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Studies on the entry of alphaviruses into BHK-21 cells Talbot, Pierre 1981

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STUDIES ON THE ENTRY OF ALPHAVIRUSES INTO BHK-21 CELLS by PIERRE TALBOT B.Sc, U n i v e r s i t e Laval, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1981 © P i e r r e Talbot, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements 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 Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of 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 allowed without my w r i t t e n p e r m i s s i o n . Department of Q, QtktM / ' i J f V The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DE-6 (2/79) i i ABSTRACT The nature of the infectious mechanism- of entry of alphaviruses into cultured BHK-21 c e l l s was studied. The importance of cellular lysosomes i n a productive Sindbis virus i n f e c t i o n was determined. Chloroquine ( 1 0 0 pM) and NH^Cl ( 1 0 mM), known i n h i b i t o r s of lysosomal function, decreased the production of in f e c t i o u s Sindbis virions in BHK-21 c e l l s by 10- and 1 2 - f o l d respectively, when present during the 1 h i n f e c t i o n period of a plaque assay. There were no apparent toxic effects on c e l l s exposed to these chemicals, with the exception of a rev e r s i b l e 2 - f o l d i n h i b i t i o n of c e l l u l a r protein synthesis by chloroquine. In order to determine i f the decrease i n the virus t i t e r was correlated with a si m i l a r decrease i n the number of virus particles, the reduction i n the formation of Sindbis v i r i o n s was monitored by 35 incorporation of L.-C S]methionine and shown to be 2 - f o l d at 8 and 11 h p o s t - i n f e c t i o n . Shedding of envelope glycoproteins into the culture medium was indire c t l y demonstrated by the fact that much more of these proteins were released that can be accounted for by progeny virions. i i i Finally, the importance of a lysosomal pathway of infection was studied i n more d e t a i l s . C e l l s were i n f e c t e d with Sindbis virus 35 labelled with L.-L S]methionine and the fate of radiolabeled v i r a l p r o t e i n s i n t o s u b c e l l u l a r f r a c t i o n s f o l l o w e d up to 120 min pos t - i n f e c t i o n . Viruses were t r a n s i e n t l y associated with a c e l l fraction enriched i n lysosomes. Capsid proteins were preferentially released into the cytoplasm af t e r 60 min post - i n f e c t i o n and v i r a l proteins started to be s i g n i f i c a n t l y degraded between the f i r s t and second hour a f t e r i n f e c t i o n . Chloroquine produced an i n i t i a l accumulation of viruses i n the lysosomes at 20 min post-infection and these trapped p a r t i c l e s appeared to be degraded t h e r e a f t e r . Unexpectedly, NH^Cl blocked an early i n f e c t i o n step between binding of the vir u s on the c e l l surface and penetration into lysosomes, possibly receptor clustering into coated p i t s or receptor recycling. Results were identical with virus preparations which contained either 147 or 36 particles per infective unit. In conclusion, the results of these studies strongly suggest the involvement of lysosomes and thus a receptor-mediated endocytic pathway (viropexis) as the main in f e c t i o u s mechanism for entry of Sindbis virus into BHK-21 ce l l s . i v The mechanism of entry of r a d i o l a b e l e d S e m l i k i Forest virus was also studied. A f t e r a 15 min pulse, whole v i r u s p a r t i c l e s migrated w i t h i n 60 min from a f r a c t i o n o f the c e l l s e n r i c h e d i n plasma membranes into an i n t r a c e l l u l a r f r a c t i o n (endoplasmic reticulum). This res u l t i s consistent with an endocytic mechanism of entry. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES xi LIST OF FIGURES x i i LIST OF ABBREVIATIONS xv ACKNOWLEDGEMENTS x v i i DEDICATION x v i i i INTRODUCTION 1. Why Study the Mechanism of Entry of Animal Viruses into Cells? 1 2. Structure of Alphaviruses 2 (a) Nucleocapsid 4 (b) Lipid Bilayer 4 (c) Glycoproteins 5 3. Replication of Alphaviruses 7 (a) Growth Cycle 8 (b) Transcription and Replication 9 (c) Synthesis of Vir a l Proteins i) Non-Structural Proteins 10 v i i i ) S t r u c t u r a l Proteins 11 (d) Maturation 13 4. Studies on the Entry of Animal Viruses I. Enveloped RNA Viruses (a) Introduction 15 (b) Togaviruses 18 (c) Rhabdpviruses 26 (d) Retroviruses 31 (e) Paramyxoviruses 34 (f) . Orthomyxoviruses 39 (g) Coronaviruses 43 I I . Non-Enveloped RNA Viruses (a) Reoviruses ^ (b) Picornaviruses 45 I I I . DNA Viruses 46 IV. Summary and Conclusion 48 5. Receptor-Mediated Endocytosis (a) Mechanism 49 (b) E f f e c t of Lysosomotropic Agents 51 6. Scope of the Present Work 54 MATERIALS AND METHODS 1. Chemicals and Equipment 56 v i i 2. General Methods (a) Sc i n t i l l a t i o n Counting 57 (b) Protein Assays 58 (c) SDS- Polyacrylamide Gel Electrophoresis i) Method 58 i i ) Quantitation of Radiolabeled Proteins i n Polyacrylamide Gels 1. Gel Slicing 60 2. Autoradiography 61 3. Cell Culture 61 4. Propagation of Viruses (a) Semliki Forest Virus 62 (b) Sindbis Virus 6 2 5. Plaque Assays (a) Regular Assays 63 (b) Plaque-Reduction Assays with Chloroquine or NH^Cl ... 63 (c) Plaque-Purification of Sindbis Virus 64 6. Determination of the Particle to PFU Ratio of Virus Preparations 65 7. Radiolabeling and Purification of Viruses (a) Semliki Forest Virus 65 (b) Sindbis Virus 67 v i i i 8. Enzyme Markers (a) Plasma Membranes i) 5'-Nucleotidase 68 i i ) Ouabain-Sensitive Na +, K + - ATPase 69 (b) Endoplasmic Reticulum: NADPH- Cytochrome c Reductase .. 70 (c) Lysosomes: Acid Phosphatase 71 (d) Mitochondria: Succinate Dehydrogenase 71 9. Preparation of Plasma Membranes and Endoplasmic Reticulum . 72 10. Subcellular Fractionation 73 11. Effect of Chloroquine or NH^Cl on Cellular Protein Synthesis 74 12. Effect of Chloroquine on Cell V i a b i l i t y 75 13. Effect of Chloroquine or NH^Cl on the Production of Radiolabeled Sindbis Virus Proteins and Particles 75 14. Pulse-Chase of Radiolabeled Semliki Forest Virus into Enriched Plasma Membrane and Endoplasmic Reticulum Fractions 77 15. Entry of Radiolabeled Sindbis Virus into Subcellular Fractions of BHK-21 Cells and the Effect of Chloroquine or NH4C1 78 RESULTS 1. Stock Virus Preparations 81 ix 2. Purification of Radiolabeled Viruses (a) Semliki Forest Virus 83 (b) Sindbis Virus 86 3. Preparation of Plasma Membranes and Endoplasmic Reticulum from BHK-21 Cells 89 4. Pulse-Chase of Radiolabeled Semliki Forest Virus into Enriched Plasma Membrane and Endoplasmic Reticulum Fractions of BHK-21 Cells 93 5. Effect of Chloroquine and NH^Cl on the Production of Infectious Sindbis Virions (a) Plaque-Reduction Assays 96 (b) Effect of Chloroquine or NH^Cl on Cellular Protein Synthesis or Cell V i a b i l i t y 98 (c) Effect of Chloroquine or NH^Cl on the Production of Radiolabeled Sindbis Virus Proteins and Particles .. 103 6. Subcellular Fractionation of BHK-21 Cells 113 7. Mechanism of Entry of Radiolabeled Sindbis Virus into BHK-21 Cells and the Ef f e c t of Chloroquine or NH^Cl on this Process (a) Preliminary Experiments: 37°C, no treatment 115 (b) Entry of Radiolabeled Sindbis Virus: Effect of Chloroquine 124 X (c) Entry of Radiolabeled Sindbis Virus: Effect of Nrl^Cl 130 DISCUSSION 1. Mechanism of Entry of Semliki Forest Virus 137 2. Mechanism of Entry of Sindbis Virus (a) Effect of Chloroquine and NH^Cl on the Production of Infectious Sindbis Virions 139 (b) Incorporation of Radiolabeled Sindbis Virus into Subcellular Fractions i) Preliminary Experiment 144 i i ) Chloroquine Effect 146 i i i ) NHjjCl Effect 148 3. Conclusion 150 4. Suggestions for Future Work 151 BIBLIOGRAPHY : 154 x i LIST OF TABLES Page 1. Characterization of stock virus preparations 82 2. Purification of plasma membranes and endoplasmic reticulum .. 92 3. Effect of chloroquine on c e l l v i a b i l i t y 104 4. Subcellular fractionation of BHK-21 cel l s 114 5. Association of radiolabeled Sindbis virus with subcellular fractions at 37°C 116 6. Association of radiolabeled Sindbis virus with subcellular fractions at 37 and 4°C, with or without treatment with 100 pM chloroquine 125 7. Association of radiolabeled Sindbis virus with subcellular fractions at 37 and 4°C, with or without treatment with 10 mM NH^Cl 132 x i i LIST OF FIGURES Page 1. Post-translational cleavages during the formation of alphavirus structural proteins 12 2. Two proposed mechanisms of entry of enveloped RNA viruses into animal c e l l s 17 3. Protein and autoradiographic patterns of purified radiolabeled Semliki Forest virus 84 4. Protein and radioactive profiles of purified radiolabeled Semliki Forest virus 85 5. Protein profile of purified radiolabeled Sindbis virus 87 6. Autoradiography and radioactive profile of purified radiolabeled Sindbis virus 88 7. Enzyme marker profiles for preparation of plasma membranes and endoplasmic reticulum from BHK-21 cel l s 90 8. Incorporation of radiolabeled Semliki Forest virus proteins into enriched plasma membrane and endoplasmic reticulum fractions of BHK-21 c e l l s 94 9. Effect of chloroquine or NH^Cl concentration on the t i t e r of Sindbis virus 97 x i i i 10. E f f e c t of NHjjCl c o n c e n t r a t i o n on the t i t e r of Sindbis v i r u s i n the absence or presence of chloroquine .... 99 11. Long-term e f f e c t o f c h l o r o q u i n e or NH^Cl on c e l l u l a r protein synthesis 101 12. Short-term e f f e c t o f c h l o r o q u i n e or NH^Cl on c e l l u l a r protein synthesis 102 13. E f f e c t of 100 uM chloroquine on the production of radiolabeled Sindbis v i r u s proteins 105 14. E f f e c t of 10 mM NH^Cl on the production of radiolabeled Sindbis v i r u s proteins v 108 15. Autoradiograph of radiolabeled Sindbis v i r u s p e l l e t e d by u l t r a c e n t r i f u g a t i o n 110 16. E f f e c t of 10 mM NH^Cl on the production of radiolabeled Sindbis v i r u s p a r t i c l e s 112 17. Association of radiolabeled Sindbis v i r u s with s u b c e l l u l a r f r a c t i o n s 117 18. Radioactive p r o f i l e of the lysosomes at 30 min a f t e r i n f e c t i o n with radiolabeled Sindbis v i r u s 119 19. V i r a l proteins associated with the lysosomes and release of acid-soluble r a d i o a c t i v i t y i n t o the medium 121 20. Radioactive p r o f i l e s of the cytosols at 60 and 120 min a f t e r i n f e c t i o n with radiolabeled Sindbis v i r u s 122 xiv 21. Incorporation of radiolabeled Sindbis virus into lysosomes at 37 and 4°C and the e f f e c t of chloroquine 128 22. Specific incorporation of radiolabeled Sindbis virus into lysosomes and the effect of chloroquine 129 23. Incorporation of radiolabeled Sindbis virus into lysosomes at 37 and 4°C and the e f f e c t of NH^Cl 134 24. Specific incorporation of radiolabeled Sindbis virus into lysosomes and the e f f e c t of NH„C1 135 i X V LIST OF ABBREVIATIONS asn asparagine AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate ACS aqueous counting s c i n t i l l a n t BHK baby hamster kidney 1 p C i Curie ( r a d i o a c t i v i t y ; 1 C i = 2 . 2 2 x 10 dpm) cpm counts per minute dpm d i s i n t e g r a t i o n s per minute DNA deoxyribonucleic acid dA change i n absorbance EDTA ethylene diamine t e t r a a c e t i c a c i d E e x t i n c t i o n c o e f f i c i e n t g grams .g. g r a v i t a t i o n a l f o r c e ; a l l c e n t r i f u g a t i o n f i e l d s are given for the middle o f t h e tubes (e ) Gal galactose GalNAc N-acetyl galactosamine H 20 g l a s s d i s t i l l e d water h hour IgG immunoglobulin G xvi K x 10 3 (used for M ) 1 l i t e r log logarithm (base 10) M molar (concentration in moles/1) min minute M molecular weight r NeuAc neuraminidic (sialic) acid NADPH nicotinamide adenine dinucleotide phosphate (reduced form) PFU plaque-forming units RNA ribonucleic acid SDS sodium dodecyl (lauryl) sulfate sec second ser serine thr threonine Tris tris(hydroxymethyl)aminomethane Standard Prefixes u micro (x 10~^) m m i l l i (x 10~ 3) _2 c centi (x 10 ) x v i i ACKNOWLEDGEMENTS I wish t o thank Dr. Dennis E. Vance, who has been my research s u p e r v i s o r f o r the past f o u r y e a r s . H i s enthusiasm, guidance and support were a consta n t h e l p . S p e c i a l thanks to Dr. Christopher D. Richardson, who provided me with encouragement and advice, as well as supplying the c e l l l i n e used f o r work w i t h S i n d b i s v i r u s . I am also indebted to my co l l e a g u e s Harry Paddon, J e n n i f e r Toone, Corinne Lee, Amelia Wong, Diana C r o o k a l l and Kathy Wong f o r s k i l l f u l l t e c h n i c a l help. I would l i k e to thank Dr. James B. Hudson, Dr. Robert S. Molday, Dr. Haydn P r i t c h a r d and Steve P e l e c h f o r t h e i r u s e f u l suggestions and comments d u r i n g the course of t h i s work. A s p e c i a l acknowledgement goes to Dr. Guy Bellemare, my undergraduate r e s e a r c h supervisor, who encouraged me int o graduate s t u d i e s and recommended my candidature for a studentship. I am a l s o g r a t e f u l t o the NSERC f o r t h e i r f i n a n c i a l support. F i n a l l y , my major debt i s to my wife, France Ouellet-Talbot, whose encouragement and support made i t a l l p o s s i b l e . I also wish to express g r a t i t u d e to my f r i e n d s i n t h i s department. They helped make t h i s experience even more enjoyable. x v i i i to France 1 INTRODUCTION 1. Why study the Mechanism o f E n t r y of Animal Viruses i n t o C e l l s ? Animal v i r u s e s are v e r y s m a l l and s i m p l e s t r u c t u r e s at the t h r e s h o l d o f l i f e . T h e i r main c o n s t i t u e n t i s t h e n u c l e i c a c i d r e s p o n s i b l e f o r t h e i r r e p r o d u c t i o n and u s u a l l y complexed w i t h a protein coat to form the n u c l e o c a p s i d , which i s often contained i n a l i p o p r o t e i n envelope. V i r u s e s have n e i t h e r p r o t e i n - s y n t h e s i z i n g machinery nor energy supply, which makes them s t r i c t l y dependent on a host organism f o r t h e i r r e p r o d u c t i o n . Thus, v i r u s e s are c e l l u l a r parasites of a very intimate n a t u r e . A more formal d e f i n i t i o n i s (50): "Viruses are e n t i t i e s whose genomes are elements of nu c l e i c a c i d that r e p l i c a t e i n s i d e l i v i n g c e l l s u s i n g the c e l l u l a r synthetic machinery and causing the s y n t h e s i s o f s p e c i a l i z e d elements t h a t can transf e r the v i r a l genome to other c e l l s " . In order to re a c h t h e s i t e o f r e p l i c a t i o n , v i r u s e s need to penetrate the host c e l l plasma membrane. D e s p i t e the importance of understanding t h i s e a r l y step o f a v i r a l i n f e c t i o n f o r the possible prevention of v i r a l diseases, v i r a l e n t r y remains a c o n t r o v e r s i a l area of modern v i r o l o g y . Furthermore, i t i s g e n e r a l l y accepted that most v i r u s e s i n i t i a l l y a t t a c h t h e m s e l v e s to t a r g e t c e l l s v i a s p e c i f i c receptors on the c e l l s u r f a c e , a l t h o u g h the nature of such receptors 2 i s generally unknown. The favored approach chosen by most workers f o r the study of v i r a l entry has been electron microscopy. As noted by Dales (51), there are several problems i n the i n t e r p r e t a t i o n o f experiments done with t h i s instrument and the technique w i l l not a l l o w one to d i f f e r e n t i a t e between i n f e c t i o u s and n o n - i n f e c t i o u s v i r u s p a r t i c l e s . Therefore, we decided to apply modern biochemical techniques to the i n v e s t i g a t i o n of t h i s d i f f i c u l t problem. A l p h a v i r u s e s , a genus o f the t o g a v i r i d a e f a m i l y o f enveloped animal viruses (52), were chosen as a model system because of t h e i r r e l a t i v e l y simple structure ( c f . s e c t i o n 2). The established c e l l l i n e BHK-21 (baby hamster kidney c e l l s ) was selected as a host c e l l i n view of i t s ease of c u l t i v a t i o n and h i g h y i e l d s of alphaviruses that can be obtained. 2. S t r u c t u r e of A l p h a v i r u s e s The t o g a v i r u s e s a r e a f a m i l y o f a n i m a l v i r u s e s which are t r a n s m i t t e d by b l o o d s u c k i n g a r t h r o p o d s , mostly mosquitoes, and possess the a b i l i t y to grow i n the a r t h r o p o d vector as well as i n the avian or mammalian host (23). These v i r u s e s were previously known as a r b o v i r u s e s ( a r t h r o p o d - b o r n e ) , b e f o r e i t became c l e a r t h a t a wide 3 v a r i e t y of v i r u s e s can m u l t i p l y i n and be t r a n s m i t t e d by arthropods (40). Therefore, morphological c r i t e r i a r e p l a c e d t h i s epidemiological c l a s s i f i c a t i o n , w h i c h l e a d t o t h e p o s s i b l e i n c l u s i o n o f t h e non-arthropod-borne r u b e l l a and l a c t i c dehydrogenase v i r u s e s as togaviruses (52). The togavirus f a m i l y i s d e f i n e d by two c r i t e r i a : the morphology of the v i r i o n (virus p a r t i c l e ) , which c o n s i s t s of a small nucleocapsid of cubic symmetry e n c l o s e d by a l i p o p r o t e i n envelope, and the v i r a l RNA, which can be extracted i n an i n f e c t i o u s state (52). The family i s subdivided i n t o two genera: alphaviruses (formerly group A arboviruses) and f l a v i v i r u s e s (formerly group B a r b o v i r u s e s ) . F l a v i v i r u s e s contain 33 known members which have not been w e l l c h a r a c t e r i z e d ( 2 3 ) . The alphaviruses i s a genus grouping 20 known members, which cross-react s e r o l o g i c a l l y i n complement-fixation assays but can be distinguished by s e r o l o g i c a l n e u t r a l i z a t i o n of i n f e c t i v i t y ( 7 , 2 3 ) . Of these, Semliki Forest and Sindbis v i r u s have been the most extensively characterized. Electron micrographs o f n e g a t i v e l y s t a i n e d , f i x e d alphaviruses p a r t i c l e s r e v e a l u n i f o r m s p h e r i c a l p a r t i c l e s of about 50-70 nm diameter w i t h i r r e g u l a r p r o t e a s e s e n s i t i v e and r a d i a l l y o r i e n t e d s u r f a c e p r o j e c t i o n s ( s p i k e s o r p e p l o m e r s ) up t o 10 nm l o n g . The i n t e r n a l c o r e , or n u c l e o c a p s i d , can be v i s u a l i z e d i n t h i n sections (7,52). The p a r t i c l e has a s e d i m e n t a t i o n c o e f f i c i e n t of 280 S and a density of 1 . 1 8 - 1 . 2 1 g/cm i n sucrose (7 , 2 3 ) . 4 (a) Nucleocapsid The alphaviruses contain one copy of a single-stranded RNA of molecular weight approximately 4.5 x 10^ (about 13,000 nucleotides) and sedimentation c o e f f i c i e n t of about 42 S. The v i r a l RNA genome i s plus stranded since i t i s infectious by i t s e l f ( i t acts as a messenger RNA). The 3'-end i s polyadenylated (60-80 adenine nucleotides) while the 5'-end of a t l e a s t S i n d b i s v i r u s RNA has an i n v e r t e d 7-methylguanosine cap (7,23,50). In a d d i t i o n to the RNA, the nucleocapsid contains 200-300 capsid protein molecules (C), each one with a molecular weight of about 34,000, as determined by SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the Sindbis virus capsid protein has been deduced from the nucleotide sequence of the RNA (39). I t i s 264 residues long and shows a basic region near the N-terminus which i s probably important in interacting with the virion RNA. The nucleocapsid has a diameter of 35-40 nm and i t i s generally accepted to have an icosahedral symmetry, although i t has never been unequivocally revealed by electron microscopy (40). (b) Lipid Bilayer The envelope of alphaviruses, as most enveloped animal viruses, i s derived from the host c e l l by a process known as budding ( c f . section 3 d ) . Thus, the l i p i d composition resembles that of the plasma membrane o f the h o s t , e x c e p t f o r a h i g h e r p r o p o r t i o n o f c h o l e s t e r o l t o p h o s p h o l i p i d s i n the v i r a l envelope (0.7-0.9 i n s t e a d o f 0 . 6 ) . The major p h o s p h o l i p i d s a r e p h o s p h a t i d y l c h o l i n e , s p h i n g o m y e l i n , phosphatidylserine and phosphatidylethanolamine. The l i p i d b i l a y e r of the v i r u s appears to be l e s s f l u i d than t h a t o f the c e l l , possibly because of the presence of transmembrane glycoproteins anchored to the nucleocapsid (23,76 and section 2c). (c) Glycoproteins The a l p h a v i r u s e s c o n t a i n about 200-300 molec u l e s each o f two glycoprotein s p e c i e s o f approximately 50,000 (E1,E2). E1 appears to be responsible f o r the h e m a g g l u t i n i n a c t i v i t y (64). Semliki Forest v i r u s a l s o c o n t a i n s equimolar amounts o f a t h i r d g l y c o p r o t e i n ( E 3 ; M^= 10,000), which i s n o t p a c k a g e d i n t o mature S i n d b i s v i r i o n s ( 2 3,24). The amino a c i d sequence o f these envelope glycoproteins has been deduced from the RNA sequence f o r S i n d b i s v i r u s (39) and Semliki Forest v i r u s (53). From these sequences, probable g l y c o s y l a t i o n s i t e s (type Asn-X-Ser/Thr) can be i d e n t i f i e d , as w e l l as the hydrophobic membrane spanning segments near the C - t e r m i n i of E1 and E2 (absent i n E3). I t appears that S e m l i k i F o r e s t v i r u s E3 i s not or only s l i g h t l y imbedded i n the l i p i d b i l a y e r . On the o t h e r hand, E1 and E2 of both viruses appear to span the membrane. The C-terminus exposed to the 6 inside of the virus i s 30 residues long in E 2 but only 2 residues long in E 1 . It i s reasonable to assume that E 1 and/or E 2 interact with the nucleocapsid, although only indirect evidence for this association has been obtained, for Semliki Forest v i r u s , either by cross-linking and labeling studies (54) or by detergent delipidation that l e f t the spike glycoproteins attached to the nucleocapsids (55). However, Richardson and Vance ( 1 0 1 ) could not demonstrate c r o s s - l i n k i n g of envelope glycoproteins with capsid proteins. A l l three membrane polypeptides contain carbohydrate units linked to asparagine residues (53). Overall, polysaccharides account for 8-12% of the mass of E1 and E 2 and 45S& of the mass of E3 (23). The typical sequence Asn-X-Ser or Asn-X-Thr which i s a necessary but not a sufficient condition for glycosylation i s found twice in each of E 3 and E 2 and once in E 1 of Semliki Forest virus (53). Sindbis virus E 2 and E 1 contain two possible g l y c o s y l a t i o n s i t e s each (39). These observations are consistent with the number of carbohydrate chains found in these proteins, except for Semliki Forest virus E3, which has two possible attachment sites for the single oligosaccharide unit that i t i s known to contain. The carbohydrates are found as polysaccharides of two classes. The simple chains (B-type) are 6 - 8 residues long and contain only mannose and N-acetyl glucosamine. The complex chains (A-type) average 1 6 - 2 0 residues i n length and contain galactose, fucose and s i a l i c acid, as well as mannose and N-acetyl glucosamine (23,87). Both Sindbis and S e m l i k i F o r e s t virus E 2 were shown to 7 contain one simple and one complex carbohydrate chain (56,60). Sindbis virus E1 has a s i m i l a r carbohydrate structure when grown in primary chicken cells but only contains complex chains when grown in BHK cells (57). The single carbohydrate chain of Semliki Forest virus E1 and E3 i s of the complex type (60). Like most animal viruses, alphavirus structural proteins appear to be glycosylated p r i n c i p a l l y , i f not entirely, by c e l l - s p e c i f i c enzymes (56,58,59). Recently, covalent f a t t y a c i d attachment to Sindbis virus glycoproteins was shown to be another post-translational modification (61). One or two palmitic acid molecules are attached to E1 whereas E2 contains 5 or 6 residues. Serine and threonine residues located in the transmembranous region of these glycoproteins are probable attachment sites (39). Cross-linking studies have shown that envelope glycoproteins might interact in some way with each other and perhaps form dimers E1-E2 (or trimers E1-E2-E3) (23,62,101). This generally accepted pairing was disputed by Scheefers e_t a l . (63), since they could isolate plasma membrane portions of Sindbis v i r u s - i n f e c t e d cells that only contained E1. 3. Replication of Alphaviruses 8 The early steps of an a l p h a v i r u s i n f e c t i o n , namely adsorption, penetration and uncoating w i l l be dealt with i n the next section. (a) Growth Cycle The a l p h a v i r u s e s grow w e l l over a wide range o f temperatures, between 20 and 41°C. T h e i r growth i s very r a p i d i n vertebrate c e l l s : i n f e c t i o u s v i r i o n s are r e l e a s e d i n t o the e x t r a c e l l u l a r medium as soon as 2-3 h p o s t - i n f e c t i o n . A f t e r a b r i e f e x p o n e n t i a l phase, v i r a l production continues at a more or l e s s constant rate of as much as 1000 P F U / c e l l / h up t o 10-12 h p o s t - i n f e c t i o n . T o t a l y i e l d may approach 10 10 PFU/ml of growth medium, e s p e c i a l l y i n primary c h i c k embryo c e l l s and BHK c e l l s (40). Host c e l l protein, RNA, and DNA syntheses are i n h i b i t e d w i t h i n 3-5 h p o s t - i n f e c t i o n by mechanisms s t i l l l a r g e l y unknown (7,23,75). By 10 h p o s t - i n f e c t i o n , c y t o p a t h i c e f f e c t s become apparent by l i g h t microscopy, concomitant w i t h a marked f a l l i n the rate of v i r u s production. In most vertebrate cultures, the i n f e c t i o n i s c y t o l y t i c : the c e l l s die within 10-20 h p o s t - i n f e c t i o n (7,40). However, ch r o n i c a l l y i n f e c t e d BHK and mouse c e l l c u l t u r e s have been described (40,65 ,66) . S e v e r a l mechanisms have been proposed t o e x p l a i n t h i s persistence: g e n e r a t i o n o f d e f e c t i v e i n t e r f e r i n g (DI) p a r t i c l e s and temperature-sensitive (ts.) mutants and the action of i n t e r f e r o n . In c o n t r a s t t o t h e i r u s u a l c y t o c i d a l e f f e c t on the v e r t e b r a t e c e l l s , alphaviruses r e a d i l y e s t a b l i s h a long-term persistence i n the 9 i n v e r t e b r a t e (e.g. mosquito) c e l l s such as Aedes a l b o p i c t u s cultures. Like vertebrate c e l l s , the mechanism by which persistence i s established in mosquito cells i s unknown (40). (b) Transcription and Replication After uncoating, the p o s i t i v e strand of 42 S v i r a l RNA becomes associated with polyribosomes (50) and i t s 5'-portion codes for the synthesis of at least one v i r u s - s p e c i f i c RNA-dependent RNA polymerase, which i s probably a complex of several so-called v i r a l non-structural proteins (67), although the possible contribution of host components i s unknown (7). Thereafter,-the 42 S RNA positive strand i s transcribed to a f u l l length strand of negative p o l a r i t y which is in turn replicated from i t s 3'-end to form a r e p l i c a of the o r i g i n a l positive strand of 42 S v i r a l RNA ( 2 3 ) . The latter i s either packaged into progeny virions or used as a template for synthesis of more non-structural proteins. A different i n i t i a t i o n site 2 / 3 of the way from the 3'-end of the minus strand i s used for the synthesis of 26 S RNA, which forms the major i n t r a c e l l u l a r v i r a l RNA and codes f o r v i r a l s t r u c t u r a l proteins ( 2 3 , 5 0 , 6 8 , 6 9,74). Double-stranded RNA i n t e r m e d i a t e s have been demonstrated in infected c e l l s . Replicative intermediates RIa and Rib are formed during synthesis of 42 S and 2 6 S RNA, respectively. Some authors have reported that a d i f f e r e n t polymerase was associated with each RI ( 2 3 ) . As i s the case in 42 S RNA, the 3 '-end of the 2 6 S RNA has a polyadenylic acid t r a c t of 6 0 - 8 0 residues and a capped 5'-end 10 (7,68). The maximum rate of synthesis of 42 S RNA negative strands i s reached at about 2.5 h post-infection and declines rapidly thereafter. The rate of synthesis of p o s i t i v e strands (42 S and 26 S) grows exponentially up to 3 h post-infection and then remains constant up to 6-7 h post-infection. The amount of 42 S and 26 S RNA molecules can reach values close to 200,000 per c e l l at 8 h post-infection, which significantly depletes the nucleotide pool size of the infected cells (7). (c) Synthesis of V i r a l Proteins i) Non-Structural Proteins Synthesis schemes of n o n - s t r u c t u r a l polypeptides have been suggested (7,70-72). It appears that Semliki Forest virus codes for 4 non-structural proteins: ns70, ns86, ns72 and ns60 (number indicates _3 M x 10 ), which are derived i n that order from the N-terminal of r ' a large polyprotein with a of 230-290 K. Sindbis virus has i t s counterparts i n ns60, ns89, ns82 and possibly ns60. The non-structural protein ns70 i s the major Semliki Forest v i r u s - s p e c i f i e d component associated with the r e p l i c a t i o n complex (73) and ns86 was also associated with p u r i f i e d preparations of RNA polymerizing activity (7). The functions of the other non-structural proteins remains to be 11 solved. The regulation of non-structural protein synthesis appears to be at the translational l e v e l since the maximum rate of synthesis i s reached at 3-4 h post-infection and declines continuously thereafter, v despite the increase in the amount of 42 S RNA. i i ) Structural Proteins The synthesis of alphavirus s t r u c t u r a l proteins also occurs via a polyprotein precursor with a Mr= 130,000 (ts 2 protein), which i s coded by the 26 S RNA. This precursor i s then processed by proteolytic cleavages, as shown in Figure 1. The ts 2 polypeptide i s immediately cleaved (probably while nascent since i t can only be is o l a t e d from cells infected with a virus mutant defective in the cleavage of t h i s protein (7)) into the capsid protein (C) and a polyprotein precursor to the envelope glycoproteins (B protein). This cleavage i s probably virus-specified (77) and i t has been attributed to an autoproteolytic a c t i v i t y of the capsid protein (78). Cleavage of the capsid protein apparently exposes a stretch of hydrophobic amino acids at the N-terminal of the B protein (signal sequence), which directs the binding of polysomes containing 26 S RNA to endoplasmic reticulum membranes. Thereafter, further protein synthesis leads to insertion of nascent proteins into these membranes and t h e i r sequestration i n the lumen of the endoplasmic reticulum (79,80). 12 26 S RNA M U t s 2  2 TWi  C 0 0 H B 34 K 100 K PE2 o r D62 EJ_ 62 K 50 K _E3 E_2_ 10 K 50 K 4.2 K or 6 K Figure 1. P o s t - t r a n s l a t i o n a l c l e a v a g e s during the formation of alpha-v i r u s s t r u c t u r a l proteins. The m o l e c u l a r weights i n d i c a t e d a r e f o r the mature p r o t e i n s (glycosylated i n the case o f E1 , E2 and E3). E3 i s not a s t r u c t u r a l protein of S i n d b i s v i r u s but i s r e l e a s e d i n t o the medium. The 4.2 K ( S i n d b i s v i r u s ) o r 6 K ( S e m l i k i F o r e s t v i r u s ) p r o t e i n s are not s t r u c t u r a l proteins but can be detected i n s i d e i n f e c t e d c e l l s . 13 Further cleavage of the B p r o t e i n i n t o PE2 (p62) and E1 i s apparently coupled to membrane in s e r t i o n (80). Unlike other signal sequences, the si g n a l peptide of the PE2 (p62) polypeptide i s not removed during translation (53,81). Translocation of the growing chain apparently continues u n t i l the carboxy-terminal transmembrane domain (cf. section 2c) stops transfer of the polypeptide (53,80). Renewed membrane insertion occurs when the signal peptide for E1 emerges from the ribosome. When most of the polyprotein has been translated, a presumably host-specific cleavage occurs to release PE2 (p62) and E1 (7) . The s i g n a l sequence of E1 has been suggested to be the v i r u s - s p e c i f i c 4.2 K (6 K) protein which i s detected i n the rough endoplasmic reticulum of i n f e c t e d c e l l s but i s not packaged into mature v i r i o n s (24,82). As with PE2 (p62), t r a n s l o c a t i o n of E1 continues un t i l the incorporation of the transmembranous hydrophobic domain i s completed (cf. section 2c). The cleavage of PE2 (p62) to E2 and E3 occurs later, probably at the plasma membrane (cf. section 3d). E3 i s packaged into mature Semliki Forest v i r i o n s but i s lost in the medium of Sindbis virus-infected c e l l s (23). (d) Maturation The nascent p o l y p e p t i d e c h a i n s o f PE2 (p62) and E1 are g l y c o s y l a t e d by t r a n s f e r o f p r e a s s e m b l e d m o i e t i e s from lipid-containing oligosaccharide intermediates, probably a dolichol oligosaccharide (7,86,87). As shown by the effects of the glycosylation 14 inhibitor tunioamycin, glycosylation i s not necessary for membrane ins e r t i o n and cleavage of these v i r a l p r o t e i n s ( 8 3 ) . However, conflicting reports have been published about the effect of impaired glycosylation on intracellular migration of v i r a l proteins to the c e l l surface (59 , 6 3,84,85). Some workers found non-glycosylated or incompletely glycosylated v i r a l envelope proteins on the c e l l surface (59 , 6 3 ) whereas other workers could not detect non-glycosylated proteins in the plasma membrane of infected cells (84,85). Completion of the oligosaccharides a f t e r t h e i r transfer en. bloc from a l i p i d donor occurs no sooner that 20 min af t e r the synthesis of the proteins and involves at le a s t the addition of fucose and s i a l i c acid to the complex chains (88). The i n t r a c e l l u l a r migration of the envelope glycoproteins from their s i t e of synthesis i n the rough endoplasmic reticulum to the plasma membrane presumably occurs through a series of fusion processes and clathrin-coated v e s i c l e s , as was shown by Rothman et a l . ( 8 9 ) for the G glycoprotein of vesicular stomatitis virus. On their way to the c e l l surface, the proteins become associated with the smooth endoplasmic reticulum and the Golgi apparatus (90). Glycosylation occurs along t h i s pathway, as well as fat t y acid attachment, which might provide these glycoproteins with a strong li p o p h i l i c anchor into the membrane (61,91)- On the other hand, capsid proteins probably diffuse through the cytoplasm (via microtubules?) and are very rapidly found associated with the plasma membrane (90). 15 The f i n a l s t e p o f the v i r a l m a t u r a t i o n i s a budding p r o c e s s . V i r a l envelope g l y c o p r o t e i n s i n s e r t e d i n t o the host plasma membrane displace host membrane p r o t e i n s , presumably by a s s o c i a t i n g with the v i r a l n u c l e o c a p s i d bound t o t h e c y t o p l a s m i c f a c e o f the plasma membrane. V e s i c l e s a r e formed which p i n c h out o f the c e l l surface to form mature progeny v i r i o n s ( 19,20,36). The cleavage of PE2 (p62) to E2 and E3 appears to be a p r e r e q u i s i t e f o r budding and occurs at the plasma membrane (63,90,92). The exact mechanism of budding i s unclear. Since energy metabolism i s not needed f or budding to occur (36), i t has been suggested that the b i n d i n g of the envelope glycoproteins to the nucleocapsid i s the major d r i v i n g f o r c e f o r the budding process and that l a t e r a l contacts between the g l y c o p r o t e i n s may also play a r o l e (36). I n c y t o c i d a l i n f e c t i o n o f Aedes a l b o p i c t u s c e l l s , t h e a l p h a v i r u s e s appear t o mature a t t h e i n t r a c e l l u l a r membranes or va c u o l e s , which f u s e w i t h t h e p l a s m a membrane t o r e l e a s e mature v i r i o n s (7). 4. Studies on the E n t r y of Animal Viruses I. Enveloped RNA V i r u s e s (a) Introduction 16 Two d i f f e r e n t mechanisms have been p r o p o s e d f o r e n t r y o f enveloped RNA viruses i n t o animal c e l l s , as shown i n Figure 2. Viruses could be i n t e r n a l i z e d by a receptor-mediated endocytic mechanism, a l s o termed v i r o p e x i s . I f t h i s were the case, the v i r u s p a r t i c l e s could e i t h e r escape from the e n d o c y t i c v e s i c l e or be uncoated a f t e r the fusion o f these v e s i c l e s w i t h primary lysosomes. The f u s i o n of the v i r u s envelope from w i t h i n the lysosomes was suggested r e c e n t l y by H e l e n i u s and co-workers f o r S e m l i k i F o r e s t v i r u s (29,31,32), as discussed l a t e r i n t h i s s e c t i o n . A l t e r n a t i v e l y , enveloped viruses may enter the host c e l l s by f u s i o n o f the v i r a l envelope w i t h the host plasma membrane. This pathway would l e a d to a d i r e c t i n j e c t i o n of the v i r a l n u c l e o c a p s i d i n t o the h o s t c y t o p l a s m . A few o t h e r p o s s i b l e pathways were a l s o s u g g e s t e d , s u c h as d i s s o l u t i o n o f the plasma membrane or d i r e c t p e n e t r a t i o n of the v i r u s through t h i s membrane. However, these mechanisms' were very r a r e l y proposed. The approach favored by most workers f o r the study of v i r a l entry has been electron microscopy. U n f o r t u n a t e l y , t h i s technique leads to many problems o f i n t e r p r e t a t i o n due to a r t e f a c t s . The most common problem a r i s e s from inadequate p r e s e r v a t i o n of the specimen, as noted by Dales (51) i n h i s review o f the e a r l y events i n c e l l - a n i m a l v i r u s i n t e r a c t i o n s . C u l t u r e d c e l l s a r e o f t e n f i x e d as a monolayer by gluteraldehyde and then s c r a p e d and p o s t - f i x e d with osmium te t r o x i d e . This sometimes r e s u l t s i n f u z z i n e s s i n the e l e c t r o n micrographs of membranes, whereby v i r u s p a r t i c l e s which appear t o be f r e e i n the 17 ENDOCYTOSIS Figure 2. Two proposed mechanisms of entry of enveloped RNA viruses i n t o animal c e l l s . 18 cytoplasm are a c t u a l l y i n endocytic v e s i c l e s (51). A second problem arises from the "thickness" of thin sections, which i s often equal or greater than the width of the virus under study. Thus, viruses may appear to have fused with the plasma membrane when they are in reality lying in an invagination at the c e l l surface (51). F i n a l l y , fragile virus envelopes can be damaged during purification or handling and give rise to erroneous observations i n the electron microscope, such as uncoating at the surface of the c e l l s or dissolution of the envelope at the site of contact with the host (51). Nevertheless, in the hands of experts, electron microscopy can be a useful tool in the study of v i r a l entry into animal c e l l s . However, one has to keep in mind that this technique does not d i f f e r e n t i a t e between an i n f e c t i o u s and a non-infectious virus particle. In the following sections, the present knowledge on the entry of animal viruses into c e l l s i s reviewed. A rapid summary of what i s known about virus attachment on the c e l l surface, an early event which precedes the penetration process i t s e l f , i s also provided for each family of enveloped RNA viruses. More information on virus receptors can be found in a review by Meager and Hughes (93) and proceedings of a sci e n t i f i c session headed by Choppin ( 9 4 ) . (b) Togaviruses Apparently, no work has been done on the entry of flaviviruses. 19 The only knowledge of the e a r l y events i n the i n f e c t i o n by these viruses i s that West Nile virus appeared to exhibit an unusually high divalent cation requirement for attachment to primary chick embryo cel l s (95). It i s therefore not s u r p r i s i n g that the local anesthetic procaine was shown to i n h i b i t adsorption of t h i s v i r u s onto BHK-21 cells (96), since t h i s drug apparently competes with calcium ions on the c e l l membrane (96). Alphaviruses have received more attention although l i t t l e work has been done on the entry of these viruses. Sindbis and Semliki Forest viruses are the only members of t h i s group to have been studied to some extent. The nature of the receptor for Sindbis virus on chicken cells was suggested to be chicken fetal antigens (CFA) since this virus attached to and agglutinated red blood c e l l s from fetal and developing chickens but not from adult chickens, which have l o s t CFA (97). Antiserum directed against erythrocyte CFA determinant 9 prevented attachment or hemagglutination. On the other hand, human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens were thought to be c e l l surface receptors for Semliki Forest virus (48). This finding was later questioned since cells lacking histocompatibility antigens were s t i l l susceptible to infection by this virus (49). Therefore, these antigens are unlikely to be major s p e c i f i c receptors needed by Semliki Forest virus to infect c e l l s . 20 A c h i c k e n embryo f i b r o b l a s t c e l l was shown t o c o n t a i n approximately 100,000 r e c e p t o r s f o r S i n d b i s v i r u s and these receptors c o u l d f o r m c l u s t e r s upon v i r u s b i n d i n g a t 4°C ( 9 8 ) . Re c e p t o r r e c y c l i n g was s u g g e s t e d i n c u l t u r e d n e u r o n a l c e l l s s i n c e they reappeared r a p i d l y on the c e l l s u r f a c e a f t e r p r o t e o l y t i c cleavage (99). F r i e s and H e l e n i u s (100) c a r e f u l l y q u a n t i t a t e d the binding of Semliki Forest v i r u s onto d i f f e r e n t mammalian c e l l s . Scatchard p l o t s indicated the presence of about 50,000 re c e p t o r s on each BHK-21 c e l l , although t h i s type of q u a n t i t a t i o n might not be appl i c a b l e to v i r u s e s . After a rapid i n i t i a l phase, the r a t e o f binding reached a plateau and further binding was n e g l i g i b l e a f t e r 2 h. I n t e r e s t i n g l y , these workers occasionally observed vacuoles c o n t a i n i n g v i r u s e s i n the cytoplasm of , o i n f e c t e d c e l l s a t 4 C, w h i c h s u g g e s t e d some p e n e t r a t i o n by endocytosis, despite the low temperature (100). S i m i l a r l y , Stephenson e_t al_. (28) showed some p e n e t r a t i o n of S e m l i k i F o r e s t v i r u s i n t o some preparations of chicken embryo f i b r o b l a s t s (about 50% of the rate o f p e n e t r a t i o n at 37°C), a l t h o u g h the mechanism o f e n t r y was not studied. Strangely, h y p e r t o n i c i t y ( t w i c e the i s o t o n i c concentration of 150 mM) was shown to i n h i b i t r e v e r s i b l y p e n e t r a t i o n of Sindbis v i r u s into BHK c e l l s , p o ssibly because o f unknown a l t e r a t i o n s of the plasma membrane ( 1 0 4 ) , a l t h o u g h a d i r e c t e f f e c t on t h e v i r u s was not eliminated. Regarding the actual mechanism of entry of alphaviruses, Levinthal et aX. (102) were the only workers to suggest a fusion of Sindbis virus with the plasma membrane. Their conclusion was based on the detection, by the immunofluorescence and immunoferritin techniques, of v i r a l antigens on the c e l l surface af t e r penetration. However, the absence of quantitative data or micrographs prevents a c r i t i c a l analysis of t h e i r r e s u l t s . On the other hand, Pathak and Webb (103) presented electron microscopic evidence for endocytosis of Semliki Forest virus from the vascular lumen to the endothelial c e l l s of mouse brain. Coated pits and vesicles were apparent in their micrographs. Fan and Sefton also suggested an endocytic mechanism for the entry of Sindbis virus into several types of mammalian ce l l s (105). They circumvented the p o t e n t i a l problems of electron microscopy by using complement-mediated anti-viral antibody l y s i s of c e l l s with v i r a l antigens incorporated on their surface, presumably after fusion of the v i r a l e n v e l o p e . L y s i s o f t h e c e l l s was d e t e r m i n e d by 5 1 antibody-dependent release of incorporated Cr (chromium release cytotoxic assay). Nearly complete l y s i s could be detected 5 h after infection by Sindbis v i r u s , a time when v i r a l maturation had already started, and 1 h a f t e r i n f e c t i o n with Sendai v i r u s , a paramyxovirus known to fuse with the host c e l l s ( c f . section 4e). However, the extent of l y s i s of Sindbis v i r u s - i n f e c t e d chick c e l l s was very low (about 20%) at 1 h post-infection, when the multiplicity of infection (PFU/cell) was less than 1000 and increased to more than 40% when the multiplicity was 5000. They concluded from t h e i r results that a very 22 large majority of the Sindbis v i r i o n s entered the ce l l s by some means other than membrane fusion, presumably endocytosis. However, their data do not allow the exclusion of fusion as an infectious mechanism of entry. Furthermore, s e v e r a l problems can be noted i n the i r experiments: (1) a r e l a t i v e l y high background of lys i s of uninfected cells (18%) or antibody-independent l y s i s of Sindbis virus-infected c e l l s (15$); (2) unknown p a r t i c l e / P F U r a t i o ; (3) p o s s i b i l i t y of disappearance of v i r a l antigens from the plasma membrane during the 1 h i n f e c t i o n period would l e a d to an underestimation of a fusion pathway. Therefore, these r e s u l t s do not conclusively identify the mechanism of entry of Sindbis v i r u s . Nevertheless, they indicate that both endocytosis and fusion may occur. In vitro experiments on the int e r a c t i o n of Sindbis virus with liposomes appeared to preclude the presence of membrane proteins for v i r a l attachment (106). Viruses could bind to liposomes prepared from a mixture of sheep erythrocyte phospholipids and showed a requirement for cholesterol and phosphatidylethanolamine. However, this binding might very well be non-specific, which was not determined in these experiments since penetration into the liposomes was not reported. The v a l i d i t y of t h i s c r i t i c i s m i s somewhat supported by the observed requirement for phosphatidylethanolamine, which i s hard to reconcile with the predominant localization of t h i s phospholipid on the internal surface of the erythrocyte membrane (107). 23 Recently, Coombs et. al.. (34) suggested that endocytosis i s not essential for a productive infection of BHK-21 cel l s by Sindbis virus, although they had no d i r e c t evidence f o r an a l t e r n a t e pathway (presumably fusion) . Their conclusion was based on the effect of two chemicals on the i n f e c t i o n . Cytochalasin B (10 ug/ml) completely blocked ingestion of virus p a r t i c l e s (as shown by electron microscopy) but had no effect on the a b i l i t y of the virus to infect or replicate (as shown by the y i e l d of infectious v i r u s e s ) . On the other hand, chloroquine (0.1 mM) did not i n h i b i t ingestion but greatly reduced the yields of virus produced. Previous work have shown that cytochalasin B i n h i b i t s microfilament formation and phagocytosis (34) whereas chloroquine presumably increases the intralysosomal pH (33). The cytochalasin B i n h i b i t i o n of phagocytosis was observed by Coombs and co-workers at a multiplicity of infection of 5000, which i s a very high and possibly non-physiological value (high multiplicities of infection are often needed for electron microscopic studies on v i r a l entry). Also, no mention i s made of the r e p r o d u c i b i l i t y of the results seen with the electron microscope. Moreover, possible toxic effects of the 13 h exposure of the c e l l s to chloroquine would explain the decreased virus yields. Finally, the correlation made by the authors between the absence of effect of cytochalasin B on the production of infectious virus p a r t i c l e s a f t e r i n f e c t i o n at 50 PFU/cell and the block of ingestion of viruses at 5000 PFU/cell might not be valid. With these reservations i n mind, t h e i r r e s u l t s provide some doubt on the generally postulated endocytosis pathway of infection by alphaviruses, 24 although more d i r e c t evidence f o r an alternate pathway i s needed. Re c e n t l y , H e l e n i u s and c o - w o r k e r s (27,29,31,32) pre s e n t e d morphological and b i o c h e m i c a l s t u d i e s on the e n t r y of Semliki Forest v i r u s into BHK-21 c e l l s . T h e i r r e s u l t s are so f a r c o n s i s t e n t with a receptor-mediated ( a d s o r p t i v e ) e n d o c y t o s i s p r o c e s s f o l l o w e d by an incorporation o f the v i r a l p a r t i c l e s i n t o lysosomes. They suggested that the low pH i n these c e l l u l a r o r g a n e l l e s a p p a r e n t l y t r i g g e r s a fusion of the v i r a l envelope from w i t h i n the lysosomes, which leads to i n j e c t i o n of the nucleocapsid i n t o the cytoplasm. However, evidence f o r t h i s f u s i o n i s i n d i r e c t : f u s i o n c o u l d o c c u r between v i r u s e s and liposomes (29,32) or v i r u s e s and c u l t u r e d c e l l s (31) only when the pH was below 6. E f f i c i e n t t r a n s f e r o f the v i r a l nucleocapsid occured i n both cases. In a d d i t i o n to low pH, the liposomes experiments showed that fusion r e q u i r e d the presence o f i n t a c t v i r a l g l y c o p r o t e i n s and l i p o s o m a l c h o l e s t e r o l but d i v a l e n t c a t i o n s were not e s s e n t i a l . Furthermore, both types of fusion occured over a wide temperature range (0-40°C) and were very r a p i d (about 5 sec). I n t e r e s t i n g l y , i t was r e c e n t l y shown (108) t h a t S e m l i k i Forest v i r u s and R u b e l l a v i r u s e s c o u l d i n d u c e f u s i o n and hemolysis o f erythrocytes at pH 5.8 but o n l y at temperatures between 37 and 42°C. However, the low pH o n l y appeared to enable attachment of the v i r u s , a f t e r which the pH c o u l d be r a i s e d to n e u t r a l i t y without i n h i b i t i n g t h e h e m o l y s i s . T h i s r e s u l t a p p e a r s t o c o n t r a d i c t t h e low pH 25 requirement for fusion shown by Helenius and co-workers but might be explained by the very rapid fusion which appeared to occur at pH lower than 6 (29,32). Evidence for the endocytosis pathway i t s e l f i s mainly based on electron microscopy and the e f f e c t of drugs that are thought to increase the intralysosomal pH (29) (lysosomotropic agents; c f . s e c t i o n 5 ) . 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 were made at high m u l t i p l i c i t y of i n f e c t i o n and showed i n t e r n a l i z a t i o n of virus particles at coated pits into coated v e s i c l e s which usually contained one particle each. After 1 min or longer at 37°C, viruses could be seen i n larger vacuoles which were not coated. After about 5 min, other types of vacuoles became prominent. Some of them had a lysosomal appearance and could be stained for acid phosphatase. In order to determine i f t h i s lysosomal pathway was the in f e c t i o u s mechanism of entry, f i v e lysosomotropic agents were i n turn added to uninfected cells and the yield of in f e c t i o u s v i r i o n s determined 5.5 h after the infection, which was i n i t i a t e d 30 min afte r addition of the drug. A l l five drugs s i g n i f i c a n t l y reduced the virus t i t e r , although possible side e f f e c t s on the h e a l t h of the c e l l s were not eliminated. Nevertheless, chloroquine apparently did not inhibit virus binding or uptake. Further s t u d i e s with S e m l i k i F o r e s t v i r u s l a b e l e d with 35 L_-[ S]methionine provided more quantitative values on the entry 26 process (27). Uptake was very rapid ( h a l f - l i f e 10-35 min) and studies with various drugs showed that i t was independent of cytoskeletal and lysosomal f u n c t i o n , although p a r t i a l l y dependent on oxidative phosphorylation. Strangely, 10 ug/ml cytochalasin B only inhibited 20% of the endocytosis and 60% of the y i e l d of virus whereas Coombs e_t a l . (34) claimed that i t completely blocked endocytosis and had no effect on the yield of Sindbis v i r u s , which supported their conclusion that endocytosis was not the i n f e c t i o u s mechanism of entry. This discrepancy demonstrates the need for caution in the interpretation of results obtained with drugs. As high as 1500-3000 viruses were found by Helenius and co-workers (27) to be internalized per min per c e l l , although the m u l t i p l i c i t y of i n f e c t i o n did not a f f e c t the rate of o internalization. Temperatures lower than 10 C blocked about 97% of the uptake, which contradicts the r e s u l t s obtained by Stephenson et a l . (28), who showed only 50% inhibition. In conclusion, most workers seem to agree that endocytosis i s probably the main mechanism of entry of alphaviruses into animal c e l l s . However, the p o s s i b i l i t y that fusion of a small portion of the viruses would constitute the actual infectious pathway or that both fusion and endocytosis would lead to a productive infection has not been entirely eliminated. (c) Rhabdoviruses 27 These e n v e l o p e d b u l l e t - s h a p e d p a r t i c l e s c o n t a i n a h e l i c a l nucleocapsid w i t h a n e g a t i v e s i n g l e - s t r a n d e d RNA and at l e a s t three proteins: N,L and NS. A v i r i o n - a s s o c i a t e d t r a n s c r i p t a s e a c t i v i t y i s associated with the r i b o n u c l e o p r o t e i n c o r e . Their external spikes are polymers of the g l y c o p r o t e i n G and the mat r i x M proteins form a s h e l l underneath the l i p i d b i l a y e r (52). V e s i c u l a r s t o m a t i t i s v i r u s i s the most s t u d i e d member o f t h i s f a m i l y , a l t h o u g h r a b i e s v i r u s a l s o received some a t t e n t i o n . The envelope g l y c o p r o t e i n G o f v e s i c u l a r s t o m a t i t i s v i r u s i s probably responsible f o r binding o f t h i s v i r u s to host c e l l s since i t s s e l e c t i v e r e m o v a l by p r o t e a s e s r e s u l t s i n l o s s o f i n f e c t i v i t y (109,110). V i r i o n c h o l e s t e r o l may a l s o play a r o l e since i t s depletion reduces i n f e c t i v i t y (112). Schloemer and Wagner (110) suggested a requirement f o r the s i a l i c a c i d p a r t o f the G p r o t e i n i n attachment since exposure of v i r i o n s to neuraminidase r e s u l t e d i n l o s s of t h e i r a b i l i t y to a g g l u t i n a t e goose e r y t h r o c y t e s and to a t t a c h to L c e l l s . T h i s l o s s was a l m o s t c o m p l e t e l y r e v e r s e d by s i a l y l t r a n s f e r a s e r e s i a l y l a t i o n of neuraminidase-treated v i r i o n s . Strangely, Thimmig e_t a l . (111) l a t e r found t h a t treatment o f the i s o l a t e d G p r o t e i n and i n t a c t v i r i o n s with neuraminidase d i d not s i g n i f i c a n t l y decrease t h e i r binding to BHK-21 ce l l s , a l t h o u g h neuraminidase treatment res u l t e d i n a 50 to 100-fold decrease of the i n f e c t i v i t y . Both groups o f workers found t h a t the c e l l u l a r r e c e p t o r f o r v e s i c u l a r s t o m a t i t i s v i r u s r e s i s t e d treatment w i t h t r y p s i n (110,111) and one group a l s o found 28 r e s i s t a n c e to neuraminidase (110). These r e s u l t s suggest an asialoglycolipid as c e l l u l a r receptor. Obviously, more work i s needed on the receptor binding of these viruses. Electron microscopic evidence i n i t i a l l y suggested endocytosis (viropexis) of vesicular stomatitis virus by L ce l l s in coated regions of the membrane (113). After a 1-1.5 h adsorption at 4°C, 80$ of a l l the inoculum p a r t i c l e s were engulfed within 15 min at 37°C, with no evidence of any fusion at the c e l l surface. On the contrary, Heine and Schnaitman (114) presented electron microscopic evidence for fusion of vesisular stomatitis virus envelope with the plasma membrane of L c e l l s . The c e l l s were i n f e c t e d at 4°C by sedimenting v i r u s - c e l l mixtures onto a f l a t agar surface. Fusion was observed as early as after 2 min incubation at 37°C. However, some vir u s particles could be observed in phagocytic vacuoles and some were even fused with the vacuole membrane (114), i n contrast with the process of fusion from within the lysosomes suggested by Helenius and co-workers for Semliki Forest virus (cf. section 4 I b). More evidence for fusion was obtained by the same investigators (115). They showed the presence of v i r a l antigens on the surface of infected c e l l s and a preparation of plasma membrane i s o l a t e d from phagocytosed latex beads was found to contain primarily the envelope proteins of the v i r u s . On the other hand, v i r a l nucleoproteins were released into the cytoplasmic f r a c t i o n of c e l l s broken by the gentle technique of nitrogen c a v i t a t i o n , which r e s u l t e d i n minimal lysosomal b r e a k a g e ( 1 1 5 ) . However, i n f e c t i o n was a l s o a c h i e v e d a f t e r sedimentation and the p o s s i b i l i t y o f n o n - s p e c i f i c binding of envelope proteins to the c e l l membrane a f t e r d e g r a d a t i o n of v i r i o n s could not be eliminated. In view o f t h i s c o n t r o v e r s y , D a h l b e r g (116) q u a n t i t a t i v e l y analyzed the p e n e t r a t i o n o f v e s i c u l a r s t o m a t i t i s v i r u s i n t o L c e l l s w i t h two d i f f e r e n t t e c h n i q u e s o f i n f e c t i o n . As shown by e l e c t r o n microscopy, when penetration was a n a l y z e d following adsorption i n the c o l d , v i r u s e s e n t e r e d c e l l s a l m o s t e x c l u s i v e l y by e n d o c y t o s i s (v i r o p e x i s ) , as r e p o r t e d by Simpson et. al_. (113). However, when the sedimentation i n o c u l a t i o n procedure o f Heine and Schnaitman (114,115) was used, fusion occured at a s i g n i f i c a n t l e v e l , although endocytosis was also observed. Therefore, the c e n t r i f u g a t i o n i t s e l f played a r o l e i n the incidence of f u s i o n , as was suspected by Dales (51). F i n a l l y , Fan and Sefton (105) recently suggested an endocytic mechanism f o r the e n t r y o f v e s i c u l a r s t o m a t i t i s v i r u s , as w e l l as S i n d b i s v i r u s , although t h e i r t e c h n i q u e o f complement-mediated a n t i - v i r a l antibody l y s i s o f c e l l s i n f e c t e d by f u s i o n showed i n h e r e n t problems ( c f . s e c t i o n 4 I b ) . In t h i s c a s e , e n d o c y t o s i s was o n l y i n d i r e c t l y suggested from the low l e v e l of l y s i s . The entry of r a b i e s v i r u s i n t o BHK-21 c e l l s was also studied by electron microscopy. Endocytosis appeared to be the preferred mechanism 30 of entry (117), a l t h o u g h f u s i o n c o u l d a l s o be observed, both at the c e l l s u r f a c e and w i t h i n p h a g o c y t i c v e s i c l e s , as e a r l y as 5 min pos t - i n f e c t i o n (118). An i n t e r e s t i n g study by M i l l e r and Lenard (119) recently showed that u l t r a v i o l e t l i g h t - i n a c t i v a t e d v e s i c u l a r s t o m a t i t i s v i r u s with a native G protein could i n h i b i t i n f e c t i o n by untreated v i r u s and that the s i t e o f i n h i b i t i o n was i n t r a c e l l u l a r and most l i k e l y the lysosomes. Furthermore, the lysosomal i n h i b i t o r chloroquine completely i n h i b i t e d i n f e c t i o n at a l l m u l t i p l i c i t i e s of i n f e c t i o n tested, when added with the v i r u s at a c o n c e n t r a t i o n of 100 uM. This suggested that a t a l l m u l t i p l i c i t i e s , t h e v i r u s e s go t h r o u g h t h e same chloroquine-sensitive s t e p , which cannot be bypassed. I n t e r e s t i n g l y , i r r a d i a t e d v e s i c u l a r s t o m a t i t i s v i r u s a l s o i n h i b i t e d i n f e c t i o n by Sindbis and Semliki Forest v i r u s e s , which suggests that the two types of v i r u s e s may share a common lysosomal pathway i n the i n f e c t i o u s process (119). These authors a l s o suggested from p r e v i o u s evidence t h a t a p a r t i c l e / P F U r a t i o g r e a t e r t h a n 1 i s not due to i n h e r e n t differences w i t h i n the p a r t i c l e s themselves but r a t h e r , at l e a s t i n part, to the i n e f f i c i e n c y of the c e l l u l a r uncoating process. The c e l l s apparently do not show any p r e f e r e n t i a l s e l e c t i o n of v i r i o n s that lead to a productive i n f e c t i o n (119). In summary, i t appears t h a t r h a b d o v i r u s e s e n t e r c e l l s mainly by endocytosis, although f u s i o n can sometimes be observed. The nature of 31 the i n f e c t i o u s mechanism of entry i s therefore unclear, (d) Retroviruses Retroviruses, also named oncornaviruses or leukoviruses, comprise four d i f f e r e n t subgenera. Subgenus A c o n s i s t s o f a v i a n leukosis and murine f e l i n e leukemia/sarcoma v i r u s e s w h i l e mouse mammary tumor v i r u s e s c o n s t i t u t e s u b g e n u s B. S u b g e n e r a C and D c o m p r i s e , res p e c t i v e l y , visna of sheep and the foamy agents of cats, c a t t l e and primates. The f e a t u r e s common to a l l these v i r u s e s are s t r u c t u r a l s i m i l a r i t y and the possession o f a v i r i o n - a s s o c i a t e d RNA-dependent DNA polymerase (reverse t r a n s c r i p t a s e ) (52). Retroviruses of the subgenus A, and to a l e s s e r extent B, are the most studied. Few s t u d i e s were done on the e a r l y events i n the i n f e c t i o n by 1 25 r e t r o v i r u s e s . P u r i f i e d and I - l a b e l e d envelope glycoprotein gp70 of Rauscher murine leukemia v i r u s was used to study c e l l u l a r receptors f o r the v i r u s . DeLarco and Todaro (120) found t h a t the p u r i f i e d gp70 s p e c i f i c a l l y bound to murine but not other mammalian c e l l s i n c u l t u r e . Calcium ions enhanced the binding and the presence of 530,000 receptors per NIH/3T3 mouse f i b r o b l a s t was c a l c u l a t e d . Kalyanaraman et a l . (121) studied b i n d i n g o f gp70 to a plasma membrane preparation from KA31 mouse c e l l s . Pretreatment o f the membrane f r a c t i o n s with e i t h e r chymotrypsin or p h o s p h o l i p a s e C l e a d t o a l o s s o f the r e c e p t o r a c t i v i t y , which suggests that a l i p o p r o t e i n structure i s important f o r 32 the r e c e p t o r f u n c t i o n . F i n a l l y , Robinson et. al_. (122) used water-soluble multimeric complexes of Friend murine leukemia virus envelope glycoprotein gp85 (major envelope glycoprotein composed of 2 polypeptide chains j o i n e d by one or more d i s u l f i d e bonds: the hydrophilic gp70 and the hydrophobic p15E, which i s thought to anchor the glycoprotein to the v i r a l membrane). These complexes were used to isolate by an immunoprecipitation technique the C57BL/6 mouse spleen 1 25 leukocyte r e c e p t o r s ( p r e l a b e l e d with I) f o r the v i r u s . The putative receptor was detected by SDS- g e l electrophoresis and autoradiography and had a of 14,000 (122). Interestingly, only 15,000 gp85 complexes were bound per leukocyte (122), as opposed to 530,000 gp70 isolated molecules per mouse c e l l (120), which emphasizes the d i f f i c u l t y in extrapolating the number of receptors found by these techniques to the actual number of receptors for the virus particles. Studies on the entry of retroviruses were mainly done by electron microscopy and lead to c o n f l i c t i n g r e s u l t s . Sarkar et. al. (123) i n i t i a l l y observed endocytosis (viropexis) of mouse mammary tumor virus i n t o mouse embryo c e l l s , although some f u s i o n was also suggested. On the other hand, Dales and Hanafusa (125) observed exclusively endocytosis of avian sarcoma and leukosis viruses into c h i c k embryo f i b r o b l a s t s , f r e q u e n t l y at coated p i t s s i t e s . Intracellular vacuoles, some of them coated, were rapidly detected in the v i c i n i t y of the nucleus, where transfer of the genomes probably occurs. In contradiction with previous evidence, including other types 33 of viruses, Miyamoto and Gilden (124) provided electron microscopic evidence for the entry of Rauscher murine leukemia virus into mouse embryo fibroblasts by mechanisms d i f f e r e n t from endocytosis or fusion. Penetration could apparently occur e i t h e r d i r e c t l y by the v i r u s p a r t i c l e through a break in the c e l l membrane or by simultaneous dissolution of both the v i r a l envelope and the c e l l membranes. Enzymes located in the v i r a l envelope and the c e l l membrane were suggested to be responsible for these dissolutions and would only be activated upon virus binding. Although these processes cannot be ruled out, they are very seldom, i f ever, detected by other workers. Dermott and Samuels (126) obtained electron micrographs which showed that simian foamy v i r u s could enter HEp-2 c e l l s by both endocytosis and fusion, although fusion was most frequent at earlier times post-infection. F i n a l l y , an i n t e r e s t i n g approach was used by Aboud et a l . (127) to study entry of Moloney murine leukemia v i r u s into NIH/3T3 mouse f i b r o b l a s t s . I n t r a c e l l u l a r virus was determined by assaying virus-specific reverse transcriptase a c t i v i t y in the high-speed pellet of the cytoplasmic f r a c t i o n of disrupted infected c e l l s . No activity was detected a f t e r i n f e c t i o n at 4°C, which indicated the absence of penetration. However, viruses rapidly appeared i n the cytoplasm at 37°C. They reached a maximal l e v e l within 20 min and uncoating was completed w i t h i n 80 min, as shown by disappearance of reverse 34 transcriptase activity in the high-speed pellet and i t s recovery as a soluble enzyme. Most of the v i r u s p a r t i c l e s recovered i n the high-speed p e l l e t prepared 45 min post-infection co-migrated i n a sucrose gradient with purified virus marker, although a small fraction of t h i s p e l l e t migrated i n a pattern t y p i c a l of v i r a l cores. Even though these cores might have been produced by i n t r a c e l l u l a r uncoating, they could also have been injected into the cel l s after fusion at the c e l l surface. Therefore, penetration of the whole virus p a r t i c l e appears to be the main mechanism of entry, although the possibility of fusion could not be eliminated. In summary, the mechanism of entry of retroviruses i s s t i l l unclear. E n d o c y t o s i s i s o f t e n observed but f u s i o n and d i r e c t penetration are sometimes suggested. More studies are needed to determine the nature of the infectious mechanism of entry. (e) Paramyxoviruses Most studies on paramyxoviruses have been made on three model viruses: Newcastle disease virus (NDV), simian v i r u s 5 (SV 5) and parainfluenza 1, s t r a i n Sendai (also c a l l e d Sendai v i r u s ) . Measles virus i s a possible member of t h i s family of viruses, which contain a helical nucleocapsid with a single-stranded negative strand of RNA and a v i r i o n - a s s o c i a t e d t r a n s c r i p t a s e (52). Two gly c o p r o t e i n s are associated with their envelope and form spike-like projections on the 35 virus surface. The larger glycoprotein (HN) possesses receptor binding activity (usually referred to as hemagglutinating activity because of the use of the erythrocyte as a model c e l l ) and neuraminidase activity (128,132). The second glycoprotein (F) i s necessary for infection and paramyxovirus-induced c e l l fusion and hemolysis (132), the l a t t e r involving fusion of the v i r a l and e r y t h r o c y t e membranes (133), although hemolysis i s not an automatic consequence of t h i s fusion (134). Two d i s u l f i d e - l i n k e d p o l y p e p t i d e s ( F 1 and F 2 ) can be demonstrated i n the F protein and only F^ i s anchored i n the v i r a l envelope (128). As shown for Sendai virus, this F protein i s formed by cleavage of a p r e c u r s o r p r o t e i n ( F Q ) which can take place i n  vivo with a protease a s s o c i a t e d with s u s c e p t i b l e c e l l s or in  vitro with trypsin, a process which activates v i r a l i n f e c t i v i t y and virus-induced c e l l fusion and hemolysis (128-131). These observations lead to the hypothesis that the mechanism of p e n e t r a t i o n of paramyxoviruses i n v o l v e s f u s i o n of the v i r a l envelope and c e l l membranes (adsorption of v i r i o n s that c o n t a i n the uncleaved F Q protein to receptors on the c e l l membrane occurs normally because adsorption i s mediated by the HN protein). Studies by Haywood with model membranes showed that gangliosides might act as receptors for the binding of Sendai virus (135), although the possibility of non-specific binding was not eliminated. Holmgren et a l . (138) extended these r e s u l t s by showing s p e c i f i c binding of Sendai virus to gangliosides adsorbed on p l a s t i c Petri dishes. Three 36 gangliosides which shared a common end sequence i n the oligosaccharide moiety (NeuAcoc 2 ,8NeuAcoC 2,3Ga^1 ,3GalNAc) were shown to bind the v i r u s . By v a r y i n g the c o m p o s i t i o n o f l i p o s o m e s , Haywood could demonstrate either a fusion (136) or an endocytic (137) mechanism for penetration of Sendai virus i n t o these model membranes. The absence of phosphatidylethanolamine favored endocytosis (137). The physiological s i g n i f i c a n c e o f t h e s e f i n d i n g s i s u n c l e a r . The importance of s i a l y l o l i g o s a c c h a r i d e determinants of defined sequence as receptors fo r Sendai v i r u s was a l s o suggested by other workers. Paulson et. a l . (139) showed that attachment w i t h s p e c i f i c s i a l y l t r a n s f e r a s e s of s i a l i c acid to neuraminidase-treated human erythrocytes could restore the o r i g i n a l hemagglutination l e v e l s abolished by removal of the s i a l i c a c i d r e s i d u e s , i f the added sequence (found m a i n l y on g l y c o p h o r i n , the major e r y t h r o c y t e g l y c o p r o t e i n ) was NeuAcoc 2,3Galp1 ,3GalNAc1 ,0-Thr/Ser . M a r k w e l l and P a u l s o n (140) l a t e r confirmed these r e s u l t s on Madin-Darby bovine kidney c e l l s . F i n a l l y , Yamamoto and Inoue (141) showed t h a t t r e a t m e n t of e r y t h r o c y t e s normally resistant to paramyxovirus-induced hemagglutination with the l e c t i n concanavalin A rendered them su s c e p t i b l e to the virus effects. However, b i n d i n g o c c u r e d by an u n c l e a r mechanism, which was independent of the i n t e r a c t i o n between the v i r a l hemagglutinin (HN) and receptors containing s i a l i c acid (141). Paramyxovirus-induced c e l l - c e l l f u s i o n i s a well-known process which i s probably i n i t i a t e d by f u s i o n of the v i r u s w i t h the c e l l 37 membrane. For example, Kohn (142) showed that adsorption of ultraviolet light-inactivated Sendai virus onto NIL 8 hamster ce l l s caused fusion of the c e l l s into polykaryocytes w i t h i n 2 h and that s t r u c t u r a l components of the v i r a l envelope, namely HN and F, were detected in the c e l l membrane up to 4 h p o s t - i n f e c t i o n . However, Miyake et a l . (143) demonstrated through the use of cytochalasin D that, unlike virus-cell fusion, c e l l - c e l l fusion required i n t a c t microfilaments. Therefore, a change in the c e l l u l a r cytoskeleton induced by fusion of the virus on the c e l l surface may be e s s e n t i a l for c e l l - c e l l fusion (143). Although fusion i s now recognized as the main mechanism of entry of paramyxoviruses, e a r l i e r r e p o r t s claimed the importance of endocytosis, mainly from electron microscopic evidence. For example, HeLa c e l l s were found to engulf Newcastle disease v i r u s , which appeared to be uncoated in the i n t r a c e l l u l a r vacuole, although release of the nucleocapsid could not be detected (144). Similar results were obtained with Simian virus 5 i n f e c t i o n of rhesus monkey ce l l s or baby hamster kidney c e l l s (145). In either case, fusion was never observed. However, other workers provided electron microscopic evidence for both fusion and endocytosis. For instance, Newcastle disease virus entry into epithelial c e l l s was shown to occur by either mechanism (146). Sendai virus appeared to enter mouse L c e l l s mainly by fusion, although some endocytosis also occured (147). 38 Strangely, more recent evidence ubiquitously suggested fusion of paramyxoviruses with t h e i r host c e l l s . Experiments by Fan and Sefton (105) on complement-mediated antibody l y s i s of c e l l s infected by Sendai virus suggested that fusion was the main mechanism of entry, although problems plagued this technique (cf. section 4 I b). Caldwell and Lyles (148) could demonstrate the incorporation of Sendai virus proteins into the erythrocyte membrane. As expected from a fusion mechanism, v i r a l envelope glycoproteins HN and F were incorporated as integral membrane proteins of the infected red c e l l (148). Lyles and Landsberger (149) could also follow the k i n e t i c s of this virus-cell fusion by electron spin resonance. The v i r a l envelope was labeled with nitroxide d e r i v a t i v e s of p h o s p h a t i d y l c h o l i n e and phosphatidyl-ethanolamine. A f t e r f u s i o n , the v i r a l l i p i d s d i f f u s e d into the c e l l u l a r membrane and could be followed by t h e i r s p i n - l a b l e l . The fusion process was shown to be a f i r s t - o r d e r reaction with a half-time of 7 min at 37°C (149). Indirect evidence for fusion also comes from the demonstration of increased p e r m e a b i l i t y of the host c e l l membrane that occurs concomitantly and i s e s s e n t i a l f o r v i r a l i n f e c t i o n (150-153). Reduction of the v i r a l envelope f l u i d i t y was also shown to inhibit infection (154). In summary, most workers now seem to agree that fusion i s the main mechanism for the entry of paramyxoviruses into susceptible cells and 39 that the F protein has a very important role to play in this process. Recently, Richardson et_ a_l. (155) showed that oligopeptides which structurally resembled the N-terminus region of the F^ polypeptide inhibited i n f e c t i o n by Measles, Simian virus 5 and Sendai viruses, either by d i r e c t i n t e r a c t i o n with the viru s e s or more l i k e l y by competing with v i r a l F proteins for binding sites on the c e l l surface (128,155). (f) Orthomyxoviruses Influenza virus i s the main representative of t h i s family of v i r u s e s which c o n t a i n a h e l i c a l n u c l e o c a p s i d with segmented single-stranded negative strands of RNA and a virion-associated transcriptase (52). Like the paramyxoviruses, the orthomyxoviruses (also called myxoviruses) possess two glycoproteins which are present on the v i r i o n as s p i k e - l i k e projections which are anchored i n the l i p i d bilayer by hydrophobic residues (128). These glycoproteins are the hemagglutinin (HA), which forms trimers and possesses the receptor binding a c t i v i t y (hemagglutination of red blood c e l l s ) and the neuraminidase (NA), which forms tetramers and i s responsible for the neuraminidase activity (thought to f a c i l i t a t e the spread of the virus) (128). The HA protein of orthomyxoviruses shows analogies to the F protein of paramyxoviruses. The HA protein of influenza virus was shown to be synthesized as a s i n g l e polypeptide chain which was cleaved by a cellular protease to y i e l d two disulfide-bonded subunits, 40 HA^ and HA^ (156). T h i s c l e a v a g e was shown to a c t i v a t e the infectivity of the virus ( 157,158). However, the precise mechanism of this activation i s not as clear with influenza v i r u s as i t i s with paramyxoviruses ( c f . s e c t i o n 4 I e ) . I t i n v o l v e s a step i n the ini t i a t i o n of infection beyond adsorption and before transcription of the v i r a l genome. Thus, penetration was suggested to be the lik e l y step (128). Furthermore, the N-terminal sequences of the influenza HA^ and the paramyxoviruses F ^  are very s i m i l a r and Richardson et a l . also showed a s e q u e n c e - s p e c i f i c i n h i b i t i o n of influenza virus replication by oligopeptides which resembled the N-terminal of the HA2 protein (128,155). However, f u s i o n cannot be as e a s i l y demonstrated for orthomyxoviruses and they only induce hemolysis and fusion of cells at acidic pH (159). Cell receptors for influenza viruses probably contain s i a l i c acid since neuraminidase treatment of s u s c e p t i b l e c e l l s abolished i n f e c t i v i t y , which could be r e s t o r e d by s p e c i f i c s i a l y l a t i o n , as described for Sendai virus (cf. section 4 I e and reference 139). Furthermore, glycophorin, the major sialoglycopeptide of erythrocytes, was implied as a receptor for influenza virus on human red blood cells (160). Unlike paramyxoviruses, only limited electron microscopic evidence suggests fusion of orthomyxoviruses with host c e l l s . Blaskovic et a l . (161) obtained electron micrographs which suggested fusion of 41 influenza virus with c e l l s of chick embryo tracheal organ cultures. Morgan and Rose (162) also provided evidence for fusion of influenza virus with the c h o r i o a l l a n t o i c membrane of chick embryos, with concomitant penetration of the nucleocapsid. They also observed some endocytosis, although they claimed that t h i s process was not infectious since i t mainly occured af t e r 10 min post-infection, at which time the frequency of f u s i o n s t a r t e d to decrease, and the viruses did not seem to cross the vacuole membrane, i . e . to be uncoated (162). F i n a l l y , Huang e_t aJL. (163) recently showed by electron microscopy that liposomes containing the influenza virus HA could fuse with c e l l membranes only i f HA was proteolytically cleaved. Furthermore, native virus p a r t i c l e s with the cleaved HA could be shown to fuse with liposomes which contained c e l l u l a r receptors for influenza virus (soluble components of o c t y l g l u c o s i d e - s o l u b i l i z e d chick f i b r o b l a s t s ; ) (163). However, Koszinowski e_t al.. (164) reported that reconstituted influenza envelopes containing the cleaved HA could fuse only i n the presence of Sendai v i r u s . These liposomes were prepared by a d i f f e r e n t procedure. Therefore, the physiological relevance of fusion of influenza virus remains to be determined. On the other hand, many workers provided evidence (mainly from electron microscopy) for endocytosis of influenza and, i n one case (165), fowl plague viruses. De St.Groth (166) f i r s t suggested the term viropexis for the engulfment of i n f l u e n z a virus by c e l l s of the allantoic cavity of the chick embryo. Dourmashkin and T y r r e l l (167) 42 also observed endocytosis ( v i r o p e x i s ) and questioned e a r l i e r observations of fusion by showing that some micrographs which appeared to indicate f u s i o n were the r e s u l t of t a n g e n t i a l sectioning. Appropriate t i l t i n g of the section showed that the virus was in fact superimposed onto the c e l l membrane i n t h i n section (167). This observation emphasizes the p o s s i b i l i t y of artefactual observation with the electron microscope (cf. section 4 I a). Further evidence for endocytosis was provided by Bossart e_t a l . (168) who showed engulfment of influenza virus by human and fowl erythrocytes. Dales and Pons (169) provided evidence that endocytosis of influenza v i r u s by primary c h i c k embryo f i b r o b l a s t s was the infectious mechanism. Individual active virus particles trapped within aggregates of ultraviolet light-inactivated particles were infectious. Because of the low pro b a b i l i t y for an i n t e r a c t i o n of envelopes of an active virus within an aggregate and the c e l l membrane, i t was concluded that the productive i n f e c t i o n probably involves endocytosis (viropexis) rather than fusion, as shown by electron microscopy. Electron microscopic evidence of endocytosis (viropexis) i n the presence of i n h i b i t o r s of g l y c o l y s i s , oxidative phosphorylation, protein synthesis, membrane Na + and K + transport and microtubule and microfilament function (170) suggested that viropexis was a process independent of c e l l u l a r energy and of an intact cytoskeletal system. This result i s in contrast with the generally accepted absence of virus i n t e r n a l i z a t i o n i n the cold, although one group of workers 43 d i d show p e n e t r a t i o n of i n f l u e n z a v i r u s at 4°C (28). V i r a l penetration i n the presence of agents that disrupt the c e l l u l a r c y t o s k e l e t a l system i s a l s o i n disagreement with the complete inhibition by cytochalasin B of Sindbis virus internalization shown by Coombs et a l . ( c f . section 4 I b and reference 34). Furthermore, the absence of dependence of i n f l u e n z a v i r u s entry on oxidative phosphorylation contrasts with the p a r t i a l dependence shown for Semliki forest v i r u s ( c f . s e c t i o n 4 I b and reference 27). These d i s c r e p a n c i e s a g a i n emphasize the need f o r c a u t i o n i n the interpretation of results obtained with drugs (cf. section 4 I b). Finally, Oxford et. aJL. (171) recently showed that a r t i f i c i a l l y reconstructed HA-lipid spheres ("virosomes") could penetrate Vero c e l l s by endocytosis, with no evidence of fusion, although t h i s a r t i f i c i a l situation may not be physiologically relevant. In conclusion, i t appears that endocytosis (viropexis) i s the main mechanism by which orthomyxoviruses infect susceptible c e l l s , although fusion was suggested by some workers. (g) Coronaviruses This family comprises viruses responsible for the common cold, as well as demyelinating encephalitis i n rats (172). The genome consists of single-stranded RNA and the envelope c a r r i e s c h a r a c t e r i s t i c 44 pedunculated projections (52). The exact structure of these viruses i s unclear, mainly because they are d i f f i c u l t to adapt to cultured cells (173), which probably also e x p l a i n s the lack of knowledge on the penetration of these viruses into susceptible c e l l s . Two conflicting reports have been published but neither of them was very convincing. David-Ferreira and Manaker (174) suggested endocytosis and Doughri et a l . (175) claimed that fusion could also occur. More work i s obvious-ly needed on the early events of infection by these viruses. II. Non-Enveloped RNA Viruses Although this thesis i s concerned with the mechanism of entry of enveloped RNA viruses into animal c e l l s , i t was f e l t appropriate to briefly review the studies on entry of other types of viruses. (a) Reoviruses The c h a r a c t e r i s t i c features of these viruses are a segmented genome of double-stranded RNA enclosed i n a double capsid (52). S i l v e r s t e i n and Dales (176) r e p o r t e d that r e o v i r u s type 3 was endocytosed by L c e l l s and rapidly sequestered inside lysosomes within the f i r s t hour of i n f e c t i o n . T h e i r evidence came from electron microscopy combined with biochemical analyses. V i r t u a l l y a l l of the labeled reovirus was present i n a lysosomal-mitochondrial pellet of 45 infected c e l l s at 60 min p o s t - i n f e c t i o n . Moreover, b i o l o g i c a l assays showed that r e o v i r u s e s s e q u e s t e r e d i n lysosomes were i n f e c t i o u s and not merely a d e f e c t i v e f r a c t i o n o f the inoculum. The coat proteins were digested by lysosomal h y d r o l a s e s but the RNA r e s i s t e d degradation because of i t s d o u b l e - s t r a n d e d s t r u c t u r e . However, the mechanism of r e l e a s e o f t h e v i r a l RNA from t h e l y s o s o m e s was u n c l e a r . More r e c e n t l y , Borsa et. al.. (177) p r o v i d e d more e l e c t r o n m i c r o s c o p i c evidence f o r e n d o c y t o s i s o f i n t a c t v i r i o n s . I n t e r m e d i a t e s u b v i r a l p a r t i c l e s o b t a i n e d by p r o t e o l y t i c d i g e s t i o n were a l s o shown to p e n e t r a t e d i r e c t l y t h e c e l l u l a r membrane and t h u s b y p a s s the i n e f f i c i e n t step of lysosomal uncoating. (b) Picornaviruses These non-enveloped v i r u s e s have an i c o s a h e d r a l shape w i t h a single-stranded p o s i t i v e strand ( i n f e c t i o u s ) of RNA. P o l i o v i r u s i s the most studied picornavirus (50). These v i r u s e s appear to enter c e l l s by endocytosis (viropexis) (51,178), although d i r e c t penetration was also observed (179). I t seems c l e a r t h a t t h e r e i s a p r o c e s s by which loosely bound v i r u s becomes r e s i s t a n t to e l u t i o n (183). The a b i l i t y to n e u t r a l i z e c e l l - a d s o r b e d v i r u s w i t h a n t i b o d i e s i s l o s t b e f o r e the i r r e v e r s i b l e a l t e r a t i o n o f the. v i r u s p a r t i c l e s and the uncoating of the v i r a l genome (178). However, the l o c a l i z a t i o n o f u n c o a t i n g i s s t i l l a subject of controversy (181,182). 46 I I I . DNA Viruses E a r l i e r s t u d i e s on the e n t r y o f herpes, pox, adeno and papovaviruses were reviewed by Dales (51). Endocytosis (viropexis) was the main mechanism of entry of these viruses, although fusion could sometimes be observed with herpes and poxviruses (51). More recently, an electron microscopic study suggested both fusion and endocytosis of Herpes Simplex virus and cytomegaloviruses (184). Moreover, herpesviruses-induced c e l l fusion was recently demonstrated (185-187), which provides an i n d i r e c t indication that fusion might be important in infection by these viruses. Conflicting evidence was recently obtained on the penetration of vaccinia virus (a poxvirus). Chang and Metz (188) published electron micrographs which suggested d i r e c t fusion with the plasma membrane of L cells or HeLa c e l l s grown in suspension or monolayers. Endocytosis was very rarely seen. After fusion, the v i r a l antigens detected with ferritin-conjugated antibodies appeared to disaggregate and become randomly dispersed i n the membrane. Al t e r n a t i v e l y , Payne and Norrby (189) suggested a virus-mediated viropexis mechanism, as opposed to cell-mediated endocytosis, since sodium fluoride or cytochalasin B or D did not significantly a f f e c t virus penetration, as monitored by the acquirement of serum r e s i s t a n c e . These re s u l t s were obtained for 47 extracellular enveloped vaccinia virus (EEV) , which i s presumably the virus form responsible for spreading the inf e c t i o n . On the contrary, intracellular naked vaccinia virus (INV) penetration appeared to be sensitive to inhibitors of membrane functions, which might indicate a possible fusion mechanism. Nevertheless, other workers, who are also working with INV (prepared by l y s i s of infected c e l l s ) , showed either fusion or viropexis. Therefore, unequivocal results on the penetration of vaccinia virus remain to be published. Recently, Lyon et a l . (190) confirmed that no a l t e r a t i o n of the polypeptide pattern of adenoviruses occured u n t i l the uncoating phase had occured in the v i c i n i t y of the nuclear pore complex. These observations were thought to i n d i c a t e an endocytic mechanism of penetration and the possible function of individual capsid components during internalization and v e c t o r i a l transfer of the infecting virus, possibly via microtubules. Previous studies suggested endocytosis of papovaviruses (51) and were recently confirmed by the finding that both v i r a l coat protein and DNA of polyoma vir u s arrived simultaneously i n the nucleus as early as 15 min post- i n f e c t i o n (191). A recent electron microscopic study by Maul et a l . (192) suggested that Simian v i r u s 40 (SV40) i n i t i a l l y acquired a t i g h t membrane envelope, presumably aft e r endocytosis, and was tr a n s p o r t e d to the nucleus v i a consecutive membrane fusion and fission processes. 48 Finally, Houts et a l . (193) provided evidence for endocytosis, with occasional direct penetration of Frog virus 3 (an iridovirus) and L i n s e r e_t a_l. (194) showed e n d o c y t o s i s of minute v i r u s (a parvovirus). IV. Summary and Conclusion Endocytosis (viropexis) appears to be a very widespread mechanism for the penetration of viruses into animal c e l l s . For non-enveloped viruses, i t might be the only p o s s i b i l i t y . However, enveloped viruses can also use the alternate process of d i r e c t fusion with the plasma membrane of the h o s t c e l l . I t seems w i d e l y a c c e p t e d that paramyxoviruses make use of t h i s apparently more e f f i c i e n t pathway, although they are sometimes engulfed by endocytosis. Nevertheless, endocytosis i s not r e a l l y a penetration of the membrane barrier of a c e l l . For r e p l i c a t i o n to occur, endocytosed v i r u s particles have to escape from the e n d o c y t i c v e s i c l e before i t fuses with primary lysosomes or, a l t e r n a t i v e l y , escape from the secondary lysosomes. Evidence for fusion of the v i r a l envelope with the membrane of the endocytic vesicle or the lysosomes has been presented. However, the escape of non-enveloped viruses from these intracellular vacuoles has not been very well documented. A l t e r n a t i v e l y , few workers have suggested direct penetration through breaks i n the plasma membrane. 49 Although possible, t h i s a b e r r a n t pathway i s not very l i k e l y and might have arisen from possible a r t e f a c t s of the electron microscopy. F i n a l l y , the main problem t h a t remains mostly uns o l v e d i s the d e f i n i t i o n o f the i n f e c t i o u s mechanism o f e n t r y . Only few workers provided evidence that the pathway of penetration they observed lead to the release of i n f e c t i o u s progeny v i r i o n s . Moreover, one has to keep i n mind that viruses might enter c e l l s by more than one mechanism. In t h i s eventuality, o n l y one o f these pathways c o u l d be i n f e c t i o u s but the p o s s i b i l i t y o f more than one i n f e c t i o u s r o u t e o f e n t r y cannot be discarded. 5 . Receptor-Mediated Endocytosis S i n c e r e c e p t o r - m e d i a t e d e n d o c y t o s i s c o n s t i t u t e s a p o s s i b l e mechanism of entry of enveloped RNA v i r u s e s i n t o animal c e l l s , a b r i e f review of t h i s process i s a p p r o p r i a t e a t t h i s point. Many good reviews have been published recently about endocytosis (44 ,195-198) . (a) Mechanism E n d o c y t o s i s i s a g e n e r a l term which r e f e r s t o the p r o c e s s by which c e l l s i n g e s t e x t r a c e l l u l a r m a t e r i a l s by t r a p p i n g them within inward fo l d i n g s of the plasma membrane that pinch o f f from the surface 50 to form i n t r a c e l l u l a r v e s i c l e s (endocytic v e s i c l e s ) . Phagocytosis constitutes the uptake of large p a r t i c l e s ( i . e . v i s i b l e by l i g h t microscopy). Pinocytosis describes the vesicular uptake of everything else, either in the f l u i d content of an endocytic vesicle (fluid-phase pinocytosis or micropinocytosis) or bound to the membrane (adsorptive pinocytosis). In fluid-phase endocytosis, uptake i s directly related to the concentration of solute i n the extracellular f l u i d , whereas in adsorptive endocytosis, uptake also depends on the number, af f i n i t y and function of c e l l surface receptors (receptor-mediated endocytosis). Phagocytosis, unlike fluid-phase pinocytosis, i s temperature-dependent and requires active c e l l u l a r metabolism and i n t a c t microfilaments, although microtubules do not appear to be involved. On the other hand, adsorptive pinocytosis does not appear to require microfilaments. In many cases, rapid internalization of receptor-bound solutes i s achieved in specialized regions of the plasma membrane called coated pits (from t h e i r fuzzy appearance) that i n v a g i n a t e i n t o the c e l l during endocytosis to form coated v e s i c l e s (198). The cytoplasmic coat i s thought to be composed of a protein c a l l e d c l a t h r i n (M^s 180,000). Most ligands which enter the c e l l by receptor-mediated endocytosis appear to be destined to lysosomes, although there i s evidence for efficient r e c y c l i n g of most receptors, as well as the internalized membrane. Receptor turnover i s apparently fast and continues even in the absence of specific ligand. Evidence that coated vesicles rapidly loose their clathrin coats to form uncoated vesicles or "receptosomes" was recently presented (199). 51 Receptor-mediated endocytosis of ligands into lysosomes usually leads to t h e i r degradation by lysosomal hydrolases. However, i t i s possible that the endocytic uptake of polypeptide hormones may mediate some of their effects on the cells (198). As reviewed in the previous section, many viruses appear to take advantage of the c e l l u l a r process of receptor-mediated endocytosis. Obviously, the presence of virus receptors on the c e l l surface places the host in a disadvantageous po s i t i o n for survival. Therefore, these receptors may mediate as yet unrecognized normal physiological functions (196). (b) Effect of Lysosomotropic Agents The use of inhibitors of normal lysosomal function has provided a very h e l p f u l t o o l i n the s t u d i e s o f l y s o s o m a l pathways of internalization. Brown, Goldstein and co-workers (44) developed their pioneer studies on the uptake of low-density lipoproteins (LDL) with the help of chloroquine, one of these so-called lysosomotropic agents. From a study by Ohkuma and Poole (33), who used a fluorescent probe to measure the pH inside c e l l u l a r lysosomes, i t i s widely accepted that lysosomotropic agents permeate i n t o c e l l s and p r e f e r e n t i a l l y accumulate i n the lysosomes, where they increase the intralysosomal pH and thereby inhibit acidic lysosomal hydrolases. 52 Chloroquine i s a commonly used a n t i - m a l a r i a l drug with the following structure: C H 2 - C H H-(CH 9),-N H 3 CH 2-CH 3 It i s rapidly taken up by c e l l s , probably by fa c i l i t a t e d transport (200,201) and i t s weak base nature would explain i t s rapid accumulation inside lysosomes, where i t would be trapped by protonation (200,204). Ammonia (or NH^Cl) probably acts by the same mechanism. Wibo and Poole (202) showed that c h l o r o q u i n e impaired the breakdown of rat f i b r o b l a s t p r o t e i n s af t e r they have entered the lysosomes. Lysosomal c a t h e p s i n B was found to be i n h i b i t e d . Lysosomotropic amines (chloroquine, NH^Cl and/or methylamine) could inhibit degradation of i n s u l i n (45,47,203), epidermal growth factor (205,206) and d i p h t e r i a toxin (207,208). Amenta and Brocher (209) showed that NH^Cl i n h i b i t i o n of the lysosomal mechanism of protein turnover in rat embryo f i b r o b l a s t s could be reversed after removal of the chemical. 53 Even though lysosomes are widely thought to be the target of the effect of chloroquine and other amines, some workers have presented evidence f o r d i f f e r e n t s i t e s of a c t i o n . M a x f i e l d e_t a_l. (41) suggested that methylamine and ammonium acetate inhibited clustering of o^-macroglobulin and epidermal growth factor receptors into coated pits. S i m i l a r l y , Van Leuven et a l . (43) also observed t h i s effect, although they thought receptor r e c y c l i n g was involved. In both cases, chloroquine had no effect. A si m i l a r i n h i b i t i o n of receptor recycling was also suggested by T i e t z e e_t al.. (210) for uptake of mannose glycoconjugates by macrophages, although chloroquine was also effective in t h i s case. I n h i b i t i o n of receptor-mediated uptake of lysosomal enzymes by chloroquine has been documented (211,212), as well as induction of t h e i r secretion by chloroquine (212) or primary amines (213). Moreover, chloroquine and NH^Cl could block the uptake of the toxic l e c t i n modeccin by HeLa c e l l s (214). F i n a l l y , ammonia was reported to inhibit phagosome-lysosome fusion in macrophages, although other amines such as chloroquine appeared to enhance i t (215). It i s also worthwhile to mention that chloroquine i s known to have other probably unrelated side e f f e c t s such as binding to melanin and DNA. Cl i n i c a l l y , i t also has e f f e c t s on the immune system, as well as anti-histaminic and anti-inflammatory effects (216). In summary, lysosomotropic agents are usually thought to increase the intralysosomal pH but other e f f e c t s are also possible, which 54 emphasizes the need for c a r e f u l i n t e r p r e t a t i o n of r e s u l t s obtained with such chemicals. 6. Scope of the Present Work Results from the above reviewed studies on the entry of viruses into animal ce l l s (cf. section 4) were generally inconclusive as to the infectious mechanism for penetration. It was hoped that the present investigation might c l a r i f y this situation. The problem was i n i t i a l l y attacked through the use of radiolabeled alphavirus preparations which were followed into BHK-21 c e l l s at early times p o s t - i n f e c t i o n . The a n a l y s i s of s u b c e l l u l a r f r a c t i o n s by polyacrylamide gel electrophoresis was performed to separate different v i r a l proteins present in these f r a c t i o n s at different times after the infection. We presumed that an endocytic mechanism would involve a lysosomal step and that both v i r a l glycoproteins and capsid proteins could be detected i n these organelles. On the other hand, a fusion mechanism would show an early physical separation between the two types of v i r a l proteins with only the capsid proteins entering the c e l l s . In this case, we would not expect incorporation of radioactivity in the lysosomes. It was also hoped that i f v i r a l proteins could be detected in the lysosomes, uncoating could be followed i n these organelles through the movement of capsid and glycoproteins. 55 Finally, the effect of two lysosomotropic agents, chloroquine and NHjjCl, on these events was studied to provide more evidence for a possible lysosomal pathway. Concomitantly, the effect of these drugs on the production of i n f e c t i o u s virus p a r t i c l e s , as shown by plaque assay, was investigated i n order to indicate the importance of an eventual lysosomal pathway in the productive infection. 56 MATERIALS AND METHODS 1. Chemicals and Equipment Chloroquine, ouabain, cytochrome c, bovine serum albumin, ATP, AMP, p - n i t r o p h e n y l phosphate and NADPH were o b t a i n e d from Sigma Chemical Co., St - L o u i s , MO. G l y c i n e , NH^Cl, SDS, and t r i c h l o r o a c e t i c a c i d were bought from BDH Chemicals, Vancouver, B.C.. Actinomycin D came from Calbiochem-Behring Corp., La J o l l a , CA. Sucrose (density g r a d i e n t grade) was from Schwarz/Mann, Orangeburg, NY. Bio-Rad L a b o r a t o r i e s , Richmond, CA, s u p p l i e d N,N'- m e t h y l e n e - b i s -acrylamide (BIS), bromophenol b l u e , Coomassie b r i l l a n t blue R-250, tetramethyl - ethylene - diamide (TEMED) and ammonium per s u l f a t e , as w e l l as the d y e - b i n d i n g p r o t e i n a s s a y k i t w i t h IgG as a p r o t e i n standard. Acrylamide was bought from MCB Reagents, C i n c i n n a t i , OH. The low M^ p r o t e i n s t a n d a r d s f o r e l e c t r o p h o r e s i s were purchased from Pharmacia Fine Chemicals, Piscataway, NJ . Agar noble was supplied by Difco L a b o r a t o r i e s j D e t r o i t , MI. Amersham, O a k v i l l e , Ont., provided ACS, NCS t i s s u e s o l u b i l i z e r and t h e f o l l o w i n g r a d i o c h e m i c a l s : [2- 3H]AMP (11.6 C i / m m o l e ) a n d L - [ 3 5 S ] m e t h i o n i n e (800-1300 Ci/mmole). A l l other chemicals were reagent grade. D i a l y s i s t u b i n g ( 12,000 M^ c u t - o f f ) was from F i s h e r S c i e n t i f i c Co., F a i r Lawn, NJ. Dulbecco's m o d i f i e d E a g l e medium, medium 199, 57 Earle's balanced salt solution, fungizone, streptomycin / p e n i c i l l i n , t r ypsin / EDTA and d i a l y z e d c a l f serum were from Grand Island Biological Co., Grand Island, NY. F e t a l calf serum and calf serum were obtained from Flow Laboratories Inc., Inglewood, CA. Plastic dishes for c e l l culture were bought from LUX S c i e n t i f i c Corp., Fair Lawn, NJ (large dishes, 150x15mm) or F a l c o n , Oxnard, CA (medium dishes, 100x20mm; and small dishes, 60x15mm). Spectrophotometric measurements were made on a G i l f o r d spectrophotometer equipped with a recorder and a l i n e a r transport mechanism. High-speed centrifugations were performed in a Beckman L5-65 or L8-70 ultracentrifuge and medium speed centrifugations in a Sorvall RC-5 refrigerated centrifuge. A l l centrifugations were at 4°C. 2. General Methods (a) Sc i n t i l l a t i o n Counting A l l samples were counted in 10 ml ACS in a Nuclear-Chicago Isocap 300 s c i n t i l l a t i o n counter. Counting e f f i c i e n c y was determined either by the external standard ratio (low counts) or the channel ratio (high counts i . e . above about 5,000 cpm). Chloroform-quenched standards 3 35 which contained [ H]hexadecane or ]±-[ S]methionine were counted with each set of samples. 58 (b) Protein Assays Protein determinations were made according to Lowry et. a l . (1). After an incubation of the samples i n 1.5 ml of 0.66 N NaOH at 37°C for at lea s t 4 h, 1.5 ml of the reagent (50 ml of 13%(w/v) Na 2C0 3; 1.5 ml of 4%(w/v) potassium t a r t r a t e ; 1.5 ml of 2%(w/v) C^SOjj) was added. A f t e r 10 min, 0.5 ml of 2 N Phenol Reagent Solution was added and the absorbance read at 625 nm after allowing 30 min for color development. Bovine serum albumin was used as a protein standard. In the experiments on the entry of radiolabeled Sindbis virus, a faster protein assay was used. Based on the dye-binding method of Bradford (2), i t involved the addition of 2.5 ml of 4.25-fold diluted and f i l t e r e d dye reagent concentrate to 0.5 ml of sample. The absorbance was measured in plas t i c cuvettes (to prevent dye binding to the walls) at 595 nm, after 10 to 40 min. IgG was used as a protein standard since bovine serum albumin gave a r t i f i c i a l l y high values. (c) SDS-Polyacrylamide gel electrophoresis i) Method One dimensional polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence of SDS was performed by the procedure of Laemmli (3), using a Bio-Rad Model 220 s l a b g e l apparatus. The s e p a r a t i o n g e l was 10 cm high, 14 cm wide and 1.5 mm t h i c k . A 1 cm h i g h s t a c k i n g g e l was cast on top of the separation g e l . The s e p a r a t i o n g e l was prepared from two s t o c k s o l u t i o n s : one contained 30%(w/v) a c r y l a m i d e and 0.8%(w/v) N,N'- methylene - b i s -acrylamide; the other one was 1.83 M T r i s - H C l , pH 8.8 and 0.5%(w/v) SDS. The f i n a l concentrations were: e i t h e r 11 or 155&(w/v) acrylamide, 0.366 M Tr i s - H C l , pH 8.8 and 0.1%(w/v) SDS. Aft e r degassing f o r a few minutes, the g e l was polymerized c h e m i c a l l y by the addi t i o n of 2%(w/v) ammonium p e r s u l f a t e and t e t r a m e t h y l - e t h y l e n e - diamide to a f i n a l concentration of 0.035&(w/v) and 0.025%(v/v) , r e s p e c t i v e l y . A layer of isobutanol allowed the formation o f a f l a t g e l surface. The separation gel was ready a f t e r about 20 min. The stacking g e l was a l s o prepared from two stock s o l u t i o n s : one contained 30%(w/v) a c r y l a m i d e and 1.5%(w/v) N,N'- methylene - b i s -acrylamide; the o t h e r one was 1.25 M T r i s - H C l , pH 6.8 and 1.0%(w/v) SDS. The f i n a l c o n c e n t r a t i o n s were: 4%(w/v) a c r y l a m i d e , 0.125 M Tris - H C l , pH 6.8 and 0.1%(w/v) SDS. A f t e r degassing f o r a few minutes, the g e l was p o l y m e r i z e d as d e s c r i b e d above, except t h a t the f i n a l concentration of tetramethyl - e t h y l e n e - diamide was 0.04%(v/v). The stacking g e l was ready a f t e r about 45 min. 60 The electrode buffer contained 0.025 M Tris-HCl, pH 8.3, 0.192 M glycine and 0.1%(w/v) SDS. The lyo p h i l i z e d samples were resuspended in 50-100 ul sample buffer, which consisted of 62.5 mM Tris-HCl, pH 6.8, 0. 001%(w/v) bromophenol blue, 10%(v/v) g l y c e r o l , 1%(w/v) SDS and 10%(v/v) 2-mercaptoethanol. The proteins were completely dissociated by immersing for 2 min in boiling water. Electrophoresis was carried out with a constant current of 35 mA per slab gel, with cooling, u n t i l the dye marker reached the bottom of the gel (3-5 - 4 h). The proteins were fixed in the gel and stained at the same time f o r 6 0 - 90 min w i t h a s o l u t i o n of 50%(w/v) trichloroacetic acid and 0.1%(w/v) Coomassie bri l l a n t blue R-250. The gels were d i f f u s i o n - destained by repeated washing i n 7.5%(v/v) acetic acid. If necessary, the protein pattern was scanned at 550 nm. i i ) Quantitation of Radiolabeled Proteins in Polyacrylamide Gels 1. Gel Slicing Individual lanes of a slab gel were cut out with a long razor blade, the gels were partly frozen i n a dry ice / acetone bath and sliced into either 2 or 5 mm s l i c e s with a Bio-Rad gel s l i c e r . Each s l i c e was d i g e s t e d with 0.5-1.0 ml of a 90%(v/v) NCS t i s s u e solubilizer solution i n a s c i n t i l l a t i o n v i a l at 55-60°C for 15-20 h. Digested s l i c e s were counted i n 10 ml of ACS containing 0.1 ml of 61 a c e t i c ac i d to prevent chemiluminescence. 2. Autoradiography After soaking f o r at l e a s t 2 h i n a 2%(v/v) g l y c e r o l , 10%(v/v) a c e t i c a c i d s o l u t i o n , s l a b g e l s were d r i e d onto Whatman 3MM f i l t e r paper with a Bio-Rad Model 224 g e l s l a b d r y e r and autoradiographed with 20.3 x 25.4 cm sheets of Kodak X-Omat R X-ray f i l m . 3. C e l l C u l t u r e For experiments w i t h S e m l i k i F o r e s t v i r u s , the established c e l l l i n e BHK-21 clone 13 (MacPherson and Stoker s t r a i n ) was obtained from Flow L a b o r a t o r i e s Inc., Inglewood, CA. BHK-21F c e l l s used f o r the experiments with S i n d b i s v i r u s were a g i f t of Dr. C h r i s Richardson, Rockefeller U n i v e r s i t y . The c e l l s were cultured i n Dulbecco's modified Eagle medium supplemented w i t h 5$(v/v) f e t a l c a l f serum (due to a shortage o f f e t a l c a l f serum, c a l f serum was used f o r the i n i t i a l experiments w i t h S i n d b i s v i r u s ) . A l l media c o n t a i n e d 100 mg/1 streptomycin, 100 U n i t s / 1 p e n i c i l l i n and 1.25 mg/1 fungizone. C e l l s were grown as monolayers on p l a s t i c d i s h e s at 37°C i n a humidified atmosphere containing 5%(v/v) CO,,. Stocks o f c e l l s were kept f o r 6-8 months at -70°C i n medium c o n t a i n i n g 20%(v/v) f e t a l c a l f serum and 10/&(v/v) dimethylsulfoxide. Every 2 to 3 days, c e l l s were subcultured 62 by t r y p s i n i z a t i o n (medium 199 c o n t a i n i n g 0.5%(w/v) t r y p s i n and 0.2%(w/v) EDTA). For a l l experiments, c e l l s were used between the 4th and the 25th passage. By v i s u a l i n s p e c t i o n , c e l l s were confluent when used, u n l e s s noted o t h e r w i s e . Using a hematocytometer ( c f . section 12), i t was determined t h a t l a r g e , medium and s m a l l dishes contained 7 r e s p e c t i v e l y 2, 1 and 0.3 x 10 confluent c e l l s . 4. P r o p a g a t i o n o f V i r u s e s (a) Semliki Forest Virus The t s + w i l d - t y p e s t r a i n was o b t a i n e d as d e s c r i b e d p r e v i o u s l y (4), except that the p r e p a r a t i o n from the brains of suckling mice was used f o r two s u c c e s s i v e p a s s a g e s i n BHK-21 c e l l s . For the f i n a l q passage, 1x 10 c e l l s i n 10 r o l l e r b o t t l e s were i n f e c t e d at 0.15 PFU/cell i n 10 ml o f medium 199 supplemented w i t h 2%(v/v) f e t a l c a l f serum per b o t t l e . A f t e r a 1 h a d s o r p t i o n p e r i o d , another 40 ml of the same medium was added i n each b o t t l e . At 25 h p o s t - i n f e c t i o n , c e l l debris was p e l l e t e d at 16,300 x £. f o r 20 min (GSA rotor; 10,000 rpm) and the s upernatant was f r o z e n i n a l i q u o t s at -70°C and used as a source of v i r u s . (b) Sindbis V i r u s 63 The AR339 wild-type strain was obtained from Dr. Ian Kennedy (5). A stock was grown i n BHK-21 c e l l s i n a si m i l a r manner as for Semliki g Forest v i r u s , except that 4.4 x 10 c e l l s i n large dishes were infected in 3 ml of medium per dish for 1 h, followed by the addition of another 7 ml of the same medium. The viruses were collected from the extracellular medium at 18 h post-infection. 5. Plaque Assays (a) Regular Assays Cells close to confluency on small dishes were infected for 1 h with 0.5 ml of virus i n medium 199 supplemented with 2%(v/v) fetal c a l f serum (or c a l f serum). The i n f e c t i o n medium was removed and infected c e l l s were overlaid with 4 ml of 0.9$(w/v) agar noble in Dulbecco's modified Eagle medium supplemented with 2%(v/v) fetal calf serum (or calf serum). Three days l a t e r , the agar was removed after a 10 min f i x a t i o n i n formolsaline (0.5$(w/v) NaCl; 1.5$(w/v) Na^O^ and 3-6$(v/v) formaldehyde). The c e l l sheet was stained for 60 min with 2%(w/v) c r y s t a l v i o l e t i n 20%(v/v) ethanol and the plaques were counted. The virus t i t e r was expressed as PFU per ml of virus sample. (b) Plaque-Reduction Assays with Chloroquine or NH„C1 64 Plaque assays were performed as described e a r l i e r with various concentrations of chloroquine (10-200 uM) or NH^Cl (0.5-100 mM) u s u a l l y present only during the 1 h i n f e c t i o n p e r i o d . Stock chloroquine solutions (1 and 10 mM) as well as NH^Cl (0.1, 1 and 5 M) were made fresh daily i n medium 199, neutralized with 1 N NaOH and sterilized with a Gelman Acrodisc disposable f i l t e r assembly (0.2 um). In one experiment, a plaque assay was done i n which various concentrations of NH^Cl were added i n the presence or absence of 100 uM chloroquine. Another plaque assay was performed i n which 100 uM chloroquine was also present at 60, 30 or 10 min before infection, as well as during the 1 h i n f e c t i o n p e r i o d . A l t e r n a t i v e l y , 100 uM chloroquine was added for 1 h at 1 h p o s t - i n f e c t i o n . F i n a l l y , the presence of NH^Cl or chloroquine i n the agar was also tested. (c) Plaque Purification of Sindbis Virus For some e x p e r i m e n t s , a p r e p a r a t i o n o f S i n d b i s v i r u s plaque-purified 3 times was used. Plaque assays were done as described e a r l i e r , i n i t i a l l y with the stock v i r u s . For the second and third plaque assays, the virus sample was obtained by collecting the viruses that formed one plaque (in a dish containing about 10 plaques) with a ste r i l e Pasteur pipet and suspending them i n 1 ml of medium 199. A stock of plaque-purified virus was grown i n BHK-21 cel l s as described before (cf. section 4c), except that 2.2 x 10^ c e l l s were used. 65 6. Determination of the P a r t i c l e to PFU Ratio of Virus Preparations This r a t i o was i n i t i a l l y obtained by electron microscopy. A suspension of uniform l a t e x beads of known concentration (mean diameter: 176 nm; Ernest F. Fullam Inc., Shenectady, NY) was mixed with the virus suspension and a negatively stained preparation was counted with a Philips electron microscope. The virus p a r t i c l e to PFU ratio was calculated from the average latex beads to virus particles ratio obtained from 7 electron microscope fields. Alternatively, a faster and more convenient technique was used which compared quantitatively to the previous method. The virus sample was assayed for protein with the Lowry procedure (cf. section 2b). A v i r a l protein content of 61$ and a v i r a l molecular weight of 64 x 10^ were assumed for the calculations (6,7). 7. Radiolabeling and P u r i f i c a t i o n of Viruses (a) Semliki Forest Virus Five large dishes of BHK-21 c e l l s were infected with Semliki Forest virus at 200 PFU/cell i n 3 ml of medium 199 with 2%(v/v) fetal 66 c a l f serum per d i s h . Actinomycin D (3 ug/ml) was present throughout the i n f e c t i o n to ensure immediate and complete i n h i b i t i o n of host RNA and protein synthesis (8). A f t e r a 1 h ads o r p t i o n period, 7 ml of the same medium was added i n each d i s h . At 3 h p o s t - i n f e c t i o n , the i n f e c t i o n medium was r e p l a c e d w i t h 5 ml per d i s h of methionine-free medium (Earle's balanced s a l t s o l u t i o n c o n t a i n i n g a l l the amino acids of medium 199, except m e t h i o n i n e , and supplemented wi t h 2$(v/v) dialyzed f e t a l c a l f serum). At 3.5 h p o s t - i n f e c t i o n , a t o t a l of 2 mCi 35 of L-[ Sjme t h i o n i n e was added and at 5 h p o s t - i n f e c t i o n , another 5 ml of medium 199 wit h 2$(v/v) f e t a l c a l f serum was a l s o added i n each d i s h . At 18 h p o s t - i n f e c t i o n , c e l l d e b r i s was p e l l e t e d out of the medium by c e n t r i f u g a t i o n at 16,000 x g. f o r 20 min (SS-34 rotor; 11,500 rpm). The supernatant was g r a d u a l l y brought to a concentration of 65/6(w/v) ammonium s u l f a t e over a p e r i o d o f 30 min at 4°C (430 g of s o l i d ammonium s u l f a t e were added per l i t e r of supernatant). During t h i s p e r i o d , the medium was c o n t i n u o u s l y s t i r r e d and the pH kept n e u t r a l by dropwise a d d i t i o n o f 1 N NaOH. A f t e r a f u r t h e r 60 min incubation at 4°C without s t i r r i n g , the p r e c i p i t a t e was recovered by ce n t r i f u g a t i o n a t 16,000 x g. f o r 30 min. The p e l l e t was resuspended in t o 2 ml of i c e - c o l d Dulbecco's phosphate buf f e r s a l i n e , pH 7.4 (137 mM NaCl, 2.68 mM KC1, 1.47 mM KH PO^ and 8.1 mM Na HPO^). The v i r u s e s i n t h i s ammonium s u l f a t e - c o n c e n t r a t e d preparation were p u r i f i e d by a m o d i f i c a t i o n o f t h e method o f S c h e e l e and Pfefferkorn ( 9 ) . The sample was l a y e r e d onto a three-phase gradient 67 which consisted (from bottom to top) of 1.5 ml of 50%(w/w) sucrose, 7.5 ml of a continuous gradient of 50%(w/w) to 25%(w/w) sucrose and 7.5 ml of 20%(w/w) to 5%(w/w) sucrose. The 50%(w/w) sucrose solution was made in 0.2 M CsCl and 2 mM Tris-HCl, pH 7.8, whereas the other sucrose solutions were in Dulbecco's phosphate buffer saline, pH 8.0. This gradient was centrifuged i n a Beckman SW 27.1 rotor at 25,000 rpm (81,900 x £.) f o r 4 h. V i r u s e s form an i s o p y c n i c band i n t h i s gradient at a posi t i o n corresponding to about 35%(w/w) sucrose. Fractions of 16 drops (about 0.4 ml) were collected from the bottom of the c e l l u l o s e n i t r a t e tube (Hoeffer gradient c o l l e c t o r and Gilson Micro Fractionator). The fractio n s containing the radioactive v i r a l peak were pooled, d i a l y z e d at 4°C a g a i n s t Dulbecco's phosphate buffer s a l i n e , pH 7.4 and lay e r e d onto a second s i m i l a r sucrose gradient (SW 27 rotor was used due to larger sample volume). After dialysis, the second virus pool was pelleted at 120,000 x £. for 2 h (Type 65 rotor; 43,000 rpm) and resuspended i n 3 ml of medium 199. This preparation was used for the experiments. (b) Sindbis Virus Six large dishes of BHK-21F c e l l s were infected by Sindbis virus with 30 PFU/cell i n 3 ml per dish of Dulbecco's modified Eagle medium lacking methionine and with a glucose concentration reduced to 1 g/1 (custom made by GIBCO, c f . section 1), supplemented with 2$(v/v) dialyzed calf serum. Actinomycin D (3 ug/ml) was present throughout 68 the preparation (cf. section 7a). The i n f e c t i o n medium was removed at 1 h p o s t - i n f e c t i o n and r e p l a c e d with 10 ml per dish of the same medium, without viruses. At 4 h p o s t - i n f e c t i o n , 5 ml per dish of a s i m i l a r medium c o n t a i n i n g a t o t a l of e i t h e r 3 or 15 mCi of 35 L_-[ S]methionine was s u b s t i t u t e d . A f t e r another hour, 5 ml of medium 199 supplemented with 2#(v/v) f e t a l c a l f serum was added in each dish. At 15 h p o s t - i n f e c t i o n , the viruses released into the medium were p u r i f i e d as d e s c r i b e d f o r Semliki Forest virus (cf. section 7a) except for the f o l l o w i n g modifications. The ammonium sul f a t e p e l l e t was resuspended i n t o 3 ml of i c e - c o l d buffer. The gradient consisted of 3 ml of 50%(w/w) sucrose, 14 ml of 50 to 255&(w/w) sucrose and 20 ml of 20 to 5/6 (w/w) sucrose. The gradient was centrifuged in a SW 27 rotor for 3.5 h and 40-drop fractions (about 1 ml) were c o l l e c t e d . Only one g r a d i e n t step was used and the i o radioactive virus pool was dialyzed at 4 C against medium 199. This preparation was used for the experiments. 8. Enzyme Markers Note: In a l l the following procedures, controls contained no enzyme, (a) Plasma Membranes i ) 5'- Nucleotidase 69 This enzyme was assayed by measuring the cleavage of radioactive AMP (10). The assay conditions were: 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and 20 pM [2- H]AMP ( s p e c i f i c r a d i o a c t i v i t y 6.3 Ci/mole). The reaction mixture had a f i n a l volume of 1 ml and contained 100 pi of b u f f e r (0.5 M T r i s - H C l , pH 8.0; 50 mM MgCl ) , 70 p i of 0.4 mM [2- H]AMP and 200 u l of protein. This l a t t e r component was used to start the reaction, which proceeded at 37°C for 30 min. The reaction was stopped by the addition of 0.2 ml of 0.25 M ZnSO^. Protein and unhydrolyzed AMP were p r e c i p i t a t e d with 0.2 ml of saturated Ba(0H) 2 and pelleted at 3,000 x g. f o r 5 min (SS-34 rotor; 5,000 rpm). A 250 p i a l i q u o t of the [2- H]adenosine supernatant was counted. One Unit of enzyme c a t a l y z e d the cleavage of 1 pmole of substrate per min. i i ) Ouabain- s e n s i t i v e Na + , K + - ATPase This enzyme was assayed by the method of Costantino-Ceccarini et a l . (11). The enzyme was f i r s t a ctivated i n 0.06$(w/v) sodium deoxycholate with 2 mM EDTA in 25 mM imidazole buffer, pH 7.0. The f i n a l incubation volume was 1 ml and 300 ul of protein was used. After this preincubation at room temperature for 30 min, 200 p i of this activated enzyme preparation was assayed i n a t o t a l volume of 1 ml consisting of 50 mM Tris-HCl, pH 7.4, 1.0 mM EDTA neutralized to pH 7.0, 100 mM NaCl, 20 mM KC1, 3.0 mM MgCl 2 and 3-0 mM ATP. The tubes 70 used to determine the ouabain-sensitive ATPase also contained 1.0 mM ouabain. The reaction mixtures were kept on ice u n t i l the addition of ATP, a f t e r which they were incubated at 37°C f o r 20 min. The reaction was terminated with 0.2 ml of 25$(w/v) trichloroacetic acid . The released inorganic phosphorus was quantitated by the method of Ames (12). A reaction mix was made fresh d a i l y and contained 1 part of 1056(w/v) a s c o r b i c a c i d and 6 p a r t s o f 0.42#(w/v) ammonium molybdate-tetrahydrate i n 1 N s u l f u r i c acid. To 0.7 ml of this mix, 0.3 ml of sample was added and the absorbance was measured at 820 nm aft e r a 60 min incubation at 37°C. A standard curve was obtained with Na^HPOjj and 10 nmoles of i n o r g a n i c phosphorus gave an absorbance of 0.260. One Unit of Enzyme catalyzed the cleavage of 10 nmoles of ATP per min. (b) Endoplasmic Reticulum : NADPH - Cytochrome c Reductase This enzyme was assayed spectrophotometrically by measuring the reduction of cytochrome c at 550 nm (25). The incubation mixture contained 0.1 mM KCN, 66 mM KC1, 44 mM potassium phosphate buffer, pH 7.6, 0.05 mM cytochrome c and 0.06 mM NADPH. P r a c t i c a l l y , 150 jil of protein was mixed with 150 p l of buffered substrate (0.23 mM KCN, 154 mM KC1, 103 mM potassium phosphate b u f f e r , pH 7.6 and 0.12 mM cytochrome c) and the reaction started at 37°C with 50 ;ul of 0.42 mM NADPH. The increase in absorbance at 550 nm was recorded. One Unit of enzyme catalyzed the reduction of cytochrome c at an i n i t i a l rate 71 corresponding to an increase i n absorbance of 0.01 per min ( i . e . reduction of 1 umole of substrate per min since E of reduced and oxidized cytochrome c are r e s p e c t i v e l y 27.7 x 10^ and 9.0 x 10^ cm'Vmo l e ) . (c) Lysosomes : Acid Phosphatase This enzyme was assayed by the method of Hubscher and West (13), as modified by Cohen et a l . (14). The enzyme present i n 250 p i of each fraction was assayed i n a t o t a l volume of 1 ml, which contained 15 mM p-nitrophenyl phosphate and 50 mM acetate buffer, pH 5.4. After an incubation of 30 min at 37°C, the reaction was stopped with 0.1 ml of 0.1 N NaOH and the absorbance of the released p-nitrophenol measured at 410 nm. One Unit of enzyme catalyzed the cleavage of 1 pmole of p-nitrophenyl phosphate per min ( a c t i v i t y = dA x 1.982 x -3 2 10 since the product has a E = 18.5 cm /pmole) (15). (d) Mitochondria : Succinate Dehydrogenase The assay procedure was o r i g i n a l l y designed by Pennington (16) and was m o d i f i e d by Porteous and C l a r k (17). I t i n v o l v e d the measurement of the reduction of an a r t i f i c i a l substrate of the enzyme : p-iodotetrazolium v i o l e t (INT). The 0.8 ml r e a c t i o n mixture contained 50 mM potassium phosphate buffer. pH 7.4, 0.1$(w/v) INT (2-(p-iodophenyl) - 3-(p-nitrophenyl) 5-phenyltetrazolium chloride), 72 50 mM sodium succinate, 25 mM sucrose and 2 mM EDTA. The reaction was started with 200 fil of protein and proceeded at 37°C for 30 min. After stopping the reduction with 1 ml of 10%(w/v) trichloroacetic acid , the reduced INT was extracted with 4.0 ml ethyl acetate which formed an upper phase that was measured spectrophotometrically at 490 nm (against ethyl acetate). One Unit of enzyme catalyzed the reduction of 1 umole of INT per min ( a c t i v i t y = dA x 6.6335 x 10 since the 2 product has a E = 20.1 cm /umole) (17). 9. Preparation of Plasma Membranes and Endoplasmic Reticulum Plasma membranes and endoplasmic reticulum from BHK-21 cells were prepared by a combination of the methods of Atkinson and Summers (18) and Richardson and Vance (19). About 3 x 10 c e l l s attached to plastic dishes were washed twice with ice-cold 10 mM Tris-HCl, pH 8.0. The c e l l s were scraped with a rubber policeman into 3.0 ml of the same buffer. After incubation on ice for 5 min (hypotonic swelling), the mixture was homogenized in a 7-ml Dounce homogenizer with 3 strokes of a loose-fitting pestle. The homogenate was made 0.25 M sucrose and 5 mM MgCl 2 (375 p i of 2.5 M sucrose and 375 p i of 50 mM MgCl 2) for s t a b i l i z a t i o n of the membranes. N u c l e i and unbroken c e l l s were pelleted at 1,000 x g for 1 min (SS-34 rotor; 3,000 rpm) and the supernatant was layered onto a discontinuous sucrose gradient which consisted of 4 ml of 45$(w/w) sucrose and 10 ml of 30%(w/w) sucrose, 73 both i n 50 mM Tris-HCl, pH 7.4 and 5 mM MgCl 2« The gradient was centrifuged at 7,000 x g. for 20 min (SW 27.1 rotor; 7,300 rpm) and dripped from the bottom in 16-drop f r a c t i o n s (about 0.4 ml). These fractions were assayed for 5'- Nucleotidase and NADPH - cytochrome c reductase. 10. Subcellular Fractionation One medium dish of BHK-21 c e l l s was washed 3 times with 3-5 ml of ice-cold 10 mM Tris-HCl, pH 8.0. The c e l l s were scraped with a rubber policeman into 2 ml of the same buffer and homogenized i n a 7-ml Dounce homogenizer with 3 strokes of a l o o s e - f i t t i n g pestle. The homogenate was made 0.25 M sucrose and 5 mM MgCl^ (250 u l of 2.5 M sucrose and 50 mM MgCl^) for s t a b i l i z a t i o n of membranes, a 200 ul aliquot was kept and the remaining homogenate was centrifuged at 1,000 x g. for 1 min (SS-34 rotor; 3,000 rpm). The p e l l e t of nuclei and unbroken c e l l s was resuspended i n 1 .0 ml of H,,0 and a homogeneous suspension was obtained with 10 strokes of the t i g h t - f i t t i n g pestle of a Dounce homogenizer. The post-nuclei supernatant was centrifuged at 12,000 x g. f o r 20 min (SS-34 r o t o r ; 10,000 rpm). The x lysosomes-mitochondria p e l l e t was resuspended i n 1 .0 ml of H^ O, as described above, whereas the supernatant was further centrifuged at 200,000 x z. for 34 min (SW 60 T i rotor; 44,000 rpm; this provided a centrifugal force equivalent to a centrifugation at 100,000x £ for 74 60 min, i . e . w t = 3-58 x 10 ). The microsomal p e l l e t was resuspended i n 1.0 ml of H 20, as d e s c r i b e d above. The f i n a l supernatant was assumed to be cytosol. 11 . E f f e c t of Chloroquine or NH^Cl on C e l l u l a r  Protein Synthesis Either 100 or 200 uM c h l o r o q u i n e or 10 or 100 mM NH^Cl was added to nearly confluent c e l l s i n medium dishes. After 1 h, the medium was removed, the c e l l sheet was washed and each dish received 50 uCi ( c h l o r o q u i n e experiment) or 25 uCi (NH^Cl experiment) 35 of L_-[ S ] m e t h i o n i n e d i s s o l v e d i n the same growth medium (Dulbecco's modified Eagle medium with 5/&(v/v) c a l f serum). After various incubation times i n the presence of the label, the medium was removed, the c e l l sheet was washed twice with i c e - c o l d Dulbecco's phosphate buffer saline, pH 7.4 ( c f . section 7a) and homogenized in 5 ml of the same buffer (10 strokes i n a 7 ml Dounce homogenizer with a ti g h t - f i t t i n g pestle). A 1 ml aliquot of this homogenate was saved for r a d i o a c t i v i t y and protein quantitation and the remaining 4 ml was treated with 4 ml of 10/6(w/v) t r i c h l o r o a c e t i c acid . The pellet was co l l e c t e d by centrifugation at 3,000 x & for 5 min (SS-34 rotor; 5,000 rpm), washed twice with 4 ml of 105&(w/v) trichloroacetic acid, and f i n a l l y dissolved in 1 ml of 0.1 N NaOH. 75 A similar experiment was performed i n which the radioactivity was added at the same time as c h l o r o q u i n e or NH^Cl (50 pCi for the NHjjCl or 25 uCi for the chloroquine experiment) and the c e l l s were harvested after 30 and 60 min. 12. Effect of Chloroquine on Cell Viability Ten small dishes of nearly confluent BHK-21 c e l l s were obtained and half of them were treated with 100 pM chloroquine. One h after the addition of t h i s chemical, a l l media were removed, the c e l l sheets washed and new medium added (5 ml of Dulbecco's modified Eagle medium plus 5%(v/v) c a l f serum per d i s h ) . At d i f f e r e n t times a f t e r the addition of chloroquine, two dishes (one control and one chloroquine -treated) were treated as follows. The media were removed and the c e l l s detached by trypsinizati.on ( c f . section 3) and resuspended in 2 ml of medium containing 0.04%(w/v) Trypan blue. The number of viable cells (attached to the dish and excluding the dye) was determined in each dish with an hematocytometer (Levy counting chamber, Hausser 2 Scientific, Blue B e l l , PA). Four 1 mm squares were counted in each case. 13. E f f e c t of Chloroquine or NH^Cl on the Production of Radiolabeled Sindbis Virus Proteins and Particles 76 Cells close to confluency were infected with Sindbis virus (100 PFU/cell in the chloroquine and 50 PFU/cell i n the NH^Cl experiment) i n the absence or presence of e i t h e r 100 uM chloroquine or 10 mM NH^Cl. Ac t i n o m y c i n D ( 3 ug/ml) was p r e s e n t throughout the experiments for the same reason as described previously (cf. section 7a). The infection media (5 ml per medium dish) were removed after 1 h and replaced with 5 ml of medium 199 supplemented with 2%(v/v) calf serum. At 2 h post-infection, each dish received 130 uCi (chloroquine 35 experiment) or 150 uCi (NH^Cl experiment) of J±-[ S]methionine dissolved i n 5 ml of serum f r e e - medium 199 (serum was omitted without adverse e f f e c t s to the c e l l s ; the large excess of serum proteins would have distorted the electrophoresis gels). At 3 , 5 , 8 and 11 h post-infection, the medium was removed, centrifuged for 20 min at 1 2 , 0 0 0 x £. (SS - 3 4 rotor; 1 0 , 0 0 0 rpm) and dialyzed against H^ O at 4°C. The c e l l s were washed twice with i c e - c o l d H^ O and homogenized i n 5 ml H^ O (10 s t r o k e s i n a 7-ml Dounce homogenizer with a t i g h t - f i t t i n g pestle). The c e l l homogenates and dialyzed media were concentrated by l y o p h i l i z a t i o n , the proteins were separated by gel electrophoresis ( 1 1 j(w/v) acrylamide) and the r a d i o a c t i v i t y detected by autoradiography. The autoradiographs were quantitated by scanning at 550 nm. The NHjjCl experiment was a l s o repeated with the following modification. The media at 5 , 8 and 11 h post-infection were collected 77 and a portion of each (25%) was centrifuged at 200,000 x g. for 34 min (SW 60 T i rotor; 44,000 rpm ; c f . section 10). The virus pellet was resuspended i n 0.5 ml E^O and concentrated by l y o p h i l i z a t i o n for gel electrophoresis followed by autoradiography. As a c o n t r o l , v i r u s e s were r a d i o l a b e l e d with 0.5 mCi of 35 L-l S]methionine ( c f . section 7b, except only one large dish was used). The virus supernatant obtained a f t e r elimination of the c e l l debris was directly centrifuged as above. The virus pellet was washed c a r e f u l l y with 2 ml of E^O and resuspended i n 1 ml of The supernatant was d i a l y z e d a g a i n s t H^O and the resuspended virus p e l l e t (0.6 p C i ) was a n a l y z e d by g e l e l e c t r o p h o r e s i s and autoradiography. 14. Pulse-Chase of Radiolabeled Semliki Forest Virus into Enriched Plasma Membrane and Endoplasmic Reticulum Fractions  of BHK-21 C e l l s . Three medium dishes o f BHK-21 c e l l s were i n f e c t e d with radiolabeled Semliki Forest virus (for each dish: 5 x 10^ dpm in 2 ml of medium 199 with 2%(v/v) f e t a l c a l f serum). The media were removed after a pulse of 15 min and the dishes washed with 3 ml of a similar medium which contained only unlabeled viruses (to compete for binding s i t e s with the adsorbed radiolabeled virus p a r t i c l e s ) . Two 78 dishes received another 5 ml of t h i s medium (10 PFU of unlabeled viruses per cel l ) whereas plasma membranes and endoplasmic reticulum were prepared from the other dish (chase time: 0 min) as described in 7 section 9, with the following modifications. Only 1 x 10 ce l l s were 7 infected and 2 x 10 u n i n f e c t e d c e l l s were used as c a r r i e r . The desired f r a c t i o n s were c o l l e c t e d d i r e c t l y from the discontinuous sucrose gradients with bent-tip Pasteur pipets: plasma membranes from the 30-4556(w/w) sucrose interface and endoplasmic reticulum from the top of the gradient. At chase times of 30 and 60 min, the procedure was repeated as described above. Plasma membranes and endoplasmic r e t i c u l u m f r a c t i o n s were dialyzed at 4°C against 50 mM Tris-HCl, pH 7.4 and 5 mM MgCl 2 and concentrated by l y o p h y l i z a t i o n . P r o t e i n s were separated by gel e l e c t r o p h o r e s i s (15$(w/v) a c r y l a m i d e ) and the r a d i o a c t i v i t y quantitated after the gels were sliced (2 mm sli c e s ) . 15. Entry of Radiolabeled Sindbis Virus into Subcellular Fractions  of BHK-21 C e l l s and the E f f e c t of Chloroquine or NH^Cl Cells were washed twice with either cold (4 C) or warm (37 C) medium 199 and infected at the appropriate temperature with freshly made radiolabeled Sindbis virus at 20 PFU/cell (for each medium dish: 4 to 6 x 10^ dpm i n 2 ml of medium 199), i n the presence or absence i 79 of 100 pM chloroquine (added to the vi r u s suspension at the very last minute). At 20, 40, 60 and 120 min post-infection (also 2 min in the 4°C experiment), dishes were subjected to subcellular fractionation, as described in section 10. The i n f e c t i o n media of dishes destined to be harvested at 120 min were removed at 60 min post-infection and the c e l l sheets washed twice with 5 ml of medium 199 per dish, and covered with an a d d i t i o n a l 5 ml of the same medium (at the appropriate temperature). The r a d i o a c t i v i t y present i n a l l s u b c e l l u l a r f r a c t i o n s was determined and the fractions enriched i n lysosomes were also analyzed by g e l e l e c t r o p h o r e s i s (11$(w/v) a c r y l a m i d e ) . The amount of radiolabeled v i r a l p r o t e i n s a s s o c i a t e d with these f r a c t i o n s was quantitated from gel slices (5 mm slices). The trichloroacetic acid - soluble r a d i o a c t i v i t y present in the medium at each time point was determined from the proportion of t r i c h l o r o a c e t i c acid - i n s o l u b l e r a d i o a c t i v i t y , as described i n section 11. C a r r i e r p r o t e i n (bovine serum albumin) was added to visualize the pellets. This experiment was repeated with 10 mM NH^Cl replacing the chloroquine. In a separate experiment, two medium dishes of uninfected c e l l s were homogenized as usual and radiolabeled viruses were added to the c e l l lysates (2 x 10^ dpm of r a d i o l a b e l e d v i r u s e s i n 150 p l of medium 199 were added to the homogenate from each medium dish). Subcellular fractionation was performed on these treated homogenates. In p a r a l l e l , an experiment s i m i l a r to the one described above was performed with the f o l l o w i n g m o d i f i c a t i o n s : the m u l t i p l i c i t y of 7 infection was 420 PFU/cell, each medium dish received 1.9 x 10 dpm of r a d i o l a b e l e d S i n d b i s v i r u s e s , no i n h i b i t o r was added and subcellular f r a c t i o n a t i o n was performed at 15, 30, 60 and 120 min p o s t - i n f e c t i o n (37°C o n l y ) ; c y t o s o l s at 60 and 120 min post-infection were also analyzed by gel electrophoresis. 81 RESULTS 1. Stock Virus Preparations The results shown in Table 1 summarize the characteristics of the three stock virus preparations used for the experiments described in this thesis. The virus t i t e r s were somewhat variable, possibly because of differences i n the growth s t a t e of the c e l l s u t i l i z e d for the plaque assays. As expected, the plaque-purification of Sindbis virus did reduce the p a r t i c l e to PFU r a t i o . Unfortunately, an even lower ratio, (i.e. closer to 1) was not reached, possibly because the stock virus used for the i n i t i a l i n f e c t i o n of the c e l l s had been kept at -70°C, in a l y o p h i l i z e d form, for about 5 years. Nevertheless, the t i t e r of the stock unlabeled v i r u s preparations kept at -70°C i n medium was relatively stable over a period of at least 1.5 years. An interesting observation from the plaque-purification procedure was that each plaque used for the next p u r i f i c a t i o n step contained 2.4 + 0 . 2 x 1 0 7 P F U . An aliquot of two stock virus preparations was also purified by sucrose density gradient centrifugation, as described in Materials and Methods (section 7), except that the p o s i t i o n of the virus was determined form the absorbance at 260 nm (for nucleic acid). It was determined that the Semliki Forest virus preparation made from 1 x 82 Table 1 C h a r a c t e r i z a t i o n of stock v i r u s preparations V i r u s e s were prepared as d e s c r i b e d i n M a t e r i a l s and Methods (sections 4 and 5c). The v i r u s t i t e r s were determined by plaque assays and the p a r t i c l e to PFU r a t i o by t h e e l e c t r o n microscope and/or protein assay techniques ( c f . Materials and Methods, section 6). Virus type Virus t i t e r Particle/PFU PFU/ml x 10~ 9 S e m l i k i F o r e s t 5 • 2» n . d. 1 S i n d b i s 2 • 1 5 2 134^ Sindbis (plaque-purified) 0.6 3 32° 1. Not determined 2. Determined by both methods 3. Determined by protein assay method 4. Standard deviation (n=9) 5. Standard deviation (n=3) 83 Q 10 c e l l s contained 6.7 mg of p r o t e i n , whereas the Sindbis v i r u s g preparation made from 4.4 x 10 c e l l s contained 4.0 mg of protein q (9.1 mg per 1 x 10 c e l l s ) . 2. P u r i f i c a t i o n of Radiolabeled Viruses (a) Semliki Forest Virus The radiolabeled Semliki Forest virus preparation used for the study described in section 4 was analyzed by SDS - polyacrylamide gel electrophoresis (Figure 3). The two envelope glycoproteins E1 and E2 were not resolved under the reducing conditions used in the gel since their electrophoretic mobilities are only significantly different when th e i r d i s u l f i d e bonds are i n t a c t (20,21). As expected (22), the envelope glycoprotein E3 could not be detected by protein staining but appeared on the autoradiograph. This anomaly has been attributed to the very high carbohydrate composition (45$) of t h i s protein (22). Finally, the capsid protein was also detected, on both the protein stain and the autoradiograph. A scan of the Coomassie blue-stained gel i s presented in Figure 4a and the radioactive profile obtained from gel slices i n Figure 4b. The estimated of the v i r a l p r o t e i n s confirmed published reports (21,23), i.e. 50K for E1 and E2, 34K for C and about 10K for E3-F i g u r e 3. P r o t e i n and a u t o r a d i o g r a p h i c p a t t e r n s of p u r i f i e d radiolabeled Semliki Forest virus. 35 Viruses were labeled with L_-[ Sjmethionine and p u r i f i e d by two sucrose density gradient centrifugations, as described in Materials and Methods ( s e c t i o n 7a). About 25 pg of the p u r i f i e d v i r u s preparation was subjected to SDS- polyacrylamide gel electrophoresis (15$(w/v) acrylamide) and stained with Coomassie b r i l l a n t blue R-250 (a). Autoradiography was also performed on the dried gel (b) (0.75 pCi, 24 h exposure). The migration of molecular weight markers appears on the far l e f t . The v i r a l proteins are i d e n t i f i e d : E1-E2, E3: envelope glycoproteins; C: capsid protein. The origin (o) and dye front (d) are also indicated. 85 Distance from origin (cm) Figure 4. P r o t e i n and r a d i o a c t i v e p r o f i l e s o f p u r i f i e d r adiolabeled Semliki Forest v i r u s . Viruses were prepared as described i n Figure 3- (a) Spectrophotometric scan of the g e l shown i n F i g u r e 3. (b) R a d i o a c t i v e p r o f i l e obtained from s l i c e s of a g e l as d e s c r i b e d i n F i g u r e 3, except that only 10 pg of protein was electrophoresed (0.3 p C i ) . The peaks of v i r a l proteins are i d e n t i f i e d as i n Figure 3. 86 Since only v i r a l proteins were detected as major bands by SDS -polyacrylamide gel electrophoresis and autoradiography, the virus preparation was considered pure. Even though two sucrose density gradient p u r i f i c a t i o n steps were used, i t was l a t e r determined that one gradient was s u f f i c i e n t to y i e l d a pure preparation. Of the radioactivity added to the c e l l s , 0.5% was recovered i n purified virus. (b) Sindbis Virus The radiolabeled Sindbis virus preparations had to be made fresh for each experiment since i t was found that the infectivity of a frozen preparation decreased substantially over a short period of time (e.g. 20-fold decrease of the virus t i t e r a f t e r 2 weeks storage at -70°C). Radiolabeled Sindbis virus preparations were routinely analyzed for purity by SDS - polyacrylamide g e l electrophoresis. Typical p r o f i l e s are presented i n F i g u r e 5 and 6 (each corresponds to a different preparation). As noticed for Semliki Forest virus, the two envelope glycoproteins E1 and E2 were not resolved under the reducing conditions used ( c f . section 2a). Furthermore, the smaller envelope glycoprotein E3 was not detected, either on the Coomassie blue stain p r o f i l e or on the r a d i o a c t i v e p r o f i l e (even when the acrylamide concentration was increased to 155&(w/v); r e s u l t s not shown). This 87 (a) (b) Absorbance at 550 nm 0 0 . 1 0 . 2 E1-E2 Figure 5. Protein profile of purified radiolabeled Sindbis virus. 35 Viruses were labeled with L_-[ S]methionine and p u r i f i e d by sucrose density gradient centrifugation, as described in Materials and Methods (section 7b). About 5 pg of the p u r i f i e d v i r u s preparation was subjected to SDS- po 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 (11$(w/v) acrylamide) and stained with Coomassie b r i l l a n t blue R-250. (a) Protein pattern of the g e l . (b) Spectrophotometric scan of the same gel. The v i r a l proteins are i d e n t i f i e d : E1-E2: envelope glycoproteins; C: capsid protein. The origin (o) and dye front (d) are also indicated. 88 94K 67K 43K 30K 20K 14Kfc E1E2 C Figure 6. Autoradiograph and r a d i o a c t i v e p r o f i l e of p u r i f i e d radiolabeled Sindbis virus. Viruses were prepared as described in Figure 5, except that 0.8 uCi (a) or 0.2 uCi (b) of the purified virus preparation was electrophoresed. (a) Autoradiograph of the dried gel. (b) Radioactive profile obtained from gel slices. The peaks of v i r a l proteins and the origin and dye front of the gel are i d e n t i f i e d as in Figure 5. The migration of molecular weight markers appears on the far l e f t . 89 confirms published observations that E3 i s made i n Sindbis virus -infected c e l l s but i s not packaged into progeny v i r i o n s (24). The molecular weights (M^) of the envelope glycoproteins (E1-E2) and the capsid protein (C) were found to be s i m i l a r to the corresponding proteins in Semliki Forest virus ( c f . section 2a), as expected from published r e s u l t s (23). The r a t i o E1-E2 / C calculated from the radioactive profile of Sindbis virus (Figure 6b) was about 1.7. Since only v i r a l proteins were detected as major bands by SDS -polyacrylamide gel e l e c t r o p h o r e s i s , the virus preparations were considered pure. The recovery of radioactivity in radiolabeled viruses was i n i t i a l l y 1-3$ but for some unexplained reason, this recovery was later reduced to 0.2-0.5$. Interestingly, the particle / PFU ratio of the radiolabeled v i r a l preparation was usually very similar to this ratio for the infecting virus sample (Table 1). 3. Preparation of Plasma Membranes and Endoplasmic Reticulum  from BHK-21 Ce l l s A procedure f o r the s e p a r a t i o n of plasma membranes from endoplasmic reticulum v e s i c l e s was outlined i n Materials and Methods (section 9). A t y p i c a l p r o f i l e of enzyme markers for these two i fractions i s presented i n Figure 7. The plasma membrane marker (5 -nucleotidase) appeared mostly at the 45-30$(w/w) sucrose interface 90 6 12 18 24 30 Fraction number from bottom Figure 7. Enzyme marker p r o f i l e s for preparation of plasma membranes and endoplasmic reticulum from BHK-21 c e l l s . Cells were homogenized and s u b c e l l u l a r fractions separated by sucrose density gradient centrifugation (cf. Materials and Methods, section 9). A plasma membrane marker ( 5 ' - n u c l e o t idase; # — " ^ H f c ; PM) and an endoplasmic reticulum marker (NADPH- cytochrome c reductase;A J4;ER) were assayed i n each g r a d i e n t f r a c t i o n by methods described i n Materials and Methods (section 8). 91 (lower band) and just below the top of the 30%(w/w) sucrose (upper band). On the other hand, the endoplasmic reticulum marker (NADPH -cytochrome c reductase) was concentrated at the top of the gradient. The two plasma membrane peaks appeared as cloudy areas i n the gradient. Phase c o n t r a s t microscopy showed that the lower band consisted of fragments, ghosts and/or aggregated v e s i c l e s , whereas single vesicles were the exclusive components of the upper band. The proportion of upper and lower bands could easily be varied: a gentler homogenization of the c e l l s yielded more of the lower band of plasma membrane vesicles. In order to obtain a clear separation of the latter from endoplasmic reticulum v e s i c l e s , the Dounce homogenization was optimized to get the largest proportion of the lower band. For the pulse-chase experiment described in the next section, the plasma membrane lower band and the endoplasmic reticulum fraction were col l e c t e d d i r e c t l y from the g r a d i e n t ( c f . Materials and Methods, section 13). A t y p i c a l p u r i f i c a t i o n table i s presented i n Table 2. These results were from uninfected c e l l s but infected c e l l s behaved identically. Each assay was optimized for protein concentration: an amount of protein that showed an a c t i v i t y r e s i d i n g i n the middle portion of the linear response curve was used. As shown by the specific enzyme markers, the plasma membranes were p u r i f i e d by a factor of 5-fold with respect to the c e l l lysate (51$ yield), with undetectable contamination by the endoplasmic reticulum. Similarly, the endoplasmic r e t i c u l u m v e s i c l e s were p u r i f i e d 5 - f o l d , with undetectable 92 Table 2 Purification of plasma membranes and endoplasmic reticulum Plasma membranes and endoplasmic r e t i c u l u m f r a c t i o n s were prepared on the discontinuous sucrose gradient described in Materials and Methods (sections 9 and 13) • Enzyme markers for plasma membrane (5'-nucleotidase) and endoplasmic r e t i c u l u m (NADPH-cytochrome c reductase) were assayed as described i n Materials and Methods (section 8). Cell fraction 5'-Nucleotidase NADPH-cytochrome c Reductase Activity Purification Activity Purification Units/mg Units/mg 5 x 10 Cell Lysate 2.7 1 3-6 1 1 Plasma Membranes 13 5 N.D. Endoplasmic . Reticulum N.D. - 17 5 1. Not detectable 93 contamination by the plasma membranes. 4. Pulse-Chase of Radiolabeled Semliki Forest Virus into Enriched  Plasma Membrane and Endoplasmic Reticulum Fractions  of BHK-21 C e l l s In order to distinguish between the two main possible mechanisms of entry of Semliki Forest virus into BHK-21 ce l l s , i.e. endocytosis of the whole virus particle or fusion of the v i r a l envelope with the c e l l plasma membrane, c e l l s were infected for 15 min with radiolabeled Semliki Forest v i r u s . At three d i f f e r e n t chase times, the v i r a l proteins associated with enriched plasma membrane and endoplasmic r e t i c u l u m f r a c t i o n s were s e p a r a t e d and q u a n t i t a t e d by SDS -polyacrylamide gel electrophoresis. The endoplasmic reticulum fraction was chosen to account f o r v i r u s e s i n s i d e the c e l l s (19). The radioactive gel p r o f i l e s are shown i n Figure 8. At a l l three time points, only \% of the r a d i o l a b e l added to the c e l l s was associated with these s u b c e l l u l a r f r a c t i o n s . This low incorporation could be explained either by a very low i n f e c t i v i t y of the virus preparation or by the possibility that most of the radiolabeled viruses were actually located in other subcellular fractions. The f i r s t explanation cannot be eliminated since the p a r t i c l e to PFU r a t i o of t h i s particular virus preparation was not determined. The second possibility i s more li k e l y in view of the results obtained with Sindbis virus (cf. sections 5 and 94 Distance from origin (cm) Figure 8. Incorporation of radiolabeled Semliki Forest virus proteins into enriched plasma membrane and endoplasmic reticulum fractions of BHK-21 c e l l s . 35 Semliki Forest v i r u s was radiolabeled with L.-[ S]methionine (cf.-Materials and Methods, section 7a). C e l l s were given a 15 min pulse of radiolabeled viruses at 37 C and the v i r a l proteins associated with enriched plasma membrane (PM) and endoplasmic reticulum (ER) were detected by SDS- polyacrylamide gel electrophoresis and slices from the gel (cf. Materials and Methods, section 13) after a chase of 0 min ( • • ), 30 min ( • M ) and 60 min ( •• • ). V i r a l proteins are i d e n t i f i e d : E1-E2: envelope glycoproteins; C: capsid protein. Only the top 50 mm of the 100 mm gel i s shown here since there was no significant radioactivity in the bottom part. 95 7). The e n d o p l a s m i c r e t i c u l u m f r a c t i o n may n o t have been very representative of viruses i n s i d e the c e l l s . Another complication of the experiment was that the r a d i o a c t i v i t y i n the s u b c e l l u l a r f r a c t i o n s was very low. T h e r e f o r e , a l l o f each f r a c t i o n had t o be a p p l i e d t o t h e g e l , w h i c h r e s u l t e d i n an overloading t h a t caused the v i r a l p r o t e i n s to appear c l o s e r to the o r i g i n o f the g e l than u s u a l , e s p e c i a l l y w i t h the plasma membrane fr a c t i o n s (compare w i t h the r a d i o a c t i v e p r o f i l e of pure radiolabeled v i r u s : Figure 4b). Nevertheless, the p r o f i l e s d i s p l a y e d i n F i g u r e 8 were analyzed. As expected, the v i r a l p r o t e i n s g r a d u a l l y disappeared from the plasma membrane f r a c t i o n and appeared i n the endoplasmic reticulum f r a c t i o n . The appearance o f r a d i o a c t i v i t y i n t h i s l a t t e r f r a c t i o n d i d not completely account f o r t h e l o s s from t h e former f r a c t i o n , which i n d i r e c t l y confirms the already p o s t u l a t e d non-representativity of the endoplasmic reticulum f r a c t i o n as an " i n t r a c e l l u l a r v i r u s f r a c t i o n " . The most s t r i k i n g o b s e r v a t i o n was t h a t both envelope g l y c o p r o t e i n s (E1-E2) and c a p s i d p r o t e i n s (C) appeared to move t o g e t h e r from the plasma membrane to the endoplasmic reticulum. This r e s u l t i s consistent with an endocytic mechanism but not w i t h a d i r e c t f u s i o n of the v i r a l envelope with the plasma membrane. 96 5. E f f e c t of C h l o r o q u i n e and NH^Cl on the P r o d u c t i o n  of I n f e c t i o u s S i n d b i s V i r i o n s (a) Plaque-Reduction Assays In order to determine a p o s s i b l e involvement o f lysosomes i n a productive S i n d b i s v i r u s i n f e c t i o n , the p l a q u e - r e d u c t i o n assay was used to monitor the e f f e c t o f c h l o r o q u i n e or NH^Cl on the production of i n f e c t i o u s Sindbis v i r u s p a r t i c l e s . The a d d i t i o n of chloroquine or NH^Cl to BHK-21 c e l l s d u r i n g the 1 h i n f e c t i o n p e r i o d had a marked e f f e c t on the production of i n f e c t i o u s v i r u s p a r t i c l e s (Figure 9). The v i r u s t i t e r was reduced 1 0 - f o l d by 100 pM c h l o r o q u i n e and 12-fold by 10 mM NHjjCl. The v i r u s t i t e r was s l i g h t l y v a r i a b l e as measured by d i f f e r e n t plaque assays, probably because of d i f f e r e n c e s i n the growth state of the c e l l s used. N e v e r t h e l e s s , the degree of reduction of the v i r u s t i t e r was very reproducible. There was no a p p a r e n t t o x i c e f f e c t , as determined by phase contrast microscopy, on c e l l s exposed to 100 uM chloroquine or 10 mM NHjjCl for 1 h. However, exposure p e r i o d s l o n g e r than 1 h (70, 90 and 120 min periods were t e s t e d ) w i t h 100 uM c h l o r o q u i n e or chloroquine concentrations greater than 125 pM f o r 1 h appeared somewhat toxi c to the c e l l s . T h e r e f o r e , the f u r t h e r r e d u c t i o n o f the v i r u s t i t e r with 200 pM chloroquine ( F i g u r e 9) was probably a r t e f a c t u a l . Furthermore, the presence o f 100 pM c h l o r o q u i n e o r 10 mM NH..C1 i n the agar 97 NH 4 CI c o n c e n t r a t i o n (mM) 0 50 100 150 200 C h l o r o q u i n e c o n c e n t r a t i o n (uM) Figure 9. E f f e c t o f c h l o r o q u i n e or NH^Cl c o n c e n t r a t i o n on the t i t e r of Sindbis v i r u s . Plaque-reduction assays were performed as d e s c r i b e d i n Mate r i a l s and Methods ( s e c t i o n 5b). Various c o n c e n t r a t i o n s of chloroquine ( • • ^ ^ ^ ) or NH^Cl ( O O ) were p r e s e n t d u r i n g the 1 h i n f e c t i o n p e r i o d . Each point i s the average of four dishes. 98 throughout the 3 day incubation period completely k i l l e d the c e l l s . An i n t e r e s t i n g o b s e r v a t i o n on t h e t i m i n g o f the c h l o r o q u i n e e f f e c t was t h a t t h e r e was no e f f e c t on the v i r u s t i t e r when 100 uM chloroquine was added f o r 1 h to the c e l l s at 1 h p o s t - i n f e c t i o n . In o r d e r to determine i f c h l o r o q u i n e and NH^Cl a c t e d v i a the same mechanism to reduce t h e number o f i n f e c t i o u s S i n d b i s v i r u s p a r t i c l e s , a p l a q u e a s s a y was p e r f o r m e d i n w h i c h t h e NH^Cl concentration was f u r t h e r i n c r e a s e d to 100 mM and 100 uM chloroquine was present i n o n l y one s e t of d i s h e s . The r e s u l t s are presented i n Figure 10. At low concentrations o f NH^Cl (up to 10 mM), the addition of 100 pM c h l o r o q u i n e r e s u l t e d i n a f u r t h e r r e d u c t i o n o f the v i r u s t i t e r whereas a t h i g h e r c o n c e n t r a t i o n s o f NH^Cl (50 and 100 mM), c h l o r o q u i n e d i d not markedly a l t e r t h e v i r u s t i t e r . The l a c k o f a d d i t i v e e f f e c t s o f c h l o r o q u i n e and NH^Cl when t h e c e l l s were saturated with these c h e m i c a l s suggests but does not prove that they both exert t h e i r e f f e c t s the same way. (b) E f f e c t of Chloroquine or NH^Cl on C e l l u l a r Protein Synthesis or C e l l V i a b i l i t y Even though there was no apparent t o x i c e f f e c t on c e l l s exposed to 100 uM c h l o r o q u i n e or 10 mM NH^Cl f o r 1 h ( c f . s e c t i o n 5a), the health of c e l l s exposed to these c h e m i c a l s was f u r t h e r monitored by 99 60 4 NH 4 CI c o n c e n t r a t i o n ( m M ) Figure 10. E f f e c t of NH^Cl c o n c e n t r a t i o n on the t i t e r of Sindbis virus in the absence or presence of 100 pM chloroquine. Plaque-reduction assays were performed as described in Materials and Methods (section 5b). Various concentrations of NH^Cl were present during the 1 h infection period. In one set of dishes ( ^ ), 100 pM chloroquine was present with the NH^Cl whereas in the other set of dishes ( Q O) only NH^Cl was present. The r e s u l t s are expressed as reduction of the virus t i t e r and each point i s the average of four dishes. 100 the rate at which they synthesized r a d i o l a b e l e d t r i c h l o r o a c e t i c a c i d -p r e c i p i t a b l e proteins. In one experiment, the r a d i o a c t i v i t y was added a f t e r removal of the i n h i b i t o r s ( F i g u r e 11). A 1 h exposure to 10 mM NH^Cl did not have any e f f e c t on c e l l u l a r p r o t e i n synthesis and 100 pM chloroquine only had a very s l i g h t e f f e c t . There was no l a t e e f f e c t of 100 pM c h l o r o q u i n e when the experiment was extended up to 11 h a f t e r the a d d i t i o n o f c h l o r o q u i n e , which was o n l y present during the f i r s t 1 h ( r e s u l t s not shown). However, when the c o n c e n t r a t i o n s of i n h i b i t o r s were i n c r e a s e d t o 200 pM c h l o r o q u i n e or 100 mM NH^Cl (Figure 11), t h e r e was a marked r e d u c t i o n i n the r a t e o f c e l l u l a r p r otein synthesis. Nevertheless, the c e l l s seemed to recover from the NHjjCl e f f e c t s i n c e the i n h i b i t i o n was r e v e r s e d 3 h a f t e r removal of the 100 mM NH^Cl. On the other hand, c e l l s d i d not recover from the i n h i b i t i o n by 200 pM chloroquine, at l e a s t i n the time period examined. 35 In another experiment, ]±-[ S ] m e t h i o n i n e was added wit h the i n h i b i t o r s ( F i g u r e 12). A 10 mM NH^Cl treatment s t i l l d i d not have any e f f e c t but 100 pM c h l o r o q u i n e seemed t o reduce the r a t e o f c e l l u l a r p r o t e i n s y n t h e s i s by about 2 - f o l d . T h i s e f f e c t was e a s i l y r e v e r s i b l e , as shown by t h e r e s u l t s p r e s e n t e d i n F i g u r e 11, and c e r t a i n l y does not account f o r the 1 0 - f o l d r e d u c t i o n o f the v i r u s t i t e r (Figure 9). F i n a l l y , the e f f e c t o f 100 pM c h l o r o q u i n e on c e l l v i a b i l i t y was determined with the Trypan blue dye-exclusion technique ( c f . Materials F i g u r e 11. Long-term e f f e c t o f c h l o r o q u i n e or NH^Cl on c e l l u l a r p r o t e i n synthesis. C e l l s were exposed to e i t h e r 100 pM c h l o r o q u i n e ( O""""O ) , 200 pM chloroquine ( A - A ) , 10 mM NH^Cl ( • — — • ) or 100 mM NH.C1 ( V — — * 7 ) f o r 1 h. Control d i s h e s f o r the chloroquine ( 9 # ) or the NH^Cl experiment d i d not r e c e i v e the chemical studied. A f t e r washing the c e l l sheet to remove the chemicals, each d i s h r e c e i v e d 50 ii£i ( c h l o r o q u i n e e x p e r i m e n t ) or 25 u C i (NH^Cl experiment) of L.-C S] methionine . A f t e r v a r i o u s i n c u b a t i o n times, the c e l l s were p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c a c i d , as described i n M a t e r i a l s and Methods ( s e c t i o n 11). Each point i s the average of two dishes. 102 F i g u r e 12. S h o r t - t e r m e f f e c t o f c h l o r o q u i n e or NH.C1 on c e l l u l a r p r o t e i n synthesis. The experiment was performed as d e s c r i b e d i n the legend of Figure 11, except that L-l S ] m e t h i o n i n e (25 p C i f o r the c h l o r o q u i n e or 50 p C i f o r t h e NHj.Cl e x p e r i m e n t ) was a d d e d a t t h e same t i m e as t h e i n h i b i t o r s . Only 100 pM c h l o r o q u i n e or 10 mM NhVCl was tested. 103 and Methods, s e c t i o n 12). As shown i n Table 3» c h l o r o q u i n e did not seem to a f f e c t s i g n i f i c a n t l y c e l l v i a b i l i t y . I n t e r e s t i n g l y , the c e l l s were a l l v i a b l e during the 1 h treatment with chloroquine, i n contrast with the reduction i n the r a t e o f c e l l u l a r p r o t e i n synthesis shown i n Figure 12. (c) E f f e c t of Chloroquine or NH^Cl on the Production of Radiolabeled Sindbis Virus Proteins and P a r t i c l e s In order to c o r r e l a t e the decrease i n the i n f e c t i o u s v i r u s t i t e r (Figure 9) with the number o f v i r u s p a r t i c l e s , the synthesis of v i r a l proteins was monitored at v a r i o u s times a f t e r a treatment with 100 uM chloroquine or 10 mM NH^Cl d u r i n g the i n f e c t i o n period. An experiment was performed where 100 uM chloroquine was present only during the 1 h i n f e c t i o n p e r i o d , as d e s c r i b e d i n Materials and Methods (section 13). The s y n t h e s i s o f v i r a l proteins within infected c e l l s i s presented i n F i g u r e 13a. At 3 h p o s t - i n f e c t i o n , chloroquine - treated c e l l s showed a net r e d u c t i o n i n the amount of radiolabeled v i r a l p r o t e i n s produced, but as the i n f e c t i o n c y c l e continued, the d i f f e r e n c e f r o m c o n t r o l c e l l s was p r o g r e s s i v e l y a b o l i s h e d . Furthermore, as shown i n F i g u r e 13b, c h l o r o q u i n e - t r e a t e d c e l l s released 4-fold l e s s r a d i o l a b e l e d v i r a l p r o t e i n s (incorporated i n t o progeny v i r i o n s ? ) i n t o the medium a t 5 h p o s t - i n f e c t i o n but t h i s reduction was only 2-fold at 8 h p o s t - i n f e c t i o n and n e g l i g i b l e at 11 h 104 Table 3 Effect of chloroquine on c e l l v i a b i l i t y C e l l s were treated with 100 uM chloroquine for 1 h. Control dishes did not receive the chemical. At different times after addition of chloroquine, the number of viable c e l l s on each dish was determined as described i n Materials and Methods (section 12). The standard deviation (n=4) i s indicated. Time after addition Number of viable c e l l s (x10 ) of chloroquine Control Chloroquine-treated 0 1 .6 + 0.2 -0.5 2.3 + 0.7 2.4 + 0.5 1 2.0 + 0.3 1.9 + 0.3 2 2.4 + 0.3 1 .9. + 0.2 7 2.9 + 0.2 2.4 + 0.4 105 Figure 13. E f f e c t of 100 p.M chl o r o q u i n e on the production of radiolabeled Sindbis virus proteins. Cells were infected with Sindbis virus (100 PFU/cell) in the absence (-) or presence (+) of 100 pM chloroquine. Actinomycin D (3 Mg/ml) was present throughout the experiment. The i n f e c t i o n mfidia were removed afte r 1 h and each dish received 130 pCi of L-l S]methionine i n serum-free medium at 2 h p o s t - i n f e c t i o n . At 3, 5, 8 and 11 h post-infection, the media were removed and dialyzed and the c e l l s were homogenized in H p0. The c e l l homogenates (100 pg/lane; (a)) and the dialyzed media (D) were analyzed by gel electrophoresis followed by 24 h autoradiography of the dried gels. D e t a i l s of the procedure are provided in Materials and Methods (section 13). The times of harvesting ( h p o s t - i n f e c t i o n ) are i n d i c a t e d and the v i r a l p r o t e i n s are identified: C: capsid protein; E1-E2: two envelope glycoproteins; PE2: precursor of E2 and E3; B: precursor of E1 and PE2; E3: small v i r a l glycoprotein. The origin (o) and dye front (d) of the gels are apparent at the top and bottom, respectively. 106 post-infection. Apparently, the choroquine inhibition of v i r a l protein synthesis could be reversed about 10 h a f t e r removal of the chemical from the medium. The accumulation of capsid proteins i n the c e l l s (Figure 13a) has also been reported in BHK-21 c e l l s infected with Semliki Forest virus (19,20). As expected from the growth cycle of Sindbis virus (23), practically no v i r a l proteins were released into the medium at 3 h post-infection and the amount of radiolabeled v i r a l proteins (progeny virions?) gradually increased as the i n f e c t i o n progressed (Figure 13b). As mentioned previously ( c f . section 2b), the protein E3 i s not associated with the virion, but has been shown to be released into the medium of Sindbis virus - infected c e l l s (24). Since E3 was detected in the e x t r a c e l l u l a r medium (Figure 13b), some of the other v i r a l proteins released into the medium may not have been associated with progeny virions. This possibility i s discussed later in this section. Another puzzling observation i s the presence of a protein with a M of about 40,000 which was more r a d i o l a b e l e d i n chloroquine-r treated c e l l s (Figure 13a). It i s most l i k e l y a cellular protein since the r a d i o a c t i v e background of the o r i g i n a l autoradiograph of chloroquine-treated cells was enhanced compared to control c e l l s . This might reflect the incomplete i n h i b i t i o n by the virus of host RNA and protein synthesis upon i n f e c t i o n of chloroquine-treated c e l l s . Interestingly, the o v e r a l l protein p r o f i l e s were exactly reproduced 107 when actinomycin D was omitted i n the experiment (results not shown). The exact nature of the 40K protein i s unclear; i t i s heavily stained w i t h Coomassie b l u e w i t h r o u g h l y the same i n t e n s i t y i n chloroquine-treated and c o n t r o l c e l l s and i s a l s o observed i n NH^Cl-treated c e l l s ( c f . Figure 14a). One obvious p o s s i b i l i t y i s that t h i s protein i s a c t i n , which i s apparently one of the l a s t cellular protein for which the synthesis i s blocked by Sindbis virus infection (Dr. D.C. Johnson, Washington University School of Medicine, St-Louis, MO, personnal communication). A similar experiment was done where 10 mM NH^Cl was present only during the 1 h infection period. The synthesis of v i r a l proteins within infected c e l l s i s shown i n Figure 14a: the r e s u l t s were similar to those in the chloroquine experiment. The release of radiolabeled v i r a l proteins into the e x t r a c e l l u l a r medium (Figure 14b) was inhibited to the same extent as i n the c h l o r o q u i n e experiment: s i g n i f i c a n t reduction i n NH^Cl-treated c e l l s at 5 h p o s t - i n f e c t i o n but only 2-fold at 8h p o s t - i n f e c t i o n and n e g l i g i b l e d i f f e r e n c e at 11 h p o s t - i n f e c t i o n . T h e r e f o r e , the NH^Cl i n h i b i t i o n could also be reversed about 10 h after removal of the chemical from the medium. The omission of actinomycin D y i e l d e d i d e n t i c a l r e s u l t s ( r e s u l t s not shown). As observed in the chloroquine experiment, the possibility that not a l l the v i r a l proteins released into the extracellular medium were 10* Figure 14. E f f e c t of 10 mM NH^Cl on the production of radiolabeled Sindbis virus proteins. The experiment was s i m i l a r to the one described i n Figure 13 with the following m o d i f i c a t i o n s : 10 mM NH^Cl was used; the m u l t i p l i c i t y of i n f j e . c t i o n was r e d u c e d t o 50 P F U / c e l l and t h e amount o f L.-[ S]methionine increased t o 150 uCi per d i s h ; the d r i e d g e l of the c e l l homogenates (a) was autoradiographed f o r 18 h and the dried gel of the media (b) was autoradiographed f o r 58 h. The f i g u r e i s labeled as i n Figure 13. 109 incorporated i n progeny v i r i o n s was suggested by two observations from Figures 13b and 14b. F i r s t l y , the unpackaged small glycoprotein E3 was d e t e c t e d i n t h e medium. S e c o n d l y , t h e r a t i o o f E1-E2 t o C was a p p a r e n t l y much h i g h e r ( i . e . 11-20) than i n p u r i f i e d r a d i o l a b e l e d Sindbis v i r u s p a r t i c l e s ( c f . F i g u r e 6 b ; r a t i o E1-E2 / C = 1.7), which suggested a r e l e a s e o f f r e e envelope g l y c o p r o t e i n s (E1-E2) in t o the medium. I n o r d e r t o i n v e s t i g a t e t h i s p o s s i b i l i t y , t h e NH^Cl experiment was repeated except t h a t a portion of the media at 5, 8 and 11 h p o s t - i n f e c t i o n was c e n t r i f u g e d i n order to p e l l e t the v i r u s p a r t i c l e s , as described i n Materials and Methods (section 13). We tested the e f f i c i e n c y and v a l i d i t y of t h i s u l t r a c e n t r i f u g a t i o n procedure with a p u r i f i e d p r e p a r a t i o n o f r a d i o l a b e l e d Sindbis v i r u s . The radioactive p r o f i l e o b t a i n e d ( F i g u r e 15) compared very well with the p r o f i l e of sucrose g r a d i e n t - p u r i f i e d v i r u s (Figure 6 ) . A scan of t h i s autoradiograph gave a r a t i o E1-E2 / C o f 2, i n good agreement w i t h t h e p r e v i o u s l y o b t a i n e d r a t i o o f 1.7. However, a f t e r u l t r a c e n t r i f u g a t i o n o n l y 56% o f the r a d i o l a b e l was r e c o v e r e d when compared to the d i a l y z e d s u p e r n a t a n t . Nevertheless, t h i s recovery was most l i k e l y s i m i l a r when t h i s p r o c e d u r e was a p p l i e d t o the media covering NH^Cl-treated or c o n t r o l c e l l s . Furthermore, i t might have been underestimated i n view o f a probable incomplete d i a l y s i s of the supernatant, which might s t i l l have c o n t a i n e d a s i g n i f i c a n t amount of 35 f r e e L_-[ S ] m e t h i o n i n e . 110 Figure 15. Autoradiograph of radiolabeled Sindbis virus pelleted by ultracentrifugation. 3 5 V i r u s e s were l a b e l e d with L_-[ S]methionine and p e l l e t e d by ultracentrifugation, as described i n Materials and Methods (section 13). The resuspended vir u s p e l l e t (0.6 uCi) was subjected to SDS-polyacrylamide gel electrophoresis (11$(w/v) acrylamide) and the dried gel was autoradiographed for 20 h. The peaks of v i r a l proteins and the origin and dye front of the gel are identified as in Figure 13. 111 The r e s u l t s presented i n F i g u r e 14b were exactly reproduced (re s u l t s not shown) but when the v i r u s e s i n the 11 h media were pelleted by ultracentrifugation and analyzed by gel electrophoresis and autoradiography (Figure 16), the n e g l i g i b l e difference (Figure 14b) between NH^Cl-treated and c o n t r o l c e l l s was replaced by a 2-fold reduction i n the amount of v i r u s p a r t i c l e s r e l e a s e d i n t o the ex t r a c e l l u l a r medium at 11 h post - i n f e c t i o n . The 2-fold reduction shown at 8 h post - i n f e c t i o n (Figure 14b) was also reproduced when pelleted virus particles were studied (Figure 16). However, much less virus particles were released at 8 h post-infection (Figure 16) than expected from the p r o f i l e of r a d i o a c t i v e v i r a l proteins shown i n Figure 14b. An even lower amount was detected at 5 h post-infection (results not shown). Therefore, i t appears that the NH^Cl i n h i b i t i o n of the release of virus particles into the medium was in fact not reversed completely 10 h a f t e r removal of the chemical from the medium. However, this 2 - f o l d r e d u c t i o n i n the r e l e a s e o f v i r u s p a r t i c l e s at 11 h post-infection i s d i f f i c u l t to correla t e with the 12-fold reduction of the number of i n f e c t i o u s virus p a r t i c l e s a f t e r 3 days, as shown by plaque-reduction assay ( c f . F i g u r e 9). As expected, the small glycoprotein E3 was not detected i n the progeny virions (Figure 16), which confirms the previously c i t e d published report that i t i s not packaged into mature v i r i o n s (24). Furthermore, the ratio E1-E2 / C, which was abnormally high i n Figure 14b (and 13b), was reduced to a 11 Figure 16. E f f e c t of 10 mM NHj^Cl on the production of radiolabeled Sindbis virus particles. The experiment was similar to the one described in Figure 14 with the f o l l o w i n g m o d i f i c a t i o n s : p o r t i o n s of the media at 8 and 11 h po s t - i n f e c t i o n were u l t r a c e n t r i f u g e d and the resuspended p e l l e t s a n a l y z e d by SDS- 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 and autoradiography (cf. Materials and Methods, section 13). The dried gel was autoradiographed for 9 days. The figure i s labeled as in Figure 14. 113 consistent value of about 2. Thus, these r e s u l t s suggest a previously unreported shedding of S i n d b i s v i r u s envelope glycoproteins E1 and E2 (or e i t h e r one of them) i n t o the e x t r a c e l l u l a r medium. I n t e r e s t i n g l y , the r e s u l t s shown i n Figure 16 were e x a c t l y reproduced i n the absence of actinomycin D ( r e s u l t s not shown). 6. S u b c e l l u l a r F r a c t i o n a t i o n of BHK-21 C e l l s The next step i n these studies was the use of radiolabeled Sindbis v i r u s to follow the movement o f the v i r a l p r o t e i n s i n t o s u b c e l l u l a r f r a c t i o n s of the c e l l s . The e s t a b l i s h e d d i f f e r e n t i a l c e n t r i f u g a t i o n method (26) was chosen f o r i t s s i m p l i c i t y and r a p i d i t y . Table 4 shows the r e s u l t s of a t y p i c a l s u b c e l l u l a r f r a c t i o n a t i o n . As expected, the lysosomes - mitochondria f r a c t i o n was enriched i n these organelles, as judged by a 4-fold p u r i f i c a t i o n o f each s p e c i f i c enzyme marker: a c i d phosphatase (lysosomes) and s u c c i n a t e dehydrogenase (mitochondria). S u r p r i s i n g l y , unlike BHK-21 c e l l s o btained from Flow Laboratories, 5'-nucleotidase a c t i v i t y c o u l d not be d e t e c t e d i n BHK-21F c e l l s , which were used because S i n d b i s v i r u s formed b e t t e r plaques i n the plaque a s s a y s . T h e r e f o r e , Na +-K +-ATPase a c t i v i t y was measured and found almost e x c l u s i v e l y i n the microsomal p e l l e t , which suggested the presence o f s m a l l plasma membrane v e s i c l e s , s i n c e they were not p e l l e t e d w i t h the lysosomes and m i t o c h o n d r i a . As judged by phase contrast microscopy, g r e a t e r than 85% o f the c e l l s were broken by the 114 Table 4 Subcellular f r a c t i o n a t i o n of BHK-21 cells Subcellular f r a c t i o n s from one medium d i s h (100x20mm) were prepared as described i n Materials and Methods (section 10). Three enzyme markers were assayed (cf. Materials and Methods, section 8): Na* -K+-ATPase to detect plasma membranes, acid phosphatase for lysosomes and succinate dehydrogenase for mitochondria. Cell fraction Na +-K +-ATPase Acid phosphatase Succinate dehydrogenase Activity Purif. Activity Purif. Activity Purif. Total Units Units /mg Total Units Units /mg (x10 3) Total Units Units /mg (x10 3) Homogenate Lysosomes-Mitochondria Microsomes Cytosol 53 7.7 n.d.1 22 30 3.9 6.1 1.9 0.25 168 24.3 -69 99.2 4.1 2.9 12.2 .50 62 19-5 .80 33 4.9 12 17.4 3.6 0.84 3.5 0.71 n.d. ^  1. n.d. : not detectable 115 h o m o g e n i z a t i o n p r o c e d u r e . T r e a t m e n t o f t h e c e l l s w i t h 100 juM c h l o r o q u i n e or 10 mM NH^Cl f o r 1 h d i d not a l t e r the s u b c e l l u l a r f r a c t i o n a t i o n pattern presented i n Table 4. F i n a l l y , f o r the purpose of t h e s e s t u d i e s the e l i m i n a t i o n o f mitochondrial contamination o f the lysosomes was judged unnecessary. However, one has t o keep i n mind t h a t the s u b c e l l u l a r f r a c t i o n from now on c a l l e d "lysosomes" did also contain mitochondria. 7. Mechanism of Entry of R a d i o l a b e l e d Sindbis Virus into BHK-21 C e l l s and the E f f e c t of C h l o r o q u i n e or NH^Cl on t h i s Process (a) Preliminary Experiments : 37°C, no treatment 35 V i r a l p r o t e i n s were r a d i o l a b e l e d w i t h L_- [ S]methionine i n order to follow t h e i r f a t e i n the e a r l y s t e ps a f t e r i n i t i a t i o n of the i n f e c t i o n . The r a d i o l a b e l e d v i r u s p r e p a r a t i o n was found t o have a p a r t i c l e / PFU r a t i o s i m i l a r t o t h e s t o c k v i r u s u s e d f o r the i n f e c t i o n , i . e . about 162. P r e l i m i n a r y experiments were performed at o 37 C w i t h o u t t r e a t m e n t w i t h c h l o r o q u i n e o r NH^Cl. T a b l e 5 and Figure 17 show the a s s o c i a t i o n o f r a d i o l a b e l e d v i r a l p r o t e i n s with d i f f e r e n t c e l l u l a r compartments a t e a r l y t i m e s p o s t - i n f e c t i o n . I n t e r e s t i n g l y , 50 to 60% o f t h e r a d i o a c t i v i t y found i n the c e l l l y s a t e s was a s s o c i a t e d w i t h l y s o s o m e s a s e a r l y a s 15 min 116 Table 5 Association of radiolabeled Sindbis virus  with s u b c e l l u l a r f r a c t i o n s at 37 C o Cells„were infected at 37 C with radiolabeled Sindbis virus (1.9 x 10 dpm per medium d i s h (100x20mm), 420 P F U / c e l l ) and subcellular f r a c t i o n a t i o n performed at 1 5, 30, 60 and 120 min post-infection, as described i n Materials and Methods (section 15). The r a d i o a c t i v i t y associated with each f r a c t i o n i s shown here at different times pos t - i n f e c t i o n . The proportion of radioactivity in each f r a c t i o n , as compared to the c e l l l y s a t e , i s shown between parentheses (percentage value). Each value i s the average of two parallel experiments. Cell fraction Radioactivity (dpm x _4 10 ) associated at various times post-•infection (min) 15 30 60 120 2 Control Cell lysate^ 86.7 129 187 80.8 110 Lysosomes 47.2 75.8 99.8 36.3 52.8 (54) (59) (53) (45) (48) Cytosol 5.21 12.4 38.3 35.7 12.7 (6) (10) (20) (44) (12) Microsomes 14.8 20.8 33.9 8.73 32.7 (17) (16) (18) (11) (30) 1. After subtracting r a d i o a c t i v i t y i n 1,000 x g. pellet (nuclei and unbroken c e l l s ) . 2. Radiolabeled virus (2 x 10 dpm) added to the c e l l lysate from one medium dish (100x20mm). 117 1 • " • 1 — 1 0 30 60 90 120 T ime post- infect ion (min) Figure 17. A s s o c i a t i o n of r a d i o l a b e l e d S i n d b i s v i r u s with s u b c e l l u l a r f r a c t i o n s . The experiment was performed as described i n the legend of Table 5. The r a d i o a c t i v i t y associated with lysosomes ( % # ), c y t o s o l s ( O Q ) and microsomes ) i s p r e s e n t e d . Each p o i n t i s the average of two p a r a l l e l experiments. 118 post-infection and up to at le a s t 120 min post-infection. However, a similar association of r a d i o a c t i v i t y with lysosomes was obtained when radiolabeled viruses were mixed with homogenized uninfected c e l l s and the subcellular fractionation was performed as usual (Table 5). This observation suggested a n o n - s p e c i f i c association of radiolabeled viruses with subcellular f r a c t i o n s . Viruses adsorbed to receptors on the plasma membrane might have been detached upon homogenization, n o n - s p e c i f i c a l l y adsorbed to lysosomes (and other c e l l u l a r compartments) and ca r r i e d through the f r a c t i o n a t i o n procedure. The radiolabeled viruses associated with the c e l l lysates represented 5-11$ of the v i r u s e s used f o r the i n f e c t i o n . The recoveries of radioactivity after subcellular fractionation of the c e l l lysates were in the range 77-90% and when a l l media and washes were accounted for, the o v e r a l l recoveries were 80-95$, with respect to the amount of radioactivity used for the infection. The r e l a t i v e l y high proportion of r a d i o a c t i v i t y found i n the cytosol at 120 min p o s t - i n f e c t i o n (Table 5) seemed to suggest a release of v i r a l proteins (or degradation products) into this cellular compartment. The significance of t h i s observation i s suggested by the fact that i t was not observed in the control situation (Table 5). The lysosomes were analyzed by gel electrophoresis and the v i r a l proteins quantitated from gel s l i c e s . Figure 18 shows a typical gel profile. The v i r a l proteins E1-E2 and C were well separated and the 119 71 4 4 3 0 2 2 17 14 11 M r x 1 0 " 3 F i g u r e 18. R a d i o a c t i v e p r o f i l e o f t h e l y s o s o m e s a t 30 min a f t e r i n f e c t i o n w i t h r a d i o l a b e l e d S i n d b i s v i r u s . The experiment was performed as des c r i b e d i n the legend o f Table 5. The l y s o s o m a l f r a c t i o n o f i n f e c t e d c e l l s was a n a l y z e d by SDS -polya 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 and the g e l s were s l i c e d i n t o 5 mm s l i c e s and the r a d i o a c t i v i t y q u a n t i t a t e d as d e s c r i b e d i n M a t e r i a l s and Methods ( s e c t i o n 2 c ) . The c o r r e s p o n d i n g m o l e c u l a r w e i g h t s (M ) were deduced from t h e p a r a l l e l m i g r a t i o n o f m o l e c u l a r w e i g h t markers (a s t r a i g h t l i n e was obtained when the m o b i l i t y o f each marker was p l o t t e d a g a i n s t l o g M ). E a c h v a l u e i s t h e a v e r a g e o f two p a r a l l e l e x p e r i m e n t s . The v i r a l p r o t e i n s a r e i d e n t i f i e d : E1-E2: e n v e l o p e g l y c o p r o t e i n s ; C: c a p s i d p r o t e i n . 120 remaining portions of the gel showed very low background radioactivity. Moreover, the p r o f i l e s from two duplicate experiments were almost superimposable. Therefore, each type of v i r a l proteins could be quantitated from such gel pr o f i l e s and the results are shown in Figure 19. As expected from the r e s u l t s presented i n F i g u r e 17, the radiolabeled v i r a l proteins were t r a n s i e n t l y associated with the lysosomes in the f i r s t hour af t e r i n f e c t i o n . The radioactivity which disappeared from the lysosomes between 60 and 120 min post-infection was quantitatively accounted for i n the ext r a c e l l u l a r medium and was mostly (80$) t r i c h l o r o a c e t i c acid - soluble r a d i o a c t i v i t y , probably 3 5 L.-C S]methionine. The most i n t e r e s t i n g observation i s that the envelope glycoproteins (E1-E2) seemed to peak i n the lysosomes at 60 min post-infection whereas capsid proteins (C) seemed to peak earlier, apparently at 30 min p o s t - i n f e c t i o n . T h i s suggested a physical separation of these two types of v i r a l proteins i n the lysosomes. However, i t i s possible that both proteins actually peaked between 30 and 60 min post-infection. The recoveries of r a d i o a c t i v i t y from the electrophoresis gels were around 8 5 % . Only cytosols at 60 and 120 min post-infection contained enough r a d i o a c t i v i t y to y i e l d an i n t e r p r e t a b l e radioactive p r o f i l e upon electrophoresis of the v i r a l proteins (unlike lysosomes, the high content of cellular proteins i n the cytosols only allowed the analysis of a 25% portion of t h i s subcellular f r a c t i o n ) . These p r o f i l e s are presented in Figure 20. As for lysosomes, radiolabeled v i r a l proteins 121 0 3 0 6 0 9 0 1 2 0 < T i m e p o s t - i n f e c t i o n ( m i n ) Figure 19. V i r a l proteins a s s o c i a t e d w i t h the lysosomes and release of aci d - s o l u b l e r a d i o a c t i v i t y i n t o the medium. The experiment was performed as d e s c r i b e d i n the legend of Table 5. Radiolabeled v i r a l proteins a s s o c i a t e d with lysosomes were quantitated a f t e r SDS- 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 ( c f . F i g u r e 18). The envelope glycoproteins E1-E2 ( ••"•H) ) and capsid proteins C O) are i d e n t i f i e d . Also shown i s the p r o p o r t i o n of t r i c h l o r o a c e t i c a c i d -soluble r a d i o a c t i v i t y present i n the e x t r a c e l l u l a r medium at the same times ( • •HB) . Each point i s the average of two p a r a l l e l experiments. 122 8 6 0 6 0 m i n E 1 E 2 1 2 0 m i n E 1 E 2 7 4 4 7 3 3 2 4 1 8 1 4 12 M r x 1 0 " 3 Figure 20. Radioactive p r o f i l e s of the c y t o s o l s at 60 and 120 min a f t e r i n f e c t i o n with r a d i o l a b e l e d Sindbis v i r u s . The experiment was performed as described i n the legend of Table.5. The c y t o s o l f r a c t i o n s a t 60 and 120 min p o s t - i n f e c t i o n were analyzed as described i n the legend of F i g u r e 18 f o r lysosomes. Each value i s the average o f two p a r a l l e l experiments. The f i g u r e i s l a b e l e d as f o r Figure 18. The r a d i o a c t i v i t y r e c o v e r e d was m u l t i p l i e d by 4 since only 25% of each c y t o s o l sample, could be analyzed on a g e l . 123 E1-E2 and C could be detected. However, a larger proportion of capsid proteins compared to envelope g l y c o p r o t e i n s was present i n the cytosols (ratio E1-E2 / C was 1.7-2.8 i n the lysosomes but was around 1.0 i n the c y t o s o l s ) . Furthermore, s i g n i f i c a n t r a d i o a c t i v i t y was detected at positions on the g e l s which corresponded to lower Mp (about 20,000). They most l i k e l y were degradation products of v i r a l proteins. In order to quantitate experimentally the postulated non-specific adsorption of bound viruses with subcellular fractions, especially the lysosomes, a control s i t u a t i o n whereby viruses would not enter the cells throughout the course of the experiment had to be devised. Marsh and Helenius (27) showed that less than 3% of infecting Semliki Forest viruses were internalized by BHK-21 c e l l s at temperatures lower than 10°C. Although exceptions to t h i s u s u a l l y observed (or assumed) absence of penetration of viruses into c e l l s at 4°C do exist (28), this low temperature penetration occurs at a lower rate compared to o what goes on at 37 C. Therefore, i t was decided that i f experiments on the entry of radiolabeled Sindbis virus into BHK-21 c e l l s were performed i n p a r a l l e l a t 37°C and 4°C, the a s s o c i a t i o n of radiolabeled v i r a l proteins with subcellular f r a c t i o n s at 4°C would account for at least the non-specific adsorption described above. It was assumed that any p e n e t r a t i o n at 4°C would occur by the same mechanism as at 37°C, but to a much lower extent. 124 (b) Entry of Radiolabeled Sindbis Virus : Effect of Chloroquine For the reasons d i s c u s s e d above, s i m i l a r experiments were performed at 37 and 4°C and the e f f e c t of the presence of 100 uM chloroquine during the 1 h i n f e c t i o n p e r i o d was a l s o studied. Radiolabeled Sindbis virus was prepared from plaque-purified stock virus and found to have a p a r t i c l e / PFU r a t i o of about 30. The results of the subcellular f r a c t i o n a t i o n are presented i n Table 6. Surprisingly, a lower association of radiolabeled v i r a l proteins with the c e l l lysates was found (1.8-3-5$ i n s t e a d of the previously obtained 5-11$). However, the p r e v i o u s l y postulated non-specific association of the r a d i o l a b e l e d v i r a l p r o t e i n s with d i f f e r e n t subcellular fractions was strongly suggested in this experiment by the fact that the same amount of r a d i o a c t i v i t y was associated with the c e l l lysates a f t e r a 2 min i n f e c t i o n at 4°C as a f t e r 20 min at the same temperature. Therefore, i t i s probably correct to assume that most of the radioactivity associated with the c e l l s after the infection at 4°C represented attachment of v i r u s e s to the c e l l surface. Given this assumption, one can observe that the chloroquine treatment did not affect virus attachment, since the same amount of radioactivity was associated with treated and c o n t r o l c e l l s throughout the 4°C infection. Interestingly, about 70$ of the maximum amount of viruses eventually found associated with the c e l l s at 4°C was bound by 2 min post-infection, which i n d i r e c t l y suggests a half-time for binding of even less than 2 min. The amount of r a d i o a c t i v i t y associated with the 125 Table 6 Association of radiolabeled S i n d b i s v i r u s with s u b c e l l u l a r f r a c t i o n s  at 37 and 4 C, with or without treatment w i t h 100 pM chloroquine C e l l s ^were infected at 37 or 4°C wi t h r a d i o l a b e l e d Sindbis v i r u s (5 . 6 x 10 dpm p e r medium d i s h (100x20mm), 20 P F U / c e l l ) . The r a d i o a c t i v i t y a s s o c i a t e d w i t h each s u b c e l l u l a r f r a c t i o n at d i f f e r e n t times p o s t - i n f e c t i o n i s shown here. C h l o r o q u i n e (100 pM) was present i n h a l f the dishes ( + ) d u r i n g the 1 h i n f e c t i o n period, as described i n Materials and Methods (section 15). R a d i o a c t i v i t y a s s o c i a t e d (dpm x 10 ) Time p o s t -i n f e c t i o n Tempe-r a t u r e Chloro-quine 2 - C e l l ^ Lysosomes Lysate 2 C y t o s o l Microsomes^ min °C 100 pM 2 4 + 10 .8(2.0) 10.9(2.0) 4.8(44) 5.0(46) 0.75(7) 0.55(5) 1.4(13) 1.3(12) 20 4 + 10 .6(1 .9) 12 .4(2.2) 5.2(49) 6.2(50) 0.52(5) 0.58(5) 1.8(17) 1.5(12) 37 + 14.1(2 .5) 13.8(2.5) 7.4(52) 8.3(60) 0.32(2) 0.34(2) 1.1(8) 1.4(10) 40 4 + 16.0(2 .9) 13.3(2 .4) 8.4(52) 6.4(48) 0.65(4) 0.54(4) 3.0(19) 1.7(13) 37 + 19.5(3.5) 15.8(2.8) 12.5(64) 8.0(51) 0.81(4) 0.63(4) 2 .9(15) 2.2(14) 60 4 + 14.2(2.5) 14.7(2.6) 6.4(45) 7.2(50) 0.68(5) 0.70(5) 2.7(19) 2.7(19) 37 + 17.2(3.1) 17.1(3-1) 9.4(54) 9.4(55) 0.83(5) 0.86(5) 1 .9(11) 1.7(10) 120 4 + 3.6(0 .6 ) 3.5(0.6) 2.3(64) 2.0(58) 0.12(3) 0 . 12n) 0.45(12) 0.44(13) 37 + 10 .5(1 .9) 10.0(1.8) 5.8(55) 5.4(54) 1.0 (9) 0.61(6) 0.78(7) 0.61(6) 1. Aft e r s u b t r a c t i n g r a d i o a c t i v i t y i n 1,000 x £. p e l l e t (nuclei and unbroken c e l l s ) ; values between parentheses indi c a t e the proportion of r a d i o a c t i v i t y compared to i n f e c t i n g v i r u s {%). 2. Values between parentheses i n d i c a t e the proportion of radio-a c t i v i t y compared with c e l l l y s a t e . 126 c e l l s at 4 WC decreased d r a m a t i c a l l y between 60 and 120 min p o s t - i n f e c t i o n . This r e d u c t i o n was 5.1-fold as opposed to about 1.7-fold at 37°C. This observation seemed to suggest either release of the bound viruses or a possible degradation of the viruses even when they are not internalized. The amount of radioactivity associated with the c e l l s at 37°C was maximum at 40 min p o s t - i n f e c t i o n , at which point i t was slightly lower (20$) i n chloroquine-treated c e l l s . At a l l other time points, both treated and control c e l l s contained the same amount of radiolabel. As expected, the majority of radiolabeled v i r a l proteins were found associated with the lysosomes, at a l l time points, even at 4°C, which again provides more s t r e n g t h to the assumption of non-specific adsorption of bound viruses to these cellular organelles. However, a higher proportion of r a d i o a c t i v i t y as compared to c e l l lysates (3-12$ more) was found i n the lysosomes at 37°C, compared to 4°C at a l l time points up to 60 min post-infection. This i s probably due to the actual penetration of the viruses into the lysosomes. Strangely, the large incorporation of radiolabeled v i r a l proteins into the cytosols that was observed previously (Table 5) could not be reproduced in this experiment (Table 6; untreated c e l l s ) . Instead, the proportion of radioactivity associated with this cellular compartment remained at a low level of 3-7$ of the viruses used for the infection t h r o u g h o u t the 4°C e x p e r i m e n t and at 2-5$ up to 60 min 127 p o s t - i n f e c t i o n at 3 7 VC. The o n l y i n t e r e s t i n g o b s e r v a t i o n i n the c y t o s o l s was at 120 min p o s t - i n f e c t i o n i n t h e 37°C experiment, whereby c o n t r o l c e l l s showed 33$ more r a d i o a c t i v i t y associated with t h e i r c y t o s o l s . U n f o r t u n a t e l y , t h e r e was not enough r a d i o a c t i v i t y i n t h e s e f r a c t i o n s t o o b t a i n an i n t e r p r e t a b l e p r o f i l e upon g e l electrophoresis. F i n a l l y , the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o the microsomal p e l l e t was v a r i a b l e and the c h l o r o q u i n e treatment did not seem to a f f e c t i t . A l l l y s o s o m a l f r a c t i o n s w e r e f u r t h e r a n a l y z e d by g e l electrophoresis. V i r a l g l y c o p r o t e i n s (E1-E2) and capsid proteins (C) were found to be always present i n the same proportion ( r a t i o E1-E2 / C = 1 .7-2.1), except a t 120 min p o s t - i n f e c t i o n (4°C experiment only) where t h i s r a t i o was reduced t o 1.4-1.5 i n the lysosomes o f both chloroquine-treated and c o n t r o l u n t r e a t e d c e l l s . This co-migration of envelope glycoproteins and c a p s i d p r o t e i n s suggested the i n t r a c e l l u l a r m i g r a t i o n o f whole v i r a l p a r t i c l e s . T h e r e f o r e , the r e s u l t s were expressed as the sum of E1-E2 and C, as shown i n Figures 21 and 22. Figure 21 shows t h a t i n u n t r e a t e d c e l l s , the amount of viruses o which became a s s o c i a t e d w i t h lysosomes a t 37 C g r a d u a l l y increased up to 60 min p o s t - i n f e c t i o n whereas t h i s amount was constant at 4°C and reached as e a r l y as 2 min p o s t - i n f e c t i o n . C h l o r o q u i n e - t r e a t e d c e l l s showed a r e l a t i v e l y s i m i l a r p a t t e r n at 4°C. T h e r e f o r e , the previous s u g g e s t i o n o f n o n - s p e c i f i c a d s o r p t i o n o f v i r u s e s with the 128 Figure 2 1 . Incorporation o f r a d i o l a b e l e d S i n d b i s v i r u s i n t o lysosomes at 37 and 4 C and the e f f e c t o f chloroquine. The experiment was performed as d e s c r i b e d i n the legend of Table 6. Radiolabeled v i r a l proteins a s s o c i a t e d with lysosomes were quantitated a f t e r SDS- 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 ( c f . F i g u r e 1 8 ) . The amount o f v i r u s e s was assumed t o be t h e sum o f t h e e n v e l o p e glycoproteins (E1-E2) and the c a p s i d p r o t e i n s (C). The experiment was done at 3 7°C ( ' ) and 4°C ( ). H a l f the d i s h e s ( • ) received 100 pM chloroquine (ch) up to 60 min p o s t - i n f e c t i o n . Control (co) dishes ( • ) d i d not receive t h i s chemical. 129 2 0 4 0 6 0 8 0 1 0 0 1 2 0 T i m e p o s t - i n f e c t i o n ( m i n ) Figure 22. Specific incorporation of radiolabeled Sindbis virus into lysosomes and the effect of chloroquine. The experiment was performed as described in the legend of Table 6 and Figure 21. From the latte r figure, the amount of radiolabeled viruses incorporated into lysosomes at 37 C was corrected by subtracting this amount at 4 C. The figure i s labeled as in Figure 21. 130 lysosomes was assumed to be correct. The actual penetration of viruses into the lysosomes was obtained by subtracting the amount of viruses associated with these c e l l u l a r organelles at 4°C from the results obtained at 37°C. These r e s u l t s are presented i n F i g u r e 22. Interestingly, viruses seemed to accumulate i n the lysosomes of chloroquine-treated cells at 20 min post-infection (4-fold increase). However, the amount of v i r u s e s a s s o c i a t e d with lysosomes was subsequently reduced so that there was no difference with control c e l l s at 40 min p o s t - i n f e c t i o n and a 3- f o l d reduction at 60 min post-infection. The accumulation of t r i c h l o r o a c e t i c acid - soluble radioactivity i n the e x t r a c e l l u l a r medium was i d e n t i c a l as i n the previous experiment (Figure 19) and chloroquine treatment did not markedly alter i t . Interestingly, removal of chloroquine from the medium at 60 min post-infection yielded a s i g n i f i c a n t increase in the incorporation of viruses with lysosomes (Figure 22, chloroquine-treated c e l l s , 120 min post-infection). (c) Entry of Radiolabeled Sindbis Virus : Effect of NH^Cl The experiment described i n the preceeding section was repeated with NHjjCl instead of chloroquine. Radiolabeled Sindbis virus was also prepared from plaque-purified stock virus and had a simil a r p a r t i c l e / PFU r a t i o ( i . e . 36). The r e s u l t s of the subcellular 131 fractionation are shown i n Table 7. The association of radiolabeled v i r a l proteins with the c e l l lysates was s l i g h t l y higher than in the chloroquine experiment (2.3-5.6$ i n s t e a d of 1.8-3.5$). These variations might l i e in a s l i g h t difference in the radiolabeled virus or more l i k e l y i n the growth state of the c e l l s , which i s probably impossible to reproduce exactly from one experiment to the other. If i t i s again assumed that most of the radioactivity associated with the c e l l s a f t e r i n f e c t i o n at 4°C represents attachment of viruses to the c e l l s u rface, NH^Cl d i d not markedly a f f e c t t h i s binding, since roughly the same amount of radioactivity was associated with treated and control c e l l s up to 60 min p o s t - i n f e c t i o n . The apparent increased binding of viruses on treated c e l l s at 120 min post-infection has an unclear meaning. The amount of radioactivity associated with untreated c e l l s at 37°C was maximum at 60 min post-infection (as opposed to 40 min i n the previous experiment). However, N H ^ C 1 - t r e a t e d c e l l s showed a maximum at 20 min post-infection and this amount gradually decreased thereafter. Except at 20 min p o s t - i n f e c t i o n , where NH^Cl-treated c e l l s incorporated s l i g h t l y more radiolabeled viruses, the NH^Cl treatment decreased t h i s o v e r a l l i n c o r p o r a t i o n (about 2 - f o l d at 60 and 120 min post-infection). The subcellular distribution of radioactivity was comparable with 1 3 2 Table 7 Association of radiolabeled Sindbis virus with subcellular fractions  at 37 and 4 C f with or without treatment with 10 mM NH^Cl C e l l s were Anfected at 37 or 4°C with radiolabeled Sindbis virus (4.65 x 10 dpm per medium dish ( 100x20mm), 20 PFU/cell). The radioactivity associated with each sub c e l l u l a r fraction at different times post-infection i s shown here. NH^Cl (10 mM) was present i n half the dishes ( + ) during the 1 h i n f e c t i o n period, as described in Materials and Methods (section 15). Radioactivity associated (dpm x 10~ ) Time post-infection Tempe-rature NH^Cl 2 C e l l Lysosomes Lysate 2 Cytosol Microsomes^ min °C 10 mM 2 4 + 13.2(2.8) 12.8(2.8) 6.7(51) 6.4(50) 0.40(3) 0.41(3) 1.6 (12) 1.1 (9) 20 4 + 15.3(3-3) 17.7(3.8) 7.6(49) 8.1(46) 0.47(3) 0.61(4) 2.2 (14) 2.0 (11) 37 + 14.1(3.0) 17.4(3.7) 7.8(55) 9.4(54) 0.48(3) 0.77(4) 0.78(6) 1.3 (7) 40 4 + 14.4(3.1) 15.1(3.2) 6.7(47) 8.3(55) 0.27(2) 0.32(2) 2.0 (14) 1.3 (9) 37 + 21.4(4.6) 15.3(3.3) 10.8(50) 8.1(52) 0.54(2) 0.40(3) 1.8 (8) 1.8 (11) 60 4 + 11.1(2.4) 11.6(2.5) 5.3(48) 5.0(43) 0.25(2) 0.28(2) 0.78(7) 1.1 (10) 37 + 26.0(5.6) 13.5(2.9) 13.8(53) 7.0(51) 1.1 (4) 0.33(2) 0.98(4) 0.51(4) 120 4 + 1.8(3.9) 2.3(4.9) 0.9(48) 1.2(54) 0.04(2) 0.04(2) 0.16(8) 0.07(3) 37 + 1.9(4.0) 1.0(2.3) 5.8(31) 4.0(38) 1.1 (6) 0.37(4) 1.3 (7) 0.70(7) 1. After subtracting r a d i o a c t i v i t y i n 1,000 x g_ pellet (nuclei and unbroken c e l l s ) ; values between parentheses indicate the proportion of radioactivity compared to infecting virus (SO. 2. Values between parentheses indicate the proportion of radio-activity compared with c e l l lysate. 133 the r e s u l t s obtained i n the previous experiment with chloroquine. However, the p r o f i l e of presumed actual penetration of the viruses into the lysosomes (proportion of radioactivity found in the lysosomes at 37°C, compared to 4°C) was less clear, which sheds some doubt on the significance of these values. When the l y s o s o m a l f r a c t i o n s were a n a l y z e d by g e l electrophoresis, the p r e v i o u s l y observed constant proportion of envelope glycoproteins (E1-E2) to capsid proteins (C) was reproduced, except at 120 min post-infection (4°C), where the ratio E1-E2 / C was reduced from 1.7-2.0 to 0.7 i n the lysosomes of the control c e l l s only. The same r e d u c t i o n occured i n NH^Cl-treated c e l l s but at 37°C r a t h e r than 4°C. As shown i n Fi g u r e 23, the p r o f i l e of gradual incorporation of radiolabeled viruses i n the lysosomes of untreated c e l l s was reproduced, i n an even more striking way (Figure 21). However, t h i s p r o f i l e from NH^Cl-treated c e l l s at 37°C was surprisingly similar to the p r o f i l e s obtained at 4°C in both control and treated c e l l s . When the non-specific association i s accounted for, as shown i n F i g u r e 24, the r e s u l t s a r e even more s t r i k i n g . Radiolabeled viruses did not seem to penetrate the lysosomes of treated c e l l s , at le a s t up to 40 min pos t - i n f e c t i o n . However, there seemed to be a very low incorporation at 60 min post-infection. Upon removal of the chemical from the e x t r a c e l l u l a r medium, a significant burst of r a d i o a c t i v i t y into the lysosomes was observed (Figure 24, NH,.Cl-treated c e l l s , 120 min p o s t - i n f e c t i o n ) , such that there 134 1 ' ' • . 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 T i m e p o s t - i n f e c t i o n ( m i n ) Figure 23. Incorporation of r a d i o l a b e l e d Sindbis virus into lysosomes at 37 and M C and the e f f e c t of NH^Cl. The experiment was performed as descri b e d i n the legend of Table 7. Radiolabeled v i r a l , proteins a s s o c i a t e d with lysosomes were quantitated a f t e r SDS- polyacrylamide g e l e l e c t r o p h o r e s i s ( c f . F i g u r e 18). The f i g u r e i s l a b e l e d as i n F i g u r e 21, e x c e p t t h a t 10 mM NH^Cl (NH) replaced chloroquine. 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 T i m e p o s t - i n f e c t i o n ( m i n ) Figure 24. S p e c i f i c i n c o r p o r a t i o n o f r a d i o l a b e l e d Sindbis v i r u s i n t o lysosomes and the e f f e c t o f NH^Cl. The experiment was performed as d e s c r i b e d i n the legend of Table 7 and Figure 2 3 . From the l a t t e r f i g u r e , £he amount of radiolabeled viruses i n c o r p o r a t e d i n t o lysosomes at 3 7 C was c o r r e c t e d by s u b t r a c t i n g t h i s amount a t 4 C. The f i g u r e i s l a b e l e d as i n F i g u r e 2 1 , except that 10 mM NH^Cl (NH) r e p l a c e d chloroquine. 136 appeared to be no d i f f e r e n c e from c o n t r o l c e l l s at 120 min post-infection. The accumulation of t r i c h l o r o a c e t i c acid - soluble radioactivity in the extracellular medium was i d e n t i c a l as i n previous experiments (Figure 19) and NH^Cl treatment did not significantly alter i t . Finally, i t i s very interesting to note that this experiment was also performed with radiolabeled Sindbis virus which had a much higher particle / PFU r a t i o ( i . e . 147), since i t was made from the regular stock of virus (Table 1). Even though a p a r a l l e l experiment at 4°C was not done, the p r o f i l e of incorporation of radiolabeled viruses into lysosomes at 37°C was very s i m i l a r with the p r o f i l e presented i n Figure 2 3 ( p a r t i c l e /PFU = 36). Furthermore, the subcellular distribution of r a d i o a c t i v i t y shown i n Table 7 was also reproduced (results not shown). 137 DISCUSSION 1. Mechanism of Entry of Semliki Forest Virus The pulse-chase experiment of radiolabeled Semliki Forest virus into enriched plasma membrane and endoplasmic reticulum fractions of BHK-21 cel l s (Figure 8) showed that the envelope glycoproteins (E1-E2) and capsid proteins (C) appeared to migrate together from the plasma membrane to the endoplasmic reticulum f r a c t i o n i n the f i r s t hour of the i n f e c t i o n . Apparently, whole v i r u s p a r t i c l e s were shortly associated with the plasma membrane and were rapidly transfered to the inside of the c e l l s . This r e s u l t i s consistent with an endocytic mechanism. A fusion pathway for entry would show a physical separation of the two types of v i r a l proteins. The envelope glycoproteins would be incorporated in the plasma membrane while the capsid proteins would enter the cytoplasm and thus g r a d u a l l y disappear from the plasma membrane fraction, to which they would only be transiently associated. Therefore, the co-migration of the v i r a l proteins associated with the v i r a l envelope with those a s s o c i a t e d with the core of the virus particle suggests the penetration of the intact v i r i o n . This could occur directly through the plasma membrane, or more l i k e l y as a result of a receptor-mediated endocytosis, as has been suggested recently by Helenius and co-workers (27,29). Obviously, the p o s s i b i l i t y exists that the v i r a l envelope glycoproteins incorporated into the plasma 138 membrane as a r e s u l t o f a f u s i o n p r o c e s s were r a p i d l y degraded and, thus, appeared to leave the plasma membrane with the capsid proteins. However, i f t h i s were o c c u r i n g , t h e g r a d u a l appearance o f these envelope g l y c o p r o t e i n s i n the endoplasmic r e t i c u l u m would not have been observed. Even though these r e s u l t s are consistent with an endocytic pathway fo r entry of Semliki Forest v i r u s , they do not conclusively show that endocytosis i s the main i n f e c t i o u s mechanism of penetration. F i r s t l y , the p a r t i c l e to PFU r a t i o o f t h e v i r u s p r e p a r a t i o n i s not known. Secondly, o n l y \% o f t h e r a d i o l a b e l u s e d d u r i n g t h e p u l s e was subsequently found i n the plasma membrane and endoplasmic reticulum f r a c t i o n s , p r o b a b l y due to the u n f o r t u n a t e c h o i c e o f endoplasmic reticulum as a f r a c t i o n designed to represent the i n s i d e of the c e l l s . I f r e c e p t o r - m e d i a t e d e n d o c y t o s i s were t h e main pro c e s s by which Semliki F o r e s t v i r u s gained access to the c e l l s , o t h e r s u b c e l l u l a r f r a c t i o n s would be e x p e c t e d t o show a s s o c i a t e d v i r u s p a r t i c l e s , presumably e n d o c y t i c v e s i c l e s , lysosomes and the c y t o s o l . F i n a l l y , the p o s s i b i l i t y t h a t v i r u s e s adsorbed on the plasma membrane were detached upon h o m o g e n i z a t i o n o f t h e c e l l s and n o n - s p e c i f i c a l l y a d s o r b e d t o t h e e n d o p l a s m i c r e t i c u l u m was n o t e l i m i n a t e d . Nevertheless, c r o s s - c o n t a m i n a t i o n o f plasma membrane and endoplasmic r e t i c u l u m i s u n l i k e l y ( F i g u r e 7 and T a b l e 2 ) . U n f o r t u n a t e l y , r e s t r i c t i o n s on the use o f S e m l i k i F o r e s t v i r u s f o r r e s e a r c h were brought about by a r e p o r t on t h e p o s s i b i l i t y t h a t t h i s v i r u s was 139 responsible f o r a f a t a l case o f human e n c e p h a l i t i s ( 3 0 ) . Therefore, further i n v e s t i g a t i o n on the mechanism o f e n t r y o f t h i s v i r u s i n t o BHK-21 c e l l s had to be halted. 2 . Mechanism of E n t r y of Sind b i s Virus (a) E f f e c t of Chloroquine and NH^Cl on the Production of Infectious Sindbis Vi r i o n s The plaque-reduction assay ( F i g u r e 9 ) , demonstrated c l e a r l y that chloroquine and NH^Cl have a very s i g n i f i c a n t i n h i b i t o r y e f f e c t on the p r o d u c t i o n o f i n f e c t i o u s S i n d b i s v i r u s p a r t i c l e s . Furthermore, chloroquine apparently acts at an e a r l y stage of i n f e c t i o n since there was no e f f e c t on the v i r u s t i t e r when 1 0 0 pM chloroquine was added f or 1 h at 1 h p o s t - i n f e c t i o n . However, these chemicals were t o x i c to the c e l l s when p r e s e n t at h i g h enough c o n c e n t r a t i o n and/or f o r a long enough time ( c f . Results, section 5 a ) . H e l e n i u s e_t a_l. ( 2 9 ) f o u n d a r e d u c t i o n o f t h e y i e l d o f i n f e c t i o u s S e m l i k i F o r e s t v i r u s when BHK -21 c e l l s were treated with 1 0 0 pM c h l o r o q u i n e or 1 0 mM NH^Cl. However, i n t h e i r a s s a y s , the c e l l s were i n c o n t a c t w i t h these c h e m i c a l s f o r 6 h, a c o n d i t i o n we found would k i l l the c e l l s w i t h i n 3 days. I n t e r e s t i n g l y , they claimed from t h e i r o b s e r v a t i o n s on the e f f e c t o f d i f f e r e n t l y s o s o m o t r o p i c 140 agents that these weak bases i n h i b i t e d the i n f e c t i v e pathway by increasing the pH in, the lysosomes and thus blocking the fusion from within the lysosomes that would release the nucleocapsid into the cytosol. This conclusion was mainly based on evidence from Ohkuma and Poole (33) t h a t l y s o s o m o t r o p i c a g e n t s a c t u a l l y r a i s e d the intralysosomal pH from 4.7-4.8 to 5.4-6.3. However, the potency of each drug to r a i s e the intralysosomal pH did not d i r e c t l y correlate with i t s inhibitory e f f e c t on the y i e l d of infectious Semliki Forest viruses. For example, 1 mM tributylamine or 0.5 mM amantadine rapidly raised the intralysosomal pH to 5.6 and 5.4, respectively (33). Therefore, one would expect tributylamine to be equivalent or more potent to block the v i r a l i n f e c t i o n . S t r a n g e l y , Helenius and co-workers found that 1 mM tributylamine only halved the virus yield whereas 0.5 mM amantadine reduced i t by a f a c t o r of 10-fold. Similarly, 10 mM methylamine, 100 uM chloroquine and 10 mM NH^Cl a l l produced a similar rapid raise of the intralysosomal pH to 6.2-6.3 but reduced the virus yield to d i f f e r e n t degrees (interestingly, we found 100 uM chloroquine and 10 mM NH^Cl to produce a similar reduction of the v i r u s t i t e r ) . Nevertheless, Helenius and co-workers recently provided more i n d i r e c t evidence on t h e i r hypothesis of fusion from within the lysosomes (31,32). They showed a pH-dependent fusion of Semliki Forest virus with e i t h e r liposomes or c e l l s i n culture. However, some d i r e c t evidence i s s t i l l needed to confirm t h i s interesting pathway. . 141 Coombs et a l . (34) also showed a s i g n i f i c a n t reduction in the yield of Sindbis virus by 100 uM chloroquine. However, the cells were in contact with the chemical for 13 h (cytotoxic according to our results). Moreover, cytochalasin B did not reduce the virus yield but blocked ingestion of virus p a r t i c l e s , as shown by electron microscopy. Thus, they claimed that endocytosis i s not essential for infection of cel l s by this virus (as well as by vesicular stomatitis virus). We found that the rate of c e l l u l a r protein synthesis was reduced 2-fold when 100 uM chloroquine was present i n the culture medium of uninfected c e l l s (Figure 12). T h i s r e s u l t suggested that, unlike NHjjCl, chloroquine might have side e f f e c t s on the c e l l s , even at a concentration which d i d not seem t o x i c to the c e l l s (Table 3). Although u n l i k e l y , i t i s p o s s i b l e that chloroquine i n h i b i t e d the synthesis of a specific set of cellular proteins that are essential for the infection. F i n a l l y , the r e s u l t s shown i n Figures 13 to 16 provided more insight on the e f f e c t of chloroquine and NH^Cl on the production of Sindbis virus proteins and p a r t i c l e s . These experiments confirmed that these chemicals i n i t i a l l y reduced the amount of radiolabeled v i r a l proteins produced within the c e l l s . However, this reduction was shown to be re v e r s e d by 11 h p o s t - i n f e c t i o n ( F i g u r e s 13 and 14). Interestingly, the release of v i r u s particles was only reduced 2-fold, 10 h after removal of the 10 mM NH^Cl from the medium (Figure 16), 142 whereas the i n f e c t i v i t y was reduced 12-fold af t e r 3 days (Figure 9). This discrepancy i s d i f f i c u l t to explain. One p o s s i b i l i t y i s that a greater p r o p o r t i o n of the v i r u s e s produced a f t e r a NH^Cl (or chloroquine) treatment are defective than in the normal situation. The plaque-reduction assay i s a very sen s i t i v e monitor of the infection and any slight alteration of the normal pathway w i l l show up strongly. On the other hand, the radiolabeling of v i r a l proteins might not pick up these differences to a s i m i l a r extent since v i r a l proteins may be synthesized but not packaged i n t o i n f e c t i o u s progeny v i r i o n s . Furthermore, experiments on the i n c o r p o r a t i o n of radiolabeled precursors r e l y on the i m p l i c i t assumption that i n f e c t i o n does not al t e r the size of precursor pools or k i n e t i c s with which they are labeled (40). Nevertheless, the most important observation from these experiments is that well-known lysosomotropic agents did block to a certain extent the infectious route of Sindbis virus infection, which strongly suggests the involvement of lysosomes in this process. Other interesting observations were made i n these experiments. F i r s t l y , the overproduction of capsid proteins i n the infected cells that was previously observed (19,20) was reproduced. Secondly, a protein with a of about 40,000 was more radiolabeled in the cells treated with either lysosomotropic agent. Presumably, this reflected an incomplete i n h i b i t i o n of host RNA and protein synthesis upon infection of drug-treated c e l l s . It was suggested that this protein was probably actin, although the importance of this finding i s remote 143 to the purpose of these studies. Lastly, a previously unreported shedding of Sindbis virus envelope glycoproteins (E1 and/or E 2 ) i n t o the e x t r a c e l l u l a r medium was i n d i r e c t l y suggested. A s i m i l a r phenomenon has been reported i n vesicular stomatitis virus-infected c e l l s (35). G proteins appeared to be cleaved at the c e l l surface, e s p e c i a l l y when the M protein was unstable or synthesized by a temperature-sensitive mutant. Since the M protein i s essential for the budding mechanism (36), these results suggested release of the externally exposed portion of G proteins mainly in the absence of budding. S i m i l a r l y , murine leukemia virus -specific antigens were also detected in the c e l l culture medium (37) or in the serum of infected mice (38). Release of such v i r a l antigens, even in abortive infections, might-play a role in v i r a l pathogenesis. No reduction in the M of E1 and/or E 2 was observed, unlike the r ' cleavage of G protein, which y i e l d e d a fragment of G with a = 54,000 (M p of G = 65,000). However, i t has been shown that only a few amino acid residues of E2 and e s p e c i a l l y E1 are exposed on the internal side of the l i p i d b i l a y e r (39). Therefore, cleavage of these glycoproteins at the c e l l surface would only very s l i g h t l y reduce their and this might not have showed up upon gel electrophoresis. The exact mechanism of shedding of Sindbis virus envelope glycoproteins was not examined. 144 (b) Incorporation of Radiolabeled Sindbis Virus into Subcellular Fractions A very surprising observation was made when ce l l s from a different source were used: even though they were a l l BHK-21 c e l l s , 5'-nucleotidase a c t i v i t y could only be detected i n c e l l s obtained from Flow L a b o r a t o r i e s ( c f . R e s u l t s , s e c t i o n 6 ) . The most l i k e l y explanation for this r e s u l t i s that even though both types of BHK-21 cells i n i t i a l l y originated from the same strain (clone 13, MacPherson and Stoker), they have mutated d i f f e r e n t l y upon continuous subculture in different laboratories. Therefore, the necessity of using the same batch of ce l l s for a complete set of experiments i s obvious. i ) Preliminary Experiment When c e l l s were infected with r a d i o l a b e l e d Sindbis virus at 37°C, most of the r a d i o a c t i v i t y was apparently associated with the lysosomes as early as 15 min post-infection (Table 5 and Figure 17). As shown in Figure 19, the v i r a l proteins were gradually associated with t h i s s u b c e l l u l a r f r a c t i o n . Furthermore, an apparent physical separation of the capsid proteins (C) from the envelope glycoproteins (E1-E2) was observed (E1-E2 and C peaked at 60 and 30 min, respectively). Moreover, Figure 20 showed that the proportion of capsid proteins i n the cytosol at 60 and 120 min post-infection was higher than in the lysosomes or in t a c t viruses. At the same time, a 145 higher proportion of r a d i o a c t i v i t y was found i n the cytosol (Table 5 and Figure 17). These r e s u l t s are c o n s i s t e n t with the lysosomal uncoating mechanism of fusion of the v i r a l envelope from within these organelles, as proposed by Helenius and co-workers (27,29,31,32). However, the presence of envelope glycoproteins i n the cytosols (Figure 20) i s not accounted for by t h i s hypothesis but may be the result of some shedding from the lysosomal membrane, as was observed from the plasma membrane. Unfortunately, the physical separation of capsid from envelope glycoproteins in the lysosomes of Sindbis virus-infected cells could not be reproduced in later experiments, which suggests that in Figure 19, both types of proteins a c t u a l l y reached a peak between 30 and 60 min post-infection. Another problem i s that a similarly high proportion of r a d i o a c t i v i t y i n the lysosomes was obtained when radiolabeled viruses were mixed with homogenized u n i n f e c t e d c e l l s and the s u b c e l l u l a r f r a c t i o n a t i o n performed as usual (Table 5 ) . This observation, as well as r e s u l t s obtained at 4°C (viruses bound to the c e l l s at t h i s temperature were ca r r i e d through the homogenization procedure; cf. Results, sections 7b and c), suggested a non-specific association of radiolabeled viruses with subcellular fractions. In spite of a l l these reservations, the accumulation of capsid proteins i n the cytosol at 60 and 120 min p o s t - i n f e c t i o n (Figure 20) i s probably a significant observation, even though lower incorporation of r a d i o a c t i v i t y i n l a t e r experiments did not allow us to study this 146 process in more details. Finally, i t i s i n t e r e s t i n g to note that v i r a l proteins appeared to be degraded a f t e r 60 min p o s t - i n f e c t i o n (Figure 19). The t r i c h l o r o a c e t i c a c i d - s o l u b l e r a d i o a c t i v i t y ( p r o b a b l y 35 L_-[ S]methionine) l i b e r a t e d by t h i s degradation was immediately r e l e a s e d i n t o the c u l t u r e medium. However, i t could not be demonstrated i f a l l v i r a l proteins were degraded simultaneously and i f this a l l happened i n the lysosomes. Presumably, i f the nucleocapsid were released into the cytoplasm, the v i r a l RNA would have to be freed of i t s capsid p r o t e i n coat i n order f o r r e p l i c a t i o n to s t a r t . Therefore, the capsid proteins might be degraded in the cytoplasm. i i ) Chloroquine Effect The preliminary experiment discussed above was performed with a virus preparation that contained 162 non-infectious virus particles for each infectious v i r i o n . Therefore, the results obtained from such an experiment could ultimately represent a non-infectious pathway of entry which might only lead to degradation of the infecting viruses. Thus, viruses were prepared which had a lower particle to PFU ratio of 30. Although better, this r a t i o was s t i l l r elatively high but was not reduced further. As mentioned i n the Results section, s p e c i f i c v i r u s entry at 147 o 37 C was o b t a i n e d a f t e r c o r r e c t i n g f o r bound v i r u s e s t h a t were c a r r i e d through the homogenization procedure, as determined by the r e s u l t s o f t h e e x p e r i m e n t p e r f o r m e d a t 4 ° C . A c o - m i g r a t i o n o f envelope g l y c o p r o t e i n s and c a p s i d p r o t e i n s was observed i n the lysosomes, which suggested the i n t r a c e l l u l a r migration of whole v i r u s p a r t i c l e s . However, t h i s o b s e r v a t i o n does not exclude the p o s s i b i l i t y of lysosomal uncoating: capsid proteins released i n t o the cytosol might have attached to the outside of the lysosomes. A l t e r n a t i v e l y , physical s e p a r a t i o n o f the c a p s i d p r o t e i n s from the envelope g l y c o p r o t e i n s might have occured i n the lysosomes a f t e r f u s i o n from w i t h i n these o r g a n e l l e s but enough e n v e l o p e g l y c o p r o t e i n s were shed i n t o the cytoplasm to y i e l d an o v e r a l l E1-E2 / C r a t i o t h a t was only s l i g h t l y reduced ( c f . Results, section 7b: a t 120 min p o s t - i n f e c t i o n , E1-E2 / C = 1.4-1.5 instead of 1.7-2.1). With these remarks i n mind, the experiment appeared to confirm the postulated mechanism o f a c t i o n o f chloroquine as a lysosomotropic agent. The i n c o r p o r a t i o n o f v i r u s e s i n the lysosomes o f untreated c o n t r o l c e l l s probably represented an average value which corresponded to a concomitant entry of some v i r u s e s i n t o and e x i t of other viruses (or only t h e i r nucleocapsid) from the lysosomes. As expected, viruses accumulated i n the lysosomes o f c h l o r o q u i n e - t r e a t e d c e l l s at 20 min p o s t - i n f e c t i o n (Figure 22), possibly because they could not escape from these organelles. However, something seemed t o happen to the viruses accumulated i n the l y s o s o m e s o f t r e a t e d c e l l s at 40 and 60 min 148 post-infection so that their number appeared to decrease (Figure 22). This observation might be explained i n two ways: either the viruses trapped i n the lysosomes were somehow degraded or chloroquine accelerated the entry of viruses so that the peak of v i r a l proteins in the lysosomes occured at 20 rather than 60 min post-infection. In view of the r e s u l t s discussed i n section 2a, the l a t t e r p o s s i b i l i t y i s unlikely. Therefore, we must assume that degradation of viruses in the lysosomes of chloroquine-treated c e l l s i s possible even though the increase of the intralysosomal pH probably inactivated many lysosomal hydrolases. Given t h i s c o n c l u s i o n , the s i m i l a r accumulation of trichloroacetic acid - soluble r a d i o a c t i v i t y in the medium of treated or control c e l l s (cf. Results, section 7b) has to be explained by the fact that a s i m i l a r amount of v i r a l proteins were degraded i n the control situation (uncoating?) than viruses degraded in the lysosomes of treated c e l l s . The s i g n i f i c a n t i n c r e a s e of i n c o r p o r a t i o n of viruses with lysosomes after removal of chloroquine from the medium (Figure 22, 120 min post-infection) might be the r e s u l t of a renewed burst of entry that followed release of the viruses which were s t i l l trapped in the lysosomes. i i i ) NHjjCl E f f e c t When the previous experiment was repeated with NH„C1 instead of chloroquine, unexpected r e s u l t s were obtained. NH^Cl did not seem to alter binding of the viruses on the c e l l surface (Table 7 and Results, section 7c) but apparently prevented penetration of viruses into the lysosomes, as shown i n Figure 24. Upon removal of the chemical from the medium, a burst of penetration was observed (Figure 24, 120 min post-infection). Therefore, NH^Cl appeared to block a step between binding on receptors on the plasma membrane and penetration into the lysosomes, which puts some doubt on the interpretation of the results presented i n Figure 10 (which were thought to indicate a s i m i l a r mechanism of action of c h l o r o q u i n e and NH^Cl since there was no a d d i t i v e e f f e c t of these two chemicals on the v i r u s t i t e r ) . I n t e r e s t i n g l y , M a x f i e l d e_t al. (41) showed that i n c u l t u r e d f i b r o b l a s t s , the c l u s t e r i n g of receptors to coated p i t s (42) was e s s e n t i a l f o r e n d o c y t o s i s to o c c u r . They found t h a t f o r o<2-macroglobulin and epidermal growth factor (EGF) , this clustering (and thus r e c e p t o r - m e d i a t e d e n d o c y t o s i s ) was blocked i n a dose-dependent fashion by various amines. The inhibition was complete when the amines, i n c l u d i n g ammonium acetate (NH^* C00~), were present at a concentration of 10-30 mM. Chloroquine (100 pM) did not have any e f f e c t on t h i s process. Van Leuven e_t a_l. (43) also observed an i n h i b i t i o n of uptake of o< 2-macroglobulin by primary amines and not chloroquine. However, their evidence pointed towards an inhibition of receptor recycling rather than clustering of receptors. By analogy with these r e s u l t s , i t i s plausible that NH^Cl inhibits Sindbis virus i n f e c t i o n of hamster f i b r o b l a t s (BHK-21 c e l l s ) by a 150 similar mechanism, rather than acting as a lysosomotropic agent, as was postulated by Helenius e_fc. JLL. (29). F i n a l l y , very s i m i l a r r e s u l t s were obtained when the virus preparation had a particle to PFU r a t i o increased to 147, rather than 36. This fact sheds some doubt on the possible c r i t i c i s m that the lysosomal pathway observed with radiolabeled Sindbis virus might not be the infectious pathway and strengthens the r e s u l t s discussed in section 2a. 3. Conclusion The r e s u l t s presented i n t h i s thesis strongly suggest that a productive Sindbis virus (and possibly Semliki Forest virus) infection requires functional lysosomes. Thus, receptor-mediated endocytosis (viropexis) i s probably the main infectious mechanism for entry of this virus into BHK-21 c e l l s . Various macromolecules such as lipoproteins (42,44) and peptide hormones (45-47) u t i l i z e a receptor-mediated lysosomal pathway for entry into the c e l l . It seems l o g i c a l that a c e l l u l a r parasite such as a v i r u s might also e x p l o i t t h i s route. Nevertheless, our r e s u l t s do not exclude the p o s s i b i l i t y that alphaviruses might actually u t i l i z e more than one route of entry. 151 4. Suggestions for Future Work The main problem with these studies has been the necessity of obtaining a radiolabeled virus preparation with a very high specific radioactivity and low p a r t i c l e / PFU r a t i o , in order to significantly follow the fate of v i r a l proteins a f t e r internalization of the virus p a r t i c l e s . Marsh and Helenius (27) claimed that they obtained radiolabeled Semliki Forest virus preparations which contained only 2.4-2.8 particles per infectious unit (PFU). Such a preparation would be a definitive asset i n the int e r p r e t a t i o n of results and should be sought further. Moreover, the s p e c i f i c r a d i o a c t i v i t y of the virus 125 35 could probably be improved by using I instead of S. V i r u s e s could a l s o be made from c e l l s p r e l a b e l e d with a phospholipid precursor such as choline. Upon budding, viruses would pick up labeled phospholipid (e.g. phosphatidylcholine). Infection of unlabeled c e l l s with such a virus preparation would provide insight on the fate of th i s phospholipid of the v i r a l envelope. Similarly, the v i r a l RNA could also be radiolabeled. A l t e r n a t i v e l y , v i r a l envelope glycoproteins could be l a b e l e d with a sugar such as [ H]mannose (unglycosylated capsid proteins would not be labeled) and a l l v i r a l 35 proteins with L±-[ S]methionine as usual. Therefore, the fate of capsid proteins and envelope glycoproteins could be distinguished more clearly. 152 The nature of Sindbis virus receptors on the c e l l surface could be studied. Histocompatibility antigens were i n i t i a l l y claimed to be the receptors for Semliki Forest virus i n mouse and human c e l l s (48). However, c e l l s lacking these antigens were s t i l l susceptible to infection by this virus (49). Therefore, histocompatibility antigens are probably not s p e c i f i c receptors. The use of antibodies against Sindbis v i r u s for the i s o l a t i o n of virus-receptor complexes would provide a very helpful tool in such an investigation. Viruses could be bound to c e l l surface receptors and treated with antibodies linked to a voluminous p r o t e i n such as Staphylococcus aureus protein A. After extraction of the protein A-antibodies-virus-receptor complexes with detergent, the receptor could be analyzed by different techniques such as polyacrylamide gel electrophoresis. Furthermore, monoclonal antibodies against d i f f e r e n t Sindbis virus proteins could be used to study i n more details the fate of these proteins inside the c e l l s , as well as the presumed shedding of envelope glycoproteins into the culture medium. Finally, i t would be very i n t e r e s t i n g to investigate the nature of the defect that makes so many v i r u s p a r t i c l e s non-infectious (particle / PFU greater than 1). When we used two radiolabeled Sindbis virus preparations with widely d i f f e r e n t particle / PFU ratios (36 and 147; c f . s e c t i o n 2 b i i i ) , a s i m i l a r lysosomal pathway was quan t i t a t i v e l y reproduced, which suggests that the nature of the defect does not l i e in the fact that non-infective particles enter the 153 c e l l s v i a a d i f f e r e n t , d e g r a d a t i v e r o u t e . However, -the use of v i r u s preparations w i t h a p a r t i c l e / PFU r a t i o c l o s e r to 1 would provide more unequivocal r e s u l t s . Studies on v i r a l entry i n t o c e l l s are a very important, although d i f f i c u l t , area of modern v i r o l o g y . A m u l t i d i s c i p l i n a r y approach w i l l be e s s e n t i a l f o r the s o l u t i o n of the remaining problems and biochemists can c e r t a i n l y have a very s i g n i f i c a n t r o l e i n t h i s t a s k . 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