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Radioactive pulse chase experiments concerning the mechanism of entry of Semliki Forest viru Grossi, Romeo 1977

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RADIOACTIVE PULSE CHASE EXPERIMENTS CONCERNING THE MECHANISM OF ENTRY OF SEMLIKI FOREST VIRUS  by Romeo G r o s s i B.Sc.  U n i v e r s i t y of B r i t i s h Columbia, 1975  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES In the Department of Biochemistry  We accept t h i s t h e s i s as conforming to the required standard for the degree of MASTER OF SCIENCE  The U n i v e r s i t y of B r i t i s h Columbia December 1977 ©  Romeo G r o s s i , 1977  In  presenting  an  advanced degree  the I  Library  further  for  shall  agree  scholarly  by  his  of  this  written  thesis  in  at  University  make  that  it  thesis  partial  freely  permission  for  It  is  financial  for  University  gain  Biochemistry of  British  April 18, 1978  of  of  Columbia,  British  Columbia  for  extensive by  the  understood  permission.  of  fulfilment  available  p u r p o s e s may be g r a n t e d  2075 Wesbrook P l a c e Vancouver, Canada V6T-1W5  D a t e  the  representatives.  Department The  this  shall  requirements  reference copying  Head o f  that  not  the  copying  be a l l o w e d  agree  and  of my  I  this  that  study. thesis  Department or  for  or  publication  without  my  i ABSTRACT  The mechanism of v i r a l penetration f o r Semliki Forest Virus i n t o BHK-21 c e l l s was i n v e s t i g a t e d through a s e r i e s of r a d i o a c t i v e pulse-chase experiments.  Entry of an enveloped v i r u s such as SF Virus  can be v i s u a l i z e d to enter host c e l l s both by pinocytosis (viropexis) or by f u s i o n of the v i r a l envelope and plasma membrane. Preliminary experiments were performed to obtain optimum conditions of v i r a l adsorption to host c e l l s .  The conditions  considered  included temperature, time of i n f e c t i o n , m u l t i p l i c i t y of i n f e c t i o n and i o n i c strength of the inoculum. In subsequent experiments BHK-21 c e l l s were i n f e c t e d one h a l f 35 hour with :..:S-Methionine-labeled  Semliki Forest V i r u s . 35  time points a f t e r removal of unadsorbed  At various  S-Met-SF V i r u s , c e l l s were  harvested and f r a c t i o n a t e d i n t o plasma membrane and endoplasmic reticulum fractions.  The f r a c t i o n s were subjected to SDS polyacrylamide g e l  electrophoresis and analyzed f o r component proteins of SF V i r u s . Maximum l e v e l s of r a d i o a c t i v i t y corresponding to the envelope proteins (E^ , E ^ and nucleocapsid p r o t e i n (NC) were found i n the PM f r a c t i o n at zero minutes of chase.  Both  decline during the chase period (approximately  E^ and NC were found to 90% w i t h i n 60 minutes of  chase). On the other, .hand, high l e v e l s of only nucleocapsid  protein  were observed associated with the endoplasmic reticulum f r a c t i o n although no general pattern of incorporation was i n d i c a t e d f u r i n g the  ii  experiment. throughout  (There were h i g h l e v i e s of NC p r e s e n t i n the ER f r a c t i o n the chase p e r i o d ) .  The r e s u l t s of these s t u d i e s a r e g e n e r a l l y as they can be r a t i o n a l i z e d b o t h by the v i r o p e x i s  inconclusive  and f u s i o n mechanisms.  The l o s s of b o t h E^ , E^ and NC from the PM suggest t h a t v i r o p e x i s  is  the  mechanism of e n t r y , however, f u s i o n i s n o t e l i m i n a t e d as an a l t e r n a t i v e s i n c e the envelope p r o t e i n s i n the PM may be degraded a f t e r of the v i r u s  internalization  c o r e by h o s t p r o t e a s e s .  A l t h o u g h no c o n c l u s i o n s have been drawn, t h i s study has demons t r a t e d t h a t r a d i o a c t i v e p u l s e - c h a s e experiments can be performed t o augment e l e c t r o n m i c r o s c o p y d a t a c o n c e r n i n g the mechanism of penetration i n t o animal c e l l s .  viral  iii  TABLE OF CONTENTS PAGE INTRODUCTION Mechanism of V i r a l P e n e t r a t i o n ( V i r o p e x i s  1 or Fusion?)  3  Speciman P r e s e r v a t i o n  4  I n t e r p e t a t i o n of Images  5  N a t u r e of the Inoculum  7  R e p l 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 PENETRATION OF VARIOUS VIRUS CLASSES  12 13  Picornaviruses  13  Adenoviruses  15  Papovaviruses  17  Reoviruses  18  Poxviruses  19  Herpesviruses  • • • 22  Rhabdoviruses  24  Myxoviruses  26  Paramyxoviruses  31  Oncornaviruses....  33  N u c l e a r I n s e c t Agents  34  Coronaviruses  34  Togaviruses  35  THE PRESENT INVESTIGATION  36  iv PAGE 39  MATERIALS AND METHODS Chemicals  :  39  I s o l a t i o n of Radioactively l a b e l l e d E x t r a c e l l u l a r Virus  40  Plaque Assays  Al  P u r i f i c a t i o n of Plasma Membrane and Endoplasmic Reticulum  ,41  SDS Acrylamide Electrophoresis  44  Gel S l i c i n g and S c i n t i l l a t i o n Counting of Gel Samples  44  A n a l y s i s of SDS Electrophoresis P r o f i l e s .  45  P r o t e i n Assays  45  EXPERIMENTAL RESULTS  46  Preparation of R a d i o a c t i v e l y l a b e l l e d SF Virus  46  Standard V i r u s Gel  47  I s o l a t i o n of Plasma Membrane and Endoplasmic Reticulum  49  Preliminary experiments  50  0 - 2 Hour PulseCChase'i Experiment  56  0-60 Minute Pulse Chase Experiment  60  DISCUSSION  64  BIBLIOGRAPHY  69  V  FIGURES PAGE  1. 2.  Schematic R e p r e s e n t a t i o n of Some P o s s i b l e Pathways of Virus Penetration  Non-Enveloped  Schematic R e p r e s e n t a t i o n of Some P o s s i b l e Pathways o f  Enveloped  2  Virus Penetration  2  3.  S i n g l e Reovirus In A Vacuole  4  4. 5.  A group of R e o v i r u s P a r t i c l e s I n A C e l l Sampled Soon A f t e r I n o c u l a t i o n . . 4 V i r o p e x i s of VSV P a r t i c l e By An L C e l l I s E v i d e n t 10 M i n u t e s A f t e r I n i t i a t i o n Of P e n e t r a t i o n 5  6.  I l l u s t r a t i o n s : o f Images R e s u l t i n g From T a n g e n t i a l S e c t i o n s  5  7.  Illustrations  5  8.  Diagrammatic R e p r e s e n t a t i o n of the T a n g e n t i a l and L o n g i t u d i n a l S e c t i o n s That Can Be Made Through A V i r u s - C e l l Complex  5  A c t i v e V i r u s On CAM  6  10.  C e l l - A s s o c i a t e d NDV P o s s e s s i n g Ruptured E n v e l o p e . . . .  7  11.  Shapes and R e l a t i v e S i z e s Of The Major F a m i l i e s Of V i r u s e s  9  12.  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  13.  P o s t T r a n s l a t i o n a l Cleavage I n The F o r m a t i o n Of SF V i r u s Proteins  9.  14.  15. 16.  of Images R e s u l t i n g From T a n g e n t i a l S e c t i o n s  11 Structural 12  A Deep I n v a g i n a t i o n of the PM of a H e l a C e l l C o n t a i n i n g a GroupJof7 P o l i o v i r u s Type 1 P a r t i c l e s Sampled 10 M i n u t e s A f t e r I n i t i a t i o n Of P e n e t r a t i o n  14  I n d i v i d u a l P o l i o v i r u s P a r t i c l e s W i t h i n P h a g o c y t i c V a c u o l e s C l o s e To The C e l l S u r f a c e  14  I n d i v i d u a l P o l i o v i r u s P a r t i c l e s W i t h i n P h a g o c y t i c V a c u o l e s C l o s e To the C e l l S u r f a c e . . .  14  vi  PAGE 17. 18.  E l e c t r o n M i c r o s c o p y A u t o r a d i o g r a p h y of T h i n - S e c t i o n e d P r i m a r y Baby Mouse K i d n e y C e l l s I n f e c t e d w i t h H - D N A - L a b e l l e d V i r i o n s  16  E l e c t r o n M i c r o s c o p y A u t o r a d i o g r a p h y of T h i n - S e c t i o n e d P r i m a r y Baby Mouse K i d n e y C e l l s I n f e c t e d w i t h % - A m i n o A c i d - L a b e l l e d V i r i o n s  16  19.  Model of Polyoma V i r i o n A t t a c h m e n t , P e n e t r a t i o n , And N u c l e a r E n t r y  17  20.  F e r r i t i n - L a b e l l e d V i r u s A t A L a t e r Stage Of F u s i o n W i t h L C e l l s  20  21.  F e r r i t i n - L a b e l l e d V i r u s At A L a t e r Stage Of F u s i o n W i t h L C e l l s  20  22.  F e r r i t i n - L a b e l l e d V i r u s A t A L a t e r Stage Of F u s i o n W i t h L C e l l s  20  23. 24.  L a b e l l e d V a c c i n i a P a r t i c l e Fused W i t h H e l a C e l l s A f t e r F u s i o n t h e V i r u s Core M i g r a t e s I n t o Host Cytoplasm and the V i r u s Envelope L a b e l l e d W i t h F e r r i t i n i s I n s e r t e d i n t o the C e l l Membrane  20  25.  21  An Amsacta P o x v i r u s P a r t i c l e Among M i c r o v i l l i a t The Apex of a Columnar C e l l  22A  26.  F u s i o n of a V i r u s P a r t i c l e w i t h the M i c r o v i l l u s  22A  27.  T r a n s v e r s e S e c t i o n of a M i c r o v i l l u s C o n t a i n i n g a V i r u s Core and P o r t i o n of the L a t e r a l Body V i r u s Core i n the P r o c e s s of E n t e r i n g A M i c r o v i l l u s a f t e r S i d e - t o - S i d e F u s i o n of V i r a l Envelope and M i c r o v i l l u s Membrane  28.  22A 22A  29.  Microvillus-Associated Virus  22A  30.  V i r u s P a r t i c l e w h i c h Appears t o be Fused w i t h the T i p of a M i c r o v i l l u s . . 22A  31.  A n o t h e r V i r u s P a r t i c l e w h i c h Appears t o be A s s o c i a t e d w i t h the T i p A Microvillus  of 22A  32.  A l i g n m e n t of the V i r u s P a r t i c l e s A l o n g , But Not I n C o n t a c t w i t h the C e l l Surface 27  33.  Engulfment of a S i n g l e P a r t i c l e by P i n o c y t o s i s  27  34.  Adherence of V i r u s P a r t i c l e s t o the Host C e l l at the C e l l S u r f a c e  27  vii  PAGE 35.  F u s i o n o f V i r u s Envelope and  Plasma Membrane of P i n o c y t o t i c V e s i c l e . . 27  36.  A c t i v e V i r u s on CAM, Warmed t o 35° C. f o r 5 M i n u t e s  29  37.  A c t i v e V i r u s on CAM, Incuabed w i t h F e r r i t i n and Warmed t o 35° C. f o r 10 M i n u t e s  29  38.  A c t i v e V i r u s on CAM, Warmed t o 35° C. f o r 30 M i n u t e s  30  39.  Schematic R e p r e s e n t a t i o n of P o s s i b l e Routes of SF V i r u s E n t r y A) P i n o c y t o s i s and B) F u s i o n  37  40.  I s o l a t i o n o f V i r u s By S u c r o s e G r a d i e n t C e n t r i f u g a t i o n  40  41.  D i s c o n t i n o u s G r a d i e n t f o r S e p a r a t i o n of Plasma Membrane and Endoplasmic R e t i c u l u m  42  42.  R a d i o a c t i v e P r o f i l e o f a T y p i c a l P r e p a r a t i v e Sucrose G r a d i e n t  46  43.  Coomassie B l u e Scan of S t a n d a r d V i r u s G e l  47  44.  R a d i o a c t i v e P r o f i l e of S t a n d a r d  45.  Dependence of SF V i r u s A d s o r p t i o n on Temperature and Time of Incubation  52  46.  E f f e c t of I o n i c S t r e n g t h of Medium on SF V i r u s A d s o r p t i o n  53  47.  The E f f e c t of M u l t i p l i c i t y of I n f e c t i o n on SF V i r u s A d s o r p t i o n  55  48.  0 - 2 Hour Chase V i r u s G e l s  49.  Time Course of SF V i r u s I n f e c t e d Plasma Membrane and E n d o p l a s m i c Reticulum.  58  50.  0' -  61-63  51.  Time Course of SF V i r u s I n f e c t e d Plasma Membrane and Endoplasmic Reticulum  3 H-Amino A c i d - h a b e l l e d A S F ' V i r u s . G e l . . . 48  6 0 ' Chase V i r u s G e l s  .57  63  viii TABLES  PAGE TABLE 1.  TABLE 2 .  Morphology and C o m p o s i t i o n of the Major F a m i l i e s o f A n i m a l Viruses A c t i v i t i e s of Plasma Membrane and Endoplasmic R e t i c u l u m Marker Enzymes  . 10 50  ix  LIST OF ABBREVIATIONS  J.T'BHK  Baby hamster'l.kidney  ds  double stranded  ss  s i n g l e stranded  SF Virus  Semliki Forest Virus  E^  Envelope p r o t e i n (MW 49,000) of S e m l i k i Forest Virus  E  2  Envelope p r o t e i n (MW 52,000) of S e m l i k i Forest Virus  E  3  Envelope p r o t e i n (MW 10,000) of Semliki Forest Virus  NC  nucleocapsid p r o t e i n of Semliki Forest V i r u s  63" PE2(pr .'NVP ' ) precursor p r o t e i n to E^ E^ E^  Combined envelope proteins  and E^ which often do not  resolve by SDS electrophoresis g i v i n g the impression of one p r o t e i n . NVP 165  Non-virion precursor p r o t e i n (MW 165,000)  NVP 97  Non-virion precursor p r o t e i n (MW 97,000)  PM  Plasma membrane as de_f^.ned by c l a s s i c a l marker enzymes (5' nucleotidase, Na K ATPase, and a l k a l i n e phosphatase)  ER  Endoplasmic reticulum as defined by c l a s s i c a l marker enzymes (NADPH cytochrome c reductase, glucose - 6 phosphatase, and NADH diaphorase)  VSV  v e s i c u l a r stomatitus v i r u s  SDS  Sodium dodecyl s u l f a t e  AiCl  Micro c u r i e  nm  Nanometer  CPM  Counts per minute  DPM  d i s i n t e g r a t i o n s per minute  X  A"*^  O p t i c a l Absorbance a t 550 nanometers  cm  Centimeters ionic  strength  MW  M o l e c u l a r Weight  CAM  C h o r i o a l l a n t o i c membrane  M199  199 maintenance medium  pfu  plaque forming u n i t s  mCi  millicurie  FCS  f e t a l c a l f serum  mmole  millimole  aa  amino a c i d  PC  phosphatidyl  Choi  cholesterol  NANA  N-acetyl-neuraminic  choline  acid  xi  ACKNOWLEDGEMENTS The author wishes to express h i s most sincere appreciation to Dr. D.E. Vance f o r h i s continual advice and encouragement. Appreciation i s also given to Mrs. N. Grossi f o r her painstaking work i n typing t h i s t h e s i s .  - 1 -  INTRODUCTION  Two d i f f e r e n t mechanisms have been proposed f o r v i r a l entry i n t o host c e l l s . ( 2 ) .  One has been imagined to be a p i n o c y t o t i c process  (also termed " v i r o p e x i s " ) whereby whole v i r u s p a r t i c l e s are incorporated i n t o cytoplasmic vacuoles and subsequently  transported to t h e i r s i t e of  replication.  Up to 1965, v i r o p e x i s was thought to be the only mode of  v i r u s entry.  However, evidence i n the l a s t decade has i n d i c a t e d that  enveloped v i r u s e s may enter host c e l l s by f u s i o n of the v i r a l envelope w i t h the host plasma membrane. A t h i r d p o s s i b l e mechanism of entry i s that the v i r u s e s may be uncoated at the c e l l surface and only the v i r a l genome penetrates the host c e l l .  However, there i s as yet no evidence that i n d i c a t e s t h i s i s a  s i g n i f i c a n t route f o r i n f e c t i o n i n animal c e l l s .  Figures l i a n d 2  demonstrate p o s s i b l e routes of entry of non-enveloped and enveloped animal viruses. Once the v i r u s or s u b - v i r a l p a r t i c l e enters the c e l l , the inoculum must s t i l l be transported to i t s ultimate s i t e of a c t i o n and i t s genome must be released p r i o r to r e p l i c a t i o n .  These p a r t i c l e s may  be d e l i v e r e d w i t h i n p i n o c y t o t i c vacuoles or moved through the cytoplasm directly.  A l t e r n a t i v e l y the v i r a l genomes may be released  immediately  a f t e r i n t e r n a l i z a t i o n and t r a n s f e r r e d to t h e i r s i t e s of r e p l i c a t i o n . Much work also has been done to unravel the events of transport and uncoating, however, t h i s t h e s i s w i l l be l i m i t e d to the a c t u a l penetration of the v i r i o n i n t o the host c e l l .  -21.  Uptake of v i r a l n u c l e i c acid.  V i r a l Replication  PM 2.  Viropexis (pinocytosis)  V i r a l uncoating v i a lysosomes? to nucleus? to cytoplasm?  Fig.  1  Schematic Representation of Some P o s s i b l e Pathways Of Non-enveloped v i r u s penetration.  1.  Viropexis (pinocytosis)  2.  Fusion of v i r a l envelope with plasma membrane.  viral  Fig.  2  Schematic Representation Of Some P o s s i b l e Pathways Of Enveloped Virus Penetration.  uncoating  -3MECHANISM OF VIRAL PENETRATION  ( V i r o p e x i s or F u s i o n ? )  The problem of e x a c t l y how v i r u s e s e n t e r t h e i r h o s t c e l l s has been the s u b j e c t of much r e s e a r c h i n r e c e n t y e a r s but the methods used t o d e t e c t p e n e t r a t i o n are l i m i t e d and a m a t t e r of u n c e r t a i n t y . p e n e t r a t i o n has been measured has been by t h e l o s s of v i r a l s e n s i t i v i t y of i n f e c t i v e c e n t e r s . t h i s method.  It  One way  antibody  However, some problems can a r i s e w i t h  i s p o s s i b l e t h a t a n t i b o d i e s may not be a b l e t o  r e a c t w i t h t h e v i r a l a n t i g e n s at the c e l l s u r f a c e .  For example, phage  0X 174 cannot be n e u t r a l i z e d by a n t i b o d y a f t e r i t has a t t a c h e d t o t h e E. C o l i c e l l w a l l s (.81) .  Thus i n some way phage attachment a t t h e c e l l  w a l l has s h i e l d e d t h e v i r u s from n e u t r a l i z a t i o n .  In a d d i t i o n , a  t r a n s i e n t enhancement of v i r a l n e u t r a l i z a t i o n by a n t i b o d y has o c c a s i o n a l l y been observed a t the c e l l s u r f a c e which i s l o s t o n l y a f t e r incubation ( l ) .  Also,  (and perhaps most i m p o r t a n t l y )  i t is possible  t h a t t h e r e c o u l d be a l o s s of a n t i b o d y s e n s i t i v i t y even i f remains o u t s i d e the c e l l .  prolonged  the v i r u s  These v i r u s e s have been d e s c r i b e d as  undergoing " i r r e v e r s i b l e a l t e r a t i o n " (  82-84  ).  These v i r u s e s  a l t e r e d i n such a way t h a t they become a n t i b o d y r e s i s t a n t .  are  An example  of t h i s a r e the p i c o r n a v i r u s e s which a r e t r a n s f o r m e d t o " A " p a r t i c l e s a t the c e l l s u r f a c e . The " A " p a r t i c l e s l a c k a v i r a l p r o t e i n (VP4)  but  r e t a i n a f u l l complement of RNA ( 85 ) . Another method t o d e t e c t p e n e t r a t i o n , the one most w i d e l y used, i s e l e c t r o n microscopy.  However, t h e r e a r e many problems w i t h  t h e i n t e r p r e t a t i o n and e v a l u a t i o n of such s t u d i e s because of the a r t i f a c t s t h a t can a r i s e .  We w i l l now c o n s i d e r the most common problems t h a t  c o n f r o n t the e l e c t r o n m i c r o s c o p i s t .  -4-  1.  Speciman Preservation By f a r , most of the problems a r i s e from inadequate preservation  of the specimen.  Dales (2) has noticed that most d i f f i c u l t y occurs  when cultured c e l l s are f i x e d as a monolayer by glutaraldehyde then scraped and p o s t f i x e d with osmium t e t r o x i d e .  and  The scraping of  c e l l s and "inadequate osmication" also has a d e l e t e r i o u s e f f e c t on the sharpness of the l i n e s d e l i n e a t i n g a phase separation by membranes between c e l l u l a r compartments.  Figs. 3 and 4 demonstrate t h i s e f f e c t .  They are e l e c t r o n micrographs of r e o v i r u s i n f e c t e d L c e l l s . difference between them i s that the l a t t e r was with osmium t e t r o x i d e .  The  only  inadequately f i x e d  C l e a r l y , the v i r u s p a r t i c l e appears as i f i t  l i e s free i n the cytoplasm.  However, F i g . 3 reveals that the reovirus  i s i n fact enclosed w i t h i n a p i n o c y t o t i c vacuole.  Fig. 3  Single reovirus i n a vacuole. evident. X120,000.  The enveloping membrane i s c l e a r l y  Fig. 4  A group of reovirus p a r t i c l e s i n a c e l l sampled soon a f t e r i n o c u l a t i o n . X120,000. (2)  -5-  2.  INTERPRETATION OF The  Since  IMAGES.  w i d t h of  thin sections  this widtli i s greater  t h a n the  i n some c a s e s i m a g e s w h i c h a p p e a r integrity they are  and  l y i n g i n an  demonstrated  Fig.  Fig  Fig.  5  have merged w i t h  the  5-8  and  diameter of  t o show t h e  f r o m 500  most v i r u s e s  viruses  losing  p l a s m a membrane when i n  i n v a g i n a t i o n at  i n Figures  range i n t h i c k n e s s  the  Figure  cell  surface.  t o 1000  there their  This  effect  is  9.  V i r o p e x i s o f VSV p a r t i c l e by an L c e i l i s e v i d e n t 10 m i n . after i n i t i a t i o n of p e n e t r a t i o n . T h i s s e c t i o n was p r o b a b l y c u t n o r m a l l y ( A - A j d i r e c t i o n i n t h e d i a g r a m Fip,. 8 ) , c l a r i f y i n g t h e s e p a r a t i o n b e t w e e n t h e v i r i o n and e n v e l o p i n g membrane. X 1A0,000.  D i a g r a m m a t i c r e p r e s e n t a t i o n of the t a n g e n t i a l o r L o n g i t u d i n a l s e c t i o n s t h a t can be made t h r o u g h a v i r u s - c e l l c o m p l e x o f t h e t y p e shown i n F i g . 5 - 7 . (2)  be  morphological  actuality  6 and 7 The same p r e p a r a t i o n as i n F i g . 5 i l l u s t r a t i n g i m a g e s r e s u l t i n g f r o m t a n g e n t i a l s e c t i o n s ( c u t i n R-B, direction i n t h e d i a g r a m , F i g . 8) X 140,000. 8  may  nm.  -6-  Kip.  9  Ac L i v e v i r u s o.n CAM, warmed l o 35 C for f i v e minutes. In (a) v i r u s appear?; Lo be p e n e t r a t i n g membrane. A f t e r t i l t i n g through 45 C , t h e same p a r t i c l e i n ( b ) i s s e e n t o he on s u r f a c e c f membrane. (22).  -7In Figure 9a i t appears that the v i r a l envelope i s a c t u a l l y " f u s i n g " w i t h the plasma membrane as the r e s t of the v i r u s enters the c e l l . simply by t i l t i n g the speciman through 45° ( F i g . 9b) reveals  However, that the  v i r u s i s a c t u a l l y s t i l l l y i n g on the surface of the membrane. 3. Nature of the Inoculum When working w i t h membrane bound v i r u s e s , m i s i n t e r p r e t a t i o n can a r i s e i f during some stage i n the p u r i f i c a t i o n and handling of the v i r u s e s , the envelopes become damaged. Figure 10 demonstrates such an example.  Fig.  10  C e l l - a s s o c i a t e d NDV possessing ruptured envelope. Arrow i n d i c a the extended nucleocapsid external to the plasma membrane. (2)  -8-  T h i s f i g u r e demonstrates a c e l l - a s s o c i a t e d damaged N e w c a s t l e D i s e a s e Particle.  The s i t e of r u p t u r e o c c u r s at the p o i n t of a t t a c h m e n t .  can c o n c l u d e ( i n c o r r e c t l y ) the plasma membrane.  t h a t the p r o c e s s of u n c o a t i n g o c c u r s  at  One  -9-  The major c l a s s e s of enveloped and n o n - e n v e l o p e d a l o n g w i t h some df t h e i r i n Table. relative  i m p o r t a n t c h a r a c t e r i s t i c s have been l i s t e d  1. A diagram ( F i g . 11) i s a l s o g i v e n d e m o n s t r a t i n g t h e shapes and s i z e s of t h e major a n i m a l v i r u s e s .  1 0 ( 1  Mill  KNA  Fig.  viruses  VIKUSI.S  11. Diagram i l l u s t r a t i n g t h e shapes and r e l a t i v e f a m i l i e s of v i r u s e s . (86)  s i z e s of t h e major  TABLE 1 MORPHOLOGY & COMPOSITION OF THE MAJOR FAMILIES OF ANIMAL VIRUSES  FAMILY  NO. OF DIFFERENT PROTEINS  SYMMETRY  6  Icosahedral  72  9  Icosahedral  252  100  12-24  Icosahedral  162  DNA  160  >30  SS  RNA  2-3  4  Icosahedral  +  SS  RNA  4  3  Icosahedral  80-120  +  SS  RNA  9  16  80-120  +  SS  RNA  5  Helical  +  SS  RNA  4  Helical  DS  RNA  15  SS  RNA  7  SHAPE  DIAMETER (nm)  Papovaviridae  Spherical  45-55  DS  DNA  Adenoviridae  Spherical  70-80  DS  DNA 20-25  Herpetoviridae  Spherical  150  DS  DNA  Poxviridae  Brick-. ... Shaped 100X240X300  DS  Picornaviridae  Spherical  20-30  Togaviridae  Spherical  40-60  Coronaviridae  Spherical  Mxyoviridae  Spherical or filamentous  Rhabdoviridae  BulletShaped  70X180  Reoviridae  Spherical  50-80  Paramyxoviridae  Spherical or Filamentous 100-200  ENVELOPE  NUCLEIC ACID CONFIGURATION MW  (86)  +  3-5  NO. OF CAPSOMERS (IF ICOSAHEDRAL)  o i  Helical  Icosahedral Helical  60  -11-  Slnce the experimental work of t h i s t h e s i s was performed with Semliki Forest V i r u s , I would l i k e to b r i e f l y review the s t r u c t u r e and r e p l i c a t i o n of SF V i r u s . SF Virus c o n s i s t s of a s i n g l e strand of RNA enclosed w i t h i n an icosahedral nucleocapsid which i n turn i s surrounded by a l i p i d bilayer.  SF v i r u s i s made up of 4 p r o t e i n s , nucleocapsid p r o t e i n  plus three glycoproteins denoted as E^, E^, E^, s i t u a t e d i n the envelope. The phospholipid to c h o l e s t e r o l r a t i o i s 1:1 and the glycoproteins are present i n equal amounts (73).  A diagrammatic representation of SF  Virus i s shown i n F i g . 12.  Nucleocapsid P r o t e i n  No. of Molecules per V i r i o n Envelope Proteins  Nucleocapsid F i g . 12  Protein  49,000 MW  190  52,000 MW  190  10,000 MW' 34,000 MW  190 194  Structure of Semliki Forest Virus  -12-  The replication of Semliki Forest Virus takes place i n the cytoplasm of the host c e l l and involves the formation of a multiple-stranded replicative intermediate.  The 42S RNA genome directs the synthesis of at  least two RNA-dependent RNA polymerases.  These enzymes aid i n the  synthesis of a complimentary 42S RNA strand (negative strand).  In turn,  this complimentary RNA strand serves as a template for the synthesis of 2 positive RNA strands having sedimentation co-efficients of 42S and 26S respectively.  Close to 200,000 molecules of each RNA species are synthesized  in baby hamster kidney c e l l s .  The v i r i o n structural proteins on the 42S  strand are duplicated on the 26S strand, and i t i s mainly from the 26S strand that the structural protein genes are translated (80). A l l the virus proteins are f i r s t translated as one polypeptide. The nucleocapsid protein at the N-terminal end i s followed by the enveloped proteins E^, E^,  • Post-translational cleavage yields large  precursor proteins and ultimately the f i n a l structural proteins are formed. A sequence for the post-translational cleavage i s shown below i n Fig. 13. (79). 26S RNA i  NVP165 NVP127 +?  NC  I  PE  NVP97  2  i  (NVP63)  E  2  E  3  Fig. 13 Post Translational Cleavage i n the Formation of SF Virus Structural Proteins  -13-  The assembly of the nucleocapsid p r o t e i n i n t o i t s icosahedralshaped sphere begins by a s s o c i a t i o n of the capsid p r o t e i n with the 42S strand which i s s t i l l serving as a messenger. maturation  The f i n a l process i n v i r u s  involves the nucleocapsid budding through the host plasma  membrane, thus forming a v i r a l envelope around the nucleocapsid. proteins are concentrated  Envelope  at the v i r a l budding s i t e s w i t h the r e s u l t that  a v i r a l envelope i s formed which i s devoid of host c e l l p r o t e i n s . At the end of the growth c y c l e , 5,000 to 20,000 v i r i o n s are produced per c e l l (80). We w i l l now discuss the up-to-date information  concerning  penetration according to the various v i r u s c l a s s e s . PART A: 1.  NON-ENVELOPED VIRUSES  Picornaviruses Picornaviruses (from 'pico' (small) and "RNA") are non-enveloped  s i n g l e stranded RNA v i r u s e s . shaped capsid.  The RNA i s contained w i t h i n an icosahedral-  Diseases these v i r u s e s are responsible f o r include foot  and mouth disease i n animals, herpangina, meningitis and p o l i o m y e l i t i s i n man. The evidence concerning s t i l l inconclusive.  the penetration of picornaviruses i s  There i s i n d i r e c t evidence that p o l i o v i r u s enters the  host c e l l by v i r o p e x i s .  Penetration and l o s s of antibody s e n s i t i v i t y of  p o l i o v i r u s occurs at the same r a t e at 37° C. (58-59) but at 25° C i n f e c t i v e centers escape n e u t r a l i z a t i o n before a l l c e l l - a s s o c i a t e d i n f e c t i v e v i r u s has been l o s t .  This l o s s of s e n s i t i v i t y to n e u t r a l i z a t i o n  -14-  before v i r u s i s l o s t is. most l i k e l y due to entry of v i r u s i n t o p i n o c y t o t i c vesicles.  However i t i s possible that the loss of s e n s i t i v i t y may  r e s u l t of t i g h t binding of the v i r u s to the c e l l membrane. explanation may  be  Another  be that not a l l c e l l - a s s o c i a t e d v i r u s need be located  a pathway leading to an i n f e c t i v e center and therefore the v i r u s may recovered a f t e r c e l l  the  on be  lysis.  Electron microscopy data (2) of an i d e n t i c a l system has confirmed Manel's proposal of v i r o p e x i s  (Fig. 14-16)  Fig. 14. A deep invagination of the PM of a Hela c e l l containing a group of p o l i o v i r u s 1 p a r t i c l e s sampled 10 minutes a f t e r i n i t i a t i o n of penetration. X180,000. Fig. 15 & 16. I n d i v i d u a l p o l i o v i r u s p a r t i c l e s w i t h i n phagocytic vacuoles close to the c e l l surface. X80,000 (2)  Dunneback et al.(60) have presented e l e c t r o n micrographs of p o l i o v i r u s - i n f e c t e d c e l l s which appear to show d i r e c t penetration through the c e l l membrane.  However t h i s f i n d i n g has yet to be confirmed.  -15-  2.  Adenoviruses  Adenoviruses are large  (80 nm)  icosahedral-shaped v i r u s e s .  T h e i r genomes c o n s i s t of d o u b l e - s t r a n d e d DNA.  These agents are u s u a l l y  a s s o c i a t e d w i t h i n f e c t i o n s of the r e s p i r a t o r y t r a c t , , a n d the eye i n mammalian and a v i a n h o s t s .  occasionally  (  A d e n o v i r u s e s have been shown to e n t e r h o s t c e l l s by v i r o p e x i s i n s e v e r a l independent was  demonstrated  studies  (50-52).  By s y n c h r o n i z i n g the i n f e c t i o n s , i t  t h a t the v i r u s e s remain i n the p i n o c y t o t i c v a c u o l e s f o r  only a short period  (51) .  The a b i l i t y of adenovirus 5 to escape out of  p i n o c y t o t i c v a c u o l e s and g a i n a c c e s s to the cytoplasm was dependent on temperature; b e i n g h i g h l y e f f i c i e n t efficient  at temperatures 12° or 20° C.  shown to be  a t 37° C. and  D a l e s and Chardonnet  less  have  concluded t h a t the a b i l i t y t o g a i n access to the cytoplasm i s a p r o p e r t y of  the v i r u s e s themselves.  Heat-denatured  i n o c u l a were r a p i d l y  taken  i n t o h o s t c e l l s by v i r o p e x i s but were not r e l e a s e d i n t o the cytoplasm very e f f i c i e n t l y  (53).  Some evidence has a l s o been p r e s e n t e d t h a t suggests t h a t may  pass d i r e c t l y  adenoviruses  through the c e l l membrane i n t o the cytoplasm without  b e n e f i t of p i n o c y t o s i s  (-41).  Brown and Burlingham  (54) showed Adenovirus  2 i n r e p l i c a s of f r e e z e - e t c h e d i n f e c t e d c e l l s t r a n s v e r i n g d i r e c t l y the plasma membrane.  the  through  V i r u s p a r t i c l e s were a l s o seen i n i n t r a c y t o p l a s m i c  v e s i c l e s but o n l y o c c a s s i o n a l l y .  -16-  F i g . 17 & 18. E l e c t r o n microscopy autoradiography of thin-sectioned primary baby mouse kidney c e l l s at 15 min. p o s t i n f e c t i o n using H-labeled polyoma v i r i o n s . (17) H-DNA-labeled v i r i o n s ; (18) %-amino a c i d labeled v i r i o n s . Non-grain-producing v i r u s can also be seen (arrows). The designations Nu f o r nucleus and Cy f o r cytoplasm are i n d i c a t e d .  3.  Papovaviruses  Papovaviruses are non-enveloped, double-stranded c i r c u l a r DNA viruses.  These viruses are tumorigenic and cause l a t e n t and chronic  i n f e c t i o n s i n mammalian hosts. I t i s w e l l documented that the mode of entry of papovaviruses i s v i a v i r o p e x i s (11, 46-48).  Recently MacKay and C o n s i g l i confirmed  that  v i r o p e x i s i s the mode of entry (49) using optimal conditions of adsorption, e l e c t r o n microscopy and autoradiographic  techniques.  They also demonstrated  that the v i r a l coat proteins and DNA a r r i v e simultaneously i n the nucleus as e a r l y as 15 minutes p o s t i n f e c t i o n (Figs. 17, 18). This i n d i c a t e s that v i r u s uncoating i s an event subsequent to nuclear entry.  They formulated a model of polyoma v i r u s attachment,  penetration and nuclear entry ( F i g . 19 ).  Fig. 19. Model of polyoma v i r i o n attachment, penetration, and nuclear entry.  -18-  4.  1)  attachment a t e i t h e r the v i r u s t w o f o l d o r f i v e f o l d a x i s , ( a t 4°C o f 37° C)  2)  C e l l membrane u n d u l a t i o n t o a l l o w v i r u s t o have c o n t a c t on i t s t h r e e f o l d a x i s s i g n a l l i n g penetration  3)  p e n e t r a t i o n of v i r u s ( v i r o p e x i s )  4)  p i n o c y t o t i c v e s i c l e migrates to nucleus  5)  e n t r y o f v i r u s i n t o n u c l e u s d e v o i d o f an  6)  v i r u s i s uncoated w i t h i n nucleus  7)  MacKay and C o n s i g l i n o t e d t h a t v i r i o n c a p s i d s devoid of n u c l e i c a c i d entered host c e l l s w i t h i n l a r g e p h a g o c y t o t i c v a c u o l e s . These d e f e c t i v e v i r u s e s were n o t seen to e n t e r the n u c l e u s .  envelope  and v i r a l DNA i s r e l e a s e d  Reoviruses R e o v i r u s e s a r e non-enveloped v i r u s e s c o n t a i n i n g genomes  c o n s i s t i n g o f segmented d o u b l e - s t r a n d e d been found i n many d i s e a s e d organs,  RNA.  A l t h o u g h these agents have  they have n o t been a s s o c i a t e d w i t h any  s p e c i f i c p a t h o l o g i c a l p r o c e s s i n man. R e o v i r u s has been shown t o e n t e r r a p i d l y i n t o h o s t c e l l s by v i r o p e x i s (48,182). w i t h i n lysosomes.  U n l i k e o t h e r a n i m a l v i r u s e s , r e o v i r u s i s uncoated  .The p i n o c y t o t i c v a c u o l e s f u s e w i t h lysosomes and then  the uncoating process begins.  I t i s i n t e r e s t i n g to note t h a t i n g e n e r a l  the l y s o s o m a l pathway p a r t i c i p a t e s i n d e f e n d i n g the h o s t c e l l s from v i r a l pathogens b u t the r e o v i r u s uses t h i s pathway f o r i t s own However, as w i t h o t h e r v i r u s e s , i t has been suggested  replication.  that reoviruses  can p e n e t r a t e d i r e c t l y i n t o h o s t c e l l s as w e l l as by p i n o c y t o s i s ( 5 7 ) .  -19-  PART B:  1«  ENVELOPED VIRUSES  Poxviruses Poxviruses are the l a r g e s t and most complex of the animal v i r u s e s .  Their genomes c o n s i s t of double-stranded DNA. agents are the cause of small pox i n man.  Among other diseases,  these  Poxviruses should be an i d e a l  t o o l to i n v e s t i g a t e i n t e r n a l i z a t i o n because they are large i n s i z e , morphologically  d i s t i n c t and also because they have a r a t i o of PFU.to p h y s i c a l  p a r t i c l e s approaching u n i t y .  V a c c i n i a v i r u s enters host c e l l s very r a p i d l y  and shuts down host p r o t e i n synthesis w i t h i n 20 minutes p o s t - i n f e c t i o n . Virus RNA  synthesis, polyribosome formation and  detectable w i t h i n 30 minutes.  proteinisynthes.is;.are  Thus the processes of adsorption,  penetration  and uncoating of the v i r u s p a r t i c l e s must be completed w i t h i n t h i s time period.  Early studies by Dales (3,4) showed that the v i r u s enters  c e l l by v i r o p e x i s .  the  However, more r e c e n t l y Armstrong, Metz and Young (5) ,  using e l e c t r o n microscopy have reported that v a c c i n i a v i r u s enters cultured L c e l l s by a process i n v o l v i n g d i r e c t f u s i o n of the v i r u s envelope with the plasma membrane of the c e l l w i t h i n minutes a f t e r adsorption.  This r e s u l t has been confirmed and extended using immuno-  f e r r i t i n conjugates to l o c a t e the v i r u s antigens on the host c e l l surfaceo.(6)  Figures 20 to 23 show the f e r r i t i n - l a b e l l e d v a c c i n i a p a r t i c l e s  i n the e a r l y stages of attachment and f u s i o n .  Figure 24 demonstrates  the migration of the v i r u s core i n t o the cytoplasm while the conjugated envelope antigens remain at the plasma membrane.  ferritin-  -20-  F i g . 20-23 Thin sections of v a c c i n i a infected c e l l s reacted with antibody conjugate.  ferritin-  F i g . 20  F e r r i t i n - l a b e l l e d v i r u s p a r t i c l e s adsorbed on the surface of L c e l l s . The v i r u s at the top shows an early stage of f u s i o n with the c e l l membrane  F i g . 21 & 22. cells. F i g . 23  F e r r i t i n - l a b e l l e d v i r u s at a l a t e r stage of f u s i o n with L L a t e r a l bodies (LB) are beginning to disperse i n F i g . 21.  Labelled v a c c i n i a p a r t i c l e fused with HeLa c e l l s .  (6)  -21-  f e r r i t i n (F) Is inserted i n t o the c e l l membrane (CM) (6)  A l s o , freeze etching has shown that the components of the v i r u s envelope become r a p i d l y dispersed i n the plasma membrane.  Under t h e i r  experimental conditions, at l e a s t , v i r o p e x i s does not appear to make an important c o n t r i b u t i o n to v a c c i n i a i n f e c t i o n .  These r e s u l t s leave l i t t l e  doubt that f u s i o n i s responsible,at l e a s t i n p a r t , f o r penetration and uncoating of v a c c i n i a v i r u s .  -22-  A l s o a pox  r e l a t e d i n s e c t v i r u s , Amsacta m o o r e i , was shown t o  p e n e t r a t e the i n s e c t i n t e s t i n a l e p i t h e l i u m v i a a f u s i o n mechanism ( 7 ) . Figs.  2 5 - 3 1 i l l u s t r a t e the v i r u s a t v a r i o u s stages of e n t r y  microvilli.  into  The r e g i o n of j u n c t i o n between the c e l l membrane and  the v i r u s envelope was o f t e n d i f f u s e ( F i g s . shown i n F i g . 2 5 .  clearly  Upon f u s i o n of the v i r u s envelope and the m i c r o v i l l u s  membrane, the v i r u s microvillus (Fig.  29-31) but i s  c o r e and p o r t i o n s of the l a t e r a l b o d i e s e n t e r  27).  It  i s apparent t h a t t h e v i r u s envelope was  l o s t a t t h e t i m e of the e n t r y p r o c e s s .  I n over 2,000 s e c t i o n s examined,  v i r o p e x i s was not observed t o be i n v o l v e d i n the e n t r y of v i r u s It  the  particles  i s i n t e r e s t i n g t o n o t e t h a t the m i c r o v i l l i have a s m a l l e r d i a m e t e r  t h a n the v i r u s c o r e . As the v i r u s c o r e " m i g r a t e s " toward the c e l l c y t o p l a s m , the m i c r o v i l l i e n l a r g e i n advance of the c o r e .  The mechanism  t h i s s w e l l i n g i s unknown. 2.  Herpesvirus The h e r p e s v i r u s e s a r e l a r g e (140-170 nm), l i p i d - c o n t a i n i n g ,  d o u b l e - s t r a n d e d DNA v i r u s e s .  These v i r u s e s a r e r e s p o n s i b l e f o r a  number of p e r s i s t a n t i n f e c t i o n s such as c h i c k e n p o x , c o l d s o r e s , i n f e c t i o u s m o n o n u c l e o s i s and s h i n g l e s .  They have a l s o been i m p l i c a t e d  as e t i o l o g i c agents of c e r v i c a l carcinoma and B u r k i t t ' s lymphoma. The p e n e t r a t i o n of h e r p e s v i r u s i n t o a n i m a l c e l l s i s a s u b j e c t of g r e a t c o n t r o v e r s y .  still  S e v e r a l e a r l y s t u d i e s have r e p o r t e d  t h a t h e r p e v i r u s e n t e r s e x c l u s i v e l y o r almost e x c l u s i v e l y by v i r o p e x i s (8-13).  T\Tithin a few m i n u t e s a f t e r e n g u l f m e n t , t h e v i r a l envelopes <['  -22A-  31  F i g . 25. An Amsacta poxvirus p a r t i c l e among m i c r o v i l l i at the apex of a columnar c e l l . X 82,000. F i g . 26 Fusion of a v i r u s p a r t i c l e with the m i c r o v i l l u s . X 82,000. F i g . 27 Transverse section of a m i c r o v i l l u s containing a v i r u s core and p o r t i o n of the l a t e r a l body. X82,000. F i g . 28 F i g . 29 F i g . 30 F i g . 31  Virus core i n the process of entering a m i c r o v i l l u s a f t e r s i d e - t o side f u s i o n of v i r a l envelope and m i c r o v i l l u s membrane. X 82,000. M i c r o v i l l u s - a s s o c i a t e d v i r u s . The v i r i o n appears to be attached l a t e r a l l y to the m i c r o v i l l u s . X 82,000. Virus p a r t i c l e which appears to be fused with the t i p of a m i c r o v i l l u s . X 82,000. Another v i r u s p a r t i c l e which appears to be associated with the t i p of a m i c r o v i l l u s . X 82,000. O)  -23-  d i s a p p e a r , a l l o w i n g the r e l e a s e of the i n n e r core i n t o the c y t o p l a s m . Viropexis  i s a l s o s u p p o r t e d from the e v i d e n c e t h a t t h e r e i s l o s s  in  h e r p e s v i r u s a n t i b o d y - n e u t r a l i z i n g a c t i v i t y concommitant w i t h the uptake of v i r u s viropexis  i n t o phagocytic vacuoles.  T h i s i s r a t h e r good e v i d e n c e  i s t h e predominant mode of e n t r y of h e r p e s v i r u s e s .  that  Interestingly,  n u c l e o p r o t e i n c o r e s , w h i c h can be p r e p a r e d i n pure form a l s o have the a b i l i t y t o be p h a g o c y t i z e d by h o s t Morgan e t a l . ( 1 3 ) v a c u o l e s however,  cells.  a l s o found h e r p e s v i r u s w i t h i n  phagocytic  they have proposed an a l t e r n a t e mechanism of  They suggested t h a t a f t e r a d s o r p t i o n of t h e v i r u s  entry.  t o the c e l l u l a r membrane,  the v i r u s e n z y m a t i c a l l y d i g e s t s b o t h t h e v i r a l envelope anduhost membrane, a l l o w i n g the v i r u s c o r e to e n t e r the  cell.  However, Morgan f i n a l l y conceded t h a t e l e c t r o n m i c r o s c o p y a l o n e cannot d i s t i n g u i s h w h i c h of two v i r a l p a r t i c l e s , e n t e r i n g by two d i f f e r e n t mechanism, a c t u a l l y i n f e c t s the h o s t c e l l and t h a t o t h e r methods a r e needed t o s o l v e the p r o b l e m .  More r e c e n t l y ,  Smith and DeHarven  (14) have c a r r i e d out an a l t r a s t r u c t u r a l s t u d y of v i r a l p e n e t r a t i o n of b o t h herpes s i m p l e x v i r u s and human c y t o m e g a l o v i r u s f u s i o n and v i r o p e x i s were o b s e r v e d .  (CMV).  Both  Subsequent t o the f u s i o n of  CMV, the c a p s i d s f r e e i n the c y t o p l a s m were coated w i t h a f i n e material. Also,  the  fibrillar  T h i s c o a t i n g was not observed w i t h the herpes s i m p l e x v i r u s .  Smith and DeHarven n o t e d t h a t enveloped v i r u s e s t a k e n i n by p i n o ^  c y t o s i s , were a b l e t o e g r e s s from the c y t o p l a s m i c v a c u o l e s by f u s i o n of t h e i r envelope w i t h the v a c u o l e membrane.  -24-  3.  Rhabdoviruses The R h a b d o v i r u s e s a r e enveloped s i n g l e - s t r a n d e d RNA v i r u s e s  w i t h a unique b u l l e t - s h a p e d  structure.  There have been c o n t r a d i c t o r y r e p o r t s on the p e n e t r a t i o n of rhabdoviruses i n t o host c e l l s e s p e c i a l l y w i t h V e s i c u l a r Stomatitus V i r u s (VSV).  VSV has been r e p o r t e d t o p e n e t r a t e by v i r o p e x i s  in a careful  study i n w h i c h a l a r g e number of s e c t i o n s were examined, 0 , 1 , 3 , 5 , 10 minutes and more a f t e r c e l l s i n f e c t e d at 4° C. were warmed t o 37° C. I n f e c t i o n was performed at 4° C. i n o r d e r to a c h i e v e p e n e t r a t i o n when the temperature was r a i s e d to 37° C.  At 4° C ,  (15)  synchronously  p e n e t r a t i o n does not  o c c u r , but attachment i s u n a f f e c t e d . On t h e o t h e r hand, another s t u d y of VSV p e n e t r a t i o n has r e p o r t e d f u s i o n of v i r i o n e n v e l o p e s w i t h c e l l plasma membranes, a l t h o u g h v i r o p e x i s was a l s o o b s e r v a b l e ( 1 6 ) .  In t h i s study t h e r e was no e v i d e n c e of  a n t i g e n s at the plasma membrane.  However, i n the subsequent s t u d y  viral (17),  t h e s e workers p r e s e n t e d i m m u n o l o g i c a l and r e l a t e d e v i d e n c e t o show the p r e s e n c e of VSV g l y c o p r o t e i n s a t the host c e l l membrane.  The problem  w i t h i n t e r p e t i n g t h e r e s u l t s from t h e s e two r e p o r t s i s t h a t t h e (16, 17) employed h i g h speed c e n t r i f u g a t i o n t o i n i t i a t e r a p i d contact.  As a r e s u l t  latter  cell-virus  i t was d i f f i c u l t to d e t e r m i n e whether t h e d i f f e r e n c e s  between t h e two s t u d i e s are due t o problems i n i n t e r p e t a t i o n of  electron  m i c r o s c o p y images or due t o the h i g h speed c e n t r i f u g a t i o n . R e c e n t l y D a h l b e r g (18) u n d e r t o o k t o q u a n t i t a t i v e l y compare  -25-  t h e d i f f e r e n t methods and determine whether the d i f f e r e n t mechanismsof p e n e t r a t i o n c o u l d be accounted f o r i n the d i f f e r e n c e s i n h a n d l i n g the c e l l s and v i r u s . D a h l b e r g a l s o sought to determine what e f f e c t s the presence, or absence of serum would have on the a d s o r p t i o n and p e n e t r a t i o n VSV L-929 c e l l s .  into  When p e n e t r a t i o n was a n a l y z e d f o l l o w i n g a d s o r p t i o n i n the  c o l d , VSV e n t e r e d c e l l s almost e x c l u s i v e l y by v i r o p e x i s . q u a n t i t a t i v e r e l a t i o n s h i p between the d i s a p p e a r a n c e of a t t a c h e d v i r u s and the appearance of v i r u s somewhat l a t e r ,  There was a extracellular  in invaginations,  i n small i n t r a c e l l u l a r vacuoles.  and,  The d a t a was q u a n t i t a t e d  by e x p r e s s i n g each c a t e g o r y i n v i r u s p a r t i c l e s per c e l l p r o f i l e , k e e p i n g the s e c t i o n t h i c k n e s s as c o n s t a n t as p o s s i b l e , and a n a l y z i n g a l a r g e number of s e c t i o n s observed.  (approximately  200) f o r each s a m p l e .  F u s i o n was o n l y  rarely  When the m u l t i p l i c i t y of i n f e c t i o n was lowered to 1 0 - 1 5  p a r t i c l e s per c e l l ,  the same f i n d i n g was o b t a i n e d .  When a d s o r p t i o n was  a c h i e v e d by c e n t r i f u g i n g v i r u s - c e l l m i x t u r e s a t 18,000 X g f o r 15 m i n u t e s , v i r o p e x i s was a g a i n the predominant mode of p e n e t r a t i o n , but f u s i o n occured at a s i g n i f i c a n t l e v e l , i n d i c a t i n g t h a t c e n t r i f u g a t i o n r o l e i n t h e i n c i d e n c e of f u s i o n . of v i r u s  I n a l l c a s e s , serum reduced t h e amount  a d s o r b e d , but d i d not a f f e c t the mode of Another r h a b d o v i r u s ,  p e n e t r a t e by v i r o p e x i s that rabies v i r u s  (19).  'per se' played a  rabies virus,  penetration.  has a l s o been r e p o r t e d  to  However, I w a s a k i e t a l . ( 2 0 ) have shown  a l s o has t h e a b i l i t y to f u s e w i t h the plasma membrane of  a baby hamster k i d n e y c e l l l i n e (BHK-21) b e s i d e s b e i n g t a k e n up by  pinocytotic vacuoles.  B e s i d e s f u s i o n w i t h t h e plasma membrane, f u s i o n  was a l s o observed t o o c c u r w i t h t h e membrane of the p h a g o c y t i c v a c u o l e s w i t h the subsequent r e l e a s e of the v i r u s c o r e i n the c y t o p l a s m ( F i g .  32-35.)  In  a d d i t i o n , v i r u s p a r t i c l e s a t d i f f e r e n t s t a g e s o f d e g r a d a t i o n were a l s o seen i n p h a g o c y t i c v a c u o l e s .  Clearly,  the mechanism of r h a b d o v i r u s  entry  w h i c h l e a d s to i n f e c t i o n i s s t i l l a m a t t e r of c o n f u s i o n . 4.  Myxoviruses M y x o v i r u s e s are g e n e r a l l y s p h e r i c a l a g e n t s , c o n t a i n i n g s i n g l e  s t r a n d e d RNA genomes.  The most w i d e l y s t u d i e d myxovirus i s the i n f l u e n z a  virus. As w i t h o t h e r v i r u s e s , v i r o p e x i s was r e a d i l y observed myxoviruses i n s e v e r a l systems.  The term " v i r o p e x i s " was f i r s t  by Fazekas De S t . G r o t h i n 1948 (21) t o d e s c r i b e the e n t r y of v i r u s i n t o the c e l l s of the c h o r i o a l l a n t o i c membrane.  for introduced  influenza  Virion antigen,  d e t e c t e d by r e a c t i o n w i t h a n t i - v i r a l a n t i b o d y , was l o s t a f t e r  intro-  d u c t i o n of v i r u s e s i n t o the egg, i n d i c a t i n g that the v i r u s e s are taken up whole i n t o the c e l l s .  EM s t u d i e s have shown t h a t i n f l u e n z a v i r u s  i n t e r a c t s by p i n o c y t o s i s w i t h BHK-21 hamster c e l l s , WSN and E h r l i c h ascites cells.  I n a more r e c e n t s t u d y , Dourmaskin and T y r r e l l  i n c u b a t e d an egg adapted s t r a i n of I n f l u e n z a membrane. 4°~C.  w i t h patches of  (22) chorioallantoic  The v i r u s was a l l o w e d t o a t t a c h t o the c e l l s by i n c u b a t i n g a t  f o r one h o u r .  When the temperature was e l e v a t e d t o 37° C ,  was seen to e n t e r by v i r o p e x i s .  virus  S u b s e q u e n t l y the v i r u s e s were seen to  e g r e s s from the p i n o c y t o t i c v a c u o l e s and u n c o a t i n g of the n u c l e o p r o t e i n  -27-  Fig. Fig. Fig. Fig. Fig.  32-35 BHK-21 c e l l s 5 minutes a f t e r i n f e c t i o n with ERA s t r a i n s of rabies v i r u s (20) 32 Alignment of the v i r u s p a r t i c l e s along, but not i n contact with the c e l l surface. No morphologic a l t e r a t i o n can be seen on e i t h e r the v i r u s p a r t i c l e or the c e l l membrane at t h i s stage. X100,000. 33 Engulfment of a s i n g l e p a r t i c l e by p i n o c y t o s i s . Several p a r t i c l e s are engulfed and intermingled with amorphous e l e c t r o n dense material w i t h i n the phagocytic vacuole. X 53,000. 34 Adherence of v i r u s p a r t i c l e s to the host c e l l at the c e l l surface (arrow) X 100,000. 35 Fusion of v i r u s envelope and plasma membrane of p i n o c y t o t i c v e s i c l e . One v i r u s p a r t i c l e i s fused, and and the other p a r t i a l l y degraded p a r t i c l e i s free i n t h i s v e s i c l e . X 100,000  -28-  c o r e was observed ( F i g .  36-38) w i t h i n the c y t o p l a s m .  Occasionally  e l e c t r o n m i c r o g r a p h s were observed t h a t suggest t h a t f u s i o n was a l s o occuring.  However, f u r t h e r a n a l y s i s by means of t i l t i n g the s e c t i o n s  r e v e a l e d t h a t the " f u s i n g " v i r u s envelopes a c t u a l l y remained d i s t i n c t from t h e c e l l membrane d u r i n g t h e s t a g e s of v i r u s  penetration.  These u l t r a s t r u c t u r a l s t u d i e s were c o r r o b o r r a t e d by s t u d i e s on the c l o s e l y r e l a t e d f o w l p l a g u e v i r u s  (23).  The i n o c u l u m was prepared  w i t h a n e u t r a l , r e d - d y e l a b e l and was p e r m i t t e d to uncoat i n c h i c k e n embryo cells.  R e l e a s e of t h e RNA genome from the v i r u s and thus s e p a r a t i o n from the  dye can be m o n i t o r e d by the l o s s i n p h o t o s e n s i t i v i t y  of the  component, whereas removal from the s u r f a c e of the v i r i o n s by a c q u i s i t i o n of a n t i - s e r u m s e n s i t i v i t y  infectious is  determined  and l o s s of h e m a g l u t i n i n .  r e s u l t s i n d i c a t e t h e mode o f e n t r y o f f o w l p l a g u e v i r u s i s  The  viropexis  and the v i r u s becomes uncoated w i t h i n the c y t o p l a s m . More r e c e n t l y ,  R e i n a c h e r and Weiss (24) performed  electron  m i c r o s c o p i c s t u d i e s w i t h f o w l plague v i r u s i n c h i c k embryo c e l l s . T h e i r study showed most of v i r i o n s p r e s e n t w i t h i n c y t o p l a s m i c v a c u o l e s (more o r l e s s d i s i n t e g r a t e d ) .  On the o t h e r hand, o n l y s e l d o m l y were  v i r i o n s a t t a c h e d a t t h e plasma membrane a l t e r e d . fowl plague v i r u s  i s t a k e n up v i a v i r o p e x i s  This i n d i c a t e d  and r e l e a s e  that  their  g e n e t i c i n f o r m a t i o n w i t h i n the c y t o p l a s m i c v a c u o l e s . Others workers have r e p o r t e d t h a t f u s i o n does i n d e e d o c c u r w i t h c h o r i o a l l a n t o i c membrane c e l l s and c h i c k embryo t r a c h e a l c e l l s infected with influencza virus  (.25, 2 6 , 2 7 ) .  e t a l . (.28) s u p p o r t t h i s t h e o r y .  The f i n d i n g s of K r i s a n o v a  D u r i n g i n c u b a t i o n of i n f l u e n z a  virus  -29-  Fig.36 . Active v i r u s on CAM, warmed to 35 C. f o r 5 min. Many p a r t i c l e s are taken up i n cytoplasmic vacuoles. Fig.37.. Active v i r u s on CAM, incubated with f e r r i t i n and warmed to 35 C. for 10 min. (.22)  -30-  F i g . 38.. (a; A c t i v e v i r u s on CAM, warmed t o 35 C. f o r 30 min. V i r u s p a r t i c l e s a r e seen p e n e t r a t i n g w a l l of v a c u o l e ( V ) . There i s a continuous, s i n g l e l a y e r o f membrane s u r r o u n d i n g the v i r u s , (b) As i n ( a ) : p a r t i c l e s a r e p o s i t i o n e d around w a l l s ot v a c u o l e s (V) and a r e i n a p a r t i a l s t a t e ot d e g r a d a t i o n . (22)  -31-  w i t h chick embryo plasma membrane preparations, v i r a l g-antigen reacting i n the complement f i x a t i o n t e s t with antiserum was released.  Levels  of free g-antigen increased according to the duration of incubation. Their r e s u l t s l e d them to conclude that the v i r u s envelope and plasma membrane fuse p r i o r to the release of the nucleoprotein core. And f i n a l l y , Stephenson and Dimmock (29) have r e c e n t l y made an i n t e r e s t i n g observation with i n f l u e n z a - i n f e c t e d chick embryo c e l l s . They noted that i n f l u e n z a v i r u s was able to i n f e c t the c e l l s at 4° C. This observation, r a i s e s questions about the way v i r u s penetrates the plasma membrane since neither f u s i o n nor pinocytosis has been shown to „ occur at 4/ o C.  5.  Paramyxoviruses  Paramyxoviruses are larger and more pleomorphic than myxoviruses. These agents are s i n g l e stranded RNA v i r u s e s which cause diseases such mumps, measles, Newcastle Disease and Canine distemper.  I t i s worthy  to note that these v i r u s e s include the most powerful " c e l l f u s i n g " and hemolytic agents.(36). C e l l b i o l o g i s t s have used paramyxoviruses to produce f u n c t i o n a l heterokaryons by fusing c e l l s from d i f f e r e n t species. The f u s i o n of paramyxovirus envelopes with host membranes have been shown unambiguously  (30-35).  A number of workers have shown that  Newcastle Disease V i r u s (NDV) and Sendai v i r u s envelopes begin to fuse  -32-  w i t h h o s t c e l l membranes w i t h i n minutes of the s t a r t of i n f e c t i o n ( 3 2 , 33,.34).  A u t o r a d i o g r a p h i c methods have been employed t o demonstrate  the r e t e n t i o n o f v i r a l p r o t e i n s on the c e l l u l a r membranes f o l l o w i n g the p e n e t r a t i o n o f v i r a l RNA.  A l s o , when s t u d i e s were done w i t h n o n -  d e f o r m a b l e c e l l membranes such as human e r y t h r o c y t e s  ( 4 0 , 41) and c i l i a  ( 3 0 ) , S e n d a i v i r u s was found t o f u s e w i t h the c e l l u l a r membranes w i t h the subsequent r e l e a s e of n u c l e o c a p s i d from the s u r f a c e . On the o t h e r hand, o t h e r workers have r e p o r t e d t h a t the mechanism of e n t r y of NDV and S e n d a i v i r u s i s v i a v i r o p e x i s  (37-39),  a l t h o u g h some f u s i o n was observed a t the same t i m e . Work has a l s o been performed to s t u d y the p r o p e r t i e s a membrane must have to promote i n g e s t i o n of S e n d a i v i r u s  which  (42).  Sendai  v i r u s e s were i n c u b a t e d w i t h l i p o s o m e s ( v e s i c u l a r model membranes) c o n s i s t i n g of p h o s p h a t i d y l c h o l i n e , c h o l e s t e r o l and g a n g l i o s i d e s . E l e c t r o n m i c r o g r a p h s were t a k e n t h a t resembled the i n g e s t i o n s t e p s of phagocytosis.  I n t e r e s t i n g l y , o n l y the l i p o s o m e s ( F i g .  39A) c o n t a i n i n g  g a n g l i o s i d e s e n v e l o p e d the v i r u s e s i n d i c a t i n g t h a t the g a n g l i o s i d e s may s e r v e as v i r u s If,  receptors.  i n a d d i t i o n to the l i p i d s used i n t h e s e experiments p h o s -  p h a t i d y l - ethano l a m i n e (and s p h i n g o m y e l i n )  a r e used to make l i p o s o m e s ,  the v i r u s membrane appears t o f u s e w i t h the l i p o s o m e s (43) and t o some extent,  p i n o c y t o s i s also takes p l a c e .  These r e s u l t s suggest t h a t  the  mode of e n t r y of p a r a m y x o v i r u s e s may be dependent on the c o m p o s i t i o n of the h o s t membrane a t the s i t e of a t t a c h m e n t .  -32A-  Fig. 38A. I n t e r a c t i o n of Sendai Virus with V e s i c u l a r Model (i)  Membranes.  Model membranes made from 4;j/moles phosphatidyl choline (PC), 2>^moles c h o l e s t e r o l (Chol) and 0.84 mgms gangliosides, containing 0.3^Umoles NANA.  ( i i ) Model membranes made from 1 ^ m o l e PC, .5//mole Chol and 0.44 mg gangliosides, containing 0 . 2 p m o l e s NANA. ( i i i ) and ( i v ) Model membranes made from 1 ^ m o l e PC, 0.5jU mole Chol and .22 mg gangliosides, containing 0.1// mole NANA.  -33-  6.  Oncornaviruses  The o n c o r n a v i r u s e s  (RNA Tumor V i r u s e s or L e u k o v i r u s e s )  enveloped v i r u s e s t h a t a r e i n f e c t i o u s i n many v e r t e b r a t e  are  species.  They i n c l u d e the l e u k e m i a , sarcoma and mammary tumor v i r u s e s of m i c e , c a t s , and sub-human p r i m a t e s . D a l e s and Hanafusa (44) have performed a number of u t i l i z i n g e l e c t r o n microscopy, .autoradiography  and p h y s i c a l - c h e m i c a l  t e c h n i q u e s t o f o l l o w the p e n e t r a t i o n of a v i a n oncorna v i r u s e s host c e l l s .  experiments  into  The r e s u l t s of these experiments i n d i c a t e d t h a t t h e v i r u s e s  e n t e r by v i r o p e x i s and s u b s e q u e n t l y m i g r a t e t o the v i c i n i t y of the n u c l e u s where v i r u s - s p e c i f i c RNA and DNA s y n t h e s i s o c c u r s .  V i r o p e x i s has a l s o  been shown t o occur w i t h the mouse mammary tumor agent and t o a s m a l l e x t e n t f u s i o n at the plasma membrane was a l s o o b s e r v e d . Rauscher l e u k e m i a v i r u s has been r e p o r t e d t o i n t e r a c t w i t h mouse embryo f i b r o b l a s t s i n t h r e e d i f f e r e n t f a s h i o n s  (45):  1)  F u s i o n of v i r u s envelopes w i t h the plasma membrane a l l o w i n g the n u c l e o c a p s i d t o p e n e t r a t e the c e l l .  2)  s i m u l t a n e o u s d i s s o l u t i o n of v i r u s envelopes and plasma membrane a l l o w i n g the n u c l e o c a p s i d a l o n e t o p e n e t r a t e ; the c e l l .  3)  D i s s o l u t i o n of t h e plasma membrane w i t h o u t a f f e c t i n g the v i r u s e s , w h i c h then passes u n a l t e r e d i n t o the cytoplasm.  The p h y s i o l o g i c a l s i g n i f i c a n c e of each of t h e s e i n t e r a c t i o n s i s y e t be d e t e r m i n e d .  to  -34-  7.  N u c l e a r I n s e c t Agents  A l o n g w i t h the i n s e c t p o x v i r u s e s ,  o t h e r i n s e c t v i r u s e s can  a l s o p e n e t r a t e i n t o h o s t c e l l s b o t h by v i r o p e x i s the membranes of m i c r o v i l l i ( 6 5 - 6 7 ) .  ( 6 1 - 6 4 ) and f u s i o n w i t h  Thus, we have a r e p e t i t i o n  of  the p a t t e r n of v i r o p e x i s by n o n - d i f f e r e n t i a t e d , deformable membranes and f u s i o n w i t h d i f f e r e n t i a t e d , n o n - d e f o r m a b l e membranes.  V i r o p e x i s has  r e c e n t l y been demonstrated a g a i n w i t h N u c l e a r P o l y h e d r o s i s V i r u s i n a continous c e l l c u l t r u e d e r i v e d from pupal o v a r i e s of Spodoptera frugiperda  8.  (68).  Coronavirus C o r o n a v i r u s e s a r e enveloped s i n g l e s t r a n d e d RNA v i r u s e s  a p p r o x i m a t e l y 120 nm i n d i a m e t e r .  C o r o n a v i r u s e s mature by b u d d i n g i n t o  the c i s t e r n a e o f the endoplasmic r e t i c u l u m and c y t o p l a s m i c v e s i c l e s but not from the plasma membrane.  These agents a r e r e s p o n s i b l e f o r  several  d i s e a s e s i n c l u d i n g human c o l d s , mouse h e p a t i t u s , i n f e c t i o u s a v i a n b r o n c h i t i s and swine h e m a g g l u t i n a t i n g e n c e p h a l o m y e l i t i s and g a s t r o e n t e r i t i s . Mouse h e p a t i t u s v i r u s has been shown t o e n t e r h o s t c e l l s by viropexis  (69) .  R e c e n t l y work was performed on v i r u s s t r a i n L Y - 1 3 8  i n c u b a t e d w i t h i n t e s t i n a l e p i t h e l i a l c e l l s from newborn c a l v e s . C o u g h r i e t a l . (70) r e p o r t e d t h a t uptake ov v i r u s o c c u r r e d through  fusion  -35-  of v i r a l envelopes w i t h the plasma membrane of the m i c r o v i l l i or by i n t e r a c t i o n w i t h the l a t e r a l c e l l membranes of adjacent epithelial cells.  intestinal  However, t h e i r e l e c t r o n micrographs are f a r from  convincing.  9.  Togaviruses  Togaviruses  of which Semliki Forest Virus i s a member, represent  the smallest of the enveloped RNA v i r u s e s . are encephalitogenic.  These agents i n many cases  The p r o t e i n composition i s r e l a t i v e l y simple  compared to some other RNA v i r u s e s as there are only four s t r u c t u r a l proteins.  One i s associated w i t h the i n t e r n a l core s t r u c t u r e while the  other three are glycoproteins which c o n s i t i t u t e the spikes on the surface of the v i r i o n . There i s very l i t t l e published work on the entry of S e m l i k i Forest Virus or other togaviruses i n t o host c e l l s .  Pathek and Webb  (71) studied by e l e c t r o n microscopy p o s s i b l e mechanism by which S e m l i k i Forest V i r u s may be transported from the lumen of the blood v e s s e l i n t o the parenchymal c e l l s of the mouse c e n t r a l nervous system.  They  suggest SF Virus i s taken i n by v i r o p e x i s i n t o e n d o t h e l i a l c e l l s . The v i r u s p a r t i c l e s then migrate through the c e l l s towards the basement membrane.  There the v i r u s - c o n t a i n i n g vacuoles fuse with the adjoining  c e l l membrane and the v i r u s i s released into the basement membrane.  -36-  Then through p r e s s u r e g r a d i e n t s v i r u s appears to be d i s p e r s e d i n t o e x t r a c e l l u l a r spaces around b r a i n c e l l s . virus  These c e l l s a d j a c e n t t o the  then absorb the v i r u s p a r t i c l e s by p i n o c y t o s i s . A c t i v e l y m i g r a t i n g c e l l s c o n t a i n i n g v i r u s such as mononuclear  cells,  p o l y m o r p h o n u c l e a r l e u k o c y t e s and m i c r o g l i a l c e l l s may a l s o  p l a y some r o l e i n the t r a n s p o r t o f SF V i r u s .  THE PRESENT INVESTIGATION  The r e s u l t s o f 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 c o n c e r n i n g p e n e t r a t i o n are g e n e r a l l y i n c o n c l u s i v e .  The purpose o f t h i s t h e s i s was to s t u d y the  mechanism o f p e n e t r a t i o n o f S e m l i k i F o r e s t V i r u s t h r o u g h a s e r i e s o f r a d i o a c t i v e p u l s e chase e x p e r i m e n t s . These experiments i n v o l v e d t h e use o f r a d i o a c t i v e l y - l a b e l l e d Semliki Forest Virus.  The n u c l e o c a p s i d and envelope p r o t e i n s were  l a b e l l e d to d i f f e r e n t e x t e n t s depending on t h e amino a c i d c o m p o s i t i o n s . The e x t e n t o f l a b e l l i n g can be determined by s e p a r a t i o n of n u c l e o c a p s i d and envelope p r o t e i n s by 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 .  The method we  used t o d i s c r i m i n a t e between v i r o p e x i s and f u s i o n i s demonstrated i n F i g . 39 35 M o n o l a y e r s of BHK-21 c e l l s were i n f e c t e d w i t h Virus  ( p u l s e time was 30 m i n u t e s ) .  S-Met-SF  A t c e r t a i n chase t i m e s , c e l l s were  f r a c t i o n a t e d and t h e plasma membrane (PM) and endoplasmic r e t i c u l u m (ER)  f r a c t i o n s were p u r i f i e d .  These f r a c t i o n s were a n a l y z e d f o r p r o t e i n  and r a d i o a c t i v i t y a f t e r r u n n i n g them on p o l y a c r y l a m i d e g e l s .  Fig.39.. Schematic R e p r e s e n t a t i o n of P o s s i b l e p i n o c y t o s i s and b) f u s i o n .  Routes of SFV e n t r y , a)  -38-  If  f u s i o n were the p r i m a r y mechanism of i n t e r n a l i z a t i o n , we  p r e d i c t t h a t the r a d i o a c t i v i t y c o r r e s p o n d i n g to the envelope p r o t e i n s found i n the PM would remain r e l a t i v e l y c o n s t a n t v a r i o u s chase p e r i o d s .  throughout'.the  On the o t h e r h a n d , the r a d i o a c t i v i t y c o r r e s p o n d i n g  to n u c l e o c a p s i d p r o t e i n would be seen to d e c l i n e as the chase t i m e s i n c r e a s e s i n c e the NC i s e x p e c t e d t o e n t e r the If  cell.  the mode o f e n t r y was v i a v i r o p e x i s  then we would expect  t h a t b o t h the n u c l e o c a p s i d and envelope p r o t e i n s would o n l y  transiently  l a b e l the plasma membrane and a l l r a d i o a c t i v i t y i n t h e PM would d e c r e a s e as the chase t i m e i n c r e a s e s .  F u r t h e r m o r e , we would a l s o expect t h a t the  r a t i o of t h e 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 t o envelope p r o t e i n s at the PM s h o u l d remain c o n s t a n t . 35 ie.  S i n NC S i n EP  ^  Constant  The i s o l a t i o n of e n d o p l a s m i c r e t i c u l u m was performed t o demonstrate the e n t r y o f v i r i o n s or s u b - v i r a l p a r t i c l e s i n t o the h o s t  cells.  -39-  MATERIALS AND METHODS CHEMICALS 35 L  3 S - M e t h i o n i n e (530.46 Ci/mmole),  H-AMP ( 1 1 - 2 5 Ci/mmole) 3  were o b t a i n e d from New England N u c l e a r and _L (25 Ci/mmole) was o b t a i n e d by Amersham/Searle.  H-Amino-acid mixture Sucrose (RNAse f r e e )  was p u r c h a s e d from Schwarz. Mann, and T r i s - H C L , B o v i n e Serum Albumin and cytochrome C were o b t a i n e d from Sigma. 199 maintenance medium, m o d i f i e d E a g l e s medium A , and E a r l e s b a s i c s a l t s medium,  penicillin-streptomycin  and f e t a l c a l f serum came from Grand I s l a n d B i o l o g i c a l Company.  Media  of h i g h i o n i c s t r e n g t h were p r e p a r e d by a d d i t i o n s of a p p r o p r i a t e amounts of sodium c h l o r i d e .  Sodium c h l o r i d e , magnesium a c e t a t e , p o t a s s i u m c y a n i d e ,  b a r i u m h y d r o x i d e , sodium l a u r y l s u l f a t e and sodium phosphate ( d i b a s i c and monobasic) were a l l purchased from F i s h e r .  N i t r o c e l l u l o s e tubes were o b t a i n e d  from Beckman, NADPH from P . L . B i o c h e m i c a l s and a c r y l a m i d e from M a t h e s o n , Coleman and B e l l .  Coomassie B l u e and m e t h y l e n e b i s a c r y l a m i d e were o b t a i n e d  from B i o r a d L a b o r a t o r i e s and g l a c i a l a c e t i c a c i d from A l l i e d C h e m i c a l s .  -40-  ISOLATION OF RADIOACTIVELY LABELLED EXTRACELLULAR VIRUS  V i r u s r e l e a s e d i n the e x t r a c e l l u a r medium was i s o l a t e d by the method of S c h e e l e and P f e f f e r k o r n (77) .  P l a t e s <(or r o l l e r b o t t l e s )  BHK c e l l s 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 p l u s 2% FCS c a l f serum (<~v^ 100 p f u ' s / c e l l ) .  confluent  (made up to 10 mis w i t h M199  Three hours a f t e r a d d i t i o n of  the medium was r e p l a c e d w i t h 5 m i s . (7 mis f o r r o l l e r b o t t l e s ) Salts  of  virus,  of E a r l e s B a s i c  s o l u t i o n p l u s 2% d i a l y z e d c a l f serum i n o r d e r to s t a r v e t h e c e l l s 3  of amino a c i d s . At 3 ' 1 / 2 h o u r s , 100 u l of r a d i o a c t i v e m a t e r i a l (  H-amino  35 a c i d m i x t u r e or .  S - m e t h i o n i n e ; l vmCi/ml, 1 mmole/ml) was added. v  5 hours a f u r t h e r 5 mis of M199 p l u s 2% FCS (10 mis f o r r o l l e r  At  bottles)  was added and the c e l l s were l e f t 12 hours to a l l o w time f o r v i r u s m a t u r a t i o n . At t h i s p o i n t , the medium was drawn o f f w i t h a p a s t e u r p i p e t and l a y e r e d onto a t h r e e - p h a s e g r a d i e n t  (shown i n F i g . .'AO  ).  14 mis V i r u s Sample  10 mis 5-20% Sucrose i n PBS pH8  10 mis 25% Sucrose i n PBS pH8 - 50% Sucrose i n .002 M T r i s , pH7.8 and . 2 M C s C l  2 mis  Fig.40  .  50% Sucrose i n .002 M T r i s , pH7.8 and .2 M C s C l  I s o l a t i o n of V i r u s by Sucrose G r a d i e n t  Centrifugation  -41-  These gradients were centrifuged at 116,000 X g (25,000 rpm) f o r 4 hours i n art SW'27 r o t o r .  The gradients were then dripped from the bottom and  0.5 ml. f r a c t i o n s c o l l e c t e d . A l i q u o t s from each f r a c t i o n were removed and analyzed f o r r a d i o a c t i v i t y , as noted above, i n an Isocap 300 s c i n t i l l a t i o n counter.  Fractions containing r a d i o a c t i v e v i r u s were pooled and stored a t  -70° C. A l i q u o t s was saved f o r plaque assays and r a d i o a c t i v e counting.  PLAQUE ASSAYS  200 u l . of v i r u s were .diluted at 10 ^ by s e r i a l d i l u t i o n . A l l v i r u s d i l u t i o n s (1 ml/plate) were added to small plates of 1/2 confluent BHK c e l l s .  A f t e r one hour of i n f e c t i o n , the medium was drawn o f f and  replaced with 5 mis of equal volumes of 2% (w/v) agar and 2X Medium A plus 4 % FCS. The p l a t e s were l e f t f o r two days to allow plaque formation. At t h i s p o i n t , the c e l l s were f i x e d with 5 mis of formyl s a l i n e f o r 10 minutes.  The agar was peeled o f f and. 5 mis of 2% (w/v) c r y s t a l v i o l e t  were added f o r one hour to allowing f i x i n g .  A f t e r removal of the c r y s t a l  v i o l e t and washing of the p l a t e s , the plaques were counted.  PURIFICATION OF PLASMA MEMBRANE AND ENDOPLASMIC RETICULUM  Plasma membrane and endoplasmic reticulum were prepared by the procedure described by Richardson and Vance (.73).  Three plates (150 mm  X 15mm) of confluent BHK c e l l s were washed twice with 10 mis of cold 10 mM  -42-  T r i s , pH 7 8 .  The c e l l s u s p e n s i o n was homogenized w i t h 10 s t r o k e s of a  l o o s e f i t t i n g V i t r o "Dounce" homogenizer.  N u c l e i and whole c e l l s were  c e n t r i f u g e d from the monogenate at 1000 X g f o r one minute ( 3 , 0 0 0 RPM on a S o r v a l l SS-34 h e a d ) . a d d i t i o n of  S u s p e n s i o n o f the membranes was e f f e c t e d by  . 1 volume of 100 mM N a C l and 30 mM MgCl2>  The homogenate was  then p l a c e d on a d i s c o u n t i n o u s s u c r o s e g r a d i e n t c o n s i s t i n g of (7mls each) of 30% (w/v) 5mM M g C l  2  S u c r o s e i n 5 mM M g C l  and c e n t r i f u g e d a t 7000 X g (5800 rpm  head) f o r 20 minutes ( F i g .  ER  2  , 45% (w/v)  layers  sucrose  in  on a SW 27 c e n t r i f u g e  41).  4 mis Homogenate  7 mis 30% Sucrose i n 5mM MgCl^  PM Bands Here 7 mis 45% S u c r o s e i n 5mM M g C l ,  Fig.  41.  D i s c o n t i n o u s G r a d i e n t f o r S e p a r a t i o n of plasma membrane and endoplasmic r e t i c u l u m .  -43-  A f t e r c e n t r i f u g a t i o n , t h e g r a d i e n t was d r i p p e d from t h e bottom r o t o r and c o l l e c t e d i n 0 . 5 m l f r a c t i o n s .  The PM and ER f r a c t i o n s were  c e n t r i f u g e d a t 105,000 X g (Beckman T i 6 5 ) f o r 1 hour and the p e l l e t s r e suspended i n 300 u l of d i s t i l l e d H 0 . 2  The f r a c t i o n s were t h e n assayed  f o r NADPH-dependent cytochrome C r e d u c t a s e (a marker enzyme f o r endop l a s m i c r e t i c u l u m ) and 5 ' - n u c l e o t i d a s e (a marker enzyme f o r plasma membrane). NADPH-dependent cytochrome C r e d u c t a s e was assayed (74) by m o n i t o r i n g the r e d u c t i o n of cytochrome C s p e c t r o p h o t o m e t r i c a l l y i n a G i l f o r d Spectrophotometer a t 550 nm a t 25° C.  The i n c u b a t i o n mix c o n -  s i s t e d of t a k i n g . 2 5 mis of c o c k t a i l mix ( 0 . 1 mM KCN, 0 . 6 6 mM KC1, .044 M phosphate ph 7 . 6 , 0 . 5 mM cytochrome C).to w h i c h 50 u l of enzyme was added.  The r e a c t i o n was s t a r t e d by a d d i t i o n of 50 u l of 0 . 6 mM NADPH. NADPH-dependent cytochrome C r e d u c t a s e a c t i v i t y was c a l c u l a t e d  as  follows: E"*"^ Reduced cytochrome C = 2 7 . 7 X 10^ cm / mole 2  E  550  O x i d i z e d cytochrome C = 9 . 0 X 1 0  ,550 A ±= E dt Red. C y t .  d  Red.  Cyt. C  6  cm / mole 2  _F Oxid. Cyt. 5 5 0  C d _ :[Red. C y t . dt  dA = —  C]  C d_ dt  [Red.  5 5 0  1 8 . 7 x 1 0 ml/mole 6  The 5 ' - n u c l e o t i d a s e a s s a y (75) was performed by i n c u b a t i n g the a s s a y mix (100 u l of 0 . 5 M T r i s - H C l ,  pH 7 . 5 and 0 . 2 M M g C l , 50 u l of 2  Cyt,  -440 . 4 mM AMP, 20 jal of  3  H-AMP, 0 . 6 3 ml o f H 0 and 200 u l of each f r a c t i o n ) 2  a t 37° C f o r 30 m i n u t e s .  The r e a c t i o n was stopped by f i r s t a d d i n g 0 . 2  mis of 0 . 2 5 M ZnSO^ and then adding 0 . 2 mis of 0 . 2 5 M Ba ( 0 H ) t o p r e c i p i t a t e the u n r e a c t e d ATP.  2  in  order  The assay mixes 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 desk top c e n t r i f u g e f o r 5 minutes at s e t t i n g 4 .  0 . 5 mis  o f s u p e r n a t a n t from each f r a c t i o n were p l a c e d i n s c i n t i l l a t i o n v i a l s  to  3 w h i c h 10 mis of ACS were added and then m o n i t o r e d f o r  H-adenCsine by  c o u n t i n g i n an I s o c a p 300 s c i n t i l l a t i o n c o u n t e r . SDS ACRYLAMIDE  ELECTROPHORESIS  C e l l p r o t e i n s and v i r u s samples were e l e c t r o p h o r e s e d on 7.5% a c r y l a m i d e SDS g e l s as d e s c r i b e d by Weber and Osborne (72) . a c r y l a m i d e was used as t h e c r o s s l i n k i n g a g e n t .  Methylenebis-  A l l samples were p r e -  e l e c t r o p h o r e s e d a t 50 mV, 2 . 5 mA/gel f o r 30 minutes and then e l e c t r o p h o r e s e d 150 mV, 8 . 0 mA/gel f o r 2 1/2 h o u r s .  The g e l s were then f i x e d i n 7.5%  a c e t i c a c i d f o r one hour a t 60° C p r i o r t o g e l s l i c i n g .  I n some c a s e s ,  the g e l s were s t a i n e d i n Coomassie B l u e f o r one hour a t 60° C. p r i o r  to  t r e a t m e n t w i t h 7.5% a c e t i c a c i d .  GEL SLICING AND SCINTILLATION COUNTING OF GEL SAMPLES G e l s were f r o z e n i n a dry i c e - a c e t o n e b a t h and cut i n t o 1 mm slices: using a Bio-Rad gel s l i c e r .  Three s l i c e f r a c t i o n s were p l a c e d i n  s c i n t i l l a t i o n v i a l s and were d i g e s t e d f o r one hour w i t h . 5 mis o f 2% periodic acid.  The samples were then suspended i n 10 mis of ACS and  counted i n a New England N u c l e a r I s o c a p 300 s c i n t i l l a t i o n c o u n t e r .  -45-  ANALYSIS OF SDS ELECTROPHORESIS PROFILES  Gels that were previously stained were scanned at 550 nm using a G i l f o r d Spectrophotometer. Subsequently the gels were s l i c e d and counted as previously outlined.  P l o t s of absorbance versus length of g e l and counts  per minute versus length of g e l were p l o t t e d .  Points i n each r a d i o a c t i v i t y  peak were summated a f t e r subtracting the basal r a d i o a c t i v i t y from each value.  PROTEIN ASSAYS  P r o t e i n assays were performed as described by Lowry (76). L i p i d - c o n t a i n i n g p r o t e i n samples were incubated overnight i n 0.66 N NaOH at 37° C. p r i o r to analysis. Bovine serum albumin was used as a standard.  -46-  EXPERIMENTAL RESULTS  PREPARATION OF RADIOACTIVELY-LABELED SF VIRUS  R a d i o a c t i v e l y - l a b e l e d SFV  was prepared as described i n the  Methods section. B r i e f l y , t h i s method involves a preparative sucrose gradient 25,000 rpm f o r four hours . These gradients were dripped from the bottom i n 0.5 ml f r a c t i o n s and a l i q u o t s (50 jul) were assayed f o r r a d i o a c t i v i t y i n 10 mis. of ACS.  The r a d i o a c t i v i t y p r o f i l e i s shown i n Figure 42 . 3 s i n g l e peak corresponds to H-a.a.-SF V i r u s .  The  25 Counts Per 20  Minute  ( X 10 ) -3  15  10  10 F i g . 42  20  30  F r a c t i o n Number  Radioactive P r o f i l e of a T y p i c a l Preparative Sucrose Gradient,  _47-  STANDARD VIRUS GEL  P u r i f i e d r a d i o a c t i v e l y - l a b e l e d SF V i r u s was e l e c t r o p h o r e s e d w i t h 60Qvgms of n o n l a b e l e d SF V i r u s s t a n d a r d ) -  9 . 5 cm i n l e n g t h .  (along  on 7.5% SDS g e l measuring  The Coomassie B l u e p r o t e i n s were scanned u s i n g the  G i l f o r d Spectrophotometer. was t h e n s l i c e d and c o u n t e d .  T h i s s c a n i s shown i n F i g . 4 3 .  The g e l  The g e l r a d i o a c t i v i t y p r o f i l e i s shown  i n F i g u r e 44.  Fig.  43.  Coomassie B l u e Scan of Standard V i r u s G e l (Absorbance = 550 nm)  -48-  44 .  R a d i o a c t i v e P r o f i l e of Standard  H-amino a c i d - l a b e l l e d  SF V i r u s G e l .  -49-  The l e f t peak of the p r o t e i n scan represents the  and E^ proteins and the  r i g h t peak represents the nucleocapsid proteins (73).  The f a c t the E^  and E^ are not resolved i n t h i s g e l system i s not s u r p r i s i n g considering t h e i r nearly i d e n t i c a l molecular weights (see page 11).  Separation of  and  E^ can be achieved by using a discontinuous b u f f e r - e l e c t r o p h o r e s i s system. Another i n t e r e s t i n g point i s that E^ i s not stained at a l l , presumably due to i t s high carbohydrate content. The r a d i o a c t i v i t y p r o f i l e c l e a r l y demonstrates that the t r i t i a t e d amino acids become s p e c i f i c a l l y associated with the v i r a l p r o t e i n s .  Summation  of the counts per minute under each peak (minus basal background l e v e l s ) reveals that the r a t i o of r a d i o a c t i v i t y i n the E^ E^ peak to that i n the nucleocapsid p r o t e i n ( i e . cpm i n E^ E^  )  i s 2.52. This value i s  cpm i n NC very much i n agreement with the t h e o r e t i c a l value of 2.78 derived from the amino a c i d composition of SF V i r u s . ( 7 8 ) .  ISOLATION OF PLASMA MEMBRANE AND ENDOPLASMIC RETICULUM  The i s o l a t i o n of PM and ER of mock-infected performed as o u t l i n e d i n the Methods s e c t i o n .  BHK c e l l s were  The 5'-nucleotidase (PM  marker) and NADPH-dependent cytochrome C reductase  (ER marker) a c t i v i t i e s  of the c e l l l y s a t e , PM p e l l e t and ER p e l l e t were measured and summarized i n Table 2.  -50-  TABLE 2 ACTIVITIES OF PLASMA MEMBRANE AND ENDOPLASMIC RETICULUM MARKER ENZYMES CELL FRACTION  5'-NUCLEOTIDASE ACTIVITY  PURIFICATION  NADPH-DEPENDENT CYTOCHR OME C SE REDUCTA PURIFICATION ACTIVITY  C e l l Lysate  1.2  1  .04.  1  Endoplasmic Reticulum  30  25  7.2  180  .54  45  .07  1.75  Plasma membrane  The endoplasmic reticulum was enriched 180-fold compared to the c e l l l y s a t e however the ER appears to be contaminated with 5' nucleotidase activity.  The plasma membrane appeared to be p u r i f i e d 45-fold with minimal  contamination from endoplasmic reticulum.  The y i e l d of PM from the c e l l  l y s a t e was 28%. I t i s c l e a r that separation of plasma membrane from endoplasmic reticulum has been achieved.  These values are comparable to  those obtained by Richardson and Vance (73).  PRELIMINARY EXPERIMENTS  A s e r i e s of c o n t r o l experiments were undertaken i n order to determine the optimal conditions of v i r u s attachment to BHK c e l l s . I n i t i a l l y , the optimal time of adsorption was determined by i n f e c t i n g BHK  -51-  c e l l s with  H-aa-SF Virus (1X10 p f u ' s / c e l l ) at 37°C and 4°C.  At desired  time intervals.unadsorbed v i r u s was removed by washing w i t h 2 X 4 mis of high i o n i c strength Medium A ( u = .25) . The c e l l s were then scraped o f f the p l a t e s w i t h 5 mis of H^O, placed i n t o s c i n t i l l a t i o n v i a l s containing 10 mis of ACS and counted i n the Isocap 300 s c i n t i l l a t i o n counter.  The  r e s u l t s of t h i s experiment are shown i n F i g . 45. I t i s evident that the adsorption of v i r u s i s greatly f a c i l i t a t e d at 37° C rather than 4° C and continues to increase up to at l e a s t three hours of incubation.  A l l subsequent experiments were performed at 37° C.  Another study was performed to determine the e f f e c t the i o n i c strength of the inoculum on v i r u s adsorption.  Solutions of d i f f e r e n t  i o n i c strengths were made by making appropriate d i l u t i o n s of the high i o n i c 3  strength Medium A. 200 u l of H-aa-SF V i r u s were added to 5 mis of appropriate medium and incubated w i t h p l a t e s (100mm X 15 mm) of BHK c e l l s f o r 60 minutes. counted as before.  At t h i s p o i n t , the c e l l s were washed, scraped and  A summary of the r e s u l t s are shown i n F i g . 46.  The important point to note i s that the v i r u s adsorption i s g r e a t l y f a c i l i t a t e d at low i o n i c strengths. strength of approximately  Medium A has an i o n i c  .13 and as can be seen, the v i r a l adsorption i s  decreased by more than 1/2 the value at i o n i c strength .10. A l l subsequent experiments were performed w i t h inoculums of i o n i c strength .10.  -52-  25  20  1.5  1.0  .5  5 10  30  60  120  Time of I n c u b a t i o n  Fig. 45.  Dependence of SF V i r u s A d s o r p t i o n on Temperature and Time of Incubation.  180  (minutes)  Fig.46  .  Effect  of  Ionic Strength  o f Medium on  SF V i r u s  Adsorption,  -54-  Two experiments a r e p r e s e n t e d i n F i g . 4 6 .  The " f i l l e d - i n " l i n e  r e p r e s e n t s an experiment i n w h i c h a low i o n i c s t r e n g t h s o l ' n ( u = . 1 0 ) was used t o wash the c e l l s .  The d o t t e d l i n e r e p r e s e n t s an experiment  u s i n g a h i g h i o n i c s t r e n g t h wash ( AI = . 2 5 ) .  It  was o r i g i n a l l y  thought  t h a t t h e h i g h i o n i c s t r e n g t h wash would be more e f f e c t i v e i n washing away " l o o s e l y bound" v i r u s .  T h i s was based on the assumption t h a t the SF V i r u s  c e l l i n t e r a c t i o n i s of an i o n i c n a t u r e however i t i s c l e a r t h a t  the  i o n i c s t r e n g t h of the wash d i d not have a s i g n i f i c a n t e f f e c t on v i r u s adsorption. The next c o n t r o l experiment s t u d i e d t h e e f f e c t f 6 r m i n g : ' - u n i t s per c e l l (or " m u l t i p l i c i t y of i n f e c t i o n " ) adsorption.  have on v i r u s  D i f f e r i n g amounts of v i r u s were d i a l y z e d a g a i n s t 2 l i t e r s  low i o n i c s t r e n g t h s o l u t i o n ( AX = . 1 0 ) cells  thevplaque  f o r t h r e e hours a t 4°C.  of  BHK  ( 100 nm X 15 nm ) were then i n f e c t e d f o r 60 minutes w i t h t h e s e  v i r u s samples made up t o 5 mis w i t h s o l u t i o n xi = . 1 0 . h a r v e s t e d and counted as b e f o r e . shown i n F i g u r e 4 7 .  C e l l s were washed,  The r e s u l t s of t h i s experiment  T h i s f i g u r e c l e a r l y shows a l i n e a r  are  relationship  between v i r u s a d s o r p t i o n and the m u l t i p l i c i t y of i n f e c t i o n up to 8,000 plaque forming u n i t s / c e l l .  Subsequent experiments were c a r r i e d out a t  5000 p l a q u e f o r m i n g u n i t s per  cell.  -55-  -56-  0-2 HOUR PULSE CHASE EXPERIMENT A f t e r completion of the preliminary experiments we decided to t r y a 2 1/2 hour pulse-chase experiment.  This experiment consisted of a 30  35 minute pulse with  S-labeled SFV ( 5 mis, 5000 p f u ' s / c e l l , i o n i c strength  yu = .10 ) and chase periods of 1, 60, and 120 minutes a f t e r removal of the radioactive virus.  Three plates (150 mm X 15mm) of BHK c e l l s were used per  time p o i n t . B r i e f l y , r a d i o a c t i v e v i r u s c o l l e c t e d from the sucrose gradients, was dialyzed at 4° C against d i l u t e d Medium 199 CjJ = .10 ). A f t e r d i a l y s i s the v i r u s was divided i n t o a l i q u o t s , made up to 5 mis with Medium 199 CP = .10) and allowed to adsorb to a p l a t e of BHK c e l l s f o r 30 minutes at 37° C.  A f t e r the end of the pulse time, the c e l l s were washed twice with 5  mis. of high i o n i c strength Medium A (jj distilled ^ 0 .  = .25") and once w i t h 5 mis of  5 mis of Chase medium c o n s i s t i n g of Medium 199 plus 2% f e t a l ..  serum was then added to the c e l l s .  At the end of the appropriate chase  periods, c e l l s were washed twice with 5 mis of d i s t i l l e d ^ 0 , scraped from t h e i r p l a t e s w i t h a rubber policeman and placed i n a loose f i t t i n g V i t r o "Dounce" homogenizer.  C e l l s were lysed and the PM and ER f r a c t i o n s were  i s o l a t e d as o u t l i n e d i n the M a t e r i a l s and Methods s e c t i o n except that the PM and ER bands i n the sucrose gradient were extracted d i r e c t l y from the sides of the c e n t r i f u g e tube using a syringe. The two f r a c t i o n s were then p e l l e t e d by r e c e n t r i f u g a t i o n at 105,000 X g (45,000 rpm) f o r 60 minutes on a Beckman T i 65 r o t o r .  The p e l l e t s were resuspended i n 300 p.1 of d i s t i l l e d  A l i q u o t s (25 ill)  H^O.  were saved f o r p r o t e i n determination and the remainder  electrophoresed on 7.5% SDS acrylamide gels and analyzed as o u t l i n e d i n the M a t e r i a l s and Methods s e c t i o n .  F i ~ . 48 ,  O - 2 Hour  Chase Virus Gels  ('Results exDressed i n terms of 2 mems D r o t e i n )  -58-  I  o XI  ft  60'  12-<D»  Time of Chase  F i g . -49.;  (minutes)  Time Course of SF V i r u s I n f e c t e d Plasma Membrane and Endoplasmic R e t i c u l u m . ( 0 - 2 Hour Chase)  -59-  The r a d i o a c t i v e p r o f i l e s of these gels are shown i n Figure 48. a-f , which represent plasma membrane f r a c t i o n s at 0, 60, and 120 minute chase times respectively.  Figure 48. a-c c l e a r l y demonstrates the v i r a l proteins associated  with the plasma membrane.  The r a d i o a c t i v i t y i n the PM i s reduced by approx-  imately 90% by 60 minutes of chase and t h i s drop i n r a d i o a c t i v i t y can be a t t r i b u t e d to a l o s s of envelope and nucleocapsid p r o t e i n . the 120 minute chase p o i n t , approximately  However, at  the same amount of r a d i o a c t i v i t y  remains i n the plasma membrane as at the 60 minute chase • Figures 48. d-f 120 minutes of chase.  represent the ER f r a c t i o n s at 0, 60, and  Virus p r o t e i n i s shown to enter the c e l l and be-  come associated with the ER however i t i s i n t e r e s t i n g to note that only the nucleocapsid p r o t e i n becomes associated while the envelope proteins are completely  lost.  T o t a l r a d i o a c t i v i t y of the v i r u s - s p e c i f i e d proteins associated w i t h the PM and ER were determined and graphed versus chase time (Fig.'49 The r e s u l t s show that the v i r a l proteins are r a p i d l y l o s t from the PM w i t h i n 60 minutes f o l l o w i n g the pulse.  On the other hand, the amount of  v i r a l proteins i n ER increase up to 60 minute of chase and then d e c l i n e again at the 120 minute chase p o i n t .  The decrease i n r a d i o a c t i v i t y i n the  ER could be due to turnover of the nucleocapsid w i t h i n the c e l l . These r e s u l t s are consistent with the theory of a precursorproduct r e l a t i o n s h i p between the v i r a l proteins i n the PM and the v i r a l proteins i n the ER.  -600 - 6 0 MINUTE PULSE CHASE EXPERIMENT Based on the p r e v i o u s e x p e r i m e n t , i t was d e c i d e d t o r e f i n e experiment by d o i n g , 0 , 2 0 , 40 and 60 minute chase time p o i n t s .  the  The  experiment was performed as d e s c r i b e d above and the r e s u l t s are shown i n F i g u r e 50 a - h . F i g u r e 50 a - d show a p r o g r e s s i v e l o s s i n r a d i o a c t i v i t y the plasma membrane over the 60 minute c h a s e .  from  The ER p l o t s ( F i g u r e 50 e - h )  do not e x h i b i t any d e f i n i t e t r e n d of i n c r e a s e o r decrease i n  radioactivity.  I n t h i s e x p e r i m e n t , a second (minor) peak o f r a d i o a c t i v i t y o c c u r s i n the 2 0 , 4 0 , and 60 minute chase p l o t s .  T h i s peak r e p r e s e n t s the envelope p r o t e i n s  of SF V i r u s and i t s p r e s e n c e i n t h e ER f r a c t i o n i s presumably due to c o n t a m i n a t i o n by plasma membrane.  The major peak, as b e f o r e ,  represents 35  the n u c l e o c a p s i d p r o t e i n .  F i g . 51f r e p r e s e n t s 50 u l of s t a n d a r d  S-SF  Virus. The t o t a l v i r u s - s p e c i f i e d r a d i o a c t i v i t y were determined and p l o t t e d v e r s u s chase time ( F i g .  51).  These r e s u l t s show a s t e a d y l o s s of  r a d i o a c t i v i t y i n the plasma membrane throughout  the 60 minute chase w h i l e  the r a d i o a c t i v i t y i n the ER remains r e l a t i v e l y c o n s t a n t throughout experiment.  the  F u r t h e r d i s c u s s i o n and c o n c l u s i o n s of the above r e s u l t s  w i l l be p r e s e n t e d i n t h e n e x t s e c t i o n .  10  20  30  40  10  20  F r a c t i o n No. F i g . 50  .  0' - 60'  Chase Virus Gels  30  40 F r a c t i o n Y.o.  ( Results expressed i n terns of  2 mgms protein)  e.  f.  ER - 0' Chase  F r a c t i o n No. F i g . 50  Con't  ER - 20  :  Chase  F r a c t i o n No.  -63-  i.  5&>|  35  S - SF Virus  F r a c t i o n No. F i g . 50. Con't  ro ! O  (E ' E  PM  and NC)  a ft  o  Time of Chase (minutes) 51  Time Course of SF Virus Infected Plasma Membrane and Endoplasmic Reticulum. (0 --60' Chase) ?  DISCUSSION  The m a j o r i t y of e v i d e n c e i n d i c a t e s t h a t v i r o p e x i s  i s the p r e -  d o m i n a n t , but not e x c l u s i v e , mechanism of v i r u s e n t r y i n t o h o s t c e l l s . I n most c a s e s , when f u s i o n was o b s e r v e d by e l e c t r o n m i c r o s c o p y , a s i g n i f i c a n t l e v e l of v i r o p e x i s was a l s o n o t e d .  However e v i d e n c e i n the l a s t  several  y e a r s has u n q u e s t i o n a b l y c o n f i r m e d f u s i o n as the mode of e n t r y of V a c c i n i a , S e n d a i , N e w c a s t l e D i s e a s e and some I n s e c t agents i n c e r t a i n h o s t c e l l s . If  t h e r e i s a d e f i n i t e p a t t e r n t o the mechanism of  of e n v e l o p e d v i r u s e s ,  It  internalization  i s that f u s i o n takes place p r e f e r e n t i a l l y  with  h o s t c e l l s e n c l o s e d by r e l a t i v e l y n o n - d e f o r m a b l e membranes such as erythrocytes, It  c i l i a and m i c r o v i l l i . i s w o r t h y to n o t e t h a t i f v i r u s e s e n t e r v i a  viropexis,  g e n e r a l l y i t must s t i l l f u s e w i t h t h e membrane of the c y t o p l a s m i c v a c u o l e i n o r d e r t o a l l o w the r e l e a s e of the v i r a l c o r e i n t o the c y t o p l a s m . T h i s may l e a d us t o t h e o r i z e t h a t enveloped v i r u s e s may have the p o t e n t i a l to e n t e r h o s t c e l l s by b o t h f u s i o n and p i n o c y t o s i s .  E x a c t l y what o c c u r s  d u r i n g each i n f e c t i o n may be the r e s u l t of two independent  factors.  P i n o c y t o s i s may o n l y be a c e l l u l a r response due to p r e s e n c e of a f o r e i g n , m a c r o m o l e c u l a r agent on i t s s u r f a c e .  F u s i o n , on the o t h e r hand,  may be s o l e l y d ue to!r.the p r o p e r t i e s of the v i r i o n s .  That i s ,  lysins  on t h e v i r a l s u r f a c e s somehow a l t e r the plasma membrane, a l l o w i n g f u s i o n to occur.  P e n e t r a t i o n may t h e r e f o r e be e n v i s a g e d as a s o r t of " r a c e "  -65-  between the f u s i o n of t h e membranes and the p h a g o c y t i c response of host c e l l .  the  T h i s w o u l d e x p l a i n why f u s i o n t a k e s p l a c e w i t h c e l l s h a v i n g  r e l a t i v e l y n o n - d e f o r m a b l e c e l l u l a r membranes and the o b s e r v a t i o n f u s i o n a t t h e c e l l membrane w i t h i n p a r t i a l i n v a g i n a t i o n s .  of  This theory  could a l s o e x p l a i n the c o n t r a d i c t o r y r e s u l t s obtained w i t h V e s i c u l a r Stomatitus Virus  ( 1 5 , 16) where f u s i o n w i t h L c e l l s was observed  rather  t h a n v i r o p e x i s , when h i g h speed c e n t r i f u g a t i o n was employed t o i n i t i a t e rapid v i r u s - c e l l contact. "tight"  It  c o u l d be i n o r d e r to e f f e c t f u s i o n , a  i n t e r a c t i o n must occur w h i c h i s f a c i l l i t a t e d by c e n t r i f u g a t i o n , _  thus n o t a l l o w i n g enough time f o r p i n o c y t o s i s t o t a k e p l a c e . S i n c e p i n o c y t o s i s i s a temperature-dependent s t e p and can be i n h i b i t e d by m e t a b o l i c p o i s o n s such as a m a n a t i d i n e , t h i s t h e o r y c o u l d p o s s i b l y be t e s t e d .  For example, t h i s t h e o r y would : b e s s u p p o r t e d : i . i f f f i u s i o n  was observed i n the p r e s e n c e of a m a n t i d i n e . The work of Chang and Metz (6) may have a l s o opened up a new d i r e c t i o n s i n the s t u d y of v i r a l p e n e t r a t i o n .  By p r e p a r i n g the same type  of i m m u n o - f e r r i t i n agents f o r o t h e r v i r u s s p e c i e s , v i r a l a n t i g e n s can be r e a d i l y i d e n t i f i e d on the c e l l s u r f a c e and thus c o n f i r m one way or another i f  fusion occurs. A n o t h e r p o s s i b l e experiment w h i c h c o u l d be attempted i s t o l a b e l  the v i r u s e s w i t h a f l u o r e s c e n t compound such as f l u o r e c e i n and d i r e c t l y v i s u a l i z e t h e p e n e t r a t i o n of t h e v i r u s e s through e l e c t r o n m i c r o s c o p y . If  the v i r i o n s were to e n t e r p r i m a r i l y by f u s i o n , we would e x p e c t ,  -66-  th e f l u o r e s c e n t marker to remain a t the c e l l membrane w h i l e the r e s t the v i r u s e n t e r s the c e l l .  of  S i n c e t h i s experiment appears to be f a c i l l e  and has y e t t o be done, I would s u s p e c t t h a t t h e r e are some t e c h n i c a l d i f f i c u l t i e s unaware t o t h i s  writer.  R e t u r n i n g t o the p r e s e n t i n v e s t i g a t i o n , i t was our  intention  t o d e t e r m i n e by b i o c h e m i c a l methods t h e mode of p e n e t r a t i o n of SF V i r u s 35 i n t o BHK-21 c e l l s .  The h o s t c e l l s were i n f e c t e d w i t h  S - l a b e l e d SF V i r u s  and the v i r a l p r o t e i n s were chased i n t o the plasma membrane and endoplasmic reticulum fractions.  If  f u s i o n were the mode of e n t r y , we would expect c  t h a t the SF V i r u s envelope p r o t e i n s w i l l remain i n the plasma membrane. The PM/ER p u l s e chase s t u d i e s ( F i g .  50 a - d )  c l e a r l y show b o t h the envelope  p r o t e i n s and n u c l e o c a p s i d b e i n g chase out of t h e plasma membrane f r a c t i o n d u r i n g the c o u r s e of the e x p e r i m e n t .  Furthermore,  the r a t i o o f  radio-  a c t i v i t y of envelope p r o t e i n s to n u c l e o c a p s i d remained a p p r o x i m a t e l y same throughout  t h e chase p e r i o d .  These r e s u l t s l e a d us t o the c o n c l u s i o n  t h a t SF V i r u s e n t e r s BHK c e l l s v i a v i r o p e x i s . g i v e n w i t h some r e s e r v a t i o n .  It  the  T h i s p r o p o s a l , however,  i s p o s s i b l e t h a t the t r u e l y  r o u t e t a k e n by SF V i r u s i s by f u s i o n .  is  infectious  T h i s may o c c u r w i t h o n l y a minor  f r a c t i o n of the t o t a l i n o c u l u m w h i l e the r e s t of the v i r u s e n t e r s by pinocytosis  ( s o l e l y a c e l l u l a r response due to the p r e s e n c e of a f o r e i g n  agent of i t ' s s u r f a c e ) .  A l t h o u g h i t may not be a l i k e l y p o s s i b i l i t y ,  t h i s type of p e n e t r a t i o n and p i n o c y t o s i s a r e i n d i s t i n g u i s h a b l e u s i n g our methods.  T h i s p o s s i b i l i t y w i l l have t o be f u r t h e r examined i n  experiments.  future  -67-  The one apparently s u r p r i s i n g r e s u l t involves the accumulation of nucleocapsid p r o t e i n i n the endoplasmic reticulum f r a c t i o n ( F i g . 50 e-h). The manner i n which the nucleocapsid i s transported to the ER and the r e l a t i o n s h i p t h i s phenomenon has to the uncoating process i s s t i l l unknown. On the other hand, t h i s phenomenon may i n fact be an a r t i f a c t . Nucleocapsid p r o t e i n from the cytoplasm could possibly be p r e c i p i t a t e d with the ER by the 100,000 X g c e n t r i f u g a t i o n . A number of c r i t i c i s m s of these experiments can be made.  Firstly,  there i s a problem i n that high l e v e l s of r a d i o a c t i v i t y become associated with the ER f r a c t i o n very q u i c k l y , even at zero minutes of chase. This s i t u a t i o n can be p a r t i a l l y a l l i e v a t e d by using shorter pulse period (eg. 10-15 minutes).  However, by p u l s i n g f o r a shorter period of  time, we may cause another problem i n v o l v i n g the attainment of enough r a d i o a c t i v i t y i n t o the plasma membrane to v i s u a l i z e on acrylamide gels. At any rate i t most l i k e l y w i l l not a l t e r the conclusions reached i n t h i s i n v e s t i g a t i o n since the v i r u s i n the PM continued to be chased out throughout the experiment,  even though the ER was h e a v i l y l a b e l l e d .  Another c r i t i c i s m of our experiments involves the high m u l t i p l i c i t y of i n f e c t i o n the host c e l l s were exposed to during the experiment (5,000 p f u ' s / c e l l and 1,000 p f u ' s / c e l l ) .  The high m u l t i p l i c i t y of i n f e c t i o n  may somehow,-alter the p h y s i o l o g i c a l response of the c e l l s i n contact with SF V i r u s , although the pfu's do not appear r i d i c u l o u s ; f o r example, the number of adenovirus receptors i s i n the range of 10,000 receptors per RHK c e l l .  A lower m u l t i p l i c i t y of i n f e c t i o n can be effected by producing  v i r u s of higher s p e c i f i c a c t i v i t y . 125 I-iodinated SF V i r u s .  This might be done by preparing  -68-  It  i s c l e a r t h a t the v i r a l e n v e l o p e s , c o a t s and enzymatic  a c t i v i t i e s of the i n t e r a c t i n g p a r t i c l e s have a fundamental r o l e the p e n e t r a t i o n and u n c o a t i n g of the p a r e n t a l genome.  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