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Radioactive pulse chase studies concerning the synthesis of viral proteins : and the membrane assembly… Richardson, Christopher Donald 1976

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RADIOACTIVE PULSE CHASE STUDIES CONCERNING THE SYNTHESIS OF VIRAL PROTEINS AND THE  MEMBRANE ASSEMBLY OF SEMLIKI FOREST VIRUS by Christopher Donald Richardson B. Sc. University of Bri t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Biochemistry We accept this thesis as conforming to the required standard for the degree of MASTER OF SCIENCE The University of British Columbia December 1975 (°&\ Christopher Donald Richardson, 1976 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co l umb i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date £) P /Ll J— /Gt /f/6 i ABSTRACT The mechanism of membrane assembly for Semliki Forest Virus, a Group A Togavirus, was investigated through a series of radioactive pulse chase experiments. I n i t i a l l y a time course for the appearance of virus specified proteins in the microsomal fraction of infected BHK (baby hamster kidney) cells and mature virions was performed. Infected cells were harvested and fractionated at 0, 1, 2, 4> 6, 3 and 11 hours post-infection. Plaque assays were performed on the virus released into the growth medium at these times. It was found that virus production was maximal between 4 and 6 hours. Nucleocapsid was clearly evident at 6, $ and 11 hours when the microsomal proteins were separated by SDS polyacrylarnide gel electrophoresis. In the next set of experiments infected cells were pulsed 3 for 20 minutes at 5 hours post-infection with H-Leu. Microsomes were prepared from the cells at 0 min., 20 min., 40 min. and 60 min. after removal of H-Leu and subjected to SDS polyacrylarnide gel electrophoresis. Virus was also isolated from the c e l l medium by sucrose gradient centrifugat-ion. Nucleocapsid protein radioactivity was at levels much greater than the combined peaks of radioactivity due to the membrane proteins E-^  and Eg. L i t t l e i f any radioactive virus was released into the media during this time of chase. A similar experiment to the one just outlined was performed except that the radioactive chase was extended over the range of 0 hrs., 0.75 hrs., 1.50 hrs., 2.25 hrs. and 3.00 hrs. i i Levels of H- labelled nucleocapsid were again i n i t i a l l y higher' than those of the combined E-^  and Eg radioactive peak. The radioactivity of E-^  Eg plateaus between 0.75 hrs. to 3.00 hrs. while that in the nucleocapsid continued to increase. This data appears to support the contention that nucleocapsid is synthesized prior to the v i r a l membrane proteins. 3 In hope of chasing the H-Leu label into and then out of the microsomes, infected BHK cells were pulsed at 3 hours and chased for 0, 1, 2, 3, 4> 5, and 6 hours after removal of 3 labelled medium. Levels of H-Leu increased in both the nucleocapsid and E-^  Eg protein bands of the SDS acrylamide gels u n t i l about 2 hours and then declined over the following time 3 range. Loss of H-Leu in the microsomes appeared to correlate with the increase of label incorporated into the virus. Finally, after devising a method for separating plasma membrane (PM) ghosts and endoplasmic reticulum (ER) fragments, another pulse chase experiment was performed. Infected BHK cells were again radioactively pulsed at 3 hours infection and the level chased for 0, 1, 2, 4» 6, £ and 11 hours follow-3 ing removal of the H-Leu. At the various time points labelled cells were harvested and fractionated into PM and ER. The samples of ER and PM were applied to SDS acrylamide gels and the radioactivity incorporated into the virus protein band was quantitated. Virus released into the medium was purified 3 by sucrose gradient sedimentation, assayed for H-Leu, and also fractionated by SDS electrophoresis. Label.was i n i t i a l l y very high in the ER in the form of precursor proteins ( N V P I 6 5 , i i i NVP97, PE2^' e n v e l o P e Proteins (E^, Eg), and nucleocapsid protein. This radioactivity was chased from the ER to the PM and then into mature virus. These results appear to indicate that Semliki Forest Virus nucleocapsid does indeed "bud" from the host c e l l membrane, thus obtaining i t s envelope. iv ACKNOWLEDGEMENTS The author wishes to express his sincere thanks and appreciation to Dr. D. E. Vance for his continual advice and encouragement. It i s also a pleasure to thank Miss Juliana Lam and Miss Karen Catherwood for their technical assistance. Special appreciation goes to Mrs. N. L. Richardson for her painstaking work in typing this thesis. V TABLE OF CONTENTS Page INTRODUCTION 1 Lipid Viruses 3 Structure of Group A Togaviruses 15 Growth of Group A Togaviruses 20 EM Observations of Togavirus Assembly 21 Replication of Viral RNA 23 Translation of Virus Specific RNA 23 Post-Translational Cleavage of Large Precursor Peptides , 31 Evidence for Maturation of Enveloped Viruses Through Budding From the Plasma Membrane 37 The Present Investigation 40 MATERIALS AND METHODS 42 Chemicals and Isotopes 42 SDS Acrylamide Electrophoresis 43 Gel Slicing and Sc i n t i l l a t i o n Counting of Gel Samples 43 SFV-Infected Microsome Time Course 44 Microsomal Pulse Chase Experiments 45 Isolation of Virus 4$ Separation of PM From ER. 49 0 - 1 1 Hour PM/ER Pulse Chase Experiment 52 VI Page EXPERIMENTAL RESULTS 54 Virus Gels and Protein Standards 54 Microsomal Time Course and Virus Production... 55 0'- 60• Microsomal Pulse Chase Experiment 60 0 - 3 Hr. Microsomal Pulse Chase Experiment..• 69 0 - 6 Hr. Microsomal Pulse Chase Experiment..• 74 Isolation of BHK Cell PM and ER 82 0 - 1 1 Hr. PM/ER pulse Chase Experiment 87 DISCUSSION 98 BIBLIOGRAPHY 107 v i i TABLES Page 1. Groups of Lipid Containing Viruses.... 3 2. Family: Togaviridae 14 3. Moles CHO Residue Per Mole Protein 17 4. Lipid Class Composition of SFV Shown as Mole Ratio Relative to Phospholipids 17 5. 5% Fatty Acid Composition of SFV PM and ER of Infected BHK21 Cells IS 6. Fatty Acid Class of Sphingolipids 19 7. Number of Different Molecules in SFV 19 £. Standard Proteins and Gel Mobilities.... 54 9. Leu Composition of SFV ' 64 10. Enzyme Purification During ER/PM Purification 33 V 1 X 1 FIGURES Page 1. Pathway of Picornavirus Morphogenesis 1 2. Structure of Vaccinia Virus 3 3. Assembly of Vaccinia.. 4 4. Structure of Herpes Virus 5 5. Assembly of Herpes Viruses 5 6. Structure of PM2 Phage 6 7. Structure of Influenza Virus 7 8. Assembly of Influenza 8 9. Structure of Paramyxoviruses 9 10. Structure of Vesicular Stomatitis Virus 10 11. Structure of Leukoviruses (Mouse Leukemia Virus) 11 12. Budding of Leukovirus 12 13. Structure of Semliki Forest Virus 15 14. Time Course of Virus Production (37) in BHK 21 Cells 20 15. Replicative Model of Simons and Strauss 24 16. Replicative Model of Martin and Burke 25 17. Sum of 3 2 P Label in +ve Strand, -ve Strand, and Virus RNA 26 18. Pulse Chase of Virus Proteins in Cell and Extracellular Virus 31 19. Viral Polypeptides in CEF and BHK Cells. 33 20. The Effect of TPCK on Virus-Specified Protein Synthesis in BHK-21 Cells . 35 21. The Effect of TPCK on Virus-Specified Protein Synthesis in CEF Cells 35 Page 22. Post-Translational Cleavage in Formation of SFV Structural Proteins 36 23. Pulse Chase Experiment 46 24. Preparation of Microsomes 47 25. Isolation of Virus by Sucrose Gradient Centrifugation. '. 43 26. Discontinuous Gradient for Separation of PM and ER 50 27. Purified Virus and Protein Standards 55 23. Photograph of Infected Microsome Gels: 0 - 1 1 Hours Infection 56 29. Coomassie Blue Scans of Microsomal Time Course Gels 57 30. Virus Production Over 0 - 1 1 Hours Infection . 59 31. 0' - 60' Chase Microsomal Protein....... 6l 32. 0' - 601 Microsomal Pulse Chase Virus Gradients 66 33. Time Course of SFV Infected Microsomal Proteins (0' - 60' Chase) 63 34. 0 - 3 Hr. Chase Microsomal Protein 70 35. Virus Produced Over 0 - 3 Hours Chase as Sedimented with Unlabelled Carrier Virus 71 36. Time Course of SFV Infected Microsomal Proteins ( 0 - 3 Hour Chase) 73 37. 0 - 6 Hour Chase Microsomal Protein SDS gels. 76 33. 0 - 6 Hours Chase Virus Gradients 73 39. Radioactivity Profiles' of'SDS Gels for 0 - 6 Hours Chase Virus 30 3 40. Time Course of H-Leu Labelled Microsomal Proteins Over 0 - 6 Hours Chase 31 41. Photographs of ER/PM Gradients from (a). Mock Infected and (b) Infected Cells 32 X Page 42. a) Profile of PM/ER Fractionation of Non-infected BHK Cells 84 b) Profile of PM/ER Fractionation of SFV-Infected BHK Cells 85 43. Sample Gels of ER and PM from SFV-Infected (1) and Mock Infected (M) BHK Cells ' 86 44. Profiles of ER SDS Acrylamide Gels 89 45. Profiles of PM SDS Acrylamide Gels 91 46. 0 - 1 1 Hours Chase Virus Gradients 93 47. 0 - 1 1 Hours Chase Virus Gels 95 3 48. Time Course of H-Leu Labelled Virus Specified Proteins in PM and ER 97 49. Scheme for Regulations of Translation of Structural Proteins 99 50. Moles of Virus Proteins Formed in ER and PM of Infected Cells 100 51. Schematic of SFV Biogenesis 102 x i LIST OF ABBREVIATIONS BHK baby hamster kidney ds double stranded ss single stranded DNA deoxyribonucleic acid RNA ribonucleic acid HA hemagglutinin NA neuraminidase SFV Semliki Forest Virus E-, envelope protein (MW 49,000) of Semliki Forest Virus E0 envelope protein (MW 52,000) of Semliki Forest ^ Virus E 3 envelope protein (MW 10,000) of Semliki Forest Virus NC nucleocapsid protein of Semliki Forest Virus PEg (or NVP ) precursor protein to Eg EnEp combined envelope proteins and Eg which often do not resolve by SDS electrophoresis giving the impression of one protein. NVP 165 non-virion precursor protein (MW 165,000) NVP 97 non-virion precursor protein (MW 97,000) NVP 78 non-virion protein (MW 78,000) which may be replicase or RNA polymerase for SFV PE phosphatidyl ethanolamine PC phosphatidyl choline PS . phosphatidyl serine PI phosphatidyl inositol PM plasma membrane as defined by classical marker enzymes (5f nucleotidase, Na +K + ATPase, and alkaline phosphatase) x i i ER CEF RI RF CPV-1 CPV-2 VSV SDS DATD MBA PPO POPOP mCi uCi nm CPM DPM A550 cm. endoplasmic reticulum as defined by classical marker enzymes (NADPH cytochrome c reductase, glucose - 6 - phosphatase, and NADH diaphorase) chick embryo fibroblast cells replicative intermediate replicative form cytopathic vacuoles type 1 cytopathic vacuoles type 2 vesicular stomatitus virus sodium dodecyl sulfate diallyltartardiamide methylenebisacrylamide 2,6-diphenyl oxazole 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene millicurie microcurie nanometer counts per minute disintegrations per minute optical absorbance at 550 nanometers centimeters 1 INTRODUCTION A great deal of information has accumulated concerning the modes of assembly of viruses and bacterial phage. Much of these studies has been summarized recently in a number of reviews (1 - 4). Helical ssRNA viruses such as tobacco mosaic virus, barley stripe mosaic virus, potato X and Y viruses, and clover yellow mosaic virus have been shown to proceed through an equilibrium self-assembly process whereby 4S pentameric units associate step by step with the RNA i n i t i a t i o n complex. The icosahedral single stranded RNA viruses -spherical ssRNA plant viruses (chlorotic mottle virus, cucumber mosaic virus, turnip yellow mosaic virus, cowpea mosaic virus), ssRNA phages (R17, f r , MS2, QB), and animal picornaviruses (polio, coxsackie, ECHO, encephalomyocarditis, Mengo, rhino-viruses) - a l l form by the step by step complexing of capsomeres. The mode of assembly for polio virus shown in Fig. 1. The assembly involves post-translational cleavage of a precursor peptide NCP1 to shorter peptides - VP1, VP3, and VPO. 35S RNA o Cleavage 5S 100,000 MW © 14 S 500,000 MVi 73S 6,000,000HW I25S I50S 8,000,r.;0 MA _^_VP1 VP1 NCVP1_. VP3 VP3.. _ ~"^~-_VP0 VPO VP1 VP1 _VPI (60) VP3 _VP3 VP3(60) VPO VPO .VPO (2) Fig. 1 Pathway of Picornavirus morphogenesis (1) 2 The dsDNA viruses in the Adenovirus group and Papovaviradae (polyoma, SV40, papilloma virus) are also non-lipid containing icosahedral viruses. These viruses are formed by the associat-ion of capsomeres to yield an "empty" capsidinto which DNA is then inserted. The diplornaviridae (of which reovirus, wound tumor virus, rice dwarf virus, and cytoplasmic polyhedris virus are members) consist of a dsRNA genome enclosed in a double shell of protein. The virion enters the c e l l , is uncoated by lysozyme to yield the inner core RNA polymerase, the mRNA becomes associated with the proteins i t specifies to form a new core, and f i n a l l y matures by synthesis of a compli-mentary strand of RNA in the immature core. By far, assembly mechanisms are best understood for the dsDNA bacteriophage.such as the E. c o l i phage (T4,*, P2, T7, T3 and T5), Salmonella  typhimurium phage ?22> a n ^ Bacillus subtilus phage 29. Bacter-iophage heads and t a i l s generally assemble independently and then join to form complete virions. The head assembly proceeds through a prophage stage (lacking DNA) into which the genome is injected during maturation. The viruses afore mentioned contain no l i p i d . It goes without saying that assembly processes are best understood for these viruses. However, a great number of virions contain l i p i d . It i s to this class that Semliki Forest Virus belongs and i t s assembly w i l l be the topic of this thesis. 3 Lipid Containing Viruses The various classes of l i p i d viruses are presented in Table 1. TABLE 1 Groups of Lipid Containing Viruses Group Nucleic Acid Virion Shape Type , MW xl0° Size X Pox Virus ds DNA 160 Herpes Virus . ds DNA 100 PM2 Phage ds DNA 6 Togavirus ss RNA 4 Myxovirus ss RNA 4 Paramyxovirus ss RNA 7 Rhabdovirus ss RNA 6 RNA tumor virus ss RNA 12 Arenovirus ss RNA Coronavirus ss RNA 1 Leukovirus ss RNA i3 brick shaped spherical spherical spherical spherical/filamentous spherical/filamentous bullet shaped spherical spherical spherical cubic 3000x2000 1200 600 700 1000 1200 700x1750 1200 600-1200 800-1200 1000 The pox viruses are the largest of a l l vertebrate viruses. This group consists of over 25 viruses which include variola (small pox), vaccinia, cowpox, rabbit pox, etc. The structure of these viruses has been deduced almost exclusively through electronmicroscopy and is shown in Figure 2. Fig. 2. Structure of Vaccinia Virus (/=*•) 4 The pox viruses are assembled entirely in the cytoplasm of the host c e l l at a factory (independent of the PM and ER) in which new v i r a l DNA and protein are assembled as shown in Figure 3. The virus contains i t s own transcriptase and replicase. Fig. 3. Assembly of Vaccinia (10) Recently Stern and Dales (11) have implicated the role of phospholipid exchange proteins in the transfer of phospholipid from microsomes for the assembly of vaccinia envelopes. Herpes viruses are another group of large dsDNA viruses which are responsible for such diseases as chicken pox, cold sores, infectious mononucleosis, and possibly cervical cancer. The virus consists of a 3 layer core and 2 envelopes (Fig. 4). The virus appears to be assembled in the nucleus and obtains i t s envelope by budding through the nuclear envelope (this remains to be thoroughly substantiated). This completed virion is then believed to be transported to the plasma membrane by cytoplasmic membrane channels which prevent degradation of 5 the virus membranes. This process is shown in Figure 5. j Piter Envelope xx{]hner Envelope Outer Capsid_ /;.;:WJ&Cap.sid_ /. !JTnner Capsid \ I 5" n v n D S brJ Pi Fig. 4. Structure of Herpes Virus The Herpes viruses contain at least 23 proteins per virion. Of these proteins, 13 are associated with the nucleocapsid and about 10 are found in the envelope. Fig. 5. Assembly of Herpes Viruses (1) 6 PM2 phage consists of an outer protein shell encompassing a lipoprotein bilayer which surrounds the inner dsDNA genome. The virus i s icosahedral in shape (Fig. 8) and contains 1+ proteins (I, II, III and IV). The assembly of this virus is s t i l l under study but i t appears to mature independent of the PM and ER at cytoplasmic factories. Phospholipid exchange proteins have again been implicated in i t s synthesis. The myxovirus or influenza virus polypeptides consists of 7 proteins - P (probably polymerase), hemagglutinin (which dissociates to form HA-^  and HAg), neuraminidase, nucleoprotein, membrane or matrix protein, and nonstructural protein. The structure of influenza i s shown in Fig. 7. .PROTEIN 1 Fig. 6. Structure of PM2 Phage (13) 7 20 40 60 80 100 Froction No. Polypeptide Function MW "P HA polymerase hemagglut in in 90,000 80,000 HA]_ 55,000 HA2 25,000 NA neuraminidase 65,000 NP nucleoprotein 60,000 M membrane protein 25,000 NS nonstructural protein 25,000 Fig. 7 Structure of Influenza Virus (5) 8 NS may act to shut down host protein synthesis and accumulates in the nucleolus. The genome of influenza consists of 3 strands of ssRNA associated with NP. The virion enters the host c e l l , is uncoated, and v i r a l RNA is transcribed to complementary RNA mediated by v i r a l transcriptase (NP). The complementary RNA i s translated to give HA, NA, NP, M, NS, and v i r a l replicase. This complementary RNA then replicates to form new virion RNA. Envelope proteins are inserted into the PM of the host c e l l , the nucleocapsid and M protein align adjacent to these proteins, and the virus matures by budding through the PM. This process i s shown in Fig. 8. ENTRY TRANSCRIPTION TRANSLATION (POLYRIBOSOMES) Fig. 8 Assembly of Influenza (7) 9 The paramyxoviruses which include parainfluenza, mumps, Newcastle Disease, measles and canine distemper are structur-a l l y similar to myxoviruses and mature in an identical manner. These viruses are however somewhat larger and more pleomorphic than myxoviruses and contain only one strand of ssRNA. The structure of a paramyxovirus i s shown in Fig. 9. item Molecular Weight SV5 Sendai NDV NP 61,000 60,000 56,000 HA 67,000 65,000 74,000 NA 56,000 53,000 56,000 M 41,000 38,000 41,000 P 76,000 69,000 62,000 NS 50,000 58,000 53,000 46,000 45,000 Fig. 9. Structure of Paramyxoviruses (7) 10 These viruses also bud through the PM as the f i n a l stage of maturation. Rhabdoviruses include rabies virus, vesicular stomatitis virus, bovine ephermeral fever, and hemorrhagis septicemia. VSV virus contains 5 major proteins - a glycoprotein compris-ing the peplomers (G), nucleoprotein (N), a non glycosylated membrane protein (M), a high molecular weight protein (L) ' associated with nucleocapsids, and another nucleocapsid protein NS. Virion transcriptase is associated with the nucleoprotein core. Ttie structure of VSV i s shown in Fig. 10. This virus has an outer lipoprotein envelope and a nucleo-protein consisting of a single strand of RNA. Fig. 10. Structure of Vesicular Stomatitis Virus (14) 11 Upon entry into the c e l l , virus RNA i s transcribed to comple-mentary RNA by the nucleoprotein associated transcriptase into two forms of mRNA - 283 and 1 3 - 1 5S which break down to form monocistronic messengers. There i s no evidence for post-translational cleavage of precursor peptides. Replication from long strands of complementary RNA may serve to synthesize new virion RNA. Virus appears to mature by budding through the c e l l plasma membrane. The leukoviruses (RNA tumor viruses or oncornaviruses) are comprised of the leukosis-leukemia-sarcoma viruses, mammary tumor virus, progressive pneumonia-visna viruses, and foamy agent viruses. The purified virions of a l l leukoviruses contain 7 or 8 polypeptides. Two of these are glycoproteins (peplomers in v i r a l envelope) and are designated m-^  and nig. There are a number of group specific proteins associated with the nucleoid (gsl-gs4) together with a nonspecific protein p 5 . These proteins have not yet been assigned to the EM structure shown in Fig. 1 1 . S u b u n i t s I 1 0 0 nm Fig. 1 1 . Structure of Leukoviruses ( 1 4 ) (Mouse Leukemia Virus) 12 The genome of leukoviruses consists of 60-70S RNA (virus genome), 4S RNA (may act as primer for DNA synthesis from v i r a l RNA template), 5-7S RNA (function unknown), IB and 28S RNA (contaminants from host ribosomes), 7S DNA (homologous to cellular DNA, function unknown). Leukoviruses contain an RNA dependent - DNA polymerase which transcribes DNA from v i r a l RNA. mRNA and virion RNA i s then transcribed from this proviral DNA by cellular DNA-dependent RNA polymerase in the nucleus. Leukoviruses assemble at the plasma membrane and are released by budding. Electron microscopy shows 2 modes of assembly indicated in Fig. 12. C e l l - o s s o c i a t e d Ext race l lu lar o Enve loped A Subgenero B and D A par t ic le A part ic le par t ic le Mature 3 Mouse m a m m a r y ( intracytoplasmic) ( a c q u i r i n g ("immature 8 pa r t i c le t u m c v i rus ; e n v e l o p e ) D a r i i c l e " ) ( ? d e g e n e r a t i o n ) Foamy agents C port ic le No intracytoplasmic . during structure seen envelopment C par t ic le S u b g e n e r o A end C L e u k e ~ > • s c r c o m o v i rus ; V i s n c virur-Fig. 12. Budding of Leukovirus ( 1 4 ) Arenaviruses include such viruses as lyphocytic choriomeningitis virus, Lassa virus, and "Tacaribe complex". These virions are very pleomorphic and vary in diameter from 100 to 300 nm with a heavy unit membrane bearing closely spaced peplomers 6 nm long. They contain a number of electron-dense granules which may be ribosomes of host c e l l origin since 13 18 S and 28S ribosomal RNA's are found in extracts of purified viruses. The viruses contain about 4 polypeptides, 2 of which are glycopeptides. L i t t l e i s known concerning the assembly of this l i p i d virus. Virions of.a great variety of shapes and sizes bud from those areas in the plasma membrane where v i r a l peplomers have been incorporated. Coronaviruses comprise the group of viruses causing human colds, mouse hepatitis, infectious avian bronchitis, swine gastroenteritis and hemagglutinating encephalomyelitis of pigs. These viruses are pleomorphic enveloped particles 1 2 0 nm in diameter with large petal-shaped peplomers. The peplomers project 1'rom a thick shell that consists of an outer double membrane and an inner protein layer. The viruses contain 6 different polypeptides, 2 of which are glycoprotein peplomers. The genome consists of ssRNA but i t s size and structure have not been determined. Coronaviruses mature by budding into the cisternae of the ER and cytoplasmic vesicles, but not from the PM. Togaviruses, of which Semliki Forest Virus i s a member, are comprised of 2 serological groups - Group A (alphaviruses) and Group B (flaviviruses) - and representative types are shown in Table 2 . 14 TABLE 2 Family: Togaviridae Genus Representative Viruses Alphavirus Flavivirus Sindbis, Semliki Forest Virus, Western Equine Encephalitis, Eastern Equine Encephalitis, Venezuelan Equine Encephalitis, Chikuns.-unya, rubella (most likely) Dengue types 1-4, yellow fever, St. Louis encephalitis, Japanese encephalitis, West Nile encephalitis, Murray Valley encephalitis, Russian tick-borne encephalitis The remainder of this introductory review deals with the structure and assembly of togaviruses. 15 The Structure of Group A Togaviruses The structures of a l l the Group A Togaviruses are almost i f not entirely identical. These viruses, of which Semliki Forest Virus and Sindbis Virus are the most studied, consist of an icosahedral nucleocapsid surrounded by a spherical l i p i d envelope. Three glycoproteins denoted as E]_, E2 and E3 are situated in the envelope and l i e in close proximity to the virus nucleocapsid (15)- The structure of Semliki Forest Virus is shown diagrammatically in Fig. 13. The virus genome con-sists of a single strand of 42S RNA. 1 1+ I 1o Fig. 13. Structure of Semliki Forest Virus 16 The structural proteins of Semliki Forest Virus have been resolved using SDS polyacrylarnide gel electrophoresis. Originally studies were performed using the gel system of Weber and Osborne (16). E-^  and Eg were not resolved in the studies of Hay, Skehel and Burke (17), Kaariainen et al.(18), and Acheson and Tamm (21). Nucleocapsid protein was clearly evident while no trace of_E^ was found on the gel and i t s existence remained unknown. More recently the discontinuous buffer SDS gel electrophoresis systems of Neville ( 1 9 ) and Laemmli (20) were applied to purified virus preparations by Simons (22) and Pfferkorn (9). These results show the molecular weights of E-^ , Eg and nucleocapsid to be 52,000, 49,000 and 34,000 respectively. The existence of E^ was not evident u n t i l very recently (23). Garoff and Simons showed that although E^ cannot be detected on 7-5$ and 10$ SDS acrylamide gels by cla s s i c a l staining techniques, the small protein could be detected /. when -^S-Met labelled SFV was applied to 10$ SDS gels - the gels were then sliced'and assayed for radioactivity. More conclusive evidence for the existence of E^ was presented when delipidated membrane protein was eluted from an SDS hydroxy-lapatite column. E^, Eg, E^, and nucleocapsid proteins appear in equimolar amounts in the mature virion and constitute 35-7$, 35.7$, 4.9$, and 23.7$ of the total protein respectively. A l l three SFV membrane proteins are glycosylated. Residues of. N-acetylglucosamine, mannose, galactose, fucose, and s i a l i c acid appear in a l l three proteins. 17 TABLE 3 (23) Moles CHO Residue Per Mole Protein Protein Nacetyl-glucosamine Mannose Galactose Fucose Sia l i c Acid Total CHO # by weight E l 7 5 3 1 2 7.5fo E2 8 12 3 1 4 1 1 . 5 $ E 3 9 4 4 2 3 45.195 The l i p i d s of the v i r a l membrane consist of 32$ neutral li p i d s , 61$ phospholipids and 7$ glycolipids ( 2 4 ) . The neutral l i p i d fraction of SFV consists almost exclusively of free cholesterol while the main components of the phospholipids are sphingomyelin, phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl serine. The glycolipid fraction contains almost exclusively s i a l i c - l a c t o s y l ceramides. The distribut-ion of the various l i p i d types i s shown as mole ratios in Table 4 ( 2 4 ) . TABLE 4 Lipid Class Composition of SFV shown Mole Ratio Relative to Phospholipids as (24) Lipid Class Mole Ratio Cholesterol Glycolipids Phospholipids PE PC PS PI Sphingomyelin 0.99 0.08 1.00 0.23 0.33 0.13 0.02 0.20 18 It i s believed that the l i p i d class composition resembles that of the host plasma membrane. Such a relationship is also reflected in the fatty acid composition of the phospholipids in the virus and PM of infected BHK 21 cells as shown in Table 5 ( 2 4 ) . TABLE 5 ( 2 4 ) 5$ Fatty Acid Composition of SFV PM and ER of Infected BHK21 cells Fatty Acid  PC Ps PI PE SFV PM ER SFV PM ER SFV PM ER SFV PM EK 16 :0 40 32 22 4 4 4 8 6 3 5 5 5 16 :1 7 7 •7 3 3 1 3 2 1 2 3 4 18 :0 7 8 6 32 28 27 42 3 9 3 7 5 8 12 18 :1 33 4 4 50 4 8 55 43 16 3 1 29 4 7 5 2 - 56 1 8 : 2 7 5 8 8 5 4 4 3 4 13 7 9 c 2 0 ~ c 2 4 2 2 2 2 4 9 27 17 25 16 8 6 19% or the phospholipids of SFV are alkyl or alkenyl ethers. The sphingolipid fatty acids are characterized by their high content of saturated and monoenoic acids and of C^o -acids, and by the absence of polyenoic acids. The fatty acid composition of the various sphingolipids in SFV, of. infected BHK cells is shown in Table 6. 19 TABLE 6 ( 2 4 ) Fatty Acid Class of Sphingolipids Gangliosides Sphingomyelins Fatty Acid SFV PM ER SFV PM ER 1 6 : 0 35 4 1 46 76 69 78 1 6 : 1 6 4 6 1 1 1 8 : 0 10 6 8 1 8 : 1 10 6 5 2 0 : 0 1 1 1 2 2 : 0 6 5 7 6 1 0 7 23 : 0 2 2 4 2 4 : 0 7 8 7 6 6 2 4 : 1 14 16 10 11 22 8 In summary the single Semliki Forest virus contains the number of d i f f e r e n t molecules l i s t e d i n Table 7 ( 2 4 ) . TABLE 7 ( 2 4 ) Number of Different Molecules i n SFV Constituent Number of Molecules Per V i r i o n RNA 1 Nucleocapsid 2 0 0 Membrane proteins 550 Cholesterol 1 5 , 0 0 0 1 6 , 0 0 0 Polar l i p i d s PE • 3 , 5 0 0 PC 6 , ' 4 0 0 PS 2 , 0 0 0 PI 200 Sphingomyelins 2 , 4 0 0 Gangliosides 1 , 0 0 0 20 Growth of Group A Togaviruses The growth of group A togaviruses i s rapid. After 2 hours infection, vertebrate cells are beginning to produce virus at 37° C. By 5 hours virus production is maximal and continues at a constant rate up to 1 0 - 1 2 hours infection. Virus product-ion may approach 1 0 0 0 PFU/cell/hr. and the total yield may approach 1 0 ^ ® PFU/ml. Highest t i t e r s are achieved in CEF and in BHK ce l l s . t)>d_, , r—, . , r , . • 0 1 2 3 4 5 6 7 8 9 TIME iHOURS) Fig. 1 4 . Time Course of Virus Production ( 3 7 ) in BHK 21 cells At 11 hours infection v i r a l cytopathic effects become apparent by light microscopy and the rate of virus production f a l l s markedly. Vertebrate cells are ultimately destroyed although persistent (chronic) infection occurs in the presence of interferon (35)» Group B togaviruses grow much slower in vert-'ebrate cells without cytopathic effect. Group A togaviruses naturally infect arthropod c e l l lines such as mosquito (Aedes albopictus) and tick and produce t i t e r s comparable to those of arthropod ce l l s . Infected cells show cytopathic effects such as cytolysis , syncytia and phagocytosis 21 but chronic persistent infections may also result. These persistent infections may also be induced by the presence of interferon. Electronmicroscopic Observations of Togavirus Assembly Electronmicroscopy discloses extensive changes in the cytoplasm of infected c e l l s . Virions appear to be produced by . budding of nucleocapsids through areas of c e l l membranes modified by the insertion of virus specified envelope proteins. This appears to occur at 2 places: 1) the plasma membrane (39, 40) 2) intracellular vacuoles (Type 2 cytopathic vacuoles) ( 4 1 , 42) Nucleocapsids are seen closely aligned to the cytoplasmic side of the membrane, and often in the process of budding through i t . Cytopathic vacuoles (CPV-2) which presumably contain mature virus have been thought to fuse with the host c e l l PM with the release of virus into the medium. Freeze etching and EM studies using f e r r i t i n - l a b e l l e d a n t i v i r a l antibody--(119, 120) show that envelope proteins are inserted: into localized regions of the plasma membrane. Grimley et a l . (42, 43) described another type of cytopathic vacuole (CPV-1) which occurs early in the exponential phase of SFV growth. :The vacuoles are 0.6-2 urn. in diameter and bear 50 nm.membranous nodules projecting from their interior surfaces. The nodules are neither virions nor nucleocapsids.. Formation of the nodules i s independent of cellular RNA synthesis since actinomycin D does not effect their appearance. Antimetablotites blocking v i r a l RNA or protein synthesis prevent 22 the formation of CPV-l's. Autoradiographic experiments performed in the presence of actinomycin D seem to indicate that the CPV-1 i s a site of v i r a l RNA synthesis. Cells infected with group B togaviruses show somewhat different cytopathic changes ( 4 4 , 4 5 , 4 6 ) . I n i t i a l l y there is a proliferation of cytoplasmic vacuoles, of smooth and rough endoplasmic reticulum, and Golgi membranes. However, there is l i t t l e concrete evidence as to the mechanism for the envelopment of group B viruses. Ota ( 4 4 ) suggested that v i r a l morphogenesis occurs by budding of these particles through cytoplasmic membranes. Lining the cytoplasmic side of many of the vacuoles and cisternae were ill-defined, electron-dense, round ragged structures 2 6 - 2 8 nm in diameter which could be nucleocapsids. No virions have ever been observed in the process of budding. Yasuzumi et a l . ( 4 7 , 4 ^ , 4 9 ) have suggested nuclear involvement in the morphogenesis of group B virions. Murphy et a l . ( 4 6 ) also indicated the presence of intranuclear virions and nucleocapsids. Membrane-enclosed virions may migrate from the perinuclear region of the c e l l and approach the plasma membrane to be released by exocytosis through narrow caniculi or through fusion of virus-containing vacuoles with the plasma membrane. Immunofluorescence studies show that group B antigens are present in the cytoplasm of infected cells ( 5 0 , 5 1 , 5 2 ) and contend that the absence of immunofluorescence at the plasma membrane of infected cells reflects that v i r a l maturation does not take place at the c e l l surface. 23 Replication of Vira l RNA  Togavirus RNA synthesis i s easily observed in infected cells with or without actinomycin D since the virus represses host c e l l specified RNA synthesis. Group A v i r a l RNA synthesis reaches levels detectable by about 2 hours after infection ( 5 3 ) which rises to maximal rates by 3 hours and continues through-out the period of virus release. The 2 principal forms of single-stranded RNA found in group A togavirus-infected cells are virion RNA ( 4 2 S ) and interjacent RNA ( 2 6 S ) (54, . 5 5 ) . These are the forms of virus mRNA. Minor ssRNA with sedi-mentation coefficients of 3$S and 33S also occur. Replication of the RNA of togaviruses proceeds through a multiple-stranded replicative intermediate similar to that described for picornaviruses and RNA bacteriophage. On extract-ion of infected cells with phenol and separation by CFll c e l l -ulose chromatography, 2 different types of double stranded RNA can be isolated: 1) replicative intermediates (RI) - consists of partially double stranded RNA with single stranded non-hydrogen bonded regions 2 ) replicative forms (RF) - extensively hydrogen bonded double stranded RNA. These double stranded RNA's were originally isolated by Pf eff erkorn, Burge and Coady ( 5 6 ) and Friedman ( 5 7 ) . Free single-stranded RNA of negative polarity (ie. complementary to virus genome) was not isolated from infe.cted c e l l s . Simmons and Strauss ( 5 8 ) , Segal and Sreevalsan ( 5 9 ) , and Martin and Burke ( 6 0 ) identified three forms of RF. It i s believed 24 that RF i s an intermediate in the formation of RI. Simmons and Strauss (5#) purport the existence of two RI's while Martin and Burke ( 6 0 ) contend that only one RI i s present. A schematic of these hypotheses i s shown in Fig. 15 and Fig. vw\ j 3 5 Fig. 1 5 . Replicative Model of Simons and Strauss ( 5 $ ) 25 Fi g . 1 6 . Replicative Model of Martin and Burke ( 6 0 ) Simmons and Strauss (58) and Segal and Sreevalsen (59) support the view that 1+2S and 26S RNA are synthesized from d i f f e r e n t RI's. Recently Bruton and Kennedy (61) have shown that the RFI consists of +ve 42S RNA (genome) and -ve 42S RNA which constitutes 80$ of the r e p l i c a t i v e forms. RFI was shown to contain non-hydrogen bounded poly A at the 3' end of the 42S po s i t i v e strand i d e n t i c a l i n length to that on the v i r a l genome. No poly U was located on RFI and neither was poly A located on 26 the minus strand just the p o s i t i v e strand. The k i n e t i c s of p o s i t i v e and negative strand synthesis were investigated dur-ing virus m u l t i p l i c a t i o n . Negative strand synthesis reaches a maximum 2-g- hours post i n f e c t i o n and thereafter r a p i d l y f a l l s , The rate of p o s i t i v e strand synthesis increases r a p i d l y up to 3 hour post - i n f e c t i o n and then remains constant f o r a further 3 to 4 hours. * c 1 S M """"/ > ^ / / 1 ' \ -I 1 1 / r I i 1 i 1 t l / O.HA I I \ t • + v C 5 + l-« n d -\ t P . N f i A - - - - V i r « S 1 CS-t 2 3 4 r 6 2. t 8 \ 7 5 F i g . 17. Sum of 3 2 P Label i n +ve Strand, -ve Strand, and Virus RNA V i r a l r e p l i c a t i o n i s reputed to occur i n d i r e c t association with CPV-1. A membrane-associated r e p l i c a t i o n complex contain-ing v i r a l RNA polymerase and r e p l i c a t i v e intermediate was 3 i d e n t i f i e d by pulse l a b e l l i n g infected c e l l s with H-uridine. P a r t i a l p u r i f i c a t i o n of t h i s structure resulted i n a concentrat-ion of CPV-1. (42, 43, 63) At 5-5 hours post i n f e c t i o n c e l l s were lysed and homogenized and centrifuged f o r 10 min. at 800 xg. The supernatant was centrifuged at 13,000 xg f o r 15 minutes;. 27 the pellet was resuspended in hypotonic buffer and resedimented at 800 xg for 5 minutes. The supernatant containing CPV-1 was centrifuged again at 1 3 , 0 0 0 xg for 15 min. to obtain the pelleted vacuoles. The isolated vesicles were membrane limited and lined by regular membranous sphericles measuring 50 nm in diameter - these were neither virus cores or virions. CPV-1 appearance coincided with virus production in BHK, CEF, and L cells infected with SFV, Sindbis and Western Equine Encephalitis viruses. These vacuoles appeared to arise from the Golgi apparatus based on assays using the Golgi marker enzyme, acid phosphatase. Actinomycin D had no effect upon the appearance of CPV-1. Cycloheximide treatment (which inhibits SFV protein synthesis almost immediately) prior to 2 hours infection completely prevented biogenesis of CPV-1; however, addition of cycloheximide at 2 or 3 hours post infection did not prevent the appearance of CPV-1. Guanidine (which inhibits RNA synthesis up to 9 0 $ within 1 0 min. of treatment) inhibited formation of CPV-1 when applied at 2 hours infection but caused l i t t l e inhibition when added later. In another experiment, a cytoplasmic extract was purified on a discontinuous sucrose gradient. A band containing RNA polymerase activity, H-uridine, and CPV-1 (visible by EM) was obtained. Similar results have been obtained with poliovirus ( 6 4 , 6 5 ) . Michel and Gomatos ( 6 6 ) indicated that RF and RI RNA was associated with such membranous structures. At present, no preparation of togaviral polymerase has been significantly purified. The role of cellular protein in 28 RNA r e p l i c a t i o n i s unknown. It w i l l be i n t e r e s t i n g to see how SFV r e p l i c a s e and RNA polymerase compare with the correspond-ing host c e l l enzymes. Translation of Vi r u s - S p e c i f i c mRNA Several investigators have i s o l a t e d polyribosomal mRNA. It i s generally agreed that 26S RNA i s messenger but 4 2 S RNA i s also associated with polysomes. Simmons and Strauss state that 26S RNA (M.W. 1.6 x 1 0 6 ) constitutes 9 0 $ by weight of the mRNA i n infected c e l l s , and i s thought to specify the s t r u c t u r a l proteins of the v i r u s . On the other hand, 42 S RNA, (M.W.4.3xlO ) which i s the v i r a l genome, constitutes approximately 1 0 $ of the t o t a l mRNA and i s thought to supply the remaining v i r a l functions ( 6 8 ) such as RNA polymerase a c t i v i t y . Wengler and Wengler however contend that 4 2 S RNA constitutes about 5$ of polysome messenger. Kennedy (71), Mowshowitz (72) and Simmons and Strauss (73) have also noted the association of small amounts of 33S RNA with polysomes. Hybridization-competition experiments showed that 90$ of the base sequences i n 33S RNA are also present i n 26S RNA. After reaction with formaldehyde, 33S RNA i s completely converted to 26S RNA. A precursor r o l e has been suggested f o r 33S RNA. Hybridization studies have shown ( 6 l , 6 9 ) that 26S RNA consists of 2/3 of the base sequence information of 42S RNA. Thus 26S RNA represents a unique f r a c t i o n of the v i r a l genome. The mechanism by which 26S RNA i s derived a f t e r i n f e c t i o n with 42S RNA i s not known to date. 4 2 S RNA is o l a t e d from polysomes 29 and incompletely assembled ribonucleotide nucleocapsid particles (RNP) have been shown to be identical in structure to virus RNA ( 7 4 ) through infectivity, sedimentation behaviour and in vitro protein synthesis. Most recently studies have been directed towards isolation of these messengers and using them in c e l l free protein synthesis (75 - 8 1 ) . The f i r s t of such studies were performed by Cancedda and Schlesinger using Krebs II ascites c e l l and rabbit reticulocyte extracts. Translation of Sindbis 2 6S RNA resulted in the formation a protein identical to capsid protein (as shown by tryptic peptide mapping and SDS polyacrylamide electrophoresis). Only traces of E-^  and Eg protein were obtained and larger M.W. proteins (which could be precursors) were also absent. These investigators seem to believe that their findings may support evidence that Sindbis virus capsid is formed from a polycistronic mRNA as the f i r s t polypeptide translated from the 5 ' end of the RNA, and that capsid i s released as free protein before translation of the polycistronic RNA i s completed. Clearly protein synthesis in this system i s far from complete - possibly premature termination or slow down in translation i s occurring. It is interesting also that in vivo there often appears to be about 4 - 5 times as much nucleocapsid protein produced as E-^  and Eg ( 7 7 ) . Using a temperature sensitive mutant of Sindbis which has RNA which f a i l s to produce nucleocapsid at the non-permissive temperature, i t was shown that a large molecular weight polypeptide accumulates in vitro when using 42S RNA at the non-permissive temperature. 30 Clegg and Kennedy ( 7 $ , 7 9 ) have also performed studies using 26S RNA from cells infected with SFV as messenger using an extract of LS ce l l s . They also indicate that the major peptide formed was nucleocapsid and that 42S RNA functioned poorly as messenger - probably due to i t s secondary structure. In their most recent studies ( 7 9 ) , however, Clegg and Kennedy-showed that at long periods of synthesis (100 min. compared to 30 min.) E-^  and Eg could be found in c e l l free systems in a non-glycosylated form. There was, however, no evidence for precursor polypeptide as has been shown with polio virus messenger ( 9 3 - 9 6 ) . Addition of TPCK (chymotrypsin inhibitor) or PMSF (a serine protease inhibitor) did not yield precursor polypeptides in the c e l l free system. Thus, there i s some question as to how 26S RNA, a polycistronic messenger, can be translated to produce the 3 discrete products. A model must also be proposed for the selective formation of nucleocapsid protein. Translation may be initiated at a single site (near the beginning of the nucleocapsid cistron) and terminated at the end of the second envelope cistron; specific gene products may arise by a series of rapid cleavages of the primary gene product or by a cleavage of the nascent polypeptide chain as ribosomes move from one cistron to the next. Premature termination could occur at the terminus of the nucleocapsid cistron. Another possibility i s that each cistron has i t s own i n i t i a t i o n site. This model is without precedent in eucaryotic systems but does occur in procaryotes. 31 Formation of Virus Proteins - Post-Translational Cleavage of  Large Precursors Just as the structural proteins of picornaviruses appear to arise from cleavage of a large polypeptide (82, 83) so do those of Group A togaviruses. This precursor phenomena was f i r s t observed by Burrell, Martin and Cooper (84) in BHK cells infected with SFV. Detailed precursor studies were carried out by Schlesinger and Schlesinger on Sindbis virus. Their f i r s t studies showed the existence of a precursor to Eg (P^g) of molecular weight 68,000. PEg was shown to be closely related to Eg by tryptic fingerprinting. The appearance of virus proteins in the total c e l l extract and virus over time was also presented and quantitated in Fig. 18. 0, 0 ) 20 40 60 TIME AFTErt CHAS2 (MIN) Fig. 18. Pulse Chase of Virus Proteins in Cell and Extracellular Virus ('86) 32 Pactamycin was found to inhibit nucleocapsid synthesis prior to an effect on membrane protein synthesis. Since this antibiotic prevents i n i t i a t i o n of protein synthesis this was taken to indicate that nucleocapsid was translated prior to the membrane proteins. PEg was also shown to be glycosylated. In a later study (85) a temperature sensitive mutant Sindbis ts2 which i s defective in nucleocapsid assembly was used. A large molecular weight precursor accumulated at the non-permissive temperature. Two dimensional tryptic mapping showed this large protein to contain E-^ , Eg, and nucleocapsid. When the infected cells were shifted to the permissive temperature radioactivity was chased from the large protein (M.W.97,000) to PEg, E^, Eg, and nucleocapsid. Jones, Waite, and Bose (87, 88) performed a similar experiment with cells infected with a temperature sensitive mutant of Sindbis defective in cleavage of PEg at the non-permissive temperature (42° C). A shift to the permissive temperature saw a decrease in PEg and increase in Eg formation. Preliminary experiments showed PEg might be associated with the PM of the host c e l l . Further evidence for post-translational cleavage of precursor proteins in Sindbis virus infected cells has been provided through use of TPCK (tosylphenylalanine chloro-methylketone) which is an inhibitor of chymotrypsin (8.9). A large molecular weight protein (approximately 97,000 M.W. ) accumulated in radioactively labelled cells infected with Sindbis and TPCK. 33 Semliki Forest Virus Similar sorts of experiments as those performed on Sindbis have been applied to Semliki Forest Virus. Simons, Keranen, and Kaariainen (90) f i r s t demonstrated the existence of the precursor to E 2 ( P E 2 ) in cells infected with a temperature sensitive mutant of SFV which was defective in P E 2 cleavage at the non-permissive temperature. Pulse chase studies and tryptic fingerprinting indicated that PEg was indeed a precursor to Eg. Further work was reported by Morser and Burke (91, 92). CEF cells and BHK cells were infected with wild type virus in the presence of actinomycin D and the cells labelled between 6 and 7 hours infection. Total protein from the infected cells was extracted and analyzed on 10$ SDS - polyacrylamide gels. Their results are shown in Fig. 19 F i g . f J . I n f e c t e d c l i i c k e m b r y o ce l l s ( O — O ) a n d B H K - 2 1 ce l l s ( • — • ) w e r e l a b e l l e d w i t h i o / ' C i / c u l t u r e o f l ] - v a l i n e a n d 5 /<Ci ; ' cu l turc o f [ "C]-% a l i n e r e s p e c t i v e l y b e t w e e n 6 a n d 7I1 a f ter i n f e c t i o n . T h e s a m p l e s w e r e . m i x e d b e f o r e e x t r a c t i o n o f the p r o t e i n s a n d a n a l y s i s b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . (9i) 3 4 The same results were obtained when amino acid analogues (to inhibit specific proteolytic cleavage) were used — fluoro-phenylalanine, canavanine, azetidine - 2 - carboxylic acid, ethionine, and azatryptophan. Temperature jump studies where infected cells were raised to 4 2 . 5 ° C to inhibit protein synthesis and then restored to 3 7 ° C with addition of label also confirmed this. Pulse chase studies in the presence of inhibitors of i n i t i a t i o n of protein synthesis (sodium fluoride, n-butanol, aurintricarboxylic acid) showed that when the inhibitor was removed NVP 97 f e l l rapidly with a rapid increase in labelling of nucleocapsid followed by E-^  and Eg. NVP 63 also f e l l but NVP 78 continued to increase slightly. It i s believed (on the basis of tryptic mapping) NVP 78 i s probably a non-structural protein required for virus replication. In a recent paper ( 9 6 ) , Clegg has demonstrated the order of peptide translation in SFV infected BHK cel l s . Translation of mRNA was i n i t i a l l y repressed with medium containing an elevated concentration of NaCl. Restoring the medium to isotonicity induced synchronous i n i t i a t i o n of protein synthesis. Radioactivity was apparent in capsid protein after 2 min. of restoring isotonicity while radioactive precursors or envelope proteins did not occur u n t i l after 5 -6 min. Further work showed the order of labelling to be capsid, NVP 9 7 , NVP 6 3 , Ep Eg-E^. Work with temperature sensitive mutants of SFV was also performed by Keranen and Kaariainin ( 9 7 ) . These workers demonstrated 5 non-structural proteins in cells infected with these mutants - NVP 1 3 0 , NVP 9 7 , NVP 8 6 , NVP 7 8 , and NVP 6 3 . 35 NVP 63 corresponds to PE 2 found in Sindbis and NVP 97 i s the high molecular weight precursor found by Schlesinger and Schlesinger. Using labelling studies with ^H-glucosamine, i t was shown that PE 2, E]_, and E 2 were the only glycoproteins in infected c e l l s . Over longer labelling periods (3 to 12 hours infection) there was an absence of larger molecular weight proteins and a much higher proportion of E]_, E 2 and nucleocapsid. In chase studies where radioactive medium i s replaced with non-radioactive medium, label in the high molecular weight proteins appears to move into the lower molecular weights envelope proteins. TPCK addition was also shown to result in the accumulation of large molecular weight proteins. These large proteins disappeared over a 1 hour chase when the radioactive medium was removed, as shown in Fig. 20 and Fig. 21. BHK CEF 0 3 20 40 60 80 100 120 Fraction number T NVP- NVP-127 97 I • NVP- N v p NVP-.165 , „ r 7S 105 , ~i i 1 r NVP- n , c „ 63 E, + E2 C •+- -t- - f - -+-NVP- NVP- c . c , . * NVP- * i Piii-ia i 7K <-nn-i£ 20 40 60 XO 100 120 Fraction number Fig-* I Fig. j o Fig. 2<f*The effect of TPCK on virus-specified protein synthesis in BHK-21 cells. TPCK (20/ig/ml) was added 55 h after infection, and at 6 h a pulse-chase experiment was carried out as described in the legend to Fig. 4. The samples from (a) the short pulse, (6) the pulse-chase and (c) the 1 h pulse were analysed on 12 cm polyacrylamide gels. TPCK. was present throughout. Fig.JLlA similar experiment to that shown in Fig. 7, but using chick embryo cells, showing (a) a 7 min pulse, (b) a 7 min pulse followed by a 53 min chase and (<•) a 1 h pulse. ^ <fx) 36 Pulse-chase experiments suggested NVP 130, NVP 97, NVP 86, and NVP 62 were precursors for the structural proteins. NVP 78 was not affected by the chase and could be the replicase or RNA polymerase. Based on the various evidence, Morser and Burke have proposed the following model for post-translational cleavage of precursor polypeptides shown in Fig. 22. 26S RNA * NVP165 J NVP127 +? NC I NVP97 (NVP63) E l E2 E 3 Fig. 22. Post-Translational Cleavage in Formation of SFV Structural Proteins 37 Evidence for Maturation of Enveloped Viruses Through Budding  from the Plasma Membrane Group A Togaviruses Original evidence concerning the manner in which SFV obtained i t s envelope came through the electronmicroscopic studies of Acheson and Tamm (21) where nucleocapsids could be seen "budding" through the plasma membrane and thereby aquiring their envelopes. Freeze-fracture studies on Sindbis also confirm this (100). The next piece of evidence indicating that the PM was the source of v i r a l envelope came with l i p i d analysis of SFV grown in various hosts. Renkonen et a l . (9&, 99) originally showed that the composition of virus l i p i d s resembled that of i t s host c e l l plasma membrane. SFV grown in BHK21 cells and Aedes albopictus cells have only 36$ of their phospholipids in common. Similar studies have also been conducted on Sindbis (101, 102). More conclusive biochemical evidence for budding of togaviruses has been presented by Jones et al.. ( £ 6 , 87). This group has shown that E]_, E 2, nucleocapsid and possibly PE 2 are present in the plasma membrane of the infected host c e l l . To date, i t has not been clearly demonstrated that Group A togaviruses "bud" through the plasma membrane of the host c e l l . Myxoviruses and Paramyxoviruses Electronmicroscopic studies also indicate that myxoviruses arid paramyxoviruses bud from the plasma membrane. This was f i r s t shown in 1955 by Hotz and Schafer (103) for influenza and later for paramyxoviruses by Choppin (104). Nucleocapsid may 38 be seen aligning with specific patches of the inner PM..prior to' budding; these patches may be labelled with fer r i t i n - l a b e l l e d antiviral antibody. Klenk and Choppin (105,'106) f i r s t showed that SV5 envelope l i p i d s resembled those of the host c e l l PM. Further studies in comparing the glycolipids of virus grown in MK and BK host cells further verified this (107). The concept that envelope components migrate from the c e l l interior to the surface has been confirmed by c e l l fractionation and analysis of the various proteins found in the different cytoplasmic fractions. It has been suggested that HA and possibly the other envelope proteins are synthesized on the rough ER (108, 109). Pulse chase experiments show that a few minutes later HA i s present in the membranes of the smooth ER (108, 109) and then the PM (110). Virus proteins are always membrane associated during this migration and are never detected in the soluble fraction. Alan J. Hay (111) reported similar results for fowl plague influenza virus but found the M protein was synthesized close to the PM and aligns next to the NA and HA as the f i n a l stage in virus assembly. As yet no experiments have been performed which have chased the myxovirus protein from the PM to the free virions. Rhabdoviruses Klenk and Choppin (113) have again shown that VSV envelope li p i d s resemble, those of the host c e l l . Cohen et a l . (114) originally showed that VSV proteins were associated with the PM fraction of infected HeLa cells. In a pulse chase experiment 39 of VSV infected HeLa cells, David ( 1 1 5 ) showed that after a 30 second pulse G and M proteins were already associated with the PM but maximal levels of incorporation were not reached u n t i l 2 minutes chase. N and NS polypeptides were found only in the soluble cytoplasm (116). At the end of a 5 min. pulse period the plasma membranes contain substantial amounts of the carbohydrate-free envelope protein and of the glycoprotein of the spikes, but not yet nucleocapsid protein. With chase times approaching 60 min., the amount of nucleocapsid protein found in the plasma membranes increases, although after about 20 min. the amount of envelope proteins attached to the plasma membrane remains approximately the same ( 1 1 7 ) . 40 The Present Investigation It was the intent of this thesis to prove through bio-chemical means that SFV matures by budding from the plasma membrane of the host c e l l . Through this process the naked nucleocapsid acquires envelope l i p i d s and proteins. Previous evidence to support this hypothesis i s derived from electronmicroscopic observations ( 2 1 , 1 0 0 ) and l i p i d class composition studies conducted on ER, PM, and purified virus ( 9 8 , 9 9 ) . The l i p i d s of the virus envelope resemble most closely those of the host c e l l PM. Similar lines of investigat-ion have been conducted on other enveloped viruses such as myxoviruses and paramyxoviruses (105-107) and rhabdoviruses ( 1 1 3 ) . Results from the above studies are far from.conclusive — the electronmicroscopic evidence could be based on art i f a c t s incurred through sample preparation and many irregularities exist in the l i p i d composition studies. It was hoped that the present investigation might c l a r i f y this situation. The problem was attacked through use of radioactive pulse 3 chase experiments. Infected ce l l s were to be pulsed with H - L e u and the label was to be followed from the membrane fractions of the host c e l l into mature virus. Preliminary experiments of this type have been performed on influenza ( 1 0 8 - 1 1 1 ) and vesicular stomatitis ( 1 1 4 , 1 1 5 ) viruses but much of this data i s incomplete and the complex nature of these viruses makes the data d i f f i c u l t to interpret. I n i t i a l l y these studies were to b e applied to SFV infected B H K c e l l microsomal fractions and liberated virus; the membranes of infected cells were then 41 to, be fractionated into ER and PM. It was the aim of these experiments to show a clear chase of radioactively labelled virus proteins from ER to PM and then into free virus. This would support the contention that virus proteins were synthesized in the ER and inserted into the PM just prior to virus maturation. 4 2 METHODS AND MATERIALS Chemicals and Isotopes D, L ^H - 4 , 5 - Leucine ( 4 5 Ci./mmole), L - 4 , 5 - Leucine ( 6 0 Ci./mmole), and ^H AMP ( 1 1 . 2 5 Ci./mmole) were obtained from New England Nuclear. Sucrose (RNAse free) was purchased from Schwarz. Mann. 1 9 9 maintenance medium, modified Eagles medium, Earles basic salts medium, p e n i c i l l i n -streptomycin from Grand Island Biological Company. The L-amino acid kit and Tris-HCl came from Sigma; magnesium acetate, EDTA sodium chloride, sodium lauryl sulfate and sodium phosphate (dibasic and monobasic) were a l l bought from Fisher. Nitro-cellulose tubes were purchased from Beckman while NCS came from New England Nuclear.' Diallyltartardiamide (DATD-) and iodoacetic acid were purchased from Bio-Rad and Eastman Kodak respectively. Toluene ( s c i n t i l l a t i o n grade), PPO and POPOP were obtained from Fisher, Triton X - 1 0 0 came from Rohm and Haas or Sigma. Acrylamide and methylenebisacrylamide were purchased from Matheson, Coleman, and B e l l . Cytochrome c (horse heart) came from Sigma and NADPH from P. L. Biochemicals. Buffer Solutions The following buffer solutions with designated abbreviations were employed throughout these studies. 1 ) PBS - phosphate buffered saline pH 7 . 4 2 ) STM - 0 . 2 5 M sucrose, 5 0 mM Tris-HCl pH 7 . 4 , 1 . 0 mM MgAc 3 ) TM - ImMTris-HCl pH8.6, 0 . 5 mM MgAc2 4 ) TNE - 5 0 mMTris pH7-4, ImM NaCl, ImM EDTA 43 SDS Acrylamide Electrophoresis Cell proteins and virus samples were electrophoresed on SDS gels consisting of 7 .5$ acrylamide according to Weber and Osborne ( 16 ) . Gels for the 0 ' - 60 ' and 0 - 3 hour pulse chase experiments were prepared using methylenebisacrylamide as crosslinking agent while the remaining gels contained diallyltartardiamide according to Anker (124) . This last modification was instituted to f a c i l i t a t e more economical counting of the gel slices. After protein samples were layered onto the gels, they were pre-electrophoresed at 50 mV,. 2. 5mA/gel for -g- hour and then electrophoresed at 150 mY, 8.3 mA/gel for 2 hours. The gels were then stained in Coomassie Blue for 1 hour at 6 0 ° C and then destained by either diffusion (using 7 .5$ acetic acid, 5$ methanol) or by electrophoresis in 7 .5$ acetic acid. Gel Slicing and Sc-intillation Counting of Gel Samples v Gels were frozen in a dry ice acetone bath and sliced into 1 mm slices using a Bio-Rad gel s l i c e r . Each s l i c e was placed in a s c i n t i l l a t i o n v i a l and digested in either 0 .5 ml of NCS: water (9:1) or 0 .5 ml of 2$ periodic acid depending upon whether MBA or DATD respectively "was used as a crosslinking agent. Digestion was complete after 1 hour in each case. To the NCS digested slices 10 ml of toluene based s c i n t i l l a t i o n f l u i d (4 gm/l PP0, 0.03 mg/l POPOP) was added. Periodate digested slices were suspended in 10 ml of a 25$ Triton X -100/75$ Toluene (5 gm/l PPO, 0.3 gm/l POPOP mixture. Tritium samples were counted in New England Nuclear Isocap 300 s c i n t i l l a t i o n counter. 44 SFV-Infected Microsomal Time Course 16 large (150 mm. x 15 mm.) plates of 3/4 confluent BHK cells were infected with 20 PFU/cell of wild type SFV in a t o t a l volume of 3 ml.- 199 per plate. Virus was allowed to adsorb for 1 hour (plates were tipped back and forth at the half hour). After adsorption, each plate was made up to 10 ml. with 199 medium (2$ f e t a l calf serum). This was 0 hours infection. Cells were harvested at 0, 1, 2, k, 6, 8, and 11 hours post-infection and the c e l l media containing virus was also removed and stored at -70°C. The infected cells at each time point were washed 2 times with 10 ml. of ice cold PBS. The cells on each plate were scraped into 5 ml. PBS.buffer and placed in a 12 ml. capacity centrifuge tube. 5 ml. PBS buffer was. added to the plate again and rescraped; the washings were combined with the previous 5 ml. of cells and sedimented by centrifuging at 1500 xg for 5 min. using a Sorval SS-34 head. The pelleted cells were resuspended in 5 ml. of STM buffer and homogenized with 25 strokes of a.tight f i t t i n g "Vitro" Dounce homogenizer. The homogenate was centrifuged at 9200 xgj using a Sorval SS-34 for 10. minutes to remove mitochondria, nuclei, and whole c e l l s . Following centrifugation the super-natant was retained and the pe.Het suspended in STM and recentrifuged at 9200 xg and the supernatant combined with the previous one. The combined supernatants were centrifuged at 100,000 xg for 60 min. on a Beckman T i 65 rotor to obtain the ; i microsomal pellet. Microsomal pellets were suspended in 0.5 ml. of TM buffer. 100 ug. protein of these preparations was applied to each gel. This was approximately 100 u l . of the suspension. . 45 Microsomal Pulse Chase Experiments The microsomal pulse chase experiments were performed in a manner similar to the microsomal time course outlined previously. For the 0' - 60* pulse chase, cells were infected for 4i? hours after which the 1 9 9 medium was replaced with 5 ml. per plate of Earles basic salts (2$ dialyzed calf serum; dialyzed to remove amino acids) starvation medium. At 5 hours the medium was replaced with 5 ml. labelling medium -Earles basic salts (2$ dialyzed calf serum) enriched with 50 mg/l of a l l unlabelled amino acids except Leu. To each plate 3 0 0 uCi. of 3 H - D, L-Leu were added for a pulse of 20 min. Finally at 5 hours 20 min. infection the labelling media of the 0' chase plate was removed and frozen at - 7 0 ° C for virus analysis. The cells of the 0' chase were harvested and the microsomal homogenate was obtained and stored in an ice bath u n t i l the other samples were ready for ultracentrifugation. Also at 0' chase, the 20', 40*, and 60' time point plates were replaced with 5 ml. chase media consisting of 1 9 9 main-tenance medium enriched with 0.5 mg./ml. of unlabelled L-Leu. When i t came time to harvest the 20 ' , 40*, and 60' chase plates, chase media was retained and frozen at - 70° C for virus analysis. Cells were harvested and microsomal pellets prepared as outlined in the previous section. A 0 - 3 hour pulse chase experiment was performed in a manner identical to the 0* - 60' chase experiments except that cells and virus were harvested at 0 hrs., 0.75 h r s . , 1 . 5 0 hrs., 46 2.25 h r s . and 3.00 hrs. Also only 100 u C i . of ^ H-D, L-Leu was added per p l a t e during pulse l a b e l l i n g . A f i n a l microsomal pulse chase experiment was performed over a chase i n t e r v a l of 0 - 11 h r s . I n f e c t e d BHK c e l l s were starved at 2-g- hrs. i n f e c t i o n and l a b e l l e d w i t h 100 u C i . / p l a t e of D, L-Leu f o r 20 minutes. L a b e l l i n g media was rep l a c e d w i t h chase media at 3 hours 20 min. and the 0 hrs. chase media and c e l l s were harvested as before. R a d i o a c t i v i t y was chased i n the remaining c e l l s f o r 1, 2, 4, 6, 8 and 11 hours; the c e l l s and v i r u s supernatants were harvested at these times and the microsomal homogenates prepared immediately a f t e r harvest. Microsomal p e l l e t s were obtained and p e l l e t e d as before. BHK" c e l l s i n f e c t e d (2 p l a t e s per time p o i n t ) I v i r u s adsorbed 1 hr. volume of each p l a t e made to 10 ml. ( t h i s i s 0 hrs. i n f e c t i o n ) 2-g- or 4"i" hours i n f e c t i o n allowed to proceed c e l l s starved f o r -g- hour; at 5 hours c e l l s were l a b e l l e d w i t h 3H-Leu f o r 20 min. a f t e r 20* l a b e l l i n g the r a d i o a c t i v e medium was removed and replaced w i t h Leu enriched chase media i n f e c t e d c e l l s and v i r u s supernatant harvested at various chase times (2 plates/chase time) • K . p r e p a r a t i o n of microsomes F i g . 23. Pulse.Chase Experiment 47 2 plates of infected BHK cells were harvested for each time point using rubber policemen I cells were pelleted at 1500 xg pelleted cells were suspended in STM and homogenized with 20 strokes of a tight f i t t i n g Dounce homogenizer homogenate was centrifuged at 10,000 xg to sediment nuclei, mitochondria and whole cells supernatant was centrifuged at 100,000 xg for 1 hr. to obtain a microsomal pellet microsomal pellet was suspended in 0.5 ml. TM buffer Fig. 24. Preparation of Microsomes 48 Isolation of Virus A method for isolating microgram amounts of radioactively labelled virus was derived from Pfefferkorn's work (125). A 3 phase gradient as shown in Fig. 25 was prepared. 10 ml. virus sample 5-20$ sucrose in PBS pH8 25$ sucrose (PBS pH8) -50$ sucrose (in 0.2M CsCl, 0.002M Tris pH7.8) 50$ sucrose (0.2M CsCl, 0.002M~ Tris pH7.8 Fig. 25. Isolation of Virus by Sucrose Gradient Centrifugation Virus gradients were centrifuged at 25,000 RPM on a SW 27 rotor for 4 hours. Gradients were then dripped from the bottom and 0.5 ml. fractions were collected. Aliquots (50 ul.) 3 were removed and assayed for H-Leu in Triton X-100, Toluene (1:3) s c i n t i l l a t i o n f l u i d . Fractions containing radioactive virus were pooled and dialyzed 2x against 4 l i t e r s of PBS at 4°G The dialyzed samples were lyophilized overnight and suspended in 100 u l . of gel electrophoresis sample buffer and gels were run. ' • Virus for the 0 - 3 hour microsomal chase was prepared in a different manner. Unlabelled SFV was partially purified 4ml Virus banded here 4ml 49 from vacuum dialyzed supernatant by the method.of Kennedy (29). 100 ug. of unlabelled SFV. was placed in each polycarbonate centrifuge tube together with the respective labelled virus chase supernatant. The samples were centrifuged at 47,000 RPM in a Beckman Ti 75 head for 2 hours. The virus pellets were suspended in 1 ml. TNE. 100 u l . of this suspension was placed on each 7.5$ SDS gel. Separation of PM from ER Plasma membrane and•endoplasmic reticulum were prepared by combining the methods of Atkinson and Summers (128) and Warren (129). Three plates (150 mm. x 15 mm.) of 3/4 confluent BHK cells were each washed 2x with 10 ml. of 10 mM Tris. Cells were scraped with rubber policemen into 2.5 ml./plate of 10 mM Tris. A total of 7.5 ml. of c e l l suspension was homogenized with 5 strokes of a loose f i t t i n g Vitro "Dounce" homogenizer. The homogenate was immediately made 0.25 M in sucrose (using 65$ sucrose) and 5 mM in Mg Clg (using.50 mM Mg Clg) to stabilize nuclei. Nuclei and whole cells were then centrifuged from the homogenate at 1000 xg (3000 RPM on a Sorval SS-34 head) for 10 min. The supernatant was then placed on a discontinuous gradient as shown in Fig. 26 and centrifuged at 7000 xg (5800 RPM on a SW 27 centrifuge head) for 20 minutes. 50 ER PM PM 3 ml. homogenate 3 ml. 20$ w/w sucrose 5mM MgCl 2 3 ml..30$ w/w sucrose 5mM MgCl 2 3 ml. 4 0 $ w/w sucrose 5mM MgCl 2 3 ml. 50$ w/w sucrose 5mM MgCl 2 3 ml. 6 0 $ w/w sucrose 5mM MgCl 2 Fig. 2 6 . Discontinuous Gradient for Separation of PM and ER The gradient was dripped from the bottom and 0 . 5 ml. fractions collected. The fractions were assayed for NADPH cytochrome c reductase (an ER marker enzyme) and 5 1-nucleotidase (a PM marker enzyme). NADPH cytochrome c reductase was assayed ( 1 2 6 ) by measur-ing the reduction of cytochrome c spectrophotometrically on a Gilford spectrophotometer at 550 nm. and 2 5 ° C. The incuba-tion mix consisted of taking 0 . 2 5 0 ml. of a cocktail ( 0 . 1 mM KCN, 0 . 0 6 6 mM KC1, 0 . 0 4 4 M phosphate pH 7 . 6 , 0 . 0 5 mM cytochrome c) to shich 5 0 u l . of enzyme was added. In order to start the reaction, 5 0 u l . of 0 . 0 6 mM NADPH was added to the enzyme and cocktail. The activity of the enzyme was calculated as follows: 51 F 5 5 ° 6 2 Reduced Cytochrome c = 27.7 x 10 cm. mole E550 6 2 Oxidized Cytochrome c = 9.0 x 10 cm. mole ^ = ERed. Cyt. c ^ [Red. Cyt. c] - Ej£° d > cyt.^CRed.Cyt.c] d [Red. Cyt. c] = d A 5 5 0 / 18.7 x 10 6 ml aT dt mole 5' nucleotidase was assayed by the method of Avruch and Wallach (127). The assay mix consisted of 100 u l . 0.5 M Tris-HCl pH 7-5 and 0.2 M MgClg, 50 u l . 0.4 mM AMP, 20 u l . of 3H-AMP, 0.630 u l . HgO, and 200 u l . of each fraction. The mix was incubated for 30 min. at 37° C. The reaction was stopped by adding f i r s t 0.200 ml. 0.25 M ZnSO^ and then the unreacted ATP was precipitated with 0.200 ml. of 0.250 M Ba(0H)2« The tubes were centrifuged on an International desk top centrifuge for 5 min. at setting 4- A 0.5 ml. aliquot 3 of the supernatant was assayed for H - adenosine by monitoring the aliquot (using 10 ml./vial of Triton X 100; Toluene(l:3) s c i n t i l l a t i o n fluid) on an Isocap 300 s c i n t i l l a t i o n counter. In order to increase plasma membrane yields in pulse chase experiments (where i t was unnecessary to assay marker enzymes) lysing buffer and sucrose gradient solutions were made 10 mM in sodium azide and 15 mM in iodoacete (127). 52 0 - 1 1 Hours PM/ER Pulse Chase Experiment This experiment was carried out in an almost identical manner to the 0 - 1 1 microsomal pulse chase experiment. 24 plates were infected, 3 plates being used for each chase point. Beginning at 2-g- hours infection the cells were starved, at 3 3 hours they were pulsed with 100 uCi L- H-Leu/150 mm. x 15 mm. plate for 20 minutes, and f i n a l l y cells and virus supernatant was harvested at 0, 1, 2, 4, 6, and 11 hours. A mock infected plate was also carried through the procedure and harvested as outlined in the previous section. Membrane gradients were dripped and assayed for radio-activity which corresponded to membrane bands. A membrane band at the 40 - 50$ sucrose interface was the major-band obtained from infected c e l l s . This band contained the greater 3 amount of H-Leu. The membrane band at the 30 - 40$ sucrose interface was not nearly so evident and contained a much 3 lesser amount of H-Leu. It was decided to pool the lower PM band fractions of each gradient, dialyze each 2X against TM buffer and then lyophilize the fractions. These fractions were reconstituted with d i s t i l l e d water (200 ul./pooled fractions) and 50 u l . was removed and assayed for protein. 100 ug. protein (which consisted almost entirely of the re-mainder of the 200 u l . after removal for protein determination) was applied to 7.5$ SDS acrylamide gels (DATD as cross-linker). The gels wsre sliced as before. 53 A mock infected BHK c e l l preparation was also pulsed at 3 hours and chased f o r 11 hours. The 3 plates of c e l l s were harvested, fractionated into PM/ER, and the gradients dripped. In t h i s case a l l the r a d i o a c t i v i t y was located i n the 30 - 40$ sucrose band which was the major PM band. L i t t l e or no membrane was present at the 40 - 50$ sucrose interface. 100 ug. protein of the upper PM band was electrophoresed as above and then the gels were s l i c e d . ER f r a c t i o n s were obtained from the top of each gradient ( f r a c t i o n #36). 100 ug. protein were placed on each gel, electrophoresed, and s l i c e d . Analysis of SDS Electrophoresis P r o f i l e s The radioactive SDS gels were scanned at 550 nm. using a G i l f o r d spectrophotometer, s l i c e d into 1 mm. pieces, digested, and counted as previously outlined. The absorbance at 550 nm. 3 and counts per minute (H-Leu) were plotted versus length of the gel (s t a r t i n g from the o r i g i n ) . Radioactivity from the points i n each peak was summated a f t e r subtracting the basal (background) r a d i o a c t i v i t y from each value. Protein Assays Protein was assayed by the method of Lowry (130). Lipoprotein samples were incubated overnight i n 0.66 N NaOH at 37° C. p r i o r to analysis. BSA was used as a standard. 5 4 EXPERIMENTAL RESULTS Virus Gels and Protein Standards Purified SFV was electrophoresed on 6 cm (before stain-ing) 7 . 5 $ SDS gels (with DATD as cross-linking agent) together with a number of protein standards. The v i r a l protein bands were compared by calculating band mobilities with those of the protein standards to determine molecular weights of the virus bands, both in this experiment and the following ones. TABLE 8 Standard Proteins and Gel Mobilities Standard M.W. Mobility Log M.W. Phosphorylase a 9 0 , 0 0 0 0 . 3 9 4 4 . 9 5 4 2 Bovine Serum 6 8 , 0 0 0 0 . 5 2 5 4 . 8 3 2 5 albumin Ovalbumin . 4 3 , 0 0 0 0 . 6 8 8 4 . 6 3 3 5 Trypsin 23,000 0 . 8 8 0 4 . 3 6 1 7 Cytochrome c 1 1 , 7 0 0 1 . 0 4 4 . 0 6 8 E x 5 2 , 4 8 0 0 . 5 7 2 4 . 7 2 E 2 4 8 , 9 8 0 0 . 6 1 0 4 . 6 9 Nucleocapsid 3 3 , 8 8 0 0 . 7 7 5 4 . 5 3 55 Fig. 27. Purified Virus and Protein Standards Ei and E 2 are clearly resolved on this gel system as seen in Fig. 27. E3 is not stained probably due to i t s large carbohydrate content. Microsomal Time Course and Virus Production In order to determine the time interval in which virus proteins could f i r s t be observed in the membrane, BHK cells were infected and gels run on microsomal proteins at 0, 1, 2, 4, 6, 8 and 11 hours infection. 100 ug. of protein was applied to each gel. The Coomassie Blue stained protein bands were scanned using the Gilford Spectrophotometer. These scans are presented in Fig. 29 and an actual photograph of these gels in Fig. 28. Clearly envelope and nucleocapsid proteins become 5 6 evident f i r s t at 4 and 6 hours infection. Envelope proteins and particularly nucleocapsid increased in quantity right up to 11 hours infection. ••••*•••• 0 | 2. ^ 6 * »> S F V *rv Fig. 28. Photograph of SDS Acrylamide Gels of Microsomal Proteins From Infected BHK Cells: 0-11 Hours Infection Virus production was also measured using both a Linbro and plaque assay. These results are portrayed in Fig. 30 and agree qualitatively with results obtained by Acheson and Tamm (39) and Wengler and Wengler (37). SFV MICROSOME TIME COURSE 3.0 ••.4.oy-'-\-*» • j j : 0 • .; I.o I • • • i . o i • jJ . O j - - - i ;• I, cm. gel c m - 9 e l Fig. 29. Coomassie Blue Scans of Microsomal Time Course Gels. 58 55 o Cm, gel cm. gel Cm. ge| Cm. gel F i g . 29 ( Continued) Fig. 30. Virus Production Over 0 - 11 Hours Infection 60 0' - 60* Microsomal Pulse Chase Experiment Based on the previous experiment i t i s clear that maximum virus production occurs between 4 and 7 hours infection. In order to determine a time relationship between virus specified protein appearance in the total membrane fraction of infected cells and free virions, BHK cells were infected, then pulsed with H-Leu at 5 hours infection and chased from 0' to 60'. The pulse consisted of 300 uCi. D, L Leu per 150 x 15 mm. plate for a period of 20 minutes. 0' chase was considered to be the point when the labelling media was removed following the 20 minute radioactive pulse. 100 ug. of microsomal protein (out of a total of 500 ug. microsomal protein) from each chase time was applied to each 7.5% SDS acrylamide gel (using N,N'*-methylenebisacrylamide as a cross-linking agent). Virus was harvested from the growth medium which was harvested at the 0', 2 0 4 0 f , and 60' chase times using the sucrose gradient procedure outlined in Materials and Methods. The SDS gels were sliced into 1 mm.pieces and assayed for •^ H-Leu using a s c i n t i l l a t i o n counter. These results are shown in Fig. 31. 0'- 6 0 ' C H A S E MIC R 0 S 0 M E G E L S era. gel Fig. 31. 0' - 60' Chase: Microsomal Protein . (Coomassie Blue Scans and 3 H - L e u Labelled Protein) ~ ^.Coomassie B l u e ; H - L e u 62 Fig. 31. (Continued) 6 3 The virus gradients (Fig. 32) i l l u s t r a t e that incorporation 3 of H-Leu into mature virus was absolutely negligible. Although labelled virus specific proteins were present in the microsomal pellet, the radioactivity had not yet been chased into free virus released into the culture medium. Taking the amino acid compositions of E-^ , Eg and nucleo-capsid published by Garoff et a l . (23) i t can be shown that nucleocapsid is produced in excess of E-^  and Eg based on the calculation shown in Table 9. Also nucleocapsid is produced before E-^  and Eg. This is very much in agreement with previously published results (85, 96, 97). Clegg (96) reports that nucleocapsid appears in the c e l l almost immediately after addition of "^C label. We find the same result. . Radioactively labelled nucleocapsid i s present i n i t i a l l y 60 times in excess of E-^  Eg. The activity of both proteins continues to increase during the course of the experiment. At 60' chase, nucleo-capsid molecules are s t i l l 5 times in excess of E-^  Eg. These calculations were based on the following data in Table 9« 64 TABLE 9 Leu Composition of SFV Proteins (23, 29) Protein Mole $ Leu M.W. of Protein (moles Leu/100 amino acids) E x 5.80 52,000 E 2 5-99 49,000 Nucleocapsid 3.78 34,000 moles Leu in NC _ mole jo Leu in NC moles Leu in E - ^ mole fo Leu in E-jtfg M.W. of NC  (M.W. of E ± + M.W. of E 2; 3.78 Y 34,000 _ n ? 1 " $770 A 101,000 - U , ( C ± Using this gel system i t i s evident that the membrane proteins E-^  and E 2 are not resolved, This fact i s not. unexpected since, these proteins are very similar in molecular weight and a" discontinuous buffer SDS acrylamide gel system must be used to. separate them (86., 90). If one views the 0' c/hase #rtaph of Fig. 31, one observes that nucleocapsid protein i s i n i t i a l l y ••• labelled much more heavily than E-^  and E,>. Also precursor to E 2 (PEg) i s present and appears to decrease over the following chase. Levels of E^, E 2 and nucleocapsid ,labelled 65 proteins increase over the remaining chase. These r e s u l t s are summarized i n F i g . 33. The r a d i o a c t i v i t y i n each peak of F i g . 31 was summated and a background consisting of the basal l e v e l s subtracted from each point. .... 66 0' - GO" VIRUS GRADIENTS Fig. 32. 0' - 60' Microsomal Pulse Chase Virus Gradients. 67 F i g . 3 2 ( C o n t i n u e d ) 69 0 - 3 Hrs. Microsomal Pulse Chase Experiment 3 In an attempt to chase the pulse of H-Leu into and then out of the microsomal proteins, the previous experiment was repeated over a longer chase period. Infected BHK cells were pulsed at 5 hours as before except that 100 uCi. D, L-Leu per 150 mm. x 15 mm. plate was used. This time, however, labelled virus from the c e l l medium at the 0, 0.75, 1.50, 2.25 and 3.00 hour chase times was prepared by sedimentation with unlabelled carrier virus. Carrier and labelled virus were then electro-phoresed on 7.5$ SDS acrylamide gels (using N, N'-methylenebis-acrylamide as cross-linking agent), sliced, and counted for 3H-Leu. Results were quite similar to those reported in the 0' - 60* microsomal pulse chase experiment. It was immediately evident that the experiment had not succeeded in showing a rise and f a l l of radioactivity in the microsomal proteins with an 3 accompanying rise of H - Leu in the virus. Microsomal protein gels are illustrated in Fig. 34. An aliquout of 3 sedimented virus was removed and assayed for H-Leu using Triton X 100-Toluene s c i n t i l l a t i o n f l u i d . It was again 3 evident that incorporation of H-Leu into free virus had not yet reached maximal levels. This was also evident from the gels (Fig. 35). 70 0-3 HR. PULSE CHASE MICROSOMES CP1VU x i d * 7 . 0 6 . 0 5 . 0 4 . 0 3 . 0 2 . 0 1 . 0 0 . 0 lilllU.O H R . C H A S E ] ; ; ; . i.v. i t!:l cm. .^el-J i g . 3 4 . (Continued) 0-3 HR. PULSE CHASE VIRUS ii " ~ It! O HR. C H A S E CPM ITS 0-75 HR. CHASE !|pll cm. gel cm. gel Fig. 3 5 . Virus Produced Over 0 - 3 Hours Chase as Sedimented-with Unlabelled Carrier Virus (.---Coomassie Blue H-Leu) Fig. 35 (Continued) 73 Fig. 36. Time Course of SFV Infected Microsomal Proteins ( 0 - 3 Hour Chase). (I* Nucleocapsid was again produced in excess in the microsomes. This fact i s clearly evident when the relative amounts of radioactivity in Eg and nucleocapsid in harvested virus are compared. From amino acid composition data one would 3 expect 1 / 5 the amount of H-Leu in nucleocapsid as compared to that in the E^ Eg peak. This appears to be the case in Fig. 3 5 • These results are summarized in the time graph of Fig. 3 6 . Nucleocapsid i s again i n i t i a l l y , very high and con-tinues to increase gradually over the experiment, while the radioactivity in E^ Eg was i n i t i a l l y low, increased between 0 and.2.25 hours., after which a plateau was reached. It i s interesting to note that 1 / 3 the total radioactivity of the pulse in the 0 ' - 6 0 ' microsomal pulse chase experiment was used in this experiment; correspondingly only 1 / 3 the levels 3 of H-Leu in E^ Eg and nucleocapsid were reached. 0 - 6 Hours Microsomal Pulse Chase Experiment 3 In an attempt to pulse the H-Leu label hot only into, but also from the microsomes and into virus, the chase time was extended over 0 - 6 hours. The pulse of 3H-D, L Leu ( 1 0 0 uCi./ 1 5 0 mm. x 1 5 mm., plate) was administered at 3 hours post-infection rather than 5 hours. This modification assured that the chase times spanned the period of maximum virus production. Microsomes and virus were harvested as before and electrophoresed on 7 . 5 $ acrylamide SDS gels (DATD cross-linker) as before. 1 0 0 ug. from a total of approximately 5 0 0 ug. microsomal protein were applied to each gel. The radioactive leucine distribution in the microsome 75 gels i s shown in Fig. 3 7 . Virus was fractionated by sucrose gradient centrifugation and the results are described in Fig. 3 8 . The total virus samples from pooled gradient fractions were electrophoresed and the gels sliced as shown in Fig. 3 9 . Again i t i s highly evident that nucleocapsid i s present at much higher levels in the microsomes than in free labelled virus. Based on amino acid analysis results one would expect the total radioactivity in Eg to be 5X that of nucleocapsid in the virus. Results of the virus gels indicate that this i s approximately so. The excess production of nucleocapsid in the c e l l has been observed by a number of investigators and i t s significance has not been determined. It i s interesting that in vitro studies using SFV and.Sindbis mRNA are successful in translating predominantly nucleocapsid ( 7 5 - 8 0 ) . Perhaps nucleocapsid also plays another role in. Togavirus reproduction — such as inhibition of host protein synthesis. A summary of the 0 - 6 Hr. microsomal pulse chase experi-ment is presented in Fig. 4 0 . Radioactivity in the microsomes appears maximal at 1 - 2 hours chase while that in the virus 3 occurs at 4 - 5 hours. H-Leu in the nucleocapsid f a l l s only slightly in the 1 - 3 Ha chase interval; the nucleocapsid protein appears relatively stable in the microsomes with a low turn-over rate. U - b t i r o . v n A o t • ivn^rcuauivin b t L b " ?e Fig. 37. 0 7 6V Hour Chase Microsjoraal Protein SDS gels 7 7 •cm, g e l Fig. 3 7 . (Continued) 7S Fraction # Fraction # Fig. 38. 0 - 6 Hours Chase Virus Gradients 79 .0-6 HRS. CHASE VIRUS CPM -x 10 -GPM , x 1CT 1.5 1 . 0 0 . 5 - . CFM ~ x 1CT 0 Hrs. Chase y« i m 4o i-a crin geT " 1.5 1.0.-CPM. x 1 0 * 0 . 5 1 , 1 Hrs. Chase y* ••' !•* cm. gel 1.5 h 1.0 L 0 . 5 - . ^ 0; 1.5 1 . 0 : CPM o x i c r \ o ? 5 0 cm . gel ') 1 1.5 , 4 ' Hrs . Chase 1 . 0 : ^ W • ( . . i: 1 0 . 5 . 0 "'. • • • • . i , • • (• ** y.m »•• "3 H r s . ' C h a s e i - t. «• • • • ? ,. ,.. *« *» _ I.. ». cm. gel CPM x 103 cm. gel cm. gel Fig. 3 9 . Radioactivity Profiles of SDS Gels f o r 0 - 6 Hours Chase Virus. 81' Mock ...... •• •. ••. -. •• i '. : v.; • I cm. g e l ' cm. g e l F i g . 39 (continued) Fig. 4 0 . Time Course of *H-Leu Labelled Microsomal Proteins Over 0 - 6 Hours Chase. 82 Isolation of BHK Cell Plasma Membrane and Endoplasmic Reticulum The membranes of both mock and infected BHK cells were ' fractionated as outlined in the Methods section. Preliminary-experiments were performed in order to establish the positions of the PM and ER fractions of the gradients shown in Fig. 4 1 . These gradients were each prepared from 5 plates ( 1 5 0 mm.x 15mm.) of confluent BHK cells. The infected cells were harvested after 8 hours infection with SFV ( 2 0 PFU/cell). (a) (b) Fig. 4 1 . Photographs of ER/PM Gradients from (a) Mock Infected and (b) Infected Cells. 83 In order to establish the position of PM and ER on the discontinuous sucrose gradients, the tubes were dripped from the bottom and 0.5 ml. fractions collected. These fractions were then assayed for 5'-nucleotidase (PM marker) and NADPH cytochrome c reductase (ER marker). The results of these assays are shown in Fig. 42 and Fig. 43. It i s evident that PM marker i s present at both the 40-50$ and 30-40$ sucrose interfaces. However, the distribution of PM may be greater in the lower band for infected cells than mock cel l s . The mean-ing of this observation is not immediately evident. However, when the SDS electrophoretic patterns of upper and lower PM bands are compared, the protein composition appears identical for both. ER marker enzyme activity appears confined solely to the top of the gradient. TABLE 10 Enzyme Purification During ER/PM Purification Enzyme Activity Purification Enzyme Homogenate ER PM Homogenate ER PM 5 1 Nucleotidase (nmoles/ 5.20 3.67 184 1.00 0.706 35.4 NADPH Cytochrome c Reductase (nmoles/mg./min.) 0.0234 1.44 0.0741 1.00 62 3.19 84 0-6 F i g . 4 2 . a ) P r o f i l e o f P M / E R F r a c t i o n a t i o n o f N o n - I n f e c t e d B H K C e l l s '• . ( N A D P H C y t o c h r o m e c R e d u c t a s e , 5' N u c l e o t i d a s e ) $5 3 n l 3x1 1 m l ] ml t o 7 - ^ >-l • to. F R « C T I O N / I T . | O N '•I / NftOPH E \ "I OS • J f >i ( i (J ii> i . \t £ i at at f.% 5 0 3 » 3 * 3 < 3 8 ^ 0 Fig. 42. b) Profile of PM/ER Fractionation of SFV-Infected BHK Cells. 86 X ft S * *• Fig. 43. Sample Gels of ER and PM from SFV Infected (1) and Mock Infected (M) BHK Cells. 37 0 - 1 1 Hour PM/ER Pulse Chase Experiment An experiment identical in form to the 0 - 6 hour micro-somal pulse chase experiment was performed using the PM/ER fractionation technique. Infected BHK cells were pulsed (100 uCi. L-Leu per 150 mm. x 15 mm. plate) and chased for 0, 1, 2 , 4 , 6, 8, and 11 hours. Three plates were harvested for each time point and fractionated into PM/ER as outlined in Methods. Virus was harvested and isolated on a sucrose gradient as before. The results of the PM/ER gels are shown in Figures 4 4 and 4 5 . Gels of the endoplasmic, reticulum show the existence of precursors reported by Karanen and Kaariainen (97) and Morser and Burke ( 9 2 ) in total protein extracts. Proteins correspond-ing to NVP165, NVP97, and PEg were evident at 0 hours chase. In addition E-^ , Eg, and nucleocapsid were clearly present. In the following chase, precursor activity and E-^  Eg activity diminished. Again nucleocapsid was present in excess of and Eg and failed to diminnsh.over the 11 hours of chase — in fact the radioactivity in the nucleocapsid continued to rise slightly. There was, however, a decrease in nucleocapsid specific radio-activity since the intensity of the Coomassie blue peak (corresponding to total nucleocapsid) increased to a greater extent than labelled nucleocapsid. From the PM gels i t i s evident that there was a rise in labelled E-^ , Eg, and nucleocapsid over the span of 0-4 hours chase. This rise was followed by a gradual drop in radioactivity as -labelled virus began to be produced. The rise and f a l l of 88 E-j_ Eg and nucleocapsid radioactivity appeared to flow in parallel indicating that a l l 3 proteins may be inserted into the PM at the same time. It i s interesting to note the absence of precursor in the PM (particularly PEg) since i t has been reported by Jones _et_al. { 8 7 , 8 8 ) that PEg might be associated with the PM where i t is cleaved to Eg. This aspect bears further investigation with studies using cleavage inhibitors such as TPCK. Virus purification gradients indicated that labelled virus began to be produced at about 2 hours (Fig. 46). Maximal rates of virus production, however, were not reached u n t i l 6-8 hours. The total virus was then analyzed by SDS electrophoresis (Fig.47) and i t was apparent that labelled nucleocapsid was not"present in excess but at a level of about 1/5 the total radioactivity in E^ and Eg corresponding to distribution of Leu in purified virus. The results of this 0 - 1 1 hour pulse experiment are summarized in Fig. 48. A pleasing relationship between E-^  Eg in the endoplasmic reticulum, plasma membrane, and free virus is evident. E-^  Eg label decreases maximally in the ER between 0 - 4 hours chase while i t increases over the same time interval in the PM. Upon reaching a maximal level at 4 hrs. in the PM, 3 H- E-^  Eg then f a l l s rapidly as virus is released. We contend that this relationship supports the concept that SFV matures by budding from the plasma membrane. We do not at present under-stand the significance of the low turnover rate of nucleocapsid protein in the endoplasmic reticulum. $9 0.0-:.--:-|.--:-.|::::|. G P M . •x i d 3 2.0 : 1 • i : : i viii^J ;biii,L l_:.Hr..;'ChaSG \ii.L-ii,;.!;.dd;iJLLilJ ii!.:;.!" -| l i p . cm. gel cm. gel CPM cm. gel cm. gel Fig. 44. Profiles of ER SDS Acrylamide Gels L—Coomassie Blue 3n-Leu) i i i •!li]iT:'i rrrr iiiliili: . . . . . f ^ \ - -7-T_" : ; ! ! ! : ! —rf-H ' I . I iii i cm. gel cm. g e l CPM v xlO cm. gel cm. gel F i g . 44 (Continued) "91 CPM cm. gel c m* g e l Fig. 4 5 . Profiles of PM SDS Acrylamide Gels ( Coomassie Blue H-Leu) 92 F i g . 45. (Continued) 9 a.. 6.u 3... 2-1, 0 , 0 Hrs. Chase. *. ». .. « i . ... J % „ u , f f < 9 CPM 4 K101 7*-6 -5 -3>-2 -1 . 0 . 1 Hrs. Chase 9 4 F i g . 4 6 . (Continued) 95 3 . 0 2 . 0 CPM o 1 . 0 1 Hr. Chase i i i t i | « l - C i.O J" *.o ((•,„ c - , „ -J.f, 1,o 9 , 3 ; 0 2 . 0 CPM -x i 0 j 1 . 0 cm. gel 2 Hrs. Chase ' ' . 1 „• ' ' - ' i . . 0 I C - a o a-0 -to g.c c-,o 7 , c .go ? 0 cm. gel 3 . 0 P U T " ^ • 0 10'' 1 . 0 Fig. 4 7 . 0 - 1 1 Hrs. Chase Virus Gels 96 3.0 2.0 3.0 2.0 CPM-x i d 3 l . o 0 6 Hrs. Chase cm. g e l cm. g e l CPM ' x. i d 3 3.0 2.0 1.0 0 11 Hrs. Chase ' . . . i »<>-l*.m* //,•»• SooO • < w »••» *<>•• i*** /*** Endoplasmic Reticulum .... — /vc 4. • (Time (Hrs.) /••• Plasma Membrane • , €.0. 7-4 Time (Hrs.; a* Fig. 48. Time Course of ^ H-Leu Labelled Virus Specified Proteins in PM and ER. 98 DISCUSSION The results presented in this thesis appear to support the concept of SFV obtaining i t s membrane by budding from the plasma membrane. This is to say the virus glycoprotein are inserted into the host PM, the nucleocapsid aligns with these proteins and buds outwards into the extracellular medium to form mature virus containing v i r a l proteins and host PM l i p i d . Microsome studies showed that i t takes a relatively long 3 time ( 4 - 6 hours) to chase a 20 minute pulse of H - Leu from the 100,000 xg microsomal membrane fraction into free virus. Nucleocapsid was always present in excess when compared to the theoretical levels found in free virus. The degree to which the capsid protein was present appeared to vary with the time of infection at which the pulse of H - Leu was applied; when applied at 5 hours infection nucleocapsid was i n i t i a l l y present at levels .60 times those of Eg while i f applied at 3 hours infection i t was only 1.4 - 2.5 times in excess of virus levels. This may reflect a form of regulation in the translation of structural proteins for SFV. Regulation in the translation of polio and Mengo virus proteins has recently been suggested by Paucha and Colter (131). These viruses are also ssRNA viruses which produce their proteins through post-translational cleavage of a precursor peptide. Structural proteins were found to increase progressively in relation -to non-structural proteins from early to late log phase — premature termination 99 of protein synthesis was occurring. It i s known that for RNA phages structural proteins can bind to mRNA and hinder trans-lation; this has been suggested as a method of regulation both for phage and picornaviruses (83). We must also explain the s t a b i l i t y of nucleocapsid protein in microsomes and ER of infected c e l l s . It was established that nucleocapsid was always present in excess of envelope proteins in the ER fraction; such was not the case in-PM and free virus. A low turnover of nucleocapsid could be postulated i f premature termination occurred to produce excess capsid protein which could not be u t i l i z e d completely during virus assembly (Fig. 49). Replicase RNA Polymerase Structural Proteins Regulatory Proteins 5» . : . V 42S RNA NC E-, E 9/E. 5 • . -* , .3' 26S RNA regulatory point and point of premature termination Fig. 49. Scheme for Regulation of Translation of Structural Proteins The advantages of excess production of nucleocapsid would be a complete u t i l i z a t i o n of virus glycoprotein inserted into the PM of the host c e l l . The ER/PM pulse chase studies demonstrated a clear chase of labelled envelope proteins from ER to PM of Infected cells and then into virus (Fig. 48). This suggests that the virus obtains i t s envelope by budding from the PM of the host c e l l . The time course of virus molecule production based upon the data in Fig. 48 is shown in Fig. 50. Counting efficiency for H3-Leu was 30$. The moles of virus protein produced were calculated as follows: ^ . r DPM -i v r moles Leu - i - l moles virus protein = L s p e c i f i c a c t i v i t y J X Lmole virus protein J of Leu moles Leu _ moles Leu x no. of amino acids mole virus protein ~ 100 amino acids mole virus protein no.'of amino acids _ molecular weight of virus protein mole virus protein "(average molecular weight of amino acids - molecular weight of water) Eg. Nucleocapsid j- DPM -j 1.32 X 10i7DPM/mole Leu" r 3.78 moles Leu Y ' 34,000 gm/mole NC -i ' 100 moles amino (135-18)gm/mole ..amino acid J • " acids;. " -DPM ' Eg. E X E 2 1.45 X 1 0 1 8 DPM/mole NC r DPM : -j 1.32 X 10i7DPM/mole Leu r 5.68 moles Leu Y 101,000 gm/mole EjE2 ' -, L100 moles amino (133.-18)gm/mole amino acid -I acids • ! DPM 100B E g i P E 2 DPM r J J r i » l - i • [ rrf^ J 1.32 X 10 1 DPM/mole Leu f5.99 moles Leu Y 68,000 gm/mole PE2 ] 100 moles amino (133-18) gm/mole amino acid acids = D^PM  4.68 X 10 1 8 DPM/mole PEg Eg. Virus DPM 1.32 X 1 0 1 7 DPM/mole Leu ] r'3.78 moles Leu X 34,000 gm/mole NC ] X 190 mole NC 100 moles amino (135-18) gm/mole amino acid mole virus acids-+ f5.68 moles Leu X 101,000 gm/mole E1E2 ] X 190 mole E ± E 2 100 moles amino (133-18) gm/mole amino acid mole virus acids r9.58 moles Leu X 10,000 gm/mole E3 ] X 190 moles E 3 100 moles amino ( i 3 i_ig) gm/mole amino acid mole virus aicds DPM I".74 X 10 2 1 DPM/mole virus 100C 18 16 H H X 10 co o S ENDOPLASMIC RETICULUM — —• 2 J-0 16 14 t I—I I o 12 H M 1 Q 8 co CD o s o 6 4 CN H w 2 • PE, E 1 E 2 — NO 1 t 5 S~ 7 Time (Hrs.) PLASMA MEMBRANE. E 1 E 2 NC Jipu-s-"? 0 ^ 35 30 3 Time (Hrs.) Fig. 50. Moles of Virus Proteins Formed in ER and PM of Infected Cells ' 1 0 1 Membrane assembly by budding from the plasma membrane i s not unique to just the togaviruses. Rhabdoviruses, myxoviruses, paramyxoviruses, and oncornaviruses are a l l reputed to obtain their membranes by budding from the plasma membrane. Upon infection host c e l l protein and RNA synthesis are inhibited by a s t i l l unknown process. The nucleocapsids are assembled in the cytoplasm whereas the glycoproteins become membrane-bound soon after translation and are apparently glycosylated by host c e l l enzymes in the endoplasmic reticulum (predominantly in the smooth membrane). Our results indicate nucleocapsid protein may also be membrane bound (based on microsomal pellet and ER studies) in SFV infected c e l l s . The role of free poly-somes and membrane polysomes in v i r a l protein synthesis has yet to be elucidated for Group A togaviruses. Results presented in this thesis suggest that post-translational cleavage of the large precursor polypeptide for SFV proteins occurs in associat-ion with the ER. The v i r a l glycoproteins are inserted into the plasma membrane through an unknown mechanism - perhaps some transport or carrier protein i s involved. Host c e l l proteins are not found'in the v i r a l membranes of mature virus particles in most cases. An exception to this rule l i e s in the case of RNA tumor viruses. Virus membranes must be derived from segments of the plasma membrane from which host c e l l proteins are excluded. The envelope proteins are believed to aggregate by a process of lateral diffusion and form patches once they are inserted into the plasma membrane and thereby exclude a l l other proteins. The next event appears to be the alignment of 102 of. nucleocapsid with the virus protein patches in the PM apparently in a stoichiometry similar to that in the mature virus - one nucleocapsid per molecule of E^, Eg, and E^. Electronmicrographs show an outward bulge of the PM as the virion matures and buds into the extracellular medium taking with i t host PM l i p i d , envelope proteins, and nucleocapsid. A schematic of this process i s shown in Fig. 51. Extracellular Virus PM Nucleocapsid Glycosylation (Smooth ER) (Rough ER and free polysomes) NC E i E2/E3 26S RNA NC El E2/E3 Polymerase Replicase ,42S RNA Fig. 51. Schematic of SFV Biogenesis. 103 One of the chief areas of question in the assembly of Group A togaviruses l i e s in the significance of CPV-2. To date these vacuoles have only been demonstrated to exist through electron-microscopy, there i s absolutely no biochemical evidence for the existence of CPV-2. These so-called cytopathic vacuoles may merely be artifacts incurred during EM sample preparation. Studies similar to the ones outlined in this thesis have been carried out using myxoviruses, paramyxoviruses, and rhabdoviruses but they are incomplete and in many instances unsatisfactory. Investigators using vesicular stomatitus virus (a rhabdo-virus) have shown that these virus proteins are associated with the membrane fractions of infected cells. Wagner et a l . (132, 133) showed that after a 1.5 hour pulse with radioactive amino acids, v i r a l proteins G and M are associated with smooth or rough ER and PM whereas proteins N and NS are i n i t i a l l y found in the membrane-free cytoplasmic fraction. Cohen ejb a l . (114) and David (115) further showed, using short pulses with radio-active amino acids, that newly synthesized G and M proteins become rapidly associated with the host c e l l plasma membrane (within a matter of 20 minutes), whereas the N protein only becomes associated with PM after a much longer period. Budding then occurs. These groups contend that mRNA's' (there are 5 sep-arate ones—28S and 4 species of 13S -15S RNA) for G and M proteins are located on or near host c e l l membranes and, after translat-ion, these proteins are inserted directly into the c e l l plasma membrane where virus maturation occurs. No clear time course of 104 v i r a l protein migration from ER to PM and then into free virus was presented yet the rate of incorporation of label and the effect of addition of unlabelled amino acids appeared much more rapid than with togaviruses. Studies with VSV have now shifted to intracellular compartmentalization and the synthesis of virus protein. The membrane glycoprotein G has now been shown to be synthesized only on membrane bound polysomes while M, N, NS, and L proteins are synthesized on both free and membrane bound polysomes (134, 135)« RNA analysis also shows that the messenger for G protein was missing in the free polysome fract-ion but was present in the membrane bound polysomes. The role of free compared to membrane bound polysomes has not yet been investigated with togaviruses. Pulse-chase studies have been initiated to some degree in influenza. The results are often confusing and incomplete. I n i t i a l l y Compans (108) fractionated infected cells and revealed that the smooth cytoplasmic membranes contained large amounts of v i r a l glycoprotein (HA and NA), as well as the nonglycosylated polypeptide (M). Rough membranes also contained the uncleaved hemagglutinin (HA), and results of pulse-chase studies suggested a migration of this polypeptide from rough ER (where i t was synthesized) to smooth ER". The nucleocapsid protein (NP) was mainly associated with the soluble cytoplasm while the non-structural protein (NS) was associated with the polysomes (both free and membrane bound). HA was not cleaved to HA-^  and HAg in the intracytoplasmic membranes. Unfortunately, PM was not isolated in these experiments. Similar work with identical 105 results was performed by Klenk et a l . (109) using the same c e l l fractionation technique. P protein was found in the cytosol after long labelling periods along with NP. These workers demonstrated the migration of non-glycosylated HA from rough ER to smooth ER, where most of glycosylation occurred. Work closest to that presented in this thesis was performed independ-ently by Hay (111) and Meier-Ewert and Compans (112). Hay succeeded in chasing influenza proteins from rough ER to PM over a span of 1 hour but he did not chase the label from the PM into the virus. Interestingly the M protein appears at constant amounts from 0 - 60 minutes chase in a l l membrane fractions and appears to be inserted instantaneously into the host c e i l PM even before HA and NA. Hay did not follow the fate of nucleo-capsid or the non-structural protein. Meier-Ewert and Compans performed pulse chase studies which were again incomplete in that virus peptides were not chased out of the PM into the virus only from the rough ER to Smooth ER over a period of 0-11 hours. No attempt was made to isolate PM from infected cells and characterize v i r a l proteins which i t contained. Investigators working with the enveloped viruses have not offered clear evidence concerning virus morphogenesis through pulse chase experiments up un t i l now. Further work must be performed to prove that virus envelope li p i d s are derived from the host PM in the process of budding. In ;future studies the-PM of the host c e l l w i l l be radioactively labelled with the aid of phospholipid exchange proteins and the incorporation of radioactive phospholipid into virus 106 measured. It would also be valuable to determine the role of membrane bound and free polysomes in SFV production. A time course of virus protein migration from rough ER to smooth ER and then to PM would yield insight into the process of glyco-sylation of SFV envelope proteins. 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