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Studies concerning the mechanism for the membrane assembly of Semliki Forest virus Richardson, Christopher Donald 1978

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STUDIES CONCERNING THE MECHANISM FOR THE MEMBRANE ASSEMBLY OF SEMLIKI FOREST VIRUS CHRISTOPHER DONALD RICHARDSON B.Sc, University of B r i t i s h Columbia, 1973 M.Sc, University'of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA © February, 1978 Christopher D. Richardson m 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 requ i rement s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that 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 tudy . I f u r t h e r agree 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 purposes 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 . It i s u n d e r s t o o d that 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 thout 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 lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date /Vlatch [> / f 7 ? 6 ABSTRACT The data from chemical studies and e l e c t r o n microscopy-suggest that S e m l i k i Forest v i r u s obtains i t s envelope by budding i n t o the medium from the plasma membrane of the host c e l l . Biochemical evidence f o r ' t h i s phenomenon, however, has not been published. Therefore, we undertook a s e r i e s of pulse-chase s t u d i e s so that we might q u a n t i t a t i v e l y evaluate the importance of the budding mechanism i n the morphogenesis of Semliki Forest v i r u s . Baby hamster kidney c e l l s (clone 13) were grown i n c u l t u r e and i n f e c t e d w i t h S e m l i k i ' F o r e s t v i r u s . The c e l l s were exposed "to 14;„5-HJ l e u c i n e f o r 20 min and the subsequent i n c o r p o r a t i o n of the l a b e l i n t o v i r u s p r o t e i n s a s s o c i a t e d w i t h cytoplasmic membrane and e x t r a c e l l u l a r v i r u s was determined. I n i t i a l experiments had been conducted p r e v i o u s l y w i t h microsomes and a precursor-product r e l a t i o n s h i p demonstrated between v i r a l p r o t e i n s i n the microsomes and e x t r a c e l l u l a r v i r u s ( C D . Richardson and D.E. Vance, J . B i o l . Chem. 251, 55LL-5550). Further s t u d i e s were performed w i t h endoplasmic r e t i c u l u m and plasma membrane preparations. Maximum i n c o r p o r a t i o n of [ JH]leucine was observed i n the v i r a l p r o t e i n s l o c a t e d i n the endoplasmic r e t i c u l u m at the end of a 20-min pulse period; greater than 50fo of t h i s r a d i o a c t i v i t y had disappeared w i t h i n 2 hr The plasma membrane f r a c t i o n contained no r a d i o a c t i v i t y at the end of the pulse period; subsequently, maximal l a b e l i n g of the v i r a l p r o t e i n s i n the plasma membrane occurred 3 hr i n t o the chase pe r i o d , and these l a b e l e d p r o t e i n s disappeared from t h i s membrane 8.5 hr afte r the pulse. At 8.5 hr chase," maximum incorporation of the labeled proteins into e x t r a c e l l u l a r virus was observed. These data are consistent with a precursor-product r e l a t i o n s h i p between v i r a l proteins i n the endoplasmic reticulum, plasma membrane, and e x t r a c e l l u l a r media. V i r a l proteins migrate to the plasma membrane and are subsequently incorporated into e x t r a c e l l u l a r v i r u s . A l l the r a d i o a c t i v i t y i n the e x t r a c e l l u l a r virus appears to have been derived from v i r a l proteins associated with the plasma membrane of the c e l l . Therefore, mechanismsffor^the"morpho-genesis of Semliki Forest v i r u s ( i n baby hamster kidney c e l l s ) , other than budding from the plasma membrane, are u n l i k e l y to.be of quantitative importance. The p o s s i b i l i t y that an i n t a c t cytoskeletal system might be required f o r the assembly of Semliki Forest virus was inves-tigated. The microtubules and microfilaments of baby hamster kidney c e l l s (BHK-21)_ were disassembled with s p e c i f i c drugs and the eff e c t on production of e x t r a c e l l u l a r v i r u s was deter-mined. Colchicine, Nocodazole, dibucaine, and cytochalasin B reduced the production of e x t r a c e l l u l a r virus by 75-90$. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [-^Hlleucine of [-^S] methionine. At various times a f t e r addition of one of these drugs, the incorporation of the labeled pre-cursors into v i r a l proteins associated with endoplasmic reticulum or plasma membrane of the c e l l was evaluated. The res u l t s c l e a r l y show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the c e l l where they accumulated. These studies strongly suggested that the cytoskeletal system was involved i n the f i n a l stages of membrane assembly of Semliki Forest v i r u s at the plasma membrane. Studies were also performed with the cross-linking agents -dimethylsuberimidate (DMS), dith i o b i s ( s u c c i n i m i d y l propionate) (DSP), and dimethylthiobi's( propionimidate) (DTBP) . The proteins of p u r i f i e d v i r u s and infected c e l l s reacted with these agents and the cross-linked proteins were evaluated by one- and two-dimensional SDS electrophoresis. Nucleocapsid protein cross-linked to form up to pentameric complexes, and envelope proteins reacted to y i e l d dimeric species. Nucleocapsid protein did not cross-l i n k with envelope proteins. Cross-linking agents were also u t i l i z e d to determine the effects of colch i c i n e and dibucaine on the proximity of v i r a l proteins to each other i n the plasma membrane of the host c e l l . Colchicine (which disrupts microtubules) appeared to have no effect on the degree to which"f-^s]-labeled virus proteins reacted with the agents, while dibucaine (which supposedly disrupts both microtubules and microfilaments) abolished envelope protein dimers dramatically. This r e s u l t was interpreted to mean that microtubules may not be required f o r the formation of patches of virus proteins i n the plasma membrane pr i o r to budding, while microfilaments may pOiay a more dominant r o l e i n t h i s process. i v TABLE OF CONTENTS Page ABSTRACT • 1 TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES v. . . i x ABBREVIATIONS xv ACKNOWLEGEMENTS x v i i i DEDICATION xix INTRODUCTION 1 A. Mechanism of Membrane Assembly for Enveloped Viruses 1 1. The Structure of Group A Togaviruses . . . . 3 2. Replication of Togaviruses 7 (a) Growth Cycle of Group A Togaviruses . . 7 -(b) Replication of V i r a l RNA 8 (c) Translation of V i r u s - S p e c i f i c mRNA . . . 11 (d) Synthesis and Insertion of V i r a l Glycoproteins into the I n t r a c e l l u l a r Membranes of the Host C e l l 13 (e) Formation of Virus Proteins (Post-" Translational Cleavage of Large Precursors) 15 3. Maturation and Membrane Assembly of Togaviruses 22 (a) Electronmicroscopic Observations of Togavirus Assembly 22 (b) Chemical Evidence f o r Maturation of Group A Togaviruses at the Plasma Membrane . . . 24 k. Assembly of Vesicular Stomatitis Virus . . . 25 (a) Structure 25 V (b) Membrane Assembly of Vesicular Stomatitis Virus 26 5 . Assembly of Myxoviruses and Paramyxoviruses 30 (a) Structure of Myxoviruses and Paramyxoviruses 30 (b) Membrane Assembly of Myxoviruses and Paramyxoviruses 31 B. Cytoskeletal Components of Membrane Systems and Their Involvement i n the Membrane Assembly of Viruses 32 1. Microtubules 33 2 . Microfilaments 36 3 . Microtubule disruptors 37 k. The disruption of Microfilaments kl 5 . Local Anesthetics - Disruptors of Both Microtubules and Microfilaments k2 6. Microtubule and Microfilament Function Within the c e l l ^3 (a) Cytoskeletal Transmembrane Control . . . . ^3 (b) C e l l Secretion and the Cytoskeletal System 48 (c) I n t r a c e l l u l a r Movement and the C. 1 " Cytoskeletal System k9 7. Previous Evidence f o r Cytoskeletal Involvement i n the Morphogenesis of Viruses 51 C. Chemical Cross-Linking of V i r a l Membrane Proteins 52 1. Chemistry of Cross-Linking Agents 52 2 . Cross-Linking of V i r a l Proteins 52 (a) Vesicular Stomatitis Virus 52 (b) Semliki Forest Virus $k (c) Adenovirus . . . . . 55 v i i D. Cytoskeletal Disruptors and Their Effects Upon Virus Assembly 90 1. The Effect of Colchicine on Virus Maturation 90 2. The Effe c t of Dibucaine on Virus Maturation 113 3. The Effe c t of Cytochalasin B on Virus Maturation '122 E. Two-Dimensional Cross-Linking Studies Performed Upon P u r i f i e d Virus 133 F. Two-Dimensional Cross-Linking Studies Performed on Plasma Membranes from Infected C e l l s . . . . 139 G. Two-Dimensional Cross-Linking Studies Performed on Plasma Membranes from Infected C e l l s which were Treated With Colchicine and Dibucaine . . 1^0 DISCUSSION , -l ; 4 f BIBLIOGRAPHY ,159 v i i i LIST OF TABLES Page 1. Groups of L i p i d Containing Viruses 1 2. Family: Togaviridae 2 3. Moles of Carbohydrate Per Mole of Protein i n Semliki Forest Virus 5 4-. L i p i d Class Composition of Semliki Forest Virus . . . 6 5. Number of Different Molecules i n Semliki Forest Virus 6 6. Representative Agents Used for Cross-Linking Proteins ,;i 53 7. A c t i v i t i e s of Enzymes Ch a r a c t e r i s t i c of Endoplasmic Reticulum or Plasma Membrane 80 8. Effects of Various Concentrations of Colchicine on C e l l V i a b i l i t y 91 9. Effects of Various Concentrations of Dibucaine on C e l l V i a b i l i t y 113 10. Incorporation of yK Leucine into T r i c h l o r o a c e t i c • •. : Acid P r e c i p i t a b l e Material from BHK C e l l s i n the Presence or Absence of Dibucaine 121 11. Effects of Various Concentrations of Cytochalasin B on C e l l V i a b i l i t y 122 i x LIST OF FIGURES Page 1. The Structure of Semliki Forest Virus 3 2 . Time Course of Virus Production i n BHK-21 C e l l s . . 7 3 . Replicative Model of Simons and Strauss . . . . . . 9 k. Model f o r the Synthesis of Sindbis V i r i o n Proteins, Subsequent Nascent Cleavages, and Sequestering of the Envelope Proteins Ik 5 . An SDS Polyacrylamide Gel Showing the Radioactive Polypeptides Which Are Present i n C e l l s Infected With t s 2 Mutants of Sindbis and HR (Wild Strain) Sindbis 16 6 . Post-Translational Cleavage i n Formation of SF v i r u s Structural Proteins . . 19 7 . Scheme f o r the Post-Translational Processing of Precursors to the Ndn-Structural Proteins of a Temperature Sensitive Mutant of Semliki Forest Virus 29 8 . Formation of Non-Structural Proteins by Post-Translational Cleavage of Precursors i n Ce l l s Which Were Infected With Semliki Forest Virus 21 9. Formation of Non-Structural Proteins by Post-Translational Cleavage of Precursors In C e l l s Which Were Infected By Sindbis Virus 21 1 0 . Structure and Composition of Vesicular Stomatitis Virus 26 1 1 . Kinetics I l l u s t r a t i n g the Association of Vesicular Stomatitis Virus With C e l l Supernatant and Membrane Fractions During a Pulse-Chase Experiment 28 1 2 . Maturation of Vesi c u l a r Stomatitis Virus Within the C e l l 29 1 3 . Structure and Protein Composition of Myxoviruses . . 3 ° Ik. Structure and Protein Composition of Paramyxoviruses 30 X 1 5 . The Basic Composition of a Microtubule 33 1 6 . Fibroblasts Labeled With Fluorescent Anti-Tubulin 3 4 1 7 . Structures of Drugs Which Disrupt Microtubules . . 38 18. The Chemical Structure of Lumicolchicine and Its Absorption Spectrum As Compared To That of Colchicine 39 1 9 . Chemical Structure of Cytochalasin B 41 2 0 . Structure of T e r t i a r y Amine Anesthetics 43 2 1 . E f f e c t of Drugs on Lymphocytes Which Were In Contact With Ahti-Ig and/or Con A 45 2 2 . E f f e c t of Drugs on 3T3 Fibroblasts Which Were In Contact With Anti-Ig and/or Con A ,, 46 23- Proposed Interaction of Microtubules and Micro-filaments With Membrane Proteins 47 24. Autoradiograph of 2-Dimensional SDS Polyacrylamide Gels Which I l l u s t r a t e the Cross-Linked Proteins of Vesicular Stomatitis Virus 53 25- P u r i f i e d Semliki Forest Virus and Standard Proteins Which Were Subjected to Electrophoresis on 7-5% Polyacrylamide Gels i n the Presence of Sodium Dodecyl Sulfate 75 26v Absorbance Scan of Semliki Forest-Virus Proteins Which Were Separated on a 7 >5f° Polyacrylamide SDS g e l 76 2 7 . Proteins From SF Virus Which Were Subjected to SDS Electrophoresis on 6 . 5 $ Polyacrylamide Slab Gels i n the Presence and Absence of 10% (v/v) B-Mercaptoethanol 28. P r o f i l e s of Enzymatic A c t i v i t i e s From a Dis-continuous Sucrose Gradient Used For I s o l a t i o n of Endoplasmic Reticulum and Plasma Membrane . . . . 79 x i 29. I n c o r p o r a t i o n o f [ J H ] J . L e u c i n e I n t o t h e P r o t e i n s Of E n d o p l a s m i c R e t i c u l u m O v e r a 0-8.5 H o u r C h a s e P e r i o d .82. _ 30. I n c o r p o r a t i o n o f ,[ H]_ L e u c i n e I n t o t h e P r o t e i n s Jfo'f ¥ h e P l a s m a Membrane 0-8.5 H o u r s A f t e r a 20 M i n P u l s e 8 4 ' -31. T i m e C o u r s e f o r I n c o r p o r a t i o n o f [^H]^ L e u c i n e I n t o Y i r u s - S p e c i f i e d P r e c u r s o r P r o t e i n s A s s o c i a t e d W i t h The E n d o p l a s m i c R e t i c u l u m 86 -32. T i m e C o u r s e f o r I n c o r p o r a t i o n o f [ ^ H ] i L e u c i n e I n t o V i r u s - S p e c i f i e d P r o t e i n s A s s o c i a t e d W i t h t h e E n d o p l a s m i c R e t i c u l u m 87* 33- T i m e C o u r s e f o r I n c o r p o r a t i o n o f L e u c i n e I n t o V i r u s - S p e c i f i e d P r o t e i n s A s s o c i a t e d W i t h P l a s m a Membrane a n d E x t r a c e l l u l a r V i r u s 88/:' 34. P r o f i l e s o f S u c r o s e G r a d i e n t s C o n t a i n i n g R a d i o a c t i v e E x t r a c e l l u l a r V i r u s W h i c h Was P r o d u c e d O v e r a 0-8.5 H o u r C h a s e 8 9 ' 35« P r o f i l e s o f S u c r o s e G r a d i e n t s C o n t a i n i n g R a d i o a c t i v e E x t r a c e l l u l a r V i r u s P r o d u c e d b y C e l l s i n t h e P r e s e n c e a n d A b s e n c e o f C o l c h i c i n e . . 92* 36. P r o f i l e s o f S u c r o s e G r a d i e n t s C o n t a i n i n g R a d i o a c t i v e E x t r a c e l l u l a r V i r u s P r o d u c e d b y C e l l s i n t h e P r e s e n c e a n d Ab sence^ o f C o l c h i c i n e 9 3 37. P h o t o g r a p h o f S u c r o s e G r a d i e n t s W h i c h C o n t a i n V i r u s P r o d u c e d i n E i t h e r t h e A b s e n c e o r P r e s e n c e o f C o l c h i c i n e 94' 38. A b s o r b a n c e S p e c t r u m f o r C o l c h i c i n e a n d L u m i c o l c h i c i n e a t Room T e m p e r a t u r e . S:£f 39• E f f e c t s o f L u m i c o l c h i c i n e a n d C o l c h i c i n e o n t h e P r o d u c t i o n o f R a d i o a c t i v e E x t r a c e l l u l a r V i r u s . . . . 9.6' 4 0 . E f f e c t o f N o c o d a z o l e on t h e P r o d u c t i o n o f R a d i o a c t i v e E x t r a c e l l u l a r V i r u s 97' 4 1 . I n c o r p o r a t i o n o f X^H ] L e u c i n e I n t o T r i c h l o r o a c e t i c A c i d (TCA) P r e c i p i t a b l e M a t e r i a l f r o m BHK C e l l s i n t h e P r e s e n c e o r A b s e n c e o f C o l c h i c i n e 98 x i i 42. P r o f i l e s of Sucrose Gradients Containing Radioactive E x t r a c e l l u l a r Virus Produced by C e l l s Which Were Continuously Labeled with [3H]Leucine i n the Presence and Absence of Colchicine .100 4 3 . P r o f i l e s of SDS Gels Which Contained Plasma Membrane Proteins from C e l l s Which Were Continuously Labeled i n the Presence and Absence of Colchicine 101 4 4 . Incorporation of c[-^H]Leucine into the V i r a l Proteins of the Plasma Membrane of C e l l s i n the Presence and Absence of Colchicine 103 4 5 . Plasma Membrane Proteins Which Were Labeled with [/HJLeucine During a Pulse-Chase Experiment Which Was Performed i n the Presence and Absence of Colchicine. 104 4 6 . Radioactive E x t r a c e l l u l a r Virus Isolated During a Pulse-Chase Experiment Which Was Performed i n the Presence and Absence of Colchicine 106 4 7 . Time Course f o r Incorporation of [ vH]Leucine into V i r u s - S p e c i f i e d Proteins Associated with the Plasma Membrane During a Pulse-Chase Experiment Which Was Performed i n the Presence and Absence of Colchicine. 107 3 ^  48. Autoradiogram of .Jj^s ]-Labeled Plasma Membrane Proteins During a Pulse-Chase Experiment Performed i n the Presence and Absence of Colchicine 108 49- Autoradiogram of [-^ S ]-Labeled Endoplasmic Reticulum Proteins from a Pulse-Chase Experiment Performed i n the Presence and Absence of Colchicine 109 5 0 . Time Course f o r the Incorporation of ["^SjMethionine into the V i r a l Proteins of the Plasma Membrane from C e l l s Which Were Pulse-Labeled i n the Presence or Absence of Colchicine 110 5 1 . P r o f i l e s of [- ^ S]-Labeled E x t r a c e l l u l a r Virus Produced During a Pulse-Chase Experiment Which Was Performed i n the Presence and Absence of Colchicine 112 5 2 . P r o f i l e s of Sucrose Gradients Containing Radioactive E x t r a c e l l u l a r Virus Produced by C e l l s i n the Presence and Absence of Dibucaine 114 53- Photograph of Sucrose Gradients Which Contain Virus Produced i n Either the Absence or Presence of Dibucaine 115 5 4 . P r o f i l e s of SDS Polyacrylamide Gels Which Contained Plasma Membrane Proteins from C e l l s Which Were Continuously Labeled i n the Presence and Absence of Dibucaine 116 x i i i 55' Accumulation of V i r a l Protein i n the Plasma Membranes of C e l l s Treated with Dibucaine .. 119 56. Autoradiogram of [-^S]-Labeled Proteins from the Endoplasmic Reticulum "of BHK C e l l s Exposed to Dibucaine 118 57. P r o f i l e s of Sucrose Gradients Which Contain Radioactive E x t r a c e l l u l a r Virus from a Pulse-Chase Experiment Performed i n the Presence and Absence of Dibucaine . 120 58. P r o f i l e s of Sucrose Gradients Containing Radioactive E x t r a c e l l u l a r Virus Produced by C e l l s i n the Presence ,-a. and Absence of Cytochalasin B 123 59. P r o f i l e s of SDS Polyacrylamide Gels Which Contained Plasma Membrane and Endoplasmic Reticulum Proteins from C e l l s Which Were Continuously Labeled i n the Presence and Absence of Cytochalasin B 1?'5 60. Electronmicrograph of Infected BHK C e l l s Which Portrays Virus Budding at the Plasma Membrane i n the Absence of Colchicine and Dibucaine .128 61. Electronmicrograph of Infected BHK C e l l s Showing Accumulation of Nucleocapsids at the Plasma Membrane i n the Presence of Colchicine .129 62. Electronmicrograph of Infected BHK Cell-s showing Accumulation of Nucleocapsids at the Plasma Membrane i n the Presence of Dibucaine 13° 63. Electronmicrograph of a Cytopathic Vacuole Found i n Infected BHK C e l l s Which Were Treated with Dibucaine ^ l 6 4 . Electronmicrograph of a Cytopathic Vacuole Found i n Infected BHK C e l l s Which Were Treated with Dibucaine .132 65. P u r i f i e d SF Virus Which Was Cross-Linked with DMS and DSP and Electrophoresed on 3-5% Polyacrylamide C y l i n d r i c a l Gels 133 66. Absorbance Scans of 3>5% Polyacrylamide Gels Containing Proteins Which Were Cross-Linked with DMS and DSP 13^ 67. P u r i f i e d SF v i r u s Which Was Cross-Linked with DTBP and Subjected to Electrophoresis on 6.-^Polyacrylamide Slab Gels v 135 68. P u r i f i e d SF vi r u s Which Was Cross-Linked with DSP and Subjected to Electrophoresis on 6.5$ Polyacrylamide Slab Gels .136 xiv 6 9 . Two-Dimensional Electrophoresis of SF Virus Proteins Which Had Been Cross-Linked with DSP 138 7 0 . Two-Dimensional Autoradiogram of Plasma Membrane Proteins from SF Virus-Infected C e l l s Which Had Been Cross-Linked with DSP 140 7 1 . Two-Dimensional Autoradiogram of Plasma Membrane Proteins from SF virus-Infected C e l l s Which Had Been Cross-Linked with DTBP 141 7 2 . One-Dimensional Autoradiogram of DTBP Cross-Linked Plasma Membrane Proteins from Infected C e l l s Cultured i n the Presence or Absence of Dibucaine and Colchicine 143 7 3 . Two-Dimensional Autoradiograms of DTBP Cross-Linked Plasma Membrane Proteins from Infected C e l l s Which Were Cultured i n the Presence or Absence of Dibucaine and Colchicine 144 XV LIST OF ABBREVIATIONS SF v i r u s Semliki Forest virus E-^ , Eg i E^ envelope proteins of Semliki Forest v i r u s NC nucleocapsid protein EnE„ combined envelope proteins E i and E2 which often do not resolve by SDS electrophoresis; t h i s gives the impression of one protein NVP 130 (or ts-2 protein) non-virion precursor protein (molecular weight 130,000) NVP 98 ( or non-virion precursor protein (molecular weight B protein) 98,000) NVP 68 (or non-virion precursor protein (molecular weight PE 2) 68,000) nsp non-structural protein p precursor protein PE phosphatidyl ethanolamine PC phosphatidyl choline PS phosphatidyl serine PI phosphatidyl i n o s i t o l PM plasma membrane ER endoplasmic reticulum BHK baby hamster kidney BK bovine kidney MK monkey kidney CEF chick embryo f i b r o b l a s t PFU plaque forming unit Leu leucine Met methionine xvi' liCi microcurie mCi m i l l i c u r i e mRNA messenger RNA ssRNA single stranded RNA dsRNA double stranded RNA RF r e p l i c a t i v e form RI r e p l i c a t i v e intermediate CPV-1 cytopathic vacuole,type 1 CPV-2 cytopathic vacuole,type 2 ts temperature sens i t i v e mutant G, M, N, NS, ve s i c u l a r stomatitis virus proteins L, P HN, F, F 0 paramyxovirus proteins HA;, NA, M influenza v i r u s proteins TPCK tosylphenylalanine chloromethylketone M.W. molecular weight Cyt. c' cytochrome c Ig immunoglobulin mamps milliamperes ml m i l l i l i t e r s u l m i c r o l i t e r s hr hours cm centimeters mm millimeters nm nanometers mM millimolar uM micromolar x v i i CPM counts per minute SDS sodium dodecyl su l f a t e A550 o p t i c a l absorbance at 550 nanometers Con A concanavalin A TDA t a r t a r y l diazide DMS dimethylsuberimidate DTBP dimethyl thiobispropionimidate DSP d i t h i o b i s (succinimidyl propionate) MMB methyl - 4 - mercaptobutyrimidate x v i i i ACKNOWLEDGEMENTS I wish to thank Dr. Dennis E. Vance f o r his guidance, support, and encouragement throughout the course of thi s work. I am also indebted to Karen Catherwood, Harry Paddon and Jennifer Toone f o r t h e i r excellent technical assistance. The electronmicrographs were performed i n the laboratory of Dr. W. Ovalle by Ms. Susan Shinn. Special thanks goes to Mrs. N. Richardson and Mrs. F. Dowling for helping type t h i s thesis and to Jennifer Toone f o r preparing some of the figures. I also wish to express my grat-itude to the friends which I made i n thi s department. x i x DEDICATION TO MY PARENTS I. INTRODUCTION A. Mechanisms of Membrane Assembly f o r Enveloped Viruses (1-9) A large proportion of v i r u s e s possess l i p o p r o t e i n envelopes as indicated i n Table 1. TABLE I'. Groups of L i p i d Containing Viruses (5) Group Nucleic Acid V i r i o n Shape Size A Type , MW x 10 Pox Virus ds DNA 160 b r i c k shaped 3 0 0 0 x 2 0 0 0 Herpes Virus ds DNA 100 s p h e r i c a l 1200 PM2 Phage ds DNA, 6 s p h e r i c a l 600 Togavirus ss RNA k s p h e r i c a l 700 Myxovirus ss RNA k spherical/filamentous 1000 Paramyxovirus ss RNA 7 spherical/filamentous 1200 Rhabdovirus ss RNA. 6 b u l l e t shaped 700x1750 RNA tumor v i r u s ss RNA 12 s p h e r i c a l • 1200 Arenavirus ss RNA ? s p h e r i c a l 6 0 0 - 1 2 0 0 Coronavirus ss RNA 7 s p h e r i c a l 8 0 0 - 1 2 0 0 The manner i n which various groups of viruses obtain t h e i r membranes v a r i e s . For example, pox viruses are assembled e n t i r e l y i n the cytoplasm of the host c e l l at a "factory" which i s inde-pendent of the plasma membrane and endoplasmic reticulum ( 3 , 8 ) . Recently Stern and Dales (10) have implicated the r o l e of phos-p h o l i p i d exchange proteins i n the t r a n s f e r of phospholipid from microsomes during the assembly of v a c c i n i a envelopes. Herpes viruses, on the other hand, appear to be assembled i n the nucleus and obtain t h e i r envelopes from the nuclear membrane (t h i s must be more thoroughly substantiated). The completed v i r i o n i s -2-probably transported to the plasma membrane by cytoplasmic membrane channels which prevent degradation of vi r u s membranes. Coronaviruses. appear to mature and obtain t h e i r membranes as they pass into the cisternae of the endoplasmic reticulum and cytoplasmic v e s i c l e s . The majority of viruses which possess membranes appear to obtain t h e i r envelopes from the plasma membrane of the host c e l l . Myxoviridae, paramyxoviridae, rhabdoviridae, r e t r o v i r i d a e , arenaviridae, and togaviridae are a l l reputed to mature i n t h i s fashion. The £ami'lty^fogavifidae of which Semliki Forest virus i s a member, - comprises two-ser o l o g i c a l groups - Group A (or alphaviruses) and Group B (or f l a v i v i r u s e s ) . Representative viruses of this family are l i s t e d i n Table 2. TABLE 2 Family: Togaviridae (2) Genus Representative Viruses Alphavirus Sindbis, Semliki Forest Virus, Western Equine Encephalitis, Eastern Equine Encephalitis, Venezuelan Equine Encephalitis, Chikungunya, r u b e l l a (most l i k e l y ) F l a v i v i r u s Dengue types 1-4, yellow fever, St. Louis encephalitis, Japanese encephalitis, West Nile encephalitis, Murray Valley encephalitis, Russian tick-borne encephalitis - 3 -1. The Structure of Group A. Togaviruses The structures of a l l the Group A Togaviruses are almost i f not e n t i r e l y i d e n t i c a l (1, 2). These viruses, of which Semliki Forest v i r u s 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-^ , Eg and E^ are situated i n the envelope and l i e i n close proximity to the virus nucleocapsid (11). The structure of Semliki Forest virus i s shown diagrammatically i n F i g . 1. The virus genome consists of a single strand of 4-2S RNA. £.j MW 52,000 Cholesterol VP ^ 3 n\ &.ut M W 10.000 PL Eg MW 49,000 SEMLIKI FOREST VIRUS F i g . 1. The structure of Semliki Forest v i r u s . The s t r u c t u r a l proteins of Semliki Forest v i r u s have been resolved with SDS polyacrylamide gel electrophoresis. O r i g i n a l l y studies were performed using the gel system of Weber and Osborne (13)' E-^  and Eg were not resolved i n the studies of Hay, Skehel and Burke ( 1 4 ) , Kaariainen et a l . (15)> and Acheson and Tamm (16). Nucleocapsid protein was c l e a r l y evident while no trace of E^ was found on the gels and i t s existence remained unknown. More recently the discontinuous buffer SDS gel electrophoresis systems of Neville (17) and Laemmli ( 1 8 ) were applied to p u r i f i e d v i r u s preparations by Simons (19) and Pfefferkorn (2). These re s u l t s show the molecular weights of E-^ , Eg and nucleocapsid to be 52.000, 49,000 and 34,000 respectively. The existence of E^ i n Semliki Forest virus was not evident u n t i l very recently (12). As yet, t h i s protein has not been demonstrated to be present i n Sindbis v i r u s . Garoff and Simons showed that although E^ cannot be detected on 7.5$ and 10$ SDS acrylamide gels by c l a s s i c a l staining techniques, the small 3 S" protein could be detected with[-^SjMet l a b e l l e d SFV was applied to 10$ SDS gels - the gels were then s l i c e d and assayed for radio a c t i v i t y . More conclusive evidence for the existence of E^ was presented when delipidated membrane protein was eluted from an SDS hydroxylapatite column (12). E-^ , Eg. , and nucleocapsid proteins appear i n equimolar amounts i n the mature v i r i o n and constitute 35-7$. 35.7$.4.9$. and 23.7$ of the t o t a l protein respectively. -5-A l l three SFV membrane pr o t e i n s are g l y c o s y l a t e d . Residues of N-acetylglucosamine, mannose, galactose, fucose, and s i a l i c a c i d appear i n a l l three p r o t e i n s ( 1 2 ) . The carbohydrate sequence f o r some of the g l y c o p r o t e i n s of S e m l i k i Forest v i r u s has r e c e n t l y been proposed ( 2 0 ) . TABLE 3 ( 1 2 ) Moles CHO Residue Per Mole P r o t e i n P r o t e i n -N-acetyl- Mannose Galactose Fucose S i a l i c T o t a l CHO % glucosamine Ac i d by weight E-L 7 5 3 1 2 7-5% E 2 8 1 2 3 1 ^ H-5% 9 L 4 2 3 k5.1% The l i p i d s of the v i r a l membrane c o n s i s t of 32% n e u t r a l l i p i d s , 6l% phospholipids and 7%> g l y c o l i p i d s ( 2 1 ) . The n e u t r a l l i p i d f r a c t i o n of SFV c o n s i s t s almost e x c l u s i v e l y of f r e e c h o l e s t e r o l while the main components of the phospholipids are sphingomyelin, phosphatidyl ethanolamine, phosphatidyl-c h o l i n e and phosphatidyl s e r i n e . The g l y c o l i p i d f r a c t i o n con-t a i n s almost e x c l u s i v e l y s i a l i c - l a c t o s y l ceramides. The d i s -t r i b u t i o n of the var i o u s l i p i d types i s shown as mole r a t i o s i n Table 4 ( 2 1 ) . -6-TABLE 4 L i p i d Class Composition of SFV shown as Mole Ratio Relative to Phospholipids (21) L i p i d Class Mole Ratio Cholesterol G l y c o l i p i d s Phospholipids 0.99 0.08 1.00 0.23 0.33 0.13 0.02 0.20 PE PC PS PI ;Sph i'r.g ojny .ei i n I t i s believed that the l i p i d class composition resembles that of the host plasma membrane. Such a r e l a t i o n s h i p i s also r e -f l e c t e d i n the f a t t y acid composition of the phospholipids i n the virus and plasma membranes of infected BHK-21 c e l l s (21). In summary, a single p a r t i c l e of Semliki Forest virus contains the molecular composition l i s t e d i n Table 5« TABLE 5 (12, 20) Number of Different Molecules i n SFV Constituent Number of Molecules Per V i r i o n RNA, Nucleocapsid Membrane proteins 1 200 550 15,ooo 16,000 Cholesterol Polar l i p i d s PE PC PS PI 3,500 6,400 2,000 200 Sphingomyelins Gangliosides 2,400 1,000 -7-2. Replication of Togaviruses (a) Growth Cycle of Group A Togaviruses The growth of group A togaviruses i s rapid (2). After 2 hours of i n f e c t i o n at 37° vertebrate c e l l s begin to produce v i r u s . Virus production may approach 1000 PFU/cell/hr and the t o t a l y i e l d may approach 10"1"0 PFU/ml growth media. High-est t i t e r s are achieved i n chick embryo f i b r o b l a s t and i n BHK-21 c e l l s . The growth curve f o r Semliki Forest virus i n BHK-21 c e l l s i s shown i n Fig. 2. n'1-—.—.— . 0 ' 2 1 4 5 6 7 8 9 TIME (HOURS) F i g . 2. Time course of virus production i n BHK-21 c e l l s . By 11 hours of i n f e c t i o n cytopathic effects become apparent by l i g h t microscopy and the rate of vi r u s production f a l l s markedly. Vertebrate c e l l s are ultimately destroyed although persistent (chronic) i n f e c t i o n occurs i n the presence of interferon (2, 23). -8-Group A togaviruses i n f e c t arthropod c e l l l i n e s such as those derived from the mosquito (Aedes albopictus) and the t i c k , but chronic persistent infections may also r e s u l t . These persistent infections may also be induced by the presence of interferon (2). (b) Replication of V i r a l RNA Togavirus RNA synthesis i s e a s i l y monitored...in infected c e l l s with or without actinomycin D since the virus represses host c e l l s p e c i f i e d RNA and DNA synthesis (1, 24). Group A v i r a l RNA synthesis reaches detectabite^'levels hy about 2 hours a f t e r i n f e c t i o n , r i s e s to maximal rates by 3 hours, and con-tinues through the period of virus release (25). The 2 p r i n c i p a l forms of single-stranded RNA found i n group A togavirus-infected c e l l s are v i r i o n RNA (42S) and interjacent RNA (26s) (1, 2, 26, 2?). These are the forms of v i r u s mRNA. Minor ssRNA with sedimentation c o e f f i c i e n t s of 38S and 3 3 S also exist. Replication of the RNA of togaviruses proceeds through a multiple-stranded r e p l i c a t i v e intermediate (which i s associated with membranes) sim i l a r to that described f o r picornaviruses and RNA bacteriophage (28). On extraction of infected c e l l s with phenol and separation by CF11 c e l l u l o s e chromatography, 2 d i f f e r e n t types of double stranded RNA can be i s o l a t e d : 1) r e p l i c a t i v e intermediates (RI) - consists of p a r t i a l l y double stranded RNA with single stranded non-hydrogen bonded regions 2) r e p l i c a t i v e forms (RF) - extensively hydrogen bonded double stranded RNA which remains aft e r t o t a l c e l l u l a r RNA i s digested with pancreatic ribonuclease - 9 -These double stranded RNA.1 s were o r i g i n a l l y i s o l a t e d by Pfefferkorn, Burge and Coady (29) and Friedman ( 3 ° ) • Free single-stranded RNA of negative p o l a r i t y ( i e . complementary to virus genome) was not i s o l a t e d from infected c e l l s . Simmons and Strauss ( 3 1 ), Segal and Sreevalsan ( 3 2 ) , and Martin and Burke (33) i d e n t i f i e d three r e p l i c a t i v e forms afte r c e l l u l a r RNA was extracted and digested with pancreatic ribonuclease ( 3 2 ) . Simmons and Strauss (31)> Martin and Burke ( 3 3 ) , and Segal and Sreevalsen purport the existence of 2 intermediates (Fig. 3) which d i f f e r i n the s i t e at which r e p l i c a t i o n i s i n i t i a t e d . Strauss (1) postulates that a regulatory protein binds to template RNA (negative stranded 42S RNA) and allows the 26S RNA to be transcribed from an i n t e r n a l s i t e on the RNA (Fig. 3 ) . j - 2 6 s (|.5*I06 daltons) (4.4 x 1 0 s doltons) Fig. 3 . Replicative Model of Simmons and Strauss (31) -10-Recently Bruton and Kennedy (3^) 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 bonded poly A at the 3 ' end of the 42S p o 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 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 i n -vestigated during virus m u l t i p l i c a t i o n . Negative strand syn-thesis reached a maximum rateo2fshours. post infection'and'thereafter r a p i d l y fells,- The rate of p o s i t i v e strand synthesis increases r a p i d l y up to 3 hr post-infection and remains constant over 8 hr. 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 i i n t r a c e l l u l a r cytopathic vacuoles (designated type 1) (2). A membrane-associated r e p l i c a t i o n complex which contained v i r a l RNA polymerase and r e p l i c a t i v e intermediate was i d e n t i f i e d a f t e r pulse-labeling infected c e l l s with [^H]uridine. P a r t i a l p u r i f i -cation of t h i s structure resulted i n a concentration of CPV-1 ( 3 5 i 3 6 , 3 7 ) . At 5 - 5 hours post i n f e c t i o n c e l l s were lysed, homogenized, and a f r a c t i o n enriched f o r cytopathic vacuoles was i s o l a t e d . The i s o l a t e d v e s i c l e s were membrane li m i t e d and l i n e d by regular membranous sphericles measuring 50 nm i n diameter- these were neither v i r u s cores or v i r i o n s . CPV-1 appearance coincided with RNA production i n BHK, CEF, and L c e l l s infected with SF v i r u s , Sindbis, and Western equine encephalitis viruses. These vacuoles appeared to a r i s e from the Golgi -11-apparatus based on assays using the Golgi marker enzyme, acid phosphatase. In another experiment, a cytoplasmic extract was p u r i f i e d on a discontinuous sucrose gradient. A band contain-ing RNA polymerase a c t i v i t y , L"3H] uridine and type 1 cytopathic vacuoles ( v i s i b l e by electronmicroscopy) was obtained. Similar r e s u l t s have been obtained with poliovirus (38, 39)- Michel and Gomatos (4-0) indicated that r e p l i c a t i v e form and r e p l i c a t i v e intermediate RNA was associated with such membranous structures. Semliki Forest virus RNA polymerase has recently been is o l a t e d and p a r t i a l l y characterized (41, 42). The polymerase was s o l u b i l i z e d from a 15(000 x;g_ membrane p e l l e t with T r i t o n N-101 and p u r i f i e d on an a f f i n i t y column which contained 42S v i r a l RNA as the ligand. Three polypeptides were found to be present i n the p u r i f i e d enzyme - 2 were virus s p e c i f i e d (M.W. 90,000 and M.W. 63,000) and one (M.W. 40,000) appeared to be host-specified or possibly a contaminant. The 2 virus speci-f i e d polypeptides are derived from 3 larger precursors of molecular weights - 200,000, 184,000 and 50,000 (42). (c) Translation of V i 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 42S RNA i s also associated with polysomes. Simmons and Strauss (43) state that 26s RNA (M.W. 1.6 x 10 ) constitutes 90% by weight of the mRNA i n infected c e l l s , and i t 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, 42S RNA, (M.W. 4.3 x 10^) which i s i d e n t i c a l to the v i r a l genome, -12-constitutes approximately 5 - 1°$ of the t o t a l mRNA and i s thought to code f o r the remaining v i r a l functions such as RNA polymerase a c t i v i t y (44). •Kennedy (45). Mowshowitz (46) and Simmons and Strauss (43) have also noted the association of small amounts of 3 3S RNA with polysomes. Hybridization-competition experiemtns showed that 90$ of the base sequences i n 3 3 S 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 or an altered conform-ation has been suggested f o r 33S RNA. Hybridization studies have shown that 26s RNA consists of 2/3 of the base sequence information of 42S RNA (31, 34, 47). 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. 42S RNA i s o l a t e d from polysomes has been shown to be i d e n t i c a l i n structure to virus RNA (48) through i n f e c t i v i t y , sedimentation behaviour and i n v i t r o protein synthesis. Most recently studies have been directed towards i s o l a t i o n of these messengers and using them i n c e l l free protein synthesis (49 - 55)- The f i r s t of such studies was performed by Cancedda and Schlesinger i n Krebs II ascites c e l l and rabbit r e t i c u l o c y t e extracts. Translation of Sindbis 26s RNA resulted i n the formation of a protein i d e n t i c a l to capsid protein (as shown by t r y p t i c peptide mapping and SDS polyacrylamide ^electrophoresis)>., Only traces of E-^ and E 2 protein were obtained and larger molecular weight proteins (which could be precursors) were also absent. - 1 3 -Clegg and Kennedy ( 5 2 , 53) have also performed studies using 26S RNA1. from c e l l s infected with SFV as messenger using an extract of L c e l l s . They also indicate that the major peptide formed was nucleocapsid and that 4-2S RNA functioned poorly as messenger - probably due to i t s secondary structure. In t h e i r most recent studies ( 5 3 ) . however, Clegg and Kennedy showed that a f t e r long periods of synthesis ( 1 0 0 .min. compared to 30 min.) from 2 6 s messenger, E-^  and Eg could be found i n c e l l free systems i n a non-glycosylated form. There was, however, no evidence for precursor polypeptides,acohtrary^to- similar•work with polio v i r u s messenger (56 - 5 9 ) . Addition of TPCK (chymotrypsin i n h i b i t o r ) or PMSF (a serine protease i n h i b i t o r ) did not y i e l d precursor polypeptides i n the c e l l free system. Thus, there i s some question as to how 2 6 s RNA, a monocistronic messenger, can be translated to produce the 3 discrete products i n v i t r o . A model must also be proposed for the sel e c t i v e formation of nucleocapsid protein. Possibly t r a n s l a t i o n of nucleocapsid i s most e f f i c i e n t from ribosomes which are not bound to mem-branes and t r a n s l a t i o n of envelope proteins occurs only i n association with membrane bound ribosomes. '''' (d) Synthesis and Insertion of V i r a l Glycoproteins into  the I n t r a c e l l u l a r Membranes of the Host C e l l Iiodish's laboratory has recently demonstrated the mode f o r t r a n s l a t i o n of 26S RNA. within c e l l s which were infected with Sindbis v i r u s ( 6 0 ) . The 2 6 s RNA was shown to be almost exclusively associated with i n t r a c e l l u l a r membranes while 4-2S RNA was found not to be membrane-bound. Newly synthesized nucleocapsid protein was l o c a l i z e d on the cytoplasmic side of - 1 4 -e n d o p l a s m i c r e t i c u l u m membranes a n d c l e a v e d w h i l e n a s c e n t . S i n d b i s membrane p r o t e i n s p e n e t r a t e d i n t o t h e l u m e n o f t h e e n d o p l a s m i c r e t i c u l u m v i a t h e a m i n o t e r m i n a l s e q u e n c e o f B p r o t e i n ( p r e c u r s o r t o E-^, E 2 a n d E^) . The e n v e l o p e p r o t e i n s a r e s e q u e s t e r e d i n t o t h e l u m e n , become g l y c o s y l a t e d , a r e c l e a v e d , a n d r e a c h t h e p l a s m a - membrane i n a p r o c e s s s i m i l a r t o t h a t o f s e c r e t o r y p r o t e i n s s u c h a s i n s u l i n a n d p a n c r e a t i c a m y l a s e . The p r o p o s e d i n t r a c e l l u l a r membrane a s s o c i a t i o n w i t h v i r a l p r o t e i n s i s s hown i n F i g . 4 . F i g . 4 . M o d e l f o r t h e s y n t h e s i s o f S i n d b i s v i r u s p r o t e i n s , s u b s e q u e n t c l e a v a g e o f n a s c e n t p e p t i d e s , a n d s e q u e s t e r i n g o f e n v e l o p e p r o t e i n s i n t o t h e l u m e n o f t h e e n d o p l a s m i c r e t i c u l u m . Some g l y c o s y l a t i o n o f t h e e n v e l o p e p r o t e i n s a p p e a r s t o o c c u r b e f o r e r e l e a s e f r o m t h e p o l y s o m e , a n d t h i s g l y c o s y l a t i o n i n -v o l v e s t r a n s f e r o f a l a r g e 1800 m o l e c u l a r w e i g h t o l i g o s a c c h a r i d e t o t h e p o l y p e p t i d e c h a i n s ( 6 1 ) . The d o n o r o f t h e o l i g o s a c c h a r i d e i s t h o u g h t t o be a l i p i d ( 6 l ) . -15-(e) Formation of Virus Proteins - Fost-Translational  Cleavage of Large Precursors ( i ) S t r uctural Proteins Just as the s t r u c t u r a l proteins of picornaviruses appear to arise from cleavage of a large polypeptide (62, 63) so do those of Group A togaviruses. This precursor phenomenon was f i r s t observed by B u r r e l l , Martin and Cooper ( 6 4 ) i n BHK c e l l s infected with SFV. Detailed precursor studies were c a r r i e d out by Schlesinger and Schlesinger on Sindbis v i r u s . Their f i r s t studies showed the existence of a precursor to Eg (PE 2) of molecular weight 68,000 (65) PE 2 was shown to be c l o s e l y related to E 2 by t r y p t i c peptide maps. PE 2 was also shown to be glycosylated. In a l a t e r study (66) a temperature sensitive mutant Sindbis ts2 which i s defect-ive i n nucleocapsid assembly was used. A large molecular weight precursor (ts2 protein) M.W. 130,000 accumulated at the non-permissive temperature. Two dimensional t r y p t i c peptide mapping showed t h i s large protein to contain E-^ , Eg, and nucleocapsid. When the infected c e l l s were s h i f t e d to the permissive tempera-ture r a d i o a c t i v i t y was chased from the large protein to another protein c a l l e d B protein (M.W. 97,000) and then into PEg, E-^ Eg and nucleocapsid. These proteins are portrayed on a SDS poly-acrylamide gel shown i n Figure '5 • -IN-FRACTION NUMBER F i g . 5. An SDS polyacrylamide g e l showing the radioactive polypeptides which are present i n c e l l s infected with ts2 mutants of Sindbis and HR (Wild Strain) Sindbis,(1). Jones, Waite, and Bose (67, 68) performed a s i m i l a r experiment with c e l l s infected with a temperature sens i t i v e mutant of Sindbis (ts 20) defective i n cleavage of PEg at the non-permissive temperature (42° C). A s h i f t to the permissive temperature saw a decrease i n PEg and increase i n Eg formation. Preliminary experiments showed PEg might be associated with the PM of the host c e l l . Further evidence f o r post-translational cleavage of pre-cursor proteins i n Sindbis v i r u s infected c e l l s has been - 1 7 -provided through use of TPCK".(tosylphenylalanine chloro-methylketone) which i s an i n h i b i t o r of chymotrypsin ( 6 9 ) . A large molecular weight protein (approximately 9 7 . 0 0 0 M.W.) accumulated i n r a d i o a c t i v e l y labeled c e l l s infected with Sindbis and TPCK. Similar experiments to those performed on Sindbis have been applied to Semliki Forest v i r u s . Simons, Keranen, and Kaariainen ( 7 0 ) f i r s t demonstrated the existence of the pre-cursor to Eg ( pEg) ^ n c e l l s infected with a temperature sensi-t i v e mutant of Semliki Forest v i r u s (ts - 1 ) which was defective i n PEg cleavage at the non-permissive temperature. Pulse chase studies and t r y p t i c peptide maps indicated that PEg was indeed a precursor to Eg. Further work was reported by Morser and Burke ( 7 1 , 7 2 ) . NVP 63 (non-virion protein, M.W. 6 3 , 0 0 0 ) corresponded to PEg found i n Sindbis and NVP 97 (non-virion protein, M.W. 9 7 , 0 0 0 ) to the high molecular weight precursor found by Schlesinger and Schlesinger(-65)JU"sing l a b e l i n g studies with -^H-glucosamine, i t was shown that PEg, E-^  and Eg were the only glycoproteins i n infected c e l l s . In chase studies where radioactive medium was replaced with non-radioactive medium, l a b e l i n the high molecular weight proteins appeared to move into the lower molecular weight, envelope proteins. TPCK (an i n h i b i t o r of chymotypsin) addition was also shown to r e s u l t i n the accumulation of large molecular weight proteins. The same re s u l t s were obtained when amino acid analogues (to i n h i b i t s p e c i f i c p r o t e o l y t i c cleavage) were used -fluorophenylalanine, canavanine, azetidine - 2 - carboxylic acid, -18-ethionine, and azatryptophan. Pulse chase studies i n the presence of i n h i b i t o r s of i n i t i a t i o n of protein synthesis (sodium f l u o r i d e , n-butanol, a u r i n t r i c a r b o x y l i c acid) showed that when the inhibitor'was removed NVP 97 decreased r a p i d l y with a rapid increase i n l a b e l i n g of nucleocapsid followed by E-j^  and Eg. Work with temperature sens i t i v e mutants of SFV was also performed by Keranen and Kaariainin (73)- These workers demon-strated 5 non-structural proteins i n c e l l s infected with these mutants - NVP 1 3 0 , NVP 9 7 . NVP 8 6 , NVP 7 8 , and NVP 6 3 . Pulse-chase experiments suggested NVP 1 3 0 , NVP 9 7 . NVP 8 6 , and NVP 62 were precursors for the s t r u c t u r a l proteins. NVP 86 and NVP 78 were not affected by the chase and were thought to be replicase or RNA polymerase. In a recent paper (7^) Clegg has demonstrated the order of peptide t r a n s l a t i o n i n SFV infected BHK c e l 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 i s o t o n i c i t y induced synchronous i n i t i a t i o n of protein synthesis. Radio-a c t i v i t y was apparent i n capsid protein a f t e r 2 min of restoring i s o t o n i c i t y while radioactive precursors or envelope proteins did not occur u n t i l a f t e r 5 - 6 min. Further work showed the order of l a b e l i n g to be capsid, NVP 9 7 . NVP 6 3 , E-^ Eg-E^. Based on the preceding evidence, the following model fo r p o s t - t r a n s l a t i o n a l cleavage of precursor polypeptides has been -proposed ( 7L> 75) • /s2 protein or NVP 130 NH - C O O H 130,000 i B protein or NVP 98 30,000 98,000 PE2or NVP 62 54,000 48,000 t E3 E2 5600 48,000 F i g . 6 . P o s t - t r a n s l a t i o n a l c l e a v a g e d u r i n g t h e f o r m a t i o n o f SF v i r u s s t r u c t u r a l p r o t e i n s . r e m a i n s a s s o c i a t e d w i t h S e m l i k i F o r e s t v i r u s , b u t i s a p p a r e n t l y l o s t f r o m S i n d b i s v i r u s . C l e a v a g e o f NVP 127 ( o r t s 2 p r o t e i n ) t o c a p s i d p r o t e i n a n d B p r o t e i n i s r a p i d a n d c l e a v a g e o f B p r o t e i n (NVP 97) ( w h i c h i s n o t g l y c o s y l a t e d ) p r o b a b l y o c c u r s w h i l e t h e p e p t i d e i s s t i l l n a s c e n t l y a t t a c h e d t o t h e r i b o s o m e s . C l e a v a g e o f PEg t o Eg a n d Ej i s s l o w , r e -q u i r e s a t l e a s t 30 m i n , a n d a p p e a r s t o be a s s o c i a t e d w i t h t h e f i n a l s t e p s o f v i r u s m a t u r a t i o n ( 1 , 6 7 ) . ( i i ) N o n - S t r u c t u r a l P r o t e i n s A g r e a t d e a l o f w o r k h a s a c c u m u l a t e d r e c e n t l y w h i c h d e a l s w i t h t h e a p p e a r a n c e o f n o n - s t r u c t u r a l p r o t e i n s ( n s p ) a n d t h e i r p r e c u r s o r s ( p ) i n c e l l s i n f e c t e d w i t h S e m l i k i F o r e s t v i r u s ( 4 2 , 7 6 - 7 8 ) . N o n - s t r u c t u r a l p r o t e i n s a r e p r o d u c e d i n s m a l l - 2 0 -amounts early i n i n f e c t i o n . These studies are f a r from complete as yet. Lachmi and Kaarianen (76) found 4 non-structural pro-teins and 2 precursor polypeptides i n c e l l s infected with a temperature sens i t i v e mutant of Semliki Forest v i r u s . A scheme which they proposed f o r the post-translational cleavage of the precursors i s presented i n F i g . "?• p 1 5 5 , 0 0 0 p 1 3 5 , 0 0 0 1 i . nsp .70,000 nsp 8 6 , 0 0 0 nsp 7 8 , 0 0 0 nsp 6 0 , 0 0 0 Fig.- ',7. Scheme for the post-translational processing of precursors (p) to the non-structural proteins (nsp) of a temp-erature s e n s i t i v e mutant (ts - 1 ) of Semliki Forest v i r u s . More complete studies have been performed by Clegg, Brzeski, and Kennedy (42) on both Semliki Forest virus and Sindbis virus ( 7 7 ) • These involved radioactive pulse-chase experiments which were performed early i n i n f e c t i o n , synchronous t r a n s l a t i o n to de-termine gene order and t r y p t i c peptide analysis of the gene products to elucidate the association between the non-structural proteins and t h e i r precursors. The cleavage patterns determined f o r Semliki Forest virus and Sindbis v i r u s are i l l u s t r a t e d i n Figures 8 and 9 respectively. -21-p 200,000 I p 184,000 P 150,000 Fig. 8. Formation of non-structural proteins (nsp) by post-t r a n s l a t i o n a l cleavage of precursors (p) i n c e l l s which were infected with Semliki Forest virus (42). 230,000 p 150,000,,.. p 7.6,000 p 215.000 _ - - - - - • — j p 60,000 nsp 89,000 nsp 82,000 nsp 80,000 p 76,000 — •••• v nsp 82,000 F i g . 9. Formation of non-structural proteins (nsp) by post-t r a n s l a t i o n a l cleavage of precursors (p) i n c e l l s which were infected by Sindbis v i r u s . * denotes a conformational change i n the precursor protein-; (77) . The non-structural proteins probably serve i n " RNA synthesis. Indeed nsp 63,000 and nsp 90,000 have already been shown to be components of the virus s p e c i f i e d RNA polymerase i n c e l l s which were infected with Semliki Forest virus (41). Functions for the other proteins have yet to be assigned. -22-3. Maturation and Membrane Assembly of Togaviruses (a) Electronmicroscopic Observations of Togavirus Assembly Electronmicroscopy discloses extensive changes i n the cytoplasm of infected c e l l s . V i r i o n s appear to be produced by budding of nucleocapsids through areas of c e l l membranes modified by the i n s e r t i o n of vi r u s envelope proteins. This appears to occur at 2 places: 1) the plasma membrane (16, 79, 80) 2) i n t r a c e l l u l a r vacuoles (Type 2 cytopathic vacuoles) ( 3 5 , 81) Nucleocapsids were seen c l o s e l y aligned to the cytoplasmic side of the membrane, and,often i n the process of budding. Cyto-pathic 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 electronmicroscopy studies have u t i l i z e d f e r r i t i n - l a b e l e d v i r a l antibody to show that envelope proteins were inserted into l o c a l i z e d regions of the plasma membrane (82, 8 3 ) . Electronmicroscopy of c e l l s infected with temperature sensi t i v e mutants of Sindbis v i r u s also seemed to indicate that the plasma membrane i s the f i n a l s i t e of vi r u s maturation (1, 84, 8 5 ) . C e l l s which were infected with t s - 2 0 mutant contain s t r i k i n g arrays of nucleocapsid just beneath the plasma membrane at the non-permissive temperature and the c e l l membrane was demonstrated to contain envelope proteins (1, 8 5 ) . The ts - 2 3 mutant of Sindbis did not inse r t envelope proteins into the plasma membrane at the non-permissive temperature, nucleocapsid did - 2 3 -not associate with the outer c e l l membrane, and mature virus was not formed ( 1 , 84, 8 5 ) . Grimley et a l . ( 3 5 , 36) described another type of cytopathic vacuole (CPV-1) which occurs early i n the exponential phase of SFV growth. The vacuoles were 0 . 6 - 2 um i n diameter and bore 50 nm membranous nodules which project from the i n t e r i o r sur-faces. The nodules were neither v i r i o n s nor nucleocapsids. Formation of the nodules i s independent of c e l l u l a r RNA synthesis since actinomycin D does not a f f e c t t h e i r appearance. Antimeta-b o l i t e s which block v i r a l RNA or protein synthesis prevented the formation of CPV-1 's. Autoradiographic experiments performed i n the presence of actinomycin D seem to indicate that the CPV-1 i s a s i t e of v i r a l RNA synthesis. C e l l s infected with group B togaviruses show somewhat dif f e r e n t cytopathic changes (86 - 8 8 ) . I n i t i a l l y there was a p r o l i f e r a t i o n of cytoplasmic vacuoles, of smooth and rough endoplasmic reticulum, and Golgi membranes. However, there was l i t t l e concrete evidence as to the mechanism fo r the envelopment of group B viruses. Ota (86) suggested that v i r a l morphogenesis occurred by the budding of these p a r t i c l e s through cytoplasmic membranes. Lining the cytoplasmic side of many of the vacuoles and cisternae were i l l - d e f i n e d , electron-dense, round ragged structures 2 6 - 2 8 nm In diameter which could be nucleocapsids. No v i r i o n s have ever been observed i n the process of budding. Yasuzumi et"-al. ( 8 9 - 9 1 , 49) have suggested nuclear involvement i n the morphogenesis of group B v i r i o n s . Murphy et a l . (88) also indicated the presence of intranuclear v i r i o n s and nucleo--24-capsids. Membrane-enclosed v i r i o n s may migrate from the perinuclear region of the c e l l and approach the plasma membrane to be released by exocytosis through narrow c a n i c u l i or through fusion of virus-containing vacuoles with the plasma membrane. Immunofluorescence studies showed that group B antigens were present i n the cytoplasm of infected c e l l s ( 9 2 - 9 4 ) and various workers contended that the absence of immunofluorescence at the plasma membrane of infected c e l l s . meant ' . that v i r a l maturation did not take place at the c e l l surface. Evidence obtained- through electronmicroscopy i s somewhat confusing. I n t r a c e l l u l a r vacuoles could well be an a r t i f a c t of sample preparation - perhaps they were cross-sections of surface invaginations of the c e l l . Also the dramatic difference between the' maturation of Group A and B togaviruses i s somewhat suspect. The r o l e of i n t r a c e l l u l a r membranes i n virus maturation also requires further c l a r i f i c a t i o n . (b) Chemical Evidence for Maturation of Group A Togaviruses  at the Plasma Membrane Chemical evidence which indicated that the plasma membrane was the source of v i r a l envelope o r i g i n a l l y came from the l i p i d analysis of Semliki Forest (21, 95) and Sindbis ( 9 6 , 9 7 , 98) viruses which were grown in- various hosts. Semliki Forest viruses which were grown i n BHK-21 and Aedes albopictus c e l l s had only 36% of t h e i r phospholipids i n common. In each case, the l i p i d com-pos i t i o n of the virus resembled that of the host c e l l membrane. More d i r e c t chemical evidence that v i r a l proteins were present i n the plasma membrane of the host c e l l came from experi-ments i n which lactoperoxidase was u t i l i z e d to l a b e l c e l l surface - 2 5 -proteins with ( 9 9 ) . Amazingly, Sindbis membrane proteins were labeled p r e f e r e n t i a l l y over those of the host c e l l . Also, i n f e c t i o n with Sindbis virus was demonstrated to increase dras-t i c a l l y the concanavalin A a g g l u t i n a b i l i t y of the host c e l l s ; the change i n surface sugar composition appeared to indicate the presence of v i r a l glycoproteins i n the plasma membrane. Even more conclusive evidence f o r budding of togaviruses at the plasma membrane was presented by Jones et a l . ( 6 7 ) . This group has demonstrated that E-^ , Eg, nucleocapsid protein, and PEg were present i n plasma membrane fractio n s p u r i f i e d from infected host c e l l s . PEg cleavage appeared necessary f o r virus to be released into the e x t r a c e l l u l a r f l u i d . Evidence i s presented i n t h i s thesis which c l e a r l y demon-strates that Group A- togaviruses "bud" exclusively through the plasma membrane of the host c e l l . 4 . Assembly of Vesicular Stomatitis Virus (A Rhabdovirus) (a) Structure Vesicular stomatitis v i r u s consists of 5 proteins (100) -G (molecular weight 6 9 , 0 0 0 ) , M (molecular weight 2 9 , 0 0 0 ) , N (molecular weight 5 0 , 0 0 0 ) , L (molecular weight 1 9 0 , 0 0 0 ) , and NS (molecular weight 40 , 0 0 0 - 4 5 , 0 0 0 ) . The d i s t r i b u t i o n and function of these proteins are shown i n Fig.. 1 0 . - 2 6 -Protein Molecular Wt. Function G 6 9 , 9 9 9 envelope protein, v i r u s - c e l l i n t e r -action., M 2 9 , 0 0 0 membrane matrix protein, organizes G proteins into patches N 5 0 , 0 0 0 'nucleocapsid protein which i s t i g h t l y associated with RNA genome; involved i n tr a n s c r i p t i o n and r e p l i c a t i o n L 1 9 0 , 0 0 0 associated with nucleocapsid and may be c a t a l y t i c sub-unit of RNA polymerase NS 40,0 0 0 J associated with 5 0 , 0 0 0 nucleocapsid and i s required f o r r e p l i c a t i o n and tr a n s c r i p t i o n Fig . 10,. Structure and composition of ve s i c u l a r - s t o m a t i t i s v i r u s The v i r u s has a simple genome which consists of one piece of single stranded RNA of molecular weight 3 - 5 x 10 daltons. Vesicular stomatitis v i r u s i s a negative strand virus and i t contains i t s own RNA-dependent RNA polymerase. (b) Membrane Assembly of Vesicular Stomatitis Virus Rhabdoviruses are reported to acquire t h e i r envelopes from the plasma membrane or i n some cases the membranes of intracytoplasmic v e s i c l e s of the host c e l l ( 1 0 0 ) . This e v i -dence i s based upon the electronmicroscopy of Zee et a l . ( 1 0 1 ) . The l i p i d of the v i r a l membrane appears to mimic that of the - 2 7 -host c e l l plasma membrane ( 1 0 2 ) . However, Tiffany and Blough have recently published evidence which contradicts t h i s f a c t ( 1 0 3 ) . They found the l i p i d of the virus to be of a character intermediate to that of the plasma membrane and endoplasmic reticulum. Cohen et a l . (104) o r i g i n a l l y showed that v e s i c u l a r stoma-t i t i s v i r u s proteins were associated with the plasma membrane of infected HeLa c e l l s . In a pulse-chase experiment with v e s i c u l a r stomatitis c e l l s , David (105) demonstrated that a f t e r a 3 ° second pulse, G and M proteins were already associated with the plasma membrane, but maximal l e v e l s of incorporation were not reached u n t i l 2 min chase. N and NS polypeptides were found only i n the soluble cytoplasm. At the end of a 5 min pulse period the plasma membrane contained substantial amounts of M and G proteins, but no nucleocapsid protein. With chase times approaching 60 min, the amount of nucleocapsid protein found i n the plasma membrane increased, although aft e r about 20 min the amount of envelope proteins attached to the plasma membrane remained approximately the same. Knipe, Baltimore and Lodish ( 107) recently performed a more elegant pulse-chase experiment with Chinese hamster ovary c e l l s which were infected with v e s i c u l a r stomatitis v i r u s . Infected c e l l s were pulsed f o r 2 . 5 min with [ S]-Met and the l a b e l was chased over 90 min. Plasma membrane, smooth and rough endoplasmic reticulum fractions were harvested at 0 , 1 0 , 2 0 , 3 0 , 60 and 90 min chase and the r a d i o a c t i v i t y i n the c e l l supernatant and membrane fra c t i o n s were quantitated by auto--23-radiography. These workers succeeded i n following the r a d i o -a c t i v i t y from the c e l l supernatant and rough endoplasmic reticulum to the smooth endoplasmic reticulum and then the plasma membrane. F i n a l l y l a b e l was chased into the extra-c e l l u l a r v i r u s ( F i g . 11). M I N U T E S O F C H A S E ' F i g . 11. Kinet i c s i l l u s t r a t i n g the ass o c i a t i o n of v e s i c u l a r stomatitis v i r u s with c e l l supernatant and membrane f r a c t i o n s during a pulse-chase experiment. Fractions 1 and 2 corres-pond to rough endoplasmic reticulum, 3 a n d 4 to smooth endo-plasmic reticulum, ' 5 and 6 to plasma membrane, and S to the c e l l supernatant. V denotes r a d i o a c t i v e e x t r a c e l l u l a r v i r u s . - 2 9 -Knipe, Baltimore and Lodish ('109') have also u t i l i z e d temperature sensitive mutants of v e s i c u l a r stomatitis v i r u s to investigate the mechanisms of virus assembly. Studies which involved surface iodination and c e l l f r a c t i o n a t i o n were per-formed on several: well defined mutants at t h e i r non-permissive temperatures. Mutations i n G protein prevented Its migration to the c e l l surface, did not allow either M or N protein to become associated with the plasma membrane, and blocked the formation of e x t r a c e l l u l a r v i r u s . Mutations i n either M or N prevented association of both these proteins with G protein which was s t i l l inserted into the c e l l membrane. These workers provided a model fo r assembly which showed the interdependence of G, M and N proteins i n "budding" (Fig . / 1 2 . ) . Plasma Membrane Fig. . 1 2. Maturation of v e s i c u l a r stomatitis v i r u s within the c e l l ( 1 0 9 ) . -30-5. Assembly of'Myxoviruses and Paramyxoviruses (a) Structure of Myxoviruses and Paramyxoviruses Myxoviruses and paramyxoviruses have-the structures shown i n Figures 13 and I r r e s p e c t i v e l y . Both types of virus con-t a i n negative strand genomes and contain t h e i r own transcriptase. The genome of myxoviruses consists of 8 separate segments of single-stranded RNA while that of para-myxoviruses i s a large single segment of single stranded RNA. Hemagg lu t i n in - , 1 N e u r a m i n i d a s e , L i p i d m e m b r a n e i Matr ix p ro te in N u c l e o p r o t e i n , P prote ins R N A F i g . 13. Structure and protein composition of myxoviruses. MP 'NA J I I I ' F i g . 14. Structure and protein composition of paramyxoviruses. - 3 1 -(t>) Membrane Assembly of Myxoviruses and Paramyxoviruses Electronmicroscopic studies also indicate that myxoviruses and paramyxoviruses bud from the plasma membrane. This was f i r s t shown i n 1955 by Hotz and Schafer (110) f o r influenza and l a t e r f o r paramyxoviruses by Choppin ( 1 1 1 ) . Nucleocapsid aligns with s p e c i f i c patches of the inner PM pr i o r to budding; these patches may be i d e n t i f i e d with f e r r i t i n -labeled a n t i v i r a l antibody. Klenk and Choppin ( 1 1 2 , 113) 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. This was further substantiated by comparison of the g l y c o l i p i d s from virus grown i n MK and BK host c e l l s (114). The concept that envelope components migrate from the c e l l i n t e r i o r to the surface has been confirmed by c e l l f r a c t i o n -ation and analysis of the various proteins found i n the d i f f e r e n t cytoplasmic f r a c t i o n s . I t has been suggested that HA and pos-s i b l y the other envelope proteins are synthesized on the rough ER ( 1 1 5 » 116). Pulse chase experiments show that a few minutes l a t e r HA i s present i n the membranes of the smooth ER ( 1 1 5 , 116) and then the PM ( 1 1 7 ) . Virus proteins are always membrane associated during t h i s migration and are never detected i n the soluble f r a c t i o n . Hay (118) reported s i m i l a r r e s u l t s f o r fowl plague v i r u s 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 i n v i r u s assembly. As yet no experiments have been performed which have chased the myxovirus protein from the PM to the e x t r a c e l l u l a r v i r u s . - 3 2 -Klehk's laboratory (119)has presented pulse-chase studies which concern the membrane assembly of Newcastle Disease v i r u s (a paramyxovirus). Infected c e l l s were pulse-labeled f o r 10 min and chased over 60 min. The c e l l s were fractionated into plasma membrane ghosts and smooth and rough endoplasmic reticulum at 0 , 20 and 60 min chase. The glyco-proteins HN and P 0 were synthesized on the rough endoplasmic reticulum and transferred from there v i a smooth i n t r a c e l l u l a r membranes to the plasma membrane and into v i r i o n s . In the course of migration, F Q i s converted to F. M protein was found to reach the plasma membrane more r a p i d l y than the labeled glycoproteins. The chief c r i t i c i s m of t h i s paper i s that r a d i o a c t i v e l y labeled proteins were not chased over longer periods into e x t r a c e l l u l a r v i r u s . Another pulse-chase experi-ment (120) performed with v i r a l proteins from a t o t a l c e l l homogenate, demonstrated that i t was possible to chase M pro-t e i n from the c e l l over 8 hours but that nucleocapsid accumu-lated i n excess within the c e l l . Very low incorporation of l a b e l into F , F and HN protein prevented a decisive chase of these proteins from the c e l l . B. Cytoskeletal Components of Membrane Systems and Their  Involvement i n the Membrane Assembly of Viruses (I30-I37) The cytoskeletal system consists of a vast network of microtubules and microfilaments dispersed throughout the c e l l . These structures have been demonstrated to be i n t r i n s i c a l l y associated with c e l l membranes ( 1 3 1 , 136 , 1 3 7 ) - Translational mobility of c e r t a i n classes of i n t e g r a l membrane proteins -33-appears to be regulated by these cytoplasmic elements -membrane proteins may be linked i n some way to microtubles and microfilaments. Nicolson (136, 137) has termed this concept as transmembrane cytoskeletal control. 1. Miiareifubules Microtubules are large, c y l i n d r i c a l structures of an outer diameter of 24 +•2 nm and a hollow core approximately 15 nm i n diameter (130 - 135)' Each microtuble i s composed of 13 proto-filaments (each 5 nm i n diameter) which run the length of the tubule. The protofilament i s i n turn composed of a globular protein named tubulin (molecular weight 103,000) which i n i t s e l f consists of 2 subunits - (molecular weight 53»°0°) and (molecular weight 56,000). The basic composition and the r e l a t i o n of the various elements which make up the microtuble are i l l u s t r a t e d i n F i g . 15. 51A CLEFT REGION Fig. 15. The basic structure of microtubules. - 3 4 -Several high molecular weight proteins also co-purify with brain tubulin ( 1 3 5 , 1 3 8 , 1 3 9 ) . These proteins are ca l l e d microtuble associated proteins (MAPS) and may regulate-assembly of tubulin dimers (this protein has been termed tau), be present as side-arms, or possess ATPase and protein kinase a c t i v i t y . Microtubles have c l a s s i c a l l y been shown to be associated with the spindle apparatus (for chromosome movement) and f l a g e l l a (for locomotion). However, research over the l a s t 10 years has proven that these organelles have much more diverse functions than previously r e a l i z e d . C e l l shape has been shown to be maintained by microtubules. Numerous studies demon-strated that disruption of cytoplasmic microtubules with either cold, hydrostatic pressure, or drugs, r e s u l t s i n a rev e r s i b l e loss of normal c e l l shape. These structures are responsible for the formation of pseudopodia, nerve axons, and the spread-ing of c e l l s . The extent to which microtubles permeate the c e l l can be seen i n studies where c e l l u l a r microtubles are labeled with fluorescent antibodies (140) to tubulin (Fig. 15) and by high-voltage electron microscopy (141). These photo-graphs were published by the laboratories of Weber and Porter, respectively (140, 141) . -35-F i g . 16. Fibroblasts labeled with fluorescent a n t i - t u b u l i n (140). Much work indicates that microtubules are associated with membranes. Stadler and Franke (142) measured the co l c h i c i n e -binding properties of rat and mouse l i v e r membranes. Both nuclear and microsomal membrane fractions demonstrated appreci-able binding to the drug. The fac t that the synaptosomes of brain homogenate contain tubulin i s thoroughly documented (143 - 146). More recently Bhattacharyya and Wolf demonstrated membrane-bound tubulin i n brain and thyroid tissue (147). In most cases the c r i t e r i a f or the presence of membrane-associated tubulin are colchicine or podophylotoxin binding, electrophoretic mobility of membrane proteins, electronmicroscopy with f e r r i t i n conjugated antibodies to tubulin, and antibody t i t r a t i o n s . The tubulin associated with plasma membrane may contain carbohydrate since tubulin ( s o l u b i l i z e d from membranes) stains with periodic acid S c h i f f base reagent (148,149). - 3 6 -2. Microfilaments Much les s i s known of the r o l e f o r microfilaments i n non-muscle c e l l s . They are responsible for many types of c e l l movement and have been implicated i n such functions as cyto-plasmic streaming, cytokinesis, secretion, blood c l o t r e t r a c t i o n , phagocytosis, and the mobility of various c e l l surface proteins ( I 3 6 , 137 , 1 5 0 - 1 5 2 ) . Microfilaments have been shown to consist of t h i n filaments of 6 to 8 nm diameter (composed of actin) and thick filaments of 10 nm diameter (composed of myosin) which are comparable to the filaments of muscle c e l l s . The filaments of non-muscle c e l l s are not nearly as well characterized as those of muscle systems. In muscle c e l l s , troponin, v i a tropomyosin, regulates the a b i l i t y of the actins along the length of one tropomyosin to in t e r a c t with myosin. Act i n and myosin have been found i n every type of non-muscle c e l l i n which they have been sought ( 1 5 0 - 1 5 2 ) . The existence of tropomyosin and troponin i n non-muscle c e l l s has not been established d e f i n i t i v e l y . However, proteins s i m i l a r to tropomyosin have been is o l a t e d and characterized from several non-muscle vertebrate tissues eg. p l a t e l e t s ( 1 5 3 ) , brain neurons ( 1 5 4 ) , brain synaptosomes ( 1 5 5 ) . blood and pancreas ( 1 5 6 ) , and f i b r o b l a s t s ( 1 5 7 ) -Weber's group have u t i l i z e d fluorescently labeled a n t i -bodies to v i s u a l i z e a c t i n and myosin within f i b r o b l a s t s . The d i s t r i b u t i o n of these structures throughout the cytoplasm, and plasma membrane i s extensive. Actin, myosin, a protein cofactor and another high-molecular weight protein - 3 7 -which binds to a c t i n , have been p u r i f i e d as a c o n t r a c t i l e complex from the 1 0 0 , 0 0 0 x_g supernatants of rabbit alveolar macrophages ( 164) and Acanthamoeba ( 1 6 2 ) . A c t i n and the high-molecular weight binding protein can aggregate to form a gel; addition of myosin and ATP causes contraction which i s accel-erated by the cofactor. In non-muscle c e l l s , a c t i n and myosin filaments f r e -quently assemble .into bundles which appear to be associated with the plasma membrane ( 1 6 4 ) . The acrosomal process of sperm (163) and f i b r o b l a s t s ( 160) are p a r t i c u l a r l y r i c h i n aggre-gated microfilaments. A c t i n and myosin bundles are p a r t i c -u l a r l y prevalent i n c e l l s which adhere to a substratum. Treat-ment of f i b r o b l a s t s with T r i t o n X-100 releases most of the c e l l proteins but leaves a c t i n attached to the substratum i n the form of s k e l e t a l bundles ( 1 5 1 ) . Bundles form i n the presence of ImM M'g-. ATP and contract on the addition of luM C a + + . 3. [Microtubule' - Disruptors A number of drugs have been shown s p e c i f i c a l l y to in t e r a c t with .m'icrotubules( 1 3 1 , I 6 5 - I 6 7 ) • These include colchicine, v i n b l a s t i n e , v i n c r i s t i n e , podophyllotoxin, g r i s e o f u l v i n and iNox-odazolej the chemical structures of these compounds are shown i n F i g . 17• H N ocodazole F i g . 17 . " Structures of drugs which irisru'pt-mrcrotubul-esJ. Colchicine i s the prototype of microtubule disruptors and has been shown to bind t i g h t l y to the tubulin dimer.. Bind-ing i s dependent upon pH (optimum i s pH7.0) but independent of s a l t concentration; since c o l c h i c i n e - t u b u l i n complexes can occur over a wide s a l t range ( 5 - 5 0 ° mM NaCl) the a t t r a c t i o n i s not e l e c t r o s t a t i c . Maximum colchicine uptake by p u r i f i e d tubulin occurs by 1 hour. Another feature of the colchicine binding reaction i s that the rate of complex formation i s strongly temperature dependent - almost no colchicine binding a c t i v i t y can be detected at 0i9c. However, once the complex i s constituted at 37'0C. -it i s then stable at 0'°C. Colchicine binds almost i r r e v e r s i b l y at 37 GC and only 1 molecule of t h i s drug reacts with 1 molecule of tubulin. I t i s thought that the colchicine binding s i t e i s one of the protein-protein i n t e r a c t i o n s i t e s between tubulin molecules; attachment of colchicine to - 3 9 -t h i s s i t e p r e v e n t s n o r m a l a s s e m b l y ( F i g . 1 4 ) . T h u s , t h e a b i l i t y o f c o l c h i c i n e t o d i s r u p t a s s e m b l e d m i c r o t u b l e s i s d e p e n d e n t o n t h e s t a b i l i t y a n d r a t e o f t u r n o v e r o f p o l y m e r -i z e d m i c r o t u b u l e s . F o r t h i s r e a s o n c o l c h i c i n e d i s r u p t s m i c r o t u b u l e s s l o w l y ( o f t e n t a k i n g u p t o 4 h o u r s ) ; p r e - i n c u b a t i o n o f t h e d r u g w i t h o n e ' s c e l l s y s t e m p r i o r t o t h e e x p e r i m e n t i s t h u s a n a b s o l u t e r e q u i r e m e n t ( 1 6 8 ) . The s e n s i t i v i t y o f v a r i o u s c e l l t y p e s t o c o l c h i c i n e d o e s v a r y a n d i s t h o u g h t t o be due t o d i f f e r e n t t u b u l i n t u r n o v e r r a t e s a n d b i n d i n g c o n s t a n t s o f t u b u l i n w i t h i n t h e c e l l ( 1 6 8 , 1 6 9 ) . C o l c h i c i n e d o e s h a v e a n i m p o r t a n t s i d e e f f e c t w h i c h may o r may n o t b e r e l a t e d t o t h e d i s r u p t i o n o f m i c r o t u b u l e s - a t h i g h c o n c e n t r a t i o n s ( g r e a t e r t h a n o r e q u a l t o 250 uM) n u c l e o s i d e t r a n s p o r t i s i n h i b i t e d ( 1 7 0 ) . L u m i c o l c h i c i n e i s f o r m e d b y i r r a d i a t i o n o f c o l c h i c i n e w i t h u l t r a v i o l e t l i g h t (165) w h i c h r e s u l t s i n t h e c o n v e r s i o n o f r i n g C t o t w o s m a l l e r r i n g s ( F i g . 18 ). , .NH-C-CH, co lch ic ine CH^ O P ond y - l u m i c o l c h i c i n e s 1 ( l ) C o l c h i c i n e \( 5 ) L u m i c o l c h i c i n d 250 300 350 W A V E LENGTH 400 F i g . 1 8 . The c h e m i c a l s t r u c t u r e o f l u m i c o l c h i c i n e a n d i t s a b s o r p t i o n s p e c t r u m a s c o m p a r e d t o t h a t o f c o l c h i c i n e . -40-Lumicolchicine derivatives are inactive as microtubule-binding agents but possess colchicine's side effects - i t i s just as potent i n i n h i b i t i n g nucleoside transport. Vinca alkaloids (vinblastine and v i n c r i s t i n e ) interact with tu b u l i n to cause d i s s o l u t i o n of microtubules and the formation of highly regular c r y s t a l s . One mole of v i n b l a s t i n e i s bound per mole of tubulin with the release of 1 of the 2 moles of GTP which are bound to tubulin. Vinblastine s t a b i l -izes c o l c h i c i n e binding a c t i v i t y and hence occurs at an a l t e r n a t i v e s i t e on the dimer. The vinca alkaloids are believed to act as cations ( i n many cases they mimic Ca + +) which pre-c i p i t a t e tubulin to y i e l d large c r y s t a l l i n e arrays. G r i s e o f u l v i n , a mold metabolite, has been shown to possess antimitotic a c t i v i t y , but i t does not prevent the polymerizat-ion of tubulin as do the other i n h i b i t o r s (165, 166). It does not influence the binding of colchicine or the vinca alkaloids and i t s mechanism remains obscure. Nocodazole (R1793^) i s a microtubule disruptor which has recently been synthesized by De Brabander et a l . (167). It i s a s p e c i f i c a n t i t u b u l i n compound which interferes with the structure and function of microtutubles through i n h i b i t i o n of tubulin polymerization into normal microtubles. The binding of Nocodazole to tubulin i s r e v e r s i b l e and the drug possesses no known side effects at the concentrations used. The drug has also shown great promise as an anti-tumor agent. -41-4 . The Disruption of Microfilaments Cytochalasin B , a metabolite of the fungus, Helmintho-sporium dematiodium has been shown to have a wide effect on c e l l u l a r a c t i v i t i e s ( 1 7 1 ) . The compound has been shown to stop cytokinesis with the production of multinucleated c e l l s ( 1 7 1 ) , i n h i b i t hexose transport ( 1 7 2 ) , i n t e r f e r e with c e l l -u l a r secretion ( 1 7 3 ) , induce nuclear extrusion ( 1 7 1 ) , i n h i b i t c e l l movement ( 1 7 4 ) , prevent phagocytosis (175) and stop the beating of embryonic cardiac muscle c e l l s ( 1 7 6 ) . Although the evidence i s not e n t i r e l y conclusive, cytochalasin B appears to disrupt microfilaments (177)-.. Recently Weihing (I78) and Hartwig and Stossel (179) have shown that t h i s drug prevents the gelation of p u r i f i e d a c t i n with actin-binding protein and also depolymerizes a c t i n . However, there i s s t i l l some dispute as to the actual targets of t h i s drug ( 1 8 0 ) . Cytochalasin A has been demonstrated to bind free sulfhydryl groups of tubulin and a c t i n monomers and thus prevent polymerization; cytochalasin B, on the other hand, does not react with tubulin and i t s i n t e r a c t i o n with actomyosin has not been c l a r i f i e d (181). The chemical structure f o r cytochalasin B i s shown i n Fig. 19". CH 2—CH 2 P I l:,C—CH CH CH. \ HC. HC F i g . 1 9 .v The structure of cytochalasin B. -42-5. Local Anesthetics - Disruptors of Both Microtubles and  Microfilaments Local anesthetics have recently been demonstrated to d i s -rupt the cytoskeletal organization i n c e l l s ( 1 3 6 , 137 , I 8 I - I 8 3 ) and cause the c e l l s to become more rounded. 'Quaternary amine anesthetics appear to mimic the effects of colchicine and cytochalasin B, when both are administered simultaneously. At high anesthetic concentrations (greater than ImM) l o c a l anesthetics produce molecular disordering i n l i p i d b i l a y e r s , enhance f l u i d i t y of phospholipids i n membranes and cause b i l a y e r expansion (184). At lower concentrations, microtubules and microfilaments are affected primarily and the t e r t i a r y amine l o c a l anesthetics (dibucaine, procaine, and tetracaine) i n h i b i t c e l l agglutination, endocytosis, exocytosis, and the mobiliti e s of surface receptor molecules ( 1 8 2 , I 8 3 . 1 8 5 - 1 8 7 ). Local anesthetics i n t e r a c t hydrophobically and e l e c t r o s t a t i c a l l y with membrane l i p i d s ( i n p a r t i c u l a r the anionic groups of acid phos-phatides) and are able to displace Ca from membranes (188, I 8 9 ) . These drugs cause the plasma membrane to crenate; the t e r t i a r y amine anesthetics are thus known as "cup-forming" anesthetics ( 1 9 0 ) . This group of anesthetics i s thought to in t e r c a l a t e within the inner monolayer of the membrane. The mechanism of l o c a l anesthetic action i s unclear but they may displace membrane Ca and increase i n t r a c e l l u l a r Ca concentrations to le v e l s s u f f i c i e n t to depolymerize microtubles ( I 3 6 ) . The manner i n which increased i n t r a c e l l u l a r l e v e l s of C a + + might affect'acto-myosin complexes or Ca dependent ATPase i s not cle a r ( 1 3 6 ) . - 4 3 -Structures f o r the t e r t i a r y amine anesthetics are.presented i n Fi g . 20. H 2 N H ^ ^ - C O O C H 2 C H 2 N ( C H 2 C H 3 ) 2 . p r o c a i n e H 2 N H Q K C b e n z o c a i n e • C H 3 ( C H 2 ) 3 N H H ^ J ^ - C O O C H J C H J N ( C H 3 ) , t e t r a c a i n e ' N ^ , 0 ( C H 2 ) 3 C H 3 C O N H C H 2 C H 2 N ( C H 2 C H 3 ) 2 d i b u c a i n e F i g . 2 0 . Structures of the t e r t i a r y amine anesthetics. 6. Microtuble and Microfilament Function Within the C e l l Microtubles and microfilaments are not merely associated with the spindle apparatus (for chromosome movement and c e l l d i v i s i o n during cytokinesis) and f l a g e l l a ( f o r locomotion). Research over the l a s t decade has proven that these organelles have many more diverse functions than previously r e a l i z e d . (a) Cytoskeletal Transmembrane Control A r e l a t i v e l y recent concept i s that microtubules may con-t r o l membrane f l u i d i t y or the t r a n s l a t i o n of proteins about the two dimensional surface of a membrane. I n i t i a l l y V a s i l i e v et a l . (191) noted that normal f i b r o b l a s t s (which are usually contact i n h i b i t e d and possess a random d i s t r i b u t i o n of c e l l surface proteins) behaved l i k e transformed c e l l s when treated with -LL-colcemid i e . contact i n h i b i t i o n was relieved and a cl u s t e r i n g of l e c t i n - b i n d i n g s i t e s was apparent. These investigators f i r s t proposed that microtubules s t a b i l i z e d the non-active state of the c e l l surface as a submembranous framework. The i n t e r -action between microtubules and i n t e g r a l membrane proteins was formally introduced by B e r l i n et a l . (192) and Edelman et a l . (193)• B e r l i n and his collaborators gathered extensive evidence to indicate that the microtubule system can exert transmembrane control over a v a r i e t y of surface receptor and capping phenomena (192, 196). Edelman and Yahara (193}, 197) have proposed a model for the modulation of lymphocyte receptors by a transmembrane control system which involved the microtubule-microfilament systems. They postulated the existence of 2 d i f f e r e n t classes of surface receptors - some of which are immobile and anchored (A-state) to microtubules and others which are free and not associated with microtubules (F-state). Capping of membrane proteins occurred under the guidance of microfilaments when multivalent ligands ( l e c t i n s or a n t i Ig) associated with receptors i n the free state. This theory explains the observations summar-ized i n F i g . 21'. -1*5-F i g . 2 1 . E f f e c t of drugs on lymphocytes which were i n contact with anti-Ig and/or Con A. Q Con A receptor; o a n t i Ig receptor. ^ Capping of anti-Ig receptors occurs presumably because surface Ig i s not associated with microtubules. Concanavalin A l e c t i n would not cause capping of membrane receptors unless the l i n k s between the lect i n - b i n d i n g protein and the microtubule were severed. Addition of concanavalin A to lymphocytes prevented anti-Ig capping since the l e c t i n acted to immobilize the surface antibody by binding i t to the concanavalin A receptor (which i s anchored). Microfilaments were demonstrated to modulate the capping process since cytochalasin B (which disrupts m i c r o f i l a -ments) and dibucaine (which presumably disrupts microfilaments and microtubules) prevents cap formation of concanavalin A receptors. - 4 6 -The Edelraan and Yahara theory also provides explanation for the transmembrane control of f i b r o b l a s t receptors. Nicolson, Poste, and Paphadjopoulos ( 1 3 6 , I 3 7 , 1 8 2 , I83) have made the observations outlined i n Fig. 22 with mouse 3^3 f i b r o -blasts which have been treated with concanavalin A. Normally concanavalin A does not cause capping or patch formation of membrane proteins; however, colch i c i n e addition caused capping while cytochalasin B prevented cap formation. The addition of dibucaine (which destroyed both microtubules and microfilaments) allowed small patches of concanavalin A receptors to form - the receptors were no longer anchored, they diffused l a t e r a l l y , and self-aggregated. untreated 2 colchicine 3 cytocholosin B 4 colchicine plus cytocholaiin B dibucaine F i g . 2 2 . E f f e c t of drugs on 3^3 f i b r o b l a s t s which were i n contact with anti-Ig and/or Con A. -47-I t would thus appear that microtubules and microfilaments have opposing roles i n membrane function. Microtubules serve to anchor i n t e g r a l proteins while microfilaments possess a c o n t r a c t i l e function which controls the movement of proteins over the c e l l surface (136,137). Metabolic i n h i b i t o r s (KCN, NaN^» DNP) i n h i b i t microfilament function presumably due to the ATP requirement f o r contraction (136, 137). A word of caution i s perhaps required before one t o t a l l y accepts this scheme. Some confusing evidence has appeared i n other laboratories using d i f f e r e n t c e l l l i n e s (198,199). I t can, however, be concluded that microtubules and microfilaments interact with membrane proteins and control t h e i r movements i n some manner. Nicolson has proposed the following scheme f o r i n t e r a c t i o n of membrane proteins with microtubules and microfilaments (Fig. 23). ACTIN FILAMENTS MYOSIN MOLECULES < ® MICROTUBULES F i g . 23. Proposed i n t e r a c t i o n of microtubules and microfilaments with membrane proteins (137). - 4 8 -(b) C e l l Secretion and the Cytoskeletal System Microtubules and microfilaments have also been impli-cated i n the i n t r a c e l l u l a r transport of secretory v e s i c l e s to the c e l l ' s periphery. Lacy (200) o r i g i n a l l y proposed a microtubule-microfilament system which linked the i n s u l i n secretory v e s i c l e s with the plasma membrane. Secretion i n -volved "contraction" of the cytoskeletal system with resultant transport to the c e l l surface. Forbes and Dent (201) proposed a similar hypothesis for gonadotrophic c e l l s . Colchicine has been demonstrated to i n h i b i t secretion of a- amylase by the parotid ( 2 0 2 ) i n s u l i n i n the pancreas ( 2 0 3 , 204) catecholamines i n the adrenal ( 2 0 5 ) , thyroxine from the thyroid ( 2 0 6 ) , serum proteins by hepatocytes ( 2 0 7 ) , and lysozyme by polymorphonuclear leukocytes ( 2 0 8 ) . Microtubules arid microfilaments have also been found to bind some secretory granules,.' i n v i t r o ( 2 0 9 , 2 1 0 ) . Secretory proteins are synthesized on polysomes attached to the membranes of the endoplasmic reticulum and are v e c t o r i a l l y discharged into the lumen of the rough endoplasmic reticulum. They then follow an i n t r a c e l l u l a r pathway which leads them to the smooth endoplasmic reticulum, to the Golgi complex, and to the Golgi-derived secretory v e s i c l e s which, when f i l l e d with secretory proteins, fuse with the plasma membrane. Glycosylation of secretory proteins occurs i n a stepwise fashion and terminal addition of sugars occurs at the Golgi ( 2 1 1 ) . Most recently, Malaise et a l . have considered the role of microtubules as guiding elements fo r the transport of secretory granules which contain i n s u l i n ; microfilaments, on the other hand, - 4 9 -w e r e c o n s i d e r e d t o c o n t r o l t h e a c c e s s o f s e c r e t o r y g r a n u l e s t o s i t e s o f e x o c y t o s i s a t t h e p l a s m a membrane ( 2 1 2 ) . C y t o c h a l -a s i n B f a c i l i t a t e d i n s u l i n r e l e a s e w h i l e c o l c h i c i n e a n d v i n c a a l k a l o i d s i n h i b i t e d g l u c o s e - i n d u c e d i n s u l i n r e l e a s e . E l e c t r o n -m i c r o s c o p y s howed t h e i n s u l i n g r a n u l e s t o b e i n c l o s e c o n t a c t w i t h m i c r o t u b u l e s a n d t h e s e w o r k e r s b e l i e v e d a d e n s e web o f m i c r o f i l a m e n t s p r e v e n t e d t h e p a r t i c l e s f r o m n o r m a l l y r e a c h i n g t h e p l a s m a membrane . M i c r o t u b u l e d i s r u p t o r s h a v e a l s o b e e n d e m o n s t r a t e d t o i n h i b i t c a t e c h o l a m i n e r e l e a s e b y t h e a d r e n a l m e d u l l a ( 2 0 5 ) , t h y r o i d h o r m o n e r e l e a s e ( 2 0 6 ) , a n d c a u s e a n a c c u m u l a t i o n o f n e u r o s e c r e t o r y g r a n u l e s i n t h e p i t u i t a r y ( 2 1 3 ) . W i l l i a m s a n d L e e (168) r e c e n t l y f o u n d c o l c h i c i n e , t e t r a c a i n e ( a l o c a l a n e s t h e t i c ) a n d t r a n q u i l i z e r s i n h i b i t p a n c r e a t i c a m y l a s e r e -l e a s e f r o m mouse p a n c r e a s . Redman a n d P a l a d e ( 2 0 7 , 2 14 ) a n d P a t z e l t e t a l . (102) s u g g e s t t h a t m i c r o t u b u l e d i s r u p t o r s do n o t h i n d e r t h e m i g r a t i o n o f t h e s e c r e t o r y p r o t e i n f r o m t h e r o u g h e n d o p l a s m i c r e t i c u l u m ( w h e r e i t i s s y n t h e s i z e d ) t o s m o o t h e n d o p l a s m i c r e t i c u l u m o r G o l g i a p p a r a t u s . T h e y f e e l t h e p o i n t o f i n h i b i t i o n i s j u s t p r i o r t o t h e f u s i o n o f t h e G o l g i v e s i c l e s ( w h i c h c o n t a i n t h e s e c r e t o r y p r o t e i n ) w i t h t h e p l a s m a membrane a f t e r g l y c o s y l a t i o n i s c o m p l e t e d . M i c r o t u b u l e i n h i b i t o r s c a u s e d a n a c c u m u l a t i o n o f a l b u m i n and . a - a m y l a s e i n G o l g i - d e r i v e d v e s i c l e s f r o m t h e l i v e r a n d t h e p a r o t i d r e s p e c t i v e l y . ( c ) I n t r a c e l l u l a r Movement a n d t h e C y t o s k e l e t a l S y s t e m M i c r o t u b u l e s h a v e a l s o b e e n i m p l i c a t e d i n a r o l e w h i c h f a c i l i t a t e s t h e movement o f g r a n u l e s w i t h i n t h e c e l l . F o r - 5 0 -example, lysosome, melanin, and intraxonal p a r t i c l e s are believed to be c l o s e l y associated with microtubules. Wilson's laboratory- • (215) has presented evidence f o r the involvement of microtubules i n the action of vasopressin. Microtubule disruptors i n h i b i t e d vasopressin-stimulated water movement across e p i t h e l i a l membranes. Mem-brane permeability was believed to be regulated by the migration (under the control of microtubules) and fusion of membrane-limited granules with the plasma membrane; the new membrane patches were believed to possess a greater permeability to water (2 1 5 ) • Malawista (216) has ascribed a r o l e to microtubules i n which microtubules f a c i l i t a t e the i n t e r a c t i o n of lysosome granules and phagocytotic vacuoles i n polymorphonucleocytes. Microtubule i n h i b i t o r s appear to i n h i b i t the movements of the lysosomes within the c e l l and prevent t h e i r degranulization which occurs on contact with phagoeytized material. Several investigators ( 2 1 7 - 2 2 0 ) have reported that micro-tubules and microfilaments p a r t i c i p a t e i n the dispersion of melanin granules i n melanocytes. Microfilaments appear to control dispersion of melanin granules (results i n skin darken-ing) while microtubules appear to dir e c t the aggregation of these p a r t i c l e s (which causes skin l i g h t e n i n g ) . The exact nature of the mechanism for t h i s process i s unclear. Microtubules, but not microfilaments, have been reported to be associated with intraxonal transport ( 2 2 1 ) . Electron-micrographs have portrayed an association between microtubules - 5 1 -and mitochondria or other membrane limi t e d v e s i c l e s (221). In some instances small cross-bridges can be seen to i n t e r - l i n k these organelles. Dahlstrom e_t a l . (222) have demonstrated that microtubule disruptors i n h i b i t the transport of noradren-a l i n e granules i n adrenergic nerves and acetylcholine granules i n cholinergic nerves. Axelrod's laboratory (223) has reported that co l c h i c i n e , v i n b l a s t i n e , and cytochalasin B i n h i b i t the release of neurotransmitters. 7. Previous Evidence f o r Cyto.skeletal Involvement i n .the  Morphagenesis of Virus The r o l e of microtubules and microfilaments i n virus assembly has received minimal attention (224). Microfilaments have been implicated i n some respect to the budding of Herpes viruses from the nuclear membrane ( 2 2 5 , 226) but the effects of cytochalasin B on t h i s process was unclear. Weihing's laboratory (227, 228) has demonstrated a close association between adeno-viruses and microtubules i n vivo and i n v i t r o and has surmised that these structures may be important i n the transport of vi r u s to and from the nucleus. In another study, Stokes (229) purports that the i n t r a c e l l u l a r movement and release of va c c i n i a v i r u s requires a host c e l l cytoplasmic network that involves microfilaments for s t a b i l i t y . High-voltage electronmicroscopy demonstrated that microfilaments were associated with the cyto-plasmic f a c t o r i e s where v a c c i n i a was manufactured and also with m i c r o v i l l i along the c e l l periphery where the virus was released. Two reports have implicated microfilaments i n the budding of RNA tumor viruses from the plasma membrane of the host c e l l . - 5 2 -Damsky et al.(230) found a c t i n associated with p u r i f i e d v i r u s and Panem (231) discovered that cytochalasin B i n h i b i t e d the release of murine Sarcoma-leukemia v i r u s at an apparently l a t e stage of. v i r a l assembly (electronmicroscopy did not show budding p a r t i c l e s but release of viruses was almost immediate when cytochalasin B was removed). C. Chemical Cross-Linking of V i r a l Membrane Proteins Chemical c r o s s - l i n k i n g agents have been u t i l i z e d on a v a r i e t y of systems - ribosomes, histones, red blood c e l l s , mitochondria, E. c o l i membranes, sarcoplasmic reticulum, and numerous enzymes - i n order to determine the proximity of one protein to another. C r o s s - l i n k i n g agents have been the subject of an excellent review (232). Results with these compounds can be very elegant but one must always remember that i n t e r p r e t a t -ion of these r e s u l t s i s l i m i t e d by the a v a i l a b i l i t y of f u n c t i o n a l groups on the protein f o r c r o s s - l i n k i n g . 1. Chemistry of Cross-Linking Agents Chemical c r o s s - l i n k e r s f a l l i nto a number of categories--a l k y l imidates (imidoesters), a c y l azides, s u l f h d r y l cross-l i n k e r s , and dialdehydes. Representative members of these cate-gories are shown i n Table 6 . 2. ' Cross-Linking of V i r a l Proteins (a) V e s i c u l a r Stomatis Virus Dubovi and Wagner ('233) r e c e n t l y cross-linked the proteins of p u r i f i e d v e s i c u l a r s t o m a t i t i s v i r u s with TDA, DSP, and MMB. The cross-linked products were resolved by two-dimensional -53-Table 5 Representative Chemical C r o s s - L i n k e r s CROSS-LINKER FORMULA ABBREVIATION MECHANISM dimethylsuberimidate CH^-C-CCH -^C-OCH, Nil dimethyl 3,3' - t h i o b i s CHp-c-fcH^ -s-s-fcH^ - CI-OCH, (propionimidate) II d i t h i o b i s ( s u c c i n - (>o-c-CHtCH,-5-s-CHJ-cHrc-o )^ i m i d y l propionate) o ° glut a r a l d e h y d e CHO-<tll^ -CHO OMS D T B P R-C-OR + N l l , -I "P H + -N-C-R • ROH (as above) O o -N-C-R d o i CHj IjfcHjCH |c-(CH^ CH jc-fcttf-t 1* - h l ^ +CH-{CH£CH - C -(CH^ CH « t a r t r y l d i a z i d e ( r e v e r s i b l e on a d d i t i o n of period a t e ) * O OM OH O Nfc'-iH-CH-H-W, TOA O OH OH a - C - C M C H - C - N j * 1 0 OM OH O H U I , H -N-C-CH-CH-C-N-c u p r i c d i ( l , 1 0 -phenanthroline) complex N,N'(4 a z i d o - 2 - n i t r o -phenyl)cystamine d i o x i d e ( r e v e r s i b l e on a d d i t i o n of BME) methyl-3-mercapto-propion i m i d a t e ^ © N H - C H J C H - S - S - C H J C H - N M ^ N , NO, II C H - O C - C H C M - S H M M P o x i d i z e s -SH o II N.-R.-.S-S-R. + HS-O i o x i d i z e SH- w i t h H2O2 a f t e r imido e s t e r has r e a c t e d w i t h p r o t e i n -54-electrophoresis (234, 235)' Major proteins of th i s virus are M, N, and G (see section I.A.4). These workers were able to detect dimers and trimers of M and G proteins, an M-N complex, and i n some cases (with TDA and MMB only) a G-M dimer. A photo-graph of t h e i r 2 dimensional slab gels i s shown i n Figure 24. Cross-linked products are i l l u s t r a t e d on horizontal l i n e s while monomeric or uncross-linked species l i e on a diagonal which runs from l e f t to r i g h t . The i n a b i l i t y of DSP to form high oligomers was f e l t to be due to a lesser permeability of the membrane to t h i s agent. First Dimension —• • p I M l ip»7| 0 N M F i g . 24. Autoradiograph of 2-dimensional SDS polyacrylamide gels which i l l u s t r a t e s the cross-linked species of ves i c u l a r stomatitis v i r u s . Virus was cross-linked with (A)TDA, (B)MMB, and (C)DTBP. -55-(b) Semliki Forest Virus Semliki Forest virus has been cross-linked with DMS and subjected to electrophoresis i n one dimension by Garoff and Simons (236, 237). They subjected 50 ug of vi r u s protein to th i s agent at a concentration of 3 mg/ml f o r 2 hr at 20°C. Envelope proteins aggregated into complexes which contained 2-6 components which were resolved by one dimensional SDS acrylamide electrophoresis. Nucleocapsid proteins were said to be p r e f e r e n t i a l l y cross-linked under these conditions and stayed at the top of the gel . Evidence f o r a nucleocapsid-envelope linkage could not be detected d i r e c t l y from these gels but disruption of the membranes of cross-linked virus with Tr i t o n X-100 f a i l e d to release the envelope proteins (236). Large membrane protein—nucleocapsid complexes were is o l a t e d on sucrose gradients and viewed by electronmicroscopy. -56-(c) Adenovirus The only other v i r u s for which cross - l i n k i n g studies have been reported i s adenovirus type 2. E v e r i t t , Lutter and Philipson (238) u t i l i z e d TDA to cross-link p u r i f i e d adenovirus. Two-dimensional electrophoresis was used to establish the topography of i t s proteins. - 5 7 -D. The Present Investigation It was the intent of t h i s thesis to prove through bio-chemical means that Semliki Forest virus matures by budding from the plasma membrane of the host c e l l . Through t h i s process the naked nucleocapsid acquires envelope l i p i d s and proteins. We also were concerned with the mechanism by which budding occurs. Previous evidence to support this hypothesis i s derived from electronmicroscopic observations ( 1 6 , 7 9 , 80) and l i p i d class composition studies conducted on ER. PM, and p u r i f i e d v i r u s (21, 9 5 - 9 8 ) . The l i p i d s of the virus envelope resemble most c l o s e l y those of the host c e l l PM. Similar l i n e s of inve s t i g a t i o n have been conducted on other enveloped viruses such as myxoviruses and paramyxoviruses (110-113) and rhabdo-viruses (101, 102). Results from the above studies are f a r from conclusive--the electronmicroscopic evidence could be based on a r t i f a c t s incurred through sample preparation and many i r r e g u l a r i t i e s exist i n the l i p i d composition studies. I t was hoped that the present i n v e s t i g a t i o n might c l a r i f y t h i s s i t u a t i o n . The problem was attacked through use of radioactive pulse-chase experiments. Infected c e l l s were to be pulsed with [•^H]lieu and the l a b e l was to be followed from the membrane fraction s of the host c e l l into mature v i r u s . Preliminary experiments of t h i s type have been performed on influenza •• (115-120) and v e s i c u l a r stomatitis (104-107) viruses but much of t h i s data i s incomplete and the complex nature of these - 5 8 -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 applied to microsomal fract i o n s and ex t r a c e l l u l a r v i r u s from BHK c e l l s which were infected with Semliki Forest v i r u s (.254); the membranes of infected c e l l s were then to be fractionated into ER and PM. I t was the aim of these experiments to show a cle a r chase of r a d i o a c t i v e l y labeled v i r u s proteins from ER to PM and then into e x t r a c e l l u l a r v i r u s . This would support the contention that virus proteins were synthesized i n the ER and inserted into the PM just p r i o r to virus maturation. The objective of the second h a l f of the thesis was to elucidate the mechanism of budding. We were interested i n the manner i n which v i r a l glycoproteins and nucleocapsid were trans-ported to and inserted into the plasma membrane. The aggregation of v i r a l proteins into d i s t i n c t patches at the c e l l surface, followed by bud formation and extrusion of vi r u s into extra-c e l l u l a r media was of intense i n t e r e s t to us. Lodish regarded the steps of vi r u s envelope formation as a "secretory" process (60) i n which glycoproteins are synthe-sized on membrane bound ribosomes and transported to the plasma membrane i n a manner comparable to i n s u l i n (212) and pancreatic amylase (202) release. Microtubules are reputed to be involved i n secretion of proteins ( 1 3 2 ) . A r o l e for microtubules and microfilaments i n organizing proteins of the plasma membrane has also been proposed ( I 3 6 , 1 3 7 ) ' With these views i n mind, we wished to te s t the effects of cytosk e l e t a l disruptors on vir u s maturation. Microtubule disruptors had previously been -59-shown to i n h i b i t secretion of glycoproteins and some sort of organizational system for v i r a l proteins at the c e l l surface seemed necessary.-The strategy behind these studies was to measure the incorporation of radioactive v i r a l proteins into plasma mem-brane, endoplasmic reticulum and e x t r a c e l l u l a r virus i n the presence of colc h i c i n e , nocodazole, dibucaine, and cytochal-asin B. We also wished to investigate the effects of these drugs on the proximity of vi r u s membrane proteins to each oth during v i r u s maturation through use of protein c r o s s - l i n k i n g agents. -6o-I I . METHODS AND MATERIALS A. Reagents and Equipment L - [ 4 , 5 3H] Leucine ( 4 5 - 6 0 Ci/mmol) and L - [ 3 5 s ] methionine ( 4 0 0 - 5 0 0 Ci/mmol) were purchased from New England Nuclear Cor-poration. Sucrose (RNAse free) was bought from Schwarz/Mann. Acrylamide came from Matheson, Coleman and B e l l ; methyl-enebisScrylamide and d i a l l y i t a r t a r d i a m i d e were supplied by Bio-Rad Laboratories. Sodium dodecyl sulphate was purchased from B r i t i s h Drug House. Actinomycin D came from Merck, Sharpe, and Dohme. Colchicine was bought from Sigma and dibucaine hydrochloride was obtained from ICN/K & K Laboratories. Aldrich supplied the cytochalasin B and Nocodazole. Chemical cross-linkers were purchased from Pierce. A l l other chemicals were reagent grade. Electrophoresis was done with tube and slab electrophoresis c e l l s from Bio-Rad Laboratories (Models 150A and 220 r e s p e c t i v e l y ) . B. Virus and C e l l s The o r i g i n and c u l t i v a t i o n of SF virus has previously been described (241). BHK-21 (Stoker and Macpherson strain) c e l l s were obtained from Microbiological Associates and grown as monolayer cultures on 150 mm X 15 mm p l a s t i c dishes (LUX Scien-t i f i c Corporation). Ce l l s were cu l t i v a t e d i n Dulbecco's Modi-f i e d Eagle's Medium supplemented with 5$ f e t a l c a l f serum while virus infections were performed i n 199 maintenance medium with 2fo f e t a l c a l f serum. - 6 l -C. Polyacrylamide Gel Electrophoresis ( C y l i n d r i c a l Gels) Proteins from c e l l u l a r membranes and e x t r a c e l l u l a r v i rus were analyzed by electrophoresis on 7-5f° acrylamide gels i n the presence of sodium dodecyl sulfate according to Weber and Osborne (24-2) except that d i a l l y l t a r t a r d i a m i d e (243) was u t i l -ized mole fo r mole i n place of me th,yleneb i s acrylamide. The gels were cast to a height of 7 cm from the following stock solutions: 22.2% (w/v) acrylamide, 0 . 8 8 $ d i a l l y l t a r t a r d i a m i d e - 13-5 ml 1.5% (w/v) ammonium persulfate - 2 . 0 ml 7 . 8 gm/l N a 2 P 0 n H 2 0 , 3 8 . 6 gm/l N a 2 H P 0 ^ . 7 H 2 0 , 2 gm/l sodium doaecyl su l f a t e - 2 0 . 0 ml d i s t i l l e d water 4 . 5 ml tetramethylethylenediamine 3 ° u l Membrane samples were s o l u b i l i z e d i n an equal volume of sample buffer (O .386 gm/l NaH 2P0^2.09 gm/l Na^PQ^. 7H 2 0 , 1% (w/v) SDS, 10%> (v/v) g l y c e r o l , 0.1% (w/v) bromphenol blue, 1% (v/v) '3-mercaptoethanol f o r 5 min at 100°C. The 2 compartments of the electrophoresis apparatus were f i l l e d with reservoir buffer ( 3 . 9 gm/l NaH2P0/j, HgO, 19-3 gm/l N a 2 H P 0 ^ 7H 20, 1 gm/l SDS). Electrophoresis was performed at a constant current of 8 mamps per gel with the pos i t i v e electrode i n the lower chamber for 2 . 5 hr. The gels were stained with Coomassie blue solu t i o n ( 0 . 2 5 $ (w/v) Coomassie blue, 4 5 . 4 % (v/v) methanol, 9.2% (v/v) g l a c i a l acetic acid) at 60°C for 1 hr and then destained by either d i f f u s i o n i n 7>5% (v/v) acetic acid, 5%> (v/v) methanol or by electrophoresis ( i n 7• 5%> acetic acid) at 3 - 4 mamps per gel . -62-D. Polyacrylamide Gel Electrophoresis (Slab Gels) One dimensional electrophoresis i n the presence of SDS was performed by the procedure of Laemmli and Favre (244) f o r slab gels. The separation gel was 8 . 5 cm high, 14 cm wide and either 0 . 7 5 mm or 1 .5 mm thick. A 1 cm high stacking gel which contained ten 8 mm wide sample s l o t s was cast on top of the separation g e l . The separation gel consisted of a spec i f i e d percentage of acrylamide and.the stacking gel was 3 . 0 $ (w/v) acrylamide, 0.15% (w/v) methylenebisacrylamide, 0 . 1 2 5 M Tris-HCl pH 6 . 8 , and 0 .1% SDS sulfat e . Gels were made from the following stock solutions: separation Vol  gel (ml) 30% (w/v acrylamide, X 0 . 8 % methylenebisacryl-amide I . 8 3 M Tris-HCl pH 8 . 8 5 - 0 0.5% sodium dodecyl sulfate 1.0% (w/v) ammonium 0 . 7 5 persulfate d i s t i l l e d water 25-X tetramethylethyl- . 10 u l enediamine stacking Vol  gel (ml) 30% acrylamide, 2 . 5 1.5% methylenebis-acrylamide 1 . 2 5 M Tris-HCl 2 . 5 p H 6 . 8 , 1.0% sodium dodecyl su l f a t e 1.0% ammonium 0 . 7 5 persulfate d i s t i l l e d water 1 9 . 2 5 tetramethylethyl- 10 u l enediamine The reservoir buffer consisted of 0 . 0 2 5 M Tris-HCl pH 8.3, 0 . 1 9 2 M glycine, 0.1% SDS while sample buffer was composed of 10% (v/v) g l y c e r o l , 1% (w/v) SDS, 0 . 0 6 2 5 M Tris-HCl,pH 6 .8 , 0.1% (w/v) bromphenol blue, and 10% (v/v) B'-mercaptoethanol ( t h i s l a s t -6 3 -ingredient was not present i n the f i r s t dimension when samples were cross-linked with DSP or DTBP). Electrophoresis was per-formed at a constant current of 3° mAmps:per slab gel f o r either 1 hr (0.75 mm thick gels) or 2 hr (1.5 mm thick gels. Strips (0.75 mm thick by 4 mm wide) of gel were cut from the slab f o r electrophoresis i n the second dimension and the re-mainder of the gel was stained. Electrophoresis i n the second dimension was performed with a separation gel (9 cm high, 14 cm wide, and 1:5 mm thick) on top of which was a 2 cm stacking gel. The separation and stack-ing gels were of the same composition as i n the f i r s t dimension. The s t r i p of gel (0.75 mm thick and 4 mm wide) which contained the separated proteins i n one dimension was placed on top of the stacking gel and overlaid with warm (60 C) buffer which con-tained 1$ (w/v) agarose, 10$ (v/v) B-mercaptoethanol, 0.125 M Tris-HCl pH 6.8, and 0.1$ SDS. Electrophoresis was performed at a constant current of 30 mAmps per slab gel for 3 hr. Slab gels were stained with a so l u t i o n of 0.1$ (w/v) Coomassie blue and 50$ (w/v) t r i c h l o r a c e t i c acid for a period of 0.75 hr and destained overnight with 7.5$ (v/v) acetic acid. E. Quantitation of Radioactivity Contained by Proteins on " ' - - Polyacrylamide Gels C y l i n d r i c a l gels were frozen i n a dry ice/acetone bath and s l i c e d into 1 mm s l i c e s with a Bio-Rad gel s l i c e r . Each s l i c e was placed i n a s c i n t i l l a t i o n v i a l and digested with 0.5 ml 2$ (w/v) periodic acid. Digestion was complete a f t e r 1 hr. -64-Periodate digested s l i c e s were suspended i n 10 ml of ACS s c i n t i l l a t i o n f l u i d (Amersham-Searle) and counted i n a Nuclear Chicago Isocap 300 s c i n t i l l a t i o n spectrophotometer. Slab gels that contained proteins l a b e l l e d with[ slMet were dried under vacuum on Whatman #1 f i l t e r paper and auto-radiographed on Kodak RP Royal X-Omat RP/R14 X-ray f i l m . Radioactive bands were cut from the gels and digested over-night i n 0 . 5 ml of IN NaOH. G l a c i a l acetic acid (100 ul) was added f o r the prevention of chemiluminescence. Ten ml of ACS s c i n t i l l a t i o n f l u i d was added to each sample and the radio-a c t i v i t y was quantitated on a Nuclear Chicago Isocap 300 counter. F. I s o l a t i o n of Radioactively Labeled E x t r a c e l l u l a r Virus Virus released into the growth medium was is o l a t e d by the method of Scheele and Pfefferkorn ( 2 4 5 ) . Virus (10 ml) from the microsomal experiments was layered onto a three-phase gradient that consisted (from bottom to top) of 2 ml of 50$ (w/w) sucrose ( i n 0.2 M CsCl and 0 . 0 0 2 M Tris/HCl, pH 7.8), 4 ml of a continuous gradient that consisted of 25$ (w/w) -sucrose ( i n O . I37 M NaCl, 2.68 mM KCl, 1 .47 mM KHgPO^, and 4.29 mM NagHPO^, adjusted to pH 8 with 1 N NaOH) to 50$ (w/w) sucrose ( i n 0.2 M CsCl and 0 . 0 0 2 M Tris/HCl, pH 7.8), and 4 ml of 5 to 20$ sucrose ( i n O . I37 M NaCl, 2.68 mM KCl, 1.47 mM KH2P0^, and 4.29 mM NagHPO^, adjusted to pH 8 with 1 N NaOH). These gradients were centrifuged at 116 ,000 x g f o r 4 hi-in an SW 27 rotor. -65-Virus ( i n 18 ml of medium) released during the plasma membrane-endoplasmic reticulum pulse-chase experiements was isol a t e d i n a si m i l a r manner except that the volumes of the two upper phases were increased 2.5 times. G. Preparation of Plasma Membrane and Endoplasmic Reticulum Plasma membrane and endoplasmic reticulum from BHK c e l l s were prepared by combining the methods of Atkinson and Summers (251) and Warren (252, 253). Three dishes of nearly confluent BHK c e l l s were each washed twice with 10 ml of cold 10 mM Tris/HCl (pH 8.0). C e l l s were scraped with rubber policemen with 1.5 ml/dish of the 10 mM T r i s buffer. A t o t a l of 4.5 ml of c e l l suspension was homogenized with f i v e strokes of a loose f i t t i n g Dounce homogenizer. Nuclei and whole c e l l s were c e n t r i -fuged from the homogenate at 1000 x g f o r 1 min and the absence of these p a r t i c l e s was v e r i f i e d by phase contrast microscopy. The supernatant was made 0.25 M i n sucrose and 5 mM i n MgClg for s t a b i l i z a t i o n of the membranes. This 1000 x g supernatant (3 "to 4 ml) was placed on a discontinuous gradient that consisted of 45% (w/w) sucrose (3 ml) and JOfo (w/w) sucrose (10 ml) i n 50 mM Tris/HCl, pH 7.4, and 5 mM MgClg. The gradient was c e n t r i -fuged at 7000 x g f o r 20 min and dripped from the bottom i n 0.5-ml f r a c t i o n s . The fra c t i o n s were assayed f o r NADPH cyto-chrome c reductase and glucose -6- phosphatase (endoplasmic reticulum marker enzymes) and 5'-nucleotidase (a plasma membrane marker enzyme). -66-NADPH cytochrome c reductase was assayed by measuring the reduction of cytochrome c (246) on a G i l f o r d spectro-photometer at 550 nm and 25° C. The incubation mixture con-si s t e d of 0.25 ml of buffered substrate (0.1 mM KCN, 0.066 M KCl, 0.044 M phosphate buffer pH 7.6, 0.05 mM cytochrome c) to which 50 u l of enzyme was added. In order to s t a r t the reaction, 50 u l of 0.06 mM NADPH was added. The a c t i v i t y of the enzyme was calculated as follows-. E 5 5 ° 6 2 Reduced Cytochrome c = 27.7 x 10 cm mole E 5 5 ° 6 2 Oxidized Cytochrome c = 9.0 x 10 cm mole 550 550 d • . 550 d - E R e d C y t c ^ [ R e d Cyt c ] - E Q x i d C y t [ R e d C y t c ] 5 5 0 , 6 d Red Cyt c = d_A / 18.7 x 10 ml (0.35 ml) dt dt mole 5'-nucleotidase was assayed by the method of Avruch and Wallach (247). The assay mix consisted of 100 u l 0.5 M Tris-HCl pH 7-5 and 0.2mM MgClg, 50 u l 0.4 mM AMP, 20 u l of C^HlAMP, 0.630 u l H20, and 200 u l of each f r a c t i o n . The mixture was incubated f o r 3° min at 37° C. Enzyme which was boiled p r i o r to the addition of the assay mixture was used as a control.. The re-r a c t i o n was stopped by adding f i r s t 0.200 ml 0.25 M ZnS04 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 - 67 -centrifuge for 5 min at 1000 RPM. A 0.5 ml aliquot of the supernatant was assayed for C3H] adenosine by monitoring the aliquot (using 10 ml/vial of T r i t o n X 100: Toluene (1:3) s c i n t i l l a t i o n f l u i d ) on an Isocap JQ0 s c i n t i l l a t i o n counter. Glucose - 6 - phosphatase was monitored by the method of Baginski et a l . (248) as modified by Hubscher and West (249). - 6 8 -H. Pulse-Chase Experiments with Enriched Fractions of Endo- plasmic Reticulum and Plasma Membrane These experiments were performed i n a s i m i l a r manner to a 6 hr chase study performed on microsomes of infected c e l l s ( 2 5 4 , 2 5 5 ) . BHK c e l l s (24 P e t r i dishes) were infected with virus (20 plaque-forming u n i t s / c e l l s ) i n 199 medium which con-tained 2% f e t a l c a l f serum; the c e l l s were starved at 3|- hr af t e r infection-medium i n each dish was replaced with 10 ml. Earles basic s a l t s medium which contained 2% dialyzed c a l f serum,... At 4 hr they were pulsed with 100 uCi of L - £ 4 , 5 3H]-leucine/dish ( i n 5 ml Earles basic s a l t s medium) fo r 20 min. Subsequently, the l a b e l i n g medium from 3 dishes at 0-hours chase was removed and frozen at -70°C and l a t e r u t i l i z e d f o r i s o l a t i o n of v i r u s . Also at 0-hours chase, the l a b e l i n g medium i n the dishes (3 per time point) f o r the 1- , 2 - , 3 _ » 4 - , 6 . 5 - , and 8 .5-hour chase times was replaced with 7-5 ml of 199 medium which was enriched with 0 . 6 g / l of unlabeled leucine. C e l l s and e x t r a c e l l u l a r v i r u s were harvested at the designated times. Plasma membrane and endoplasmic reticulum were prepared as outlined i n the previous section. Membrane bands were c o l l e c t e d with a Pasteur pipette from the 3 ° "to 45% sucrose interface and the top 3 ml of the gradient. Plasma membrane' and endoplasmic reticulum fractions were centrifuged a t 1 0 5 , 0 0 0 x £ for. 1 hr and the. p e l l e t s were resuspended i n 250 u l d i s t i l l e d water; 200 ug of protein were analyzed by electrophoresis. The gels were' s l i c e d and assayed f o r 3H -leucine as previously outlined. -69-I. Measurement of E x t r a c e l l u l a r Virus Production i n the  Presence of Cytoskeletal Disruptors I n i t i a l l y , BHK c e l l s were infected with 20 plaque-forming units of SFV per c e l l . Preliminary experiments were performed i n which infected BHK c e l l s were labeled at 5 hr af t e r i n f e c t i o n with 50 uCi [ 3H] leucine ( i n 5 ml Earles basic s a l t s medium which contained 2% dialyzed f e t a l c a l f serum) per ;150 x 15 mm culture dish. After 1 hr the radioactive medium was removed and replaced with 10 ml of 199 maintenance medium (with 2% f e t a l c a l f serum) which contained 5° uM co l c h i c i n e . Tritium-labeled virus was harvested from 3 dishes at 10 hr af t e r i n f e c t i o n and i s o l a t e d as described previously; the grad-ient consisted (from bottom to top) of 4 ml of 5°% (w/w) sucrose, 10 ml of 50% (w/w) to 25% .(w/w) sucrose, and 10 ml of 20% (w/w) to 5%,(w/w) sucrose. Other experiments were per-formed i n a s i m i l a r manner except that the cytoskeletal d i s -ruptor (colchicine or dibucaine) was added to the c e l l s at 3 hr af t e r i n f e c t i o n followed by the addition of [ 3H] leucine 4 hr ^after i n f e c t i o n . The l a b e l i n g medium was di l u t e d with an equal volume (5 ml) of 199 maintenance medium (with 2% f e t a l c a l f serum) a f t e r 1 hr. Radioactive e x t r a c e l l u l a r virus was quanti-tated 10 hr l a t e r . In a l l cases, l a b e l i n g and post-labeling media contained the disrupting drug. Control experimentswere performed i n an i d e n t i c a l fashion with lumicolchicine. Lumicolchicine or colch i c i n e i n 20 u l ethanol was added to c e l l cultures infected with SF vitfus at 3 hr post-infection. Ethanol (20 ul) was also added to infected -70-cell.s which were not treated with either colchicine or lumi-c o l c h i c i n e . C e l l s were labeled between 4 and 5 hr of i n f e c -ion. Maintenance medium (5 ml) was subsequently added, and the virus was harvested a f t e r another k hr. Lumicolchicine was prepared from 10 mM c o l c h i c i n e i n ethanol by i r r a d i a t i o n of the s o l u t i o n with UV l i g h t (366 nm) for a period of 15 hr (165). Conversion of c o l c h i c i n e to lumicolchicine was monitored spectrophotometrically. Another microtubule i n h i b i t o r Nocodazole (dissolved i n dimethylsulfoxide so as to y i e l d a f i n a l concentration of 300 uM i n the medium) was also administered at 3 hr a f t e r i n f e c t i o n . The infected c e l l s were labeled withl^H] leucine between k and 5 hr post-infection, and the virus was harvested and quantitated a f t e r a further 10 hr. The control dishes (minus Nocodazole) also contained 1$ dimethylsulfoxide. J. E f f e c t of Colchicine on the Incorporation of !^H] Leucine into Acid Precipitable Protein BHK c e l l s on 15 mm x 60 mm culture dishes were incubated for 1 hr i n Earles basic s a l t s solution/199 maintenance medium (1:1) which contained 2% dialyzed c a l f serum. Colchicine (50 uM) was administered to h a l f the plates during t h i s hour. [-^ H] Leucine (25 uCi/dish) was then added to a l l the dishes. C e l l s were harvested at 0, 0.25, 1, 3, 5 and 7 hr a f t e r ad-d i t i o n of l a b e l . The c e l l s were washed 3 times with 10 ml phos-phate buffered s a l i n e (5), scraped into 2.5 ml of the buffer (50 u l aliquots were removed fo r protein determination) and pr e c i p i t a t e d by addition of 0.5 ml of 10$ (w/v) t r i c h l o r a c e t i c - 7 1 -acid. Precipitates were sedimented at 1 , 0 0 0 x g for 5 min, the supernatants were aspirated and discarded, and the p e l l e t s suspended i n 11 ml of ACS s c i n t i l l a t i o n f l u i d . K. Continuous Labeling Experiments Experiments were performed i n a manner sim i l a r to the previous experiments. Colchicine (50 uM) was administered to BHK c e l l s at 3 hr post-infection. I3H] Leucine (50 -uCi/150 mm x 15 mm dish) i n 5 ml of Earles medium (which contained 2% dialyzed c a l f serum) was added at 4 hr, and supplemented with 5 ml 199 maintenance medium/dish at 5 hr. Plasma membranes and e x t r a c e l l u l a r virus were harvested at 5 i 6 , 8 and 10 hr a f t e r i n f e c t i o n . The membrane samples were subjected "to sodium dodecyl s u l f a t e polyacrylamide electrophoresis on c y l i n d r i c a l 7.5% acrylamide gels which were cross-linked with d i a l l y l -tartardiamide; the gels were s l i c e d and s o l u b i l i z e d with 2% periodic acid. Radioactive e x t r a c e l l u l a r virus was quantitated a f t e r p u r i f i c a t i o n by v e l o c i t y gradient sedimentation. L. Pulse-Chase Experiments Performed i n the Presence of Colchicine and Dibucaine Studies were performed i n which colchicine was added at 3 hr post-infection. At k hr a f t e r i n f e c t i o n , the c e l l s were incubated i n Earles basic s a l t s medium (• 2% dialyzed. c a l f ... serum^ for 3 ° min. Subsequently, a pulse of either [^H ] leucine t o r [ 3•%']methionine i n Earle's basic s a l t s solution was applied (50 uCi isotope per 150 mm x 15 mm culture dish). T h i r t y min l a t e r , the l a b e l i n g medium was removed and replaced with 199 -72-maintenance medium which was supplemented with either non-radioactive leucine or methionine (0.5 mg/ml). Actinomycin D (1 g/ml) was present i n a l l media throughout the experiment. Plasma membrane ghosts, endoplasmic reticulum fragments and e x t r a c e l l u l a r v i r u s were prepared from c e l l s harvested at the s p e c i f i e d times. Radioactive membrane proteins were subjected to SDS polyacrylamide electrophoresis. Incorporation of [-^ H] leucine or [-^S] methionine was quantitated by auto-radiography or elution of the proteins from the gel and determination of r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n counting. M. P u r i f i c a t i o n of Semliki Forest Virus for Cross-Linking Studies Ten r o l l e r ^ bottles of confluent BHK-21 c e l l s (clone 13 purchased from Flow laboratories) were infected with SF virus at a m u l t i p l i c i t y of i n f e c t i o n of 0.1 - 0.2 plaque forming units per c e l l i n 10 ml of 199 maintenance media [2% f e t a l c a l f serum). Q Each b o t t l e contained 1-2 x 10 c e l l s . Following 1 hour adsorption, 40 ml of 199 maintenance media (2fo f e t a l c a l f serum) was added to each b o t t l e . Virus was harvested from the c e l l media a f t e r 24 hours of i n f e c t i o n . The media was centrifuged at 15,000 x g (10,000 RPM on a Sorval GSA rotor) for 15 min i n order to sediment detached c e l l s and debris. Soli d ammonium sulfa t e was added to the supernatant gradually over a h a l f hour to y i e l d 65$ saturation at 0°C (390 gm of ammonium sulf a t e were added per l i t e r of supernatant). The medium was s t i r r e d over this half hour and a pH of about 7.4 was maintained with dropwise addition of 0.66 N NaOH. The preparation was allowed to -73-p r e c i p i t a t e (without s t i r r i n g ) f o r 1 hour at 0°C and the pre-c i p i t a t e was recovered hy centrifugation at 15,000 x g f o r 30 min. The ammonium sulf a t e p e l l e t which was o r i g i n a l l y obtained from 10 bottles of infected c e l l s was suspended i n 20 ml of phosphate buffered saline pH 7-^ (see section II.F) and centrifuged at 15,000 x g f o r 2 min i n order to remove part i c u l a t e material. The resuspended p e l l e t was layered onto two gradients which consisted of 28 ml of 10-45% (w/w) potassium t a r t r a t e ,and was centrifuged at 65,000 x g (25,000 RPM on a Beckman SW27 centrifuge head) f o r 3 hr. Two bands were v i s i b l e - the lower one consisted of c e l l debris while the upper one was p u r i f i e d v i r u s . The vi r u s band was diluted three times with phosphate buffered saline and either placed on another potassium t a r t r a t e gradient f o r further p u r i f i c a t i o n , or centrifuged at 105,000 x g for 2 hr i n order to obtain a virus p e l l e t . N. Cross-Linking Studies Performed'.'on P u r i f i e d Semliki Forest  Virus or Virus-Infected C e l l s SF virus was cross-linked with DMS, DSP, and DTBP i n a manner si m i l a r to that of Garoff and Simons (236,237). Virus (100 ug protein i n 50 u l of 0.15 M NaCl) was subjected to cross-l i n k i n g by addition of 50 u l of either DMS, DSP, or DTBP i n 0.2 M triethanolamine buffer pH 8.5• . DSP was f i r s t dissolved i n dimethylsulfoxide to y i e l d a f i n a l concentration of 2% (v/v) dimethylsulfoxide i n the cr o s s - l i n k i n g reaction (250). The reaction was either terminated with the addition of IM Tris-HCl pH 8.5 (20 ul) or by immediate addition of sample buffer for gel electrophoresis (50 u l ) . The cross-linked v i r u s was subjected to electrophoresis on either 3-5% acrylamide c y l i n d r i c a l gels or 6.5% acrylamide slab gels. -74-Whole c e l l s which were infected with Semliki Forest virus were also cross-linked with DSP and DTBP. BHK (Stoker McPherson strain) c e l l s were pulse-labeled f o r \ hr with r^SjJmethionine (100 uCi/plate) at 4.5 hours a f t e r i n f e c t i o n with SF v i r u s . Radioactivity was chased f o r 2 hr i n methionine enriched 199 maintenance media. The c e l l s were washed twice with cold phosphate buffered saline pH 7.4 (10 ml/dish); 5 ml of 0.15 M NaCl and 5 ml of triethanolamine buffer pH 8.5 which contained cr o s s - l i n k e r was added to each plate. The reaction was allowed to proceed f o r . 0.5 hr or 1 hr and was terminated by the addition of 1 ml M Tris-HCl (pH 8.5) and plasma membrane ghosts were prepared. Preparations i n which the effects of colchicine and dibucaine were measured contained the drug throughout the cros s - l i n k i n g procedure. -75-I I I . RESULTS A. Resolution of V i r a l Proteins on Polyacrylamide Gels i n the Presence of Sodium Dodecyl Sulfate Semliki Forest v i r u s was c u l t i v a t e d and p u r i f i e d as described i n Section II.M. I n i t i a l l y v i r a l proteins were resolved upon the polyacrylamide gel system of Weber and Osborn (13). The samples were incubated i n buffer which contained 0.5$ g-mercaptoethanol and 0.5$ SDS. A photograph of c y l i n d r i c a l gels which consisted of 7-5$ acrylamide i s presented i n F i g . 25. F i g . 25- P u r i f i e d Semliki Forest v i r u s and standard proteins which were subjected to electrophoresis on 7-5$ polyacrylamide gels i n the presence of sodium dodecyl s u l f a t e . Virus was p u r i f i e d as outlined i n "Methods and Materials" and s o l u b i l i z e d i n sample buffer which contained a f i n a l concentration of 0.5$ g-mercaptoethanol and 0.5$ SDS. V i r u s (10 and 20 ug protein) and standard proteins (phosphorylase a, bovine serum albumin,trypsin, and cytochrome c) were separated on c y l i n d r i -c a l gels which consisted of 7-5$ polyacrylamide. Proteins were stained with Coomassie blue. Envelope proteins are designated El32 since they resolve poorly i n t h i s system and nucleocapsid protein i s denoted NC. E3 was not detected on these gels. Protein standards f o r molecular weight c a l i b r a t i o n were also subjected to electrophoresis. An absorbance scan of the gel on which 10 ug of p u r i f i e d virus was fractionated i s shown i n Fi g . 26. The v i r a l proteins, E-^  and Eg» were not c l e a r l y resolved i n t h i s system. 0 1 •distance from origin (cm) Fig. 26. Absorbance scan of Semliki Forest virus proteins which were separated on a 7-5% polyacrylamide SDS gel. The gel i l l u s t r a t e d i n Fig. 25 (10 ug of SF virus protein) was scanned at 550 nm wavelength to y i e l d an absorbance p r o f i l e . Envelope proteins are designated E1E2 and nucleocapsid i s denoted NC. P u r i f i e d virus was also fractionated upon slab gels which were prepared according to Laemmli and Favre (244). The presence of 8-mercaptoethanol affected the separation of vir u s proteins dramatically as i l l u s t r a t e d i n Fig. 27. -77-r ~* L E , E 2 N C — IMC Fig. 27. Proteins from SF virus were subjected to SDS electro-phoresis on 6.5% polyacrylamide slab gels i n the presence and absence of 10% (v/v) (3-mereaptoethanol. Purified virus (20 ug protein) was subjected to electrophoresis on slab gels which consisted of 6.5% polyacrylamide. Samples which were run i n the l e f t gel were solubilized in sample buffer which contained 10%(v/v) (5-mercaptoethanol while those on the right gel were solubilized in sample buffer in which 3-mercaptoethanol was absent. V i r a l proteins which were electrophoresed in the presence of 10% 8-mercaptoethanol showed absolutely no separation of E, and E 2 proteins while the absence of g-mercaptoethanol yielded two distinct protein bands, g-mercaptoethanol apparently reduced -78-intramolecular d i s u l f i d e linkages and resulted i n a slower migration of the v i r u s proteins. Two-dimensional gels presented i n Section III.F indicate that the protein band which i s designated Eg co-migrates with E-^ i n the presence of 10% (v/v) B-mercaptoethanol. Garoff, Simons, Renkonen (12) have performed amino acid analyses which indicate that. E-^  and Eg d i f f e r i n composition and not merely i n t h e i r conformation due to the arrangement of d i s u l f i d e bonds. B. I s o l a t i o n of Plasma Membrane Fragments and Endoplasmic Reticulum From BHK-21 C e l l s A procedure f o r separation of endoplasmic reticulum vesicles-and plasma membrane ghosts from BHK c e l l s was outlined i n Section II.G.. There was no s i g n i f i c a n t difference i n the separation on the discontinuous gradient of these membranes from mock and infected c e l l s . Marker enzymes (5 '-nucleotidase f o r plasma membranes and NADPH cytochrome c reductase f o r endoplasmic reticulum) were assayed for the fractio n s from the gradient and the p r o f i l e s of enzyme a c t i v i t y are presented i n F i g . 28. < It i s evident that the plasma membrane marker (5'-nucleotidase) was separated from the endoplasmic reticulum marker (NADPH-cytochrome c reductase). Spe c i f i c a c t i v i t i e s and p u r i f i c a t i o n r a t i o s were calculated f o r "'.-the marker enzymes on other gradients prepared from mock infected BHK c e l l s . Enzymes associated with endoplasmic reticulum (NADPH-cytochrome c reductase and glucose-6-phosphatase) and plasma membrane (5'-nucleotidase) were assayed. The a c t i v i t i e s were measured from c e l l lysate, the plasma membrane 1.5 3 c 11 -S o * E 2 c o z "in E 1.0 + 0.5 PM / ER GRADIENT FRACTIONS f " " /ER +0. ^*X***.KJ.*.^-...*...<i....Ji...J.Ji.^..». 5 10 15 20 +0.4 +0 5 f D o CD ^ 3 3 3 3 CD 3 0 t 30 ~ CD 0.2 xg. _i o o <-*• AS* row CD 0.1 • 30% ER 45% PM Ghosts & Fragments F i g . 28. P r o f i l e s of enzymatic a c t i v i t i e s from a discontinuous sucrose gradient used f o r i s o l a t i o n of endoplasmic reticulum and plasma membrane. Baby hamster kidney c e l l s were lysed and centrifuged at 1000 x g_ to remove nuc l e i . The supernatant was layered onto a discontinuous sucrose gradient (as described i n Section I I . G ) and centrifuged at 7000 x g f o r 20 min. This gradient was dripped from the bottom of the tube, 0.5 ml fra c t i o n s were c o l l e c t e d and enzymes assayed. • : : • , 5' -nucleotidase (marker f o r plasma membrane);" • , NADPH-cyochrome c reductase (marker f o r endoplasmic reticulum). -80-f r a c t i o n , and endoplasmic reticulum f r a c t i o n (both the 100,000 x g p e l l e t and supernatant). The r e s u l t s are presented i n Table a S CQ cd H ft U o 3 H O •H -P ^ O •H E CQ cd H ft o CD O •H -P 0 CQ H •H XI M Crf CD b-l -P O cd H cd o CQ CD fl 0 ' H o CQ CD •H -P •rH J> •H •P O <: CD fl cd U o x> e CD S CD XI •P fl O T} CD cd ft U ft CD d U • CD H £ H CQ fl O • H -P O cd C/2 H CD fl a u x> s CD S CQ CD Cd TJ X> H S O H b H CD M CD 0 CQ £ O CD 3 CQ TJ Cd 0 T j <-l -H -P O O CD 0 H S O O ^ U fl -fl I O - H O U > H f ^d) >> O CD fl cd H X! e CD T 3 S 0 U cd 3 S CQ co cd cd 0 X o o o o o H 0 .fl -p x! •P o X> 0 -P cd CQ >> fl H O •H -P o cd H tH o B o tH H O •H •P 0 u ft CQ cd •P fl 0 ft-H TD 1 fl cd 3 H o •H -P 0 H O •H a CQ cd H ft o O O Ti CQ •p 0 T ) ! H fl cd o •H ^ e 0 ^ CQ CQ W)cd cd H -P ft cd o T3 fl 0 •P i •H VO £ I 0 CO o o 3 0 •H •P •H ^•H ft-P CQ O o cd XJ ft 0 CQ 0 H £ ft o CQ fl 0 - 0 fl H 0 cd cd X> CQ CQ CQ fl CQ cd cd cd fl o •H •P O cd U tH 13 fl -P fl cd -p cd fl 0 ft CQ 0 cd -P 0 0 ft > a . LU CO < r -< X a. co o J: i LU co o O L A LU < C • H p-< <_> c • H < CJ Z> Q_ s: >• • 1- cn - e r - H O 0 LTl O J Csl CD CO C D o C D C O L A O J r A co < CO >-_1 LU C_5 -81-Plasma membrane appeared to be p u r i f i e d 37-fold with minimal contamination due to endoplasmic reticulum. The y i e l d of plasma membrane from the o r i g i n a l lysate was 35•3% £ 5'58% (S.D., N' = 3)- The endoplasmic reticulum was highly enriched as judged by the NADPH-cytochrome c reductase a c t i v i t y ( 157-fold pu r i f i c a t i o n ) but less enriched according to the r e s u l t s with glucose-6-phosphatase (6-fold). In addition the endoplasmic reticulum was contaminated with 5'-nucleotidase a c t i v i t y . C. Radioactive Pulse-Chase Experiments with Endoplasmic Reticulum and Plasma Membranes In an e a r l i e r study we demonstrated a precursor-product r e l a t i o n s h i p between the v i r a l proteins i n the microsomes and i n ex t r a c e l l u l a r v i r u s ( 254). We wanted to re f i n e the experiment to see whether such a r e l a t i o n s h i p might exist between the v i r a l proteins i n the plasma membrane and released v i r i o n s . An experiment s i m i l a r to the microsomal experiment with a 6-hour chase was performed except that frac t i o n s enriched with plasma membrane and endoplasmic reticulum were i s o l a t e d from the c e l l s . Proteins from the membranes and vi r u s were analyzed by electrophor-esis on 7-5% polyacrylamide gels i n the presence of sodium dodecyl s u l f a t e . P r o f i l e s showing [ 3H]leucine incorporation and Coomassie blue scans were plotted f o r the endoplasmic reticulum and plasma membrane fractio n s at 0, 1, 2, 3» >^ 6.5, and 8.5 hours chase (Figs. 29 and 3°)• A. radioactive p r o f i l e of the proteins from endoplasmic reticulum at 0-hours chase shows the existence of v i r a l precursor proteins - NVP 130, . - 8 2 -0 - 8 . 5 H R C H A S E E R G E L S F i g . 2 9 - Incorporation of E3H"]leucine into the proteins of endoplasmic reticulum over a 0 - 8 . 5 hour chase period. Infected baby hamster kidney c e l l s were pulsed f o r 20 min with pHJleucine 4 hours a f t e r i n f e c t i o n . Endoplasmic reticulum and plasma membrane were separated and fractionated as shown i n Fig. 28 at 0 , 1 , 2 , 3 » ^ » 6 . 5 , and 8 . 5 hours following removal of [ 3H]leucine. Precursor proteins (NVP 1 3 0 , NVP 9 8 , and PE2), envelope proteins ( E l and E2), and nucleocapsid protein (NC) were c l e a r l y evident. • • , [3H]leucine, counts per min; , A550, Coomassie blue s t a i n . - 8 3 -1 2 3 4 5 6 7 D i s t a n c e f r o m O r i g i n ( c m ) 2 3 4 5 6 D i s t a n c e f r o m O r i g i n ( c m } 'o 31 X e 3 2| •x1 ER -N C 6.5 h o u r s c h a s e /A • V i {""N A • \ /1 f\ j Ma ' \ \ 1 \l il t i L \ 2 3 4 5 6 D i s t a n c e f r o m O r i g i n ( c m ) Fig. 29. Incorporation of [ ^ H]leucine into the proteins of endoplasmic reticulum over a 0-8.5 hour chase period (continued). - 8 4 -0 - 8 . 5 H R C H A S E P M G E L S Distance from Origin (cm) Distance from Origin &m) F i g . 30. Incorporation of [^HJileucine into the proteins of the plasma membrane 0-8.5 hours a f t e r a 20 min pulse. The experiment described i n Fig. 2 9 was performed and plasma membrane was is o l a t e d from the JO-k^fo sucrose interface of the discontinuous gradient used f o r separation of plasma membrane and endoplasmic reticulum. After u l t r a c e n t r i f u g a t i o n the plasma membrane proteins were subjected to sodium dodecyl s u l f a t e -acrylamide gel elctrophoresis, scanned for Coomassie blue, and assayed f o r [3H]leucine. • ; • , [3H]leucine counts per min; , A550 of Coomassie blue. - 8 5 -2 3 4 5 6 7 Distance from Origin (cm) 2 3 4 5 6 Distance from Origin (om) mi rap PM . 8.5 hours chase • / NC \ / MK. K. \ 2 3 4 5 6 Distance from Origin (cm) Fig. 3 0 . Incorporation of [ 3H]leucine into the proteins of the plasma membrane 0 - 8 . 5 hours after a 20 min pulse (continued). - 8 6 -NVP ' " 9 8 , and PEg - which have been detected by other laboratories (Fig. 29) ( 6 5 - 7 4 ) . In addition envelope proteins (E-j^ and Eg) and nucleocapsid were c l e a r l y present. During the subsequent chase period, r a d i o a c t i v i t y associated with precursor and envelope proteins diminished, whereas the r a d i o a c t i v i t y i n the nucleocapsid protein f a i l e d to decline over the 8 . 5 hour chase period. There was, however, a decrease i n s p e c i f i c r a d i o a c t i v i t y of the nucleocapsid protein since the i n t e n s i t y of the Coomassie blue peak that corresponded to nucleocapsid protein increased during the chase period. These r e s u l t s were quantitated i n Figs. 31 and 3 2 . Time After Pulse (Hours) F i g . 31- Time course f o r incorporation of T^Hlleucine into v i r u s - s p e c i f i e d precursor proteins associated with the endoplasmic reticulum. Endoplasmic reticulum was i s o l a t e d from infected c e l l s at various times a f t e r a pulse of [3H]lleucine and t o t a l r a d i o a c t i v i t y i n the v i r a l proteins was plotted against time, o o, PE2 ; A A, NVP 9 8 ; 0 O, NVP 1 3 0 . Fig. 32. Time .course for incorporation of [3}i]leucine into v i r u s -s p e c i f i e d proteins associated with the endoplasmic reticulum. Endoplasmic reticulum was is o l a t e d from infected c e l l s at various times a f t e r a pulse of. I3H]leucine and t o t a l r a d i o a c t i v i t y i n the v i r a l proteins was plotted against time. • • ', envelope proteins (Ej and E 2 ) ; A A, nucleocapsid (NC) protein. Analysis of the proteins from the plasma membrane indicated that there was a dramatic increase i n labeled envelope proteins (E-^ and Eg) and nucleocapsid protein during the f i r s t 3 hours of the chase period (Figs. 30 and 33). Subsequently a decrease i n the labeled plasma membrane proteins (E-^, Eg, and nucleocapsid) occurred with a concomitant increase i n labeled virus i n the c e l l medium (Fig. 33)' P r o f i l e s f o r radioactive e x t r a c e l l u l a r virus which were plotted for the appropriate chase times are shown i n Fig. 34.. - 8 8 -Time After Pulse (Hours) F i g . 33- Time course f o r incorporation of T^H] leucine into v i r u s -s p e c i f i e d proteins associated with plasma membrane and extrac e l l u -l a r v i r u s . Plasma membrane and virus were i s o l a t e d from infected c e l l s at various times a f t e r the pulse of [3H]leucine and the t o t a l r a d i o a c t i v i t y f o r the v i r a l proteins was plotted against time. Plasma membrane y i e l d was estimated to be 35*3 + 5 ' 5 8 $ (S.D., N = 3) and only three-fourths of the plasma membrane i s o l a t e d was applied to each gel. In order to normalize 1 the data for the ex t r a c e l l u l a r v i r u s with the data f o r the plasma membrane, the t o t a l r a d i o a c t i v i t y associated with e x t r a c e l l u l a r virus was multiplied by (O .353 x 0 . 7 5 ) . • • , Envelope proteins ( E i and E 2 ) ; * nucleocapsid ( NC) protein; • • , v i r u s . A precursor-product r e l a t i o n s h i p between envelope proteins (E-^ and E^) i n the endoplasmic reticulum, plasma membrane, and ext r a c e l l u l a r v i r u s i s evident from Figs. 3 1 , 3 2 , and 3 3 . The major decrease i n r a d i o a c t i v i t y i n the envelope proteins of the -89-8.5 HR CHASE 15 S) 3B F i g . Jk. P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r v i rus which was produced over a 0-8.5 hour chase. The experiment described i n Fig. 28 was performed and e x t r a c e l l u l a r v i r u s was harvested from the medium as described i n Section II.F at 0,1,2,3,4,6.5. and 8.5 hours chase. Media from 3 dishes (18 ml) was placed on the gradient which was then centrifuged. The tube was then dripped from the bottom and 0.25 ml fractions were col l e c t e d and 5° u l aliquots were assayed f o r t r i t i u m by s c i n t i l -l a t i o n counting. -90-endoplasmic reticulum occurred between 0 and 3 hours while these labeled proteins increased over the same i n t e r v a l i n the plasma membrane. Between 4 and 8 .5 hours a f t e r the pulse of r a d i o a c t i v i t y , there was a rapid decline i n radioactive v i r a l proteins which were associated with the plasma membrane, while there was a marked increase i n the r a d i o a c t i v i t y i n the extra-c e l l u l a r v i r u s . The r i s e and f a l l of r a d i o a c t i v i t y of the enve-lope proteins (E-^ and Eg) and nucleocapsid protein of the plasma membrane appeared to be p a r a l l e l . D. Cytoskeletal Disruptors and Their Effects Upon Virus Assembly 1) The Effect of Colchicine on Virus Maturation We tested the possible involvement of microtubules i n the budding process by addition of colc h i c i n e to BHK c e l l s infected with Semliki Forest v i r u s . An appropriate dosage was determined on the basis of previous studies with f i b r o b l a s t s (137) and observations through a phase-contrast microscope over a period of 24 hours; c e l l s i n contact with c o l c h i c i n e (at concentrations of 20uM - 100 uM) appeared v i a b l e f o r at l e a s t 15 hours (Table 8). C e l l s i n contact with colc h i c i n e demon-strated l i t t l e i f any rounding i n t h i s concentration range. I n i t i a l l y , the infected c e l l s were incubated with [ 3H] leucine f o r 1 hour at 5 hours a f t e r i n f e c t i o n with Semliki Forest v i r u s . Subsequently, the medium was removed and replaced with maintenance medium which contained 50 uM colchicine. Labeled v i r u s was harvested at 10 hours a f t e r i n f e c t i o n and i s o l a t e d by v e l o c i t y gradient centrifugation. Virus production was i n h i b i t e d by 73% ( F i g . 3 5 ) - Since c o l c h i c i n e disrupts microtubules -91-Table 8 Effects of various concentrations of colchicine on c e l l shape (As determined by phase contrast microscopy). BHK-21 c e l l s were incubated i n 199 maintenance media which contained 0-400 uM col c h i c i n e . Observations of the c e l l condition were made at 0, 15 min, 3° min, 1 hr, 2 hr, 4 hr, 10 hr, 15 hr, and 24 hr. x denotes that the c e l l s were detached from the surface of the culture dish, r denotes that the c e l l s were rounded, - designates no drug effect.. Incubation Time With Colchicine (Hr) colchi c i n e uM Q Q ^ Q ^ Q 1 > Q 2.0 k.O 10.0 15.O 24.0 400 200 100 75 50 25 10 5 2 0.5 0.25 0.10 r x- ' x r r x - - r - - r - - r -92-1.5 + Control 1.0 + - 0.5 E a o 3 CD X 1.0 + 0.5 10 15 20 25 Colchicine 50uM 10 15 20 FRACTION NO. Fig. 35- P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r virus produced by c e l l s i n the presence and absence of col c h i c i n e . Infected c e l l s were incubated with [3}i]leucine for 1 hour at 5 hours post-infection with SF v i r u s . The medium was removed and replaced with maintenance medium which contained 50 uM colch i c i n e . Labeled virus was harvested at 10 hours a f t e r '"' i n f e c t i o n and i s o l a t e d by v e l o c i t y gradient centrifugation. The tube was dripped from the bottom i n 0.25 ml fractions and 50 u l aliquots were assayed f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. -93-slowly (168), subsequent experiments were performed i n which the drug was added at 3 hours and [ 3H] leucine ..'at 4 hours a f t e r i n f e c t i o n . In th i s instance production of [ % ] - labeled virus was reduced 86$ with 50 uM colch i c i n e 10 hours a f t e r the addition of l a b e l ( Fig. 36). Fract ion No. F i g . 36. P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r v i rus produced by c e l l s i n the presence and absence of colchicine. Colchicine (50 uM) was added to infected c e l l s at 3 hours and [3H]leucine at 4 hours a f t e r i n f e c t i o n . Media from these c e l l s (18 ml) was placed on a sucrose gradient a f t e r 14 hours i n f e c t i o n , centrifuged, and the tube was dripped from the bottom i n 0.25 ml fra c t i o n s . Aliquots (200 ul) were assayed f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. - 9 4-This inhibitory effect was directly observable on sucrose gradients which were layered with media from infected c e l l s (Fig. 37); the virus band obtained from the colchicine-treated cells is markedly fainter than the band from untreated c e l l s . Control • Colchicine Fig. 3 7 . Photograph of sucrose gradients which contain virus produced i n either the absence or presence of colchicine (50 uM) . BHK ce l l s were infected with 20 plaque-forming units of SF virus per c e l l . Colchicine was added to the 199 maintenance medium after 3 hours infection, extracellular virus was harvested at 14 hours post-infection. Media (18 ml) from three 15 mm x 150 mm plates was layered onto each sucrose gradient. - 9 5 -I t was necessary f o r us to establish as f a r as possible that the i n h i b i t i o n was d i r e c t l y a t t r i b u t a b l e to microtubule disruption and not due to a side e f f e c t of the drug (such as a possible i n h i b i t i o n of. nucleoside transport). Lumicolchicine was prepared from colchicine (as outlined i n Section II.I) and the conversion was monitored spectrophotometrically over 12 hours (Fig. 3 8 ) . 1.0 + 0.8 + a, °'6 + o c CO -Q o 0.4 w < 0.2 + 200 Lumico l ch i c ine Co lch ic ine 300 400 wavelength nm. Minimum! 500 F i g . 3 8 . Absorbance spectrum f o r colchicine and lumicolchicine at room temperature. Lumicolchicine was prepared from colchicine as outlined i n Section I I , I . The change i n absorbance spectrum was monitored a f t e r colchicine was i r r a d i a t e d with u l t r a v i o l e t l i g h t f o r 15 '• hours at room temperature. -96-Subsequently, lumicolchicine, which i s unable to disrupt microtubules ( 165) and colchicine were administered at 20 uM. There was l i t t l e or no effect of lumicolchicine on the production of labeled v i r u s (Fig. 39). 4 8 Fraction No . 1 8 2 2 F-ig. 39 • Effects of lumicolchicine and colchicine on the production of radioactive e x t r a c e l l u l a r v i r u s . Lumicolchicine (20 uM) and colchicine (20 uM) were administered to infected c e l l s at 3 hours i n f e c t i o n . The c e l l s were then labeled with [3H"]leucine at 4 hours i n f e c t i o n . Radioactive medium was removed and [ 3H]-labeled virus was harvested at 8 hours i n f e c t i o n . The vi r u s was is o l a t e d on sucrose gradients and the tubes were dripped from the bottom into O.30 ml fr a c t i o n s . Aliquots ( 50 ul) were assayed f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. - 9 7 -Nocodazole ( 1 6 7 ) , at 300 uM reduced labeled virus production by 85% (Fig. 40). Control Nocodazole 300uM Fraction Number (p.25ml/tube) Fig. 4 0 . Effect of Nocodazole on the production of radioactive e x t r a c e l l u l a r v i r u s . Nocodazole ( 3 0 0 uM) was administered to infected c e l l s at 3 hours in f e c t i o n ; c e l l s were then labeled with [3H]leucine at 4 hours i n f e c t i o n . Radioactive medium was supplemented with an equal amount of maintenance medium a f t e r 1 hour. [3H']-Labeled v i r u s was harvested at 10 hours i n f e c t i o n and is o l a t e d on sucrose gradients. Aliquots (200 ul) of the 0 . 2 5 ml fracti o n s were assayed f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. We also wanted evidence that colchicine was not a f f e c t i n g protein synthesis or the transport of [3H]leucine into the c e l l . BHK c e l l s were labeled with [3H]leucine ( i n the presence or absence of 5° uM colchicine) and the incorporation into material that was precipitated by t r i c h l o r o a c e t i c acid was monitored. -98-There was no observable e f f e c t by colchicine on [^Hjleucine incorporation over 8 hours (Fig. 41). -i i i i i • i 0 1 2 3 4 5 6 7 Time of Incubation With (3H)~Leu (Hours) Fig. 41 Incorporation of {3rl]leucine into t r i c h l o r o a c e t i c acid (TCA) pre c i p i t a b l e material from BHK c e l l s i n the presence or absence of colc h i c i n e . C e l l s were i n i t i a l l y incubated for 1 hour i n Earle's basic s a l t s solution/199 maintenance medium (1:1); one-half the dishes contained colchicine (50 uM) . . [3rl]Leucine (25 uCi/dish) was addied to a l l the dishes and c e l l s were harvested at 0, 0.25, 1, 3, 5, and 7 hours. The labeled c e l l s were suspended i n 2.5 ml buffer and precipitated by addition of 10$ (w/v) t r i c h l o r o a c e t i c acid. Precipitates were resuspended i n s c i n t i l l a t i o n f l u i d . C e l l s treated with (v v) or without colchicine (• • ). -99-A l l the above data indicate that colchicine i n h i b i t s virus production by disruption of microtubules. The next question was at what stage of vi r u s assembly did the i n h i b i t o r act. Hence, we performed a series of continuous l a b e l i n g and pulse-chase experiments. For the continuous l a b e l i n g experiment, colchicine (50 uM) was added at 3 hours a f t e r i n f e c t i o n . The £ 3Hjleucine i n Earle's medium was added at 4Jr hours, followed by supplementation with maintenance medium at 5 hours. Plasma membrane and e x t r a c e l l u l a r virus were harvested at 0 , 1, 3 , and 5 hours post-labeling. F i g . 42 shows the amount of r a d i o a c t i v i t y i n e x t r a c e l l u l a r virus i s o l a t e d on sucrose gradients from colchicine-treated and control c e l l s . I n h i b i t i o n of virus production occurred over the entire labeling period. .The incorporation of I^H]leucine into v i r a l proteins which were present i n the plasma membrane at the various l a b e l l i n g times i s shown i n Fig. 43- An accumulation of radioactive v i r a l proteins occurred i n the plasma membranes of colchicine-treated c e l l s (Fig. 4 4 ) . These data suggest that virus proteins reach the c e l l surface, and that colchicine i n h i b i t i o n occurs at a l a t e r stage of morphogenesis. This p o s s i b i l i t y was tested further by pulse-chase studies i n which colch i c i n e was added at 3 hours a f t e r i n f e c t i o n with subsequent addition of either [ 3H]leucine or [ 3^S]methionine at 4g- hours f o r a pulse period of 30 min. Medium enriched with either non-radioactive leucine or methionine was added and the plasma membrane and e x t r a c e l l u l a r virus were harvested at sp e c i f i e d times during the chase period. F i g . 45 i l l u s t r a t e s -100-Control Colchicine 50uM 0 hours 0 hours 10 post-label post-label 5 - -I o 1 hour 1 hour T - 10 post-label X post-label £ a 5 -u O _J 10 3 hours 3 hours . post-label post-label 1 \ 5 \ -10 h 5 hours 5 hours • l l post-label post-label 5 J S . i i l T I - l — * * * -»«»*»«*««* ' ' " ' 10 20 30 40 10 20 30 40 Fraction Number (o.25ml/tube) F i g . 42. P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r v irus produced by c e l l s which were continuously labeled with [3H]leucine i n the presence and absence of colchicine. Colchicine (50 uM) was administered to BHK c e l l s at 3 hours post-infection. [3H] leucine (50 uCi) i n 5 ml of Earle's basic s a l t s medium was added to each dish at 4 hours, and supplemented with 5 ml 199 maintenance medium at 5 hours. Plasma., membranes and e x t r a c e l l u l a r v i r u s were harvested at 5» 6, 8, and 10 hours a f t e r i n f e c t i o n . E x t r a c e l l u l a r v i r u s was harvested from untreated c e l l s (control) and colchicine-treated c e l l s at 0, 1, 3> and 5 hours l a b e l l i n g and i s o l a t e d on sucrose gradients. -101-Distance from Origin (cm) F i g . 4 3 , P r o f i l e s of sodium dodecyl sulfate gels which contained plasma membrane proteins from c e l l s which were continuously labeled i n the presence and absence of colchicine. Plasma membranes were i s o l a t e d from infected c e l l s which were incubated i n the presence and absence of colchicine as described i n F i g . 4 2 . The proteins were fractionated on 7-5% polyacrylamide els which were s l i c e d , s o l u b i l i z e d , and the incorporation of 3H]leucine was quantitated by s c i n t i l l a t i o n counting. . Precursor protein (PEg), envelope proteins ( E l and E2)» and nucleocapsid protein (NC) were present. • • , [3H]leucine, counts per min; , A55° Coomassie blue stain. -102-* a ^ 6 6 r 1 i 5 5 i S e J B Distance from Origin (cm) Distance from Origin (cm) F i g . 4 3 . P r o f i l e s of sodium dodecyl sulfate gels which contained plasma membrane proteins from c e l l s which were continuously labeled i n the presence and absence of colchicine (continued). -103-o control Hours Post-Labelling F i g . 44. Incorporation, of [3H]leucine into the v i r a l proteins of the plasma membrane of c e l l s i n the presence and absence of colchicine. Colchicine (50 uM) was administered to BHK c e l l s at 3 hours post-infection, [3rl]leucine (50 uCi) i n 5 m l of Earle's medium was added at 4 hours, and supplemented with 5 ml 199 maintenance medium at 5 hours. Plasma membranes and ex t r a c e l l u l a r v i rus were harvested at 5. °» 8, and 10 hours a f t e r i n f e c t i o n . Membrane samples were subjected to sodium dodecyl sulfate polyacrylamide electrophoresis and the radioactive v i r a l proteins were quantitated. Envelope proteins ( E l and E2) i n the plasma membrane i n the presence of colch i c i n e (v v) and without the drug (• • •) . Nucleocapsid i n the plasma membrane i n the presence (• •) and absence (o- o) of colchicine. -104-Pig. 4 5 . Plasma membrane proteins which were labeled with I3H"]leucine during a pulse-chase experiment which was performed i n the presence and absence of colchicine. Colchicine was added to infected BHK c e l l s at 3 hours a f t e r i n f e c t i o n with subsequent addition of [3H]leucine at 4 f hours post-infection for a pulse period of 30 min. Medium enriched with non-radioactive leucine was added and plasma... membranes and e x t r a c e l l u l a r v i r u s were harvested at 2 , 4 , 6 , and 10 hours chase. The proteins were fractionated on c y l i n d r i c a l polyacrylamide (2-5%) gels which were s l i c e d , s o l u b i l i z e d and assayed f o r X^H]leucine by s c i n t i l l a t i o n counting. • • , £3H]leucine, counts per min; , A55° Coomassie blue. - 1 0 5 -F i g . 45._ Plasma membrane proteins which were labeled with p H ] l e u c i n e during a pulse-chase experiment which was performed i n the presence and absence of colchicine.(continued). - 1 0 6 -the p r o f i l e s a f t e r electrophoresis of the plasma membrane proteins prepared 2 , 4, 6, and 10 hours a f t e r the [ 3H]leucine l a b e l was removed while F i g . 46 demonstrates the i n h i b i t o r y e f f e c t of colchicine on the production of e x t r a c e l l u l a r v i r u s . Extracellular virus 2 3 4 5 6 7 8 9 10 C h a s e T i m e (hours) F i g . 46. Radioactive e x t r a c e l l u l a r virus i s o l a t e d during a pulse-chase experiment which was performed i n the presence and absence of c o l c h i c i n e . * • , colchicine( 50uM) • • , control. E x t r a c e l l u l a r virus was harvested from medium of the pulse-chase exp riment described i n F i g . 45.. The v i r u s was i s o l a t e d on sucrose gradients which were dripped i n 0.25 ml f r a c t i o n s . Aliquots ( 2 0 0 ul) were assayed f o r [3H]leucine and the r a d i o a c t i v i t y i n the virus peaks was summated and plotted versus chase time. - 1 0 7 -I t i s apparent that there i s an accumulation of v i r a l s t r u c t u r a l proteins i n the plasma membrane of the colchicine-treated c e l l s (Fig.4-7). Plasma Membrane 10h I i i • 2 4 6 8 10 Chase Time (hours) F i g . 4 7 . Time course f o r incorporation of "[3H-]leucine into v i r u s - s p e c i f i e d proteins associated with the plasma membrane during a pulse-chase experiment which was performed i n the presence and absence of col c h i c i n e . Plasma membranes were i s o l a t e d and subjected to SDS electrophor as described i n Fig. 4 5 . Radioactive v i r a l proteins (envelope proteins, E 1 E 2 ; nucleocapsid, NC) were quantitated and plotted against the chase time. • •, proteins labeled i n the presence of colchicine; • • , proteins labeled i n the absence of co l c h i c i n e . -108-0 1 2 3 5 7 A * . l 0 1 2 3 5 7 c 3 F i g . 48. A u t o r a d i o g r a m o f [35s]-labeled plasma membrane p r o t e i n s d u r i n g a p u l s e - c h a s e experiment performed i n t h e p r e s e n c e and absence o f c o l c h i c i n e . C o l c h i c i n e was added t o BHK c e l l s a t 3 hours p o s t - i n f e c t i o n . These c e l l s were p u l s e d a t 4.5 hours a f t e r i n f e c t i o n f o r 30 min. R a d i o a c t i v e medium was removed and r e p l a c e d w i t h medium e n r i c h e d w i t h n o n - r a d i o a c t i v e m e t h i o n i n e . Plasma membrane, endoplasmic r e t i c u l u m , and e x t r a c e l l u l a r v i r u s were h a r v e s t e d a t 0, 1, 2, 3> 5, and 7 hours chase. Plasma membrane p r o t e i n s were s u b j e c t e d t o e l e c t r o p h o r e s i s on 7-5% p o l y a c r y l a m i d e s l a b g e l s i n t h e p r e s e n c e o f 10% ^ - m e r c a p t o e t h a n o l and 0.1% SDS (as d e s c r i b e d i n S e c t i o n I I . ). The s l a b g e l s were s t a i n e d w i t h Coomassie b l u e , d r i e d under vacuum, and a u t o r a d i o g r a p h e d 3 days. The g e l s a r e o f plasma membrane p r o t e i n s 0, 1, 2, 3, 5. and 7 hours a f t e r t h e p u l s e . Plasma membrane p r o t e i n s from c o l c h i c i n e - t r e a t e d c e l l s a r e p r e s e n t e d i n t h e upper p a n e l (A) w h i l e t h o s e from u n t r e a t e d c e l l s appear i n t h e l o w e r p a n e l (B) . -109-0 1 2 3 5 7 A NVP130 N V P 9 8 tag — = — 0 1 2 3 5 7 F i g . 49. Autoradiogram of [ 3 5 s]-labeled endoplasmic r e t i c u l u m p r o t e i n s from a pulse-chase experiment performed i n the presence and absence of c o l c h i c i n e . Endoplasmic r e t i c u l u m was i s o l a t e d i n the experiment described i n F i g . 48. The membranes were s o l u b i l i z e d i n sample b u f f e r which contained 10$ (v/v) 3-mercaptoethanol and electrophoresed on 7 -5$ polyacrylamide s l a b gels i n the presence of sodium dodecyl s u l f a t e . The p r o t e i n s (on 2 slab gels) were elctrophoresed simultaneously i n the same apparatus. Gels were s t a i n e d , d r i e d under vacuum and autoradiographed 3 days. Endoplasmic r e t i c u l u m p r o t e i n s from c o l c h i c i n e - t r e a t e d c e l l s are presented i n the upper panel (A) wh i l e those from untreated c e l l s appear i n the lower panel (B). The gels are of endoplasmic r e t i c u l u m p r o t e i n s 0, 1, 2 , 3 . 5 , and 7 hours a f t e r the pulse. -110-This experiment was repeated with [^ vS]methidnine as the labeled precursor, plasma membranes and endoplasmic reticulum were harvested at 0, 1, 2, 3» 5, and 7 hours chase. The membrane samples were subjected to electrophoresis on 7-5% acrylamide slab gels and autoradioagraphed as shown i n Figures 48 and 49. An accumulation of radioactive v i r a l proteins i n the plasma membrane of colchicine-treated c e l l s was observed when the r a d i o a c t i v i t y i n the vi r u s bands was quantitated and plotted versus time (Fig.5°) • P la sma Membrane Chase Time (hours) F i g . 50 Time course f o r the incorporation of "{^^S^methionine into the v i r a l proteins of the plasma membrane from c e l l s which were pulse-labeled i n the presence or absence of colchicine. Colchicine was added to BHK c e l l s at 3 hours post-infection. These c e l l s were pulsed at 4.5 hours aft e r i n f e c t i o n f o r 30 min. At various times a f t e r the pulse, incorporation of • [ 3^S]methionine into the v i r a l proteins from the plasma membrane was determined a f t e r electrophoresis on SDS poly-acrylamide gels. Incorporation of r a d i o a c t i v i t y into envelope proteins ( E l , E 2 ) , nucleocapsid, and precursor to E2 ( P E 2 ) i n the presence (• •) or absence (••—•' —•) of colchic i n e (100 uM). - 1 1 1 -Again the production of e x t r a c e l l u l a r virus was i n h i b i t e d ( F i g Furthermore, the cleavage of PEg (a precursor to Eg) was also inhibited; t h i s p o s t - t r a n s l a t i o n a l cleavage may be a pre-r e q u i s i t e f o r budding ( 6 7 , 1 2 1 ) . When endoplasmic reticulum enriched fract i o n s were subjected to electrophoresis, l i t t l e difference was observed between the colchicine-treated and control preparations. It would appear that the c o l c h i c i n e -mediated i n h i b i t i o n of SF virus maturation occurs a f t e r the v i r a l proteins reach the plasma membrane of the infected c e l l . - 1 1 2 -Control Colchicine 100/JM Ohours chase 1 hour chase 2hours chase 3 hours chase Ohours chase i 1 hour chaseH 2hours chase 5 hours chase 3hours chase 7 hours chase 5 hours chase 7hours chasei 20 30 10 20 30 Fraction Number (p.25ml tube) Pig. 5 1 . P r o f i l e s of J^'Sl-labeled e x t r a c e l l u l a r virus produced during a pulse-chase experiment which was performed i n the presence and absence of colchicine. E x t r a c e l l u l a r v i r u s was is o l a t e d on sucrose gradients f o r the experiment described i n F i g . 48. Medium (18 ml) was placed on each gradient, the tube was centrifuged and dripped from the bottom i n O .30 ml fr a c t i o n s . Aliquots of 2 0 0 u l were assayed for r a d i o a c t i v i t y over the 0 , 1, 2 , 3 , 5 , and 7 hour chase times. - 1 1 3 -2) The Effect of Dibucaine on Virus Maturation We tested the combined involvement of microtubules and microfilaments i n the budding process by addition of dibucaine to BHK c e l l s infected with SF v i r u s . The appropriate dosage was again determined on the basis of previous studies with f i b r o -blasts (137) and observations through a phase-contrast microscope over a period of 2k hours; c e l l s i n contact with dibucaine (50 - 200 uM) became spherical but were viable at least 15 hours (Table 9) . Table 9 Effects of various concentrations of dibucaine on c e l l shape, (as determined by phase contrast microscopy). BHK-21 c e l l s were incubated i n 199 maintenance media which contained 0 - 2000 uM dibucaine. Observations on the c e l l condition were made at 0 min, 15 min, 1 hr, 2 hr, k hr, 10 hr, 15 hr, and 2k hr. x denotes that the c e l l s were detached, r denotes that the c e l l s were rounded, - designates no drug eff e c t . Incubation Time With Dibucaine (Hr) Dibucaine uM 0 0 . 2 5 0 . 5 1 2 5 15 2k 2000 - - r r r r r r 1000 - - - r r r r r 700 - - - - r r r r 500 - - - - r r r r 200 - - - - r r r 100 - - - - r r r 80 _ _ _ _ _ _ R R 60 _ _ _ _ _ _ r r I4.O _ _ _ _ _ _ _ r 20 _ _ _ _ _ _ _ _ 1 0 - _ _ _ _ _ _ _ -114-Dibucaine, which i s thought to disrupt both microtubule and microfilament systems, also i n h i b i t e d virus production dramatically. The drug was incubated with c e l l s 3 hours a f t e r i n f e c t i o n , [ 3H] leucine was added at 4 hours and additional maintenance medium was added at 5 hours. The e x t r a c e l l u l a r virus was har-vested a f t e r 10 hours i n f e c t i o n . Dibucaine (100 uM) i n h i b i t e d production of labeled virus by 90% ( F i g . 52). 5 1 5 2 5 Fraction No. Fig. 52. P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r v i r u s produced by c e l l s i n the presence and absence of dibucaine. Dibucaine ( 100 uM) was added to infected c e l l s at 3 hours and [ 3H]leucine at 4 hours a f t e r i n f e c t i o n . Media from these c e l l s (18 ml) was placed on a sucrose gradient aft e r 14 hours i n f e c t i o n , centrifuged, and the tube was dripped from the bottom i n 0.25 ml f r a c t i o n s . Aliquots (200 ul) were assayed for r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. -115-The inhibition was also evident when the gradients were viewed directly (Fig. 53). Control • Dibucaine Fig. 53' Photograph of sucrose gradients which contain virus produced in either the absence or presence of dibucaine (100 uM) . BHK cells were infected with 20 plaque-forming units of SF virus per c e l l . Dibucaine was added to the 199 maintenance medium after 3 hours infection, extracellular virus was harvested at Ik hours post-infection. Medium (18 ml) from three 15mm x 150mm plates was layered onto each sucrose gradient. - 1 1 6 -Plasma membrane was also i s o l a t e d a f t e r 10 hours of i n f e c t i o n and membranes from dibucaine-treated c e l l s contained increased l e v e l s of radioactive v i r a l proteins. Hence, the effect of dibucaine on virus production appeared to be si m i l a r to that of colchicine (Fig. 5^)• Distance From Origin cm. F i g . 54. P r o f i l e s of SDS polyacrylamide gels which contained plasma membrane proteins from c e l l s which were continuously labeled i n the presence and absence of dibucaine. Dibucaine ( 1 00 uM) was administered to BHK c e l l s at 3 hours post-infection. r3H]leucine ( 5 0 u C i ) i n 5 ml of Earle.'s basic s a l t s medium was added to each dish at 4 hours, and supplemented with 5 ml 199 maintenance medium at 5 hours. Plasma membranes were harvested a f t e r 14 hours i n f e c t i o n . Proteins were f r a c t i o n -ated on 7.$% polyacrylamide c y l i n d r i c a l gels which were s l i c e d , s o l u b i l i z e d and quantitated f o r [3H]leucine by s c i n t i l l a t i o n counting. Envelope proteins ( E l and E 2 ) and nucleocapsid protein (NC) were present. • • , [3H]leucine, counts per min; , A550 Coomassie blue s t a i n . -117-Further support for that conclusion came from a pulse-chase experiment with dibucaine, i d e n t i c a l i n form to that performed with c o l c h i c i n e . BHK-21 c e l l s were infected with SF v i r u s , pulsed with [-^S]methionine and fractionated into plasma membrane and endoplasmic reticulum at 0, 1, 2, 3» 5, and 7 hour chase times. Autoradioagraphy was again performed a f t e r the membrane proteins were separated by SDS polyacrylamide electrophoresis. Again we observed an accumulation of radioactive v i r a l proteins i n the plasma membrane of the drug-treated c e l l s (Fig. 55) • Cleavage of PEg to Eg did not seem to be i n h i b i t e d to a s i g n i f i c a n t extent by dibucaine, i n contrast to the pulse-chase experiment with c o l c h i c i n e . As observed with colchicine, there does not appear to be a dramatic contrast between radioactive proteins found i n the endoplasmic reticulum prepared from c e l l s with or without dibucaine. However, conversion of some of the non-virion proteins to envelope and nucleocapsid proteins may be i n h i b i t e d " s l i g h t l y i n the dibucaine sample (Fig. 5&)• The production of radioactive e x t r a c e l l u l a r v i r u s was also profoundly i n h i b i t e d (90$) by t h i s drug (Fig. 57)• Thus, i t seems that dibucaine ( l i k e colchicine) exerts i t s i n h i b i t o r y effect on virus production afte r the v i r a l proteins reach the plasma membrane of the BHK c e l l . Dibucaine did not a f f e c t the incorporation of f/HJleucine into c e l l proteins precipitated by t r i c h l o r o a c e t i c acid during a control experiment which was si m i l a r to that performed with colch i c i n e (Table 1 6 ) . O -118-N C B P E 2 E , E 2 F i g . 55- Accumulation of v i r a l p r o t e i n s i n the plasma membranes of c e l l s t r e a t e d with d i b u c a i n e . A pulse-chase experiment i d e n t i c a l to t h a t d e s c r i b e d i n F i g . 4 8 was performed i n which d i b u c a i n e r e p l a c e d c o l c h i c i n e . Plasma membranes were i s o l a t e d at v a r i o u s times a f t e r the chase and the p r o t e i n s s o l u b l e i n SDS were s u b j e c t e d to p o l y a c r y l a m i d e e l e c t r o p h o r e s i s . The s l a b g e l was d r i e d and autoradl-ographed 3 days. Plasma membrane p r o t e i n s from d i b u c a i n e - t r e a t e d c e l l s a re presented i n the upper panel (A) while those from u n t r e a t e d c e l l s appear i n the lower panel ( B ) . The g e l s are of plasma membrane p r o t e i n s 0, 1, 2, 3, 5, and 7 hours a f t e r the p u l s e . -119-0 1 2 3 5 7 B NVP130 N V P 98 w 0 1 2 3 5 7 Fig. 56. Autoradiogram of [ 35 s j-labeled proteins from the endoplasmic reticulum of BHK cells exposed to dibucaine. A pulse-chase experiment was performed i n the presence or absence of dibucaine (as described in Fig. 5 5 ) - Endoplasmic reticulum was harvested at 0, 1, 2, 3> 5, and 7 hours into the chase period and the membrane proteins were subjected to polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Autoradiography was again performed for 3 days. The top panel (A) is an autoradiograph of the proteins from dibucaine-treated c e l l s while the lower panel (B) i s an autoradiograph from non-treated c e l l s . Precursor proteins are designated as non-virion proteins (NVP) followed by their estimated molecular weights (x 10 -3) . -120-C o n t r o l 2.5 2.5 o 5 X E cu 10 m 15 10 D i b u c a i n e 1 0 0 u M 0 hours chase 0 hours chase -1 hour chase 1 hour chase -2 hours chase ; L,. 2 hours cha se 3 hours chase A 3 hours chase 5 hours chase 5 hours chase j 7 hours chase 7 hours chase 5 10 15 2 0 5 10 15 2 0 F r a c t i o n N u m b e r ( o . 5 m l / t u b e ) F i g . 57 • P r o f i l e s of sucrose gradients which contain radioactive e x t r a c e l l u l a r v i r u s from a pulse-chase experiment performed i n the presence and absence of dibucaine. E x t r a c e l l u l a r v i r u s was is o l a t e d during the pulse-chase experiments outlined i n Figures 55 and 56 at 0, 1, 2, 3, 5, and 7 hours chase. Media (18 ml) was placed on each gradient which was centrifuged, dripped into 0.5 ml fractions and 200 u l aliquots were assayed f o r [35s]methionine. - 1 2 1 -Table 10 Incorporation of [ 3H]leucine into t r i c h l o r o a c e t i c acid (TCA) p r e c i p i t a b l e material from BHK c e l l s i n the presence or absence of dibucaine. C e l l s were i n i t i a l l y incubated f o r 1 hour i n Earle's basic s a l t s solution / 1 9 9 maintenance medium ( 1 : 1 ) ; two-thirds the dishes contained dibucaine (50 uM and 100 uM). [3H]Leucine (25 uCi/dish) was added to a l l the dishes and c e l l s were.-harvested at 0 , 0 . 5 . 1, 3 , 5 , and 7 hours. The labeled c e l l s were suspended i n 2 . 5 ml buffer and precipitated by addition of 10$ .(w/v) t r i c h l o r o a c e t i c acid. Precipitates were resuspended i n s c i n t i l l a t i o n f l u i d . Time of Incubation P H ] L e u Incorporated Into TCA P e l l e t With [ 3 H]Leu (Hr) (cpm x 10-°/mg protein) Control 50 uM Dibucaine 100 uM Dibucaine 0 0 . 0 2 0 3 0 . 0 2 5 1 0.0218 o . 5 0 . 2 5 2 0.214 O . 1 9 6 1.0 0 .356 0.424 0.408 3-0 0 . 7 9 8 O . 7 2 0 0 . 7 6 5 5-0 1.16 1.20 1.13 7.0 1.41 1.42 1 .37 -122-3) The Eff e c t of Cytochalasin B on Virus Maturation The effects of the microfilament disruptor, cytochalasin B, on v i r u s assembly were also investigated. The correct dosage was estimated from previous work with f i b r o b l a s t s (137) and by observations through a phase contrast microscope over 24 hours. C e l l s i n contact with cytochalasin B (1-50 uM) appeared to become spherical but were v i a b l e at least to 15 hours (Table 10)i Table 11 E f f e c t s of various concentrations of cytochalasin B on c e l l v i a b i l i t y (as determined by phase contrast microscopy). BHK-21 c e l l s were incubated i n 199 maintenance media which contained 0 - 200 uM cytochalasin B. Observations on the c e l l condition were made at 0, 5 min, 15 min, 3° min, 1 hr, 2 hr, 4 hr, 10 hr, 15 hr, and 24 hr. x denotes that the c e l l s were detached, r denotes that the c e l l s were rounded, - designates no drug e f f e c t . Incubation Time With Cytochalasin B (Hr) 0.083 0.25. ;;0.50 1.0 2.0 4.0 12.0 24.0. 200 r r r r r X X X 100 r r r r r r r r X 75 r r r r r r r r r 50 - - r r r r r r 20 - - - r r r r r r 10 - - - - r r r r r 5 - - - - r r r r r 2.5 - - - - - - r r r 1.0 - - - - - - r r r 0.5 - - - - - - - - r Cytochalasin B uM - 1 2 3 -Coneentrations of 10 uM and 20 u'M' i n h i b i t e d the production of radioactive e x t r a c e l l u l a r virus by 90$ (Fig. 5 8 ) . no 8 + CONTROL Cytochalasin B F r a c t i o n Pig- 5 8 . P r o f i l e s of sucrose gradients containing radioactive e x t r a c e l l u l a r virus produced by c e l l s i n the presence and absence of cytochalasin B. Cytochalasin B (10 uM) was added to infected c e l l s at 3 hours and [ 3 H]leucine at k hours a f t e r i n f e c t i o n . Media from these c e l l s (18 ml) was placed on a sucrose gradient a f t e r Ik hours i n f e c t i o n , centrifuged, and the tube was dripped from the bottom i n 0 . 2 5 ml fr a c t i o n s . Aliquots (200 ul) were assayed f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n counting. -12:4-However, the synthesis of radioactive v i r a l proteins was s i g n i f -i c a n t l y lower i n the c e l l s which were treated with the drug. Levels of [ H]leucine labeled envelope and nucleocapsid proteins were diminished by 6ofo i n cytochalasin B - treated c e l l s (Fig.v>59). Further studies were not performed with t h i s drug due to i t s seemingly deleterious effects on c e l l v i a b i l i t y . - 1 2 5 -Distance from Origin (cm| Distance from Ori. F i g . 5 9 - P r o f i l e s of SDS polyacrylamide gels which contained plasma membrane and endoplasmic reticulum proteins from c e l l s which were continuously labeled i n the presence and absence of cytochalasin B . Cytochalasin B (10 uM) was administered to BHK c e l l s a t 3 hours post-infection. [3H]leucine ( 5 0 uCi) i n 5 ml of Earle's basic s a l t s medium was added to each dish at 4 hours, and supplemented with 5 ml 1 9 9 maintenance medium at 5 hours. Plasma membranes and endoplasmic reticulum were harvested a f t e r 14 hours i n f e c t i o n . Proteins were fractionated on 7 . 5 ^ polyacrylamide c y l i n d r i c a l gels which were s l i c e d , s o l u b i l i z e d and quantitated for[3H]leucine by s c i n t i l l a t i o n counting. Envelope proteins ( E i and E2) and nucleocapsid protein (NC) were present. • • "", [3H]leucine, counts per min; , A 5 5 0 Coomassie blue s t a i n . -126-4) Electronmicrographs of C e l l s Infected with Semliki Forest  Virus i n the Presence of Colchicine and Dibucaine BHK-21 c e l l s were infected with Semliki Forest virus i n the presence and absence of 100 uM colchicine and 100 uM dibucaine. The drugs were administered at 2 hours i n f e c t i o n and the c e l l s were harvested at 10 hours, suspended i n phosphate buffered saline, and prepared for transmission electronmicroscopy. The control samples contained a c t i v e l y budding virus -mature v i r i o n s were present on the outside portions of the plasma membrane and p a r t i c l e s were seen to extend into the e x t r a c e l l u l a r media ( F i g . 60). Other portions of the plasma membrane showed very few underlying nucleocapsids. Infected c e l l s which were treated with colc h i c i n e exhibited dense patches of nucleocapsids underlying the plasma membrane but no a c t i v e l y budding virus appeared to be present (Fig. 6l). Microfilaments were associated with these p a r t i c l e s to some extent but i t i s hard to attach any sign i f i c a n c e to t h e i r presence. Dibucaine also seemed to cause an accumulation of nucleo-capsids beneath the c e l l surface, though the patches were not as dense as those produced with colchicine. Budding virus p a r t i c l e s were again absent. This drug has been demonstrated to impair both microtubule and microfilament function (139) yet microtubules and microfilaments did not appear to depolymerize as they are s t i l l seen associated with the c e l l membrane (Fig. 62). These structures must s t i l l be affected dramatically since the c e l l s were severely rounded - microtubules and microfilaments - 1 2 7 -are known to regulate c e l l shape. Cytopathic vacuoles (both type 1 and type 2 ) appeared to be quite frequent i n these c e l l s but the l a t t e r type could well be indentations of the plasma membrane which have been cross-sectioned (Figs. 63 and 64). Electronmicroscopic observations appear to -corroborate the biochemical evidence - colchi c i n e and dibucaine appear to cause an accumulation of v i r a l proteins at the plasma membrane pr i o r to the f i n a l stages of v i r u s maturation. These studies were d i f f i c u l t to assess with ce r t a i n t y (due to the l i m i t a t i o n s of electronmicroscopy), but taken together with the previous data they appear to indicate that microtubules and microfilaments may be involved i n the budding mechanism. - 1 2 8 -Fig. 60. Electronmicrograph of infected BHK cells which portrays virus budding at the plasma membrane in the absence of colchicine and dibucaine (105,000 x magnification). BHK cells were infected with SF virus at 20 plaque forming units per c e l l . Cells were harvested at 10 hours infection, suspended in phosphate buffered saline, and fixed in glutar-aldehyde followed by 1% osmium tetroxide. -129-Colchicine Fig. 6 l . Electronmicrograph of infected BHK cells showing accumulation of nucleocapsids at the plasma membrane in the presence of colchicine (70,000 x magnification). BHK cells were infected with Semliki Forest virus at 20 plaque forming units per c e l l . Colchicine (100 uM) was administered at 2 hours infection and cells were harvested at 10 hours infection and fixed and stained as in Fig. 60. -130-Fig. 62. Electronmicrograph of infected BHK cells showing accumulation of nucleocapsids at the plasma membrane in the presence of dibucaine (70,000 x magnification). BHK cells were infected with Semliki Forest virus at 20 plaque forming units per c e l l . Dibucaine (50 uM) was administered at 2 hours infection and cells were harvested, fixed, and stained as in Fig. 60. -131-Fig. 63. Electronmicrograph of a cytopathic vacuole found in infected BHK cells which were treated with dibucaine (50 uM) (105,000 x magnification). -1 3 2 -Fig. 64. Electronmicrograph of a cytopathic vacuole found i infected BHK cells which were treated with dibucaine (50 uM) (105,000 x magnification). ; -133-E. Two-Dimensional Cross-Linking Studies Performed Upon P u r i f i e d Virus P u r i f i e d v i r u s was reacted with b i f u n c t i o n a l c r o s s - l i n k i n g agents - dimethylsuberimidate (DMS) and d i t h i o b i s ( s u c c i n i m i d y l propionate) (DSP). I n i t i a l l y samples were reacted with the appropriate c r o s s - l i n k e r according to the procedure of Simons and Garoff (23&) and subjected to one-dimensional electrophoresis on 3-5$ acrylamide c y l i n d r i c a l gels. Both c r o s s - l i n k e r s appeared to y i e l d s i m i l a r patterns ( F i g . 65). D S P D M S 0.5 i 2 1 2 3 F i g . 65. P u r i f i e d SF vi r u s which was cross-linked with DMS and DSP, and subjected to electrophoresis on 3-5$ polyacrylamide c y l i n d r i c a l gels. P u r i f i e d v i r u s ( 1 0 0 ug protein) was suspended i n 0.15 M NaCl and cross-linked with DMS ( 1 . 0 mg/ml, 2 . 0 mg/ml, and 3 .0 mg/ml) and DSP (0.5 mg/ml, 1 .0 mg/ml, 2 . 0 mg/ml) f o r 2 hours. The cross-linked proteins were separated on 3-5^ polyacrylamide c y l i n d r i c a l gels i n the presence of SDS, stained with Coomassie blue, and destained by diffusion i n 7-5^ acetic a c i d . Nucleo-capsid monomer (NC), dimer ( N C 2 ) i trimer ( N C 3 ) , tetramer (NCZj.), pentamer ( N C 5 ) , and hexamer (NC6) were evident. Envelope monomer ( E 1 E 2 ) was present but dimer could not be detected. - 1 3 . 4 -Gel scans of virus which was cross-linked with DMS ( 3 . 0 mg/ml) and DSP ( 1 . 0 mg/ml) are presented i n F i g . 6 6 . Higher cross-linked species of envelope proteins were not obtained as Garoff and Simons reported under these conditions ( 2 3 6 ) . 2-1 2 3 4 6 6 7 8 9 10 Distance of Migration (cmi 2 3 4 5 6 7 8 9 10 Distance of Migration (cm) F i g . 6 6 . Absorbance scans of 3 - 5 $ polyacrylamide gels containing proteins which were cross-linked with DMS and DSP. Pu r i f i e d v i r u s which was cross-linked with DMS ( 3 - 0 mg/ml) and DSP ( 2 . 0 mg/ml) f o r 2 hours (as described i n Fig . 6 5 ) was scanned f o r absorbance at 5 5 0 nm. Nucleocapsid monomer (NC), dimer (NC£)» trimer ( N C 3 ) , tetramer (NCzj,), pentamer ( N C 5 ) , and hexamer (NC6) are indicated on the scans. Envelope monomer ( E 1 E 2 ) was also designated. - 1 3 5 -Virus was cross-linked with dimethylthiobis(propionimidate) (DTBP) and dithiobis(succinimidyl propionate) (DSP) at a range of concentrations ( 0 . 5 , 1.0, and 2.0 mg/ml) for a period of 2 hours and electrophoresed on 6.5$ acrylamide slab gels in the absence of 8-mercaptoethanol (Figs. 67 and 68) . ,0.5h ih 2h,0.5h 1h 2h. 0.5h 1h 2h ( — 2 mg/ml 1 mg/ml 0.5mg/ml Fig. 67. Purified SF virus which was cross-linked with DTBP and subjected to electrophoresis on 6.5$ polyacrylamide slab gels. Purified virus (100 ug protein) was suspended in 0.15 M NaCl and cross-linked with DTBP (0.5 mg/ml, 1 mg/ml, and 2 mg/ml) for 0.5 , 1, and 2 hours. The cross-linked proteins were separated on 6.5$ polyacrylamide slab gels in the presence of SDS, stained with Coomassie blue, and destained by diffusion in 7-5$ acetic acid. Noncross-linked virus (50 ug protein ) which had been purified on 1 gradient was also electrophoresed as a reference. B-mercapto-ethanol was not present during electrophoresis. Nucleocapsid monomer (NC), dimer (NC2), and trimer (NC3) were evident as was envelope monomer (Ei and E2) and dimer (E-E). - 136 -DSP r\ic3 E-E N O , NC , 0.5h 1h 2h|0.5h 1h 2h |0.5h 1h 2h , -2 mg/ml 1 mg/ml 0.5mg/ml F i g . 6 8 . P u r i f i e d SF v i r u s w h i c h was c r o s s - l i n k e d w i t h DSP and s u b j e c t e d t o e l e c t r o p h o r e s i s on 6.5% p o l y a c r y l a m i d e s l a b g e l s . P u r i f i e d v i r u s (100 ug p r o t e i n ) was c r o s s - l i n k e d w i t h DSP (0.5 mg/ml, 1 mg/ml, and 2 mg/ml) and e l e c t r o p h o r e s e d as d e s c r i b e d i n F i g . 6 7 . N u c l e o c a p s i d monomer ( N C ) , d i m e r (NC2), and t r i m e r (NC3) were e v i d e n t as was e n v e l o p e monomer ( E i and E2) and d i m e r ( E - E ) . N u c l e o c a p s i d was f o u n d t o y i e l d up t o p e n t a m e r i c s p e c i e s w h i l e d i m e r i c e n v e l o p e p r o t e i n s were e v i d e n t . The c a p s i d p r o t e i n has been shown t o be l y s i n e r i c h a nd i t i s t h u s u n d e r s t a n d a b l e as t o why l o w e r m o l e c u l a r w e i g h t s p e c i e s o f t h i s p r o t e i n ( i e . monomer and -137-diraer) disappear at longer c r o s s - l i n k i n g times or at higher concentrations of DSP or DTBP. Again envelope proteins which were cross-linked to a greater degree than dimer were not c l e a r l y evident. Semliki Forest v i r u s was then cross-linked with either DTBP ( 2 mg/ml) or DSP ( 1 mg/ml) for a period of 1 hour and f r a c -tionated by the two-dimensional c r o s s - l i n k i n g system of Wang and Richards ( 2 3 5 ) . V i r t u a l l y i d e n t i c a l r e s u l t s were achieved i n each case. The vi r u s was subjected to electrophoresis i n the f i r s t dimension i n the absence of ^ -mercaptoethanol and then the second dimension i n the presence of 1 0 $ (v/v) :g-mercaptoethanol (Fi g . 6 9 ) . Monomeric or noncross-linked species l a y on a diagonal running from l e f t to r i g h t on the gel, while cross-linked species appeared on horizontal l i n e s adjacent to the monomers. Nucleocapsid was cross-linked to y i e l d up to tetramers while envelope proteins joined to y i e l d only dimers. Three d i s t i n c t spots were present. These could correspond to the combinations E-^ -E-^ , E-^-Eg, and Eg-Eg but one should be cautious i n assessing t h i s s i t u a t i o n since varying degrees of intramolecular d i s u l f i d e bonding w i l l y i e l d multiple protein bands when such proteins are electro-phoresed i n the absence of g-mercaptoethanol ( 2 5 1 ) . Nucleocapsid , ' . did not appear to cr o s s - l i n k with envelope proteins i n any instance - even when higher cross - l i n k e r concentrations and longer incubation times were employed. These data agree with some interpretations of X-ray crystallography ( 1 , 2 5 2 ) and other, recent c r o s s - l i n k i n g studies performed on SF virus and Sindbis v i r u s ( 2 5 3 ) - However, our data do*. disagree with the re s u l t s of -138-NC3 E-E NC 2 E, E 2 IMC II I IMC, NC F i g . 6 9 . Two-dimensional electrophoresis of SF v i r u s proteins which had been cross-linked with DSP. P u r i f i e d v i r u s (150 ug protein) was suspended i n 0.15 M NaCl and cross-linked with DSP (-1 mg/ml) f o r 1 hour. The cross-linked proteins were separated on 7.0$ polyacrylamide gels ( i n the presence of 10$ (v/v) 3-mercaptoethanol for the second dimension. Monomeric (or noncross-linked proteins) l a y on a diagonal l i n e running from l e f t to r i g h t while cross-linked species l a y on horizontal l i n e s which were adjacent to the monomers. Nucleocapsi monomer (NC) , dimer ( N C 2 ) , trimer ( N C 3 ) , tetramer (NCZf) • and envelope protein monomers (Ej_ and Eg) and dimers (E-E) are evident -139-Simons and Garoff ( 2 3 6 ) , who stated that DMS yielded nucleocapsid bound to envelope proteins even when virus was delipidated with Tri t o n X-100. However, these l a s t workers did not provide evidence f o r nucleocapsid-envelope protein i n t e r a c t i o n based upon t h e i r polyacrylamide gels. F. Two-Dimensional Cross-Linking Studies Performed on Plasma Membranes from Infected C e l l s The two-dimensional system u t i l i z e d i n the c r o s s - l i n k i n g of p u r i f i e d v i r u s , was applied to the plasma membranes of infected c e l l s which had been labeled with [ - ^ s|methionine. Infected c e l l s were labeled f o r j/k hour at 4 hours i n f e c t i o n and chased with non-radioactive methionine enriched media for a period of 2 hours and cross-linked with either DSP or DTBP for a period of 1 hour. Plasma membrane ghosts were is o l a t e d and the proteins were resolved by two-dimensional electrophoresis (Figs. 70 and 71). Nucleocapsid tetramers were c l e a r l y resolved while envelope dimer was again present as three species. E^ did not appear to c r o s s - l i n k to any degree with any other v i r a l proteins. PEg> however, does appear to pa r t i c i p a t e i n c r o s s - l i n k i n g -perhaps i t interacts with nucleocapsid protein or E-^  - but the re s o l u t i o n and the degree of accuracy for molecular weight determination i n the f i r s t dimension make t h i s d i f f i c u l t to inte r p r e t . G. Two-Dimensional Cross-Linking Studies Performed on Plasma Membranes from Infected C e l l s which were Treated  With Colchicine and Dibucaine Similar two-dimensional c r o s s - l i n k i n g studies were performed on infected c e l l s which had been treated with either 100 uM -140-Fig. 70 . Two-dimensional autoradiogram of plasma membrane proteins from SF virus-infected c e l l s which had been cross-linked with DSP. Infected c e l l s were labeled with [35s] methionine for 3/4 hour at 4 hours infection and chased with non-radioactive methionine enriched media for a period of 2 hours. Cells were cross-linked with DSP (1 mg/ml) for a period of 1 hour, plasma membranes were ioslated and the proteins were resolved by two-dimensional electrophoresis. The f i r s t dimension (which contained no 6-mercaptoethanol) consisted of 7 . 0 $ acrylamide while the second dimension (which contained 10$ (v/v) B-mercaptoethanol) was 7 -5$ acrylamide. Nucleocapsid monomer (NC), dimer (NC2), trimer (NCo), tetramer (NC/j.), envelope protein monomers ( E l and E 2) , and the presumed dimers (E^-Ei, E]_-E2, and E2-E2) were present. Identities of dimer species could not be absolutely assigned since intramolecu-l a r d i s u l f i d e bonds can also y i e l d multiple spots i n the f i r s t dimension. - 1 4 1 -F i g . 71- Two-dimensional autoradiogram of plasma membrane proteins from SF v i r u s - i n f e c t e d c e l l s which had been cross-linked with DTBP. Infected c e l l s were labeled and cross-linked with DTBP (1 mg/ml) as described i n F i g . 70. Plasma membranes were i s o l a t e d and subjected to two-dimensional electrophoresis. Envelope monomers ( E i and Eg) and the presumed dimers (Ei-E]_, Ei-E? • and Eg-Eg) were evident as well as nucleocapsid monomer (NC), dimer (NCg), and trimer (NC^). - 142-colchicine or 1 0 0 uM dibucaine at 2 hours i n f e c t i o n . C e l l s were l a b e l l e d f o r ^  hour with[3^s]methionine and chased for 2 hours with non-radioactive methionine enriched media. The labeled BHK c e l l s were then fractionated into plasma membranes and electrophoresed under conditions which were i d e n t i c a l f or each sample. Envelope dimer appeared to be present i n the plasma membranes from control and colchicine-treated c e l l s but was absent i n c e l l s which had been treated with dibucaine (Figs. 72 and 7 3 )• We i n i t i a l l y thought that microtubules and m i c r o f i l a -ments might be important i n regulating the i n t e r a c t i o n of v i r a l proteins at the plasma membrane during patch formation and budding. However, colchicine (which disrupts microtubules) does not appear to a l t e r the proximity of envelope proteins to each other during the f i n a l stages of v i r a l morphogenesis while dibucaine (which disrupts microtubules and microfilaments) appears to abolish envelope dimer formation. This would seem to implicate microfilaments i n the process where envelope proteins aggregate i n the plasma membrane p r i o r to budding while microtubules may simply act to stabilize.' the membrane during the f i n a l stage of assembly. -143-Dib. Col. -Fig. 72. One-dimensional autoradiogram of DTBP cross-linked plasma membrane proteins from infected cells cultured in the presence or absence of dibucaine and colchicine. Infected BHK cells were treated with colchicine (100 uM) and dibucaine (100 uM) at 3 hours infection. Cells were labeled with[35s]methionine for \ hour at 4 hours infection and chased with non-radioactive methionine enriched media for a period of 2 hours. Cells were cross-linked with DTBP (lmg/ml) for a period of § hour. Plasma membranes were isolated and the proteins were resolved by SDS electrophoresis on 6.5$ polyacrylamide gels (^-mercaptoethanol was absent). Nucleocapsid monomers (NC), dimers (NC?)% trimers (NC3), tetramers (NC4), envelope protein monomers (El and E 2 ) and presumed dimers (Ei-E]_, E 1 - E 2 , and E 2 ~ E 2 ) were evident. Envelope dimers appeared absent in the plasma membranes from cells which were cultured in the presence of dibucaine. -144-Control Fig. 73(a). Two-dimensional autoradiograms of DTBP cross-linked plasma membrane proteins from infected c e l l s which were cultured in the presence or absence of dibucaine and colchicine. Infected BHK ce l l s were treated with colchicine (100 uM) and ucaine (100 uM) at 3 hours infection. Cellswere labeled with SMethionine for § hour at k hours infection and chased with non-radioactive methionine enriched media for a period of 2 hours. Cells were cross-linked with DTBP (lmg/ml) for a period of f hour, plasma membranes were isolated and resolved by two-dimensional electrophoresis - the f i r s t dimension was performed on 6.5$ polyacrylamide gels, the second was done on 7-5$ polyacrylamide gels. The arrow designates the region where envelope protein dimers resolve; cross-linked plasma membranes from ce l l s treated with dibucaine appeared to contain no envelope dimers. -145-F i g . 7 3 ( b ) . T w o - d i m e n s i o n a l a u t o r a d i o g r a m s o f DTBP c r o s s - l i n k e d plasma membrane p r o t e i n s f r o m i n f e c t e d c e l l s w h i c h were c u l t u r e d i n t h e p r e s e n c e o r absence o f d i b u c a i n e and c o l c h i c i n e . - 1 4 6 -Dibucaine i 1 NC Fig. 73(c). Two-dimensional autoradiograms of DTBP cross-linked plasma membrane proteins from infected c e l l s which were cultured i n the presence or absence of dibucaine and colchicine. -147-IV. DISCUSSION A. The V i r a l Envelope Originates from the Plasma Membrane of the Host C e l l From the graphs i n Figs. 3 1 > 3 2 and 3 3 we see that the movement of labeled proteins i s e n t i r e l y consistent with the hypothesis that SF virus obtains i t s envelope by budding from the plasma membrane of the BHK c e l l . Furthermore, i f we correct f o r recovery of plasma membrane as follows, the data suggest that a l l of the labeled v i r u s i n the medium originated from the plasma membrane. We might assume that our recovery of the plasma membrane was 3 5 - 3 $ as demonstrated with other experiments (see "Results"). In addition, the experiment depicted i n Fig. 3 0 used only three-fourths of the actual plasma membrane obtained (one-fourth was used f o r protein determination); therefore, the measured r a d i o a c t i v i t y i s 7 5 $ of the t o t a l radio-a c t i v i t y a c t u a l l y recovered i n the membrane f r a c t i o n . I f these corrections are made i n the data of F i g . 3 3 "the counts as-sociated with E-L and Eg at 3 hours chase would be 1 5 1 , 0 0 0 ( 4 0 , 0 0 0 / . 3 5 3 x O . 7 5 ) and 5 3 , 0 0 0 ( 1 4 . 0 0 0 / . 3 5 3 x 0 . 7 5 ) for the nucleocapsid protein. When these two values are added ( 2 0 4 , 0 0 0 ) the r e s u l t i s i n good agreement with the t o t a l counts per min ( 1 7 0 , 0 0 0 ; 4 4 , 0 0 0 / 0 . 3 5 3 x O . 7 5 ) associated with e x t r a c e l l u l a r v i r u s as 1 1 hours chase. However, several other points must be considered. (a) From the decay of r a d i o a c t i v i t y i n E-^ , Eg, and the nucleocapsid proteins of the plasma membrane 4 hours aft e r the beginning of the chase -148-(see Fig. 33) > "the h a l f - l i f e of these proteins can be estimated to be 2 . 0 hours. Hence, almost one-half of the labeled pro-teins present 2 . 0 hours aft e r the chase period began w i l l have disappeared from the plasma membrane at the time of maximal incorporation of [ 3 H ] 1 eucine ( 4 hours). (b) [-^Hlleucine associated with the envelope protein E^ i n the plasma membrane has not been included i n our c a l c u l a t i o n . Hence, the value of 2 0 4 , 0 0 0 cpm associated with the v i r a l proteins i n the plasma membrane i s a minimal estimate. (c) From the curve (Fig. 33) for r a d i o a c t i v i t y associated with e x t r a c e l l u l a r v i r u s , we can assume that 1 7 0 , 0 0 0 cpm i s also a minimal estimate for radio-a c t i v i t y associated with e x t r a c e l l u l a r v i r u s . (d) From the precision of our measurements, a l l of these calculations are subject to an error of 1 0 - 2 0 $ . Bearing these q u a l i f i c a t i o n s i n mind, we f i n d that v i r t u a l l y a l l of the r a d i o a c t i v i t y associated with e x t r a c e l l u l a r v i r u s can be accounted f o r by labeled proteins that were previously associated with the plasma mem-brane. These data and the previous studies by chemical analysis and electron microscopy ( 1 6 , 2 1 , 79> 8 0 , 95~98) make a strong case for morphogenesis of SF"Yirrus- from .the plasma • membrane of the BHK c e l l . The one apparent anomaly i n our re s u l t s was the accumulation of r a d i o a c t i v i t y i n the nucleocapsid protein associated with the endoplasmic reticulum f r a c t i o n . However, th i s agrees with the accumulation of cytoplasmic nucleoids l a t e i n i n f e c t i o n as observed by electronmicroscopists ( 1 6 ) . These nucleoids were, thought to be nucleocapsids which accumulate as pa r a c r y s t a l l i n e - 1 4 9 -arrays or i n association with cytoplasmic vacuoles. In addition, Jones et a l . (67) pulse-labeled Sindbis v i r u s - i n f e c t e d c e l l s with [-^H]leucine and showed that nucleocapsid protein was labeled f a s t e r and to a greater degree than envelope proteins. This agrees with evidence based on autoradiography of polyacry-lamide gels . ( 4 9 ) and studies on i n v i t r o protein synthesis from v i r a l mRNA ( 4 9 - 5 5 ) which suggest that nucleocapsid protein i s synthesized i n large quantities p r i o r to E-^  and Eg. Hence, we believe that accumulation of the labeled nucleocapsid protein i n the endoplasmic reticulum occurred because nucleocapsid protein was produced and labeled i n excess of envelope proteins. Therefore, a l l of the labeled nucleocapsid protein was not u t i l i z e d i n the production of e x t r a c e l l u l a r virus at the plasma membrane. Morphogenesis from the plasma membrane occurs f o r Group A togaviruses. A scheme which depicts the maturation of Semliki Forest v i r u s i s presented i n F i g . 7 4 . Maturation from the plasma membrane may not be unique to the Group A togaviruses. Rhabdo-viruses, myxoviruses, paramyxoviruses, arenaviruses, and r e t r o -viruses are a l l reputed to "bud" from the plasma membrane. This theory i s supported mainly by electron microscopy and l i p i d composition studies (101, 102, 110-114) . Pulse-chase studies have been undertaken with influenza and v e s i c u l a r stomatitis v i r u s (104-109, U 5 - H 9 ) • The pulse-chase study reported by Hay (18) showed ki n e t i c data that were consistent with a precursor-product r e l a t i o n s h i p between some v i r a l proteins i n the endo-plasmic reticulum and the plasma membrane. However, he did not - 1 5 0 -POLYHERASE REPLICASE 5' „ I 42S RNA Fi g . 7 4 . Proposed scheme fo r the maturation of Semliki Forest v i r u s i n BHK c e l l s . -151-demonstrate that these proteins i n the plasma membrane were subsequently associated with e x t r a c e l l u l a r v i rus p a r t i c l e s . Since our work was completed, s i m i l a r r e s u l t s f o r the morphogenesis of vesi c u l a r stomatitis virus were obtained by Knipe, Baltimore and Lodish ( 1 0 7 ) • B. The Role of the Cytoskeletal System i n the Membrane  Morphogenesis of Semliki Forest Virus Further work was required to elucidate the mechanism by which SF v i r u s "budded" from the plasma membrane of the host c e l l . I t remained to be seen whether or not maturation of the v i r u s from the plasma membrane was a spontaneous event, depended on s p e c i f i c host c e l l structures, or was v i r u s mediated. Studies with a temperature-sensitive mutant of Sindbis v i r u s (84) indicate that the l a t t e r may be true; nucleocapsids appear to accumulate next to the plasma membrane at the nonpermissive temperature but f a i l to bud from the plasma membrane. The r o l e of microtubules and microfilaments in,..the-budding process of Semliki Forest v i r u s were an obvious consideration. Palade, Lacy and Malaisse ( 2 0 0 , 2 0 7 , 2 1 1 , 212 ) have implicated microtubules i n the secretion of hormone granules and microtubules and microfilaments have been demonstrated to regulate capping phenomena and patch formation at the c e l l surface (I36, 137» 1 8 2 ) . Our r e s u l t s indicated that microtubule disruptors and dibucaine (a fjtert'far'y /. amine l o c a l anesthetic) i n h i b i t v i r u s production and cause an accumulation of v i r a l proteins at the plasma membrane. Many control experiments suggest that the conclusions we have reached are not a r e s u l t of secondary effects on the c e l l by - 1 5 2 -these drugs. In a l l cases, low concentration of the drugs was used so we might minimize the pot e n t i a l effects of these chemicals on c e l l v i a b i l i t y . Transport of the radioactive amino acids into the c e l l and protein synthesis were not affected by these disruptors as ascertained by incorporation of l a b e l into the t r i c h l o r o a c e t i c acid p r e c i p i t a t e s . The effects of colchicine could not be duplicated with lumicolchicine (which i s unable to disrupt microtubules) while Nocodazole produced a s i m i l a r i n h i b i t i o n of virus production. F i n a l l y , colchicine and d i -bucaine disrupt microtubules by two apparently d i s t i n c t mechanisms ( 1 3 6 , I 6 5 , 188, 189) yet had s i m i l a r effects upon vi r u s production. With these considerations i n mind, we conclude that the cyto-s k e l e t a l system i s important i n the f i n a l stages of v i r a l assembly. Neither c o l c h i c i n e nor dibucaine completely i n h i b i t e d the production of e x t r a c e l l u l a r v i r u s . This may be because the microtubules are a heterogeneous population and some appear to be less sensitive to the effects of colchicine and other disruptors ( 1 6 8 , 1 6 9 ). A l t e r n a t i v e l y , the budding mechanism of enveloped viruses may be a slow process that i s normally catalyzed by an int a c t cytoskeletal system. The mechanism by which the cytoskeletal system i s involved i n v i r us morphogenesis i s not understood. Our i n i t i a l hypothesis was that t h i s organization was required f o r transport of the v i r a l protein to the c e l l surface. This i s c l e a r l y not the case f o r SF virus i n BHK c e l l s even though microtubules may be important f o r the transport of adenovirus within HeLa c e l l s ( 2 2 7 , 2 2 8 ) . A second p o s s i b i l i t y was that the cleavage of PE ? to E9 might be - 1 5 3 -hindered, since i t appears that t h i s occurs i n the plasma membrane as well as the endoplasmic reticulum and i s a pre-r e q u i s i t e f o r budding (67, 1 2 1 ) . This mechanism also seems un l i k e l y since cleavage of PEg was not retarded by dibucaine although i t was i n h i b i t e d by c o l c h i c i n e . The i n h i b i t o r y effects of co l c h i c i n e and dibucaine might also be due to a decrease i n bound calcium. T e r t i a r y amine anesthetics ( l i k e dibucaine) are reputed to displace C a + + found i n association with phospholipids (188, I 8 9 ) and colchicine might exert the same effect by dispersion of the Ca. associated with polymerized microtubules ( 1 3 6 ) . Another p o s s i b i l i t y i s that colchicine and dibucaine may a l t e r the topology of v i r a l proteins i n the plasma membrane so that patches of envelope proteins and nucleo-capsid may not occur. We tested t h i s l a t t e r p o s s i b i l i t y through the use of protein c r o s s - l i n k i n g agents. We f e l t that c r o s s - l i n k i n g agents might inform us about the proximity of the v i r a l proteins to each other during the f i n a l stages of v i r u s assembly. Preliminary studies were performed with DMS, DSP and DTBP; p u r i f i e d virus was reacted with these reagents and subjected to two dimensional electro-phoresis. In contrast to the re s u l t s of Garoff and Simons (236 , 237) no cr o s s - l i n k i n g was observed between envelope proteins and the nucleocapsid which suggests that part of the glyco-proteins might not be close to the nucleocapsid protein. Other cr o s s - l i n k i n g studies with Sindbis and Semliki Forest virus cor-roborate t h i s r e s u l t (.253) and also suggest that envelope poly-peptides may na t u r a l l y exist as dimers which function as a - 1 5 4 -s t r u c t u r a l unit. We were able to demonstrate that envelope, proteins reacted to form dimers and i n some cases trimers with DSP and DTBP. Higher molecular weight complexes were not detected. X-ray crystallography data have also been interpreted (although t h i s i s not e n t i r e l y j u s t i f i e d ) to support a scheme where envelope proteins do not penetrate through the membrane (1, '252) as have freeze-fracture studies (1, 79, 80). Nucleo-capsid protein i s ly s i n e r i c h and reacts r e a d i l y with DMS, DSP and DTBP to y i e l d dimeric to pentameric complexes. Our cross-l i n k i n g studies appear to support the scheme where nucleocapsid proteins are c l o s e l y associated with each other - envelope proteins may in t e r a c t to form dimeric structures but do not interact with the nucleocapsid. In no case does the t h i r d en-velope protein E^ appear to cross-link with the other virus proteins which supports the view that i t i s a loosely associated protein (1). Interpretation of cr o s s - l i n k i n g studies must be approached with caution, however, since c r o s s - l i n k i n g depends upon the presence of an available reactive group on the protein. Studies with plasma membranes which contained cross-linked v i r a l proteins also yielded s i m i l a r r e s u l t s to those discussed above. Again, no di r e c t evidence f o r envelope protein-nucleocapsid i n t e r a c t i o n was evident. Nucleocapsid was presumably cross-linked because the plasma membrane was permeable to DSP and DTBP as'1 has previously been reported, by Richards ( 2 3 4 ) . An i n -teresting observation which might warrant further i n v e s t i g a t i o n i s the cr o s s - l i n k i n g of PE 2_- i t was d i f f i c u l t to assess the nature of these complexes i n our system. I t i s also worthwhile - 1 5 5 -to note that v i r a l proteins did not cross-link with host plasma membrane proteins although two-dimensional gels did demonstrate that host proteins inter a c t with each other; this was evident when the two-dimensional gels were stained with Coomassie blue. Obviously, host c e l l proteins are e f f i c i e n t l y excluded-: from areas of the c e l l membrane which contain virus proteins during the budding process. The nature of t h i s l a s t process s t i l l requires elucidation. '- • Cross-linking studies performed on infected c e l l s i n the presence of colchi c i n e and dibucaine indicated that dibucaine prevented envelope dimer formation while colchicine appeared to have no e f f e c t . One inter p r e t a t i o n of ;this r e s u l t would be that microfilaments might be required to keep the envelope proteins proximal to each other p r i o r to budding while microtubules may be needed f o r a l a t e r stage i n v i r a l morphogenesis. One pos-s i b i l i t y exists - colchicine might a l t e r or i n h i b i t a stage of glyc o s y l a t i o n of the envelope proteins p r i o r to t h e i r i n s e r t i o n into the plasma membrane. In thi s case orientation of these glycoproteins might be altered and i n h i b i t i o n of glycosylation has previously been shown to a f f e c t budding of Semliki Forest virus ( 2 5 6 ) . I t i s quite obvious that the f i n a l stages of virus maturation are complex. These studies have shown that assembly of SF vi r u s i s dependent on the state of the host c e l l - i n p a r t i c u l a r the rol e and function of microtubules i n the "budding" process. - 1 5 6 -Further studies w i l l not only provide further information on the maturation of envelope viruses but also impart knowledge as to how glycoproteins are formed and secreted from the c e l l . C. Possible Topics for Further Research Low ionic strength media has been demonstrated to i n h i b i t v i r u s formation d r a s t i c a l l y (.257, 2 5 8 ) , It would be of i n t e r e s t to determine i f C a + + were involved i n virus maturation since t h i s ion has been shown to be i m p l i c i t l y involved i n the function of microtubules and microfilaments. Studies with ethyleneglycol-bis-( g-amino-ethyl ether)N^'N^-tetraacetic acid (which chelates Ca + +) and the Ca++', ionophore A23187 might reinforce the theory that microtubules and microfilaments are involved i n budding. The mechanism fo r transport of v i r a l proteins from the endo-plasmic reticulum to the plasma membrane i s of extreme i n t e r e s t . Proteolytic i n h i b i t o r s could be u t i l i z e d and the various points within the c e l l where the large precursor proteins accumulate could be ascertained. Also, the enzymes responsible for post-t r a n s l a t i o n a l cleavage should be i d e n t i f i e d - i n p a r t i c u l a r , whether they are virus or host s p e c i f i e d . Tunicamycin, an i n h i b i t o r of glycosylation, might also be u t i l i z e d and i t s effects on the i n s e r t i o n of envelope proteins into the plasma membrane could be monitored - i t would be i n t e r e s t i n g also to see how t h i s would a f f e c t precursor cleavage. Experiments si m i l a r to those performed on v e s i c u l a r stomatitis virus by Lodish ( 2 5 9 ) would also provide insight into how SF virus inserts i t s proteins into the endoplasmic reticulum and - 1 5 7 -how they are glycosylated. Rothman and Lodish (259) performed i n v i t r o experiments i n which G protein (an unglycosylated form, G 0,and a glycosylated protein, G]_)was synthesized from v i r a l RNA i n a wheat germ c e l l - f r e e homogenate. The G protein was found to associate with added microsomal membranes spontaneously while s t i l l nascent on the ribosomes. Once the G protein was inserted into the membrane i t appeared to be glycosylated almost immediately. The glycosylated protein G^ was not susceptible to t r y p s i n digestion while G Q was t o t a l l y digested. Similar experiments could be performed u t i l i z i n g 26s RNA which was isol a t e d from c e l l s infected with SF v i r u s . Experiments could be done to monitor the association of the proteins t s 2 , B-^ , PEg, E-^ , Eg and E^ with the endoplasmic reticulum and the glyco-s y l a t i o n of v i r a l proteins at the i n t r a c e l l u l a r membranes. The packaging and transfer of v i r a l glycoproteins from the Golgi to the plasma membrane should also be considered. This mechanism may be important i n determining why host c e l l proteins are excluded i n the virus patches on the plasma membrane. Cholesterol l e v e l s are about 3°$ higher i n the vi r u s than i n the plasma membrane - the exact si g n i f i c a n c e i s unclear but i t may a l t e r membrane f l u i d i t y and f a c i l i t a t e budding. Virus proteins could be p u r i f i e d and phospholipid cholesterol binding studies could be performed to determine whether v i r a l envelope proteins might have a p a r t i c u l a r l y high a f f i n i t y f o r cholesterol. 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