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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,  U n i v e r s i t y of B r i t i s h Columbia,  1973  M.Sc,  U n i v e r s i t y ' o f 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  m  THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA  ©  February, 1978 C h r i s t o p h e r D. Richardson  6  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at  the U n i v e r s i t y  make  it  freely  that permission  purposes  may  representatives. thesis  in p a r t i a l  for  is  financial  of  British  gain  Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  /Vlatch  [  >  /f 7  ?  by  for  shall  the  that  not  requirements  Columbia,  I  agree  r e f e r e n c e and copying  t h e Head o f  understood  of  The U n i v e r s i t y  British  for extensive  permission.  Department  of  available  be g r a n t e d  It  fulfilment of  of  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT The  d a t a from c h e m i c a l s t u d i e s and e l e c t r o n microscopy-  suggest t h a t S e m l i k i F o r e s t v i r u s o b t a i n s i t s envelope by budding i n t o t h e medium from t h e plasma membrane o f t h e h o s t c e l l . Biochemical  e v i d e n c e f o r ' t h i s phenomenon, however, has n o t been  published.  Therefore,  we undertook a s e r i e s o f p u l s e - c h a s e  s t u d i e s so t h a t we might q u a n t i t a t i v e l y e v a l u a t e  t h e importance  of t h e budding mechanism i n t h e morphogenesis o f S e m l i k i  Forest  virus. Baby hamster k i d n e y c e l l s and  ( c l o n e 13)  infected with Semliki'Forest v i r u s .  were grown i n c u l t u r e 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 t h e subsequent i n c o r p o r a t i o n o f 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 c y t o p l a s m i c and  e x t r a c e l l u l a r v i r u s was determined.  membrane  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 t h e microsomes and  e x t r a c e l l u l a r v i r u s ( C D . R i c h a r d s o n and D.E. Vance, J . B i o l .  Chem. 251,  55 -5550). LL  F u r t h e r 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 p r e p a r a t i o n s .  Maximum i n c o r p o r a t i o n o f  [ H ] l e u c i n e was observed i n t h e v i r a l p r o t e i n s l o c a t e d i n t h e J  endoplasmic r e t i c u l u m a t t h e end o f a 20-min p u l s e  period;  g r e a t e r t h a n 50fo o f t h i s r a d i o a c t i v i t y had d i s a p p e a r e d w i t h i n The  plasma membrane f r a c t i o n c o n t a i n e d  2 hr  no r a d i o a c t i v i t y a t  the end o f t h e p u l s e p e r i o d ; s u b s e q u e n t l y , maximal l a b e l i n g of t h e v i r a l p r o t e i n s i n t h e plasma membrane o c c u r r e d  3 hr into  the chase p e r i o d , and t h e s e l a b e l e d p r o t e i n s d i s a p p e a r e d from  t h i s membrane 8.5 hr a f t e r the p u l s e .  At 8.5 hr chase," maximum  i n c o r p o r a t i o n of the l a b e l e d p r o t e i n s i n t o e x t r a c e l l u l a r v i r u s was observed.  These data a r e c o n s i s t e n t with a  precursor-  product r e l a t i o n s h i p between v i r a l p r o t e i n s i n the endoplasmic reticulum,  plasma membrane, and e x t r a c e l l u l a r media.  Viral  p r o t e i n s migrate t o the plasma membrane and a r e subsequently incorporated  into extracellular virus.  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 v i r u s appears to have been d e r i v e d from v i r a l p r o t e i n s a s s o c i a t e d with the plasma membrane of the c e l l .  Therefore,  mechanismsffor^the"morpho-  genesis of S e m l i k i F o r e s t v i r u s ( i n baby hamster kidney c e l l s ) , other than budding from the plasma membrane, a r e u n l i k e l y to.be of q u a n t i t a t i v e importance. The  p o s s i b i l i t y that an i n t a c t c y t o s k e l e t a l system might  be r e q u i r e d f o r the assembly of S e m l i k i F o r e s t v i r u s was i n v e s tigated.  The microtubules and m i c r o f i l a m e n t s  kidney c e l l s and  (BHK-21)_ were disassembled w i t h s p e c i f i c  the e f f e c t on p r o d u c t i o n  mined.  o f baby hamster  of e x t r a c e l l u l a r v i r u s was d e t e r -  C o l c h i c i n e , Nocodazole, dibucaine,  reduced the p r o d u c t i o n Lumicolchicine  drugs  and c y t o c h a l a s i n B  o f e x t r a c e l l u l a r v i r u s by 75-90$.  had no e f f e c t on v i r u s growth.  Other c o n t r o l  experiments showed no e f f e c t by these drugs on the i n c o r p o r a t i o n of  [-^Hlleucine  o f [-^S] methionine.  At v a r i o u s  times a f t e r a d d i t i o n  of one of these drugs, the i n c o r p o r a t i o n of the l a b e l e d  pre-  cursors  reticulum  i n t o v i r a l p r o t e i n s a s s o c i a t e d with endoplasmic  or plasma membrane of the c e l l was evaluated. show t h a t the envelope and n u c l e o c a p s i d  The r e s u l t s c l e a r l y  proteins  of the v i r u s  move to the plasma membrane o f the c e l l where they accumulated. These s t u d i e s s t r o n g l y suggested t h a t the c y t o s k e l e t a l system was  i n v o l v e d i n the f i n a l stages of membrane assembly o f S e m l i k i F o r e s t v i r u s a t the plasma membrane. S t u d i e s were a l s o performed dimethylsuberimidate  with the c r o s s - l i n k i n g agents -  (DMS), d i t h i o b i s ( s u c c i n i m i d y l  (DSP), and dimethylthiobi's( propionimidate)  (DTBP) .  propionate) The p r o t e i n s  of p u r i f i e d v i r u s and i n f e c t e d c e l l s r e a c t e d w i t h these  agents  and the c r o s s - l i n k e d p r o t e i n s were evaluated by one- and twodimensional SDS e l e c t r o p h o r e s i s . Nucleocapsid p r o t e i n c r o s s - l i n k e d to form up t o pentameric  complexes, and envelope  to y i e l d dimeric s p e c i e s .  proteins reacted  N u c l e o c a p s i d p r o t e i n d i d not c r o s s -  l i n k w i t h envelope p r o t e i n s . C r o s s - l i n k i n g agents were a l s o u t i l i z e d t o determine the e f f e c t s o f c o l c h i c i n e and d i b u c a i n e on the p r o x i m i t y o f v i r a l p r o t e i n s t o each other i n the plasma membrane o f the host  cell.  C o l c h i c i n e (which d i s r u p t s microtubules) appeared t o have no e f f e c t on the degree t o w h i c h " f - ^ s ] - l a b e l e d v i r u s p r o t e i n s r e a c t e d w i t h the agents, w h i l e d i b u c a i n e (which  supposedly  d i s r u p t s both microtubules and m i c r o f i l a m e n t s ) a b o l i s h e d envelope  p r o t e i n dimers d r a m a t i c a l l y .  T h i s r e s u l t was  i n t e r p r e t e d to mean t h a t microtubules may not be r e q u i r e d f o r the f o r m a t i o n of patches p r i o r t o budding,  of v i r u s p r o t e i n s i n the plasma membrane  while m i c r o f i l a m e n t s may pOiay a more dominant  r o l e i n t h i s process.  iv  TABLE OF CONTENTS  Page ABSTRACT  •  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES . . .  ix  v  ABBREVIATIONS  xv  ACKNOWLEGEMENTS  xviii  DEDICATION  xix  INTRODUCTION A.  1  1  Mechanism Viruses  of Membrane Assembly f o r Enveloped  1. The S t r u c t u r e o f Group A Togaviruses  1 . . . .  2. R e p l i c a t i o n o f Togaviruses (a) Growth C y c l e o f Group A Togaviruses  7 . .  (b) R e p l i c a t i o n o f V i r a l RNA (c)  3.  3  T r a n s l a t i o n of V i r u s - S p e c i f i c mRNA . . .  7 8 11  (d) Synthesis and I n s e r t i o n of V i r a l G l y c o p r o t e i n s i n t o the I n t r a c e l l u l a r Membranes o f the Host C e l l  13  (e) Formation of V i r u s P r o t e i n s (Post-" T r a n s l a t i o n a l Cleavage o f Large Precursors)  15  M a t u r a t i o n and Membrane Assembly o f Togaviruses (a) E l e c t r o n m i c r o s c o p i c Observations o f Togavirus Assembly  22 22  (b) Chemical Evidence f o r M a t u r a t i o n of Group A Togaviruses a t the Plasma Membrane . . . 24 k. Assembly of V e s i c u l a r S t o m a t i t i s V i r u s . . . (a) S t r u c t u r e  25 25  V  (b) Membrane Assembly Stomatitis Virus 5.  B.  of V e s i c u l a r 26  Assembly o f Myxoviruses and Paramyxoviruses  30  (a) S t r u c t u r e of Myxoviruses and Paramyxoviruses  30  (b) Membrane Assembly Paramyxoviruses  31  of Myxoviruses and  C y t o s k e l e t a l Components o f Membrane Systems and T h e i r Involvement i n the Membrane Assembly o f Viruses  32  1.  Microtubules  33  2.  Microfilaments  36  3.  Microtubule  37  k.  The d i s r u p t i o n of M i c r o f i l a m e n t s  kl  5.  L o c a l A n e s t h e t i c s - D i s r u p t o r s o f Both M i c r o t u b u l e s and M i c r o f i l a m e n t s  k2  6.  Microtubule  disruptors  and M i c r o f i l a m e n t F u n c t i o n  Within ^3  the c e l l (a) C y t o s k e l e t a l Transmembrane C o n t r o l (b) C e l l S e c r e t i o n and the C y t o s k e l e t a l System (c) I n t r a c e l l u l a r Movement and the C. C y t o s k e l e t a l System 7. C.  . . . .  1  "  Previous Evidence f o r C y t o s k e l e t a l Involvement i n the Morphogenesis o f V i r u s e s  Chemical C r o s s - L i n k i n g o f V i r a l  ^3 48 k9 51  Membrane  Proteins  52  1.  Chemistry o f C r o s s - L i n k i n g Agents  52  2.  C r o s s - L i n k i n g of V i r a l  52  Proteins  (a) V e s i c u l a r S t o m a t i t i s V i r u s  52  (b) S e m l i k i F o r e s t V i r u s  $k  (c) Adenovirus  . . . . .  55  vii  D.  C y t o s k e l e t a l D i s r u p t o r s and T h e i r E f f e c t s Upon V i r u s Assembly 1.  The E f f e c t of C o l c h i c i n e on V i r u s 90  Maturation 2. 3. E. F. G.  90  The E f f e c t of Dibucaine on V i r u s Maturation  113  The E f f e c t o f C y t o c h a l a s i n B on V i r u s Maturation  '122  Two-Dimensional C r o s s - L i n k i n g Upon P u r i f i e d V i r u s  Studies Performed 133  Two-Dimensional C r o s s - L i n k i n g Studies Performed on Plasma Membranes from I n f e c t e d C e l l s . . . .  139  Two-Dimensional C r o s s - L i n k i n g Studies Performed on Plasma Membranes from I n f e c t e d C e l l s which were Treated With C o l c h i c i n e and Dibucaine . .  1^0  DISCUSSION BIBLIOGRAPHY  ,  -l;4f ,159  viii  LIST OF TABLES Page 1.  Groups o f L i p i d C o n t a i n i n g V i r u s e s  1  2.  Family:  2  3.  Moles o f Carbohydrate Forest Virus  Togaviridae Per Mole o f P r o t e i n i n S e m l i k i  5  4-.  L i p i d C l a s s Composition  5.  Number o f D i f f e r e n t Molecules Virus  6.  of Semliki Forest V i r u s . . .  6  i n Semliki Forest 6  R e p r e s e n t a t i v e Agents Used f o r C r o s s - L i n k i n g Proteins ,  53  7.  A c t i v i t i e s o f Enzymes C h a r a c t e r i s t i c o f Endoplasmic Reticulum or Plasma Membrane  80  8.  E f f e c t s of Various Concentrations Cell Viability  o f C o l c h i c i n e on 91  E f f e c t s of V a r i o u s C o n c e n t r a t i o n s Cell Viability  o f Dibucaine on  ;i  9.  113  10. I n c o r p o r a t i o n of K Leucine i n t o T r i c h l o r o a c e t i c • •. : A c i d P r e c i p i t a b l e M a t e r i a l from BHK C e l l s i n the Presence or Absence of Dibucaine 121 y  11. E f f e c t s o f V a r i o u s C o n c e n t r a t i o n s on C e l l V i a b i l i t y  of Cytochalasin B 122  ix  LIST OF FIGURES  Page 1.  The S t r u c t u r e of S e m l i k i F o r e s t V i r u s  2.  Time Course of V i r u s P r o d u c t i o n  3.  R e p l i c a t i v e Model o f Simons and Strauss  . . . . . .  9  k.  Model f o r the Synthesis of S i n d b i s V i r i o n P r o t e i n s , Subsequent Nascent Cleavages, and Sequestering o f the Envelope P r o t e i n s  Ik  An SDS Polyacrylamide G e l Showing the R a d i o a c t i v e P o l y p e p t i d e s Which Are Present i n C e l l s I n f e c t e d With t s 2 Mutants o f S i n d b i s and HR (Wild S t r a i n ) Sindbis  16  P o s t - T r a n s l a t i o n a l Cleavage i n Formation of SF v i r u s S t r u c t u r a l P r o t e i n s . .  19  Scheme f o r the P o s t - T r a n s l a t i o n a l P r o c e s s i n g of P r e c u r s o r s to the N d n - S t r u c t u r a l P r o t e i n s of a Temperature S e n s i t i v e Mutant of S e m l i k i F o r e s t Virus  29  Formation of N o n - S t r u c t u r a l P r o t e i n s by PostT r a n s l a t i o n a l Cleavage of Precursors i n C e l l s Which Were I n f e c t e d With S e m l i k i F o r e s t V i r u s  21  Formation of N o n - S t r u c t u r a l P r o t e i n s by PostT r a n s l a t i o n a l Cleavage o f Precursors I n C e l l s Which Were I n f e c t e d By S i n d b i s V i r u s  21  S t r u c t u r e and Composition o f V e s i c u l a r S t o m a t i t i s Virus  26  K i n e t i c s I l l u s t r a t i n g the A s s o c i a t i o n of 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 C e l l Supernatant and Membrane F r a c t i o n s During a Pulse-Chase Experiment  28  5.  6. 7.  8.  9.  10. 11.  12.  Maturation  3  i n BHK-21 C e l l s  of V e s i c u l a r S t o m a t i t i s V i r u s  . .  7  Within 29  the C e l l 13.  S t r u c t u r e and P r o t e i n Composition of Myxoviruses  Ik.  S t r u c t u r e and P r o t e i n Composition of Paramyxoviruses  . .  3° 30  X  15.  The B a s i c Composition o f a Microtubule  33  16.  F i b r o b l a s t s Labeled Anti-Tubulin  34  With F l u o r e s c e n t  17.  S t r u c t u r e s o f Drugs Which D i s r u p t M i c r o t u b u l e s  18.  The Chemical S t r u c t u r e of L u m i c o l c h i c i n e and I t s A b s o r p t i o n Spectrum As Compared To That of  . .  38  Colchicine  39  19.  Chemical S t r u c t u r e of C y t o c h a l a s i n B  41  20.  S t r u c t u r e of T e r t i a r y Amine A n e s t h e t i c s  43  21.  E f f e c t o f Drugs on Lymphocytes Which Were I n Contact With A h t i - I g and/or Con A E f f e c t of Drugs on 3T3 F i b r o b l a s t s Which Were I n Contact With A n t i - I g and/or Con A ,,  46  23-  Proposed I n t e r a c t i o n of M i c r o t u b u l e s f i l a m e n t s With Membrane P r o t e i n s  47  24.  Autoradiograph o f 2-Dimensional SDS Polyacrylamide Gels Which I l l u s t r a t e the C r o s s - L i n k e d P r o t e i n s of V e s i c u l a r S t o m a t i t i s V i r u s  53  P u r i f i e d S e m l i k i F o r e s t V i r u s and Standard P r o t e i n s Which Were Subjected to E l e c t r o p h o r e s i s on 7-5% Polyacrylamide Gels i n the Presence o f Sodium Dodecyl S u l f a t e  75  Absorbance Scan o f S e m l i k i F o r e s t - V i r u s P r o t e i n s Which Were Separated on a 7 >5f° Polyacrylamide SDS g e l  76  22.  25-  26v  and Micro-  27.  P r o t e i n s From SF V i r u s Which Were Subjected to SDS E l e c t r o p h o r e s i s on 6 . 5 $ Polyacrylamide Slab Gels i n the Presence and Absence o f 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 D i s continuous Sucrose G r a d i e n t Used F o r I s o l a t i o n of Endoplasmic Reticulum and Plasma Membrane . . . .  45  79  xi  29.  30.  31.  32.  33-  34.  35«  36.  37.  38. 39• 40. 41.  Incorporation of [ H]J.Leucine Into the Proteins 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 Period J  Incorporation Jfo'f ¥ h e P l a s m a 20 M i n P u l s e  o f ,[ H ] _ L e u c i n e I n t o M e m b r a n e 0-8.5 H o u r s  Time Course f o r Yirus-Specified The E n d o p l a s m i c  Of .82.  the Proteins After a  84'-  I n c o r p o r a t i o n o f [^H]^ L e u c i n e Precursor Proteins Associated Reticulum  Into With  Time Course 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 V i r u s - S p e c i f i e d Proteins Associated With the Endoplasmic Reticulum  Into  Time C o u r s e f o r I n c o r p o r a t i o n o f Virus-Specified Proteins Associated Membrane and E x t r a c e l l u l a r V i r u s P r o f i l e s of Sucrose Extracellular Virus 0-8.5 H o u r C h a s e  Gradients W h i c h Was  _  86 -  87*  Leucine Into With Plasma 88/:'  Containing Radioactive Produced Over a  89'  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 C o l c h i c i n e . .  92*  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^ o f C o l c h i c i n e  93  Photograph of Sucrose Gradients Which C o n t a i n Produced i n E i t h e r the Absence or Presence of Colchicine  94'  Absorbance Spectrum a t Room T e m p e r a t u r e  for .  C o l c h i c i n e and  Virus  Lumicolchicine S£ :  E f f e c t s o f L u m i c o l c h i c i n e and C o l c h i c i n e on t h e P r o d u c t i o n of Radioactive E x t r a c e l l u l a r V i r u s . . E f f e c t of Nocodazole Extracellular Virus  on t h e  Production of  .  .  f  9.6'  Radioactive  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 ( T C A ) 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 the Presence or Absence of C o l c h i c i n e  97'  98  xii  42.  P r o f i l e s of Sucrose 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 Produced by C e l l s Which Were C o n t i n u o u s l y Labeled w i t h [3H]Leucine i n the Presence and Absence of C o l c h i c i n e .100  43.  P r o f i l e s of SDS Gels Which Contained Plasma Membrane P r o t e i n s from C e l l s Which Were C o n t i n u o u s l y Labeled i n the Presence and Absence of C o l c h i c i n e  101  I n c o r p o r a t i o n o f [-^H]Leucine i n t o the V i r a l P r o t e i n s of the Plasma Membrane o f C e l l s i n the Presence and Absence of C o l c h i c i n e  103  44.  c  45.  Plasma Membrane P r o t e i n s Which Were L a b e l e d w i t h [/HJLeucine During a Pulse-Chase Experiment Which Was Performed i n t h e Presence and Absence o f C o l c h i c i n e . 104  46.  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 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  106  Time Course 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 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 the Plasma Membrane During a Pulse-Chase Experiment Which Was Performed i n t h e Presence and Absence of C o l c h i c i n e .  107  3^ Autoradiogram o f .Jj^s ]-Labeled Plasma Membrane P r o t e i n s During a Pulse-Chase Experiment Performed i n the Presence and Absence of C o l c h i c i n e  108  Autoradiogram o f [-^S ]-Labeled Endoplasmic Reticulum P r o t e i n s from a Pulse-Chase Experiment Performed i n the Presence and Absence o f C o l c h i c i n e  109  Time Course f o r the I n c o r p o r a t i o n of ["^SjMethionine i n t o t h e V i r a l P r o t e i n s o f the Plasma Membrane from C e l l s Which Were P u l s e - L a b e l e d i n the Presence or Absence of C o l c h i c i n e  110  47.  48. 49-  50.  v  51.  P r o f i l e s of [- ^ S ] - L a b e l e d E x t r a c e l l u l a r V i r u s Produced 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 112  52.  P r o f i l e s o f Sucrose 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 Produced by C e l l s i n t h e Presence and Absence o f Dibucaine 114  53-  Photograph o f Sucrose G r a d i e n t s Which C o n t a i n V i r u s Produced i n E i t h e r the Absence or Presence o f Dibucaine  115  P r o f i l e s of SDS P o l y a c r y l a m i d e G e l s Which Contained Plasma Membrane P r o t e i n s from C e l l s Which Were C o n t i n u o u s l y L a b e l e d i n the Presence and Absence of Dibucaine  116  54.  xiii  55'  Accumulation o f V i r a l P r o t e i n i n the Plasma Membranes of C e l l s T r e a t e d with Dibucaine ..  56.  Autoradiogram o f [-^S]-Labeled P r o t e i n s from the Endoplasmic Reticulum "of BHK C e l l s Exposed t o Dibucaine  119  118  57.  P r o f i l e s o f Sucrose G r a d i e n t s Which C o n t a i n 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 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 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 Produced by C e l l s i n the Presence ,-. and Absence of C y t o c h a l a s i n B 123 a  59.  60.  61.  62.  63.  64.  65.  66.  67.  P r o f i l e s of SDS P o l y a c r y l a m i d e Gels Which Contained Plasma Membrane and Endoplasmic Reticulum P r o t e i n s from C e l l s Which Were C o n t i n u o u s l y Labeled i n the Presence and Absence o f C y t o c h a l a s i n B  1?'5  E l e c t r o n m i c r o g r a p h of I n f e c t e d BHK C e l l s Which P o r t r a y s V i r u s Budding a t the Plasma Membrane i n the Absence o f C o l c h i c i n e and Dibucaine  .128  E l e c t r o n m i c r o g r a p h of I n f e c t e d BHK C e l l s Showing Accumulation o f Nucleocapsids a t the Plasma Membrane i n the Presence of C o l c h i c i n e  .129  E l e c t r o n m i c r o g r a p h of I n f e c t e d BHK Cell-s showing Accumulation of Nucleocapsids a t the Plasma Membrane i n the Presence of Dibucaine  13°  E l e c t r o n m i c r o g r a p h of a C y t o p a t h i c Vacuole Found i n I n f e c t e d BHK C e l l s Which Were Treated with Dibucaine  ^  l  E l e c t r o n m i c r o g r a p h of a C y t o p a t h i c Vacuole Found i n I n f e c t e d BHK C e l l s Which Were T r e a t e d with Dibucaine  .132  P u r i f i e d SF V i r u s Which Was C r o s s - L i n k e d w i t h DMS and DSP and E l e c t r o p h o r e s e d on 3-5% Polyacrylamide C y l i n d r i c a l Gels  133  Absorbance Scans of 3>5% Polyacrylamide Gels C o n t a i n i n g P r o t e i n s Which Were C r o s s - L i n k e d w i t h DMS and DSP  13^  P u r i f i e d SF v i r u s Which Was C r o s s - L i n k e d w i t h DTBP and Subjected t o E l e c t r o p h o r e s i s on 6.-^Polyacrylamide Slab Gels 135 v  68.  P u r i f i e d SF v i r u s Which Was C r o s s - L i n k e d w i t h DSP and Subjected to E l e c t r o p h o r e s i s on 6.5$ Polyacrylamide Slab Gels  .136  xiv  69.  Two-Dimensional E l e c t r o p h o r e s i s o f SF V i r u s Which Had Been C r o s s - L i n k e d with DSP  70.  Two-Dimensional Autoradiogram o f Plasma Membrane P r o t e i n s from SF V i r u s - I n f e c t e d C e l l s Which Had Been C r o s s - L i n k e d with DSP  140  Two-Dimensional Autoradiogram o f Plasma Membrane P r o t e i n s from SF v i r u s - I n f e c t e d C e l l s Which Had Been C r o s s - L i n k e d with DTBP  141  71.  Proteins  138  72.  One-Dimensional Autoradiogram o f DTBP C r o s s - L i n k e d Plasma Membrane P r o t e i n s from I n f e c t e d C e l l s C u l t u r e d i n the Presence or Absence of Dibucaine and Colchicine 143  73.  Two-Dimensional Autoradiograms of DTBP C r o s s - L i n k e d Plasma Membrane P r o t e i n s from I n f e c t e d C e l l s Which Were C u l t u r e d i n the Presence or Absence of Dibucaine and C o l c h i c i n e 144  XV  LIST OF ABBREVIATIONS SF v i r u s  Semliki Forest v i r u s  E-^, Eg i E^  envelope p r o t e i n s o f S e m l i k i F o r e s t v i r u s  NC  nucleocapsid  EnE„  combined envelope p r o t e i n s E i and E2 which o f t e n do not r e s o l v e by SDS e l e c t r o p h o r e s i s ; t h i s g i v e s the impression of one p r o t e i n  NVP 130 ( o r ts-2 p r o t e i n )  protein  non-virion precursor 130,000)  p r o t e i n (molecular  weight  NVP 98 ( or B protein)  non-virion precursor 98,000)  p r o t e i n (molecular  weight  NVP 68 ( o r PE )  non-virion precursor 68,000)  p r o t e i n (molecular  weight  nsp  non-structural protein  p  precursor  PE  phosphatidyl  ethanolamine  PC  phosphatidyl  choline  PS  phosphatidyl  serine  PI  phosphatidyl  inositol  PM  plasma membrane  ER  endoplasmic  BHK  baby hamster kidney  BK  bovine kidney  MK  monkey kidney  CEF  c h i c k embryo f i b r o b l a s t  PFU  plaque forming u n i t  Leu  leucine  Met  methionine  2  protein  reticulum  xvi'  liCi  microcurie  mCi  millicurie  mRNA  messenger RNA  ssRNA  s i n g l e stranded RNA  dsRNA  double stranded RNA  RF  replicative  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  G, M, N, NS, L, P  vesicular stomatitis virus proteins  HN, F, F  paramyxovirus p r o t e i n s  0  form  s e n s i t i v e mutant  HA, NA, M  influenza virus proteins  TPCK  t o s y l p h e n y l a l a n i n e chloromethylketone  M.W.  molecular weight  Cyt. c'  cytochrome c  Ig  immunoglobulin  mamps  milliamperes  ml  milliliters  ul  microliters  hr  hours  cm  centimeters  mm  millimeters  nm  nanometers  mM  millimolar  uM  micromolar  ;  xvii  CPM  counts per minute  SDS  sodium dodecyl  A550  o p t i c a l absorbance a t 550  Con  A  sulfate nanometers  concanavalin A  TDA  tartaryl diazide  DMS  dimethylsuberimidate  DTBP  dimethyl t h i o b i s p r o p i o n i m i d a t e  DSP  dithiobis (succinimidyl  MMB  methyl-4-  propionate)  mercaptobutyrimidate  xviii  ACKNOWLEDGEMENTS I wish t o thank Dr. Dennis E. Vance f o r h i s guidance, support, and encouragement throughout t h e course of t h i s work.  I am a l s o  i n d e b t e d t o Karen Catherwood, Harry Paddon and J e n n i f e r Toone f o r t h e i r excellent technical assistance. were performed i n the l a b o r a t o r y Shinn.  The e l e c t r o n m i c r o g r a p h s  o f Dr. W. O v a l l e by Ms. Susan  S p e c i a l thanks goes t o Mrs. N. Richardson and Mrs. F.  Dowling f o r h e l p i n g type t h i s t h e s i s and t o J e n n i f e r Toone f o r p r e p a r i n g some of the f i g u r e s .  I a l s o wish t o express my g r a t -  i t u d e t o the f r i e n d s which I made i n t h i s  department.  xix  DEDICATION TO MY PARENTS  I.  A.  INTRODUCTION  (1-9)  Mechanisms of Membrane Assembly f o r Enveloped V i r u s e s  A l a r g e p r o p o r t i o n of v i r u s e s possess l i p o p r o t e i n envelopes as i n d i c a t e d  i n Table  1.  TABLE I'. Groups of L i p i d C o n t a i n i n g V i r u s e s Group  Nucleic Acid Type , MW x 10  Pox V i r u s Herpes V i r u s PM2 Phage Togavirus Myxovirus Paramyxovirus Rhabdovirus RNA tumor v i r u s Arenavirus Coronavirus  The  ds ds ds ss ss ss ss ss ss ss  DNA DNA DNA, RNA RNA RNA RNA. RNA RNA RNA  160 100 6 k k  7 6 12  ?  7  V i r i o n Shape  b r i c k shaped spherical spherical spherical spherical/filamentous spherical/filamentous b u l l e t shaped spherical spherical spherical  Size A  3000x2000 1200 600 700 1000 1200 700x1750 • 1200 600-1200 800-1200  manner i n which v a r i o u s groups o f v i r u s e s o b t a i n  membranes v a r i e s .  For  example, pox  (5)  their  v i r u s e s are assembled  entirely  i n the cytoplasm of the host c e l l a t a " f a c t o r y " which i s i n d e pendent of the plasma membrane and Recently  S t e r n and  Dales (10)  endoplasmic r e t i c u l u m  (3,  8).  have i m p l i c a t e d the r o l e of phos-  p h o l i p i d exchange p r o t e i n s i n the  t r a n s f e r of p h o s p h o l i p i d  microsomes d u r i n g the assembly o f v a c c i n i a envelopes.  from  Herpes  v i r u s e s , on the other hand, appear to be assembled i n the n u c l e u s and  o b t a i n t h e i r envelopes from the n u c l e a r membrane ( t h i s must  be more t h o r o u g h l y  substantiated).  The  completed v i r i o n i s  -2-  probably t r a n s p o r t e d  to the plasma membrane by cytoplasmic  membrane channels which prevent degradation o f v i r u s membranes. C o r o n a v i r u s e s . appear to mature and o b t a i n t h e i r membranes as they pass i n t o the c i s t e r n a e cytoplasmic v e s i c l e s .  o f the endoplasmic r e t i c u l u m and  The m a j o r i t y  o f v i r u s e s which possess  membranes appear to o b t a i n t h e i r envelopes from the plasma membrane o f the host c e l l . rhabdoviridae,  Myxoviridae, paramyxoviridae,  r e t r o v i r i d a e , arenaviridae,  a l l reputed t o mature i n t h i s f a s h i o n . of which S e m l i k i  and t o g a v i r i d a e a r e  The £ami'lty^fogavifidae  F o r e s t v i r u s i s a member,  - comprises two-  s e r o l o g i c a l groups - Group A ( o r a l p h a v i r u s e s ) flaviviruses).  Representative viruses  and Group B ( o r  of t h i s family are l i s t e d  i n Table 2.  TABLE 2  Genus  Family: T o g a v i r i d a e (2)  Representative  Viruses  Alphavirus  Sindbis, Semliki Forest Virus, Western Equine E n c e p h a l i t i s , E a s t e r n Equine E n c e p h a l i t i s , Venezuelan Equine E n c e p h a l i t i s , Chikungunya, r u b e l l a (most l i k e l y )  Flavivirus  Dengue types 1-4, yellow f e v e r , St. L o u i s e n c e p h a l i t i s , Japanese e n c e p h a l i t i s , West N i l e e n c e p h a l i t i s , Murray V a l l e y e n c e p h a l i t i s , Russian t i c k - b o r n e e n c e p h a l i t i s  -3-  1.  The  S t r u c t u r e of Group A. Togaviruses  The i f not  s t r u c t u r e s of a l l the Group A Togaviruses  e n t i r e l y i d e n t i c a l (1, 2).  S e m l i k i F o r e s t v i r u s and  These v i r u s e s , of which  S i n d b i s v i r u s are the most s t u d i e d ,  c o n s i s t of an i c o s a h e d r a l n u c l e o c a p s i d lipid  envelope.  are almost  surrounded by a s p h e r i c a l  Three g l y c o p r o t e i n s denoted as E-^, Eg and  are s i t u a t e d i n the envelope and v i r u s nucleocapsid  (11).  i s shown diagrammatically of a s i n g l e s t r a n d of 4-2S  The  l i e i n close proximity  i n F i g . 1.  The v i r u s genome c o n s i s t s  RNA. MW  52,000  Cholesterol  VP  ^  n\ &.ut  PL  SEMLIKI FOREST The  the  s t r u c t u r e of S e m l i k i F o r e s t v i r u s  £.j  F i g . 1.  to  E^  Eg  M W  MW  VIRUS  s t r u c t u r e of S e m l i k i F o r e s t v i r u s .  3  10.000  49,000  The  s t r u c t u r a l p r o t e i n s of S e m l i k i F o r e s t v i r u s have  been r e s o l v e d with SDS  polyacrylamide  gel electrophoresis.  O r i g i n a l l y s t u d i e s were performed u s i n g the g e l system of Osborne (13)'  Weber and  E-^ and  s t u d i e s of Hay,  Skehel and  and  Tamm (16).  Acheson and  Eg were not r e s o l v e d i n the  Burke ( 1 4 ) ,  Nucleocapsid  evident w h i l e no t r a c e of E^ was existence  remained unknown.  b u f f e r SDS  p r o t e i n was  clearly  found on the gels and i t s  More r e c e n t l y the  discontinuous and  were a p p l i e d to p u r i f i e d v i r u s p r e p a r a t i o n s and  P f e f f e r k o r n (2).  molecular weights of E-^, Eg and  (15)>  et a l .  g e l e l e c t r o p h o r e s i s systems of N e v i l l e (17)  Laemmli ( 1 8 ) Simons (19)  Kaariainen  by  These r e s u l t s show the nucleocapsid  52.000,  to be  49,000 and 34,000 r e s p e c t i v e l y . The  existence  of E^ i n S e m l i k i F o r e s t v i r u s was  u n t i l v e r y r e c e n t l y (12).  As yet, t h i s p r o t e i n has  demonstrated to be present  i n Sindbis v i r u s .  showed t h a t although E^ cannot be detected  not  evident  not been  G a r o f f and  on 7.5$  acrylamide g e l s by c l a s s i c a l s t a i n i n g techniques,  Simons  10$  and the  SDS  small  3 S" p r o t e i n c o u l d be detected to 10$  SDS  activity.  with[-^SjMet l a b e l l e d SFV  g e l s - the g e l s were then s l i c e d and More c o n c l u s i v e  hydroxylapatite  column (12).  E-^, Eg.  applied  assayed f o r r a d i o  evidence f o r the e x i s t e n c e  presented when d e l i p i d a t e d membrane p r o t e i n was SDS  was  of E^  e l u t e d from  , and  35-7$. 35.7$.4.9$.  respectively.  and  23.7$  an  nucleocapsid  p r o t e i n s appear i n equimolar amounts i n the mature v i r i o n constitute  was  of the t o t a l  and  protein  -5-  A l l t h r e e SFV membrane p r o t e i n s a r e g l y c o s y l a t e d . of N - a c e t y l g l u c o s a m i n e , mannose, g a l a c t o s e , a c i d appear i n a l l t h r e e p r o t e i n s  (12).  sequence f o r some o f t h e g l y c o p r o t e i n s  Residues  f u c o s e , and s i a l i c  The c a r b o h y d r a t e  of Semliki 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 R e s i d u e P e r Mole P r o t e i n P r o t e i n -N-acetylMannose G a l a c t o s e Fucose S i a l i c T o t a l CHO % glucosamine Acid by weight E-L  7  E  8  2  9 The  5 1 2 L  3  1  2  7-5%  3  1  ^  H-5%  4  2  3  k5.1%  l i p i d s o f t h e v i r a l membrane c o n s i s t o f 32% n e u t r a l  l i p i d s , 6l% p h o s p h o l i p i d s  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 o f SFV c o n s i s t s a l m o s t e x c l u s i v e l y o f f r e e c h o l e s t e r o l w h i l e t h e main components o f t h e p h o s p h o l i p i d s are s p h i n g o m y e l i n , p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l  serine.  ethanolamine,  phosphatidyl-  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 o f t h e v a r 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  Lipid  L i p i d C l a s s Composition o f SFV shown as Mole R a t i o R e l a t i v e t o P h o s p h o l i p i d s (21)  Class  Mole R a t i o  Cholesterol Glycolipids Phospholipids PE PC PS PI ;Sph i'r.g ojny .ei i n  0.99  0.08  1.00 0.23 0.33 0.13 0.02 0.20  I t i s b e l i e v e d t h a t the l i p i d  c l a s s composition  of the host plasma membrane.  Such a r e l a t i o n s h i p i s a l s o r e -  f l e c t e d i n the f a t t y a c i d composition  resembles t h a t  o f the p h o s p h o l i p i d s i n  the v i r u s and plasma membranes o f i n f e c t e d BHK-21 c e l l s ( 2 1 ) . In summary, a s i n g l e p a r t i c l e o f S e m l i k i F o r e s t v i r u s c o n t a i n s the molecular  composition  TABLE 5 (12, 20) Constituent RNA, Nucleocapsid Membrane p r o t e i n s Cholesterol Polar l i p i d s PE PC PS PI Sphingomyelins Gangliosides  listed  i n Table 5«  Number of D i f f e r e n t Molecules Number o f Molecules  3,500  6,400 2,000  200 2,400 1,000  1 200 550 15,ooo 16,000  i n SFV  Per V i r i o n  -7-  2.  R e p l i c a t i o n o f Togaviruses (a) Growth C y c l e o f Group A Togaviruses The growth o f group A t o g a v i r u s e s i s r a p i d (2).  After  2 hours o f i n f e c t i o n a t 37° v e r t e b r a t e c e l l s begin t o produce virus.  V i r u s p r o d u c t i o n may approach 1000 P F U / c e l l / h r and  the t o t a l est  y i e l d may approach 10"" PFU/ml growth media. 1  t i t e r s a r e achieved  BHK-21 c e l l s .  0  High-  i n c h i c k embryo f i b r o b l a s t and i n  The growth curve f o r S e m l i k i F o r e s t v i r u s i n  BHK-21 c e l l s i s shown i n F i g . 2.  n' -—.—.—  .  1  0 '  2  1  4  5  6  7  8  9  TIME (HOURS)  Fig.  2.  Time course  o f v i r u s p r o d u c t i o n i n BHK-21 c e l l s .  By 11 hours o f i n f e c t i o n c y t o p a t h i c e f f e c t s become apparent by l i g h t microscopy and t h e r a t e o f v i r u s p r o d u c t i o n  falls  markedly.  V e r t e b r a t e c e l l s a r e u l t i m a t e l y destroyed  although  persistent  ( c h r o n i c ) i n f e c t i o n occurs i n the presence of  i n t e r f e r o n (2, 23).  -8-  Group A t o g a v i r u s e s  i n f e c t arthropod  c e l l l i n e s such as  d e r i v e d from the mosquito (Aedes a l b o p i c t u s ) and but c h r o n i c p e r s i s t e n t i n f e c t i o n s may p e r s i s t e n t i n f e c t i o n s may  the  also r e s u l t .  a l s o be induced  those  tick, These  by the presence of  i n t e r f e r o n (2). (b) R e p l i c a t i o n of V i r a l Togavirus  RNA  RNA  s y n t h e s i s i s e a s i l y monitored...in  c e l l s with or without actinomycin host c e l l s p e c i f i e d RNA v i r a l RNA  and  DNA  infected  D s i n c e the v i r u s  synthesis  (1, 24).  represses Group A  s y n t h e s i s 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 t o maximal r a t e s by 3 hours, and t i n u e s through the p e r i o d of v i r u s r e l e a s e (25). forms of s i n g l e - s t r a n d e d RNA c e l l s are v i r i o n RNA 2?).  (42S)  and  i n t e r j a c e n t RNA  c o e f f i c i e n t s of 38S  R e p l i c a t i o n of the RNA multiple-stranded  2 principal  found i n group A t o g a v i r u s - i n f e c t e d  These are the forms of v i r u s mRNA.  sedimentation  The  con-  and  r e p l i c a t i v e intermediate  (1, 2,  26,  Minor ssRNA w i t h  3 3 S also  of t o g a v i r u s e s  (26s)  exist.  proceeds through a (which i s a s s o c i a t e d  with membranes) s i m i l a r to t h a t d e s c r i b e d f o r p i c o r n a v i r u s e s and  RNA  bacteriophage (28).  with phenol and  On e x t r a c t i o n of i n f e c t e d c e l l s  s e p a r a t i o n by CF11  d i f f e r e n t types of double stranded  c e l l u l o s e chromatography, 2 RNA  can be  isolated:  1) r e p l i c a t i v e i n t e r m e d i a t e s (RI) - c o n s i s t s of p a r t i a l l y double stranded RNA with s i n g l e stranded non-hydrogen bonded r e g i o n s 2) r e p l i c a t i v e forms (RF) - e x t e n s i v e l y hydrogen bonded double stranded RNA which remains a f t e r t o t a l c e l l u l a r RNA i s d i g e s t e d with p a n c r e a t i c r i b o n u c l e a s e  - 9 -  These double stranded RNA. s were o r i g i n a l l y i s o l a t e d by 1  P f e f f e r k o r n , Burge and Coady ( 2 9 ) and Friedman ( 3 ° ) •  Free  s i n g l e - s t r a n d e d RNA o f n e g a t i v e p o l a r i t y ( i e . complementary to v i r u s genome) was not i s o l a t e d from i n f e c t e d c e l l s .  Simmons  and Strauss ( 3 1 ) , Segal and S r e e v a l s a n ( 3 2 ) , and M a r t i n and Burke ( 3 3 ) i d e n t i f i e d three r e p l i c a t i v e forms a f t e r  cellular  RNA was e x t r a c t e d and d i g e s t e d w i t h p a n c r e a t i c r i b o n u c l e a s e ( 3 2 ) . Simmons and Strauss (31)> M a r t i n and Burke ( 3 3 ) , and Segal and S r e e v a l s e n purport the e x i s t e n c e o f 2 i n t e r m e d i a t e s ( F i g . 3) which d i f f e r i n the s i t e a t 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 )  p o s t u l a t e s t h a t a r e g u l a t o r y p r o t e i n binds to  template RNA ( n e g a t i v e stranded 42S RNA) and allows the 26S RNA to be t r a n s c r i b e d from an i n t e r n a l s i t e on the RNA ( F i g . 3 ) .  j  -  2 6 s (|.5*I0 daltons) 6  (4.4 x 1 0 doltons) s  Fig. 3 .  R e p l i c a t i v e Model o f Simmons and Strauss  (31)  -10-  R e c e n t l y Bruton and Kennedy (3^)  have shown t h a t the  (genome) and  -ve 42S RNA, which  c o n s i s t s of +ve  42S RNA.  c o n s t i t u t e s 80$  of the r e p l i c a t i v e forms.  RFI was  c o n t a i n non-hydrogen bonded p o l y A a t the 3 '  end  shown to  of the  p o s i t i v e s t r a n d i d e n t i c a l i n l e n g t h to t h a t on the genome.  No p o l y U was  RFI  42S  viral  l o c a t e d on RFI and n e i t h e r was  poly A  l o c a t e d on the minus s t r a n d , j u s t the p o s i t i v e s t r a n d . k i n e t i c s of p o s i t i v e and n e g a t i v e  The  s t r a n d s y n t h e s i s were i n -  vestigated during v i r u s m u l t i p l i c a t i o n .  Negative s t r a n d  syn-  t h e s i s reached a maximum rateo2fshours. post i n f e c t i o n ' a n d ' t h e r e a f t e r r a p i d l y fells,-  The r a t e of p o s i t i v e s t r a n d s y n t h e s i s  increases  r a p i d l y up to 3 hr p o s t - i n f e c t i o n and remains constant V i r a l r e p l i c a t i o n i s reputed  over 8 hr.  to occur i n d i r e c t a s s o c i a t i o n  with i i n t r a c e l l u l a r c y t o p a t h i c vacuoles  (designated  type 1)  A membrane-associated r e p l i c a t i o n complex which contained RNA  polymerase and r e p l i c a t i v e i n t e r m e d i a t e was  p u l s e - l a b e l i n g i n f e c t e d c e l l s with  [^H]uridine.  (35i36,37).  At 5 - 5  Partial  isolated.  The  after  purifiCPV-1  hours post i n f e c t i o n c e l l s were l y s e d ,  homogenized, and a f r a c t i o n enriched f o r c y t o p a t h i c was  viral  identified  c a t i o n of t h i s s t r u c t u r e r e s u l t e d i n a c o n c e n t r a t i o n of  (2).  vacuoles  i s o l a t e d v e s i c l e s were membrane l i m i t e d  l i n e d by r e g u l a r membranous s p h e r i c l e s measuring 50 nm  in  diameter- these were n e i t h e r v i r u s cores or v i r i o n s .  CPV-1  appearance c o i n c i d e d with RNA  p r o d u c t i o n i n BHK,  CEF,  and L  and  cells  i n f e c t e d with SF v i r u s , S i n d b i s , and Western equine e n c e p h a l i t i s viruses.  These vacuoles  appeared to a r i s e from the G o l g i  -11-  apparatus based on assays u s i n g phosphatase.  the G o l g i marker enzyme, a c i d  I n another experiment, a cytoplasmic e x t r a c t was  p u r i f i e d on a d i s c o n t i n u o u s sucrose g r a d i e n t .  A band  contain-  i n g RNA polymerase a c t i v i t y , L" H] u r i d i n e and type 1 c y t o p a t h i c 3  v a c u o l e s ( v i s i b l e by electronmicroscopy) was obtained.  Similar  r e s u l t s have been obtained w i t h p o l i o v i r u s (38, 39)- M i c h e l and Gomatos (4-0)  i n d i c a t e d t h a t r e p l i c a t i v e form and r e p l i c a t i v e  i n t e r m e d i a t e RNA was a s s o c i a t e d Semliki  with such membranous  F o r e s t v i r u s RNA polymerase has r e c e n t l y been  i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d was N-101  structures.  (41,  42).  The polymerase  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 (  and p u r i f i e d on an a f f i n i t y column which contained 4 2 S  v i r a l RNA as the l i g a n d .  Three p o l y p e p t i d e s were found t o be  present i n the p u r i f i e d enzyme - 2 were v i r u s 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 t o be h o s t - s p e c i f i e d o r p o s s i b l y a contaminant. f i e d polypeptides are derived  The 2 v i r u s  speci-  from 3 l a r g e r p r e c u r s o r s o f  molecular weights - 200,000, 184,000 and 50,000  (42).  (c) T r a n s l a t i o n of V i r u s - S p e c i f i c mRNA Several  i n v e s t i g a t o r s have i s o l a t e d p o l y r i b o s o m a l mRNA.  It i s generally  agreed t h a t 26s RNA i s messenger but 42S RNA  i s also associated  with polysomes.  Simmons and Strauss (43)  s t a t e t h a t 26s RNA (M.W. 1.6 x 10 ) c o n s t i t u t e s of the mRNA i n i n f e c t e d c e l l s , the  s t r u c t u r a l proteins  90% by weight  and i t i s thought t o s p e c i f y  of the v i r u s .  On t h e other hand, 42S  RNA, (M.W. 4.3 x 10^) which i s i d e n t i c a l t o the v i r a l genome,  -12-  c o n s t i t u t e s approximately 5 - 1°$  of the t o t a l mRNA and  thought to code f o r the remaining v i r a l f u n c t i o n s  such as  Mowshowitz (46)  and  Simmons and  of the base sequences i n 3 3 S RNA  that 90$ 26S  Hybridization-competition  converted to 26s a t i o n has  RNA.  A precursor  been suggested f o r 33S  are a l s o present i n  RNA  represents  i s completely  RNA.  of the base sequence i n f o r m a t i o n  Thus 26s  RNA  r o l e or an a l t e r e d conform-  H y b r i d i z a t i o n s t u d i e s have shown that 26s 2/3  of 42S  RNA  consists  (31,  RNA  34,  mechanism by which 26s  42S  RNA  has  been shown to be i d e n t i c a l i n s t r u c t u r e to v i r u s RNA  RNA  47).  i s derived a f t e r i n f e c t i o n with  i s not known to date.  protein  of  a unique f r a c t i o n of the v i r a l genome.  The  through i n f e c t i v i t y ,  RNA  experiemtns showed  A f t e r r e a c t i o n w i t h formaldehyde, 33S  RNA.  (43)  Strauss  have a l s o noted the a s s o c i a t i o n of s m a l l amounts of 3 3 S with polysomes.  RNA  (44).  polymerase a c t i v i t y •Kennedy (45).  is  42S  RNA  i s o l a t e d from polysomes  s e d i m e n t a t i o n behaviour and  (48)  in vitro  synthesis.  Most r e c e n t l y s t u d i e s have been d i r e c t e d towards i s o l a t i o n of these messengers and synthesis  (49  Cancedda and  - 55)-  using  The  Schlesinger  reticulocyte extracts.  them i n c e l l f r e e  first  protein  of such s t u d i e s was  performed  i n Krebs I I a s c i t e s c e l l and T r a n s l a t i o n of Sindbis  26s  rabbit  RNA  resulted  i n the f o r m a t i o n of a p r o t e i n i d e n t i c a l to c a p s i d p r o t e i n shown by t r y p t i c peptide mapping and ^electrophoresis)>., and  Only t r a c e s of E-^ and  l a r g e r molecular weight p r o t e i n s  were a l s o absent.  SDS  by  (as  polyacrylamide E  2  p r o t e i n were obtained  (which could be  precursors)  -13Clegg and Kennedy ( 5 2 , u s i n g 26S  53)  have a l s o performed s t u d i e s  RNA. from c e l l s i n f e c t e d w i t h SFV 1  an e x t r a c t of L c e l l s . peptide formed was  as messenger u s i n g  They a l s o i n d i c a t e t h a t the major  n u c l e o c a p s i d and t h a t 4-2S RNA  functioned  p o o r l y as messenger - probably due  to i t s secondary s t r u c t u r e .  I n t h e i r most r e c e n t s t u d i e s ( 5 3 ) .  however, Clegg and Kennedy  showed t h a t a f t e r l o n g p e r i o d s of s y n t h e s i s ( 1 0 0 . m i n . compared to  30 min.)  from 26s  messenger, E-^ and  Eg c o u l d be found  c e l l f r e e systems i n a n o n - g l y c o s y l a t e d form. no evidence  in  There was,  however,  f o r precursor polypeptides,acohtrary^to- similar•work  p o l i o v i r u s messenger (56  -  59).  A d d i t i o n of TPCK  i n h i b i t o r ) or PMSF (a s e r i n e protease  i n h i b i t o r ) d i d not  p r e c u r s o r p o l y p e p t i d e s i n the c e l l f r e e system. 26s  i s some q u e s t i o n as to how  RNA,  (chymotrypsin yield  Thus, t h e r e  a monocistronic  messenger,  can be t r a n s l a t e d to produce the 3 d i s c r e t e products  in vitro.  A model must a l s o be proposed f o r the s e l e c t i v e f o r m a t i o n nucleocapsid protein.  of  P o s s i b l y t r a n s l a t i o n of n u c l e o c a p s i d  i s most e f f i c i e n t from ribosomes which are not bound to membranes and t r a n s l a t i o n of envelope p r o t e i n s occurs only i n a s s o c i a t i o n with membrane bound ribosomes. ''''  (d) S y n t h e s i s and I n s e r t i o n of V i r a l G l y c o p r o t e i n s the I n t r a c e l l u l a r Membranes of the Host C e l l  into  Iiodish's l a b o r a t o r y has r e c e n t l y demonstrated mode f o r t r a n s l a t i o n of 26S with S i n d b i s v i r u s ( 6 0 ) .  the  RNA. w i t h i n c e l l s which were i n f e c t e d  The 26s  RNA  was  shown to be almost  e x c l u s i v e l y a s s o c i a t e d w i t h 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.  n u c l e o c a p s i d p r o t e i n was  Newly s y n t h e s i z e d  l o c a l i z e d on the cytoplasmic  s i d e of  with  -14-  endoplasmic Sindbis  r e t i c u l u m membranes  membrane  endoplasmic protein are  sequestered  that  of  is  2  and  lumen,  the  nascent.  lumen of  t e r m i n a l sequence  E^) .  The  envelope  s u c h as  i n a process i n s u l i n and  i n t r a c e l l u l a r membrane  shown i n F i g .  the of  B  proteins  become g l y c o s y l a t e d ,  p l a s m a - membrane  proteins  proposed  proteins  E  into the  secretory The  t h e amino  E-^,  and r e a c h the  amylase. viral  to  cleaved while  penetrated into  reticulum via  (precursor  cleaved, to  proteins  and  are similar  pancreatic  association  with  4.  F i g . 4. Model f o r the s y n t h e s i s of S i n d b i s v i r u s p r o t e i n s , subsequent 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 , and s e q u e s t e r i n g o f envelope p r o t e i n s i n t o the lumen of the endoplasmic reticulum.  Some g l y c o s y l a t i o n before  release  volves  transfer  of  the  from the of  polysome,  a large  to  the polypeptide chains  is  thought  to  envelope  be a l i p i d  1800 (61).  (6l).  proteins and t h i s  appears  donor  occur  glycosylation  molecular weight The  to  of  the  in-  oligosaccharide oligosaccharide  -15-  (e) Formation o f V i r u s P r o t e i n s - F o s t - T r a n s l a t i o n a l Cleavage o f Large P r e c u r s o r s (i) Structural Proteins J u s t as the s t r u c t u r a l p r o t e i n s o f p i c o r n a v i r u s e s  appear  to a r i s e from cleavage of a l a r g e p o l y p e p t i d e  (62, 63) so do  those of Group A t o g a v i r u s e s .  phenomenon was  first  This precursor  observed by B u r r e l l , M a r t i n and Cooper ( 6 4 ) i n BHK c e l l s  i n f e c t e d w i t h SFV. Detailed precursor and  s t u d i e s were c a r r i e d out by S c h l e s i n g e r  S c h l e s i n g e r on S i n d b i s v i r u s .  the e x i s t e n c e of a p r e c u r s o r  Their f i r s t  s t u d i e s showed  t o Eg ( P E ) o f molecular  weight  2  68,000 (65) P E was shown to be c l o s e l y r e l a t e d t o E 2  2  by t r y p t i c  p e p t i d e maps. PE (66)  2  was a l s o shown to be g l y c o s y l a t e d .  In a l a t e r  study  a temperature s e n s i t i v e mutant S i n d b i s ts2 which i s d e f e c t -  ive i n nucleocapsid precursor permissive  assembly was used.  (ts2 p r o t e i n ) M.W. temperature.  A l a r g e molecular  weight  130,000 accumulated a t the non-  Two dimensional  t r y p t i c p e p t i d e mapping  showed t h i s l a r g e p r o t e i n t o c o n t a i n E-^, Eg, and n u c l e o c a p s i d . When the i n f e c t e d c e l l s were s h i f t e d to the permissive  tempera-  t u r e r a d i o a c t i v i t y was chased from the l a r g e p r o t e i n t o another p r o t e i n c a l l e d B p r o t e i n (M.W. 97,000) and then i n t o PEg, E-^ Eg and  nucleocapsid.  acrylamide  These p r o t e i n s a r e p o r t r a y e d  g e l shown i n F i g u r e '5 •  on a SDS p o l y -  -IN-  FRACTION NUMBER F i g . 5. An SDS p o l y a c r y l a m i d e g e l showing the r a d i o a c t i v e p o l y p e p t i d e s which are present i n c e l l s i n f e c t e d with ts2 mutants of S i n d b i s and HR (Wild S t r a i n ) S i n d b i s , ( 1 ) .  Jones, Waite, and Bose (67, experiment  performed  a similar  w i t h c e l l s i n f e c t e d w i t h a temperature  mutant of S i n d b i s ( t s 20) non-permissive temperature temperature  68)  sensitive  d e f e c t i v e i n cleavage of PEg a t the (42° C ) .  A s h i f t to the p e r m i s s i v e  saw a decrease i n PEg and i n c r e a s e i n Eg f o r m a t i o n .  P r e l i m i n a r y experiments PM of the host  showed PEg might be a s s o c i a t e d with the  cell.  F u r t h e r evidence f o r p o s t - t r a n s l a t i o n a l cleavage of prec u r s o r p r o t e i n s i n S i n d b i s v i r u s i n f e c t e d c e l l s has been  -17-  p r o v i d e d through use o f TPCK".(tosylphenylalanine c h l o r o methylketone) which i s an i n h i b i t o r of chymotrypsin ( 6 9 ) . l a r g e m o l e c u l a r weight p r o t e i n ( a p p r o x i m a t e l y 9 7 . 0 0 0  A  M.W.)  accumulated i n r a d i o a c t i v e l y l a b e l e d c e l l s i n f e c t e d w i t h S i n d b i s and TPCK. S i m i l a r experiments to those performed on S i n d b i s have been a p p l i e d t o S e m l i k i F o r e s t v i r u s .  Simons, Keranen, and  Kaariainen (70) f i r s t  demonstrated the e x i s t e n c e o f the pre-  c u r s o r to Eg ( E g ) ^  c e l l s i n f e c t e d with a temperature  p  n  t i v e mutant o f S e m l i k i F o r e s t v i r u s  (ts-1)  which was  i n PEg cleavage a t the non-permissive temperature.  sensi-  defective P u l s e chase  s t u d i e s and t r y p t i c p e p t i d e maps i n d i c a t e d t h a t PEg was indeed a p r e c u r s o r t o Eg. F u r t h e r work was r e p o r t e d by Morser and Burke ( 7 1 , NVP 63 ( n o n - v i r i o n p r o t e i n , M.W.  72).  6 3 , 0 0 0 ) corresponded t o PEg  found i n S i n d b i s and NVP 97 ( n o n - v i r i o n p r o t e i n , M.W.  97,000)  to the h i g h m o l e c u l a r weight p r e c u r s o r found by S c h l e s i n g e r and Schlesinger(-65)JU"sing l a b e l i n g s t u d i e s w i t h -^H-glucosamine,  i t was  shown t h a t PEg, E-^ and Eg were the o n l y g l y c o p r o t e i n s i n i n f e c t e d cells.  I n chase s t u d i e s where r a d i o a c t i v e medium was r e p l a c e d  w i t h n o n - r a d i o a c t i v e medium, l a b e l i n the h i g h m o l e c u l a r weight p r o t e i n s appeared t o move i n t o the lower m o l e c u l a r weight, envelope p r o t e i n s .  TPCK (an i n h i b i t o r o f chymotypsin)  addition  was a l s o shown t o r e s u l t i n the accumulation o f l a r g e m o l e c u l a r weight p r o t e i n s .  The same r e s u l t s were o b t a i n e d when amino a c i d  analogues ( t o 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 -  f l u o r o p h e n y l a l a n i n e , canavanine, a z e t i d i n e - 2 - c a r b o x y l i c  acid,  -18-  e t h i o n i n e , and azatryptophan.  Pulse chase s t u d i e s 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 p r o t e i n s y n t h e s i s (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 a c i d ) showed 97 decreased r a p i d l y  t h a t when the i n h i b i t o r ' w a s removed NVP  w i t h a r a p i d i n c r e a s e i n l a b e l i n g of n u c l e o c a p s i d E-j^  and  followed  by  Eg.  Work with temperature s e n s i t i v e mutants of SFV  was  also  performed by Keranen and K a a r i a i n i n (73)-  These workers demon-  strated 5 non-structural proteins i n c e l l s  i n f e c t e d with  mutants - NVP  130,  NVP  97.  86,  NVP  chase experiments suggested NVP were p r e c u r s o r s  78,  NVP  130,  and  97.  NVP  86,  NVP  f o r the s t r u c t u r a l p r o t e i n s .  63.  NVP  NVP  these Pulse-  and  NVP  62  86 and  NVP  78  were not a f f e c t e d by the chase and were thought to be r e p l i c a s e or RNA  polymerase.  In a r e c e n t paper (7^) p e p t i d e t r a n s l a t i o n i n SFV mRNA was  has  demonstrated the order  i n f e c t e d BHK  cells.  of NaCl.  R e s t o r i n g the medium to  apparent i n c a p s i d p r o t e i n a f t e r 2 min  i s o t o n i c i t y while r a d i o a c t i v e precursors not  occur u n t i l a f t e r 5 - 6  min.  of l a b e l i n g to be c a p s i d , NVP Based on the preceding  97.  L  75) •  Radioof r e s t o r i n g  or envelope p r o t e i n s d i d  F u r t h e r work showed the NVP  evidence,  63,  E-^  order  Eg-E^.  the f o l l o w i n g model f o r  p o s t - t r a n s l a t i o n a l cleavage of p r e c u r s o r -proposed ( 7 >  elevated  isotonicity  synchronous i n i t i a t i o n of p r o t e i n s y n t h e s i s .  a c t i v i t y was  of  T r a n s l a t i o n of  i n i t i a l l y r e p r e s s e d w i t h medium c o n t a i n i n g an  concentration induced  Clegg  polypeptides  has  been  /s2  protein or NVP 130  NH  -COOH 130,000  i B protein or NVP 98 30,000  98,000  P E 2 o r N V P 62 54,000  48,000  t  F i g . 6. SF v i r u s  E2  5600  48,000  P o s t - t r a n s l a t i o n a l cleavage structural proteins.  remains apparently ts2  E3  associated lost  with Semliki  Forest  from Sindbis v i r u s .  protein ) to capsid of B protein  ( N V P 97)  probably  occurs  the peptide i s  while  the ribosomes.  quires final  at least steps  30  (ii) A great  (42,  76-78).  Cleavage  (which i s  min, and appears maturation  d e a l o f work  (p)  virus,  not  s t i l l  (1,  of  is (or  r a p i d and  glycosylated)  nascently is  attached  slow,  t o be a s s o c i a t e d  re-  with the  67).  Proteins  has accumulated r e c e n t l y which  of non-structural proteins  in cells  but  o f NVP 127  o f P E g t o E g a n d Ej  Non-Structural  w i t h the appearance precursors  Cleavage  of virus  the formation  p r o t e i n and B p r o t e i n i s  cleavage  to  during  infected with Semliki  Non-structural proteins  (nsp) Forest  are produced  in  deals  and t h e i r virus small  -20-  amounts e a r l y i n i n f e c t i o n . as yet.  Lachmi and  t e i n s and  These s t u d i e s are f a r from complete  Kaarianen (76)  2 precursor  polypeptides  found 4 n o n - s t r u c t u r a l  pro-  i n c e l l s i n f e c t e d with  a  temperature s e n s i t i v e mutant of S e m l i k i F o r e s t v i r u s .  A scheme  which they proposed f o r the p o s t - t r a n s l a t i o n a l cleavage of precursors  i s presented  i n F i g . "?•  p 155,000  p 135,000  1 . nsp .70,000  the  i nsp  86,000  nsp 7 8 , 0 0 0  nsp  60,000  Fig.- ',7. Scheme f o r the p o s t - t r a n s l a t i o n a l p r o c e s s i n g of p r e c u r s o r s (p) to the n o n - s t r u c t u r a l p r o t e i n s (nsp) of a tempe r a t u r e s e n s i t i v e mutant ( t s - 1 ) of S e m l i k i F o r e s t v i r u s . More complete s t u d i e s have been performed by Clegg, Kennedy (42)  on both S e m l i k i F o r e s t v i r u s and  Brzeski,  Sindbis v i r u s  and (77)•  These i n v o l v e d r a d i o a c t i v e pulse-chase experiments which were performed e a r l y i n i n f e c t i o n , synchronous t r a n s l a t i o n to termine gene order and  tryptic  peptide  a n a l y s i s of the gene  products t o e l u c i d a t e the a s s o c i a t i o n between the p r o t e i n s and  t h e i r precursors.  f o r S e m l i k i F o r e s t v i r u s and Figures  8 and  9 respectively.  The  de-  non-structural  cleavage p a t t e r n s  determined  S i n d b i s v i r u s are i l l u s t r a t e d i n  -21-  p  200,000  I p  184,000  P  150,000  F i g . 8. Formation of n o n - s t r u c t u r a l p r o t e i n s (nsp) by p o s t t r a n s l a t i o n a l cleavage o f p r e c u r s o r s (p) i n c e l l s which were i n f e c t e d with S e m l i k i F o r e s t v i r u s ( 4 2 ) .  230,000  p 150,000,,..  - - - - -  _ p 60,000  p 7.6,000  nsp 8 9 , 0 0 0  p  •  215.000  —  nsp 8 2 , 0 0 0  j nsp  80,000  —  p  76,000  •••• nsp  v  82,000  F i g . 9. Formation of n o n - s t r u c t u r a l p r o t e i n s (nsp) by postt r a n s l a t i o n a l cleavage o f p r e c u r s o r s (p) i n c e l l s which were i n f e c t e d by S i n d b i s v i r u s . * denotes a c o n f o r m a t i o n a l change i n the p r e c u r s o r protein-; ( 7 7 ) . The  n o n - s t r u c t u r a l p r o t e i n s probably  synthesis.  serve i n  " RNA  Indeed nsp 6 3 , 0 0 0 and nsp 9 0 , 0 0 0 have a l r e a d y been  shown to be components o f the v i r u s s p e c i f i e d RNA polymerase i n c e l l s which were i n f e c t e d with Functions  Semliki Forest v i r u s (41).  f o r the other p r o t e i n s have y e t to be assigned.  -22-  3.  M a t u r a t i o n and Membrane Assembly o f Togaviruses (a)  E l e c t r o n m i c r o s c o p i c Observations o f Togavirus Assembly  E l e c t r o n m i c r o s c o p y d i s c l o s e s extensive changes i n the cytoplasm o f i n f e c t e d c e l l s . budding  V i r i o n s appear t o be produced by  o f n u c l e o c a p s i d s through areas of c e l l membranes  m o d i f i e d by the i n s e r t i o n o f v i r u s envelope T h i s appears  proteins.  to occur a t 2 p l a c e s :  1)  the plasma membrane (16, 79, 80)  2)  i n t r a c e l l u l a r v a c u o l e s (Type 2 c y t o p a t h i c vacuoles) ( 3 5 , 81)  Nucleocapsids were seen c l o s e l y a l i g n e d t o the cytoplasmic s i d e of the membrane, and,often i n the process o f budding.  Cyto-  p a t h i c v a c u o l e s (CPV-2) which presumably c o n t a i n mature v i r u s have been thought to f u s e with the host c e l l of v i r u s i n t o the medium.  PM with the r e l e a s e  Freeze e t c h i n g and e l e c t r o n m i c r o s c o p y  s t u d i e s 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 t h a t envelope p r o t e i n s were i n s e r t e d i n t o l o c a l i z e d r e g i o n s o f the plasma membrane (82, 8 3 ) . E l e c t r o n m i c r o s c o p y of c e l l s i n f e c t e d with  temperature  s e n s i t i v e mutants of S i n d b i s v i r u s a l s o seemed to i n d i c a t e that the plasma membrane i s the f i n a l s i t e of v i r u s maturation (1, 84, 8 5 ) .  C e l l s which were i n f e c t e d with t s - 2 0 mutant c o n t a i n  s t r i k i n g a r r a y s o f n u c l e o c a p s i d j u s t beneath at the non-permissive demonstrated  temperature  the plasma membrane  and the c e l l membrane was  to c o n t a i n envelope p r o t e i n s (1, 8 5 ) .  The t s - 2 3  mutant o f S i n d b i s d i d not i n s e r t envelope p r o t e i n s i n t o the plasma membrane a t the non-permissive  temperature,  nucleocapsid d i d  -23-  not a s s o c i a t e with the outer c e l l membrane, and mature v i r u s was  not formed ( 1 , Grimley  vacuole SFV  84,  85).  et a l . ( 3 5 ,  36)  d e s c r i b e d another  type of c y t o p a t h i c  (CPV-1) which occurs e a r l y i n the e x p o n e n t i a l phase of  growth.  The v a c u o l e s were 0 . 6 - 2  um  i n diameter  and bore  50 nm membranous nodules which p r o j e c t from the i n t e r i o r s u r faces.  The nodules  Formation  were n e i t h e r v i r i o n s nor n u c l e o c a p s i d s .  of the nodules  i s independent of c e l l u l a r RNA  s i n c e a c t i n o m y c i n D does not a f f e c t t h e i r appearance. b o l i t e s which b l o c k v i r a l RNA f o r m a t i o n of C P V - 1 ' s . the presence  Antimeta-  or p r o t e i n s y n t h e s i s prevented  Autoradiographic  RNA  the  experiments performed i n  of actinomycin D seem to i n d i c a t e t h a t the  i s a s i t e of v i r a l  synthesis  CPV-1  synthesis.  C e l l s i n f e c t e d w i t h group B t o g a v i r u s e s show somewhat d i f f e r e n t c y t o p a t h i c changes (86  - 88).  Initially  there was  a  p r o l i f e r a t i o n of cytoplasmic v a c u o l e s , of smooth and rough endoplasmic r e t i c u l u m , and G o l g i membranes. little  However, there  was  c o n c r e t e evidence as to the mechanism f o r the envelopment  of group B v i r u s e s .  Ota ( 8 6 )  suggested  that v i r a l  occurred by the budding of these p a r t i c l e s through membranes.  morphogenesis cytoplasmic  L i n i n g the cytoplasmic s i d e of many of the v a c u o l e s  and c i s t e r n a e were i l l - d e f i n e d ,  electron-dense, round  s t r u c t u r e s 2 6 - 2 8 nm  which c o u l d be n u c l e o c a p s i d s .  In diameter  No v i r i o n s have ever been observed Yasuzumi et"-al.  (89-91,  49)  i n the process of budding.  have suggested  i n the morphogenesis of group B v i r i o n s . a l s o i n d i c a t e d the presence  ragged  nuclear  involvement  Murphy et a l . (88)  of i n t r a n u c l e a r v i r i o n s and  nucleo-  -24-  capsids.  Membrane-enclosed v i r i o n s may  p e r i n u c l e a r r e g i o n of the c e l l and  migrate from the  approach the plasma membrane  to be r e l e a s e d by e x o c y t o s i s through narrow c a n i c u l i or through f u s i o n of v i r u s - c o n t a i n i n g vacuoles  with the plasma membrane.  Immunofluorescence s t u d i e s showed t h a t group B antigens present  i n the cytoplasm  of i n f e c t e d c e l l s  (92-94)  were  and  various  workers contended t h a t the absence of immunofluorescence a t the plasma membrane of i n f e c t e d c e l l s . meant ' . t h a t v i r a l d i d not take p l a c e at the c e l l Evidence confusing.  maturation  surface.  obtained- through e l e c t r o n m i c r o s c o p y  I n t r a c e l l u l a r vacuoles  i s somewhat  c o u l d w e l l be an a r t i f a c t  of  sample p r e p a r a t i o n - perhaps they were c r o s s - s e c t i o n s of s u r f a c e i n v a g i n a t i o n s of the c e l l . the' maturation  A l s o the dramatic  d i f f e r e n c e between  of Group A and B t o g a v i r u s e s i s somewhat  The r o l e of i n t r a c e l l u l a r membranes i n v i r u s maturation  suspect. also  requires further c l a r i f i c a t i o n . (b)  Chemical Evidence f o r M a t u r a t i o n at the Plasma Membrane  Chemical evidence was  the source  of v i r a l  of Group A  Togaviruses  which i n d i c a t e d t h a t the plasma membrane envelope o r i g i n a l l y came from the  a n a l y s i s of S e m l i k i F o r e s t (21, 95)  and S i n d b i s ( 9 6 ,  v i r u s e s which were grown in- v a r i o u s h o s t s .  97,  i n common.  98)  Semliki Forest viruses  which were grown i n BHK-21 and Aedes a l b o p i c t u s c e l l s had of t h e i r p h o s p h o l i p i d s  lipid  In each case,  only 36%  the l i p i d com-  p o s i t i o n of the v i r u s resembled t h a t of the host c e l l membrane. More d i r e c t chemical present  evidence  t h a t v i r a l p r o t e i n s were  i n the plasma membrane of the host c e l l came from e x p e r i -  ments i n which l a c t o p e r o x i d a s e was  u t i l i z e d to l a b e l c e l l  surface  -25-  proteins with  (99).  Amazingly, S i n d b i s membrane p r o t e i n s  were l a b e l e d p r e f e r e n t i a l l y over those o f the host c e l l .  Also,  i n f e c t i o n w i t h S i n d b i s v i r u s was demonstrated to i n c r e a s e d r a s t i c a l l y the c o n c a n a v a l i n A a g g l u t i n a b i l i t y of the host the change i n s u r f a c e sugar composition appeared presence  to i n d i c a t e the  o f v i r a l g l y c o p r o t e i n s i n the plasma membrane.  Even more c o n c l u s i v e evidence f o r budding at  cells;  of togaviruses  the plasma membrane was presented by Jones et a l . ( 6 7 ) .  This  group has demonstrated t h a t E-^, Eg, n u c l e o c a p s i d p r o t e i n , and PEg were present i n plasma membrane f r a c t i o n s p u r i f i e d i n f e c t e d host c e l l s .  PEg cleavage appeared  to be r e l e a s e d i n t o the e x t r a c e l l u l a r  from  necessary f o r v i r u s  fluid.  Evidence i s presented i n t h i s t h e s i s which c l e a r l y demons t r a t e s t h a t Group A- t o g a v i r u s e s "bud" e x c l u s i v e l y through the plasma membrane of the host c e l l . 4.  Assembly o f V e s i c u l a r S t o m a t i t i s V i r u s (A Rhabdovirus) (a)  Structure  V e s i c u l a r s t o m a t i t i s v i r u s c o n s i s t s of 5 p r o t e i n s ( 1 0 0 ) G (molecular weight 6 9 , 0 0 0 ) ,  M (molecular weight  29,000),  N (molecular weight 5 0 , 0 0 0 ) ,  L (molecular weight  190,000),  and NS (molecular weight 4 0 , 0 0 0 - 4 5 , 0 0 0 ) .  The d i s t r i b u t i o n  and f u n c t i o n of these p r o t e i n s a r e shown i n F i g . . 1 0 .  -26-  P r o t e i n Molecular Wt. F u n c t i o n G  69,999  envelope p r o t e i n , virus-cell interaction.,  M  29,000  membrane m a t r i x p r o t e i n , organizes G proteins into patches  N  50,000  L  190,000  NS  F i g . 10,.  40,000 50,000  'nucleocapsid p r o t e i n which is tightly associated with RNA genome; involved i n transcription and r e p l i c a t i o n a s s o c i a t e d with n u c l e o c a p s i d and may be c a t a l y t i c sub-unit o f RNA polymerase associated with n u c l e o c a p s i d and i s required f o r r e p l i c a t i o n and transcription  J  S t r u c t u r e and composition o f v e 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 c o n s i s t s o f one p i e c e o f s i n g l e stranded RNA of molecular weight 3 - 5 x 10  daltons.  V e s i c u l a r s t o m a t i t i s v i r u s i s a n e g a t i v e s t r a n d v i r u s and i t c o n t a i n s i t s own RNA-dependent RNA polymerase. (b)  Membrane Assembly of V e s i c u l a r S t o m a t i t i s V i r u s  Rhabdoviruses  are r e p o r t e d to a c q u i r e t h e i r  envelopes  from the plasma membrane o r i n some cases the membranes o f i n t r a c y t o p l a s m i c v e s i c l e s o f the host c e l l  (100).  This e v i -  dence i s based upon the e l e c t r o n m i c r o s c o p y of Zee et a l . ( 1 0 1 ) . The l i p i d o f the v i r a l membrane appears  to mimic t h a t of the  -27-  h o s t c e l l plasma membrane ( 1 0 2 ) .  However, T i f f a n y and Blough  have r e c e n t l y p u b l i s h e d evidence which c o n t r a d i c t s t h i s (103).  fact  They found the l i p i d o f the v i r u s to be o f a c h a r a c t e r  i n t e r m e d i a t e 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 t h a t v e s i c u l a r stomat i t i s v i r u s p r o t e i n s were a s s o c i a t e d with the plasma membrane of i n f e c t e d HeLa c e l l s .  I n a pulse-chase experiment  with  v e s i c u l a r s t o m a t i t i s c e l l s , David ( 1 0 5 ) demonstrated  that  after  a 3 ° second p u l s e , G and M p r o t e i n s were a l r e a d y a s s o c i a t e d w i t h the plasma membrane, but maximal l e v e l s o f i n c o r p o r a t i o n were not reached u n t i l 2 min chase.  N and NS p o l y p e p t i d e s were found  only i n the s o l u b l e cytoplasm.  At the end of a 5 min p u l s e  p e r i o d the plasma membrane c o n t a i n e d s u b s t a n t i a l amounts of M and G p r o t e i n s , but no n u c l e o c a p s i d p r o t e i n .  With chase  times  approaching 60 min, the amount of n u c l e o c a p s i d p r o t e i n found i n the plasma membrane i n c r e a s e d , although a f t e r about 20 min the amount of envelope p r o t e i n s a t t a c h e d t o the plasma membrane remained approximately the same. Knipe, B a l t i m o r e and L o d i s h ( 1 0 7 ) r e c e n t l y performed a more e l e g a n t pulse-chase experiment  w i t h Chinese hamster ovary  c e l l s which were i n f e c t e d with v e s i c u l a r s t o m a t i t i s v i r u s . I n f e c t e d c e l l s were p u l s e d f o r 2 . 5 min w i t h l a b e l was chased over 90 min. rough 20,  endoplasmic  [ S]-Met and the  Plasma membrane, smooth and  r e t i c u l u m f r a c t i o n s were h a r v e s t e d a t 0 , 1 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 f r a c t i o n s were q u a n t i t a t e d by auto-  -23-  radiography.  These workers  a c t i v i t y from the c e l l reticulum  succeeded i n f o l l o w i n g  s u p e r n a t a n t and rough  to the smooth endoplasmic r e t i c u l u m  plasma membrane.  the r a d i o -  endoplasmic and then t h e  F i n a l l y l a b e l was chased i n t o t h e e x t r a -  c e l l u l a r v i r u s ( F i g . 11).  MINUTES  OF CHASE  '  F i g . 11. K i n e t i c s i l l u s t r a t i n g the a s s o c i a t i o n o f v e s i c u l a r s t o m a t i t i s v i r u s w i t h c e l l s u p e r n a t a n t and membrane f r a c t i o n s d u r i n g a p u l s e - c h a s e experiment. F r a c t i o n s 1 and 2 c o r r e s pond to rough endoplasmic r e t i c u l u m , 3 d 4 t o smooth endoplasmic r e t i c u l u m , ' 5 and 6 to plasma membrane, and S t o t h e 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 . a  n  -29-  Knipe, B a l t i m o r e and  L o d i s h ('109') have a l s o  utilized  temperature s e n s i t i v e mutants of v e s i c u l a r s t o m a t i t i s to i n v e s t i g a t e the mechanisms of v i r u s assembly. involved  surface  i o d i n a t i o n and  formed on several: w e l l d e f i n e d temperatures.  become a s s o c i a t e d  c e l l f r a c t i o n a t i o n were permutants at t h e i r non-permissive  d i d not  with the  a l l o w e i t h e r M or N p r o t e i n  plasma membrane, and  f o r m a t i o n of e x t r a c e l l u l a r v i r u s .  with G  i n s e r t e d i n t o the c e l l membrane.  p r o v i d e d a model f o r assembly which showed the of G,  M and  blocked  N proteins  i n "budding"  to the  Mutations i n e i t h e r M or N  prevented a s s o c i a t i o n of both these p r o t e i n s still  Studies which  Mutations i n G p r o t e i n prevented I t s m i g r a t i o n  to the c e l l s u r f a c e ,  which was  virus  protein  These workers  interdependence  (Fig./12.).  Plasma Membrane  Fig..12. the c e l l  M a t u r a t i o n of v e s i c u l a r s t o m a t i t i s v i r u s (109).  within  -30-  5.  Assembly of'Myxoviruses and Paramyxoviruses (a)  S t r u c t u r e of Myxoviruses and Paramyxoviruses  Myxoviruses and paramyxoviruses have-the s t r u c t u r e s shown i n F i g u r e s 13 and I r r e s p e c t i v e l y .  Both types o f v i r u s  con-  t a i n n e g a t i v e s t r a n d genomes and c o n t a i n t h e i r own transcriptase.  The genome o f myxoviruses c o n s i s t s of 8  separate segments o f s i n g l e - s t r a n d e d RNA while t h a t of paramyxoviruses i s a l a r g e s i n g l e segment o f s i n g l e stranded RNA.  Hemagglutinin-,  1  Neuraminidase  ,  Lipid membrane i Matrix p r o t e i n Nucleoprotein, P proteins RNA  F i g . 13.  S t r u c t u r e and p r o t e i n composition of myxoviruses. MP 'NA  JIII'  F i g . 14.  S t r u c t u r e and p r o t e i n composition of paramyxoviruses.  -31-  (t>)  Membrane Assembly  o f Myxoviruses and Paramyxoviruses  E l e c t r o n m i c r o s c o p i c s t u d i e s a l s o i n d i c a t e t h a t myxoviruses and paramyxoviruses bud from the plasma membrane.  This was  shown i n 1 9 5 5 by Hotz and Schafer ( 1 1 0 ) f o r i n f l u e n z a  first  and l a t e r f o r paramyxoviruses by Choppin  (111).  N u c l e o c a p s i d a l i g n s w i t h s p e c i f i c patches o f the i n n e r PM p r i o r t o budding; these patches may be i d e n t i f i e d w i t h f e r r i t i n l a b e l e d a n t i v i r a l antibody. Klenk and Choppin ( 1 1 2 ,  113)  first  showed t h a t SV5 envelope  l i p i d s resembled those of the host c e l l PM.  T h i s was f u r t h e r  s u b s t a n t i a t e d by comparison o f the g l y c o l i p i d s from v i r u s grown i n MK and BK host c e l l s (114). The concept t h a t envelope components migrate from the c e l l i n t e r i o r t o the s u r f a c e has been confirmed by c e l l  fraction-  a t i o n and a n a l y s i s o f the v a r i o u s p r o t e i n s 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 t h a t HA and pos-  s i b l y t h e other envelope p r o t e i n s a r e s y n t h e s i z e d on the rough ER ( 1 1 5 » 1 1 6 ) .  P u l s e chase experiments show t h a t a few minutes  l a t e r HA i s present i n the membranes o f the smooth ER ( 1 1 5 , and then the PM ( 1 1 7 ) .  116)  V i r u s p r o t e i n s a r e always membrane  a s s o c i a t e d d u r i n g t h i s m i g r a t i o n and a r e never d e t e c t e d i n the soluble fraction.  Hay ( 1 1 8 ) r e p o r t e d s i m i l a r r e s u l t s f o r f o w l  plague v i r u s but found the M p r o t e i n was s y n t h e s i z e d c l o s e t o the PM and a l i g n s next t o the NA and HA as the f i n a l assembly.  stage i n v i r u s  As y e t no experiments have been performed which have  chased the myxovirus p r o t e i n from the PM t o the e x t r a c e l l u l a r virus.  -32-  ( 1 1 9 ) h a s presented pulse-chase  Klehk's l a b o r a t o r y s t u d i e s which concern  the membrane assembly of Newcastle  Disease v i r u s ( a paramyxovirus).  I n f e c t e d c e l l s were p u l s e -  l a b e l e d f o r 10 min and chased over 60 min.  The c e l l s were  f r a c t i o n a t e d i n t o plasma membrane ghosts and smooth and rough endoplasmic r e t i c u l u m a t 0 , 20 and 60 min chase. p r o t e i n s HN and P  0  were s y n t h e s i z e d  The g l y c o -  on the rough endoplasmic  r e t i c u l u m and t r a n s f e r r e d from there v i a smooth i n t r a c e l l u l a r membranes t o the plasma membrane and i n t o v i r i o n s . course o f m i g r a t i o n ,  F  Q  i s c o n v e r t e d t o F.  I n the  M p r o t e i n was  found to r e a c h the plasma membrane more r a p i d l y than the labeled glycoproteins.  The c h i e f c r i t i c i s m of t h i s paper i s  t h a t r a d i o a c t i v e l y l a b e l e d p r o t e i n s were not chased over periods  into extracellular virus.  ment ( 1 2 0 ) performed w i t h v i r a l  longer  Another pulse-chase e x p e r i -  p r o t e i n s from a t o t a l  cell  homogenate, demonstrated t h a t i t was p o s s i b l e t o chase M prot e i n from the c e l l  over 8 hours but t h a t n u c l e o c a p s i d  l a t e d i n excess w i t h i n the c e l l .  accumu-  V e r y low i n c o r p o r a t i o n of  l a b e l i n t o F , F and HN p r o t e i n prevented a d e c i s i v e chase of these p r o t e i n s from the c e l l . B.  C y t o s k e l e t a l Components of Membrane Systems and T h e i r Involvement i n the Membrane Assembly of V i r u s e s (I30-I37) The c y t o s k e l e t a l system c o n s i s t s of a v a s t network o f  m i c r o t u b u l e s and m i c r o f i l a m e n t s  dispersed  throughout the c e l l .  These s t r u c t u r e s have been demonstrated t o be i n t r i n s i c a l l y a s s o c i a t e d w i t h c e l l membranes ( 1 3 1 , 1 3 6 , 1 3 7 ) -  Translational  m o b i l i t y o f c e r t a i n c l a s s e s of i n t e g r a l membrane p r o t e i n s  -33-  appears t o be r e g u l a t e d by these cytoplasmic  elements -  membrane p r o t e i n s may be l i n k e d i n some way t o m i c r o t u b l e s and microfilaments.  N i c o l s o n (136,  as transmembrane c y t o s k e l e t a l 1.  137)  has termed t h i s  concept  control.  Miiareifubules Microtubules  a r e l a r g e , c y l i n d r i c a l s t r u c t u r e s o f an outer  diameter  o f 24 +•2 nm and a hollow core approximately  diameter  (130  - 135)'  15 nm i n  Each m i c r o t u b l e i s composed of 13 p r o t o -  f i l a m e n t s (each 5 nm i n diameter) which r u n the l e n g t h o f the tubule.  The p r o t o f i l a m e n t i s i n t u r n composed of a g l o b u l a r  p r o t e i n named t u b u l i n (molecular weight 103,000) which i n i t s e l f c o n s i s t s o f 2 subunits - (molecular weight 53»°0°) and (molecular weight 56,000).  The b a s i c composition and the r e l a t i o n of the  v a r i o u s elements which make up the m i c r o t u b l e a r e i l l u s t r a t e d i n F i g . 15. 51A  C L E F T R E G I O N  F i g . 15.  The b a s i c s t r u c t u r e o f m i c r o t u b u l e s .  -34-  S e v e r a l h i g h molecular weight p r o t e i n s a l s o c o - p u r i f y with b r a i n t u b u l i n ( 1 3 5 , 1 3 8 , 1 3 9 ) .  These p r o t e i n s are  c a l l e d m i c r o t u b l e a s s o c i a t e d p r o t e i n s (MAPS) and may r e g u l a t e assembly o f t u b u l i n dimers ( t h i s be present as side-arms,  p r o t e i n has been termed t a u ) ,  or possess ATPase and p r o t e i n kinase  activity. M i c r o t u b l e s have c l a s s i c a l l y been shown t o be a s s o c i a t e d with the s p i n d l e apparatus flagella  ( f o r locomotion).  ( f o r chromosome movement) and However, r e s e a r c h over the l a s t 10  years has proven t h a t these o r g a n e l l e s have much more d i v e r s e f u n c t i o n s than p r e v i o u s l y r e a l i z e d . to be maintained by m i c r o t u b u l e s .  C e l l shape has been shown Numerous s t u d i e s demon-  s t r a t e d t h a t d i s r u p t i o n of c y t o p l a s m i c microtubules w i t h  either  c o l d , h y d r o s t a t i c p r e s s u r e , or drugs, r e s u l t s i n a r e v e r s i b l e l o s s o f normal c e l l shape.  These s t r u c t u r e s a r e r e s p o n s i b l e  for  the f o r m a t i o n of pseudopodia,  ing  of c e l l s .  nerve axons, and the spread-  The extent t o which m i c r o t u b l e s permeate the  c e l l can be seen i n s t u d i e s where c e l l u l a r m i c r o t u b l e s are l a b e l e d w i t h f l u o r e s c e n t a n t i b o d i e s (140) t o t u b u l i n ( F i g . 15) and by h i g h - v o l t a g e e l e c t r o n microscopy  (141).  These  photo-  graphs were p u b l i s h e d by the l a b o r a t o r i e s of Weber and P o r t e r , r e s p e c t i v e l y (140, 141) .  -35-  F i g . 16.  F i b r o b l a s t s l a b e l e d with f l u o r e s c e n t  a n t i - t u b u l i n (140).  Much work i n d i c a t e s t h a t m i c r o t u b u l e s a r e a s s o c i a t e d membranes. binding  S t a d l e r and Franke  properties  able binding  Both  membrane f r a c t i o n s demonstrated  to the drug.  b r a i n homogenate c o n t a i n (143 - 146).  (142) measured the c o l c h i c i n e -  o f r a t and mouse l i v e r membranes.  n u c l e a r and microsomal  with  appreci-  The f a c t t h a t the synaptosomes o f t u b u l i n i s thoroughly  documented  More r e c e n t l y Bhattacharyya and Wolf  demonstrated  membrane-bound t u b u l i n i n b r a i n and t h y r o i d t i s s u e (147).  In  most cases the c r i t e r i a f o r the presence o f membrane-associated t u b u l i n a r e c o l c h i c i n e or podophylotoxin b i n d i n g , m o b i l i t y o f membrane conjugated a n t i b o d i e s tubulin associated  proteins,  electrophoretic  e l e c t r o n m i c r o s c o p y with  ferritin  t o t u b u l i n , and antibody t i t r a t i o n s .  w i t h plasma membrane may c o n t a i n  The  carbohydrate  s i n c e t u b u l i n ( s o l u b i l i z e d from membranes) s t a i n s with p e r i o d i c a c i d S c h i f f base reagent  (148,149).  -36-  2.  Microfilaments Much l e s s i s known o f the r o l e f o r m i c r o f i l a m e n t s i n  non-muscle c e l l s .  They a r e r e s p o n s i b l e f o r many types o f c e l l  movement and have been i m p l i c a t e d i n such f u n c t i o n s as c y t o plasmic  streaming, c y t o k i n e s i s , s e c r e t i o n , blood  phagocytosis, (I36,  clot retraction,  and t h e m o b i l i t y o f v a r i o u s c e l l s u r f a c e  137, 150-152).  Microfilaments  proteins  have been shown to c o n s i s t  of t h i n f i l a m e n t s o f 6 to 8 nm diameter (composed o f a c t i n ) and t h i c k f i l a m e n t s o f 10 nm diameter (composed of myosin) which are comparable t o the f i l a m e n t s of muscle c e l l s .  The f i l a m e n t s  of non-muscle c e l l s a r e not n e a r l y as w e l l c h a r a c t e r i z e d as those of muscle systems.  I n muscle c e l l s ,  t r o p o n i n , v i a tropomyosin,  r e g u l a t e s the a b i l i t y of the a c t i n s along tropomyosin t o i n t e r a c t with myosin.  the l e n g t h of one  A c t i n and myosin have been  found i n every type o f non-muscle c e l l i n which they have been sought ( 1 5 0 - 1 5 2 ) .  The e x i s t e n c e  o f tropomyosin and t r o p o n i n  i n non-muscle c e l l s has not been e s t a b l i s h e d  definitively.  However, p r o t e i n s s i m i l a r t o tropomyosin have been i s o l a t e d and c h a r a c t e r i z e d from s e v e r a l non-muscle v e r t e b r a t e t i s s u e s eg. p l a t e l e t s ( 1 5 3 ) ,  b r a i n neurons ( 1 5 4 ) ,  b l o o d and pancreas ( 1 5 6 ) ,  b r a i n synaptosomes  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 f l u o r e s c e n t l y l a b e l e d a n t i bodies t o v i s u a l i z e a c t i n and myosin w i t h i n 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 s t r u c t u r e s throughout the cytoplasm, and  plasma membrane i s e x t e n s i v e .  A c t i n , myosin, a p r o t e i n  c o f a c t o r and another high-molecular weight p r o t e i n  (155).  -37-  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 r a b b i t macrophages  (164)  and Acanthamoeba ( 1 6 2 ) .  alveolar  A c t i n and the h i g h -  molecular weight b i n d i n g p r o t e i n can aggregate to form a g e l ; a d d i t i o n of myosin and ATP causes c o n t r a c t i o n which i s a c c e l erated by the c o f a c t o r . In non-muscle c e l l s ,  a c t i n and myosin f i l a m e n t s  fre-  q u e n t l y assemble .into bundles which appear to be a s s o c i a t e d with the plasma membrane ( 1 6 4 ) . (163)  and f i b r o b l a s t s ( 1 6 0 )  gated m i c r o f i l a m e n t s .  The acrosomal process of sperm  a r e p a r t i c u l a r l y r i c h i n aggre-  A c t i n and myosin bundles are p a r t i c -  u l a r l y p r e v a l e n t 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 r e l e a s e s most of the c e l l p r o t e i n s but l e a v e s a c t i n a t t a c h e d 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 c o n t r a c t on the a d d i t i o n of luM C a  3.  [Microtubule' - D i s r u p t o r s  + +  .  A number of drugs have been shown s p e c i f i c a l l y to i n t e r a c t with .m'icrotubules( 1 3 1 ,  I65-I67) •  These i n c l u d e  colchicine,  v i n b l a s t i n e , v i n c r i s t i n e , p o d o p h y l l o t o x i n , g r i s e o f u l v i n and iNox-odazolej the chemical s t r u c t u r e s of these compounds are shown i n F i g . 17•  H  N ocodazole F i g . 17.  " S t r u c t u r e s of drugs which  Colchicine and  has  i s the  irisru'pt-mrcrotubul-es . J  p r o t o t y p e of m i c r o t u b u l e  been shown to b i n d t i g h t l y to the  i n g i s dependent upon pH s a l t concentration;  binding reaction strongly  constituted  of  can  attraction  Maximum c o l c h i c i n e uptake by p u r i f i e d  1 hour. i s that  Another f e a t u r e the  of the  colchicine  However, once the  a t 37' C. - i t i s t h e n s t a b l e a t 0'°C. 0  G  colchicine  r a t e of complex f o r m a t i o n i s  d e t e c t e d at 0i9c.  almost i r r e v e r s i b l y at 37 C reacts  NaCl) the  temperature dependent - almost no  a c t i v i t y can be  Bind-  independent  s i n c e c o l c h i c i n e - t u b u l i n complexes  electrostatic.  t u b u l i n occurs by  t u b u l i n dimer..  (optimum i s pH7.0) but  occur over a wide s a l t range ( 5 - 5 0 ° mM i s not  disruptors  and  complex i s  Colchicine  binds  o n l y 1 molecule of t h i s drug  w i t h 1 molecule of t u b u l i n .  c o l c h i c i n e b i n d i n g s i t e i s one  binding  I t i s thought t h a t  of the  protein-protein  the interaction  s i t e s between t u b u l i n molecules; attachment of c o l c h i c i n e  to  -39-  this  site  ability  prevents  normal  of colchicine  dependent  assembly  to disrupt  For this  microtubules  (often taking  the drug  slowly with  thus  an absolute  cell  types  different  one's  disruption than  tubulin turnover  (168).  side  colchicine  of  polymer-  disrupts pre-incubation  The s e n s i t i v i t y and i s  is  to the experiment  thought  and b i n d i n g  169).  (168,  the  microtubles  of turnover  system p r i o r  rates  Thus,  up t o 4 hours);  t o c o l c h i c i n e does v a r y  of  various  t o be due t o  constants  Colchicine  is  of  does have  an  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  of microtubules  or equal  (170).  cell  reason  requirement  tubulin within the c e l l important  assembled  on t h e s t a b i l i t y and r a t e  ized microtubules.  of  (Fig. 14).  - at high concentrations  t o 250 uM) n u c l e o s i d e  Lumicolchicine  with  ultraviolet light  ring  C t o two s m a l l e r  is  transport  is  inhibited  formed by i r r a d i a t i o n of  (165) rings  which r e s u l t s  (greater  colchicine  i n the conversion  of  ( F i g . 18 ). , 1 (l)Colchicine  .NH-C-CH,  \( 5 ) L u m i c o l c h i c i n d  colchicine  CH^O  P ond y - l u m i c o l c h i c i n e s 250  300  350  WAVE LENGTH  F i g . 18. 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 and i t s a b s o r p t i o n spectrum as compared t o t h a t o f c o l c h i c i n e .  400  -40-  Lumicolchicine derivatives binding  agents but  are  i n a c t i v e as m i c r o t u b u l e -  possess c o l c h i c i n e ' s s i d e e f f e c t s - i t i s  j u s t as potent i n i n h i b i t i n g n u c l e o s i d e Vinca alkaloids  ( v i n b l a s t i n e and  transport.  vincristine) interact  with t u b u l i n to cause d i s s o l u t i o n of m i c r o t u b u l e s and f o r m a t i o n of h i g h l y r e g u l a r  crystals.  One  mole of  i s bound per mole of t u b u l i n w i t h the r e l e a s e moles of GTP izes  which are bound to t u b u l i n .  c o l c h i c i n e binding  a l t e r n a t i v e s i t e on the to act as c a t i o n s  a c t i v i t y and dimer.  The  the  vinblastine  of 1 of the  Vinblastine  stabil-  hence occurs at  an  v i n c a a l k a l o i d s are  ( i n many cases they mimic C a  + +  2  believed  ) which pre-  c i p i t a t e t u b u l i n to y i e l d l a r g e c r y s t a l l i n e a r r a y s . G r i s e o f u l v i n , a mold m e t a b o l i t e , has a n t i m i t o t i c a c t i v i t y , but i o n of t u b u l i n as do the the b i n d i n g  i t does not  polymerizat-  166).  I t does  of c o l c h i c i n e or the v i n c a  alkaloids  influence  and  i t s mechanism remains obscure.  (R1793^)  prevent the  other i n h i b i t o r s (165,  not  Nocodazole  been shown to possess  i s a microtubule d i s r u p t o r  r e c e n t l y been s y n t h e s i z e d by De  Brabander et a l .  which  (167).  a s p e c i f i c a n t i t u b u l i n compound which i n t e r f e r e s with structure  and  function  It i s  the  of m i c r o t u t u b l e s through i n h i b i t i o n of  t u b u l i n p o l y m e r i z a t i o n i n t o normal m i c r o t u b l e s . of Nocodazole to t u b u l i n i s r e v e r s i b l e and no  has  known s i d e e f f e c t s at the  the  The  binding  drug possesses  c o n c e n t r a t i o n s used.  a l s o shown great promise as an anti-tumor agent.  The  drug  has  -41-  4.  The  D i s r u p t i o n of  Cytochalasin  B,  Microfilaments a metabolite  of the fungus, Helmintho-  sporium dematiodium has been shown to have a wide e f f e c t on cellular activities  (171).  The  compound has been shown to  stop c y t o k i n e s i s w i t h the p r o d u c t i o n (171),  (172),  i n h i b i t hexose t r a n s p o r t  ular secretion (173), c e l l movement ( 1 7 4 ) , beating  of m u l t i n u c l e a t e d i n t e r f e r e with  extrusion ( 1 7 1 ) ,  induce n u c l e a r  prevent phagocytosis (175)  of embryonic c a r d i a c muscle c e l l s  evidence i s not  (176).  cellinhibit  stop  the  Although  the  e n t i r e l y c o n c l u s i v e , c y t o c h a l a s i n B appears to  disrupt microfilaments Hartwig and  and  cells  (177)-..  S t o s s e l (179)  Recently  Weihing (I78)  and  have shown t h a t t h i s drug prevents  the g e l a t i o n of p u r i f i e d a c t i n with a c t i n - b i n d i n g p r o t e i n a l s o depolymerizes a c t i n .  However, t h e r e i s s t i l l  as to the a c t u a l t a r g e t s of t h i s drug ( 1 8 0 ) .  and  some d i s p u t e  Cytochalasin  A  has been demonstrated to b i n d f r e e s u l f h y d r y l groups of t u b u l i n and  a c t i n monomers and  c y t o c h a l a s i n B,  thus prevent  polymerization;  on the other hand, does not r e a c t with t u b u l i n  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 s t r u c t u r e f o r c y t o c h a l a s i n B i s shown i n F i g .  19".  CH —CH 2  I l ,C—CH :  CH. \ HC.  Fig.  19.v  The  P  2  CH HC  s t r u c t u r e of c y t o c h a l a s i n  B.  -42-  5.  Local Anesthetics Microfilaments  - Disruptors  o f Both M i c r o t u b l e s and  L o c a l a n e s t h e t i c s have r e c e n t l y been demonstrated t o d i s (136, 137,  r u p t the c y t o s k e l e t a l o r g a n i z a t i o n i n c e l l s and  cause the c e l l s to become more rounded.  I8I-I83)  'Quaternary amine  a n e s t h e t i c s appear t o mimic the e f f e c t s o f c o l c h i c i n e and c y t o c h a l a s i n B, when both a r e administered At h i g h a n e s t h e t i c c o n c e n t r a t i o n s a n e s t h e t i c s produce molecular  ( g r e a t e r than ImM)  microfilaments  i n membranes and cause b i l a y e r  At lower c o n c e n t r a t i o n s ,  microtubules  (dibucaine, procaine,  c e l l a g g l u t i n a t i o n , endocytosis,  and t e t r a c a i n e )  inhibit  e x o c y t o s i s , and the m o b i l i t i e s  of s u r f a c e r e c e p t o r molecules ( 1 8 2 , I 8 3 . 1 8 5 - 1 8 7 ) . anesthetics i n t e r a c t hydrophobically  phatides)  and  a r e a f f e c t e d p r i m a r i l y and the t e r t i a r y amine  l o c a l anesthetics  membrane l i p i d s  local  disordering i n l i p i d bilayers,  enhance f l u i d i t y of p h o s p h o l i p i d s expansion (184).  simultaneously.  Local  and e l e c t r o s t a t i c a l l y  with  ( i n p a r t i c u l a r the a n i o n i c groups of a c i d phos-  and a r e able t o d i s p l a c e Ca  from membranes (188, I 8 9 ) .  These drugs cause the plasma membrane t o crenate;  the t e r t i a r y  amine a n e s t h e t i c s a r e thus known as "cup-forming" a n e s t h e t i c s (190).  T h i s group of a n e s t h e t i c s  i s thought t o i n t e r c a l a t e  w i t h i n the i n n e r monolayer o f the membrane.  The mechanism o f  l o c a l a n e s t h e t i c a c t i o n i s u n c l e a r but they may d i s p l a c e membrane Ca  and i n c r e a s e i n t r a c e l l u l a r Ca  concentrations  s u f f i c i e n t to depolymerize m i c r o t u b l e s  (I36).  which i n c r e a s e d i n t r a c e l l u l a r l e v e l s of C a myosin complexes or Ca  + +  to l e v e l s  The manner i n might a f f e c t ' a c t o -  dependent ATPase i s not c l e a r ( 1 3 6 ) .  -43-  S t r u c t u r e s f o r the t e r t i a r y amine a n e s t h e t i c s are.presented Fig.  in  20. H  N H ^ ^ - C O O C H  2  .  C H  2  N  2  ( C H  C H  2  3  )  2  procaine  H NHQKC 2  benzocaine  •C H  3  ( C H  2  )  3  NHH^J^-  C O O C H J C H J N  ( C H ) , 3  tetracaine  ' N ^ , 0 ( C H  C O N H C H  2  2  )  C H  3  2  C H  3  N ( C H  2  C H  3  )  2  dibucaine  Fig. 20.  6.  S t r u c t u r e s of the t e r t i a r y amine a n e s t h e t i c s .  Microtuble  and M i c r o f i l a m e n t  Microtubles  F u n c t i o n W i t h i n the C e l l  and m i c r o f i l a m e n t s  are not merely a s s o c i a t e d  with the s p i n d l e apparatus ( f o r chromosome movement and d i v i s i o n d u r i n g c y t o k i n e s i s ) and Research over the l a s t decade has  flagella (for  cell  locomotion).  proven t h a t these o r g a n e l l e s  have many more d i v e r s e f u n c t i o n s than p r e v i o u s l y  realized.  (a) C y t o s k e l e t a l Transmembrane C o n t r o l A r e l a t i v e l y r e c e n t concept i s t h a t microtubules 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 two (191)  dimensional  s u r f a c e of a membrane.  noted t h a t normal f i b r o b l a s t s  inhibited  and  may  con-  of p r o t e i n s about the  Initially Vasiliev  et a l .  (which are u s u a l l y c o n t a c t  possess a random d i s t r i b u t i o n of c e l l  surface  p r o t e i n s ) behaved l i k e transformed c e l l s when t r e a t e d  with  -LL-  colcemid i e . c o n t a c t i n h i b i t i o n was r e l i e v e d and a c l 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. f i r s t proposed t h a t microtubules  These i n v e s t i g a t o r s  s t a b i l i z e d the n o n - a c t i v e  of t h e c e l l s u r f a c e as a submembranous framework. a c t i o n between microtubules  state  The i n t e r -  and i n t e g r a l membrane p r o t e i n s was  f o r m a l l y i n t r o d u c e d by B e r l i n et a l . (192)  and Edelman et a l .  (193)• B e r l i n and h i s c o l l a b o r a t o r s gathered to i n d i c a t e t h a t the microtubule  extensive  evidence  system can exert transmembrane  c o n t r o l over a v a r i e t y o f s u r f a c e r e c e p t o r and capping phenomena  (192, 196).  Edelman and Yahara  f o r the modulation  (193}, 197)  have proposed a model  of lymphocyte r e c e p t o r s by a transmembrane  c o n t r o l system which i n v o l v e d t h e m i c r o t u b u l e - m i c r o f i l a m e n t systems.  They p o s t u l a t e d t h e e x i s t e n c e o f 2 d i f f e r e n t c l a s s e s  of s u r f a c e r e c e p t o r s - some of which a r e immobile and anchored (A-state) to microtubules  and others which a r e f r e e and not  a s s o c i a t e d w i t h microtubules  (F-state).  Capping of membrane  p r o t e i n s o c c u r r e d under the guidance of m i c r o f i l a m e n t s when m u l t i v a l e n t l i g a n d s ( l e c t i n s or a n t i Ig) a s s o c i a t e d with r e c e p t o r s i n the f r e e s t a t e . i z e d i n F i g . 21'.  T h i s theory e x p l a i n s the o b s e r v a t i o n s  summar-  -1*5-  Fig. 2 1 . E f f e c t of drugs on lymphocytes which were i n c o n t a c t w i t h a n t i - I g and/or Con A. Q Con A r e c e p t o r ; o a n t i I g receptor. ^  Capping Ig  of a n t i - I g r e c e p t o r s occurs presumably because s u r f a c e  i s not a s s o c i a t e d w i t h m i c r o t u b u l e s .  Concanavalin A l e c t i n  would not cause capping of membrane r e c e p t o r s unless the l i n k s between the l e c t i n - b i n d i n g p r o t e i n and the microtubule were severed.  A d d i t i o n of c o n c a n a v a l i n A t o lymphocytes  prevented  a n t i - I g capping s i n c e the l e c t i n a c t e d to immobilize the s u r f a c e a n t i b o d y by b i n d i n g i t t o the c o n c a n a v a l i n anchored).  A r e c e p t o r (which i s  M i c r o f i l a m e n t s were demonstrated to modulate the  capping process s i n c e c y t o c h a l a s i n B (which d i s r u p t s m i c r o f i l a ments) and d i b u c a i n e (which presumably d i s r u p t s m i c r o f i l a m e n t s and microtubules) prevents cap f o r m a t i o n of c o n c a n a v a l i n A receptors.  -46-  The Edelraan and Yahara t h e o r y for  also  provides explanation  the transmembrane c o n t r o l o f f i b r o b l a s t r e c e p t o r s .  N i c o l s o n , Poste, and Paphadjopoulos  ( 1 3 6 , I 3 7 , 1 8 2 , I83) have  made the o b s e r v a t i o n s o u t l i n e d i n F i g . 22 with mouse 3^3 f i b r o b l a s t s which have been t r e a t e d w i t h c o n c a n a v a l i n A.  Normally  c o n c a n a v a l i n A does not cause capping or patch f o r m a t i o n of membrane p r o t e i n s ; however, c o l c h i c i n e a d d i t i o n caused while c y t o c h a l a s i n B prevented  cap f o r m a t i o n .  capping  The a d d i t i o n o f  dibucaine (which destroyed both microtubules and m i c r o f i l a m e n t s ) allowed s m a l l patches  of c o n c a n a v a l i n A r e c e p t o r s t o form - the  r e c e p t o r s were no l o n g e r anchored,  they d i f f u s e d l a t e r a l l y , and  self-aggregated.  untreated  2  3  colchicine  cytocholosin  B  dibucaine 4  colchicine  plus  cytocholaiin B  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 c o n t a c t with a n t i - I g and/or Con A.  -47-  I t would thus appear t h a t microtubules and m i c r o f i l a m e n t s have opposing r o l e s i n membrane f u n c t i o n .  M i c r o t u b u l e s serve to  anchor i n t e g r a l p r o t e i n s while m i c r o f i l a m e n t s possess a c o n t r a c t i l e f u n c t i o n which c o n t r o l s the movement of p r o t e i n s over the c e l l s u r f a c e (136,137).  Metabolic i n h i b i t o r s (KCN,  NaN^» DNP) i n h i b i t m i c r o f i l a m e n t f u n c t i o n presumably due to the ATP requirement  f o r c o n t r a c t i o n (136, 137).  A word o f c a u t i o n  i s perhaps r e q u i r e d b e f o r e one t o t a l l y accepts t h i s scheme. Some c o n f u s i n g evidence has appeared d i f f e r e n t c e l l l i n e s (198,199).  i n other l a b o r a t o r i e s u s i n g  I t can, however, be concluded  t h a t microtubules and m i c r o f i l a m e n t s i n t e r a c t with membrane p r o t e i n s and c o n t r o l t h e i r movements i n some manner. has proposed  Nicolson  the f o l l o w i n g scheme f o r i n t e r a c t i o n of membrane  p r o t e i n s w i t h microtubules and m i c r o f i l a m e n t s ( F i g . 23).  ACTIN FILAMENTS MYOSIN MOLECULES <  ®  MICROTUBULES  F i g . 23. Proposed i n t e r a c t i o n o f microtubules and m i c r o f i l a m e n t s w i t h membrane p r o t e i n s (137).  -48-  (b) C e l l S e c r e t i o n and Microtubules cated  i n the  and  the C y t o s k e l e t a l System  microfilaments  have a l s o been i m p l i -  i n t r a c e l l u l a r t r a n s p o r t of s e c r e t o r y v e s i c l e s  to the c e l l ' s p e r i p h e r y .  Lacy (200)  microtubule-microfilament  system which l i n k e d the  o r i g i n a l l y proposed a  s e c r e t o r y v e s i c l e s w i t h the plasma membrane. volved  insulin  Secretion i n -  " c o n t r a c t i o n " of the c y t o s k e l e t a l system w i t h r e s u l t a n t  transport  to the c e l l s u r f a c e . Forbes and  Dent (201)  a s i m i l a r h y p o t h e s i s f o r gonadotrophic c e l l s .  proposed  Colchicine  been demonstrated to i n h i b i t s e c r e t i o n of a- amylase by parotid (202) i n the a d r e n a l  i n s u l i n i n the pancreas ( 2 0 3 , (205),  thyroxine  p r o t e i n s by hepatocytes ( 2 0 7 ) , leukocytes  (208).  Microtubules  204)  lysozyme by  arid  microfilaments  p r o t e i n s are s y n t h e s i z e d  the membranes of the discharged  have a l s o (209,  are  i n t o the lumen of the rough endoplasmic  reticulum. them to  to the G o l g i complex, and  s e c r e t o r y v e s i c l e s which, when f i l l e d  s e c r e t o r y p r o t e i n s , fuse with the plasma membrane.  Most r e c e n t l y , Malaise m i c r o t u b u l e s as g u i d i n g  et a l .  to  with  Glycosylation  of s e c r e t o r y p r o t e i n s occurs i n a stepwise f a s h i o n and a d d i t i o n o f sugars occurs at the G o l g i  to  vectorially  They then f o l l o w an i n t r a c e l l u l a r pathway which leads  the G o l g i - d e r i v e d  210).  on polysomes attached  endoplasmic r e t i c u l u m and  the smooth endoplasmic r e t i c u l u m ,  serum  polymorphonuclear  been found to b i n d some s e c r e t o r y granules,.' i n v i t r o Secretory  the  catecholamines  from the t h y r o i d ( 2 0 6 ) , and  has  terminal  (211).  have considered  elements f o r the t r a n s p o r t  granules which c o n t a i n i n s u l i n ; m i c r o f i l a m e n t s ,  the r o l e of of  secretory  on the other  hand,  -49-  were  considered  sites asin  to c o n t r o l the access  of exocytosis B facilitated  alkaloids  microfilaments plasma  neurosecretory  release  that  i t is  prior  to the fusion  They  protein)  completed.  from normally  reaching  facilitates  and cause  demonstrated medulla  to  (205),  an accumulation  of  Williams  (213).  tetracaine  inhibit  disruptors  (a  pancreatic  and  local  amylase  do n o t h i n d e r  t o smooth  feel  re-  migration  reticulum  reticulum or  of i n h i b i t i o n i s  vesicles  (which  membrane  inhibitors  the  endoplasmic  endoplasmic  the point  w i t h the plasma  caused  just  contain the  after  glycosylation  an accumulation  i n Golgi-derived vesicles  from the  have  Movement also  t h e movement  been  and t h e C y t o s k e l e t a l implicated in a role  of granules  within the c e l l .  of  liver  respectively.  Intracellular  Microtubules  been  ( 2 0 7 , 2 1 4 ) a n d P a t z e l t e t a l . (102)  Microtubule  the parotid  also  by the adrenal  of the Golgi  a l b u m i n and. a-amylase  (c)  contact  b e l i e v e d a d e n s e web o f  colchicine,  synthesized)  apparatus.  and  Electron-  t o be i n c l o s e  p r o t e i n from the rough  Golgi  is  release.  pancreas.  microtubule  the secretory  secretory  c o l c h i c i n e and v i n c a  i n the p i t u i t a r y  and t r a n q u i l i z e r s  f r o m mouse  (where  have  (206),  granules  Redman a n d P a l a d e  of  workers  release  r e c e n t l y found  anesthetic)  suggest  granules  to  Cytochal-  (212).  insulin  the particles  disruptors  catecholamine  (168)  while  granules  membrane.  t h y r o i d hormone  lease  and these  prevented  Microtubule  Lee  release  showed t h e i n s u l i n  with microtubules  inhibit  insulin  membrane  inhibited glucose-induced  microscopy  the  a t the plasma  of secretory  System which For  -50-  example, lysosome,  melanin, and i n t r a x o n a l p a r t i c l e s are  b e l i e v e d to be c l o s e l y a s s o c i a t e d with m i c r o t u b u l e s . • (215)  Wilson's l a b o r a t o r y evidence f o r the involvement of v a s o p r e s s i n .  has  presented  of microtubules i n the a c t i o n  Microtubule disruptors i n h i b i t e d vasopressin-  s t i m u l a t e d water movement a c r o s s e p i t h e l i a l membranes. brane p e r m e a b i l i t y was  Mem-  b e l i e v e d to be r e g u l a t e d by the  m i g r a t i o n (under the c o n t r o l of microtubules) and f u s i o n of membrane-limited granules with the plasma membrane; the  new  membrane patches were b e l i e v e d to possess a g r e a t e r p e r m e a b i l i t y to water ( 2 1 5 ) • Malawista (216)  has a s c r i b e d a r o l e t o m i c r o t u b u l e s i n  which m i c r o t u b u l e s 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 p h a g o c y t o t i c v a c u o l e s i n  polymorphonucleocytes.  M i c r o t u b u l e i n h i b i t o r s appear to i n h i b i t the movements of the lysosomes  w i t h i n the c e l l and prevent t h e i r d e g r a n u l i z a t i o n  which occurs on c o n t a c t with phagoeytized  material.  S e v e r a l i n v e s t i g a t o r s ( 2 1 7 - 2 2 0 ) have r e p o r t e d t h a t microt u b u l e s and m i c r o f i l a m e n t s p a r t i c i p a t e i n the d i s p e r s i o n of melanin g r a n u l e s i n melanocytes.  M i c r o f i l a m e n t s appear to  c o n t r o l d i s p e r s i o n of melanin granules ( r e s u l t s i n s k i n  darken-  ing) while m i c r o t u b u l e s appear to d i r e c t the a g g r e g a t i o n of these p a r t i c l e s (which causes s k i n l i g h t e n i n g ) .  The  exact  nature of the mechanism f o r t h i s process i s u n c l e a r . M i c r o t u b u l e s , but not m i c r o f i l a m e n t s , have been r e p o r t e d to be a s s o c i a t e d with i n t r a x o n a l t r a n s p o r t ( 2 2 1 ) . micrographs  Electron-  have p o r t r a y e d an a s s o c i a t i o n between microtubules  -51-  and mitochondria  or other membrane l i m i t e d v e s i c l e s  (221).  In  some i n s t a n c e s s m a l l c r o s s - b r i d g e s can be seen to i n t e r - l i n k these o r g a n e l l e s . t h a t microtubule a l i n e granules  Dahlstrom e_t a l . (222) have demonstrated d i s r u p t o r s i n h i b i t the t r a n s p o r t o f noradren-  i n a d r e n e r g i c nerves and a c e t y l c h o l i n e granules  i n c h o l i n e r g i c nerves.  Axelrod's  l a b o r a t o r y ( 2 2 3 ) has r e p o r t e d  t h a t c o l c h i c i n e , v i n b l a s t i n e , and c y t o c h a l a s i n 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 o f V i r u s The r o l e o f microtubules  and m i c r o f i l a m e n t s  assembly has r e c e i v e d minimal a t t e n t i o n (224).  i n virus Microfilaments  have been i m p l i c a t e d i n some r e s p e c t t o the budding of Herpes v i r u s e s from the n u c l e a r membrane ( 2 2 5 , 2 2 6 ) but the e f f e c t s o f c y t o c h a l a s i n B on t h i s process was u n c l e a r . (227,  Weihing's l a b o r a t o r y  228) has demonstrated a c l o s e a s s o c i a t i o n between adeno-  v i r u s e s and microtubules t h a t these  i n v i v o and i n v i t r o and has surmised  s t r u c t u r e s may be important  v i r u s to and from the nucleus.  i n the t r a n s p o r t of  I n another  study,  Stokes (229)  purports t h a t the i n t r a c e l l u l a r movement and r e l e a s e o f v a c c i n i a v i r u s r e q u i r e s a host c e l l cytoplasmic microfilaments  forstability.  network t h a t i n v o l v e s  High-voltage  electronmicroscopy  demonstrated t h a t m i c r o f i l a m e n t s were a s s o c i a t e d with the c y t o plasmic  f a c t o r i e s where v a c c i n i a was manufactured and a l s o with  m i c r o v i l l i along the c e l l p e r i p h e r y where the v i r u s was r e l e a s e d . Two r e p o r t s have i m p l i c a t e d m i c r o f i l a m e n t s  i n the budding of  RNA tumor v i r u s e s from the plasma membrane o f the host  cell.  -52-  Damsky et al.(230) found a c t i n a s s o c i a t e d w i t h 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  r e l e a s e of murine Sarcoma-leukemia v i r u s a t an a p p a r e n t l y stage of. v i r a l assembly ( e l e c t r o n m i c r o s c o p y  late  d i d not show budding  p a r t i c l e s but r e l e a s e o f v i r u s e s was almost immediate when c y t o c h a l a s i n B was removed). C.  Chemical C r o s s - L i n k i n g  o f 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, h i s t o n e s , r e d b l o o d mitochondria,  E. c o l i membranes, sarcoplasmic  numerous enzymes - i n o r d e r p r o t e i n to another.  cells,  r e t i c u l u m , and  to determine the p r o x i m i t y  o f one  C r o s s - l i n k i n g agents have been t h e s u b j e c t  of an e x c e l l e n t review (232).  R e s u l t s with these compounds can  be v e r y elegant but one must always remember t h a t i n t e r p r e t a t i o n o f 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 o f f u n c t i o n a l groups on the p r o t e i n f o r c r o s s - l i n k i n g . 1.  Chemistry o f C r o s s - L i n k i n g  Agents  Chemical c r o s s - l i n k e r s f a l l i n t o a number o f c a t e g o r i e s - 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 d i a l d e h y d e s .  Representative  members o f t h e s e  cate-  g o r i e s are shown i n Table 6 . 2. ' C r o s s - L i n k i n g (a)  of V i r a l  Proteins  V e s i c u l a r Stomatis V i r u s  Dubovi and Wagner ('233) r e c e n t l y c r o s s - l i n k e d the p r o t e i n s o f 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 The  c r o s s - l i n k e d p r o d u c t s were r e s o l v e d by two-dimensional  MMB.  -53-  Table 5 Representative Chemical Cross-Linkers FORMULA  ABBREVIATION  CH^-C-CCH^-C-OCH,  OMS  CROSS-LINKER  dimethylsuberimidate  MECHANISM  R-C-OR + N l l , I "P H+ -N-C-R • ROH  Nil  d i m e t h y l 3,3' - t h i o b i s (propionimidate) dithiobis( succini m i d y l propionate)  CHp-c-fcH^-s-s-fcH^- CI-OCH,  ( a s above)  DTBP  O  o  II  (>o-c-CH CH,-5-s-CH -cH c-o^) o ° J  t  r  -N-C-R d o  glutaraldehyde  i  IjfcHjCH |c-(CH^CH jc-fcttf-t CHj  CHO-<tll^-CHO  1*  - h l ^  +CH-{CH£CH - C -(CH^CH «  tartryl diazide ( r e v e r s i b l e on addition of periodate)  * O OM OH O  TOA  Nfc'-iH-CH-H-W,  0  1  OM OH O ,H  -N-C-CH-CH-C-N-  o x i d i z e s -SH o  NO,  methyl-3-mercaptopropionimidate  CH-C-Nj*  H U I  cupric di(l,10phenanthroline) complex N,N'(4 a z i d o - 2 - n i t r o phenyl)cystamine dioxide (reversible on a d d i t i o n o f BME)  O OH OH a -C-CM  II  ^©NH-CHJCH-S-S-CHJCH-NM^N,  II CH-OC-CH  CM-SH  N.-R.-.S-S-R. + HSO i  MMP  o x i d i z e SH- w i t h H2O2 a f t e r i m i d o e s t e r has r e a c t e d with protein  -54-  electrophoresis  (234,  235)'  Major p r o t e i n s  M, N, and G (see s e c t i o n I.A.4). detect and  dimers and t r i m e r s  o f t h i s v i r u s are  These workers were able to  of M and G p r o t e i n s , an M-N complex,  i n some cases (with TDA and MMB  only) a G-M dimer.  A photo-  graph of t h e i r 2 dimensional s l a b g e l s i s shown i n F i g u r e 24. Cross-linked  products a r e i l l u s t r a t e d on h o r i z o n t a l l i n e s w h i l e  monomeric or u n c r o s s - l i n k e d runs from l e f t  to r i g h t .  species  l i e on a diagonal  The i n a b i l i t y o f DSP to form  oligomers was f e l t t o be due t o a l e s s e r p e r m e a b i l i t y  which high o f the  membrane to t h i s agent.  First Dimension —•  • p I M l ip»7|  0  N  M  F i g . 24. Autoradiograph o f 2-dimensional SDS p o l y a c r y l a m i d e g e l s which i l l u s t r a t e s the c r o s s - l i n k e d species of v e s i c u l a r s t o m a t i t i s v i r u s . V i r u s was c r o s s - l i n k e d w i t h (A)TDA, (B)MMB, and (C)DTBP.  -55-  (b)  Semliki Forest Virus  S e m l i k i F o r e s t v i r u s has been c r o s s - l i n k e d with DMS and subjected  to e l e c t r o p h o r e s i s i n one dimension by G a r o f f and  Simons (236,  237).  They s u b j e c t e d  t h i s agent a t a c o n c e n t r a t i o n  50 ug o f v i r u s p r o t e i n to  of 3 mg/ml f o r 2 h r a t 20°C.  Envelope p r o t e i n s aggregated i n t o complexes which 2-6  contained  components which were r e s o l v e d by one dimensional  acrylamide  electrophoresis.  Nucleocapsid  SDS  p r o t e i n s were s a i d  to be p r e f e r e n t i a l l y c r o s s - l i n k e d under these c o n d i t i o n s and stayed a t the t o p o f the g e l .  Evidence f o r a n u c l e o c a p s i d -  envelope l i n k a g e c o u l d not be detected  d i r e c t l y from these g e l s  but d i s r u p t i o n of the membranes o f c r o s s - l i n k e d v i r u s with Triton  X-100 f a i l e d  to r e l e a s e the envelope p r o t e i n s  Large membrane p r o t e i n — n u c l e o c a p s i d on sucrose  g r a d i e n t s and viewed by  (236).  complexes were i s o l a t e d electronmicroscopy.  -56-  (c) The  Adenovirus o n l y other v i r u s f o r which c r o s s - l i n k i n g  have been r e p o r t e d i s adenovirus type 2. Philipson  (238)  u t i l i z e d TDA  Everitt, Lutter  and  to c r o s s - l i n k p u r i f i e d adenovirus.  Two-dimensional e l e c t r o p h o r e s i s topography of i t s p r o t e i n s .  studies  was  used to e s t a b l i s h  the  -57-  D.  The Present I n v e s t i g a t i o n I t was the i n t e n t o f t h i s t h e s i s to prove through b i o chemical means t h a t S e m l i k i F o r e s t v i r u s matures by budding from the plasma membrane of the host c e l l .  Through  this  process the naked n u c l e o c a p s i d a c q u i r e s envelope l i p i d s and proteins.  We a l s o were concerned w i t h the mechanism by which  budding o c c u r s . Previous evidence t o support t h i s h y p o t h e s i s i s d e r i v e d from e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s ( 1 6 , 7 9 , 8 0 ) and l i p i d c l a s s c o m p o s i t i o n s t u d i e s conducted on ER. PM, and p u r i f i e d virus  (21, 9 5 9 8 ) . -  The l i p i d s o f the v i r u s envelope  most c l o s e l y those o f the host c e l l  PM.  resemble  S i m i l a r l i n e s of  i n v e s t i g a t i o n have been conducted on other enveloped v i r u s e s such as myxoviruses and paramyxoviruses  (110-113) and rhabdo-  v i r u s e s (101, 102). R e s u l t s from the above s t u d i e s a r e f a r from c o n c l u s i v e - the  e l e c t r o n m i c r o s c o p i c evidence could be based on a r t i f a c t s  i n c u r r e d through sample p r e p a r a t i o n and many i r r e g u l a r i t i e s e x i s t i n the l i p i d composition studies.  I t was hoped t h a t t h e  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  situation.  The problem was a t t a c k e d through use o f r a d i o a c t i v e p u l s e chase experiments.  I n f e c t e d c e l l s were to be p u l s e d w i t h  [•^H]lieu and the l a b e l was to be f o l l o w e d from the membrane f r a c t i o n s o f the host c e l l  i n t o mature v i r u s .  Preliminary  experiments o f t h i s type have been performed on i n f l u e n z a ••• (115-120) and v e s i c u l a r s t o m a t i t i s (104-107) v i r u s e s but much of  t h i s data i s incomplete and the complex nature of these  -58-  v i r u s e s makes the data d i f f i c u l t  to i n t e r p r e t .  Initially  these s t u d i e s were a p p l i e d to microsomal f r a c t i o n s and e x 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 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 (.254);  the membranes o f i n f e c t e d c e l l s were  then to be f r a c t i o n a t e d i n t o ER and PM.  I t was the aim o f  these experiments to show a c l e a r chase o f r a d i o a c t i v e l y l a b e l e d v i r u s p r o t e i n s from ER t o PM and t h e n i n t o e x t r a c e l l u l a r v i r u s . T h i s would support the c o n t e n t i o n synthesized  t h a t v i r u s p r o t e i n s were  i n the ER and i n s e r t e d i n t o the PM j u s t p r i o r t o  v i r u s maturation. The  o b j e c t i v e of the second h a l f of the t h e s i s was t o  e l u c i d a t e the mechanism o f budding. manner i n which v i r a l g l y c o p r o t e i n s ported  We were i n t e r e s t e d i n the and n u c l e o c a p s i d  t o and i n s e r t e d i n t o the plasma membrane.  The a g g r e g a t i o n  of v i r a l p r o t e i n s i n t o d i s t i n c t patches a t the c e l l f o l l o w e d by bud formation  were t r a n s -  surface,  and e x t r u s i o n of v i r u s i n t o e x t r a -  c e l l u l a r media was of i n t e n s e  i n t e r e s t to us.  L o d i s h regarded the steps  o f v i r u s envelope f o r m a t i o n as  a " s e c r e t o r y " process ( 6 0 ) i n which g l y c o p r o t e i n s  a r e synthe-  s i z e d on membrane bound ribosomes and t r a n s p o r t e d  t o the plasma  membrane i n a manner comparable t o i n s u l i n ( 2 1 2 ) and p a n c r e a t i c amylase ( 2 0 2 ) r e l e a s e . i n s e c r e t i o n of proteins microfilaments has  Microtubules (132).  a r e reputed to be i n v o l v e d  A r o l e f o r m i c r o t u b u l e s and  i n o r g a n i z i n g p r o t e i n s of the plasma membrane  a l s o been proposed ( I 3 6 , 1 3 7 ) '  With these views i n mind,  we wished to t e s t the e f f e c t s o f c y t o s k e l e t a l d i s r u p t o r s on v i r u s maturation.  Microtubule  d i s r u p t o r s had p r e v i o u s l y been  -59-  shown t o i n h i b i t s e c r e t i o n o f g l y c o p r o t e i n s o r g a n i z a t i o n a l system f o r v i r a l  and some s o r t o f  p r o t e i n s a t the c e l l  surface  seemed necessary.The s t r a t e g y behind these s t u d i e s was to measure the incorporation of r a d i o a c t i v e v i r a l proteins brane,  endoplasmic  reticulum  i n t o plasma mem-  and e x t r a c e l l u l a r v i r u s i n the  presence o f c o l c h i c i n e , nocodazole, d i b u c a i n e , and c y t o c h a l a s i n B.  We a l s o wished  to i n v e s t i g a t e the e f f e c t s o f these  drugs on the p r o x i m i t y of v i r u s membrane p r o t e i n s  t o each o t h  during v i r u s m a t u r a t i o n through use of p r o t e i n c r o s s - l i n k i n g agents.  -6o-  II.  A.  Reagents and L-[4,5  3  H]  METHODS AND  MATERIALS  Equipment  L e u c i n e ( 4 5 - 6 0 Ci/mmol) and L - [  ( 4 0 0 - 5 0 0 Ci/mmol) were purchased from New poration.  3 5  s ] methionine  England Nuclear Cor-  Sucrose (RNAse f r e e ) 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 s u p p l i e d by Bio-Rad L a b o r a t o r i e s .  Sodium dodecyl s u l p h a t e was  from B r i t i s h Drug House.  Actinomycin D came from Merck, Sharpe,  and Dohme.  purchased  C o l c h i c i n e was bought from Sigma and d i b u c a i n e  h y d r o c h l o r i d e was  o b t a i n e d from ICN/K & K L a b o r a t o r i e s .  A l d r i c h s u p p l i e d the c y t o c h a l a s i n B and Nocodazole. c r o s s - l i n k e r s were purchased from P i e r c e . were reagent grade.  E l e c t r o p h o r e s i s was  Chemical  A l l other chemicals done w i t h tube and  s l a b e l e c t r o p h o r e s i s c e l l s from Bio-Rad L a b o r a t o r i e s  (Models  150A and 220 r e s p e c t i v e l y ) . B.  V i r u s 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 v i r u s has p r e v i o u s l y been  d e s c r i b e d (241).  BHK-21 ( S t o k e r and Macpherson s t r a i n )  cells  were o b t a i n e d from M i c r o b i o l o g i c a l A s s o c i a t e s and grown as monolayer tific  c u l t u r e s on 150 mm  Corporation).  X 15 mm  p l a s t i c d i s h e s (LUX  C e l l s were c u l t i v a t e d i n Dulbecco's  ScienModi-  f i e d E a g l e ' s Medium supplemented w i t h 5$ f e t a l c a l f serum w h i l e v i r u s i n f e c t i o n s were performed i n 199 maintenance 2fo f e t a l c a l f serum.  medium w i t h  -6l-  C.  Polyacrylamide Gel E l e c t r o p h o r e s i s ( C y l i n d r i c a l  Gels)  P r o t e i n s 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 r u s were analyzed by e l e c t r o p h o r e s i s on 7-5f° presence  acrylamide g e l s i n the  o f sodium dodecyl s u l f a t e a c c o r d i n g t o Weber and  Osborne (24-2) except t h a t d i a l l y l t a r t a r d i a m i d e ( 2 4 3 ) was u t i l i z e d mole f o r mole i n p l a c e o f me th,yleneb i s acrylamide. The g e l s were c a s t t o a h e i g h t o f 7 cm from the f o l l o w i n g stock s o l u t i o n s : 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 - 1 3 - 5 ml 1.5% (w/v) ammonium p e r s u l f a t e - 2 . 0 ml 7.8  gm/l  Na P0nH 0, 2  2  38.6  Na HP0^.7H 0,  gm/l  2  2  2 gm/l sodium doaecyl s u l f a t e d i s t i l l e d water tetramethylethylenediamine  - 2 0 . 0 ml 4 . 5 ml 3° u l  Membrane samples were s o l u b i l i z e d i n an equal volume o f sample b u f f e r ( O . 3 8 6 gm/l NaH P0^2.09 gm/l Na^PQ^. 7 H 0 , 2  2  SDS,  10%> (v/v) g l y c e r o l , 0.1% (w/v)  '3-mercaptoethanol  1% (w/v)  bromphenol b l u e , 1% (v/v)  f o r 5 min a t 100°C.  The 2 compartments o f the  e l e c t r o p h o r e s i s apparatus were f i l l e d with r e s e r v o i r b u f f e r (3.9  gm/l NaH P0/j, HgO, 2  19-3  gm/l N a H P 0 ^  E l e c t r o p h o r e s i s was performed  2  7H 0, 2  1 gm/l SDS).  a t a constant c u r r e n t o f 8 mamps  per g e l w i t h the p o s i t i v e e l e c t r o d e i n the lower chamber f o r 2.5  hr.  (0.25$  The g e l s were s t a i n e d w i t h Coomassie blue s o l u t i o n (w/v)  Coomassie b l u e , 4 5 . 4 %  (v/v) methanol, 9.2% (v/v)  g l a c i a l a c e t i c a c i d ) a t 60°C f o r 1 h r and then d e s t a i n e d by e i t h e r d i f f u s i o n i n 7>5% (v/v) a c e t i c a c i d , 5%> (v/v) methanol o r by e l e c t r o p h o r e s i s ( i n 7• 5%> a c e t i c a c i d ) a t 3 - 4 mamps per g e l .  -62-  D.  P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s (Slab Gels) One  was  dimensional e l e c t r o p h o r e s i s i n t h e presence  performed  slab gels.  by the procedure  o f Laemmli and Favre  of SDS (244) f o r  The s e p a r a t i o n g e l was 8 . 5 cm high, 14 cm wide and  e i t h e r 0 . 7 5 mm  or 1 . 5 mm t h i c k .  A 1 cm h i g h s t a c k i n g g e l  which c o n t a i n e d t e n 8 mm wide sample s l o t s was c a s t on t o p of the s e p a r a t i o n g e l . s p e c i f i e d percentage  The s e p a r a t i o n g e l c o n s i s t e d of a  of acrylamide  and.the s t a c k i n g g e l  was 3 . 0 $ (w/v) acrylamide, 0 . 1 5 % (w/v) methylenebisacrylamide, 0.125  M T r i s - H C l pH 6 . 8 , and 0 . 1 % SDS s u l f a t e .  G e l s were made  from the f o l l o w i n g stock s o l u t i o n s : separation gel  Vol (ml)  30% (w/v acrylamide, 0.8% methylenebisacrylamide  X  30% acrylamide, 1.5% methylenebisacrylamide  2.5  I . 8 3 M T r i s - H C l pH 8 . 8 0 . 5 % sodium dodecyl sulfate  5-0  1.25 M Tris-HCl p H 6 . 8 , 1.0% sodium dodecyl s u l f a t e  2.5  1.0% (w/v) ammonium persulfate  0.75  1.0% ammonium persulfate  0.75  d i s t i l l e d water  25-X  d i s t i l l e d water  19.25  10 u l  tetramethylethylenediamine  10 u l  tetramethylethylenediamine  .  stacking gel  The r e s e r v o i r b u f f e r c o n s i s t e d o f 0 . 0 2 5 0.192  Vol (ml)  M T r i s - H C l pH 8.3,  M g l y c i n e , 0 . 1 % SDS while sample b u f f e r was composed o f 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 b l u e , and 10% (v/v) B'-mercaptoethanol ( t h i s  last  -6 3  i n g r e d i e n t was not present  i n the f i r s t  were c r o s s - l i n k e d w i t h DSP or DTBP). formed a t a constant e i t h e r 1 h r (0.75 S t r i p s (0.75  current  dimension when samples  E l e c t r o p h o r e s i s was per-  o f 3° mAmps:per s l a b g e l f o r  mm t h i c k g e l s ) or 2 h r (1.5  mm t h i c k g e l s .  mm t h i c k by 4 mm wide) of g e l were c u t from the  s l a b f o r e l e c t r o p h o r e s i s i n the second dimension and the r e mainder of the g e l was s t a i n e d . E l e c t r o p h o r e s i s i n the second dimension was performed w i t h a s e p a r a t i o n g e l (9 cm high,  14 cm wide, and 1:5  top of which was a 2 cm s t a c k i n g g e l .  mm t h i c k ) on  The s e p a r a t i o n and s t a c k -  ing  g e l s were o f the same composition as i n the f i r s t  The  s t r i p of g e l (0.75  the separated  mm t h i c k and 4 mm wide) which  p r o t e i n s i n one dimension was p l a c e d  s t a c k i n g g e l and o v e r l a i d w i t h warm (60 t a i n e d 1$  (w/v) agarose, 10$  T r i s - H C l pH 6.8, and 0.1$ a constant  current  on t o p of the  (v/v) B-mercaptoethanol, 0.125  SDS.  M  E l e c t r o p h o r e s i s was performed a t  of 30 mAmps per s l a b g e l f o r 3 h r .  Coomassie blue and 50$  E.  contained  C) b u f f e r which con-  Slab g e l s were s t a i n e d w i t h a s o l u t i o n of 0.1$  of 0.75  dimension.  (w/v)  (w/v) t r i c h l o r a c e t i c a c i d f o r a p e r i o d  h r and d e s t a i n e d  overnight  w i t h 7.5$  (v/v) a c e t i c a c i d .  Q u a n t i t a t i o n of R a d i o a c t i v i t y Contained by P r o t e i n s on " ' - - P o l y a c r y l a m i d e Gels C y l i n d r i c a l g e l s were f r o z e n i n a d r y ice/acetone  s l i c e d i n t o 1 mm s l i c e s w i t h a Bio-Rad g e l s l i c e r .  bath and  Each s l i c e  was p l a c e d i n a s c i n t i l l a t i o n v i a l and d i g e s t e d w i t h 0.5 ml 2$ (w/v) p e r i o d i c a c i d . D i g e s t i o n was complete a f t e r 1 h r .  -64-  Periodate  digested  scintillation  fluid  slices  were suspended i n 10 ml of ACS  (Amersham-Searle) and counted i n a Nuclear  Chicago Isocap 3 0 0 s c i n t i l l a t i o n Slab g e l s t h a t contained  spectrophotometer.  p r o t e i n s l a b e l l e d with[  slMet  were d r i e d under vacuum on Whatman #1 f i l t e r paper and autoradiographed on Kodak RP Royal X-Omat RP/R14 X-ray Radioactive  film.  bands were c u t from the g e l s and d i g e s t e d  n i g h t i n 0 . 5 ml o f IN NaOH. added f o r the p r e v e n t i o n  over-  G l a c i a l a c e t i c a c i d ( 1 0 0 u l ) was  of chemiluminescence.  Ten ml o f ACS  s c i n t i l l a t i o n f l u i d was added t o each sample and the r a d i o activity F.  was q u a n t i t a t e d  on a Nuclear Chicago Isocap 300 counter.  I s o l a t i o n o f R a d i o a c t i v e l y Labeled  Extracellular Virus  V i r u s r e l e a s e d i n t o the growth medium was i s o l a t e d by the method of Scheele and P f e f f e r k o r n ( 2 4 5 ) .  Virus  ( 1 0 ml) from  the microsomal experiments was l a y e r e d onto a three-phase gradient  t h a t c o n s i s t e d (from bottom t o top)  of 2 ml of 5 0 $  (w/w) sucrose ( i n 0.2 M C s C l and 0 . 0 0 2 M T r i s / H C l , pH 7 . 8 ) , 4 ml o f a continuous g r a d i e n t t h a t c o n s i s t e d o f 2 5 $ (w/w) sucrose ( i n O . I 3 7 M NaCl, 2.68 mM KCl, 1 . 4 7 mM KHgPO^, and 4.29  mM NagHPO^, a d j u s t e d  t o pH 8 with 1 N NaOH) t o 5 0 $ (w/w)  sucrose ( i n 0.2 M C s C l and 0 . 0 0 2 M T r i s / H C l , pH 7.8), of 5 t o 2 0 $ sucrose ( i n O . I 3 7 M NaCl, 2.68 mM KCl, KH P0^, and 4.29 mM NagHPO^, a d j u s t e d 2  and 4 ml  1.47 mM  t o pH 8 with 1 N NaOH).  These g r a d i e n t s were c e n t r i f u g e d a t 1 1 6 , 0 0 0 x g f o r 4 hi-in an SW 27 r o t o r .  -65-  V i r u s ( i n 18 ml of medium) r e l e a s e d during the plasma membrane-endoplasmic r e t i c u l u m pulse-chase experiements  was  i s o l a t e d i n a s i m i l a r manner except t h a t the volumes of the two upper phases were i n c r e a s e d 2.5 G.  times.  P r e p a r a t i o n o f Plasma Membrane and Endoplasmic Plasma membrane and endoplasmic  were prepared by combining (251)  and Warren (252,  Reticulum  r e t i c u l u m from BHK c e l l s  the methods o f A t k i n s o n and Summers  253).  Three d i s h e s o f n e a r l y c o n f l u e n t  BHK c e l l s were each washed twice with 10 ml o f c o l d 10 T r i s / H C l (pH 8.0). with 1.5  C e l l s were scraped with rubber  m l / d i s h of the 10 mM T r i s b u f f e r .  mM  policemen  A t o t a l of 4.5  ml  of c e l l suspension was homogenized w i t h f i v e s t r o k e s of a l o o s e f i t t i n g Dounce homogenizer.  N u c l e i and whole c e l l s were c e n t r i -  fuged from the homogenate a t 1000 of  these p a r t i c l e s was v e r i f i e d by phase c o n t r a s t microscopy.  The supernatant was made 0.25 for  x g f o r 1 min and the absence  M i n sucrose and 5 mM i n MgClg  s t a b i l i z a t i o n o f the membranes.  T h i s 1000  x g supernatant  (3 "to 4 ml) was p l a c e d on a d i s c o n t i n u o u s g r a d i e n t t h a t c o n s i s t e d of  45%  (w/w) sucrose (3 ml) and JOfo  50 mM T r i s / H C l , pH 7.4, fuged a t  7000  (w/w) sucrose (10 ml) i n  and 5 mM MgClg.  The g r a d i e n t was c e n t r i -  x g f o r 20 min and d r i p p e d from the bottom i n  0.5-ml f r a c t i o n s .  The f r a c t i o n s were assayed f o r NADPH c y t o -  chrome c r e d u c t a s e and g l u c o s e -6-  phosphatase  (endoplasmic  r e t i c u l u m marker enzymes) and 5'-nucleotidase ( a plasma membrane marker enzyme).  -66-  NADPH cytochrome c r e d u c t a s e was assayed by measuring the r e d u c t i o n of cytochrome c (246) on a G i l f o r d photometer  a t 550 nm and 25° C.  spectro-  The i n c u b a t i o n mixture con-  s i s t e d of 0.25 ml o f b u f f e r e d s u b s t r a t e (0.1 mM KCN, 0.066 M KCl, 0.044 M phosphate b u f f e r pH 7.6, 0.05 mM cytochrome c) to which 50 u l of enzyme was added.  I n order t o s t a r t the  r e a c t i o n , 50 u l of 0.06 mM NADPH was added.  The a c t i v i t y o f  the enzyme was c a l c u l a t e d as follows-. E  E  550  ° Reduced  6 2 27.7 x 10 cm mole  5 5  5 5  Cytochrome  °  O x i d i z e d Cytochrome  550 - E R e d  c  =  6  c =  9.0 x 10  • . 550 ^ [ R e d Cyt c ] - E d  C  y  t  c  Q  5  d Red C y t c dt  =  d_A dt  5  0  x  i  d  2  cm mole  d C  y  t  , 6 / 18.7 x 10 ml mole  [  R  e  d C  y  t  c  ]  (0.35 ml)  5 ' - n u c l e o t i d a s e was assayed by the method of Avruch and Wallach (247).  The assay mix c o n s i s t e d of 100 u l 0.5 M  T r i s - H C l pH 7-5 and 0.2mM MgClg, 50 u l 0.4 mM AMP, 20 u l o f C^HlAMP, 0.630 u l H 0, and 200 u l o f each f r a c t i o n . 2  was i n c u b a t e d f o r 3° min a t 37° C.  The mixture  Enzyme which was b o i l e d  to the a d d i t i o n o f the assay mixture was used as a c o n t r o l . .  prior 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 u n r e a c t e d ATP was p r e c i p i t a t e d w i t h 0.200 ml of 0.250 M Ba(0H) . 2  The tubes were c e n t r i f u g e d on an I n t e r n a t i o n a l  67  -  centrifuge  f o r 5 min a t 1000 RPM.  supernatant was assayed f o r C H] 3  aliquot  (using  -  A 0.5 ml a l i q u o t of the adenosine  by m o n i t o r i n g the  10 m l / v i a l of T r i t o n X 100: Toluene  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 Glucose - 6 - phosphatase  (1:3) counter.  was monitored by the method of  B a g i n s k i et a l . (248) as m o d i f i e d by Hubscher and West  (249).  -68-  H.  Pulse-Chase Experiments w i t h E n r i c h e d F r a c t i o n s of Endoplasmic 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 (254,  255).  BHK  cells  of i n f e c t e d  cells  (24 P e t r i d i s h e s ) were i n f e c t e d w i t h  v i r u s (20 plaque-forming u n i t s / c e l l s ) i n 199 medium which cont a i n e d 2% f e t a l c a l f serum; the c e l l s were s t a r v e d at 3|- hr a f t e r infection-medium i n each d i s h was  r e p l a c e d w i t h 10 ml.  E a r l e s b a s i c s a l t s medium which c o n t a i n e d 2% d i a l y z e d serum,...  calf  At 4 hr they were p u l s e d w i t h 100 u C i o f L - £ 4 , 5 H ] 3  l e u c i n e / d i s h ( i n 5 ml E a r l e s b a s i c s a l t s medium) f o r 20 min. Subsequently, the l a b e l i n g medium from 3 dishes a t 0-hours chase was  removed and f r o z e n a t -70°C and l a t e r u t i l i z e d f o r A l s o at 0-hours chase, the l a b e l i n g  i s o l a t i o n of v i r u s .  medium i n the d i s h e s (3 per time p o i n t ) f o r the 1-, 4-,  6.5-,  3 » _  and 8 . 5 - h o u r chase times was r e p l a c e d w i t h 7-5  199 medium which was leucine.  2-,  ml of  enriched w i t h 0 . 6 g / l of u n l a b e l e d  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 h a r v e s t e d a t the  designated times.  Plasma membrane and endoplasmic r e t i c u l u m  were prepared as o u t l i n e d i n the p r e v i o u s s e c t i o n .  Membrane  bands were c o l l e c t e d w i t h a Pasteur p i p e t t e from the 3 ° "to 45% sucrose i n t e r f a c e and the top 3 ml of the g r a d i e n t .  Plasma  membrane' and endoplasmic r e t i c u l u m f r a c t i o n s were c e n t r i f u g e d 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 p r o t e i n were a n a l y z e d by electrophoresis.  The g e l s were' s l i c e d and assayed f o r  l e u c i n e as p r e v i o u s l y  outlined.  3  H  -  -69-  I.  Measurement of E x t r a c e l l u l a r V i r u s P r o d u c t i o n i n the Presence of C y t o s k e l e t a l D i s r u p t o r s Initially,  BHK c e l l s were i n f e c t e d w i t h 20  forming u n i t s of SFV per c e l l .  Preliminary  plaque-  experiments were  performed i n which i n f e c t e d BHK c e l l s were l a b e l e d a t 5 hr a f t e r i n f e c t i o n with 50 u C i [ H] l e u c i n e  ( i n 5 ml E a r l e s  3  s a l t s medium which contained ;150  x 15 mm c u l t u r e d i s h .  was removed and r e p l a c e d  2% d i a l y z e d f e t a l c a l f serum) per  A f t e r 1 hr the r a d i o a c t i v e w i t h 10 ml of 199  (with 2% f e t a l c a l f serum) which contained  a f t e r i n f e c t i o n and i s o l a t e d as d e s c r i b e d  5° uM c o l c h i c i n e .  of 20%  (w/w)  (w/w)  a t 10 hr  previously;  (from bottom to top) of 4 ml of 5°%  sucrose, 10 ml of 50%  medium  maintenance medium  T r i t i u m - l a b e l e d v i r u s was harvested from 3 dishes  ient consisted  basic  the grad-  (w/w)  to 25% .(w/w) sucrose, and 10 ml  t o 5%,(w/w) sucrose.  Other experiments were per-  formed i n a s i m i l a r manner except t h a t the c y t o s k e l e t a l d i s ruptor  ( c o l c h i c i n e or dibucaine) was added to the c e l l s a t 3 hr  a f t e r i n f e c t i o n followed ^after i n f e c t i o n .  by the a d d i t i o n of [ H] l e u c i n e 4 hr 3  The l a b e l i n g medium was d i l u t e d with an equal  volume (5 ml) of 199  maintenance medium  (with 2% f e t a l  calf  serum) a f t e r 1 h r .  Radioactive  t a t e d 10 h r l a t e r .  I n a l l cases, l a b e l i n g and p o s t - l a b e l i n g  media contained Control  quanti-  the d i s r u p t i n g drug.  experimentswere performed i n an i d e n t i c a l f a s h i o n  with lumicolchicine. ethanol  e x t r a c e l l u l a r v i r u s was  Lumicolchicine  or c o l c h i c i n e i n 20 u l  was added t o c e l l c u l t u r e s i n f e c t e d with SF vitfus a t 3 h r  post-infection.  Ethanol (20  u l ) was a l s o added to i n f e c t e d  -70-  cell.s which were not  C e l l s were l a b e l e d between 4 and  colchicine.  Maintenance medium (5  ion.  the v i r u s was was  t r e a t e d w i t h e i t h e r c o l c h i c i n e or  was  5 hr of i n f e c -  subsequently added,  h a r v e s t e d a f t e r another k hr.  prepared from 10  mM  (366  light  nm)  and  Lumicolchicine  c o l c h i c i n e i n ethanol  of the s o l u t i o n w i t h UV  (165).  ml)  lumi-  by  irradiation  f o r a p e r i o d of 15  C o n v e r s i o n of c o l c h i c i n e to l u m i c o l c h i c i n e was  hr  monitored  spectrophotometrically. Another m i c r o t u b u l e i n h i b i t o r Nocodazole ( d i s s o l v e d i n dimethylsulfoxide 300  uM  i n the medium) was  infection.  The  between k and and  so as to y i e l d a f i n a l c o n c e n t r a t i o n  a l s o a d m i n i s t e r e d at 3 hr a f t e r  i n f e c t e d c e l l s were l a b e l e d w i t h l ^ H ] l e u c i n e  5 hr p o s t - i n f e c t i o n , and a f t e r a f u r t h e r 10  quantitated  hr. 1$  (minus Nocodazole) a l s o contained J.  of  the v i r u s was The  control  harvested dishes  dimethylsulfoxide.  E f f e c t of C o l c h i c i n e on the I n c o r p o r a t i o n  of !^H]  Leucine  into Acid P r e c i p i t a b l e Protein BHK  c e l l s on 15  mm  x 60  mm  c u l t u r e dishes  were incubated  f o r 1 hr i n E a r l e s b a s i c s a l t s solution/199 maintenance medium (1:1) (50 [-^H]  which contained uM)  was  2% d i a l y z e d c a l f serum.  a d m i n i s t e r e d to h a l f the p l a t e s during  Leucine (25  uCi/dish)  C e l l s were h a r v e s t e d at 0, d i t i o n of l a b e l . phate b u f f e r e d (50  Colchicine  The  was 0.25,  t h i s hour.  then added to a l l the 1,  3,  5 and  dishes.  7 hr a f t e r  c e l l s were washed 3 times w i t h 10  s a l i n e (5),  scraped i n t o 2.5  ml of the  ml  of 10$  (w/v)  ml  phos-  buffer  u l a l i q u o t s were removed f o r p r o t e i n determination)  p r e c i p i t a t e d by a d d i t i o n of 0.5  ad-  and  trichloracetic  -71-  acid. the  P r e c i p i t a t e s were sedimented a t 1 , 0 0 0 x g f o r 5 min,  supernatants were a s p i r a t e d  and d i s c a r d e d , 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 K.  Continuous L a b e l i n g  fluid.  Experiments  Experiments were performed i n a manner s i m i l a r to the p r e v i o u s experiments.  Colchicine  BHK c e l l s a t 3 hr p o s t - i n f e c t i o n . x 15 mm dish) i n dialyzed  5 ml of E a r l e s  ( 5 0 uM) was a d m i n i s t e r e d t o I H]  Leucine ( 5 0 -uCi/150  3  (which c o n t a i n e d 2%  medium  c a l f serum) was added a t 4 h r , and supplemented with  5 ml 199 maintenance medium/dish a t 5 h r . and  Plasma membranes  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 5 i 6 , 8 and 10 hr  after infection.  The membrane samples were s u b j e c t e d "to sodium  dodecyl s u l f a t e 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 7.5% acrylamide g e l s which were c r o s s - l i n k e d tartardiamide; periodic  acid.  on c y l i n d r i c a l  with  diallyl-  the g e l s were s l i c e d and s o l u b i l i z e d w i t h 2% 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 was  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 g r a d i e n t L.  mm  quantitated  sedimentation.  Pulse-Chase Experiments Performed i n the Presence of Colchicine  and Dibucaine  Studies were performed i n which c o l c h i c i n e was added a t 3 hr p o s t - i n f e c t i o n .  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 serum^ f o r 3 ° min. tor (50  s a l t s medium  Subsequently, a pulse of e i t h e r [^H ] l e u c i n e  [ •%']methionine i n Earle's basic 3  uCi i s o t o p e  (• 2% d i a l y z e d . c a l f ...  s a l t s s o l u t i o n was  per 150 mm x 15 mm c u l t u r e  dish).  l a t e r , the l a b e l i n g medium was removed and r e p l a c e d  applied  T h i r t y min w i t h 199  -72-  maintenance medium which was  supplemented with e i t h e r non-  r a d i o a c t i v e l e u c i n e or methionine (0.5 (1 g/ml)  was  present  SDS  [-^H]  l e u c i n e or [-^S]  electrophoresis.  at  the  methionine was  I n c o r p o r a t i o n of  q u a n t i t a t e d by  auto-  or e l u t i o n of the p r o t e i n s from the g e l  determination M.  from c e l l s harvested  and  R a d i o a c t i v e membrane p r o t e i n s were s u b j e c t e d  polyacrylamide  radiography  D  experiment.  endoplasmic r e t i c u l u m fragments  e x t r a c e l l u l a r v i r u s were prepared  to  Actinomycin  i n a l l media throughout the  Plasma membrane ghosts,  s p e c i f i e d times.  mg/ml).  and  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.  P u r i f i c a t i o n of S e m l i k i F o r e s t V i r u s f o r C r o s s - L i n k i n g Ten r o l l e r ^ b o t t l e s of c o n f l u e n t BHK-21 c e l l s  (clone  Studies 13  purchased from Flow l a b o r a t o r i e s ) were i n f e c t e d with SF v i r u s 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 cell  i n 10 ml of 199  - 0.2  plaque forming  u n i t s per  maintenance media [2% f e t a l c a l f serum). Q  Each b o t t l e contained adsorption, was  1-2  40 ml of 199  added to each b o t t l e .  x 10  cells.  maintenance media (2fo f e t a l c a l f serum) V i r u s was  a f t e r 24 hours of i n f e c t i o n .  15,000 x g  F o l l o w i n g 1 hour  harvested  The media was  from the c e l l media  centrifuged at  (10,000 RPM on a S o r v a l GSA r o t o r ) f o r 15 min i n  order to sediment detached c e l l s and  debris.  s u l f a t e was  g r a d u a l l y over a h a l f hour  to  y i e l d 65$  added to the supernatant s a t u r a t i o n at 0°C  (390  added per l i t e r of s u p e r n a t a n t ) . h a l f hour and a pH of about 7.4 a d d i t i o n of 0.66  N NaOH.  The  gm  S o l i d ammonium  of ammonium s u l f a t e were  The medium was was  maintained  p r e p a r a t i o n was  s t i r r e d over t h i s  with dropwise allowed  to  -73p r e c i p i t a t e (without s t i r r i n g ) f o r 1 hour a t 0°C and the prec i p i t a t e was r e c o v e r e d hy c e n t r i f u g a t i o n a t The  15,000  x g f o r 30 min.  ammonium s u l f a t e p e l l e t which was o r i g i n a l l y obtained from  10 b o t t l e s o f i n f e c t e d c e l l s was suspended i n 20 ml o f phosphate s a l i n e pH 7-^  buffered  15,000 The  resuspended p e l l e t was l a y e r e d o f 28 ml of  centrifuged centrifuge  at  65,000  10-45%  material.  onto two g r a d i e n t s  which  (w/w) potassium t a r t r a t e ,and  (25,000  x g  head) f o r 3 h r .  consisted  virus.  and c e n t r i f u g e d a t  f o r 2 min i n order to remove p a r t i c u l a t e  x g  consisted  one  (see s e c t i o n II.F)  was  RPM on a Beckman SW27  Two bands were v i s i b l e - the lower  o f c e l l d e b r i s w h i l e the upper one was p u r i f i e d  The v i r u s band was d i l u t e d t h r e e times with phosphate  buffered  s a l i n e and e i t h e r placed  on another potassium t a r t r a t e  gradient  f o r f u r t h e r p u r i f i c a t i o n , or c e n t r i f u g e d  at  105,000  x g  f o r 2 h r i n order to o b t a i n a v i r u s p e l l e t . N.  C r o s s - L i n k i n g Studies Performed'.'on P u r i f i e d S e m l i k i V i r u s or V i r u s - I n f e c t e d C e l l s  Forest  SF v i r u s was c r o s s - l i n k e d w i t h DMS, DSP, and DTBP i n a manner s i m i l a r to that o f G a r o f f (100  ug p r o t e i n i n 50 u l of 0.15  and Simons  (236,237).  Virus  M NaCl) was subjected  to cross-  l i n k i n g by a d d i t i o n of 50 u l of e i t h e r DMS, DSP, or DTBP i n 0.2  M t r i e t h a n o l a m i n e b u f f e r pH 8.5• . DSP was f i r s t  i n dimethylsulfoxide dimethylsulfoxide  to y i e l d a f i n a l concentration  i n the c r o s s - l i n k i n g r e a c t i o n  dissolved of 2% (v/v)  (250).  The  r e a c t i o n was e i t h e r terminated w i t h the a d d i t i o n of IM T r i s - H C l pH 8.5  (20 u l ) or by immediate a d d i t i o n of sample b u f f e r f o r g e l  electrophoresis  (50  to e l e c t r o p h o r e s i s 6.5%  acrylamide s l a b  ul).  The c r o s s - l i n k e d v i r u s was  on e i t h e r 3-5% gels.  subjected  acrylamide c y l i n d r i c a l g e l s or  -74Whole c e l l s which were i n f e c t e d with S e m l i k i F o r e s t v i r u s were a l s o c r o s s - l i n k e d with DSP and DTBP.  BHK (Stoker McPherson  s t r a i n ) c e l l s were p u l s e - l a b e l e d f o r \ h r with (100  u C i / p l a t e ) a t 4.5  hours a f t e r i n f e c t i o n w i t h SF v i r u s .  R a d i o a c t i v i t y was chased f o r 2 h r i n methionine maintenance media.  r^SjJmethionine  enriched  199  The c e l l s were washed twice w i t h c o l d  phosphate b u f f e r e d s a l i n e pH 7.4  (10  m l / d i s h ) ; 5 ml of 0.15  M  NaCl and 5 ml of t r i e t h a n o l a m i n e b u f f e r pH 8.5 which c o n t a i n e d c r o s s - l i n k e r was added to each p l a t e . f o r . 0.5  to proceed  hr or 1 h r and was terminated by the a d d i t i o n  of 1 ml M T r i s - H C l (pH 8.5) prepared.  The r e a c t i o n was allowed  and plasma membrane ghosts were  P r e p a r a t i o n s i n which the e f f e c t s of c o l c h i c i n e  and d i b u c a i n e were measured c o n t a i n e d the drug throughout the cross-linking  procedure.  -75I I I . RESULTS A.  Resolution  of V i r a l P r o t e i n s  on P o l y a c r y l a m i d e G e l s i n  the Presence of Sodium Dodecyl S u l f a t e S e m l i k i F o r e s t v i r u s was described  i n S e c t i o n II.M.  c u l t i v a t e d and  contained  0.5$  The  g e l system of Weber  samples were i n c u b a t e d  g-mercaptoethanol and  and  i n b u f f e r which  0.5$  of c y l i n d r i c a l g e l s which c o n s i s t e d of 7-5$ presented i n F i g .  as  I n i t i a l l y v i r a l p r o t e i n s were  r e s o l v e d upon the p o l y a c r y l a m i d e Osborn (13).  purified  SDS.  A photograph  acrylamide i s  25.  F i g . 25P u r i f i e d S e m l i k i F o r e s t v i r u s and standard p r o t e i n s which were s u b j e c t e d to e l e c t r o p h o r e s i s on 7-5$ polyacrylamide g e l s i n the presence of sodium d o d e c y l s u l f a t e . V i r u s was p u r i f i e d as o u t l i n e d i n "Methods and M a t e r i a l s " and s o l u b i l i z e d i n sample b u f f e r which c o n t a i n e d a f i n a l c o n c e n t r a t i o n o f 0.5$ g-mercaptoethanol and 0.5$ SDS. V i r u s (10 and 20 ug p r o t e i n ) and standard p r o t e i n s ( p h o s p h o r y l a s e a, b o v i n e serum a l b u m i n , t r y p s i n , and cytochrome c) were s e p a r a t e d on c y l i n d r i c a l g e l s which c o n s i s t e d of 7-5$ p o l y a c r y l a m i d e . P r o t e i n s were s t a i n e d w i t h Coomassie blue. Envelope p r o t e i n s are designated El32 s i n c e t h e y r e s o l v e p o o r l y i n t h i s system and n u c l e o c a p s i d p r o t e i n i s denoted NC. E3 was not d e t e c t e d on these g e l s .  P r o t e i n standards f o r molecular weight c a l i b r a t i o n were a l s o subjected  to e l e c t r o p h o r e s i s .  An absorbance scan o f the g e l  on which 10 ug of p u r i f i e d v i r u s was f r a c t i o n a t e d i s shown i n Fig. 26. resolved  The v i r a l p r o t e i n s , E-^ and Eg» were not c l e a r l y i n this  system.  0  1 •distance from origin (cm)  F i g . 2 6 . Absorbance scan of S e m l i k i F o r e s t v i r u s p r o t e i n s which were separated on a 7 - 5 % p o l y a c r y l a m i d e SDS g e l . The g e l i l l u s t r a t e d i n F i g . 25 ( 1 0 ug of SF v i r u s p r o t e i n ) was scanned at 5 5 0 nm wavelength to y i e l d an absorbance p r o f i l e . Envelope p r o t e i n s are designated E1E2 and n u c l e o c a p s i d i s denoted NC. P u r i f i e d v i r u s was a l s o f r a c t i o n a t e d upon s l a b which were prepared a c c o r d i n g The  to Laemmli and Favre  gels (244).  presence of 8-mercaptoethanol a f f e c t e d the s e p a r a t i o n of  v i r u s proteins  d r a m a t i c a l l y as i l l u s t r a t e d  i n Fig. 27.  -77-  r  ~*  E,  E  L  2  NC  —  IMC  F i g . 27. Proteins from SF v i r u s were subjected to SDS e l e c t r o phoresis on 6.5% polyacrylamide slab gels i n the presence and absence of 10% (v/v) (3-mereaptoethanol. P u r i f i e d v i r u s (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 g e l were s o l u b i l i z e d i n sample buffer which contained 10%(v/v) (5-mercaptoethanol while those on the r i g h t g e l were s o l u b i l i z e d i n sample buffer i n which 3-mercaptoethanol was absent. V i r a l proteins which were electrophoresed i n the presence of 10% 8-mercaptoethanol showed absolutely no separation and E  2  of E,  proteins while the absence of g-mercaptoethanol yielded  two d i s t i n c t protein bands,  g-mercaptoethanol apparently reduced  -78-  i n t r a m o l e c u l a r d i s u l f i d e l i n k a g e s and r e s u l t e d i n a slower m i g r a t i o n o f the v i r u s p r o t e i n s .  Two-dimensional g e l s presented  i n S e c t i o n I I I . F i n d i c a t e that the p r o t e i n band which i s designated Eg co-migrates with E-^ i n the presence o f 10% (v/v) B-mercaptoethanol.  G a r o f f , Simons,  Renkonen (12) have performed  amino a c i d analyses which i n d i c a t e 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 t o the arrangement B.  of d i s u l f i d e  bonds.  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 s e p a r a t i o n o f endoplasmic r e t i c u l u m v e s i c l e s - a n d plasma membrane ghosts from BHK c e l l s was o u t l i n e d i n S e c t i o n II.G.. There was no s i g n i f i c a n t d i f f e r e n c e i n the s e p a r a t i o n on the d i s c o n t i n u o u s g r a d i e n t o f these membranes from mock and i n f e c t e d c e l l s .  Marker enzymes ( 5 ' - n u c l e o t i d a s e f o r  plasma membranes and NADPH cytochrome c reductase f o r endoplasmic r e t i c u l u m ) were assayed f o r the f r a c t i o n s from the g r a d i e n t and the p r o f i l e s o f enzyme a c t i v i t y a r e presented i n F i g . 28. < I t i s evident t h a t the plasma membrane marker ( 5 ' - n u c l e o t i d a s e ) was separated from the endoplasmic r e t i c u l u m marker  (NADPH-  cytochrome c r e d u c t a s e ) . S p e 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 c a l c u l a t e d f o r "'.-the marker  enzymes on other g r a d i e n t s prepared from mock  i n f e c t e d BHK c e l l s .  Enzymes a s s o c i a t e d w i t h endoplasmic  r e t i c u l u m (NADPH-cytochrome c r e d u c t a s e and glucose-6-phosphatase) and plasma membrane ( 5 ' - n u c l e o t i d a s e ) were assayed.  The  a c t i v i t i e s were measured from c e l l l y s a t e , the plasma membrane  PM / ER  1.5  GRADIENT FRACTIONS  f "" +0. 5 /ER  f  D  o  +0.4 3  c E 1.0 +  11 -S *  2  o  z  ER  CD ^  +0  3  3  3  3 CD 3 0  o E  t 30  30%  ~ CD  0.2 xg. _i o  c  0.5  o <-*• AS*  "in  0.1  row CD  PM Ghosts & 45% Fragments  •  ^*X***.KJ.*.^-...*...<i....Ji...J.Ji.^..».  5  10  15  20  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 d i s c o n t i n u o u s sucrose g r a d i e n t used f o r i s o l a t i o n of endoplasmic r e t i c u l u m and plasma membrane. Baby hamster kidney c e l l s were l y s e d and c e n t r i f u g e d a t 1 0 0 0 x g_ to remove n u c l e i . The supernatant was l a y e r e d onto a d i s c o n t i n u o u s sucrose g r a d i e n t (as d e s c r i b e d i n S e c t i o n I I . G ) and c e n t r i f u g e d a t 7000 x g f o r 20 min. T h i s g r a d i e n t was d r i p p e d from t h e bottom o f the tube, 0.5 ml f r a c t i o n s were c o l l e c t e d and enzymes assayed. • : : • , 5' - n u c l e o t i d a s e (marker f o r plasma membrane);" •, NADPH-cyochrome c reductase (marker f o r endoplasmic r e t i c u l u m ) .  -80-  fraction,  and  endoplasmic r e t i c u l u m f r a c t i o n  p e l l e t and s u p e r n a t a n t ) .  The r e s u l t s  a  S  CQ H ft  M 0  CD CQ £  T}  U  O CD 3 CQ TJ Cd  cd ft  3 H O  •H -P  O  •H  E  CQ H  ft o  0  U  <-l  ft  O  CD fl  O cd  U  o x> •H -P e XI Crf b-l  CQ CD  •H M  CD -P O cd H  cd  o CQ CD  -P O 0 CD d CD -P X> U • 0 H cd O CQ CD H £ H O ^ >> fl U fl H O CQ -fl I •H fl O H -P O OU>H o •H -P o cd >> H O cd C/2 O CD B tH fl o H cd  S  X!  CD fl  a u X> x>  s  H  0  'H  o  CQ CD  •H -P  •rH J>  •H •P O  <:  Csl CD  a .  LU CO < r< X  e  CO  CD  CD  T3 0  cd SS  3  U  o  a. co o J:  tH H  S  p< <_>  i  O  •H  LU  0  o  c  •H  co  •P  CD  O  u  CQ OQ H co cd C cd 0 CD Cd TJ b ft •p H CQ •P 0 T ) cd fl ! H fl 0 H 0 ft cd £ CD  S  ft-H TD  1  o ^ e 0  •H  C 0Q  fl ^ CQ CQ •H cd W)cd cd •P H -P •H  < CJ  fl 0  -P fl cd o -p T3 ft-P cd fl CQ O o cd flfl 0 o XJ  o -P  •P  ft i  •H VO  0  £ I  H  O  0  CO  CQ cd H  3  o o  a  ft o O CQ  H  •H  0  CQ •P ft O cd CQ  0  •H  Ti  o  Z> Q_  ^•H  •H  O  CO  ft cd  3  H  fl  x! •P o  -H  C •H  >  0  .fl -p  Tj  H  H  LTl  f ^d)  cd  0  OJ  S  ^  CD  i n Table  H  CD  o  <  o o o o o  CD  fl O  cd  are presented  X  CD XI •P  (both the 100,000 x g  -  LA  r-H  U tH 13 fl 0  fl  s: >• • - e  1- cn  O  LA  cd  -P  0  cd  cd X> CQ CQ CQ fl CQ  0  0  < CO >-  0  ft  cd cd cd LU  OJ  rA  co  0  _1  LU C_5  -81-  Plasma membrane appeared t o be p u r i f i e d 37-fold w i t h minimal contamination due to endoplasmic r e t i c u l u m .  The y i e l d o f  plasma membrane from the o r i g i n a l l y s a t e was  35•3% £ 5'58%  (S.D., N' = 3)-  The endoplasmic r e t i c u l u m was h i g h l y e n r i c h e d  as judged by the NADPH-cytochrome c reductase a c t i v i t y ( 157-fold p u r i f i c a t i o n ) but l e s s e n r i c h e d a c c o r d i n g t o the r e s u l t s w i t h glucose-6-phosphatase  (6-fold).  I n a d d i t i o n the endoplasmic  r e t i c u l u m was contaminated w i t h 5 ' - n u c l e o t i d a s e a c t i v i t y . C.  R a d i o a c t i v e Pulse-Chase Experiments with Endoplasmic Reticulum and Plasma Membranes I n an e a r l i e r study we demonstrated a p r e c u r s o r - p r o d u c t  r e l a t i o n s h i p between t h e v i r a l p r o t e i n s i n the microsomes and i n e x t r a c e l l u l a r v i r u s ( 254).  We wanted t o r e f i n e the experiment  t o see whether such a r e l a t i o n s h i p might e x i s t between the v i r a l p r o t e i n s i n the plasma membrane and r e l e a s e d v i r i o n s .  An  experiment s i m i l a r t o the microsomal experiment w i t h a 6-hour chase was performed except t h a t f r a c t i o n s enriched with plasma membrane and endoplasmic r e t i c u l u m were i s o l a t e d from the c e l l s . P r o t e i n s from the membranes and v i r u s were analyzed by 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 g e l s i n the presence o f sodium  dodecyl s u l f a t e .  P r o f i l e s showing [ H ] l e u c i n e i n c o r p o r a t i o n 3  and Coomassie b l u e scans were p l o t t e d f o r the endoplasmic r e t i c u l u m and plasma membrane f r a c t i o n s a t 0, 1, and 8.5  hours chase ( F i g s . 29 and 3°)•  2, 3» ^>  A. r a d i o a c t i v e  6.5,  profile  of the p r o t e i n s from endoplasmic r e t i c u l u m a t 0-hours chase shows the e x i s t e n c e o f v i r a l p r e c u r s o r p r o t e i n s - NVP  130,  .  0-8.5  HR  -82-  C H A S E  ER  GELS  F i g . 29I n c o r p o r a t i o n of E H"]leucine i n t o the p r o t e i n s of endoplasmic r e t i c u l u m over a 0 - 8 . 5 hour chase p e r i o d . 3  I n f e c t e d baby hamster kidney c e l l s were pulsed f o r 2 0 min with p H J l e u c i n e 4 hours a f t e r i n f e c t i o n . Endoplasmic r e t i c u l u m and plasma membrane were separated and f r a c t i o n a t e d as shown i n F i g . 28 a t 0 , 1 , 2 , 3» ^ » 6 . 5 , and 8 . 5 hours f o l l o w i n g removal of [ 3 H ] l e u c i n e . P r e c u r s o r p r o t e i n s (NVP 1 3 0 , NVP 9 8 , and PE2), envelope p r o t e i n s ( E l and E 2 ) , and n u c l e o c a p s i d p r o t e i n (NC) were c l e a r l y e v i d e n t . • • , [3H]leucine, counts per min; , A550, Coomassie blue s t a i n .  -83-  1  2  3 Distance  4  5  6  f r o m Origin  2  7  3 Distance  (cm)  ER  4 from  5 Origin  6 (cm}  6.5 h o u r s NC  chase  -  /A  'o 31 X  e  V  3 2|  •x  1  •  i  {•""N\ A/1  '\ \  f\ j Ma 1 \l  2  t  il  3 Distance  4 from Origin  i  L 5  \  6 (cm)  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  Distance from Origin (cm)  HR  C H A S E  P M  GELS  Distance from Origin  &m)  F i g . 30. I n c o r p o r a t i o n o f [^HJileucine i n t o the p r o t e i n s of the plasma membrane 0-8.5 hours a f t e r a 20 min p u l s e . The experiment d e s c r i b e d i n F i g . 2 9 was performed and plasma membrane was i s o l a t e d from the JO-k^fo sucrose i n t e r f a c e o f the d i s c o n t i n u o u s g r a d i e n t used f o r s e p a r a t i o n o f plasma membrane and endoplasmic r e t i c u l u m . A f t e r u l t r a c e n t r i f u g a t i o n the plasma membrane p r o t e i n s were s u b j e c t e d to sodium d o d e c y l s u l f a t e acrylamide g e l e l c t r o p h o r e s i s , scanned f o r Coomassie b l u e , and assayed f o r [ 3 H ] l e u c i n e . • ; • , [3H]leucine counts per min; , A550 of Coomassie b l u e .  -85-  2  3 4 5 6 Distance from Origin (cm)  2  7  3 4 5 6 Distance from Origin (om)  . 8.5 hours chase  PM  •  mi rap /  NC /  2  MK.  \  K.  \  3 4 5 6 Distance from Origin (cm)  Fig. 3 0 . Incorporation of [ H]leucine into the proteins of the plasma membrane 0 - 8 . 5 hours a f t e r a 20 min pulse (continued). 3  -86-  NVP  '"98,  ( F i g . 29)  and  PEg  - which have been detected by other l a b o r a t o r i e s  (65-74).  nucleocapsid  In a d d i t i o n envelope p r o t e i n s (E-j^ and  were c l e a r l y present.  envelope nucleocapsid  hour chase p e r i o d .  There  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  p r o t e i n s i n c e the i n t e n s i t y of the Coomassie blue  peak t h a t corresponded to n u c l e o c a p s i d the chase p e r i o d . and  and  whereas the r a d i o a c t i v i t y i n the  p r o t e i n f a i l e d to d e c l i n e over the 8 . 5 was,  and  During the subsequent chase  p e r i o d , r a d i o a c t i v i t y a s s o c i a t e d with p r e c u r s o r p r o t e i n s diminished,  Eg)  protein increased  during  These r e s u l t s were q u a n t i t a t e d i n F i g s .  31  32.  Time After  Pulse (Hours)  F i g . 31Time course f o r i n c o r p o r a t i o n of T ^ H l 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 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 with the endoplasmic r e t i c u l u m . Endoplasmic r e t i c u l u m was i s o l a t e d from i n f e c t e d c e l l s at v a r i o u s times a f t e r a p u l s e 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 p r o t e i n s was p l o t t e d a g a i n s t time, o o, P E 2 ; A , NVP 9 8 ; 0 O, NVP 130. A  F i g . 32. Time .course f o r i n c o r p o r a t i o n of [3}i]leucine 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 with the endoplasmic r e t i c u l u m . Endoplasmic r e t i c u l u m was i s o l a t e d from i n f e c t e d c e l l s a t v a r i o u s 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 p r o t e i n s was p l o t t e d a g a i n s t time. • • ', envelope p r o t e i n s ( E j and E 2 ) ; , n u c l e o c a p s i d (NC) protein. A  A  A n a l y s i s o f the p r o t e i n s from the plasma membrane i n d i c a t e d that there was a dramatic i n c r e a s e i n l a b e l e d envelope p r o t e i n s (E-^ and Eg) and n u c l e o c a p s i d  p r o t e i n during the f i r s t 3 hours  of the chase p e r i o d ( F i g s . 30 and 33).  Subsequently a decrease  i n the l a b e l e d plasma membrane p r o t e i n s (E-^, Eg, and  nucleocapsid)  occurred with a concomitant i n c r e a s e i n l a b e l e d v i r u s i n the c e l l medium ( F i g . 33)'  Profiles f o r radioactive extracellular virus  which were p l o t t e d f o r the a p p r o p r i a t e Fig.  34..  chase times are shown i n  -88-  Time After Pulse (Hours) F i g . 33- Time course f o r i n c o r p o r a t i o n o f T^H] 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 with plasma membrane and e x t r a c e l l u lar virus. Plasma membrane and v i r u s were i s o l a t e d from i n f e c t e d c e l l s a t v a r i o u s times a f t e r the p u l s e 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 p r o t e i n s was p l o t t e d a g a i n s t time. Plasma membrane y i e l d was estimated to be 3 5 * 3 + 5 ' 5 8 $ (S.D., N = 3) and only t h r e e - f o u r t h s o f the plasma membrane i s o l a t e d was a p p l i e d to each g e l . I n order to normalize the data f o r the e x 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 a s s o c i a t e d with e x t r a c e l l u l a r v i r u s was m u l t i p l i e d by ( O . 3 5 3 x 0 . 7 5 ) . • • , Envelope p r o t e i n s ( E i and E 2 ) ; * n u c l e o c a p s i d ( NC) protein; • • , virus. 1  A precursor-product r e l a t i o n s h i p  between envelope p r o t e i n s  (E-^ and E^) i n the endoplasmic r e t i c u l u m , plasma membrane, and extracellular virus  i s evident from F i g s . 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 p r o t e i n s o f the  -89-  8.5 HR CHASE  15  S)  3B  F i g . Jk. P r o f i l e s of sucrose 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 which was produced over a 0-8.5 hour chase. The experiment d e s c r i b e d i n F i g . 28 was performed and e x t r a c e l l u l a r v i r u s was h a r v e s t e d from the medium as d e s c r i b e d i n S e c t i o n I I . F a t 0,1,2,3,4,6.5. and 8.5 hours chase. Media from 3 d i s h e s (18 ml) was p l a c e d on the g r a d i e n t which was then c e n t r i f u g e d . The tube was then dripped from the bottom and 0.25 ml f r a c t i o n s were c o l l e c t e d and 5° u l a l i q u o t s 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 r e t i c u l u m occurred between 0 and 3 hours while these l a b e l e d p r o t e i n s i n c r e a s e d over the same i n t e r v a l i n the plasma membrane.  8.5  Between 4 and  r a d i o a c t i v i t y , t h e r e was  hours a f t e r the pulse of  a rapid decline i n radioactive v i r a l  p r o t e i n s which were a s s o c i a t e d with the plasma membrane, w h i l e there was  a marked i n c r e a s e i n the r a d i o a c t i v i t y i n the e x t r a -  cellular virus.  The  r i s e and  l o p e p r o t e i n s (E-^ and  of r a d i o a c t i v i t y of the enve-  Eg) and n u c l e o c a p s i d p r o t e i n of the plasma  membrane appeared to be D.  fall  parallel.  C y t o s k e l e t a l D i s r u p t o r s and T h e i r E f f e c t s Upon V i r u s Assembly 1) We  The  E f f e c t of C o l c h i c i n e on V i r u s  t e s t e d the p o s s i b l e involvement  Maturation  of microtubules  the budding process by a d d i t i o n of c o l c h i c i n e to BHK i n f e c t e d with Semliki Forest v i r u s .  and  o b s e r v a t i o n s through a phase-contrast  a p e r i o d of 24 hours;  hours (Table 8 ) .  strated l i t t l e Initially, [ H] 3  was  fibroblasts microscope  over  c e l l s i n c o n t a c t with c o l c h i c i n e (at  c o n c e n t r a t i o n s of 20uM - 100 uM) 15  cells  An a p p r o p r i a t e dosage  determined on the b a s i s of previous s t u d i e s with (137)  in  appeared v i a b l e f o r at l e a s t  C e l l s i n c o n t a c t with c o l c h i c i n e demon-  i f any rounding  i n t h i s c o n c e n t r a t i o n range.  the i n f e c t e d c e l l s were incubated  with  l e u c i n e f o r 1 hour at 5 hours a f t e r i n f e c t i o n with S e m l i k i  Forest v i r u s .  Subsequently, the medium was  with maintenance medium which c o n t a i n e d v i r u s was  harvested  removed and  50 uM c o l c h i c i n e .  at 10 hours a f t e r i n f e c t i o n and  Since c o l c h i c i n e d i s r u p t s  Labeled  isolated  v e l o c i t y g r a d i e n t c e n t r i f u g a t i o n . V i r u s p r o d u c t i o n was by 73% ( F i g . 3 5 ) -  replaced  by  inhibited  microtubules  -91-  Table 8 E f f e c t s of v a r i o u s c o n c e n t r a t i o n s of c o l c h i c i n e on c e l l (As determined by phase c o n t r a s t microscopy).  shape  BHK-21 c e l l s were incubated i n 199 maintenance media which  contained 0-400 uM c o l c h i c i n e . Observations of the c e l l c o n d i t i o n were made a t 0, 15 min, 3° min, 1 h r , 2 h r , 4 hr, 10 h r , 15 hr, and 24 h r . x denotes t h a t the c e l l s were detached from the s u r f a c e of the c u l t u r e d i s h , r denotes t h a t the c e l l s were rounded, - designates no drug e f f e c t . .  c o l c h i c i n e uM  Q  Q  ^  I n c u b a t i o n Time With C o l c h i c i n e (Hr) ^ .0 k.O 10.0 15.O 24.0 Q  Q  1  >  Q  2  400  r  x- '  x  200  r  r  x  -  -  r  -  -  r  -  -  r  100 75 50 25 10 5 2 0.5 0.25 0.10  -92Control  1.5 +  1.0 +  -  0.5  E a o  10  3 CD  15  20  25  Colchicine 50uM  X 1.0 +  0.5  10  15 20 FRACTION NO.  F i g . 35P r o f i l e s of sucrose 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 produced by c e l l s i n the presence and absence of c o l c h i c i n e . I n f e c t e d c e l l s were incubated with [3}i]leucine f o r 1 hour a t 5 hours p o s t - i n f e c t i o n w i t h SF v i r u s . The medium was removed and r e p l a c e d w i t h maintenance medium which contained 50 uM colchicine. Labeled v i r u s was harvested a t 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 g r a d i e n t c e n t r i f u g a t i o n . The tube was d r i p p e d from the bottom i n 0.25 ml f r a c t i o n s and 50 u l a l i q u o t 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.  -93s l o w l y (168), subsequent experiments were performed i n which the drug was added a t 3 hours and [ H] l e u c i n e ..'at 4 hours a f t e r 3  infection.  I n t h i s i n s t a n c e p r o d u c t i o n of [ % ] - l a b e l e d v i r u s  was reduced 86$ with 50 uM c o l c h i c i n e 10 hours a f t e r the a d d i t i o n of l a b e l ( F i g . 36).  Fraction  No.  F i g . 36. P r o f i l e s of sucrose 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 produced by c e l l s i n the presence and absence of c o l c h i c i n e . C o l c h i c i n e (50 uM) was added t o i n f e c t e d c e l l s [3H]leucine a t 4 hours a f t e r i n f e c t i o n . Media (18 ml) was p l a c e d on a sucrose g r a d i e n t a f t e r c e n t r i f u g e d , and the tube was d r i p p e d from the fractions. A l i q u o t s (200 u l ) were assayed f o r s c i n t i l l a t i o n counting.  a t 3 hours and from these c e l l s 14 hours i n f e c t i o n , bottom i n 0.25 ml r a d i o a c t i v i t y by  - 49  This i n h i b i t o r y e f f e c t was d i r e c t l y observable on sucrose gradients which were layered with media from infected  cells  (Fig. 37); the v i r u s band obtained from the c o l c h i c i n e - t r e a t e d c e l l s i s markedly f a i n t e r than the band from untreated c e l l s .  Control • Colchicine  F i g . 3 7 . Photograph of sucrose gradients which contain v i r u s produced i n either the absence or presence of c o l c h i c i n e ( 5 0 uM) . BHK c e l l s were infected with 20 plaque-forming units of SF v i r u s per c e l l . Colchicine was added to the 199 maintenance medium a f t e r 3 hours 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 at 14 hours p o s t - i n f e c t i o n . Media (18 ml) from three 15 mm x 150 mm plates was layered onto each sucrose gradient.  -95I t was necessary f o r us to e s t a b l i s h as f a r as p o s s i b l e 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 t o m i c r o t u b u l e d i s r u p t i o n and not due to a s i d e e f f e c t of the drug (such as a p o s s i b l e i n h i b i t i o n of. n u c l e o s i d e t r a n s p o r t ) .  L u m i c o l c h i c i n e was prepared  from c o l c h i c i n e (as o u t l i n e d i n S e c t i o n I I . I ) and the c o n v e r s i o n was monitored s p e c t r o p h o t o m e t r i c a l l y over 12 hours ( F i g . 3 8 ) .  1.0 + Lumicolchicine Colchicine  0.8 +  a, °' + o 6  c  CO -Q  o 0.4 w  <  0.2 +  200  300 400 wavelength nm.  Minimum!  500  F i g . 3 8 . Absorbance spectrum f o r c o l c h i c i n e and l u m i c o l c h i c i n e a t room temperature. L u m i c o l c h i c i n e was prepared from c o l c h i c i n e as o u t l i n e d i n Section II,I. The change i n absorbance spectrum was monitored a f t e r c o l c h i c i n e was i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t f o r 15 '• hours a t room temperature.  -96Subsequently, microtubules  lumicolchicine, ( 165)  There was l i t t l e  which i s unable to d i s r u p t  and c o l c h i c i n e were administered a t 20 uM.  or no e f f e c t of l u m i c o l c h i c i n e  on the p r o d u c t i o n  of l a b e l e d v i r u s ( F i g . 39).  4  8  Fraction N o .  1 8  2  2  F-ig. 39 • E f f e c t s of l u m i c o l c h i c i n e and c o l c h i c i n e on the production of radioactive e x t r a c e l l u l a r v i r u s . L u m i c o l c h i c i n e (20 uM) and c o l c h i c i n e (20 uM) were administered t o i n f e c t e d c e l l s a t 3 hours i n f e c t i o n . The c e l l s were then l a b e l e d with [3H"]leucine a t 4 hours i n f e c t i o n . R a d i o a c t i v e medium was removed and [ H ] - l a b e l e d v i r u s was harvested a t 8 hours i n f e c t i o n . The v i r u s was i s o l a t e d on sucrose g r a d i e n t s and the tubes were d r i p p e d from the bottom i n t o O.30 ml f r a c t i o n s . A l i q u o t s ( 50 u l ) 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. 3  -97-  Nocodazole ( 1 6 7 ) ,  a t 300 uM reduced l a b e l e d v i r u s p r o d u c t i o n by  85% ( F i g . 40).  Control  Fraction Number  Nocodazole 3 0 0 u M  (p.25ml/tube)  F i g . 4 0 . E f f e c t of Nocodazole on the p r o d u c t i o n of r a d i o a c t i v e extracellular virus. Nocodazole ( 3 uM) was administered t o i n f e c t e d c e l l s a t 3 hours i n f e c t i o n ; c e l l s were then l a b e l e d w i t h [3H]leucine a t 4 hours infection. R a d i o a c t i v e medium was supplemented w i t h an equal amount of maintenance medium a f t e r 1 hour. [3H']-Labeled v i r u s was harvested a t 10 hours i n f e c t i o n and i s o l a t e d on sucrose gradients. A l i q u o t s ( 2 0 0 u l ) o f the 0 . 2 5 ml f r a c t i 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. 0 0  We a l s o wanted evidence t h a t c o l c h i c i n e was not a f f e c t i n g protein synthesis  or the t r a n s p o r t  BHK c e l l s were l a b e l e d with  of [3H]leucine i n t o the c e l l .  [3H]leucine ( i n the presence or  absence of 5 ° uM c o l c h i c i n e ) and the i n c o r p o r a t i o n  into  material  t h a t was p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d was monitored.  -98-  There was no observable e f f e c t by c o l c h i c i n e on [^Hjleucine incorporation  over 8 hours ( F i g . 41).  0  -i i i i i • 1 2 3 4 5 6 7 Time of Incubation With ( H)~Leu (Hours)  i  3  F i g . 41 I n c o r p o r a t i o n of {3rl]leucine 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 from BHK c e l l s i n the presence or absence of colchicine. C e l l s were i n i t i a l l y incubated f o r 1 hour i n E a r l e ' s b a s i c s a l t s s o l u t i o n / 1 9 9 maintenance medium (1:1); one-half the dishes c o n t a i n e d c o l c h i c i n e (50 uM) . . [3rl]Leucine (25 u C i / d i s h ) was addied to a l l the dishes and c e l l s were h a r v e s t e d a t 0, 0.25, 1, 3, 5, and 7 hours. The l a b e l e d c e l l s were suspended i n 2.5 ml b u f f e r and p r e c i p i t a t e d by a d d i t i o n of 10$ (w/v) t r i c h l o r o a c e t i c a c i d . P r e c i p i t a t e s were resuspended i n s c i n t i l l a t i o n f l u i d . Cells t r e a t e d w i t h (v v) or without c o l c h i c i n e (• • ).  -99A l l the above data i n d i c a t e that c o l c h i c i n e i n h i b i t s v i r u s p r o d u c t i o n by d i s r u p t i o n o f m i c r o t u b u l e s .  The next q u e s t i o n was  a t what stage o f v i r u s assembly d i d the i n h i b i t o r a c t .  Hence, we  performed a s e r i e s o f 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, c o l c h i c i n e (50 uM) was added a t 3 hours a f t e r i n f e c t i o n .  The £ Hjleucine i n E a r l e ' s 3  medium was added a t 4Jr hours, followed maintenance medium a t 5 hours. v i r u s were harvested a t 0 , 1,  by supplementation with  Plasma membrane and e x t r a c e l l u l a r 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 v i r u s i s o l a t e d on sucrose g r a d i e n t s cells.  I n h i b i t i o n o f v i r u s p r o d u c t i o n occurred over the e n t i r e  l a b e l i n g period. proteins  from c o l c h i c i n e - t r e a t e d and c o n t r o l  .The i n c o r p o r a t i o n  of I ^ H ] l e u c i n e i n t o v i r a l  which were present i n the plasma membrane a t the v a r i o u s  l a b e l l i n g times i s shown i n F i g . 4 3 radioactive v i r a l proteins colchicine-treated cells v i r u s proteins  An accumulation o f  occurred i n the plasma membranes o f  (Fig. 4 4 ) .  These data suggest  reach the c e l l s u r f a c e ,  and that  that  colchicine  i n h i b i t i o n occurs a t a l a t e r stage o f morphogenesis. This p o s s i b i l i t y was t e s t e d f u r t h e r by pulse-chase studies  i n which c o l c h i c i n e was added a t 3 hours a f t e r i n f e c t i o n  w i t h subsequent a d d i t i o n o f e i t h e r [ H ] l e u c i n e or [ ^ S ] m e t h i o n i n e 3  3  a t 4g- hours f o r a pulse p e r i o d  o f 30 min.  either non-radioactive leucine  or methionine was added and the  Medium enriched with  plasma membrane and e x t r a c e l l u l a r v i r u s were harvested a t s p e c i f i e d times during the chase p e r i o d .  F i g . 45 i l l u s t r a t e s  -100-  Control  Colchicine 0 hours post-label  10  50uM 0 hours post-label  5-  -  I  o T-  X  1 hour post-label  10  1 hour post-label  £ a 5u O  _J  3 hours  10  .  h ll  5 hours post-label  1  5  10  \\  post-label  3 hours post-label  •  -  5 hours post-label  5 J  10  20  30  40  -»«»*»«*««* '  10  S. iilTI-l—***  20  ' "  30  '  40  Fraction Number (o.25ml/tube)  F i g . 42. P r o f i l e s o f sucrose 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 produced by c e l l s which were c o n t i n u o u s l y l a b e l e d with [ 3 H ] l e u c i n e i n the presence and absence o f c o l c h i c i n e . C o l c h i c i n e (50 uM) was a d m i n i s t e r e d t o B H K c e l l s a t 3 hours post-infection. [3H] l e u c i n e (50 uCi) i n 5 ml o f E a r l e ' s b a s i c s a l t s medium was added t o each d i s h a t 4 hours, and supplemented w i t h 5 ml 199 maintenance medium a t 5 hours. Plasma., membranes 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 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 u n t r e a t e d c e l l s ( c o n t r o l ) and c o l c h i c i n e - t r e a t e d c e l l s a t 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 g r a d i e n t s .  -101-  Distance from Origin  (cm)  F i g . 4 3 , P r o f i l e s of sodium dodecyl s u l f a t e g e l s which contained plasma membrane p r o t e i n s from c e l l s which were c o n t i n u o u s l y l a b e l e d i n the presence and absence of c o l c h i c i n e . Plasma membranes were i s o l a t e d from i n f e c t e d c e l l s which were incubated i n the presence and absence of c o l c h i c i n e as d e s c r i b e d i n F i g . 4 2 . The p r o t e i n s were f r a c t i o n a t e d on 7-5% p o l y a c r y l a m i d e e l s which were s l i c e d , s o l u b i l i z e d , and the i n c o r p o r a t i o n o f 3H]leucine was q u a n t i t a t e d by s c i n t i l l a t i o n counting. . Precursor p r o t e i n (PEg), envelope p r o t e i n s ( E l and E 2 ) » and n u c l e o c a p s i d p r o t e i n (NC) were present. • • , [3H]leucine, counts per min; , A55° Coomassie blue s t a i n .  -102-  *  a  ^  6 6 Distance from Origin (cm)  r  1  i  5  5  i  S  e  J  B  Distance from Origin (cm)  F i g . 4 3 . P r o f i l e s o f sodium dodecyl s u l f a t e g e l s which contained plasma membrane p r o t e i n s from c e l l s which were c o n t i n u o u s l y l a b e l e d i n the presence and absence o f c o l c h i c i n e ( c o n t i n u e d ) .  -103-  o control Hours Post-Labelling  F i g . 44. Incorporation, o f [ 3 H ] l e u c i n e i n t o the v i r a l p r o t e i n s of the plasma membrane o f c e l l s i n the presence and absence of colchicine. C o l c h i c i n e (50 uM) was a d m i n i s t e r e d t o B H K c e l l s a t 3 hours p o s t - i n f e c t i o n , [3rl]leucine (50 uCi) i n 5 l o f E a r l e ' s medium was added a t 4 hours, and supplemented with 5 ml 199 maintenance medium a t 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 a t 5. °» 8, and 10 hours a f t e r i n f e c t i o n . Membrane samples were s u b j e c t e d t o sodium dodecyl s u l f a t e 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 and the r a d i o a c t i v e v i r a l p r o t e i n s were quantitated. Envelope p r o t e i n s ( E l and E2) i n the plasma membrane i n the presence of c o l c h i c i n e (v v) and without the drug (• • •) . Nucleocapsid i n the plasma membrane i n the presence (• •) and absence (oo) of c o l c h i c i n e . m  -104-  Pig. 4 5 . Plasma membrane p r o t e i n s which were l a b e l e d with I3H"]leucine d u r i n g a pulse-chase experiment which was performed i n the presence and absence of c o l c h i c i n e . C o l c h i c i n e was added t o i n f e c t e d BHK c e l l s a t 3 hours a f t e r i n f e c t i o n with subsequent a d d i t i o n o f [ 3 H ] l e u c i n e a t 4 f hours p o s t - i n f e c t i o n f o r a p u l s e p e r i o d o f 30 min. Medium enriched w i t h n o n - r a d i o a c t i v e l e u c i n e 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 h a r v e s t e d a t 2 , 4 , 6 , and 10 hours chase. The p r o t e i n s were f r a c t i o n a t e d on c y l i n d r i c a l p o l y a c r y l a m i d e (2-5%) g e l s 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 ] l e u c i n e by s c i n t i l l a t i o n c o u n t i n g . • •, £3H]leucine, counts per min; , A55° Coomassie b l u e .  -105-  F i g . 45._ Plasma membrane p r o t e i n s which were l a b e l e d w i t h p H ] l e u c i n e d u r i n g a pulse-chase experiment which was performed i n the presence and absence of c o l c h i c i n e . ( c o n t i n u e d ) .  -106-  the p r o f i l e s a f t e r e l e c t r o p h o r e s i s of the plasma membrane p r o t e i n s prepared 2 ,  4, 6, and 10 hours a f t e r the [ H ] l e u c i n e l a b e l 3  removed while F i g . 46 demonstrates colchicine  2  the i n h i b i t o r y e f f e c t of  on the p r o d u c t i o n of e x t r a c e l l u l a r  Extracellular  3  4  5  6  was  virus.  virus  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 . * • , c o l c h i c i n e ( 50uM) • • , control. E x t r a c e l l u l a r v i r u s was harvested from medium of the pulse-chase exp riment d e s c r i b e d i n F i g . 45.. The v i r u s was i s o l a t e d on s u c r o s e g r a d i e n t s which were d r i p p e d i n 0.25 ml f r a c t i o n s . Aliquots (200 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 v i r u s peaks was summated and p l o t t e d v e r s u s chase time.  - 1 0 7 -  I t i s apparent t h a t t h e r e i s an accumulation o f v i r a l s t r u c t u r a l p r o t e i n s i n the plasma membrane o f the c o l c h i c i n e - t r e a t e d cells  (Fig.4-7).  Plasma Membrane  10h  I  2  i  4  i  6  8  •  10  Chase Time (hours) F i g . 4 7 . Time course f o r i n c o r p o r a t i o n o f "[3H-]leucine 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 with the plasma membrane d u r i n g a pulse-chase experiment which was performed i n the presence and absence o f c o l c h i c i n e . Plasma membranes were i s o l a t e d and subjected t o SDS e l e c t r o p h o r as d e s c r i b e d i n F i g . 4 5 . R a d i o a c t i v e v i r a l p r o t e i n s (envelope p r o t e i n s , E 1 E 2 ; n u c l e o c a p s i d , NC) were q u a n t i t a t e d and p l o t t e d a g a i n s t the chase time. • •, p r o t e i n s l a b e l e d i n the presence o f c o l c h i c i n e ; • • , proteins labeled i n t h e absence o f c o l c h i c i n e .  -108-  0  1  A  2  3  5  7  3  5  7  * . l  0  1  2  c  3  F i g . 48. A u t o r a d i o g r a m o f [ 3 5 s ] - l a b e l e d p l a s m a membrane p r o t e i n s d u r i n g a pulse-chase experiment performed i n t h e presence 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 a d d e d t o BHK c e l l s a t 3 h o u r s p o s t - i n f e c t i o n . T h e s e c e l l s w e r e p u l s e d a t 4.5 h o u r s a f t e r i n f e c t i o n f o r 30 m i n . R a d i o a c t i v e medium was r e m o v e d a n d r e p l a c e d w i t h medium e n r i c h e d with non-radioactive methionine. P l a s m a membrane, e n d o p l a s m i c r e t i c u l u m , a n d e x t r a c e l l u l a r v i r u s w e r e h a r v e s t e d a t 0, 1, 2, 3> 5, a n d 7 h o u r s c h a s e . P l a s m a membrane p r o t e i n s w e r e 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 o n 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 a n d 0.1% SDS ( a s 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 w e r e s t a i n e d w i t h C o o m a s s i e b l u e , d r i e d u n d e r vacuum, and a u t o r a d i o g r a p h e d 3 d a y s . The g e l s a r e o f p l a s m a membrane p r o t e i n s 0, 1, 2, 3, 5. a n d 7 h o u r s a f t e r t h e p u l s e . P l a s m a 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 f r o m u n t r e a t e d c e l l s a p p e a r 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 NVP 9  8  tag  —  0  1  =  2  —  3  5  7  F i g . 4 9 . A u t o r a d i o g r a m o f [ 3 5 s ] - l a b e l e d endoplasmic r e t i c u l u m p r o t e i n s from a p u l s e - c h a s e experiment performed i n t h e presence and absence o f 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 t h e experiment d e s c r i b e d i n F i g . 4 8 . The membranes were s o l u b i l i z e d i n sample b u f f e r which c o n t a i n e d 10$ ( v / v ) 3 - m e r c a p t o e t h a n o l and e l e c t r o p h o r e s e d 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 presence o f sodium d o d e c y l sulfate. The p r o t e i n s (on 2 s l a b g e l s ) were e l c t r o p h o r e s e d s i m u l t a n e o u s l y i n t h e same a p p a r a t u s . G e l s were s t a i n e d , 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. 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 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 ) . The g e l s a r e o f 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 t h e p u l s e .  -110T h i s experiment was r e p e a t e d with [^ S]methidnine as the l a b e l e d v  p r e c u r s o r , plasma  membranes and endoplasmic r e t i c u l u m were  h a r v e s t e d a t 0, 1, 2, 3» 5, and 7 hours chase.  The membrane  samples were s u b j e c t e d to e l e c t r o p h o r e s i s on 7-5%  acrylamide slab  g e l s and autoradioagraphed as shown i n F i g u r e s 48 and 49.  An  accumulation o f r a d i o a c t i v e v i r a l p r o t e i n s i n the plasma membrane of c o l c h i c i n e - t r e a t e d 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 v i r u s bands was q u a n t i t a t e d and p l o t t e d v e r s u s time (Fig.5°) • Plasma  Membrane  C h a s e T i m e (hours) F i g . 50 Time course f o r the i n c o r p o r a t i o n o f "{^^S^methionine i n t o the v i r a l p r o t e i n s o f the plasma membrane from c e l l s which were p u l s e - l a b e l e d i n the presence or absence of 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 pulsed a t 4.5 hours a f t e r i n f e c t i o n f o r 30 min. At v a r i o u s times a f t e r the pulse, i n c o r p o r a t i o n o f • [ ^S]methionine i n t o the v i r a l p r o t e i n s from the plasma membrane was determined a f t e r e l e c t r o p h o r e s i s on SDS p o l y acrylamide g e l s . I n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o envelope p r o t e i n s ( E l , E 2 ) , n u c l e o c a p s i d , and p r e c u r s o r t o E 2 ( P E 2 ) i n the presence (• •) or absence (••—•' — • ) of c o l c h i c i n e (100 uM). 3  -111-  Again the  p r o d u c t i o n of e x t r a c e l l u l a r v i r u s was  Furthermore, the cleavage of PEg inhibited;  (a p r e c u r s o r to Eg)  t h i s p o s t - t r a n s l a t i o n a l cleavage may  r e q u i s i t e f o r budding ( 6 7 , 1 2 1 ) .  inhibited (Fig  be  When endoplasmic  was  a prereticulum  enriched f r a c t i o n s were s u b j e c t e d to e l e c t r o p h o r e s i s , difference  was  little  observed between the c o l c h i c i n e - t r e a t e d  control preparations.  I t would appear t h a t the  also  and  colchicine-  mediated i n h i b i t i o n of SF v i r u s maturation occurs a f t e r v i r a l proteins  reach the plasma membrane of the  infected  the cell.  -112-  Colchicine 100/JM  Control  Ohours chasei  Ohours chase  1 hour chaseH  1 hour chase 2hours chase  2hours  3 hours chase  3hours chase  5 hours chase  5 hours chase  chase  7hours chasei  7 hours chase  20  30  10  20  30  Fraction Number (p.25ml tube) P i g . 5 1 . P r o f i l e s of J ^ ' S l - l a b e l e d e x t r a c e l l u l a r v i r u s produced d u r i n g a pulse-chase experiment which was performed i n the presence and absence o f c o l c h i c i n e . E x t r a c e l l u l a r v i r u s was i s o l a t e d on sucrose g r a d i e n t s f o r the experiment d e s c r i b e d i n F i g . 48. Medium (18 ml) was p l a c e d on each g r a d i e n t , the tube was c e n t r i f u g e d and d r i p p e d from the bottom i n O . 3 0 ml f r a c t i o n s . A l i q u o t s of 2 0 0 u l were assayed f o r 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.  -1132)  The E f f e c t of Dibucaine on V i r u s Maturation We t e s t e d the combined involvement  m i c r o f i l a m e n t s i n the budding BHK c e l l s  process by a d d i t i o n of d i b u c a i n e to  i n f e c t e d w i t h SF v i r u s .  a g a i n determined  of microtubules and  The a p p r o p r i a t e dosage was  on the b a s i s of p r e v i o u s s t u d i e s w i t h  fibro-  b l a s t s ( 1 3 7 ) and o b s e r v a t i o n s through a phase-contrast over a p e r i o d of 2k hours; (50  microscope  c e l l s i n c o n t a c t with d i b u c a i n e  - 200 uM) became s p h e r i c a l but were v i a b l e a t l e a s t 15  hours ( T a b l e 9) . Table 9 E f f e c t s o f v a r i o u s c o n c e n t r a t i o n s of d i b u c a i n e on c e l l shape, (as determined by phase c o n t r a s t microscopy). BHK-21 c e l l s were incubated i n 199 maintenance media which contained 0 - 2000 uM d i b u c a i n e . Observations on t h e c e l l c o n d i t i o n were made a t 0 min, 15 min, 1 h r , 2 h r , k hr, 10 h r , 15 hr, and 2k hr. x denotes t h a t the c e l l s were detached, r denotes t h a t the c e l l s were rounded, - designates no drug e f f e c t .  Dibucaine uM  I n c u b a t i o n Time With Dibucaine (Hr) 0 0.25 0.5 1 2 5 15  2k  2000  -  1000  -  700  -  -  -  -  r  r  r  r  500  -  -  -  -  r  r  r  r  -  r -  r -  r r  r  r  r  r  r  r  r  200  -  -  -  -  r  r  r  100  -  -  -  -  r  r  r  80  _  _  _  _  _  _  60  _  _  _  _  _  _  _  _  _  20  _  _  _  1 0 -  _  I4.O  _  _  _  _  _  _ _  _  _ _  R  r  r  r  _ _  R  _ _  _  -114Dibucaine, which i s thought to d i s r u p t m i c r o f i l a m e n t systems, a l s o The drug was  inhibited virus  production dramatically.  i n c u b a t e d w i t h c e l l s 3 hours a f t e r  [ H] l e u c i n e was 3  medium was  both m i c r o t u b u l e and  added at 4 hours and a d d i t i o n a l  added a t 5 hours.  v e s t e d a f t e r 10  virus  5  maintenance  The e x t r a c e l l u l a r v i r u s was  hours i n f e c t i o n .  p r o d u c t i o n of l a b e l e d  infection,  Dibucaine (100  by 90% ( F i g .  15  uM)  har-  inhibited  52).  25  Fraction No.  F i g . 52. P r o f i l e s of sucrose 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 produced by c e l l s i n the presence and absence of d i b u c a i n e . Dibucaine ( 100 uM) was added to i n f e c t e d c e l l s a t 3 hours and [ 3 H ] l e u c i n e 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 p l a c e d on a s u c r o s e g r a d i e n t a f t e r 14 hours i n f e c t i o n , c e n t r i f u g e d , and the tube was d r i p p e d from the bottom i n 0.25 ml fractions. A l i q u o t s (200 u l ) 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.  -115-  The i n h i b i t i o n was also evident when the gradients were viewed d i r e c t l y (Fig. 53).  Control • Dibucaine  Fig. 53' Photograph of sucrose gradients which contain virus produced i n either the absence or presence of dibucaine (100 uM) . BHK c e l l s 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.  -116-  Plasma membrane was a l s o i s o l a t e d a f t e r 1 0 hours of i n f e c t i o n and membranes from d i b u c a i n e - t r e a t e d c e l l s contained i n c r e a s e d levels of radioactive v i r a l proteins.  Hence, the e f f e c t o f  dibucaine on v i r u s p r o d u c t i o n appeared to be s i m i l a r to t h a t of c o l c h i c i n e ( F i g . 5^)•  Distance From Origin  cm.  F i g . 54. P r o f i l e s o f SDS p o l y a c r y l a m i d e g e l s which c o n t a i n e d plasma membrane p r o t e i n s from c e l l s which were c o n t i n u o u s l y l a b e l e d i n the presence and absence o f d i b u c a i n e . Dibucaine ( 1 0 0 uM) was administered t o BHK c e l l s a t 3 hours post-infection. r3H]leucine ( 5 0 u C i ) i n 5 ml of Earle.'s b a s i c s a l t s medium was added t o each d i s h a t 4 hours, and supplemented with 5 ml 1 9 9 maintenance medium a t 5 hours. Plasma membranes were harvested a f t e r 14 hours i n f e c t i o n . P r o t e i n s were f r a c t i o n ated on 7.$% polyacrylamide c y l i n d r i c a l g e l s which were s l i c e d , s o l u b i l i z e d and q u a n t i t a t e d f o r [3H]leucine by s c i n t i l l a t i o n counting. Envelope p r o t e i n s ( E l and E 2 ) and n u c l e o c a p s i d p r o t e i n (NC) were present. • • , [3H]leucine, counts per min; , A550 Coomassie blue s t a i n .  -117-  F u r t h e r support f o r t h a t c o n c l u s i o n came from a pulse-chase experiment  with d i b u c a i n e , i d e n t i c a l i n form t o t h a t  with c o l c h i c i n e .  BHK-21 c e l l s were i n f e c t e d with SF v i r u s ,  with [-^S]methionine endoplasmic  performed pulsed  and f r a c t i o n a t e d i n t o plasma membrane and  r e t i c u l u m a t 0, 1, 2, 3» 5, and 7 hour chase times.  Autoradioagraphy  was a g a i n performed  a f t e r the membrane p r o t e i n s  were separated by SDS p o l y a c r y l a m i d e e l e c t r o p h o r e s i s . Again we observed an accumulation  o f r a d i o a c t i v e v i r a l p r o t e i n s i n the  plasma membrane o f the d r u g - t r e a t e d c e l l s  ( F i g . 55) •  Cleavage  of PEg t o Eg d i d not seem t o be i n h i b i t e d to a s i g n i f i c a n t by d i b u c a i n e , i n c o n t r a s t to the pulse-chase colchicine.  the endoplasmic  c o n t r a s t between r a d i o a c t i v e p r o t e i n s found i n r e t i c u l u m prepared from c e l l s with or without  However, c o n v e r s i o n of some o f the n o n - v i r i o n  p r o t e i n s t o envelope slightly  with  As observed with c o l c h i c i n e , there does not appear  to be a dramatic  dibucaine.  experiment  extent  and n u c l e o c a p s i d p r o t e i n s may be i n h i b i t e d "  i n the d i b u c a i n e sample ( F i g . 5&)•  The p r o d u c t i o n of  r a d i o a c t i v e e x t r a c e l l u l a r v i r u s was a l s o p r o f o u n d l y i n h i b i t e d (90$)  by t h i s drug  ( F i g . 57)•  Thus, i t seems t h a t d i b u c a i n e  ( l i k e c o l c h i c i n e ) exerts i t s i n h i b i t o r y e f f e c t on v i r u s p r o d u c t i o n a f t e r the v i r a l p r o t e i n s r e a c h the plasma membrane o f the BHK c e l l . Dibucaine d i d not a f f e c t the i n c o r p o r a t i o n o f f / H J l e u c i n e i n t o c e l l p r o t e i n s p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d during a c o n t r o l experiment  which was s i m i l a r t o t h a t performed  c o l c h i c i n e (Table 1 6 ) .  O  with  -118-  NC  B  PE E , E  2  2  F i g . 55Accumulation o f v i r a l p r o t e i n s i n the plasma of c e l l s t r e a t e d with d i b u c a i n e .  membranes  A pulse-chase experiment i d e n t i c a l t o t h a t d e s c r i b e d i n F i g . 4 8 was p e r f o r m e d i n w h i c h 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 a t v a r i o u s t i m e s a f t e r t h e c h a s e a n d t h e 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 t o p o l y a c r y l a m i d e electrophoresis. The s l a b g e l was d r i e d and a u t o r a d l - o g r a p h e d 3 days. P l a s m a membrane p r o t e i n s f r o m d i b u c a 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 u p p e r p a n e l (A) w h i l e t h o s e f r o m u n t r e a t e d c e l l s a p p e a r i n t h e l o w e r p a n e l ( B ) . The g e l s a r e o f p l a s m a membrane p r o t e i n s 0, 1 , 2, 3, 5, a n d 7 h o u r s a f t e r t h e 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  F i g . 5 6 . Autoradiogram of [ 3 5 s j - l a b e l e d proteins from the endoplasmic reticulum of BHK c e l l s exposed to dibucaine. A pulse-chase experiment was performed i n the presence or absence of dibucaine (as described i n F i g . 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 i n the presence of sodium dodecyl s u l f a t e . Autoradiography was again performed f o r 3 days. The top panel (A) i s 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 t h e i r estimated molecular weights (x 10 3) . -  -120Control  Dibucaine  100uM  2.5  0 hours chase  0 hours chase -  2.5  1 hour chase  1 hour chase -  2 hours chase  2 hours chase  3 hours chase  3 hours chase  5 hours chase  5 hours chase  7 hours chase  7 hours chase  o  5  X  E cu  ; L,. A  10  m 15  10  j 5  10  15  Fraction  20  Number  5  10  15  20  (o.5ml/tube)  F i g . 57 • P r o f i l e s of sucrose g r a d i e n t s which c o n t a i n 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 from a pulse-chase experiment performed i n the presence and absence of d i b u c a i n e . E x t r a c e l l u l a r v i r u s was i s o l a t e d d u r i n g the pulse-chase experiments o u t l i n e d i n F i g u r e s 55 and 56 a t 0, 1, 2, 3, 5, and 7 hours chase. Media (18 ml) was p l a c e d on each g r a d i e n t which was c e n t r i f u g e d , dripped i n t o 0.5 ml f r a c t i o n s and 200 u l a l i q u o t s were assayed f o r [35s]methionine.  -121-  Table  10  I n c o r p o r a t i o n of [ 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 from BHK c e l l s i n the presence or absence of d i b u c a i n e . 3  C e l l s were i n i t i a l l y i n c u b a t e d f o r 1 hour i n E a r l e ' s b a s i c s a l t s s o l u t i o n / 1 9 9 maintenance medium ( 1 : 1 ) ; t w o - t h i r d s the d i s h e s c o n t a i n e d d i b u c a i n e (50 uM and 100 uM). [3H]Leucine ( 2 5 u C i / d i s h ) was added to a l l the dishes and c e l l s were.h a r v e s t e d a t 0 , 0 . 5 . 1, 3 , 5 , and 7 hours. The l a b e l e d c e l l s were suspended i n 2 . 5 ml b u f f e r and p r e c i p i t a t e d by a d d i t i o n of 10$ .(w/v) t r i c h l o r o a c e t i c a c i d . P r e c i p i t a t e s were resuspended in scintillation fluid.  Time of I n c u b a t i o n With [ 3 H ] L e u (Hr)  P H ] L e u I n c o r p o r a t e d Into TCA P e l l e t (cpm x 10-°/mg p r o t e i n ) Control  0  50 uM Dibucaine  100 uM  Dibucaine  0.0203  0.0251  0.0218  o.5  0.252  0.214  O.196  1.0  0.356  0.424  0.408  3-0  0.798  O.720  0.765  5-0  1.16  1.20  1.13  7.0  1.41  1.42  1.37  -122-  3) The E f f e c t of C y t o c h a l a s i n The  B on V i r u s M a t u r a t i o n  e f f e c t s of the m i c r o f i l a m e n t  d i s r u p t o r , c y t o c h a l a s i n B,  on v i r u s assembly were a l s o i n v e s t i g a t e d . estimated from previous observations  The c o r r e c t dosage was  work w i t h f i b r o b l a s t s (137)  and by  through a phase c o n t r a s t microscope over 24 hours. with c y t o c h a l a s i n B (1-50  C e l l s i n contact  uM) appeared t o  become s p h e r i c a l but were v i a b l e a t l e a s t t o 15 hours (Table 10)i Table 11 E f f e c t s of v a r i o u s c o n c e n t r a t i o n s o f c y t o c h a l a s i n B on c e l l v i a b i l i t y (as determined by phase c o n t r a s t microscopy). BHK-21 c e l l s were incubated i n 199 maintenance media which contained 0 - 200 uM c y t o c h a l a s i n B. Observations on the c e l l c o n d i t i o n were made a t 0, 5 min, 15 min, 3° min, 1 h r , 2 h r , 4 h r , 10 h r , 15 h r , and 24 h r . x denotes that the c e l l s were detached, r denotes t h a t the c e l l s were rounded, - designates no drug e f f e c t . Incubation Cytochalasin uM  B  0.083  Time With C y t o c h a l a s i n  0.25. ;;0.50 1.0  B (Hr)  2.0  4.0  12.0  r  r  X  X  X  24.0.  200  r  r  r  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  -123-  Coneentrations o f 10 uM and 20 u'M' i n h i b i t e d the p r o d u c t i o n of radioactive  extracellular virus  by 9 0 $ ( F i g . 5 8 ) .  CONTROL  Cytochalasin B  no 8 +  Fraction  Pig- 5 8 . P r o f i l e s of sucrose 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 produced by c e l l s i n the presence and absence of c y t o c h a l a s i n B. C y t o c h a l a s i n B (10 uM) was added t o i n f e c t e d c e l l s a t 3 hours and [ 3 H ] l e u c i n e a t 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 p l a c e d on a sucrose g r a d i e n t a f t e r Ik hours i n f e c t i o n , c e n t r i f u g e d , and the tube was d r i p p e d from the bottom i n 0 . 2 5 ml fractions. A l i q u o t s ( 2 0 0 u l ) 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 s y n t h e s i s  o f r a d i o a c t i v e v i r a l p r o t e i n s was  signif-  i c a n t l y lower i n the c e l l s which were t r e a t e d with t h e drug. Levels  of [ H]leucine  were diminished Further  l a b e l e d envelope and n u c l e o c a p s i d  by 6ofo i n c y t o c h a l a s i n B - t r e a t e d c e l l s  proteins  (Fig. >59).  s t u d i e s were not performed with t h i s drug due t o i t s  seemingly d e l e t e r i o u s  e f f e c t s on c e l l  viability.  v  - 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 g e l s which contained plasma membrane and endoplasmic r e t i c u l u m p r o t e i n s from c e l l s which were c o n t i n u o u s l y l a b e l e d i n the presence and absence o f cytochalasin B . C y t o c h a l a s i n B (10 uM) was administered t o BHK c e l l s a t 3 hours post-infection. [ 3 H ] l e u c i n e ( 5 0 uCi) i n 5 ml of E a r l e ' s b a s i c s a l t s medium was added t o each d i s h a t 4 hours, and supplemented with 5 ml 1 9 9 maintenance medium a t 5 hours. Plasma membranes and endoplasmic r e t i c u l u m were harvested a f t e r 14 hours i n f e c t i o n . P r o t e i n s were f r a c t i o n a t e d on 7 . 5 ^ polyacrylamide c y l i n d r i c a l g e l s which were s l i c e d , s o l u b i l i z e d and q u a n t i t a t e d f o r [ 3 H ] l e u c i n e by s c i n t i l l a t i o n c o u n t i n g . Envelope p r o t e i n s ( E i and E 2 ) and n u c l e o c a p s i d p r o t e i n (NC) were present. • • "", [3H]leucine, counts per min; , A 5 5 0 Coomassie blue s t a i n .  -1264)  Electronmicrographs o f C e l l s I n f e c t e d with S e m l i k i F o r e s t V i r u s i n the Presence o f C o l c h i c i n e and Dibucaine  BHK-21 c e l l s were i n f e c t e d with S e m l i k i F o r e s t v i r u s i n the presence  and absence o f 100  uM c o l c h i c i n e and 100  uM d i b u c a i n e .  The drugs were a d m i n i s t e r e d a t 2 hours i n f e c t i o n and the c e l l s were h a r v e s t e d a t 10 hours,  suspended i n phosphate b u f f e r e d  s a l i n e , and prepared f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . The c o n t r o l samples c o n t a i n e d a c t i v e l y budding v i r u s mature v i r i o n s were present on the o u t s i d e p o r t i o n s o f the plasma membrane and p a r t i c l e s were seen to extend i n t o the e x t r a c e l l u l a r media ( F i g . 60).  Other p o r t i o n s of the plasma membrane showed  v e r y few u n d e r l y i n g n u c l e o c a p s i d s . I n f e c t e d c e l l s which were t r e a t e d w i t h c o l c h i c i n e e x h i b i t e d dense patches  of  n u c l e o c a p s i d s u n d e r l y i n g the plasma membrane  but no a c t i v e l y budding v i r u s appeared  to be present ( F i g . 6 l ) .  M i c r o f i l a m e n t s were a s s o c i a t e d with these p a r t i c l e s to some extent but i t i s hard to a t t a c h any s i g n i f i c a n c e to t h e i r presence. Dibucaine a l s o seemed t o cause an accumulation  of n u c l e o -  c a p s i d s beneath the c e l l s u r f a c e , though the patches were not as dense as those produced  with c o l c h i c i n e .  p a r t i c l e s were a g a i n absent. to yet  Budding v i r u s  T h i s drug has been demonstrated  impair both microtubule and m i c r o f i l a m e n t f u n c t i o n  (139)  m i c r o t u b u l e s and m i c r o f i l a m e n t s d i d not appear to depolymerize  as they a r e s t i l l  seen a s s o c i a t e d with the c e l l membrane ( F i g . 62).  These s t r u c t u r e s must s t i l l be a f f e c t e d d r a m a t i c a l l y s i n c e the c e l l s were s e v e r e l y rounded - m i c r o t u b u l e s and m i c r o f i l a m e n t s  -127-  are known t o r e g u l a t e c e l l shape.  Cytopathic vacuoles  1 and type 2 ) appeared t o be q u i t e frequent i n these  (both  type  cells  but the l a t t e r type c o u l d w e l l be i n d e n t a t i o n s of the plasma membrane which have been c r o s s - s e c t i o n e d ( F i g s . 6 3 and  64).  E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s appear t o -corroborate the b i o c h e m i c a l evidence an accumulation  of v i r a l  p r i o r to the f i n a l were d i f f i c u l t of  - c o l c h i c i n e and d i b u c a i n e appear to cause p r o t e i n s a t the plasma membrane  stages of v i r u s maturation.  These s t u d i e s  t o assess with c e r t a i n t y (due to the l i m i t a t i o n s  e l e c t r o n m i c r o s c o p y ) , but taken t o g e t h e r with the previous  data they appear t o i n d i c a t e t h a t microtubules and m i c r o f i l a m e n t s may be i n v o l v e d i n the budding mechanism.  -128-  Fig. 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 (105,000 x magnification). BHK c e l l s were infected with SF virus at 20 plaque forming units per c e l l . Cells were harvested at 10 hours infection, suspended i n phosphate buffered saline, and fixed i n glutaraldehyde followed by 1% osmium tetroxide.  -129-  Colchicine  Fig. 6 l . Electronmicrograph of infected BHK c e l l s showing accumulation of nucleocapsids at the plasma membrane i n the presence of colchicine (70,000 x magnification). BHK c e l l s were infected with Semliki Forest virus at 20 plaque forming units per c e l l . Colchicine (100 uM) was administered at 2 hours i n f e c t i o n and c e l l s were harvested at 10 hours i n f e c t i o n and fixed and stained as i n F i g . 60.  -130-  Fig. 62. Electronmicrograph of infected BHK c e l l s showing accumulation of nucleocapsids at the plasma membrane i n the presence of dibucaine (70,000 x magnification). BHK c e l l s were infected with Semliki Forest virus at 20 plaque forming units per c e l l . Dibucaine (50 uM) was administered at 2 hours i n f e c t i o n and c e l l s were harvested, fixed, and stained as i n F i g . 60.  -131-  Fig. 63. Electronmicrograph of a cytopathic vacuole found i n infected BHK c e l l s which were treated with dibucaine (50 uM) (105,000 x magnification).  -1  32-  F i g . 64. Electronmicrograph of a cytopathic vacuole found i infected BHK c e l l s which were treated with dibucaine (50 uM) (105,000 x magnification). ;  -133E.  Two-Dimensional C r o s s - L i n k i n g S t u d i e s Performed Upon Purified Virus P u r i f i e d v i r u s was r e a c t e d with b i f u n c t i o n a l  agents  - dimethylsuberimidate  propionate)  (DSP).  cross-linking  (DMS) and d i t h i o b i s ( s u c c i n i m i d y l  I n i t i a l l y samples were r e a c t e d w i t h t h e  a p p r o p r i a t e c r o s s - l i n k e r a c c o r d i n g t o t h e procedure  o f Simons  and G a r o f f (23&) and s u b j e c t e d t o one-dimensional e l e c t r o p h o r e s i s on 3-5$ a c r y l a m i d e c y l i n d r i c a l g e l s . to y i e l d  Both c r o s s - l i n k e r s  appeared  s i m i l a r p a t t e r n s ( F i g . 65). DSP  0.5  i  D M S  2  1  2  3  F i g . 65. P u r i f i e d SF v i r u s which was c r o s s - l i n k e d w i t h DMS and 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 3-5$ p o l y a c r y l a m i d e cylindrical gels. P u r i f i e d v i r u s ( 1 0 0 ug p r o t e i n ) was suspended i n 0.15 M NaCl and c r o s s - l i n k e d w i t h 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 c r o s s - l i n k e d p r o t e i n s were separated on 3-5^ p o l y a c r y l a m i d e c y l i n d r i c a l g e l s i n t h e presence o f SDS, s t a i n e d w i t h Coomassie b l u e , and d e s t a i n e d by d i f f u s i o n i n 7-5^ a c e t i c a c i d . Nucleoc a p s i d monomer (NC), dimer ( N C 2 ) i t r i m e r ( N C 3 ) , t e t r a m e r (NCZj.), pentamer ( N C 5 ) , and hexamer (NC6) were e v i d e n t . Envelope monomer ( E 1 E 2 ) was p r e s e n t but dimer c o u l d not be d e t e c t e d .  -13.4-  G e l scans o f v i r u s which was c r o s s - l i n k e d with DMS ( 3 . 0 mg/ml) and DSP ( 1 . 0 mg/ml) a r e presented i n F i g . 6 6 . Higher s p e c i e s of envelope  cross-linked  p r o t e i n s were not obtained as G a r o f f and Simons  r e p o r t e d under these c o n d i t i o n s  ( 2 3 6 ) .  2-  1  2  2  3  3  4 6 6 7 8 Distance of Migration (cmi  4 5 6 7 8 9 Distance of Migration (cm)  9  10  10  F i g . 6 6 . Absorbance scans o f 3 - 5 $ p o l y a c r y l a m i d e g e l s c o n t a i n i n g p r o t e i n s which were c r o s s - l i n k e d with DMS and DSP. P u r i f i e d v i r u s which was c r o s s - l i n k e d w i t h DMS ( 3 - 0 mg/ml) and DSP ( 2 . 0 mg/ml) f o r 2 hours (as d e s c r i b e d i n F i g . 6 5 ) was scanned f o r absorbance a t 5 5 0 nm. Nucleocapsid monomer (NC), dimer (NC£)» t r i m e r ( N C 3 ) , tetramer (NCzj,), pentamer ( N C 5 ) , and hexamer (NC6) a r e i n d i c a t e d on t h e scans. Envelope monomer ( E 1 E 2 ) was a l s o designated.  -135-  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 i n the absence of 8-mercaptoethanol (Figs. 67 and 6 8 ) .  ,0.5h i h  2h,0.5h 1h  2 mg/ml  2 h . 0.5h 1h  1 mg/ml  2h  (  —  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 i n 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 i n the presence of SDS, stained with Coomassie blue, and destained by diffusion i n 7-5$ acetic acid. Noncross-linked virus (50 ug protein ) which had been purified on 1 gradient was also electrophoresed as a reference. B-mercaptoethanol 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\ic  3  E-E  NO,  NC  , 0.5h 1h 2h|0.5h 1h 2h |0.5h 1h 2h , 2 mg/ml 1 mg/ml 0.5mg/ml Fig. 68. subjected  P u r i f i e d S F 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 a n d t o e l e c t r o p h o r e s i s o n 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 u g p r o t e i n ) was c r o s s - l i n k e d w i t h D S P (0.5 m g / m l , 1 mg/ml, a n d 2 mg/ml) a n d e l e c t r o p h o r e s e d a s 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), a n d t r i m e r (NC3) w e r e e v i d e n t a s was e n v e l o p e monomer ( E i a n d E2) a n d d i m e r ( E - E ) . Nucleocapsid dimeric  was f o u n d t o y i e l d  up t o pentameric  envelope p r o t e i n s were e v i d e n t .  b e e n shown t o be l y s i n e r i c h why l o w e r  molecular  while  The c a p s i d p r o t e i n h a s  and i ti s thus  weight species  species  of this  understandable  as t o  p r o t e i n ( i e . monomer a n d  -137diraer) d i s a p p e a r a t l o n g e r c r o s s - l i n k i n g times or a t h i g h e r c o n c e n t r a t i o n s of DSP or DTBP.  Again envelope p r o t e i n s which  were c r o s s - l i n k e d t o a g r e a t e r degree than dimer were not c l e a r l y evident. S e m l i k i F o r e s t v i r u s was then c r o s s - l i n k e d w i t h e i t h e r DTBP ( 2 mg/ml) or DSP ( 1 mg/ml) f o r a p e r i o d of 1 hour and f r a c t i o n a t e d by the two-dimensional c r o s s - l i n k i n g system of Wang and Richards  (235).  i n each case. the f i r s t  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 a c h i e v e d  The v i r u s was 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 i n  dimension i n the absence of ^-mercaptoethanol and then  the second dimension i n the presence of 1 0 $ (v/v) g-mercaptoethanol :  (Fig. 6 9 ) .  Monomeric or n o n c r o s s - l i n k e d s p e c i e s l a y on a d i a g o n a l  running from l e f t t o r i g h t on the g e l , w h i l e c r o s s - l i n k e d appeared on h o r i z o n t a l l i n e s a d j a c e n t t o the monomers.  species  Nucleocapsid  was c r o s s - l i n k e d t o y i e l d up t o tetramers w h i l e envelope p r o t e i n s j o i n e d t o y i e l d only dimers.  Three d i s t i n c t spots were p r e s e n t .  These c o u l d correspond t o the combinations E-^-E-^, E-^-Eg, and Eg-Eg but one should be c a u t i o u s i n a s s e s s i n g t h i s  situation  s i n c e v a r y i n g degrees of i n t r a m o l e c u l a r d i s u l f i d e bonding  will  y i e l d m u l t i p l e p r o t e i n bands when such p r o t e i n s a r e e l e c t r o phoresed i n the absence of g-mercaptoethanol ,'  ( 2 5 1 ) .  Nucleocapsid  .  d i d not appear to c r o s s - l i n k w i t h envelope p r o t e i n s i n any i n s t a n c e - even when h i g h e r c r o s s - l i n k e r c o n c e n t r a t i o n s and l o n g e r i n c u b a t i o n times were employed.  These data agree w i t h  some i n t e r p r e t a t i o n s of X-ray c r y s t a l l o g r a p h y  (1,252)  and other,  r e c e n t c r o s s - l i n k i n g s t u d i e s performed on SF v i r u s and S i n d b i s virus  (253)-  However, our data do*.  d i s a g r e e w i t h the r e s u l t s of  -138-  NC  3  E-E  NC  2  II E,  E  2  IMC  I  IMC, NC  F i g . 6 9 . Two-dimensional e l e c t r o p h o r e s i s of SF v i r u s p r o t e i n s which had been c r o s s - l i n k e d w i t h DSP. P u r i f i e d v i r u s (150 ug p r o t e i n ) was suspended i n 0.15 M NaCl and c r o s s - l i n k e d w i t h DSP (-1 mg/ml) f o r 1 hour. The c r o s s - l i n k e d p r o t e i n s were s e p a r a t e d on 7.0$ p o l y a c r y l a m i d e g e l s ( i n t h e presence of 10$ (v/v) 3-mercaptoethanol f o r t h e second dimension. Monomeric ( o r n o n c r o s s - l i n k e d p r o t e i n s ) l a y on a d i a g o n a l l i n e running from l e f t t o r i g h t w h i l e c r o s s - l i n k e d s p e c i e s l a y on h o r i z o n t a l l i n e s which were a d j a c e n t t o t h e monomers. Nucleocapsi monomer (NC) , dimer ( N C 2 ) , t r i m e r ( N C 3 ) , tetramer (NCZf) • and envelope p r o t e i n monomers (Ej_ and Eg) and dimers (E-E) a r e e v i d e n t  -139-  Simons and G a r o f f ( 2 3 6 ) , who  s t a t e d t h a t DMS  yielded nucleocapsid  bound to envelope p r o t e i n s even when v i r u s was T r i t o n X-100.  d e l i p i d a t e d with  However, these l a s t workers d i d not p r o v i d e  evidence f o r n u c l e o c a p s i d - e n v e l o p e p r o t e i n i n t e r a c t i o n based upon t h e i r p o l y a c r y l a m i d e g e l s . F.  Two-Dimensional  C r o s s - L i n k i n g Studies Performed on Plasma  Membranes from I n f e c t e d 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 a p p l i e d to the plasma membranes of i n f e c t e d c e l l s which had been l a b e l e d with c e l l s were l a b e l e d f o r j/k  [-^s|methionine.  Infected  hour at 4 hours i n f e c t i o n and chased  with n o n - r a d i o a c t i v e methionine enriched media f o r a p e r i o d of 2 hours and c r o s s - l i n k e d with e i t h e r DSP or DTBP f o r a p e r i o d of 1 hour.  Plasma membrane ghosts were i s o l a t e d and the p r o t e i n s  were r e s o l v e d by two-dimensional e l e c t r o p h o r e s i s ( F i g s . 70 and 71).  N u c l e o c a p s i d tetramers were c l e a r l y r e s o l v e d w h i l e envelope  dimer was  a g a i n present as three s p e c i e s .  E^ d i d not appear  to c r o s s - l i n k to any degree with any other v i r a l p r o t e i n s . PEg> however, does appear to p a 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 i n t e r a c t s with n u c l e o c a p s i d p r o t e i n or E-^ - but the r e s o l u t i o n and the degree of accuracy f o r m o l e c u l a r weight d e t e r m i n a t i o n i n the f i r s t  dimension make t h i s d i f f i c u l t to  interpret. G.  Two-Dimensional C r o s s - L i n k i n g Studies Performed on Plasma Membranes from I n f e c t e d C e l l s which were Treated With C o l c h i c i n e and Dibucaine S i m i l a r two-dimensional c r o s s - l i n k i n g s t u d i e s were performed  on i n f e c t e d c e l l s which had been t r e a t e d with e i t h e r 100  uM  -140-  Fig. 7 0 . 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 c r o s s - l i n k e d with DSP. Infected c e l l s were labeled with  [35 ] s  methionine f o r 3/4 hour at 4 hours i n f e c t i o n and chased with non-radioactive methionine enriched media f o r a period of 2 hours. C e l l s were c r o s s - l i n k e d with DSP ( 1 mg/ml) f o r a period of 1 hour, plasma membranes were i o s l a t e d 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 p r o t e i n monomers ( E l and E ) , and the presumed dimers ( E ^ - E i , E]_-E2, and E2-E2) were present. Identities of dimer species could not be absolutely assigned since intramolecul a r d i s u l f i d e bonds can also y i e l d m u l t i p l e spots i n the f i r s t dimension. 2  -141  -  F i g . 71Two-dimensional autoradiogram o f plasma membrane p r o t e i n s from SF v i r u s - i n f e c t e d c e l l s which had been c r o s s - l i n k e d w i t h DTBP. I n f e c t e d c e l l s were l a b e l e d and c r o s s - l i n k e d w i t h DTBP (1 mg/ml) as d e s c r i b e d i n F i g . 7 0 . Plasma membranes were i s o l a t e d and s u b j e c t e d to two-dimensional e l e c t r o p h o r e s i s . Envelope monomers ( E i and Eg) and the presumed dimers (Ei-E]_, E i - E ? • and Eg-Eg) were evident as w e l l as n u c l e o c a p s i d monomer (NC), dimer (NCg), and t r i m e r (NC^).  - 142-  c o l c h i c i n e or 1 0 0 uM d i b u c a i n e a t 2 hours i n f e c t i o n . l a b e l l e d f o r ^ hour with[3^s]methionine with n o n - r a d i o a c t i v e methionine  and chased  e n r i c h e d media.  C e l l s were  f o r 2 hours  The  l a b e l e d BHK c e l l s were then f r a c t i o n a t e d i n t o plasma membranes and e l e c t r o p h o r e s e d under c o n d i t i o n s which were i d e n t i c a l f o r each sample.  Envelope  dimer appeared to be present i n the  plasma membranes from c o n t r o l and c o l c h i c i n e - t r e a t e d c e l l s but was absent  i n c e l l s which had been t r e a t e d w i t h d i b u c a i n e  72 and 7 3 ) •  We i n i t i a l l y thought  ments might be important  (Figs.  t h a t microtubules and m i c r o f i l a -  i n r e g u l a t i n g the i n t e r a c t i o n of v i r a l  p r o t e i n s a t the plasma membrane d u r i n g patch f o r m a t i o n and budding.  However, c o l c h i c i n e (which d i s r u p t s microtubules)  does not appear t o a l t e r the p r o x i m i t y o f envelope  proteins to  each other d u r i n g the f i n a l stages o f v i r a l morphogenesis while d i b u c a i n e (which d i s r u p t s microtubules and m i c r o f i l a m e n t s ) appears to a b o l i s h envelope  dimer formation.  T h i s would seem  to i m p l i c a t e m i c r o f i l a m e n t s i n the process where p r o t e i n s aggregate  envelope  i n the plasma membrane p r i o r to budding  while m i c r o t u b u l e s may simply a c t t o s t a b i l i z e . ' the membrane d u r i n g the f i n a l stage o f assembly.  -143-  Dib. Col. -  Fig. 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. Infected BHK c e l l s were treated with colchicine (100 uM) and dibucaine (100 uM) at 3 hours i n f e c t i o n . C e l l s were labeled with[35s]methionine f o r \ hour at 4 hours i n f e c t i o n and chased with non-radioactive methionine enriched media f o r a period of 2 hours. C e l l s 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 ( E l and E 2 ) and presumed dimers (Ei-E]_, E 1 - E 2 , and E 2 ~ E ) were evident. Envelope dimers appeared absent i n the plasma membranes from c e l l s which were cultured i n the presence of dibucaine. 2  -144-  Control  F i g . 73(a). Two-dimensional autoradiograms of DTBP c r o s s - l i n k e d plasma membrane proteins from infected c e l l s which were cultured i n the presence or absence of dibucaine and c o l c h i c i n e . Infected BHK c e l l s were treated with c o l c h i c i n e (100 uM) and ucaine (100 uM) at 3 hours i n f e c t i o n . Cellswere labeled with S M e t h i o n i n e f o r § hour at k hours i n f e c t i o n and chased with non-radioactive methionine enriched media f o r a period of 2 hours. C e l l s were cross-linked with DTBP (lmg/ml) f o r a period of f hour, plasma membranes were i s o l a t e d 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 g e l s . The arrow designates the region where envelope protein dimers resolve; cross-linked plasma membranes from c e l l s treated with dibucaine appeared to contain no envelope dimers.  -145-  Fig. 73(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 p l a s m a 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 w e r e c u l t u r e d i n t h e presence o r absence o f dibucaine and c o l c h i c i n e .  -146-  Dibucaine  i  1 NC  Fig. 73(c). Two-dimensional autoradiograms of DTBP c r o s s - l i n k e d plasma membrane proteins from i n f e c t e d c e l l s which were c u l t u r e d i n the presence or absence of dibucaine and c o l c h i c i n e .  -147-  IV.  A.  DISCUSSION  The V i r a l Envelope O r i g i n a t e s from the Plasma  Membrane  of the Host C e l l From the graphs i n F i g s . 3 1 > 3 2 and 3 3 we see that the movement o f l a b e l e d p r o t e i n s i s e n t i r e l y c o n s i s t e n t w i t h the h y p o t h e s i s t h a t SF v i r u s o b t a i n s i t s envelope by budding from the plasma membrane  o f the BHK c e l l .  Furthermore, i f we c o r r e c t  f o r r e c o v e r y of plasma membrane as f o l l o w s , the data suggest that a l l o f the l a b e l e d v i r u s i n the medium o r i g i n a t e d from the plasma membrane. plasma membrane was (see " R e s u l t s " ) .  We might assume t h a t our r e c o v e r y o f the 35-3$  as demonstrated w i t h o t h e r experiments  I n a d d i t i o n , the experiment d e p i c t e d i n  F i g . 3 0 used only t h r e e - f o u r t h s of the a c t u a l plasma  membrane  obtained ( o n e - f o u r t h was used f o r p r o t e i n d e t e r m i n a t i o n ) ; t h e r e f o r e , the measured r a d i o a c t i v i t y i s 7 5 $ o f the t o t a l a c t i v i t y a c t u a l l y r e c o v e r e d i n the membrane f r a c t i o n .  radio-  I f these  c o r r e c t i o n s a r e made i n the data o f F i g . 3 3 "the counts a s s o c i a t e d w i t h E-L and Eg a t ( 4 0 , 0 0 0 / . 3 5 3  x  O.75)  nucleocapsid protein.  and  3  hours chase would be  53,000  x  0.75)  for  When these two v a l u e s are added  the r e s u l t i s i n good agreement ( 1 7 0 , 0 0 0 ; 4 4 , 0 0 0 / 0 . 3 5 3  (14.000/.353  151,000  x  O.75)  the (204,000)  w i t h the t o t a l counts per min  a s s o c i a t e d 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, s e v e r a l other p o i n t s must be c o n s i d e r e d .  (a) From  the decay of r a d i o a c t i v i t y i n E-^, Eg, and the n u c l e o c a p s i d  proteins  of the plasma membrane 4 hours a f t e r the b e g i n n i n g o f the chase  -148-  (see F i g . 3 3 ) > "the h a l f - l i f e to be 2 . 0 hours.  o f these p r o t e i n s can be estimated  Hence, almost  one-half o f the l a b e l e d pro-  t e i n s present 2 . 0 hours a f t e r the chase p e r i o d began w i l l have disappeared from the plasma membrane a t the time o f maximal i n c o r p o r a t i o n o f [ 3 ] 1 eucine ( 4 h o u r s ) . H  a s s o c i a t e d with the envelope  (b) [-^Hlleucine  p r o t e i n E^ i n the plasma membrane  has not been i n c l u d e d i n our c a l c u l a t i o n .  Hence, the v a l u e o f  2 0 4 , 0 0 0 cpm a s s o c i a t e d with the v i r a l p r o t e i n s i n the plasma membrane i s a minimal for  estimate.  (c)  From the curve ( F i g . 3 3 )  r a d i o a c t i v i t y a s s o c i a t e d w i t h e x t r a c e l l u l a r v i r u s , we can  assume t h a t 1 7 0 , 0 0 0 cpm i s a l s o a minimal  estimate f o r 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 .  (d)  From the  p r e c i s i o n o f our measurements, a l l o f these c a l c u l a t i o n s a r e s u b j e c t to an e r r o r o f 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 t h a t v i r t u a l l y a l l o f the r a d i o a c t i v i t y a s s o c i a t e d w i t h 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 l a b e l e d  p r o t e i n s t h a t were p r e v i o u s l y a s s o c i a t e d with the plasma membrane. and  These data and the p r e v i o u s s t u d i e s by chemical  e l e c t r o n microscopy  analysis  ( 1 6 , 2 1 , 79> 8 0 , 95~98) make a s t r o n g  case f o r morphogenesis o f SF"Yi rus- from .the plasma • membrane of the r  BHK c e l l . The one apparent of  anomaly i n our r e s u l t s was the accumulation  r a d i o a c t i v i t y i n the n u c l e o c a p s i d p r o t e i n a s s o c i a t e d with  the endoplasmic the accumulation  reticulum fraction.  However, t h i s agrees  of c y t o p l a s m i c n u c l e o i d s l a t e i n i n f e c t i o n as  observed by e l e c t r o n m i c r o s c o p i s t s ( 1 6 ) . thought  with  These n u c l e o i d s were,  t o be n u c l e o c a p s i d s which accumulate as p a r a c r y s t a l l i n e  - 1 4 9 -  a r r a y s or i n a s s o c i a t i o n with cytoplasmic Jones et a l . (67) with  [-^H]leucine  .(49)  lamide g e l s  and  showed t h a t n u c l e o c a p s i d  protein  was  to a g r e a t e r degree than envelope p r o t e i n s .  evidence based on autoradiography of and  polyacry-  s t u d i e s on i n v i t r o p r o t e i n s y n t h e s i s  v i r a l mRNA ( 4 9 - 5 5 ) which suggest t h a t n u c l e o c a p s i d synthesized  In a d d i t i o n ,  pulse-labeled Sindbis v i r u s - i n f e c t e d c e l l s  l a b e l e d f a s t e r and T h i s agrees with  vacuoles.  i n l a r g e q u a n t i t i e s p r i o r to E-^ and  protein i s  Eg.  Hence,  b e l i e v e t h a t accumulation of the l a b e l e d n u c l e o c a p s i d i n the endoplasmic r e t i c u l u m o c c u r r e d because p r o t e i n was Therefore, utilized  produced and  from  we  protein  nucleocapsid  l a b e l e d i n excess of envelope p r o t e i n s .  a l l of the l a b e l e d n u c l e o c a p s i d  i n the p r o d u c t i o n  p r o t e i n was  not  of e x t r a c e l l u l a r v i r u s at the plasma  membrane. Morphogenesis from the plasma membrane occurs togaviruses.  A scheme which d e p i c t s the maturation of S e m l i k i  F o r e s t v i r u s i s presented membrane may  f o r Group A  i n F i g . 74.  Maturation  from the plasma  not be unique to the Group A t o g a v i r u s e s .  v i r u s e s , myxoviruses, paramyxoviruses, a r e n a v i r u s e s , v i r u s e s are a l l reputed  to "bud"  s t u d i e s (101,  102,  and r e t r o -  from the plasma membrane.  theory i s supported mainly by e l e c t r o n microscopy and composition  Rhabdo-  110-114).  This  lipid  Pulse-chase s t u d i e s  have been undertaken with i n f l u e n z a and v e s i c u l a r s t o m a t i t i s v i r u s (104-109, U 5 - H 9 ) • (18)  The  pulse-chase study r e p o r t e d by  showed k i n e t i c data t h a t were c o n s i s t e n t with a  precursor-  product r e l a t i o n s h i p between some v i r a l p r o t e i n s i n the plasmic  r e t i c u l u m and  the plasma membrane.  Hay  endo-  However, he d i d not  -150-  5'  I  POLYHERASE REPLICASE „ 42S  RNA  Fig. 74. Proposed scheme f o r the maturation of S e m l i k i v i r u s i n BHK c e l l s .  Forest  -151-  demonstrate t h a t these p r o t e i n s i n the plasma membrane were subsequently a s s o c i a t e d with e x t r a c e l l u l a r v i r u s p a r t i c l e s . our work was completed,  Since  s i m i l a r r e s u l t s f o r the morphogenesis of  v e s i c u l a r s t o m a t i t i s v i r u s were obtained by Knipe, B a l t i m o r e and Lodish  (107)•  B. The Role o f the C y t o s k e l e t a l System i n the Membrane Morphogenesis o f S e m l i k i F o r e s t V i r u s F u r t h e r work was r e q u i r e d t o e l u c i d a t e the mechanism by which SF v i r u s  "budded" from the plasma membrane o f t h e host  cell.  I t remained t o be seen whether or not maturation o f the v i r u s from the plasma membrane was a spontaneous event,  depended on  s p e c i f i c h o s t c e l l s t r u c t u r e s , or was v i r u s mediated.  Studies  with a t e m p e r a t u r e - s e n s i t i v e mutant o f S i n d b i s v i r u s (84) i n d i c a t e t h a t the l a t t e r may be t r u e ; n u c l e o c a p s i d s appear to accumulate next t o t h e plasma membrane a t the nonpermissive temperature  but f a i l to bud from the plasma membrane.  The r o l e o f microtubules and m i c r o f i l a m e n t s in,..the-budding process o f S e m l i k i F o r e s t v i r u s were an obvious c o n s i d e r a t i o n . Palade, Lacy and M a l a i s s e ( 2 0 0 , 2 0 7 , 2 1 1 , 2 1 2 ) have i m p l i c a t e d m i c r o t u b u l e s i n the s e c r e t i o n o f hormone granules and microtubules and m i c r o f i l a m e n t s have been demonstrated t o r e g u l a t e capping phenomena and patch f o r m a t i o n a t the c e l l  s u r f a c e (I36,  137» 1 8 2 ) .  Our r e s u l t s i n d i c a t e d 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 and d i b u c a i n e (a fjtert'far'y /. amine l o c a l a n e s t h e t i c ) i n h i b i t v i r u s p r o d u c t i o n and cause an accumulation  of v i r a l  Many c o n t r o l experiments  p r o t e i n s a t the plasma membrane.  suggest t h a t the c o n c l u s i o n s we  have reached a r e not a r e s u l t o f secondary  e f f e c t s on the c e l l by  -152-  these drugs.  In a l l cases, low c o n c e n t r a t i o n of the drugs  used so we might minimize on c e l l v i a b i l i t y .  was  the p o t e n t i a l e f f e c t s of these chemicals  T r a n s p o r t of the r a d i o a c t i v e amino a c i d s  i n t o the c e l l and p r o t e i n s y n t h e s i s were not a f f e c t e d by these d i s r u p t o r s as a s c e r t a i n e d by i n c o r p o r a t i o n of l a b e l i n t o the trichloroacetic acid precipitates.  The  e f f e c t s of c o l c h i c i n e  c o u l d not be d u p l i c a t e d with l u m i c o l c h i c i n e (which i s unable to d i s r u p t m i c r o t u b u l e s ) while Nocodazole i n h i b i t i o n of v i r u s p r o d u c t i o n .  produced  a similar  F i n a l l y , c o l c h i c i n e and d i -  bucaine d i s r u p t m i c r o t u b u l e s by two a p p a r e n t l y d i s t i n c t mechanisms (136,  I65,  188,  189)  yet had s i m i l a r e f f e c t s upon v i r u s p r o d u c t i o n .  With these c o n s i d e r a t i o n s i n mind, we conclude t h a t the c y t o 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 N e i t h e r c o l c h i c i n e nor d i b u c a i n e completely i n h i b i t e d p r o d u c t i o n of e x t r a c e l l u l a r v i r u s . m i c r o t u b u l e s are a heterogeneous  This may  (168,  169).  enveloped v i r u s e s may  p o p u l a t i o n and some appear  be a slow process t h a t i s n o r m a l l y system.  The mechanism by which the c y t o s k e l e t a l system i n v i r u s morphogenesis i s not understood. that t h i s o r g a n i z a t i o n was  p r o t e i n to the c e l l s u r f a c e . SF v i r u s i n BHK  i s involved  Our i n i t i a l h y p o t h e s i s  r e q u i r e d f o r t r a n s p o r t of the v i r a l T h i s i s c l e a r l y not the case f o r  c e l l s even though m i c r o t u b u l e s may  f o r the t r a n s p o r t of adenovirus w i t h i n HeLa c e l l s second p o s s i b i l i t y was  other  A l t e r n a t i v e l y , the budding mechanism of  c a t a l y z e d by an i n t a c t c y t o s k e l e t a l  was  the  be because the  to be l e s s s e n s i t i v e to the e f f e c t s of c o l c h i c i n e and disruptors  assembly.  that the cleavage of P E  ?  be  important  (227,  to E  9  228).  might be  A  -153-  hindered,  s i n c e i t appears t h a t t h i s occurs  i n t h e plasma  membrane as w e l l as the endoplasmic r e t i c u l u m and i s a prer e q u i s i t e f o r budding ( 6 7 , 1 2 1 ) .  This mechanism a l s o seems  u n l i k e l y s i n c e cleavage o f PEg was not r e t a r d e d 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  e f f e c t s o f c o l c h i c i n e and d i b u c a i n e might a l s o be due to a decrease i n bound calcium. dibucaine)  a r e reputed  phospholipids  T e r t i a r y amine a n e s t h e t i c s  to d i s p l a c e C a  and  dibucaine  found i n a s s o c i a t i o n with  (188, I 8 9 ) and c o l c h i c i n e might exert the same  e f f e c t by d i s p e r s i o n o f the Ca. microtubules  + +  (like  (136).  associated with  polymerized  Another p o s s i b i l i t y i s t h a t c o l c h i c i n e  may a l t e r the topology  o f v i r a l p r o t e i n s i n the  plasma membrane so t h a t patches o f envelope p r o t e i n s and nucleoc a p s i d may not occur.  We t e s t e d t h i s l a t t e r p o s s i b i l i t y  through  the use o f p r o t e i n c r o s s - l i n k i n g agents. We f e l t t h a t c r o s s - l i n k i n g agents might inform us about the p r o x i m i t y f i n a l stages  o f the v i r a l p r o t e i n s to each other during the o f v i r u s assembly.  P r e l i m i n a r y s t u d i e s were  performed with DMS, DSP and DTBP; p u r i f i e d v i r u s was r e a c t e d with these reagents and s u b j e c t e d phoresis. (236, and  to two dimensional  electro-  I n c o n t r a s t t o the r e s u l t s o f G a r o f f and Simons  237) no c r o s s - l i n k i n g was observed between envelope p r o t e i n s the n u c l e o c a p s i d  which suggests t h a t p a r t o f the g l y c o -  p r o t e i n s might not be c l o s e to the n u c l e o c a p s i d  protein.  Other  c r o s s - l i n k i n g s t u d i e s with S i n d b i s and S e m l i k i F o r e s t v i r u s c o r roborate  this result  (.253) and a l s o suggest t h a t envelope p o l y -  peptides  may n a t u r a l l y e x i s t as dimers which f u n c t i o n as a  -154-  structural unit. proteins reacted DSP and DTBP. detected. (although  We were a b l e t o demonstrate t h a t  envelope,  to form dimers and i n some cases t r i m e r s  with  Higher molecular weight complexes were not  X - r a y c r y s t a l l o g r a p h y data have a l s o been i n t e r p r e t e d 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 p r o t e i n s do not penetrate  through the membrane  (1, '252) as have f r e e z e - f r a c t u r e s t u d i e s  ( 1 , 79, 80).  Nucleo-  c a p s i d p r o t e i n i s l y s i n e r i c h and r e a c t s r e a d i l y w i t h DMS, DSP and  DTBP t o y i e l d dimeric  t o pentameric complexes.  l i n k i n g s t u d i e s appear to support the scheme where p r o t e i n s a r e c l o s e l y a s s o c i a t e d with each other p r o t e i n s may i n t e r a c t to form dimeric i n t e r a c t w i t h the n u c l e o c a p s i d .  Our c r o s s nucleocapsid  - envelope  s t r u c t u r e s but do not  I n no case does the t h i r d en-  v e l o p e p r o t e i n E^ appear to c r o s s - l i n k with the other v i r u s p r o t e i n s which supports the view t h a t i t i s a l o o s e l y a s s o c i a t e d protein (1).  I n t e r p r e t a t i o n o f c r o s s - l i n k i n g s t u d i e s must be  approached with c a u t i o n , however, s i n c e c r o s s - l i n k i n g depends upon the presence o f an a v a i l a b l e r e a c t i v e group on the p r o t e i n . S t u d i e s with plasma membranes which contained  cross-linked  v i r a l p r o t e i n s a l s o y i e l d e d s i m i l a r r e s u l t s to those above.  Again, no d i r e c t evidence f o r envelope  i n t e r a c t i o n was e v i d e n t .  Nucleocapsid  discussed  protein-nucleocapsid  was presumably c r o s s -  l i n k e d because the plasma membrane was permeable to DSP and DTBP as'  1  has p r e v i o u s l y been reported, by Richards ( 2 3 4 ) .  t e r e s t i n g observation  An i n -  which might warrant f u r t h e r i n v e s t i g a t i o n  i s the c r o s s - l i n k i n g of P E _ - i t was d i f f i c u l t 2  nature of these complexes i n our system.  to assess the  I t i s a l s o worthwhile  -155-  to note t h a t v i r a l p r o t e i n s d i d not c r o s s - l i n k with host plasma membrane p r o t e i n s although two-dimensional  g e l s d i d demonstrate  t h a t host p r o t e i n s i n t e r a c t w i t h each other; t h i s was evident when the two-dimensional  g e l s were s t a i n e d with Coomassie b l u e .  Obviously, host c e l l p r o t e i n s a r e e f f i c i e n t l y excluded-: from areas o f the c e l l membrane which c o n t a i n v i r u s p r o t e i n s d u r i n g the budding requires  process.  The nature of t h i s l a s t process  elucidation.  '- •• C r o s s - l i n k i n g s t u d i e s performed presence prevented  still  on i n f e c t e d c e l l s i n the  o f c o l c h i c i n e and d i b u c a i n e i n d i c a t e d t h a t d i b u c a i n e envelope  have no e f f e c t .  dimer f o r m a t i o n w h i l e c o l c h i c i n e appeared to  One i n t e r p r e t a t i o n o f t h i s r e s u l t would be t h a t ;  m i c r o f i l a m e n t s might be r e q u i r e d to keep the envelope p r o t e i n s proximal t o each other p r i o r t o budding w h i l e m i c r o t u b u l e s 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 e x i s t s - c o l c h i c i n e might a l t e r or i n h i b i t a stage of g l y c o s y l a t i o n o f the envelope p r o t e i n s p r i o r to t h e i r i n t o the plasma membrane.  insertion  I n t h i s case o r i e n t a t i o n o f these  g l y c o p r o t e i n s might be a l t e r e d and i n h i b i t i o n of g l y c o s y l a t i o n has p r e v i o u s l y been shown t o a f f e c t budding virus  of S e m l i k i F o r e s t  (256).  I t i s q u i t e obvious t h a t the f i n a l stages of v i r u s are complex.  maturation  These s t u d i e s have shown t h a t assembly of SF v i r u s  i s dependent on the s t a t e of the host c e l l - i n p a r t i c u l a r the r o l e and f u n c t i o n of m i c r o t u b u l e s i n the "budding"  process.  -156-  F u r t h e r s t u d i e s w i l l not o n l y provide  f u r t h e r i n f o r m a t i o n on  the  maturation of envelope v i r u s e s but a l s o impart knowledge as to how  g l y c o p r o t e i n s are formed and  C.  Possible Topics Low  for Further  s e c r e t e d from the  cell.  Research  i o n i c s t r e n g t h media has been demonstrated to  v i r u s formation  drastically  to determine i f C a  + +  (.257, 2 5 8 ) ,  inhibit  I t would be of i n t e r e s t  were i n v o l v e d i n v i r u s maturation s i n c e t h i s  i o n has been shown to be i m p l i c i t l y i n v o l v e d i n the f u n c t i o n of microtubules  and m i c r o f i l a m e n t s .  Studies with  b i s - ( g-amino-ethyl ether)N^'N^-tetraacetic C a ) and ++  plasmic  a c i d (which c h e l a t e s  the Ca ', ionophore A23187 might r e i n f o r c e the ++  t h a t microtubules The  ethyleneglycol-  and m i c r o f i l a m e n t s  theory  are i n v o l v e d i n budding.  mechanism f o r t r a n s p o r t of v i r a l p r o t e i n s from the endor e t i c u l u m to the plasma membrane i s of extreme i n t e r e s t .  P r o t e o l y t i c i n h i b i t o r s c o u l d be u t i l i z e d and w i t h i n the c e l l where the l a r g e p r e c u r s o r could be a s c e r t a i n e d .  the v a r i o u s  points  p r o t e i n s accumulate  A l s o , the enzymes r e s p o n s i b l e f o r 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 v i r u s or host  specified.  Tunicamycin,  i n h i b i t o r of g l y c o s y l a t i o n , might a l s o be u t i l i z e d and  an i t s effects  on the i n s e r t i o n of envelope p r o t e i n s i n t o the plasma membrane c o u l d be monitored - i t would be i n t e r e s t i n g a l s o to see how would a f f e c t p r e c u r s o r  this  cleavage.  Experiments s i m i l a r to those performed on v e s i c u l a r s t o m a t i t i s v i r u s by L o d i s h  ( 2 5 9 ) would a l s o p r o v i d e  i n s i g h t i n t o how  v i r u s i n s e r t s i t s p r o t e i n s i n t o the endoplasmic r e t i c u l u m  SF and  -157-  how they a r e g l y c o s y l a t e d . in vitro  Rothman and L o d i s h  ( 2 5 9 ) performed  experiments i n which G p r o t e i n (an u n g l y c o s y l a t e d  G ,and a g l y c o s y l a t e d p r o t e i n , G]_)was s y n t h e s i z e d  from v i r a l  0  i n a wheat germ c e l l - f r e e homogenate.  form, RNA  The G p r o t e i n was found  to a s s o c i a t e with added microsomal membranes spontaneously w h i l e s t i l l nascent on the ribosomes.  Once the G p r o t e i n was  i n s e r t e d i n t o the membrane i t appeared to be g l y c o s y l a t e d immediately.  The g l y c o s y l a t e d p r o t e i n G^ was not s u s c e p t i b l e  to t r y p s i n d i g e s t i o n while G  Q  was t o t a l l y d i g e s t e d .  experiments could be performed u t i l i z i n g  Experiments  be done to monitor the a s s o c i a t i o n o f the p r o t e i n s E-^, Eg and E^ with the endoplasmic r e t i c u l u m s y l a t i o n of v i r a l proteins  Similar  2 6 s RNA which was  i s o l a t e d from c e l l s i n f e c t e d with SF v i r u s .  The  almost  could  t s 2 , B-^, PEg,  and the g l y c o -  a t the i n t r a c e l l u l a r membranes.  packaging and t r a n s f e r of v i r a l g l y c o p r o t e i n s  from the  G o l g i t o the plasma membrane should a l s o be considered.  This  mechanism may be important i n determining why host c e l l  proteins  are excluded i n the v i r u s patches on the plasma membrane. C h o l e s t e r o l l e v e l s a r e about 3°$ h i g h e r i n the v i r u s t h a n i n the plasma membrane - the exact s i g n i f i c a n c e i s u n c l e a r 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. proteins  c o u l d be p u r i f i e d and p h o s p h o l i p i d  cholesterol binding  s t u d i e s c o u l d be performed t o determine whether v i r a l proteins  Virus  might have a p a r t i c u l a r l y h i g h a f f i n i t y  envelope  f o rcholesterol.  I f such were the case, t h i s would account f o r a u n i - d i r e c t i o n a l flow o f v i r a l p r o t e i n s  from the endoplasmic r e t i c u l u m  plasma membrane (which i s e n r i c h e d  f o r cholesterol).  to the Phospho-  -158.-  l i p i d and c h o l e s t e r o l v e s i c l e s might a l s o be with S e m l i k i  Forest v i r u s proteins  reconstituted  to determine i f enveloped  v i r u s e s were spontaneously produced.  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