration of the yeast RNA was adjusted to 2.5 x 10 moles of nucleotide/1. The hybridization was carried out as described in the Methods Section. The solid line represents the best f i t obtained by linear least squares minimization. .9 .B .. S \ V .7 .. £0 100 IS0 HDUR5 132s Fig. 35: Hybridization of MCMV DNA with Excess Exponential RNA  at 24 h. Post Infection (Permissive Late RNA) Exponentially growing 3T3 cells in roll e r bottles were infected at an input m.o.i. of 10 pfu/cell, and total RNA extracted at 24 h. post infection. The hybridization contained 900 ug/ml 24 h. RNA -3 5 (2.5 x 10 moles nucleotide/liter) and 2 x 10 cpm (or 5 ng/ml) 125 I-MCMV DNA in 2.0 ml 2 x SSC. The solution was sonicated, sealed in 100 ul aliquots and heat denatured. After the various incubation periods, samples were treated with s - l nuclease and TCA precipitated. A computer aided non-linear least squares reduction (Fletch) was employed to determine an, $n in equation 8, in which the number of RNA classes was assumed to be 1, 2, 3 etc. The upper (n = 1) and lower (n = 2) graphs were drawn by 'curv-plot' and the best f i t parameters are indicated. The f i t for n = 2 is clearly better than n = 1. When n was set at 3 or 4,. .0:3 was negative and therefore not significant. A l l g values are expressed with negative signs according to equation 8. The a values however, must be positive to have any meaning. Q Ordinate: — i s the fraction of DNA remaining single stranded Co Abscissa: upper - R t is the RNA concentration in moles nucleo-:tide/liter times seconds of incubation lower - hours of incubation 133 EEJEJ R 0 T - » I 2 0 0 ] | | R I = 0.H 1 70H2 B 1 = — 0.217 1 BBS . 3 1 FINBL FUNCTION VHl_UEc0.002a 1 G s \ . B . ."7 V . B s i a 1 IZIIZI i s i a . 3 . H O U R S R 1 sEJ.ElBB 1 BB B 1 = -0 .2BS0 1 B B 2 : 0 . 3 3 1 HSta B2:-IZI. EJ H B121E EI • F"INHl_ FUNCTION VBLUEs0.0B0770 13 \ . B . ."7 \ . B . ft-* H O U R S 133a F i g . 36: Hybridization of MCMV DNA with Excess Exponential RNA  at 6 h. Post I n f e c t i o n (Permissive Early RNA), The experimental d e t a i l s were the same as described i n F i g . 35. This fi g u r e i s the actual hard-copy p l o t generated by the curv-p l o t program on the Hewlett Packard 9810A system. The function value, a and 3 are indicated on the graph. The f i t f o r n = 2 n n was 4 f o l d better than n = 1 according to function evaluation (equation 9). When n was greater than 2, the r e s u l t s were meaningless. R0T-> E00 1200 ^ 0 I 0 0 | 5 0 HOURS 135 greater than 3, the a values either decreased to zero or became negative. Therefore, n >2 yielded meaningless parameters. The f i t was better for n = 2 for both early and late RNA's, according to the function values. The results of these studies are summarized in Table IX and indicate the following; (1) Approximately 27% of the v i r a l DNA serves as a template for RNA synthesized at 6 h. post infection. Assuming asymmetric transcrip-tion (i.e. from 1 strand) this corresponds to 54% of the total coding capacity of the genome. (2) Approximately 43% of the DNA was expressed as transcripts, corresponding to 86% of the asymmetric information content, at 24 h. after infection. (3) At 6 h., abundant and scarce classes were present differing by 7 fold i n concentration. The most abundant class was transcribed from 7.4% of the double stranded v i r a l DNA, and the scarce class represented approximately 20%. (4) At 24 h., the abundant class was present in a 6 fold greater concentration than the scarce class, representing 10% and 33% of the v i r a l DNA respectively. (5) The fraction of the DNA specifying the abundant RNA at 6 and 24 h. after infection did not change significantly (i.e. 7% and 10% respectively). However, the fraction coding for scarce RNA increased from 20% (6 h.) to 33% (24 h.). (6) The abundant class comprises 86 to 88% of the late and early v i r a l RNA sequences respectively and arises from 7 to 10% of the v i r a l DNA. (7) In contrast, the scarce RNA species comprise 10 to 12% of the total v i r a l transcripts, yet arise from 20 to 33% of the DNA early and late in infection respectively. 3. Analysis of v i r a l RNA present i n infected non-permissive (G-l) 3T3 c e l l s . RNA was extracted from infected G-l arrested 3T3 cells at 6 and 24 h. Fig- 37: Hybridization of MCMV DNA With Excess G-l RNA at 6 and 24 h. Post Infection G-l arrested 3T3 cells in 90 mm plates were infected at an input m.o.i. of 10 pfu/cell and RNA isolated at 6 h. (early) and 24 h. 125 (late) after infection. The RNA concentrations and input I-MCMV DNA were the same as stated in Fig. 35 legend. Computer aided, best f i t curves were evaluated for n = 1, 2, 3 etc. by Fletch and the hard-copy plots obtained by the curv-plot program. When n was greater than 1, the a n + ^ v a l u e s approached zero and therefore cancelled. A. upper panel: G-l early RNA, n = 1 B. bottom panel: G-l late RNA, n = 1 E — 1 E R R L Y R N R R 1 = 0. I S2H00 B 1 = — 0.0 I 37 I 2 FINRL. FUNCTIDN VHL.UEcH.Eia I S7S B00 I Z K Z l H O U R S 1 S 0 1200 ® © E — I LRTE RNR R I E0 .2H00S3 B I : - 0 . 0 H S 2 2 B FINRL. FUNCTIDN VRL.UEsB.BH 1 73H S E ! I 0 E J 1 5 0 H O U R S 137 Table IX: Summary of the Transcriptional Patterns in Permissive and Non-Permissive 3T3 Cells Source of unlabelled RNA: Fraction of the Relative v i r a l DNA transcribed RNA abundance a 2 E a i a 2 81 3; Ratio of abundant to scarce transcripts Permissive 3T3 cells (early) Permissive 3T3 cells (late) Non-permissive 3T3 cells (early) Non-permissive 3T3 cells (late) 0.0736 0.198 0.272 -0.1863 -0.0255 0.0982 0.331 0.429 -0.2890 -0.0481 0.1524 0 0.240 0 0.1524 -0.0197 0 0.240 -0.0452 0 7.30 6.01 a values correspond to the fraction of the double stranded genome transcribed. These values should be doubled to represent single strand equivalents. post infection and hybridized in solution. The results are presented in Fig. 37 and Table 9, and are summarized below. The kinetics of hybridization for RNA extracted at 6 h. and 24 h. indicated only a single abundance class (n = 1) at both times. Although the f i t for n = 2 resulted in a slightly lower function value, i t i s extremely unlikely that n does in fact equal two since n = 2 generates -19 a negative a value for 6 h. RNA and a very small a value (2 x 10 ) 2 2 for 24 h. RNA. Thus for n = 2 the results were nonsensical. It is concluded that, at 6 h. post infection in G-l cel l s , v i r a l RNA i s present as a single class, representing 15% of the v i r a l DNA (Fig. 37A). Analysis of the G—l, 24 h. RNA indicated a single class transcribed from 24% of the v i r a l DNA (Fig. 37B). 4. Summation hybridization analyses The observation that exponential early RNA represented 27% of the v i r a l DNA and exponential late RNA, 43%, naturally raised the question whether the early RNA was transcribed from the same or a different DNA sequence as late RNA. To answer this query, a summation experiment was performed. The concentration of each RNA preparation was adjusted to ensure that the summated R t (R t ) was the same as the R t i n the two o o sum o control hybridizations, each containing exponential early and exponential late RNA (Fig. 38). The results demonstrate that the a value' for early RNA was 27%, a for late RNA, 43%, and the summated a ( a__J equaled 31%. In theory, i f the two RNAs were actually transcribed from different regions of the genome, the predicted value for a summ would be greater than 43%. Intermediate values for d (between 27 and 43%) indicate that a l l summ 138a Fig. 38: Summation Analysis of Permissive Early and Permissive Late RNA. Purified RNAs from permissive 6 h. (early) and permissive 24 h. (late) 3T3 cells were mixed in equal proportions (0.00125 moles nucleotide/1 each) to yield a total RNA concentration of 2.5 x 10 moles nucleotide/1. The kinetics of hybridization and data reduction were carried out as described before. The a value summ (upper panel) was approximately 0.31. The best f i t was obtained _3 for n = 2 (function value (f) = 1.68 x 10 ). The R t values in o the upper panel correspond to the R t or summated R t. The o summ o lower panel illustrates the control hybridizations for permissive early and late RNAs (n = 2 for each). . 3 • B \ V .7 . B EBB 1200 R 0 T - > * 9 SUMM. 5 : 0 1 izirzi 1 srizi H D U R 5 EHHUY LRTE S I Z I I IZIIZ 1 5 : 0 H O U R S 140 sequences expressed at 6 h. are also expressed at 24 h., or early i s a subset of late transcription. The analysis of the kinetics of hybridization in solution for the non-permissive, G-l system has exposed three salient features: (1) at 6 h. post infection, the RNA present in G-l cells i s complementary to 15-16% of the v i r a l DNA; (2) at 24 h. the extent of transcription increased to 24-25%; and (3) abundant and scarce RNA classes were not observed (i.e. n = 1) at 6 h. and 24 h. Therefore, i t i s clear that transcription in G-l cells i s essentially equimolar, and the extent of transcription in G-l cells never exceeded the a value observed in the early exponential analysis. These data may logically be explained i f one assumes that only early sequences are expressed as transcripts in the G-l c e l l . Therefore, this hypothesis predicts that any DNA sequences transcribed in the G-l c e l l would be the same as or a subset to sequences expressed in the permissive system. To test this, a second summation analysis was carried out between exponential early and G-l early RNAs (Fig. 39). The a r J summ value was in between a (27%) and a„., (14%) which suggests that expo-e Gl-e 0 0 sequences transcribed in the G—l c e l l are a l l the same as in the exponential c e l l . Similar conclusions may be drawn from a third summation analysis of G-l late and exponential early (Fig. 40); viz: that the DNA ..sequences serving as template for G-l late RNA are the same as, or a subset of sequences transcribed in the exponential early system. Fig. 39: Summation Analysis of Permissive Early and Non-Permissive Early RNA The purified RNAs from permissive cells and non-permissive cells isolated at 6 h. post infection were mixed in equal proportions and -3 hybridized in solution. The f i n a l RNA concentration was 2.5 x 10 moles nucleotide/1. The a value (upper panel) was approximately summ 0.16. In this case n = 1 resulted in a better f i t than n = 2 or 3. The upper panel R Qt values correspond to 0.0025 moles nucleotide/1 (R t ). The lower panel shows the kinetics of hybridization in o summ the controls. 'Expo E' corresponds to early RNA from infected exponentially growing c e l l s . 'G-l E' corresponds to early RNA from infected G-l arrested c e l l s . 141 \ . B ."7 BB0 1200 R 0 T - » JL 5 U M M . .3 . B IS \ .-7 1 • B ETIZI i 0 0 i 5T0 H O U R S E - 1 E . E X P O E . S 0 I 0 0 H O U R S I S 0 141a Fig. 40: - Summation Analysis of Permissive Early and Non-Permissive Late RNA The two RNA species were mixed in equal proportions as before (Fig. 38 and 39). The a value was approximately 0.20. Two classes summ of RNA (n = 2) were detected in this analysis. The R t values in -3 the upper panel correspond to an RNA concentration of 2.5 x 10 moles nucleotide/1 for the combined G-l late and expo early RNA species. SIZJ i 0 0 i s i a H O U R S S T 0 1 0 0 i S 0 H O U R S 142a CHAPTER 4 DISCUSSION The primary objective of this research project was to expand on current knowledge concerning the reciprocal influence of the c e l l cycle on v i r a l replication. Toward this end, a number of interesting observa-tions were made, several of which raised more questions than they answered However, many of these observations were of peripheral interest and w i l l not be discussed at length. This discussion i s oriented around plausible data in an attempt to formulate a model which may explain the mechanism of c e l l cycle dependent replication of a herpes virus. A. A Summary of the Data The data related to the proposed models are summarized in Table I. The three c e l l systems are contrasted and each point w i l l be discussed. 1. Viral growth An early observation concerning v i r a l growth in asynchronous cells indicated that optimal yields of virus occurred only under conditions of exponential cellular growth, when the monolayers were subconfluent. The dependence of v i r a l growth yields on the physiological status of the host c e l l suggested that MCMV had a requirement' for cells in some phase of the c e l l cycle other than G—.1. The latent period of v i r a l growth in exponential 3T3 cells was approximately 12 h., in agreement with other reports (Henson et a l , 1966; Mosmann and Hudson, 1974). The latent period of v i r a l growth was protracted in synchronized 3T3 cells infected in early G-l unt i l 20-24 h., or mid- to late S-phase. Although the pro-Table I: Comparison of the Lytic, Synchronized and Latent 3T3 Cell-Virus Systems, a L y t i c Synchronized ^Latent (Non-(Permissive) Permissive) I. V i r a l Growth A. latent period (h.) II. V i r a l DNA Synthesis A. Onset (in vivo) (h.) B. In vitro + 12 + 10 + + 20-24 + 16-18 + III.rXTP pools A. Purines B. Pyrimidines CND ND 20-30% of controls 60-70% of controls ND ND IV. Vi r a l Induced Enzymes A. TK B. DNA polymerase MCMV Specific Transcription ^Number of RNA classes (n) A. B. 1. 2. early late + + 2 27% 43% + + ND ND ND + + 1 15% 24% a b c d e Exponentially growing 3T3 cells G-l arrested 3T3 cells ND = not done n was the same for both early and late RNA a corresponds to the total fraction of the double strand v i r a l DNA (abundant and scarce) which is transcribed. tracted latent period was independent of the m.o.i., i t was necessary to eliminate t r i v i a l explanations for this delay. Briefly, the following facts emerged: (1) radioactive virus uptake experiments indicated that the virus e f f i c i e n t l y entered G-l, S and exponential 3T3 ce l l s ; (2) infectious center assays from infected G-l and exponential cells demon-strated that greater than 90% of the cells were susceptible to MCMV in both cases; (3) intracellular degradation of the v i r a l genome was not significantly different in G—l and exponential 3T3 cells; (4) G—l mono-layers prior to serum-split contained predominantly viable c e l l s ; (5) the protracted latent period was not due to the trypsinization and re-plating procedure used to induce synchrony; (6) after infection of syn-chronized cells in early G-l and late G-l, progeny virus appeared at the same time in S-phase for the two separate infections, thus localizing the ' c r i t i c a l period' to events just prior to mid- or late S-phase; (7) in a l l cases, the a b i l i t y of MCMV to undergo the_'.lytic^infectipn-cycle was contingent upon entry of the host c e l l into S phase. It is conceivable that the protracted latent period of v i r a l growth and indeed, the phenomenon of c e l l cycle dependence are events mediated by v i r a l or host repressor factors, which are expressed during G-l and masked during S phase. This possibility cannot be excluded. The concept is analogous to the phage X repressor. The classical review by Jacob and Monod (1961) described the phenomenon of lysogeny as a model for gene control by repressors. It was proposed that the repressor of phage A blocked early functions necessary for DNA synthesis, rendering the l y t i c cycle reversibly arrested. A potentially l y t i c virus such as MCMV may code for a repressor which limits the expression of genes required for l y t i c infection. In vivo this would enable the virus to maintain primarily a latent state in resting differentiated c e l l s . It i s not clear i f such 146 a control i s operable in vitro or how the control works. When host c e l l cycle traverse was inhibited, v i r a l growth did not occur. This was demonstrated in G-l arrested 3T3 cells and TdR-blocked 3T3 c e l l s . In the latter, i t was not immediately obvious whether the block in v i r a l replication was due primarily to the failure of the host to enter S phase or to dTTP sensitive steps in v i r a l replication. 2. MCMV DNA synthesis V i r a l DNA synthesis began around 10 h. post infection in permissive (exponential) 3T3 cells and between 16 and 18 h. in synchronized cells infected in early G—l phase. The onset of v i r a l DNA synthesis was deduced 125 from rates of reassociation of infected c e l l DNA and I-MCMV DNA probe. The technique is very sensitive and the results are not affected by nucleotide pool changes after infection. In permissive 3T3 cells there was l i t t l e i f any delay between the i n i t i a t i o n of v i r a l DNA replication and release of progeny. However, i n synchronized 3T3 cells a considerable delay was observed, as much as 4 hours between the onset of DNA synthesis and release of the f i r s t progeny virus. In the non-permissive system, significant levels of v i r a l DNA syn-thesis did not occur, as determined by reassociation kinetics. Thus, in the absence of the ' c r i t i c a l period' in S phase v i r a l DNA synthesis cannot i n i t i a t e . The results of the reassociation kinetics with infected syn-chronized c e l l DNA, indicated that the i n i t i a t i o n of v i r a l DNA synthesis corresponds to the time when mock infected cells are In approximately early S phase. The results do not necessarily imply that host DNA synthesis i s required per se, particularly since host DNA synthesis i s probably inhibited 147 by MCMV (Moon et a l , 1976). A more likely explanation is that the host c e l l need only be 'primed' for host DNA synthesis, thereby allowing v i r a l DNA synthesis to i n i t i a t e . Once the c e l l cycle requirement for v i r a l DNA synthesis is met, the virus replicates without further regard for the host c e l l cycle. The reason for this belief is that MCMV inhibits mitosis (Tegtmeyer et a l , 1969), hence, the virus would not have access to a sub-sequent S phase 'priming' event, due to the c e l l cycle block imposed at M phase. In vitro DNA synthesis was investigated in nuclear monolayers of 3T3 cells. Nuclei, isolated from infected 3T3 cells , synthesized v i r a l DNA in vitro only when they had the capacity to do so in the intact c e l l . Although there are no direct data on this point, i t appears likely that 3 v i r a l DNA synthesis in permissive 3T3 nuclei represented H-dTMP incor-poration into pre-existing replication forks which had initiated in vivo. MCMV DNA synthesized in vitro hybridized exclusively to v i r a l DNA on f i l t e r s and did not differ, in buoyant density, from mature v i r a l DNA (1.718 g/cc, Mosmann and Hudson, 1973). In neutral sucrose gradients the sedimentation profile of total DNA synthesized in vitro was f a i r l y heterogeneous, with most of the v i r a l specific DNA banding at 24-32s (ca. 12-23 million daltons). There are three possible explanations for the size difference between mature v i r a l DNA (61.5s) and DNA synthesized in vitro (26-28s): (1) smaller molecules in vitro are a result of fragmentation of higher molecular weight DNA; (2) DNA molecules made in vitro represent replicative intermediates; (3) DNA molecules larger than 28s are just not made in vit r o . Smaller pieces of nascent MCMV DNA (14-19s, approximately 3 to 6 million daltons) were detected by sedimentation velocity in alkaline sucrose gradients. It is suggested that the in vitro synthesized DNA may be associated a l k a l i 148 s e n s i t i v e material. Similar observations have been reported for HSV DNA synthesized i n v i t r o (Biswal and Murray 1974; Shlomai et a l , 1977). In a recent report on i n v i t r o DNA r e p l i c a t i o n with the c e l l cycle dependent v i r u s SV-40 (Le Blanc and Singer, 1976), i t was observed that the addition of unlabelled dTTP to the reaction mixture greatly f a c i l i t a t e d accumulation of f u l l - l e n g t h SV-40 DNA molecules. However the addition of dTTP to the reaction mixture with MCMV had l i t t l e , i f any, influence upon s i z e or buoyant density of the product. The only r e a l difference was that the percent of sing l e strand DNA was s l i g h t l y greater i n the absence of dTTP for 'supernatant associated' DNA only, i . e . H l a b e l l e d DNA which was released from NP-40 treated n u c l e i during the 60 minute incubation period. Nuclear associated DNA was predominantly double-stranded i n the presence and absence of dTTP. I t seems l i k e l y that omission of dTTP did not introduce any a r t i f a c t s i n t o the analysis of i n v i t r o v i r a l DNA synthesis. V i r a l DNA r e p l i c a t i o n was n e g l i g i b l e i n v i t r o i n infec t e d G-l n u c l e i . This was indicated by the isopycnic analyses and DNA annealing on f i l t e r s . However, i n attempting to demonstrate the absence of a p a r t i c u l a r species by the l a t t e r technique, one must accept the 'negative' r e s u l t only with reservation. On the other hand, i t i s u n l i k e l y that s i g n i f i c a n t v i r a l DNA synthesis occurs i n v i t r o since DNA r e p l i c a t i o n i s c l e a r l y n e g l i g i b l e i n vivo as determined by DNA-DNA reass o c i a t i o n k i n e t i c a n a l y s i s . I f the 3 assumption that DNA synthesis i n v i t r o represents H-dTMP incorporation into p r e - e x i s t i n g r e p l i c a t i o n forks i s true, then DNA molecules i n non-permissive G-l n u c l e i would not r e p l i c a t e because they did not have the capacity to do so i n vivo. I n i t i a t i o n of v i r a l DNA synthesis i n v i t r o has never been reported i n any animal v i r u s - c e l l system. 149 3. Ribonucleoside triphosphate pools The nucleotide pool data are integrated only superficially into the proposed model of c e l l cycle dependent replication of MCMV for several reasons. Primarily, the details of v i r a l nucleotide metabolism represent an enigma, hence even cautious interpretation of pool studies is fraught with problems (e.g. i t is not clear i f the virus even requires nucleotide pools for replication). With these qualifications in mind, i t s t i l l seems worthwhile to mention two points: (1) the virus suppressed the rXTP pools by 30 to 70%, compared to mock infected cells; (2) sufficient rXTP pre-cursors were present in G-l cells to allow v i r a l specific RNA synthesis Thus assuming the appropriate enzyme(s) is present i n the G-l c e l l , inter-conversion of the rXTPs to yield the corresponding dXTPs i s feasible. In a non-permissive ( c e l l cycle blocked) system with HSV-2, i t was reported by Roller and Cohen (1976) that the failure of the virus to replicate was not related to a block in the pathways leading to dXTPs. Furthermore, they concluded that the block in v i r a l replication was operating at the level of v i r a l DNA synthesis. Such may be the case for MCMV in G-l (non-permissive) c e l l s . 4. V i r a l specific enzymes Recently, i t was reported that the HSV induced enzyme, TK, was not required for v i r a l replication in rapidly growing (exponential) c e l l s , but was indispensible for growth in G-l cells (Jamieson, et a l , 1974). A TK HSV mutant (HSV-B2006) was used to investigate the significance of this gene product for v i r a l replication outside S. A fundamental role for the v i r a l TK was indicated since synchronized cells infected in early 150 G-l did not allow HSV-B2006 growth until early S phase. Exponential cells allowed normal v i r a l growth patterns for the mutant. In addition, the report by Cohen et a l (1971) that wild-type HSV-1 replication i s c e l l cycle independent, was verified in serum-split synchronized c e l l s . Cell cycle independence may be linked to the a b i l i t y of HSV-1 to provide a source of dXTPs, at times when the host c e l l does not contain 'functional' levels of dXTPs (Cohen et a l , 1974). It was then demonstrated, with a TK~ 3T3 c e l l line, that: (1) MCMV 3 did not stimulate uptake and incorporation of H-TdR during S phase; (2) replication of MCMV was totally resistant to high levels of BUdR; and (3) MCMV replicated normally in TK- 3T3 ce l l s . It was also shown that MCMV did not induce any detectable increase in TK levels after infection. In fact, TK activity was inhibited compared to mock infected c e l l s . These data are consistent with the notion that, in contrast to HSV, MCMV does not code for TK and this enzyme is not required for v i r a l replication. The failure of MCMV to replicate i t s DNA, in non-permissive (G-l arrested) 3T3 cells and synchronized cells during G—l phase, raised the question whether a v i r a l specific DNA polymerase was present in these c e l l s . Since most (or a l l ) herpesviruses seem to code for their own DNA polymerase activity (Weissbach et a l , 1973; Boezi et a l , 1974; Huang, 1975; Hirai and Watanabe, 1976; Halliburton and Andrew, 1976; Miller et a l , 1977; Allen et a l , 1977), i t was not surprising to find that MCMV also induced a novel v i r a l DNA polymerase activity. The observation that the novel DNA polymerase was induced i n permissive and non-permissive cells was surprising. The presence of a MCMV coded DNA polymerase was deduced from the following evidence: (1) infected G-l and S phase nuclei synthesized more DNA in vitro than their mock infected counterparts; (2) infection of G-l or S phase cells resulted in a DNA polymerase activity distinguishable from the host enzyme by i t s high salt requirement; (3) antiserum prepared against total infected ME c e l l protein inhibited i n vitro DNA synthesis in MCMV infected nuclei; (4) DNA synthesis in nuclei taken from uninfected cells was not affected by the anti-ICP serum. Therefore, the v i r a l DNA polymerase was present in permissive and non-permissive systems; however, in the latter, v i r a l DNA was not replicated 3 and H-dTMP was incorporated into cellular DNA only. The block in v i r a l DNA synthesis in G—1 cells i s not due to the failure of MCMV to induce the v i r a l polymerase in these c e l l s . It must be concluded that v i r a l DNA synthesis cannot i n i t i a t e in G—l cells despite the presence of the v i r a l DNA polymerase. A cofactor (or co-factors), either host or v i r a l , may be required for i n i t i a t i o n of v i r a l DNA synthesis to take place. If in fact, a host co-factor is involved, then, by necessity, such a function would be absent in G-l and present in S phase. However, the block in the i n i t i a t i o n of v i r a l DNA synthesis in non-permissive cells may be a secondary effect. The primary cause may be occurring at the level of transcription or translation. Thus, the in i t i a t i o n co-factor may actually be a v i r a l gene product, the expression of which is controlled by labile host factors limited to S phase. The 'early' regions of the v i r a l DNA (ca. 3 to 5%, see below) which are not transcribed in G-l cells may code for the v i r a l co-factor(s) required for i n i t i a t i o n of DNA replication. 5. Transcription analyses In general, solution hybridization with excess RNA i s valid only i f the v i r a l transcripts arise by asymmetric transcription, i.e. from the same DNA strand. DNA which i s transcribed symmetrically would yield RNA capable of annealing with other complementary RNA strands. RNA-RNA hybrid formation could effectively compete with DNA-RNA hybridization and introduce serious error into the numerical analysis. This possibility i s , in fact, remote since double strand RNA has not been detected in infected permissive and non-permissive 3T3 cells (V. Misra, personal communication). It should also be mentioned that hybridization measures stable RNA species only. There are no data concerning the longevity of MCMV trans-cripts. This consideration i s crucial since restricted transcription in non-permissive cells may be due to rapid degradation of early or late transcripts. It has been assumed that selective loss of v i r a l RNA does not occur in the RNA purification procedure, although this has not been tested. The f i r s t major biosynthetic event in both permissive and non-per-missive cells i s probably the synthesis of v i r a l specific RNA. In the permissive c e l l at 6 h. post infection (early), 27% of the v i r a l DNA was transcribed into abundant and scarce RNA. The abundant class represented 88% of the total v i r a l transcripts and was synthesized from 7% of the DNA. The scarce class, which comprised 12% of the transcripts, was synthesized from 20% of the v i r a l genome. At late times (24 h. post infection) the fraction of the genome transcribed increased to 43%. Abundant and scarce transcripts were detected, comprising 86 and 14% of the v i r a l RNA respectively. The fraction of the v i r a l genome specifying abundant RNA was 10% (approximately the same as early), while the fraction coding for scarce RNA was 33%. In the non-permissive system at 6 h. (early) only a single class of RNA was resolved, representing 15% of the v i r a l DNA. At 24 h. post infection, the fraction of the genome which was transcribed increased to 24% and only one class of RNA was detected. 153 Comparison of the non-permissive early and late a values revealed that slightly more (10%) of the v i r a l DNA was transcribed at 24 h. (late RNA). Several poss i b i l i t i e s may explain these data: (1) early trans-cription in the non-permissive c e l l proceeds more slowly than in the permissive c e l l . For example, there may be a period of limited early transcription i n permissive c e l l s prior to 6 h., but i n non-permissive cells this period of limited transcription i s extended to 6 h. or more. (2) there is no difference between G—l early and late a values, except that at 6 h., transcripts from 10% of the DNA are present in a very low molar concentration and not detectable. (3) there i s a real difference between the two a values. (4) the transcripts from 10% of the early DNA are degraded soon after synthesis. To sum up, i t is clear that the MCMV genome in G-l cel l s , although non-replicating, i s transcriptionally active. The hypothesis that MCMV dependence upon S phase i s mediated by a transcriptase seems unlikely. The possibility that a host RNA polymerase is required for i n i t i a t i o n of v i r a l DNA synthesis s t i l l exists. However, this seems equally unlikely since rates of RNA synthesis (and therefore RNA polymerase activity) do not vary significantly in resting and serum activated 3T3 cells (Rudland, 1974). Hence, RNA polymerase is not an S phase limited host event. The transcriptional analysis of MCMV in permissive and non-permissive cells i s significant from two points of view. The f i r s t point concerns the two kinds of transcriptional control. One is an on-off temporal control, based on the increase in the a values between early (27%) and late (43%). The other is an abundant-scarce control (Frenkel and Roizman, 1972) which is operable in the permissive system only. It is at present perplexing as to how the latter control works. What is clear however is 154 that the non-permissive host c e l l has a profound affect on both control mechanisms. The second point concerns the use of the non-permissive c e l l system as a potential tool to investigate the l y t i c cycle of virus replication. In the G—1 c e l l , only parental DNA molecules are transcribed. Thus, one may follow the fate of input DNA in terms of which genes are expressed as well as assigning which region of the genome i s responsible for the early functions. The re'sults of the summation experiments indicate that only, the early region of the v i r a l DNA are transcribed in non-permissive c e l l s , and trans-cription i s equimolar (n = 1). More specifically, there are 4 alternatives which describe the relationship between permissive early and non-permissive early transcription (note - abundant and scarce regions refer to those sequences in the DNA which specify abundant or scarce RNA in permissive (early) c e l l s ) : (1) a l l of the abundant and scarce regions are transcribed in an equimolar fashion; (2) part of the abundant and a l l of the scarce regions are expressed or vice versa; (3) only scarce regions are expressed; or (4) only abundant regions are expressed. The f i r s t alternative is very improbable since the a value for non-permissive cells i s less than 27% (permissive early a i and a^) . The 4th alternative is unlikely since abundant RNA is made from only 7% of the genome, whereas 15 and 24% of the DNA i s transcribed in non-permissive cells early and late respectively. Therefore, the number of poss i b i l i t i e s i s reduced to two. The numerical computer aided analysis of the summation experiments has provided evidence that in non-permissive early RNA only scarce regions are transcribed, and in non-permissive late RNA, both abundant and scarce regions are transcribed. This interpretation i s depicted i n Fig. 41. One should realize, however, that there are several sources of potential error which 154a Fig. 41: A Hypothetical Model of MCMV Transcription in Permissive and Non-Permissive 3T3 Cells Three summation hybridization experiments are depicted. Permissive early RNA was summated with either permissive late, non-permissive early or non-permissive late RNA. The double strand v i r a l genome i s represented as two solid horizontal lines. Transcribed regions are set-off by vertical lines and v i r a l transcripts appear as the broken lines. The arrows refer to permissive early (top) abundant sequences (c^ = 0.07) and scarce sequences (a 2 = 0.20). For reasons of graphical clarity, the abundant and scarce sequences are shown as contiguous regions. There are no data which indicate this to be true. Determined from the host f i t parameters of Bi and 82 f° r n = 2. When n = 1, 81^82 is unity. k" The value of $ 1 : 8 2 i f a l l sequences in permissive early RNA are present in the other RNA in the summation. I. Permissive early (n=2), 61:62 = 7 ou CX2 .07 0.20 I. Permissive early + II. Permissive late (n=2), 61:62 = 6 'Summation 0.10 | 0.33 | I. Permissive early, + III. Non-permissive early (n=l) | | _/ Summation I 0.15 1 ' ^ ^ ^ ^ i Permissive early. + IV. Non-permissive late (n=l) 'Summation J I 0-7A I Observed Predicted Fraction of scarce 61:62 in 61:62 in DNA sequences not summation summation transcribed 5% 17.6 I-1 Ln 156 may render the numerical analysis inaccurate. First, for any number of reasons the number of terms (n) in equation (8) may be incorrect with summation data. For example, i f the molar r a t i c o f abundant to scarce RNA is less than a predicted value of 2 to 3, the computer tends to 'oversimplify' by underestimating n. Consider the case when n = 4, but 6 2 , 33 and 31+ do not differ from each other by more than 3 fold, and 3 i differs by 10 fold. Thus, the program would generate an n = 2 model by combining 32 33 3if into a single class and 3 i a second class. Secondly, the results of the summation experiments may be explained by the hypothesis that template regions for early and late RNA are on opposite strands, thus resulting in symmetrical transcripts. In this case both a and n would be spurious. Despite this note of pessimism, conservative evaluation of the summation data is feasible. The interpretation of the summation data rests upon accurate determination of 8 and n. The summation analysis between permissive early and permissive late RNA yielded an n = 2 situation and an observed 8 1 : 8 2 of 7 (column A Fig. 41). The 8 1 : 8 2 ratio did not change significantly, compared to the separate control hybridizations of permissive early and late. This result suggests that the abundant RNA at 6 h. is a subset of abundant RNA at 24 h. and similarly, 6 h. scarce is a subset of 24 h. scarce RNA. The predicted value for 8 1 : 8 2 (column B, Fig. 41) corresponds to RNA, which is transcribed predominantly from the same DNA region which specifies early abundant and early scarce RNA. Thus, i t is predicted that i f a l l of the permissive early DNA regions were transcribed in toto to yield an n = 1 class (i.e. equimolar transcription), i t follows that the 8 1 : 8 2 value would not deviate from 7 ( 8 1 : 8 2 for permissive early RNA) . 157 The summation of permissive early, and non-permissive early RNA resulted in n = 1 class (Fig. 39), which indicated the partial expression of the scarce DNA region only. A l l but 5% of the scarce DNA region was expressed at 6 h. in non-permissive cells (Fig. 41). Therefore, a class two model (permissive early) plus a class one model (non-p_ermissive early) yielded a class one model summation. It appears that the scarce transcripts i n permissive early RNA were enriched for by the addition of non-permissive early RNA, thereby driving the 8 1 : 8 2 ratio toward 1.0 or a single RNA class. On the other hand, i f a l l abundant regions and a portion of scarce regions were transcribed, the summated 8 1 : 8 2 value would increase to a value greater than 7, and two RNA classes would result. This situation was observed in the summation between non-per-missive late and permissive early RNA. The 8 1 : 8 2 increased 2 fold i n the summation. This suggests that in non-permissive c e l l s , at 24 h., transcription i s equimolar from a l l of the DNA region specifying abundant RNA, and a portion of the DNA sequences specifying scarce RNA. It i s pertinent to emphasize that the model in Fig. 41 is subject to the qualifications imposed above. Briefly, Fig. 41 may be summarized as follows: (1) In non-permissive c e l l s , at 6 h., the majority of the DNA region specifying scarce RNA is transcribed in an equimolar fashion. About 5% of the scarce region is not transcribed. (2) In non-permissive cells at 24 h., a l l abundant regions are transcribed, and a l l but 3% of scarce regions are transcribed. Trans-cription in non-permissive cells at 24 h. i s also equimolar. It is concluded that 3 to 5% of the scarce regions are never expressed i n G-l cells up to 24 h. post infection. (3) In permissive cells at 24 h. after infection, abundant and 158 scarce t r a n s c r i p t s are present represening 10 and 33% of the v i r a l DNA re s p e c t i v e l y . The summation data suggest that early abundant and early scarce RNAs are transcribed from predominantly the same DNA sequences as l a t e abundant and scarce RNAs. B. The Proposed Model to Describe C e l l Cycle Dependent Re p l i c a t i o n of  MCMV. The model i s shown i n F i g . 42. The sequence begins i n the upper left-hand corner with the entry and uncoating of the v i r a l genome, eith e r i n the nucleus or cytoplasm. The parental DNA molecules enter the nucleus and serve as templates f o r early t r a n s c r i p t i o n . There are no data concerning the nature (host or v i r a l ) of the RNA polymerase responsible f o r early t r a n s c r i p t i o n of v i r a l DNA. Before discussing the d e t a i l s of early t r a n s c r i p t i o n , i t i s necessary to dwell on the nature of v i r a l gene regulation at the t r a n s c r i p t i o n a l l e v e l . The hypothesis i s concerned with the temporal ('on-off') co n t r o l of v i r a l t r a n s c r i p t i o n . The abundant-scarce co n t r o l mechanism i s beyond the realm of speculation at present. The hypothesis f o r temporal c o n t r o l , although highly speculative and without the support of data, i s consistent with the t r a n s c r i p t i o n analyses. I t i s proposed that parental DNA i s a f f i l i a t e d with a s t r u c t u r a l p r o t e i n which binds DNA at a s i t e (or s i t e s ) analogous to an operator sequence, such that template r e s t r i c t i o n i s imposed and only early sequences are transcribed. HSV and PRV are two other herpesviruses which display temporal control of t r a n s c r i p t i o n (Roizman and Furlong, 1974; Ben-Porat and Kaplan, 1973), and both contain i n t e r n a l basic proteins i n a s s o c i a t i o n with t h e i r DNAs. These v i r a l s t r u c t u r a l proteins have 158a Fig. 42: The Proposed Model to Describe Cell Cycle Dependent Replication of MCMV The sequence of events begins in the upper l e f t corner with entry and uncoating of parental v i r a l DNA. Transcriptional events are shown in the blocked-off areas. v-RNA ' = MCMV transcripts v-DNA = MCMV DNA = hypothetical v i r a l coded DNA binding protein = double strand MCMV DNA molecule = DNA polymerase (MCMV specific) - v i r a l or host derived i n i t i a t o r factor or factors v , * ^ = MCMV transcripts CYTOPLASM NUCLEUS Early Translation DNA Polymerase S Phase Cell o V Viral or Host initiator Factor(s) or Nucleotide Precursors xn vitro H-dTMP incorporation into V-DNA Entry and uncoating of parental DNA Early Transcription — a i a.z PERMISSIVE SYSTEM (n=2) 0.07 a2= 0.20 ^aia*" Bl/B?= 7 =0.27 NON-PERMISSIVE SYSTEM (n=l) ai= 0.15 V-RNA Elongation or repair of Cellular DNA in vitro in vitro No~-^ H-dTMP In-corporation into V-DNA in vivo Initiation (and Elongation) of V-DNA Replication NON-PERMISSIVE SYSTEM (n=l) cti= 0.24 24 h.p.i. in vivo 1. no DNA replication 2. no progeny virus 3. continued Early Transcription ai q 2 i l ^ ' i w ' 1  VS. 61/62= 6 PERMISSIVE SYSTEM (n=2) ai = a?= 0.33 24 h. p. i . 0 - 1 0 Saia2=0.43 Late Transcription A Production of naked progeny DNA (i.e. no af f i l i a t i o n with binding protein) 160 been implicated in the control or inhibition of host DNA and RNA synthesis (Stevens et a l , 1964; Roizman and Furlong, 1974). However, under con-ditions of arginine deprivation, synthesis of these proteins was prefer-entially inhibited, yet inhibition of host DNA synthesis s t i l l occurred (Chantler and Stevely, 1976). Thus, rather than regulation of host macro-molecular synthesis, these basic proteins may interact with v i r a l DNA to regulate transcription. Early transcription then begins and is characterized by 27% of the DNA being transcribed in permissive cel l s , and 15% in non-permissive c e l l s . The other details of transcription have been described (see Fig. 41). In both permissive and non-permissive cell s , at least one v i r a l transcript is translated to form DNA polymerase. The v i r a l induced DNA polymerase then enters the nucleus, and under appropriate conditions (i.e. during early S phase) v i r a l DNA synthesis begins. In the S phase c e l l , the model predicts that the polymerase interacts with a host (or viral) i n i t i a t o r co-factor (or co-factors) as a prerequisite to v i r a l DNA replication. The precise nature of the co-factor is unknown; however, i f i t i s of host origin, any S phase specific function would be a candidate. Some obvious po s s i b i l i t i e s are the following: (1) host DNA polymerase, polymerase subunit, or related function (Weissbach, 1974); (2) DNA swivel enzyme or related enzymes (Bina-Stein and Singer, 1977); (3) deoxyribo-nucleotides; or (4) enzymes involved in the production of deoxyribonucleo-tides, but not TK. If the i n i t i a t i o n co-factor i s of v i r a l origin, then one must envision a labile S phase host factor which controls the trans-cription or translation of the i n i t i a t i o n co-factor. The v i r a l gene sequence which codes for the i n i t i a t i o n co-factor may, for example, be contained within the 3 to 5% region of the.early DNA sequences which are not transcribed in G-l cells (see Fig. 41). 161 Once the Initiation requirement is met, v i r a l DNA synthesis proceeds. 3 When nuclear monolayers are prepared at this point, H-dTMP i s incorporated into v i r a l DNA. The model then predicts that in vivo, as v i r a l DNA syn-thesis proceeds in a semi-conservative manner, the binding protein is eventually diluted out to the point where the majority of progeny DNA molecules are devoid of any basic protein. These may then act as templates for late transcription, the details of which have been discussed in Section A (and Fig. 41). At least one late v i r a l gene would code for new binding proteins, which may also aid in the packaging process of v i r a l DNA. 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