UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1978_A6_7 G76.pdf [ 10.33MB ]
Metadata
JSON: 831-1.0094447.json
JSON-LD: 831-1.0094447-ld.json
RDF/XML (Pretty): 831-1.0094447-rdf.xml
RDF/JSON: 831-1.0094447-rdf.json
Turtle: 831-1.0094447-turtle.txt
N-Triples: 831-1.0094447-rdf-ntriples.txt
Original Record: 831-1.0094447-source.json
Full Text
831-1.0094447-fulltext.txt
Citation
831-1.0094447.ris

Full Text

RADIOACTIVE PULSE CHASE EXPERIMENTS  CONCERNING THE MECHANISM OF  ENTRY OF SEMLIKI FOREST VIRUS by Romeo Grossi B.Sc. University 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 this thesis as conforming to the required standard for the degree of MASTER OF SCIENCE The University of B r i t i s h Columbia December 1977 © Romeo Grossi, 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f Biochemistry The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T-1W5 D a t e April 18, 1978 i ABSTRACT The mechanism of v i r a l penetration for Semliki Forest Virus into BHK-21 c e l l s was investigated through a series of radioactive pulse-chase experiments. Entry of an enveloped virus 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 fusion 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 in f e c t i o n and ionic strength of the inoculum. In subsequent experiments BHK-21 c e l l s were infected one half 35 hour with :..:S-Methionine-labeled Semliki Forest Virus. At various 35 time points after removal of unadsorbed S-Met-SF Virus, c e l l s were harvested and fractionated into plasma membrane and endoplasmic reticulum fractions. The fractions were subjected to SDS polyacrylamide gel electrophoresis and analyzed f o r component proteins of SF Virus. Maximum levels of r a d i o a c t i v i t y corresponding to the envelope proteins (E^ , E ^ and nucleocapsid protein (NC) were found i n the PM fr a c t i o n at zero minutes of chase. Both E^ and NC were found to decline during the chase period (approximately 90% within 60 minutes of chase). On the other, .hand, high levels of only nucleocapsid protein were observed associated with the endoplasmic reticulum fraction although no general pattern of incorporation was indicated furing the i i exper iment . (There were h i g h l e v i e s of NC present i n the ER f r a c t i o n throughout 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 are g e n e r a l l y i n c o n c l u s i v e as they can be r a t i o n a l i z e d both by the v i r o p e x i s and f u s i o n mechanisms. The l o s s of both E^ , E^ and NC from the PM suggest tha t v i r o p e x i s i s the mechanism of e n t r y , however, f u s i o n i s not 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 i n t e r n a l i z a t i o n of the v i r u s core by host p r o t e a s e s . A l though no c o n c l u s i o n s have been drawn, t h i s study has demon-s t r a t e d tha 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 to augment e l e c t r o n microscopy data concern ing the mechanism of v i r a l p e n e t r a t i o n i n t o an imal c e l l s . i i i TABLE OF CONTENTS PAGE INTRODUCTION 1 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 or Fus ion?) 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 Nature 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 12 PENETRATION OF VARIOUS VIRUS CLASSES 13 P i c o r n a v i r u s e s 13 Adenov i ruses 15 Papovav i ruses 17 Reov i ruses 18 P o x v i r u s e s 19 Herpesv i ruses • • • 22 Rhabdoviruses 24 Myxov i ruses 26 Paramyxovi ruses 31 O n c o r n a v i r u s e s . . . . 33 N u c l e a r I n s e c t Agents 34 Coronav i ruses 34 Togavi ruses 35 THE PRESENT INVESTIGATION 36 i v PAGE MATERIALS AND METHODS 39 Chemicals : 39 I s o l a t i o n of Radioactively labelled 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 Analysis of SDS Electrophoresis P r o f i l e s . 45 Protein Assays 45 EXPERIMENTAL RESULTS 46 Preparation of Radioactively labelled SF Virus 46 Standard Virus 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. 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 Non-Enveloped V i r u s P e n e t r a t i o n 2 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 Enveloped V i r u s P e n e t r a t i o n 2 3 . S i n g l e Reov i rus In A Vacuole 4 4 . A group of Reov i rus P a r t i c l e s In A C e l l Sampled Soon A f t e r I n o c u l a t i o n . . 4 5 . 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 Ev ident 10 Minutes 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 Sec t ions 5 7. I l l u s t r a t i o n s 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 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 9 . 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 1 1 . 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 11 1 3 . P o s t T r a n s l a t i o n a l Cleavage I n The Formation Of SF V i r u s S t r u c t u r a l P r o t e i n s 12 14 . 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 Minutes 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 1 5 . 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 Phagocy t i c Vacuoles C lose To The C e l l Sur face 14 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 Phagocy t i c Vacuoles C lose To the C e l l S u r f a c e . . . 14 v i PAGE 17. E l e c t r o n Microscopy Autoradiography of T h i n - S e c t i o n e d Pr imary Baby Mouse Kidney C e l l s I n f e c t e d w i t h H -DNA-Label led V i r i o n s 16 18. E l e c t r o n Mic roscopy Autoradiography of T h i n - S e c t i o n e d Pr imary Baby Mouse Kidney 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 At tachment , P e n e t r a t i o n , And Nuc lear Ent ry 17 20. 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 Fus ion With L C e l l s 20 2 1 . 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 Wi th 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 At A L a t e r Stage Of Fus ion Wi th L C e l l s 20 2 3 . 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 With H e l a C e l l s 20 24. A f t e r Fus ion the V i r u s Core M i g r a t e s Into Host Cytoplasm and the V i r u s Envelope L a b e l l e d Wi th 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 21 2 5 . 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 ransverse 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 22A 28. V i r u s Core i n the Process 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 Fus ion of V i r a l Envelope and M i c r o v i l l u s Membrane 22A 29 . 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 22A 30 . V i r u s P a r t i c l e which Appears to be Fused w i t h the T ip of a M i c r o v i l l u s . . 22A 3 1 . Another V i r u s P a r t i c l e which Appears to be A s s o c i a t e d w i t h the T ip of A M i c r o v i l l u s 22A 32. A l ignment of the V i r u s P a r t i c l e s A l o n g , But Not In Contact w i t h the C e l l Sur face 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 to the Host C e l l at the C e l l Sur face 27 v i i PAGE 3 5 . Fus ion 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 . . 27 36. A c t i v e V i r u s on CAM, Warmed to 35° C. f o r 5 Minutes 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 to 35° C. f o r 10 Minutes 29 38. A c t i v e V i r u s on CAM, Warmed to 35° C. f o r 30 Minutes 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 Ent ry A) P i n o c y t o s i s and B) Fus ion 37 40. I s o l a t i o n of V i r u s By Sucrose Grad ient C e n t r i f u g a t i o n 40 4 1 . D i s c o n t i n o u s Gr ad ien 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 4 2 . R a d i o a c t i v e P r o f i l e of a T y p i c a l P r e p a r a t i v e Sucrose Grad ient 46 43 . Coomassie B lue Scan of Standard V i r u s Ge l 47 3 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 - h a b e l l e d A S F ' V i r u s . G e l . . . 48 4 5 . Dependence of SF V i r u s A d s o r p t i o n on Temperature and Time of I n c u b a t i o n 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 4 7 . 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 4 8 . 0 - 2 Hour Chase V i r u s Gels .57 49. 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 . 58 5 0 . 0 ' - 60 ' Chase V i r u s Ge ls 61-63 5 1 . 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 63 v i i i TABLES PAGE TABLE 1. Morphology and Composi t ion of the Major F a m i l i e s of An imal V i r u s e s . 10 TABLE 2 . 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 50 i x LIST OF ABBREVIATIONS J.T'BHK Baby hamster'l.kidney ds double stranded ss single stranded SF Virus Semliki Forest Virus E^ Envelope protein (MW 49,000) of Semliki Forest Virus E 2 Envelope protein (MW 52,000) of Semliki Forest Virus E 3 Envelope protein (MW 10,000) of Semliki Forest Virus NC nucleocapsid protein of Semliki Forest Virus 63" PE2(pr .'NVP ' ) precursor protein to E^ E^ E^ Combined envelope proteins and E^ which often do not resolve by SDS electrophoresis giving the impression of one protein. NVP 165 Non-virion precursor protein (MW 165,000) NVP 97 Non-virion precursor protein (MW 97,000) 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 alkaline 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 vesicular stomatitus virus SDS Sodium dodecyl sulfate AiCl Micro curie nm Nanometer CPM Counts per minute DPM disintegrations per minute X A"*^ O p t i c a l Absorbance a t 550 nanometers cm Cent imeters i o n i c s t r e n g t h 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 m i l l i c u r i e FCS f e t a l c a l f serum mmole m i l l i m o l e aa amino a c i d PC p h o s p h a t i d y l c h o l i n e Choi c h o l e s t e r o l NANA N - a c e t y l - n e u r a m i n i c a c i d x i ACKNOWLEDGEMENTS The author wishes to express his most sincere appreciation to Dr. D.E. Vance for his continual advice and encouragement. Appreciation i s also given to Mrs. N. Grossi for her painstaking work i n typing this thesis. - 1 -INTRODUCTION Two different mechanisms have been proposed for v i r a l entry into host c e l l s . ( 2 ) . One has been imagined to be a pinocytotic process (also termed "viropexis") whereby whole virus p a r t i c l e s are incorporated into cytoplasmic vacuoles and subsequently transported to their s i t e of r e p l i c a t i o n . Up to 1965, viropexis was thought to be the only mode of virus entry. However, evidence i n the l a s t decade has indicated that enveloped viruses may enter host c e l l s by fusion of the v i r a l envelope with the host plasma membrane. A t h i r d possible mechanism of entry i s that the viruses 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 indicates this i s a s i g n i f i c a n t route for i n f e c t i o n i n animal c e l l s . Figures liand 2 demonstrate possible routes of entry of non-enveloped and enveloped animal viruses. Once the virus or sub-viral 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 action and i t s genome must be released prior to r e p l i c a t i o n . These p a r t i c l e s may be delivered within pinocytotic vacuoles or moved through the cytoplasm d i r e c t l y . A l t e r n a t i v e l y the v i r a l genomes may be released immediately after i n t e r n a l i z a t i o n and transferred to their 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, this thesis w i l l be limited to the actual penetration of the v i r i o n into the host c e l l . -2-1. Uptake of v i r a l nucleic 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 Possible Pathways Of Non-enveloped virus penetration. 1. Viropexis (pinocytosis) 2. Fusion of v i r a l envelope with plasma membrane. v i r a l uncoating Fig. 2 Schematic Representation Of Some Possible Pathways Of Enveloped Virus Penetration. -3-MECHANISM OF VIRAL PENETRATION ( V i r o p e x i s or Fus ion?) The problem of e x a c t l y how v i r u s e s enter t h e i r host c e l l s has been the sub jec t of much r e s e a r c h i n recent years but the methods used to 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 matter of u n c e r t a i n t y . One way p e n e t r a t i o n has been measured has been by the l o s s of v i r a l ant ibody 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 . However, some problems can a r i s e w i t h t h i s method. I t 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 to r e a c t w i t h the v i r a l an t igens 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 ant ibody a f t e r i t has at tached to the E. C o l i c e l l w a l l s (.81) . Thus i n some way phage attachment at the c e l l w a l l has s h i e l d e d the 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 ant ibody has o c c a s i o n a l l y been observed at the c e l l s u r f a c e which i s l o s t on ly a f t e r prolonged i n c u b a t i o n ( l ) . A l s o , (and perhaps most i m p o r t a n t l y ) i t i s p o s s i b l e that there cou ld be a l o s s of ant ibody s e n s i t i v i t y even i f the v i r u s remains o u t s i d e the c e l l . 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 are a l t e r e d i n such a way tha t they become ant ibody r e s i s t a n t . An example of t h i s are the p i c o r n a v i r u s e s which a re t ransformed to "A" p a r t i c l e s at 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 to de tec t p e n e t r a t i o n , the one most w i d e l y u s e d , i s e l e c t r o n microscopy . However, there are many problems w i t h the 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 that can a r i s e . We w i l l now cons ide r the most common problems tha t conf ront 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 arise 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 fixed as a monolayer by glutaraldehyde and then scraped and postfixed with osmium tetroxide. The scraping of c e l l s and "inadequate osmication" also has a deleterious effect on the sharpness of the l i n e s delineating a phase separation by membranes between c e l l u l a r compartments. Figs. 3 and 4 demonstrate t h i s effect. They are electron micrographs of reovirusinfected L c e l l s . The only difference between them i s that the l a t t e r was inadequately fixed with osmium tetroxide. Clearly, the virus p a r t i c l e appears as i f i t l i e s free i n the cytoplasm. However, Fig. 3 reveals that the reovirus i s i n fact enclosed within a pinocytotic vacuole. Fig. 3 Single reovirus i n a vacuole. The enveloping membrane i s clearly evident. X120,000. 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 inoculation. X120,000. (2) -5-2. INTERPRETATION OF IMAGES. The w i d t h of t h i n s e c t i o n s range i n t h i c k n e s s from 500 t o 1000 nm. S i n c e t h i s w i d t l i i s g r e a t e r t h a n the d i a m e t e r of most v i r u s e s t h e r e may be i n some cases images which appear to show the v i r u s e s l o s i n g t h e i r m o r p h o l o g i c a l i n t e g r i t y and have merged w i t h the plasma membrane when i n a c t u a l i t y they a r e l y i n g i n an i n v a g i n a t i o n at the c e l l s u r f a c e . T h i s e f f e c t i s d emonstrated i n F i g u r e s 5-8 and F i g u r e 9. F i g . 5 V i r o p e x i s of 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 min. 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 . 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-Aj d i r e c t i o n i n the diagram Fip,. 8), c l a r i f y i n g the s e p a r a t i o n between the v i r i o n and e n v e l o p i n g membrane. X 1A0,000. F i g 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 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 (cut i n R-B, d i r e c t i o n i n the d i a g r a m , F i g . 8) X 140,000. F i g . 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 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 through a v i r u s - c e l l complex of the t y p e shown i n F i g . 5 - 7 . (2) -6-K i p . 9 Ac L i v e v i r u s o.n CAM, warmed l o 35 C f o r f i v e m i n u t e s . 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 t h r o u g h 45 C , the same p a r t i c l e i n (b) i s seen t o he on s u r f a c e c f membrane. (22). -7-In Figure 9a i t appears that the v i r a l envelope i s actually "fusing" with the plasma membrane as the rest of the virus enters the c e l l . However, simply by t i l t i n g the speciman through 45° (Fig. 9b) reveals that the virus i s actually s t i l l l ying on the surface of the membrane. 3. Nature of the Inoculum When working with membrane bound viruses, misinterpretation can arise i f during some stage i n the p u r i f i c a t i o n and handling of the viruses, the envelopes become damaged. Figure 10 demonstrates such an example. Fig. 10 Cell-associated NDV possessing ruptured envelope. Arrow indica the extended nucleocapsid external to the plasma membrane. (2) - 8 -Th is f i g u r e demonstrates a c e l l - a s s o c i a t e d damaged Newcast le D isease P a r t i c l e . The s i t e of rupture occurs at the p o i n t of at tachment . One can conclude ( i n c o r r e c t l y ) tha t the process of uncoat ing occurs at the plasma membrane. - 9 -The major c l a s s e s of enveloped and non-enveloped v i r u s e s a long w i t h some df t h e i r important c h a r a c t e r i s t i c s have been l i s t e d i n Table. 1. A diagram ( F i g . 11) i s a l s o g iven demonstrat ing the r e l a t i v e shapes and s i z e s of the major an imal v i r u s e s . 10(1 Mill KNA V I K U S I . S F i g . 11. Diagram i l l u s t r a t i n g the 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 . (86) TABLE 1 MORPHOLOGY & COMPOSITION OF THE MAJOR FAMILIES OF ANIMAL VIRUSES (86) NUCLEIC ACID NO. OF DIFFERENT NO. OF CAPSOMERS FAMILY SHAPE DIAMETER ENVELOPE CONFIGURATION MW PROTEINS SYMMETRY (IF ICOSAHEDRAL) (nm)  Papovaviridae Adenoviridae Herpetoviridae Poxviridae Picornaviridae Togaviridae Coronaviridae Mxyoviridae Rhabdoviridae Reoviridae Paramyxoviridae Spherical Spherical Spherical Brick-. ... Shaped Spherical Spherical Spherical 45-55 70-80 150 100X240X300 20-30 40-60 80-120 Spherical or filamentous B u l l e t -Shaped Spherical 80-120 70X180 50-80 + + + + + Spherical or Filamentous 100-200 DS DNA 3-5 DS DNA 20-25 DS DNA 100 DS DNA 160 SS RNA 2-3 SS RNA 4 SS RNA 9 SS RNA 5 SS RNA 4 DS RNA 15 SS RNA 7 6 9 12-24 > 3 0 4 3 16 Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral H e l i c a l H e l i c a l H e l i c a l Icosahedral H e l i c a l 72 252 162 o i 60 -11-Slnce the experimental work of this thesis was performed with Semliki Forest Virus, I would l i k e to b r i e f l y review the structure and r e p l i c a t i o n of SF Virus. SF Virus consists of a single strand of RNA enclosed within an icosahedral nucleocapsid which i n turn i s surrounded by a l i p i d b i l ayer. SF virus i s made up of 4 proteins, nucleocapsid protein plus three glycoproteins denoted as E^, E^, E^, situated i n the envelope. The phospholipid to cholesterol 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 Fig. 12. Envelope Proteins 49,000 MW 52,000 MW 10,000 MW' 34,000 MW Nucleocapsid Protein No. of Molecules per V i r i o n 190 190 Nucleocapsid Protein Fig. 12 Structure of Semliki Forest Virus 190 194 -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 in 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 cells. The virion 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 fi n a l structural proteins are formed. A sequence for the post-translational cleavage i s shown below in Fig. 13. (79). 26S RNA NVP165 i NVP127 +? NC I NVP97 PE 2 (NVP63) i E2 E3 Fig. 13 Post Translational Cleavage in the Formation of SF Virus Structural Proteins -13-The assembly of the nucleocapsid protein into i t s icosahedral-shaped sphere begins by association of the capsid protein with the 42S strand which i s s t i l l serving as a messenger. The f i n a l process i n virus maturation involves the nucleocapsid budding through the host plasma membrane, thus forming a v i r a l envelope around the nucleocapsid. Envelope proteins are concentrated at the v i r a l budding site s with the result that a v i r a l envelope i s formed which i s devoid of host c e l l proteins. At the end of the growth cycle, 5,000 to 20,000 vi 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 virus classes. PART A: NON-ENVELOPED VIRUSES 1. Picornaviruses Picornaviruses (from 'pico' (small) and "RNA") are non-enveloped single stranded RNA viruses. The RNA i s contained within an icosahedral-shaped capsid. Diseases these viruses are responsible for include foot and mouth disease i n animals, herpangina, meningitis and poliomyelitis i n man. The evidence concerning the penetration of picornaviruses i s s t i l l inconclusive. There i s indirect evidence that poliovirus enters the host c e l l by viropexis. Penetration and loss of antibody s e n s i t i v i t y of poliovirus occurs at the same rate at 37° C. (58-59) but at 25° C inf e c t i v e centers escape neutralization before a l l cell-associated i n f e c t i v e virus has been l o s t . This loss of s e n s i t i v i t y to neutralization -14-before virus i s l o s t is. most l i k e l y due to entry of virus into pinocytotic vesicles. However i t i s possible that the loss of s e n s i t i v i t y may be the result of tight binding of the virus to the c e l l membrane. Another explanation may be that not a l l cell-associated virus need be located on a pathway leading to an i n f e c t i v e center and therefore the virus may be recovered after c e l l l y s i s . Electron microscopy data (2) of an i d e n t i c a l system has confirmed Manel's proposal of viropexis (Fig. 14-16) Fig. 14. A deep invagination of the PM of a Hela c e l l containing a group of poliovirus 1 p a r t i c l e s sampled 10 minutes after i n i t i a t i o n of penetration. X180,000. Fig. 15 & 16. Individual poliovirus particles within phagocytic vacuoles close to the c e l l surface. X80,000 (2) Dunneback et al.(60) have presented electron micrographs of poliovirus-infected c e l l s which appear to show direct penetration through the c e l l membrane. However this finding has yet to be confirmed. -15-2. Adenoviruses Adenoviruses are large (80 nm) icosahedral-shaped v i r u s e s . Their genomes consist of double-stranded DNA. These agents are usually associated with i n f e c t i o n s of the r e s p i r a t o r y tract,,and occasionally the eye i n mammalian and avian hosts. ( Adenoviruses have been shown to enter host c e l l s by virop e x i s i n several independent studies (50-52). By synchronizing the i n f e c t i o n s , i t was demonstrated that the viruses remain i n the p i n o c y t o t i c vacuoles for only a short period (51) . The a b i l i t y of adenovirus 5 to escape out of pi n o c y t o t i c vacuoles and gain access to the cytoplasm was shown to be dependent on temperature; being highly e f f i c i e n t at 37° C. and less e f f i c i e n t at temperatures 12° or 20° C. Dales and Chardonnet have concluded that the a b i l i t y to gain access to the cytoplasm i s a property of the v i r u s e s themselves. Heat-denatured inocula were r a p i d l y taken into host c e l l s by virop e x i s but were not released into the cytoplasm very e f f i c i e n t l y (53). Some evidence has also been presented that suggests that adenoviruses may pass d i r e c t l y through the c e l l membrane into the cytoplasm without the benefit of pinocytosis (-41). Brown and Burlingham (54) showed Adenovirus 2 i n r e p l i c a s of freeze-etched infected c e l l s transvering d i r e c t l y through the plasma membrane. Virus p a r t i c l e s were also seen i n intracytoplasmic v e s i c l e s but only occassionally. -16-Fig. 17 & 18. Electron microscopy autoradiography of thin-sectioned primary baby mouse kidney c e l l s at 15 min. postinfection using H-labeled polyoma v i r i o n s . (17) H-DNA-labeled v i r i o n s ; (18) %-amino acid-labeled v i r i o n s . Non-grain-producing virus can also be seen (arrows). The designations Nu for nucleus and Cy for cytoplasm are indicated. 3. Papovaviruses Papovaviruses are non-enveloped, double-stranded c i r c u l a r DNA viruses. These viruses are tumorigenic and cause latent and chronic infections i n mammalian hosts. I t i s well documented that the mode of entry of papovaviruses i s v i a viropexis (11, 46-48). Recently MacKay and Consigli confirmed that viropexis i s the mode of entry (49) using optimal conditions of adsorption, electron microscopy and autoradiographic techniques. They also demonstrated that the v i r a l coat proteins and DNA arrive simultaneously i n the nucleus as early as 15 minutes postinfection (Figs. 17, 18). This indicates that virus uncoating i s an event subsequent to nuclear entry. They formulated a model of polyoma virus attachment, penetration and nuclear entry (Fig. 19 ). Fig. 19. Model of polyoma v i r i o n attachment, penetration, and nuclear entry. -18-1) attachment at e i t h e r the v i r u s twofold or f i v e f o l d a x i s , (at 4°C of 37° C) 2) C e l l membrane undu l a t i o n to a l l o w v i r u s to have contact 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 p e n e t r a t i o n 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) entry of v i r u s i n t o nucleus devoid of an envelope 6) v i r u s i s uncoated and v i r a l DNA i s re l e a s e d w i t h i n nucleus 7) MacKay and C o n s i g l i noted that v i r i o n capsids 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 phagocytotic vacuoles. These d e f e c t i v e v i r u s e s were not seen to enter the nucleus. 4. Reoviruses Reoviruses are 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 of segmented double-stranded RNA. Although these agents have been found i n many diseased organs, they have not been as 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 process i n man. Reovirus has been shown to enter r a p i d l y i n t o host c e l l s by v i r o p e x i s (48,182). U n l i k e other animal v i r u s e s , r e o v i r u s i s uncoated w i t h i n lysosomes. .The p i n o c y t o t i c vacuoles fuse 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 that i n general the lysosomal pathway p a r t i c i p a t e s i n defending the host c e l l s from v i r a l pathogens but the r e o v i r u s uses t h i s pathway f o r i t s own r e p l i c a t i o n . However, as w i t h other v i r u s e s , i t has been suggested that r e o v i r u s e s can penetrate d i r e c t l y i n t o host c e l l s as w e l l as by p i n o c y t o s i s (57). -19-PART B: ENVELOPED VIRUSES 1« Poxviruses Poxviruses are the largest and most complex of the animal viruses. Their genomes consist of double-stranded DNA. Among other diseases, these agents are the cause of small pox i n man. Poxviruses should be an i d e a l t o o l to investigate 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 physical p a r t i c l e s approaching unity. Vaccinia virus enters host c e l l s very rapidly and shuts down host protein synthesis within 20 minutes post-infection. Virus RNA synthesis, polyribosome formation and proteinisynthes.is;.are detectable within 30 minutes. 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 within t h i s time period. Early studies by Dales (3,4) showed that the virus enters the c e l l by viropexis. However, more recently Armstrong, Metz and Young (5) , using electron microscopy have reported that vaccinia virus enters cultured L c e l l s by a process involving direct fusion of the virus envelope with the plasma membrane of the c e l l within minutes after adsorption. This result has been confirmed and extended using immuno-f e r r i t i n conjugates to locate the virus 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 vaccinia p a r t i c l e s i n the early stages of attachment and fusion. Figure 24 demonstrates the migration of the virus core into the cytoplasm while the f e r r i t i n -conjugated envelope antigens remain at the plasma membrane. - 2 0 -Fig. 20-23 Thin sections of vaccinia infected c e l l s reacted with f e r r i t i n -antibody conjugate. Fig. 20 F e r r i t i n - l a b e l l e d virus p a r t i c l e s adsorbed on the surface of L c e l l s . The virus at the top shows an early stage of fusion with the c e l l membrane Fig. 21 & 22. F e r r i t i n - l a b e l l e d virus at a la t e r stage of fusion with L c e l l s . Lateral bodies (LB) are beginning to disperse i n Fig. 21. Fig. 23 Labelled vaccinia 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 into the c e l l membrane (CM) (6) Also, freeze etching has shown that the components of the vir u s envelope become rapidly dispersed i n the plasma membrane. Under their experimental conditions, at least, viropexis does not appear to make an important contribution to vaccinia in f e c t i o n . These results leave l i t t l e doubt that fusion i s responsible,at least i n part, for penetration and uncoating of vaccinia v i r u s . - 2 2 -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 to 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 ) . F i g s . 25 -31 i l l u s t r a t e the v i r u s at v a r i o u s stages of ent ry i n t o m i c r o v i l l i . 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 . 29-31) but i s c l e a r l y shown i n F i g . 25 . 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 core and p o r t i o n s of the l a t e r a l bodies enter the m i c r o v i l l u s ( F i g . 2 7 ) . I t i s apparent tha t the v i r u s envelope was l o s t at the t ime of the ent ry p r o c e s s . In over 2,000 s e c t i o n s examined, v i r o p e x i s was not observed to be i n v o l v e d i n the entry of v i r u s p a r t i c l e s I t i s i n t e r e s t i n g to note that the m i c r o v i l l i have a s m a l l e r d iameter than the v i r u s c o r e . As the v i r u s core " m i g r a t e s " toward the c e l l c y top lasm, the m i c r o v i l l i en la rge 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. H e r p e s v i r u s The h e r p e s v i r u s e s are 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 are 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 pox, c o l d s o r e s , i n f e c t i o u s mononucleosis 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 animal c e l l s i s s t i l l a s u b j e c t of g reat c o n t r o v e r s y . S e v e r a l e a r l y s t u d i e s have repor ted that h e r p e v i r u s en te rs e x c l u s i v e l y or almost e x c l u s i v e l y by v i r o p e x i s ( 8 - 1 3 ) . T\Tithin a few minutes a f t e r engul fment , the v i r a l envelopes <[' -22A-3 1 Fig. 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. Fig. 26 Fusion of a virus p a r t i c l e with the mi c r o v i l l u s . X 82,000. Fig. 27 Transverse section of a microvillus containing a virus core and portion of the l a t e r a l body. X82,000. Fig. 28 Virus core i n the process of entering a microvillus after side-to-side fusion of v i r a l envelope and mic r o v i l l u s membrane. X 82,000. Fig. 29 Microvillus-associated 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 mi c r o v i l l u s . X 82,000. Fig. 30 Virus p a r t i c l e which appears to be fused with the t i p of a microvillus. X 82,000. Fig. 31 Another virus 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) - 2 3 -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 cy top lasm. V i r o p e x i s i s a l s o supported from the ev idence tha t there i s l o s s i n 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 i n t o phagocy t i c v a c u o l e s . Th is i s r a t h e r good evidence tha t v i r o p e x i s i s the predominant mode of ent ry of h e r p e s v i r u s e s . I n t e r e s t i n g l y , n u c l e o p r o t e i n c o r e s , which can be prepared i n pure form a l s o have the a b i l i t y to be phagocyt i zed by host c e l l s . Morgan et a l . ( 1 3 ) a l s o found h e r p e s v i r u s w i t h i n phagocy t i c vacuo les however, they have proposed an a l t e r n a t e mechanism of e n t r y . They suggested tha t a f t e r a d s o r p t i o n of the v i r u s to 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 both the v i r a l envelope anduhost membrane, a l l o w i n g the v i r u s core to enter the c e l l . However, Morgan f i n a l l y conceded that e l e c t r o n microscopy a lone cannot d i s t i n g u i s h which 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 host c e l l and tha t other methods are needed to s o l v e the problem. 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 study of v i r a l p e n e t r a t i o n of both herpes s implex v i r u s and human cy tomegalov i rus (CMV). Both f u s i o n and v i r o p e x i s were observed. Subsequent to the f u s i o n of the CMV, the caps ids f r e e i n the cytoplasm were coated w i t h a f i n e f i b r i l l a r m a t e r i a l . Th is c o a t i n g was not observed w i t h the herpes s implex v i r u s . A l s o , Smith and DeHarven noted tha t enveloped v i r u s e s taken i n by p ino^ c y t o s i s , were ab le to egress from the c y t o p l a s m i c vacuo les by f u s i o n of t h e i r envelope w i t h the vacuo le membrane. - 2 4 -3 . Rhabdoviruses The Rhabdoviruses are 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 s t r u c t u r e . 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 rhabdov i ruses 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 Stomat i tus V i r u s (VSV). VSV has been repor ted to penet ra te by v i r o p e x i s i n a c a r e f u l study i n which 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 to 37° C. (15) I n f e c t i o n was performed at 4° C. i n order to ach ieve p e n e t r a t i o n synchronously when the temperature was r a i s e d to 37° C. At 4° C , 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 the other hand, another study of VSV p e n e t r a t i o n has repor ted f u s i o n of v i r i o n envelopes w i t h c e l l plasma membranes, a l though v i r o p e x i s was a l s o observab le ( 1 6 ) . In t h i s study there was no ev idence of v i r a l an t igens at the plasma membrane. However, i n the subsequent study (17) , these workers presented immunologica l and r e l a t e d evidence to show the presence of VSV g l y c o p r o t e i n s at the host c e l l membrane. The problem w i t h i n t e r p e t i n g the r e s u l t s from these two r e p o r t s i s tha t the l a t t e r (16, 17) employed h i g h speed c e n t r i f u g a t i o n to i n i t i a t e r a p i d c e l l - v i r u s c o n t a c t . As a r e s u l t i t was d i f f i c u l t to determine whether the d i f f e r e n c e s between the two s t u d i e s are due to problems i n i n t e r p e t a t i o n of e l e c t r o n microscopy images or due to 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 Dahlberg (18) undertook to q u a n t i t a t i v e l y compare - 2 5 -the 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 cou ld 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 . Dahlberg 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 i n t o L -929 c e l l s . When p e n e t r a t i o n was analyzed 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 entered 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 . There was a 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 isappearance of e x t r a c e l l u l a r a t tached v i r u s and the appearance of v i r u s i n i n v a g i n a t i o n s , and , somewhat l a t e r , i n s m a l l i n t r a c e l l u l a r v a c u o l e s . The data was q u a n t i t a t e d by e x p r e s s i n g each category 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 , keeping the s e c t i o n t h i c k n e s s as constant 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 (approx imate ly 200) f o r each sample. Fus ion was on ly r a r e l y observed . 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 10-15 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 ach ieved by c e n t r i f u g i n g v i r u s - c e l l mix tu res at 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 aga in 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 tha t c e n t r i f u g a t i o n 'per s e ' p layed a r o l e i n the i n c i d e n c e of f u s i o n . In a l l c a s e s , serum reduced the amount of v i r u s adsorbed, but d i d not a f f e c t the mode of p e n e t r a t i o n . Another r h a b d o v i r u s , r a b i e s v i r u s , has a l s o been repor ted to p e n e t r a t e by v i r o p e x i s ( 1 9 ) . However, Iwasak i et a l . ( 2 0 ) have shown that r a b i e s v i r u s a l s o has the a b i l i t y to fuse w i t h the plasma membrane of a baby hamster k idney c e l l l i n e (BHK-21) bes ides be ing taken up by p i n o c y t o t i c v a c u o l e s . Bes ides f u s i o n w i t h the plasma membrane, f u s i o n was a l s o observed to occur w i t h the membrane of the phagocy t i c vacuo les w i t h the subsequent r e l e a s e of the v i r u s core i n the cytoplasm ( F i g . 3 2 - 3 5 . ) In a d d i t i o n , v i r u s p a r t i c l e s at d i f f e r e n t stages of degradat ion were a l s o seen i n phagocy t i c v a c u o l e s . C l e a r l y , the mechanism of rhabdov i rus en t r y which leads to i n f e c t i o n i s s t i l l a matter of c o n f u s i o n . 4. Myxov i ruses Myxov i ruses are g e n e r a l l y s p h e r i c a l agents , c o n t a i n i n g s i n g l e s t randed 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 v i r u s . As w i t h other 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 f o r myxovi ruses 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 i n t r o d u c e d by Fazekas De S t . Groth i n 1948 (21) to d e s c r i b e the ent ry of i n f l u e n z a 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. V i r i o n a n t i g e n , de tec ted 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 i n t r o -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 tha t 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 that 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 a s c i t e s c e l l s . In a more recent s tudy , Dourmaskin and T y r r e l l (22) incubated an egg adapted s t r a i n of I n f l u e n z a w i t h patches of c h o r i o a l l a n t o i c membrane. The v i r u s was a l lowed to a t t a c h to the c e l l s by i n c u b a t i n g at 4°~C. f o r one hour . When the temperature was e l e v a t e d to 37° C , v i r u s was seen to enter by v i r o p e x i s . Subsequently the v i r u s e s were seen to egress from the p i n o c y t o t i c vacuo les and uncoat ing of the n u c l e o p r o t e i n -27-Fig. 32-35 BHK-21 c e l l s 5 minutes after infection with ERA str a i n s of rabies virus (20) Fig. 32 Alignment of the virus 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 either the virus p a r t i c l e or the c e l l membrane at th i s stage. X100,000. Fig. 33 Engulfment of a single p a r t i c l e by pinocytosis. Several p a r t i c l e s are engulfed and intermingled with amorphous electron-dense material within the phagocytic vacuole. X 53,000. Fig. 34 Adherence of virus 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. Fig. 35 Fusion of virus envelope and plasma membrane of pinocytotic v e s i c l e . One virus 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 this v e s i c l e . X 100,000 - 2 8 -core was observed ( F i g . 36-38) w i t h i n the cy top lasm. O c c a s i o n a l l y e l e c t r o n micrographs were observed that suggest that f u s i o n was a l s o o c c u r i n g . 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 that 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 f rom the c e l l membrane d u r i n g the s tages of v i r u s p e n e t r a t i o n . 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 lague v i r u s ( 2 3 ) . The inoculum was prepared w i t h a n e u t r a l , red -dye 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 c e l l s . Re lease of the 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 moni tored 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 i n f e c t i o u s component, whereas removal from the s u r f a c e of the v i r i o n s i s determined 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 and l o s s of h e m a g l u t i n i n . The r e s u l t s i n d i c a t e the mode of e n t r y o f f o w l p lague v i r u s i s v i r o p e x i s and the v i r u s becomes uncoated w i t h i n the cy top lasm. More r e c e n t l y , Re inacher and Weiss (24) performed e l e c t r o n m i c r o s c o p i c s t u d i e s 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 . The i r study showed most of v i r i o n s present w i t h i n c y t o p l a s m i c vacuo les (more o r l e s s d i s i n t e g r a t e d ) . On the o ther hand, on ly se ldomly were v i r i o n s a t t a c h e d a t the plasma membrane a l t e r e d . Th is i n d i c a t e d that f o w l p lague v i r u s i s taken up v i a v i r o p e x i s and r e l e a s e t h e i r 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 indeed occur 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 i n f e c t e d w i t h i n f l u e n c z a v i r u s (.25, 26 , 2 7 ) . The f i n d i n g s of K r i s a n o v a e t a l . (.28) support t h i s t h e o r y . Dur ing i n c u b a t i o n of i n f l u e n z a v i r u s -29-Fig.36 . Active virus on CAM, warmed to 35 C. for 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; Active v i r u s on CAM, warmed to 35 C. f o r 30 min. Virus p a r t i c l e s are seen penetrating wall of vacuole (V). There i s a continuous, s i n g l e layer of membrane surrounding the v i r u s , (b) As in (a): p a r t i c l e s are positioned around walls ot vacuoles (V) and are i n a p a r t i a l state ot degradation. (22) -31-with 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 test with antiserum was released. Levels of free g-antigen increased according to the duration of incubation. Their results led them to conclude that the virus envelope and plasma membrane fuse prior to the release of the nucleoprotein core. And f i n a l l y , Stephenson and Dimmock (29) have recently made an interesting observation with influenza-infected chick embryo c e l l s . They noted that influenza virus was able to infect the c e l l s at 4° C. This observation, raises questions about the way virus penetrates the plasma membrane since neither fusion nor pinocytosis has been shown to / o „ occur at 4 C. 5. Paramyxoviruses Paramyxoviruses are larger and more pleomorphic than myxoviruses. These agents are single stranded RNA viruses which cause diseases such mumps, measles, Newcastle Disease and Canine distemper. I t i s worthy to note that these viruses include the most powerful " c e l l fusing" and hemolytic agents.(36). C e l l b i o l o g i s t s have used paramyxoviruses to produce functional heterokaryons by fusing c e l l s from different species. The fusion of paramyxovirus envelopes with host membranes have been shown unambiguously (30-35). A number of workers have shown that Newcastle Disease Virus (NDV) and Sendai virus envelopes begin to fuse - 3 2 -w i t h host 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 (32, 3 3 , . 3 4 ) . A u t o r a d i o g r a p h i c methods have been employed to demonstrate the r e t e n t i o n of 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 of 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 -deformable c e l l membranes such as human e r y t h r o c y t e s (40, 41) and c i l i a ( 3 0 ) , Sendai v i r u s was found to fuse 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 ther hand, o ther workers have repor ted that the mechanism of e n t r y of NDV and Sendai v i r u s i s v i a v i r o p e x i s ( 3 7 - 3 9 ) , a l though 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 study the p r o p e r t i e s - which a membrane must have to promote i n g e s t i o n of Sendai v i r u s ( 4 2 ) . Sendai v i r u s e s were incubated w i t h l iposomes ( 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 micrographs were taken tha t resembled the i n g e s t i o n s teps of p h a g o c y t o s i s . I n t e r e s t i n g l y , on l y the l iposomes ( 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 enveloped the v i r u s e s i n d i c a t i n g tha t the g a n g l i o s i d e s may serve as v i r u s r e c e p t o r s . I f , i n a d d i t i o n to the l i p i d s used i n these experiments phos -p h a t i d y l - ethano lamine (and sphingomyel in) are used to make l i p o s o m e s , the v i r u s membrane appears to fuse w i t h the l iposomes (43) and to some e x t e n t , p i n o c y t o s i s a l s o takes p l a c e . These r e s u l t s suggest tha t the mode of en t r y of paramyxoviruses may be dependent on the compos i t ion of the hos t membrane at the s i t e of at tachment . -32A-Fig. 38A. Interaction of Sendai Virus with Vesicular Model Membranes. (i) Model membranes made from 4;j/moles phosphatidyl choline (PC), 2>^moles cholesterol (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 0.2pmoles NANA. ( i i i ) and (iv) Model membranes made from 1 ^ m o l e PC, 0.5jU mole Chol and .22 mg gangliosides, containing 0.1// mole NANA. - 3 3 -6. Oncornav i ruses The oncornav i ruses (RNA Tumor V i r u s e s or Leukov i ruses ) are enveloped v i r u s e s that are i n f e c t i o u s i n many v e r t e b r a t e s p e c i e s . They i n c l u d e the leukemia , 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 . Dales and Hanafusa (44) have performed a number of experiments u t i l i z i n g e l e c t r o n mic roscopy , .autoradiography and p h y s i c a l - c h e m i c a l techniques to 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 i n t o host c e l l s . The r e s u l t s of these experiments i n d i c a t e d that the v i r u s e s enter by v i r o p e x i s and subsequent ly mig ra te to the v i c i n i t y of the nuc leus 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 to occur w i t h the mouse mammary tumor agent and to a s m a l l ex tent f u s i o n at the plasma membrane was a l s o observed . Rauscher leukemia v i r u s has been repor ted to 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 th ree d i f f e r e n t f a s h i o n s (45) : 1) Fus ion 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 to penet ra te the c e l l . 2) s imul taneous 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 lone to penet ra te ; the c e l l . 3) D i s s o l u t i o n of the plasma membrane wi thout a f f e c t i n g the v i r u s e s , which then passes u n a l t e r e d i n t o the cy top lasm. 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 these i n t e r a c t i o n s i s yet to be determined . - 3 4 -7. N u c l e a r I n s e c t Agents A long w i t h the i n s e c t p o x v i r u s e s , other 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 host c e l l s both by v i r o p e x i s (61-64) and f u s i o n w i t h the membranes of m i c r o v i l l i ( 6 5 - 6 7 ) . 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 , non-deformable membranes. V i r o p e x i s has r e c e n t l y been demonstrated aga in w i t h Nuc lear P o l y h e d r o s i s V i r u s i n a cont inous 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  f r u g i p e r d a ( 6 8 ) . 8 . Coronav i rus Coronav i ruses are enveloped s i n g l e s t randed RNA v i r u s e s approx imate ly 120 nm i n d iamete r . Coronav i ruses mature by budding i n t o the c i s t e r n a e of 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 are r e s p o n s i b l e f o r s e v e r a l d i seases 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 hemagg lu t inat ing 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 to enter hos t c e l l s by v i r o p e x i s (69) . Recent l y work was performed on v i r u s s t r a i n LY-138 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 . Coughr i e t a l . (70) repor ted tha t uptake ov v i r u s occur red through f u s i o n -35-of v i r a l envelopes with the plasma membrane of the m i c r o v i l l i or by interaction with the l a t e r a l c e l l membranes of adjacent 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 . However, their electron micrographs are far from convincing. 9. Togaviruses Togaviruses of which Semliki Forest Virus i s a member, represent the smallest of the enveloped RNA viruses. These agents i n many cases are encephalitogenic. The protein composition i s r e l a t i v e l y simple compared to some other RNA viruses as there are only four structural proteins. One i s associated with the internal core structure while the other three are glycoproteins which consititute 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 Semliki Forest Virus or other togaviruses into host c e l l s . Pathek and Webb (71) studied by electron microscopy possible mechanism by which Semliki Forest Virus may be transported from the lumen of the blood vessel into the parenchymal c e l l s of the mouse central nervous system. They suggest SF Virus i s taken i n by viropexis into endothelial c e l l s . The virus p a r t i c l e s then migrate through the c e l l s towards the basement membrane. There the virus-containing vacuoles fuse with the adjoining c e l l membrane and the virus i s released into the basement membrane. - 3 6 -Then through pressure 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 . These c e l l s ad jacent to the v i r u s 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 c e l l s , polymorphonuclear l eukocy tes 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 of SF V i r u s . THE PRESENT INVESTIGATION The r e s u l t s of e l e c t r o n microscopy s t u d i e s concern ing 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 of t h i s t h e s i s was to study 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 through a s e r i e s of r a d i o a c t i v e p u l s e chase exper iments . These experiments i n v o l v e d the use of r a d i o a c t i v e l y - l a b e l l e d S e m l i k i F o r e s t V i r u s . 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 ex tents depending on the amino a c i d c o m p o s i t i o n s . The extent of 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 Monolayers of BHK-21 c e l l s were i n f e c t e d w i t h S - M e t - S F V i r u s (pu lse t ime was 30 m i n u t e s ) . 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 the 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 ana lyzed 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 running them on p o l y a c r y l a m i d e g e l s . Fig.39.. Schematic Representation of Possible Routes of SFV entry, a) pinocytosis and b) fu s i o n . - 3 8 -I f f u s i o n were the pr imary 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 that the r a d i o a c t i v i t y cor responding 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 constant throughout ' . the v a r i o u s chase p e r i o d s . On the other hand, the r a d i o a c t i v i t y cor responding 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 imes i n c r e a s e s i n c e the NC i s expected to enter the c e l l . I f the mode of ent ry was v i a v i r o p e x i s then we would expect t h a t both the n u c l e o c a p s i d and envelope p r o t e i n s would only t r a n s i e n t l y 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 the PM would decrease as the chase t ime i n c r e a s e s . Furthermore, we would a l s o expect that the r a t i o of the 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 to envelope p r o t e i n s at the PM shou ld remain c o n s t a n t . 35 i e . S i n NC ^ Constant S i n EP The i s o l a t i o n of endoplasmic r e t i c u l u m was performed to demonstrate the ent ry of 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 host c e l l s . - 3 9 -MATERIALS AND METHODS  CHEMICALS 35 3 L S - M e t h i o n i n e (530.46 Ci/mmole) , H-AMP (11-25 Ci/mmole) 3 were obta ined from New England Nuc lear and _L H -Amino -ac id mix tu re (25 Ci/mmole) was obta ined by Amersham/Searle. Sucrose (RNAse f r e e ) was purchased from Schwarz. Mann, and T r i s - H C L , Bovine Serum Albumin and cytochrome C were obta ined from Sigma. 199 maintenance medium, m o d i f i e d Eag les medium A , and E a r l e s b a s i c s a l t s medium, p e n i c i l l i n - s t r e p t o m y c i n 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 prepared 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 , potass ium c y a n i d e , bar ium 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 obta ined from Beckman, NADPH from P . L . B i o c h e m i c a l s and acry lamide from Matheson, Coleman and B e l l . Coomassie B lue and methy leneb isacry lamide were obta ined 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 Chemica ls . - 4 0 -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 Scheele 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 ) of c o n f l u e n t 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 Fo res t V i r u s (made up to 10 mis w i t h M199 p l u s 2% FCS c a l f serum (<~v^  100 p f u ' s / c e l l ) . Three hours a f t e r a d d i t i o n of v i r u s , 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 ) of E a r l e s B a s i c S a l t s 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 order to s t a r v e the c e l l s 3 of amino a c i d s . At 3 '1/2 hours , 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 mix tu re or . S - m e t h i o n i n e ; l vvmCi/ml, 1 mmole/ml) was added. At 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 b o t t l e s ) 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 pasteur p i p e t and l a y e r e d onto a th ree -phase 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 CsC l 2 mis 50% Sucrose i n .002 M T r i s , pH7.8 and .2 M C s C l F i g . 4 0 . I s o l a t i o n of V i r u s by Sucrose Grad ient C e n t r i f u g a t i o n -41-These gradients were centrifuged at 116,000 X g (25,000 rpm) for 4 hours i n art SW'27 rotor. The gradients were then dripped from the bottom and 0.5 ml. fractions collected. Aliquots from each f r a c t i o n were removed and analyzed for 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 radioactive virus were pooled and stored at -70° C. Aliquots was saved for plaque assays and radioactive counting. PLAQUE ASSAYS 200 u l . of virus were .diluted at 10 ^  by s e r i a l d i l u t i o n . A l l virus 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 . After one hour of in f e c t i o n , the medium was drawn off and replaced with 5 mis of equal volumes of 2% (w/v) agar and 2X Medium A plus 4 % FCS. The plates were l e f t for two days to allow plaque formation. At this point, the c e l l s were fixed with 5 mis of formyl saline for 10 minutes. The agar was peeled off and. 5 mis of 2% (w/v) c r y s t a l v i o l e t were added for one hour to allowing f i x i n g . After removal of the c r y s t a l v i o l e t and washing of the plates, 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 - 4 2 -T r i s , pH 78. The c e l l suspens ion 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 (3,000 RPM on a S o r v a l l SS-34 head) . Suspension of the membranes was e f f e c t e d by a d d i t i o n of . 1 volume of 100 mM NaCl 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 sucrose g r a d i e n t c o n s i s t i n g of l a y e r s (7mls each) of 30% (w/v) Sucrose i n 5 mM M g C l 2 , 45% (w/v) sucrose i n 5mM M g C l 2 and c e n t r i f u g e d a t 7000 X g (5800 rpm on a SW 27 c e n t r i f u g e head) f o r 20 minutes ( F i g . 4 1 ) . ER 4 mis Homogenate 7 mis 30% Sucrose i n 5mM MgCl^ PM Bands Here 7 mis 45% Sucrose i n 5mM MgCl , F i g . 4 1 . D i s c o n t i n o u s Grad ient f o r Separa t ion of plasma membrane and endoplasmic r e t i c u l u m . - 4 3 -A f t e r c e n t r i f u g a t i o n , the g r a d i e n t was d r i p p e d from the bottom r o t o r and c o l l e c t e d i n 0 . 5 ml 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 at 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 2 0 . The f r a c t i o n s were then assayed f o r NADPH-dependent cytochrome C reductase (a marker enzyme f o r endo-p 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 reductase 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 .25 mis of c o c k t a i l mix ( 0 . 1 mM KCN, 0 .66 mM KC1, .044 M phosphate ph 7 . 6 , 0 . 5 mM cytochrome C).to which 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 reductase a c t i v i t y was c a l c u l a t e d as f o l l o w s : E"*"^  Reduced cytochrome C = 27.7 X 10^ cm 2 / mole E 550 O x i d i z e d cytochrome C = 9 . 0 X 1 0 6 cm 2 / mole ,550 A ± - = E _ F 5 5 0 dt Red. Cy t . C d _ :[Red. C y t . C] O x i d . Cy t . C d_ [Red. Cyt , d t dt d d A 5 5 0 Red. Cy t . C = — 18.7 x 1 0 6 ml/mole The 5 ' - n u c l e o t i d a s e assay (75) was performed by i n c u b a t i n g the assay 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 2 , 50 u l of - 4 4 -3 0 .4 mM AMP, 20 jal of H-AMP, 0 .63 ml of H 2 0 and 200 u l of each f r a c t i o n ) a t 37° C f o r 30 minutes . The r e a c t i o n was stopped by f i r s t adding 0 . 2 mis of 0 .25 M ZnSO^ and then adding 0 . 2 mis of 0 .25 M Ba (0H) 2 i n order to p r e c i p i t a t e the unreacted ATP. 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 of supernatant from each f r a c t i o n were p laced i n s c i n t i l l a t i o n v i a l s to 3 which 10 mis of ACS were added and then monitored f o r H-adenCsine by count ing i n an Isocap 300 s c i n t i l l a t i o n counte 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 lec t rophoresed on 7.5% acry lamide SDS g e l s as desc r ibed by Weber and Osborne (72) . M e t h y l e n e b i s -a c r y l a m i d e was used as the c r o s s l i n k i n g agent . A l l samples were p r e -e l e c t r o p h o r e s e d at 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 at 60° C p r i o r to 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 Blue f o r one hour at 60° C. p r i o r to t reatment w i t h 7.5% a c e t i c a c i d . GEL SLICING AND SCINTILLATION COUNTING OF GEL SAMPLES Gels were f r o z e n i n a dry i c e - a c e t o n e bath and cut i n t o 1 mm s l i c e s : u s i n g a B i o - R a d g e l 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% p e r i o d i c a c i d . The samples were then suspended i n 10 mis of ACS and counted i n a New England Nuc lear Isocap 300 s c i n t i l l a t i o n counter . -45-ANALYSIS OF SDS ELECTROPHORESIS PROFILES Gels that were previously stained were scanned at 550 nm using a Gi l f o r d Spectrophotometer. Subsequently the gels were s l i c e d and counted as previously outlined. Plots of absorbance versus length of gel and counts per minute versus length of gel were plotted. Points i n each r a d i o a c t i v i t y peak were summated after subtracting the basal r a d i o a c t i v i t y from each value. PROTEIN ASSAYS Protein assays were performed as described by Lowry (76). Lipid-containing protein samples were incubated overnight i n 0.66 N NaOH at 37° C. pr i o r to analysis. Bovine serum albumin was used as a standard. -46-EXPERIMENTAL RESULTS PREPARATION OF RADIOACTIVELY-LABELED SF VIRUS Radioactively-labeled SFV was prepared as described i n the Methods section. B r i e f l y , this method involves a preparative sucrose gradient 25,000 rpm for four hours . These gradients were dripped from the bottom i n 0.5 ml fractions and aliquots (50 jul) were assayed for 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 . The 3 single peak corresponds to H-a.a.-SF Virus. 25 Counts Per Minute ( X 10 - 3) 20 15 10 10 20 30 Fig. 42 Fraction Number Radioactive P r o f i l e of a Typical Preparative Sucrose Gradient, _ 4 7 -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 lec t rophoresed (a long w i t h 60Qvgms of n o n - l a b e l e d SF V i r u s standard) on 7.5% SDS g e l measuring 9 . 5 cm i n l e n g t h . The Coomassie Blue p r o t e i n s were scanned u s i n g the G i l f o r d Spectrophotometer . Th is scan i s shown i n F i g . 43 . The g e l was then s l i c e d and counted. 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. F i g . 4 3 . Coomassie B lue Scan of Standard V i r u s Ge l (Absorbance = 550 nm) -48-44 . Radioactive P r o f i l e of Standard H-amino a c i d - l a b e l l e d SF Virus Gel. -49-The l e f t peak of the protein scan represents the and E^ proteins and the right peak represents the nucleocapsid proteins (73). The fact the E^ and E^ are not resolved i n this gel system i s not surprising considering their 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 buffer-electrophoresis system. Another interesting 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 proteins. Summation of the counts per minute under each peak (minus basal background levels) 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 protein ( 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 theoretical value of 2.78 derived from the amino acid composition of SF Virus.(78). ISOLATION OF PLASMA MEMBRANE AND ENDOPLASMIC RETICULUM The i s o l a t i o n of PM and ER of mock-infected BHK c e l l s were performed as outlined i n the Methods section. 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 lysate, 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'-NUCL ACTIVITY EOTIDASE NADPH-D CYTOCHR PURIFICATION REDUCTA ACTIVITY EPENDENT OME C SE PURIFICATION C e l l Lysate 1.2 Endoplasmic Reticulum 30 Plasma membrane .54 1 .04. 25 7.2 45 .07 1 180 1.75 The endoplasmic reticulum was enriched 180-fold compared to the c e l l lysate however the ER appears to be contaminated with 5' nucleotidase a c t i v i t y . The plasma membrane appeared to be puri 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 lysate was 28%. I t i s clear 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 series of control experiments were undertaken i n order to determine the optimal conditions of virus 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 infecting BHK -51-c e l l s with H-aa-SF Virus (1X10 pfu's/cell) at 37°C and 4°C. At desired time intervals.unadsorbed virus was removed by washing with 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 off the plates with 5 mis of H^ O, placed into 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 results of th i s experiment are shown i n Fig. 45. I t i s evident that the adsorption of virus 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 least three hours of incubation. A l l subsequent experiments were performed at 37° C. Another study was performed to determine the effect the ionic strength of the inoculum on virus adsorption. Solutions of different i o n i c strengths were made by making appropriate d i l u t i o n s of the high ionic 3 strength Medium A. 200 u l of H-aa-SF Virus were added to 5 mis of appropriate medium and incubated with plates (100mm X 15 mm) of BHK c e l l s for 60 minutes. At th i s point, the c e l l s were washed, scraped and counted as before. A summary of the results are shown i n Fig. 46. The important point to note i s that the virus adsorption i s greatly f a c i l i t a t e d at low io n i c strengths. Medium A has an ionic strength of approximately .13 and as can be seen, the v i r a l adsorption i s decreased by more than 1/2 the value at io n i c strength .10. A l l subsequent experiments were performed with inoculums of ionic strength .10. - 5 2 -25 20 1 .5 1 .0 . 5 5 10 30 60 120 180 Time of I n c u b a t i o n (minutes) F i g . 4 5 . Dependence of SF V i r u s A d s o r p t i o n on Temperature and Time of I n c u b a t i o n . F i g . 4 6 . 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 , - 5 4 -Two experiments are presented i n F i g . 46. 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 which a low i o n i c s t r e n g t h s o l ' n ( u = .10) was used to wash the c e l l s . The d o t t e d l i n e rep resents 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 = .25 ) . I t was o r i g i n a l l y thought tha t the 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 . Th is was based on the assumption that 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 nature however i t i s c l e a r that 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 a d s o r p t i o n . The next c o n t r o l experiment s t u d i e d the e f f e c t thevplaque f6rming: ' -units 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 " ) have on v i r u s a d s o r p t i o n . 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 of low i o n i c s t r e n g t h s o l u t i o n ( AX = .10 ) f o r three hours at 4°C. BHK c e l l s ( 100 nm X 15 nm ) were then i n f e c t e d f o r 60 minutes w i t h these v i r u s samples made up to 5 mis w i t h s o l u t i o n xi = . 1 0 . C e l l s were washed, ha rves ted and counted as b e f o r e . The r e s u l t s of t h i s experiment are shown i n F i g u r e 47 . This f i g u r e c l e a r l y shows a l i n e a r r e l a t i o n s h i p 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 at 5000 plaque forming u n i t s per c e l l . - 5 5 --56-0-2 HOUR PULSE CHASE EXPERIMENT After completion of the preliminary experiments we decided to try 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 , ionic strength yu = .10 ) and chase periods of 1, 60, and 120 minutes after removal of the radioactive virus. Three plates (150 mm X 15mm) of BHK c e l l s were used per time point. B r i e f l y , radioactive virus collected from the sucrose gradients, was dialyzed at 4° C against diluted Medium 199 CjJ = .10 ). After d i a l y s i s the virus was divided into aliquots, made up to 5 mis with Medium 199 CP = .10) and allowed to adsorb to a plate of BHK c e l l s for 30 minutes at 37° C. After the end of the pulse time, the c e l l s were washed twice with 5 mis. of high ionic strength Medium A (jj = .25") and once with 5 mis of d i s t i l l e d ^ 0 . 5 mis of Chase medium consisting 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 their plates with a rubber policeman and placed i n a loose f i t t i n g V i t r o "Dounce" homogenizer. Cells were lysed and the PM and ER fractions were isolated as outlined i n the Materials and Methods section 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 centrifuge tube using a syringe. The two fractions were then pelleted by recentrifugation at 105,000 X g (45,000 rpm) for 60 minutes on a Beckman Ti 65 rotor. 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 H^ O. Aliquots (25 ill) were saved for protein determination and the remainder electrophoresed on 7.5% SDS acrylamide gels and analyzed as outlined i n the Materials and Methods section. F i ~ . 48 , O - 2 Hour Chase Virus Gels ('Results exDressed in terms of 2 mems Drotein) - 5 8 -I o XI ft 60' 12-<D» Time of Chase (minutes) F i g . -49.; 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 radioactive p r o f i l e s of these gels are shown i n Figure 48. a-f , which represent plasma membrane fractions 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 this drop i n r a d i o a c t i v i t y can be attributed to a loss of envelope and nucleocapsid protein. However, at the 120 minute chase point, approximately 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 represent the ER fractions at 0, 60, and 120 minutes of chase. Virus protein i s shown to enter the c e l l and be-come associated with the ER however i t i s interesting to note that only the nucleocapsid protein becomes associated while the envelope proteins are completely l o s t . Total r a d i o a c t i v i t y of the virus-specified proteins associated with the PM and ER were determined and graphed versus chase time (Fig.'49 The results show that the v i r a l proteins are rapidly l o s t from the PM within 60 minutes following 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 decline again at the 120 minute chase point. 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 within the c e l l . These results are consistent with the theory of a precursor-product relationship between the v i r a l proteins i n the PM and the v i r a l proteins i n the ER. - 6 0 -0-60 MINUTE PULSE CHASE EXPERIMENT Based on the p rev ious exper iment , i t was decided to r e f i n e the experiment by d o i n g , 0 , 2 0 , 40 and 60 minute chase t ime p o i n t s . 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 from the plasma membrane over the 60 minute chase . The ER p l o t s (F igure 50 e-h) do not e x h i b i t any d e f i n i t e t rend of i n c r e a s e or decrease i n r a d i o a c t i v i t y . In t h i s exper iment , a second (minor) peak of r a d i o a c t i v i t y occurs i n the 2 0 , 4 0 , and 60 minute chase p l o t s . Th is peak represents the envelope p r o t e i n s of SF V i r u s and i t s presence i n the ER f r a c t i o n i s presumably due to contaminat ion 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 represents 50 u l of s tandard S -SF V i r u s . 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 versus chase t ime ( F i g . 5 1 ) . These r e s u l t s show a steady 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 constant throughout the exper iment . 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 presented i n the next s e c t i o n . 10 20 30 40 10 20 30 40 Fraction No. Fraction Y.o. Fig. 50 . 0' - 60' Chase Virus Gels ( Results expressed in terns of 2 mgms protein) e. ER - 0' Chase Fraction No. Fig. 50 Con't f. ER - 20: Chase Fraction No. -63-35 i . 5&>| S - SF Virus Fraction No. Fig. 50. Con't ro ! O a ft o PM (E ' E and NC) 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 ev idence i n d i c a t e s that v i r o p e x i s i s the p r e -dominant , but not e x c l u s i v e , mechanism of v i r u s ent ry i n t o host c e l l s . In most c a s e s , when f u s i o n was observed by e l e c t r o n mic roscopy , 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 ev idence i n the l a s t s e v e r a l years has unquest ionab ly conf i rmed f u s i o n as the mode of ent ry of V a c c i n i a , S e n d a i , Newcast le D isease and some Insect agents i n c e r t a i n host c e l l s . I f there i s a d e f i n i t e p a t t e r n to the mechanism of i n t e r n a l i z a t i o n of enveloped v i r u s e s , I t i s tha t f u s i o n takes p l a c e p r e f e r e n t i a l l y w i t h host c e l l s enc losed by r e l a t i v e l y non-deformable membranes such as e r y t h r o c y t e s , c i l i a and m i c r o v i l l i . I t i s worthy to note tha t i f v i r u s e s enter v i a v i r o p e x i s , g e n e r a l l y i t must s t i l l f use w i t h the membrane of the c y t o p l a s m i c vacuo le i n order to a l l o w the r e l e a s e of the v i r a l core i n t o the cy top lasm. Th is may l e a d us to t h e o r i z e tha t enveloped v i r u s e s may have the p o t e n t i a l to en te r host c e l l s by both 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 occurs 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 f a c t o r s . P i n o c y t o s i s may on ly be a c e l l u l a r response due to presence of a f o r e i g n , macromolecular agent on i t s s u r f a c e . F u s i o n , on the o ther 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 , l y s i n s on the 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 o c c u r . P e n e t r a t i o n may t h e r e f o r e be envisaged as a s o r t of " r a c e " - 6 5 -between the f u s i o n of the membranes and the phagocyt ic response of the hos t c e l l . T h i s would e x p l a i n why f u s i o n takes p l a c e w i t h c e l l s hav ing r e l a t i v e l y non-deformable c e l l u l a r membranes and the o b s e r v a t i o n of f u s i o n at the 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 . T h i s theory c o u l d 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 obta ined w i t h V e s i c u l a r S tomat i tus V i r u s (15, 16) where f u s i o n w i t h L c e l l s was observed r a t h e r than 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 to i n i t i a t e r a p i d v i r u s - c e l l c o n t a c t . I t c o u l d be i n order to e f f e c t f u s i o n , a " t i g h t " i n t e r a c t i o n must occur which 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 not a l l o w i n g enough t ime f o r p i n o c y t o s i s to take p l a c e . S ince p i n o c y t o s i s i s a temperature-dependent step and can be i n h i b i t e d by m e t a b o l i c po isons such as amanat id ine , t h i s theory cou ld p o s s i b l y be t e s t e d . For example, t h i s theory would :bessuppor ted : i . i f f f ius ion was observed i n the presence of amant id ine . 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 study 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 ther v i r u s s p e c i e s , v i r a l ant igens 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 f u s i o n o c c u r s . Another p o s s i b l e experiment which cou ld be attempted i s to 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 the p e n e t r a t i o n of the v i r u s e s through e l e c t r o n mic roscopy . I f the v i r i o n s were to enter p r i m a r i l y by f u s i o n , we would expec t , - 6 6 -th e f l u o r e s c e n t marker to remain at the c e l l membrane w h i l e the r e s t of the v i r u s e n t e r s the c e l l . S ince t h i s experiment appears to be f a c i l l e and has yet to be done, I would suspect that there are some t e c h n i c a l d i f f i c u l t i e s unaware to t h i s w r i t e r . Retu rn ing to the present i n v e s t i g a t i o n , i t was our i n t e n t i o n to determine by b i o c h e m i c a l methods the 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 hos 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 r e t i c u l u m f r a c t i o n s . I f f u s i o n were the mode of e n t r y , we would expect c that 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 both the envelope p r o t e i n s and n u c l e o c a p s i d be ing chase out of the plasma membrane f r a c t i o n d u r i n g the course of the exper iment . Fur thermore, the r a t i o of r a d i o -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 approx imate ly the same throughout the chase p e r i o d . These r e s u l t s l ead us to the c o n c l u s i o n tha t SF V i r u s en te rs BHK c e l l s v i a v i r o p e x i s . Th is p r o p o s a l , however, i s g i v e n w i t h some r e s e r v a t i o n . I t i s p o s s i b l e tha t the t r u e l y i n f e c t i o u s route taken by SF V i r u s i s by f u s i o n . Th is may occur w i t h on ly a minor f r a c t i o n of the t o t a l inoculum w h i l e the r e s t of the v i r u s e n t e r s by p i n o c y t o s i s ( s o l e l y a c e l l u l a r response due to the presence of a f o r e i g n agent of i t ' s s u r f a c e ) . A l though 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 are i n d i s t i n g u i s h a b l e u s i n g our methods. Th is p o s s i b i l i t y w i l l have to be f u r t h e r examined i n f u t u r e exper iments . -67-The one apparently surprising result involves the accumulation of nucleocapsid protein i n the endoplasmic reticulum fraction (Fig. 50 e-h). The manner i n which the nucleocapsid i s transported to the ER and the relationship this phenomenon has to the uncoating process i s s t i l l unknown. On the other hand, this phenomenon may in fact be an a r t i f a c t . Nucleocapsid protein from the cytoplasm could possibly be precipitated with the ER by the 100,000 X g centrifugation. A number of c r i t i c i s m s of these experiments can be made. F i r s t l y , there i s a problem i n that high levels of r a d i o a c t i v i t y become associated with the ER fraction very quickly, even at zero minutes of chase. This si 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 pulsing for a shorter period of time, we may cause another problem involving the attainment of enough ra d i o a c t i v i t y into 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 in this investigation since the virus i n the PM continued to be chased out throughout the experiment, even though the ER was heavily labelled. Another c r i t i c i s m of our experiments involves the high multi-p l i c i t y of infe 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 infection may somehow,-alter the physiological response of the c e l l s i n contact with SF Virus, although the pfu's do not appear ridiculous; for 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 infection can be effected by producing virus of higher s p e c i f i c a c t i v i t y . This might be done by preparing 125 I-iodinated SF Virus. - 6 8 -I t i s c l e a r that the v i r a l enve lopes , coats 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 i n the p e n e t r a t i o n and uncoat ing of the p a r e n t a l genome. I t i s my hope that t h i s i n v e s t i g a t i o n has shown that b i o c h e m i c a l techniques can be used to study the e a r l y events i n v i r u s - h o s t i n t e r a c t i o n s . - 6 9 -BIBLIOGRAPHY Hawkes, R.A. and Lafferty, K.J., Virology 33, 250 (1967) Dales, S., Bacteriological Reviews 37, 103 (1973) Dales, S., Journal of C e l l Biology 18, 51 (1963) Dales, S. and Kajioka, R., Virology 24, 278 (1964) Armstrong, J.A., Metz, D.H. and Young, M.R., Journal of  General Virology 21, 533 (1973) Chang, A. and Metz, D.H., Journal of General Virology 32, 275, (1976) Granados, R.R., Virology 52, 305 (1973) Abodeely, R.R., Lawson, L.A. and Randall, C.C., Journal  of Virology _5, 513 (1970) Dales, S. and Siminovitch, L., J. Biophys. Biochem. Cytol. 10, 475 (1961) Garabedian, G.A. and Scott, L.V. Arch. Gesamte Virusforsch 32, 59 (1970) Hummeler, K. Tomassini, N. and Zajac, B. Journal of Virology k_, 67 (1969) Miyamoto, K., and Morgan, C. Journal of Virology 8, 910 (1971) Morgan, C, Rose, H.M. and Mednis, B. Journal of Virology 2, 507 (1968) Smith, J.D. and DeHarven, E. Journal of Virology 14, 945 -946 (1974) Simpson, R.W., Hauser, R.E. and Dales, S. Virology 37, 285 (1969) Heine, J.W. and Schnaitman, CA. Journal of Virology 3, 619 (1969) Heine, J.W. and Schnaitman, CA. Journal of Virology 9, 431 (1972) Dahlberg, J.E. Virology 58, 250 (1974) Hummeler, K., Koprowski, H. and Wiktor, T.J. Journal of Virology 1, 152 (1967) - 7 0 -I w a s a k i , Y . , W i k t o r , T . J . and Koprowsk i , H. Laboratory  I n v e s t i g a t i o n 2 8 , 142 (1973) Faze Kas De S t . G r o t h , S . Na tu re , Lond. 162, 294 (1948) Dourmashkin, R.R. and T y r r e l l , D . A . J . J o u r n a l of Genera l  V i r o l o g y 24, 129 (1974) K a t o , N. and Eggers , H. J . V i r o l o g y 3 7 , 632 (1969) R e i n a c h e r , N. and W e i s s , E. A r c h i v e s of V i r o l o g y 4 9 , 187 (1975) H o y l e , L . , H o m e , R.W. and Waterson, A . P . V i r o l o g y 1 7 , 533 (1962) Morgan, C. and Rose, H.M. J o u r n a l of V i r o l o g y 2_, 925 (1968) B l a s k o v i e , P . et a l . A r c h , ges , V i r u s f o r s c h 38 , 250 (1972) K r i s a n o v a , 0 . e t a l . A c t a . V i r o l . 1 5 , 352 (1971) Stephenson, J . R . and Dimmock, W . J . V i r o l o g y 6 5 , 77 (1975) Dourmashkin, R .R. and T y r r e l l , D . A . J . J o u r n a l of Genera l V i r o l o g y 9 , 77 (1970) Meise lman, N . , Kohn, A . and Danon, D. J o u r n a l of C e l l Sc ience 2, 71 (1967) Morgan, C. and Howe, C. J o u r n a l of V i r o l o g y 2 , 1122 (1968) L e v i n t h a l , J . Dunnebacke, T . H . and W i l l i a m s , R . C . J . U l t r a s t r u c t .  Res . 30 , 244 (1970) B a c h i , T. and Howe, C. J o u r n a l of C e l l B i o l o g y 5 5 , 10a (1972) B a c h i , T. and Howe, C. P r o c . Soc . exp . B i o l . Med. 1 4 1 , (1972) P o s t e , G. I n t . Rev. C y t o l . 33, 157 (1972) Compans, R.W. , Holmes, K . V . , D a l e s , S . and Choppin, P.W. V i r o l o g y 30 , 411 (1964) Hosaka, Y . , and K o s h i , Y . V i r o l o g y 34 , 419 (1968) -71-(39) S i l v e r s t e i n , S.C. and Marcus, P.I. Virology 23, 370 (1964) (40) Howe, C. and Morgan, C. Journal of Virology 3, 70 (1969) (41) Bachi, T. Aguet, M. and Howe, C. Journal of Virology 11, 1004 (1973) (42) Haywood, A.M. Journal of General Virology 29, 63 (1975) (43) Haywood, A.M. Journal of Molecular Biology 87, 625 (1974) (44) Dales, S. and Hanafusa, H. Virology 50, 440 (1972) (45) Miyamoto, K. and Gidden, R.V. Journal of Virology 7, 395 (1971) (46) Barbanti-Brodano, G. Possati, L. and LaPlaca, M. Journal of Virology 8, 796 (1971) (47) Fraser, K. B. and Crawford, E.M. Exp. Mol. Pathol. 4, 51 (1965) (48) Mattern, C.F.T., Takemoto, K.K. and Wendell, A.D. Virology 30, 242 (1966) (49) MacKay, R.L. and C o n s i g l i , R.A. Journal of Virology 19, 620 (1976) (50) Chardonnet, Y. and Dales, S. Virology 40, 462 (1970) (51) Chardonnet, Y. and Dales, S. Virology 40, 478 (1970) (52) Fong, CK. Y., Bensch, K.G. and Hsiung, G.D. Virology 35, 297 (1968) (53) Chardonnet, Y. and Dales, S. Virology 48, 342 (1972) (54) Brown, D.T. and Burlingham, B.T. Virology 12, 386 (1973) (55) Dales, S., Gomatos, P.J. and Hsu, K.C. Virology 25, 193 (1965) (56) S i l v e r s t e i n , S.C. and Dales, S. Journal of C e l l Biology 36, 197 (1968) (57) Morgan, C. Bact. Proc. 203, (V322) (1970) (5 8) Holland, J . J . Virology 16, 163 (1962) -72-(59) Mandel, B. Virology 31, 248 (1967) (60) Dunnebacke, T.H., Le v i n t h a l , J.D.-and Williams, R.C. Journal of Virology _4, 505 (1969) (61) Leutenegger, R. Virology 32, 109 (1967) (62) Tanada, Y. and Leutenegger, R.J. U l t r a s t r u c t . Res. 30, 589 (1970) (63) Younghusband, H.G. and Lee, P.E. Virology 40, 757 (1970) (64) Zee, Y.C. and Talens, L. Journal of General Virology 11, 59 (1971) (65) Summers, M.D. Journal of Virology 4, 188 (1969) (66) Summers, M.D. J. U l t r a s t r u c t . Res. 35, 606 (1971) (67) Kawanishi, C.Y. et a l . J . Inverteb. Pathol. 20, 104 (1972) (68) Knudson, D.L. and Harrap, K.A. Journal of Virology 17, 254 (1976) (69) David-Ferreira, J.F. and Hanaker, R.A. Journal of C e l l Biology 24, 57 (1965) (70) Doughi, A.M. et a l . Exp. Mol. Path. 25, 355 (1976) (71) Pathak, S. and Webb, H.E. J . Neurol. S c i . 23, 175 (1974) (72) Weber, K.J. and Osborne M. Journal of B i o l o g i c a l Chemistry 244, 4406 (1969) (73) Richardson, CD. and Vance, D.E.V. Journal of B i o l o g i c a l Chemistry 251, 5544 (1976) (74) Ragnotti, G., Lawfor, G.R. and Campbell, P.N. Biochem. J . 233, 334 (1971) (75) Avruch, J . and Wallach, D.F.H. Biochem. Biophys. Acta. 233, 334 (1971) (76) Lowry, O.H. et a l . Journal of B i o l o g i c a l Chemistry 193, 265 (1951) -73-(77) Scheele, CM. and Pfefferkorn, E.R. Virology 3, 369 (1969) (78) Kennedy, S.I. T. and Burke, D.C 14, 87 (1972) (79) Morser, M.J. and Burke, D.C. Journal of General Virology 22, 395 (1974) (80) Kaariainen, L. et a l . Medical Biology 53, 342 (1975) (81) Newbold, J.E. and SInsheimer, R.L.J. J. Mo. Biol. 49, 49 (1970) (82) Fenwick, M.L. and Cooper, P.D. Virology 18, 212 (1962; (83) Luria, S.E. Science 111, 507 (1950) (84) Holland, J.J. Virology 16, 163 (1962) (85) Lonberg-Holm, K. and Yin, F.H. Journal of Virology 12, 114 (1973) (86) Fenner, F.J. and White, D.O. Medical Virology, Second Edition Academic Press, New York, pg. 17-19 (1976) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094447/manifest

Comment

Related Items