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Molecular biological studies of rubella virus structural proteins Qiu, Zhiyong 1994

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MOLECULAR BIOLOGICAL STUDIES OF RUBELLA VIRUSSTRUCTURAL PROTEINSbyZhiyong QiuB.Sc., Fudan University, Shanghai, China, 1984A thesis submitted in partial fuilfifiment ofthe requirements for the degree ofDoctor of Philosophyinthe Faculty of Graduate StudiesGenetics ProgramWe accept this thesis as conformingto the required standardTheJune, 1994©Zhiyong Qiu, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of_________________The University of British ColumbiaVancouver, CanadaDate ‘(Signature)DE.6 (2/88)AbstractRubella virus (RV) is a small enveloped RNA virus in the Togaviridae family. The virion containsthree structural proteins, a capsid protein (C) associated with the genomic RNA to form the nucleocapsidand two membrane glycoproteins, El and E2. The RV structural proteins are translated as a polyproteinprecursor p110 (NH2-C-E2-E1-COOH) from a RV-speciflc 24S subgenomic RNA and derived byposttranslational processing of p110.The role of N-linked glycosylation of El and E2 on their respective biological functions has beenstudied by expressing glycosylation mutants of El and E2 generated by oligonucleotide-directedmutagenesis on coding cDNA. Expression of the E2 mutant proteins in COS cells indicated that removalof any of the glycosylation sites resulted in slower glycan processing, lower protein stability and aberrantdisulfide bonding of the mutant proteins, with the severity of defect depending on the number and locationof deleted carbohydrate sites. Expressed El glycosylation mutant proteins from vaccinia recombinants wererecognized by a panel of El-specific monoclonal antibodies, indicating that carbohydrate side chains onEl are not involved in the constitution of epitopes recognized by these monoclonal antibodies. All the Elglycosylation mutants were capable of eliciting anti-RV El antibodies in immunized mice; however, onlythe single glycosylation mutants were found to be capable of inducing viral neutralizing antibodies,suggesting that carbohydrates on El are important for maintaining proper protein folding and epitopeexposure.Assembly of RV was found to be independent of the genomic RNA but strictly dependent uponthe co-expression of C, E2 and El, in stable cell lines expressing RV structural proteins. Assembly andrelease of RV vinon was dramatically reduced in RV-infectecl cells treated with two Golgi transportinhibitors, brefeldin A and monensin, although there was no significant alteration for the expression andprocessing of the structural proteins. My finding indicates that stable association of RV El and E2 withthe intact Golgi complex is essential for efficient RV assembly.11Table of ContentsABSTRACT iiLIST of TABLES viLIST of FIGURES viiLIST of ABBREVIATIONS ixACKNOWLEDGEMENTS xi1. INTRODUCTION 11.1. N-linked glycosylation of viral glycoproteins 11.1.1. Structure and biosynthesis 11.1.2. Approaches for functional analysis of N-linked glycosylation 51.1.3. Biological functions of N-linked glycosylation 71.2. Targeting of viral membrane glycoproteins and virus assembly 151.2.1. General concepts 151.2.2. Virus assembled at the plasma membrane 191.2.3. Virus assembled in the Golgi complex 231.2.4. Virus assembled in the ER, the Golgi complexor a pre-Golgi compartment 251.2.5. Closing remarks 271.3. Rubella virus biology 291.3.1. Classification 291.3.2. Clinical aspects 291.3.3. Morphology and morphogenesis 301.3.4. Nucleic acids and genome organization 311.3.5. Non-structural proteins 321.3.6. Structural protein expression and processing 331.3.7. Posttranslational modification of RV structural proteins 351.3.8. Intracellular localization of RV structural proteins 401.3.9. Biological function of RV structural proteins 401.3.10. Immune responses to RV infection 411.3.11. Immunological determinants on RV structural proteins 431.3.12. Project rationale and thesis objectives 452. MATERIALS AND METHODS 472.1. MATERIALS and SUPPLIES 472.2. METHODS 482.2.1. Propagation of bacterial strains 482.2.2. Preparation of competent cells and transformation 482.2.3. DNA preparation and handling 492.2.4. Expression vectors 522.2.5. DNA-mediated transfection 532.2.6. Construction of vaccinia recombinants 551112.2.7. Metabolic labelling 572.2.8. Immunoprecipitation 582.2.9. Endoglycosidase digestion 582.2.10. Immunoblotting 592.2.11. Indirect immunofluorescence 592.2.12. Electrophoresis 602.2.13. RV propagation, purification and titration 612.2.14. Electronmicroscopy 632.2.15. Mice immunization 632.2.16. Enzyme linked immunoadsorbant assay (ELISA) 642.2.17. Hemagglutination and hemagglutination inhibition assays 642.2.18. Viral neutralization assay 652.2.19. Lymphoproliferative assay 663. RESULTS and DISCUSSIONS 673.1. Section I. Role of N-linked glycosylation on E2 processingand transport 673.1.1. E2 cDNAs 673.1.2. Determination of functional N-linked glycosylation sites in E2 673.1.3. Expression of E2 glycosylation mutants in COS cells 693.1.4. Formation of aberrant disuffide bonding in E2 mutant proteins 713.1.5. Glycan processing and intracellular stability of E2 proteins 743.1.6. Intracellular localization of mutant E2 proteins 763.1.7. Secretion of an anchor-free form of wild-type andmutant E2 proteins 793.1.8. Summary and Discussion for section I 813.2. Section II. Effect of Brefeldin A (BFA) and monensin on proteinprocessing and virus assembly 863.2.1. Processing of N-linked oligosaccharides on E2 863.2.2. Processing of 0-linked glycans on E2 883.2.3. Processing and secretion of an anchor-free form of E2 913.2.4. Proteolytic processing of RV structural protein precursor 913.2.5. Subcellular distribution 933.2.6. Effect of BFA and monensin on RV assembly and release 953.2.7. Assembly of virus particles 973.2.8. Summary and Discussion for section II 1013.3. Section III. Influence of N-linked glycosylation on theantigenicity and immunogenicity of El glycoprotein 1063.3.1. Construction of recombinant vaccinia viruses expressingRV El glycosylation mutants 1063.3.2. Expression and antigenicity of El glycosylation mutants. 1063.3.3. Immunogenic properties of expressed El glycosylation mutants 112iv3.3.4. Antigenic properties of deglycosylated RV El from RV virions 1133.3.5. Effect of glycosylation on El cell surface expression 1153.3.6. Summary and Discussion for section III 1183.4. Section IV. Expression and characterization of virus-likeparticles containing rubella virus structural proteins 1223.4.1. Isolation of BilK cell lines expressing RV structural proteins 1223.4.2. Expression of RV structural proteins 1253.4.3. Assembly and release of virus-like particles instable BHK-24S cells 1253.4.4. Electron-microscopic analysis of the VLPs 1283.4.5. Antigenicity of the VLPs 1313.4.6. Immunogenicity of the VLPs 1313.4.7. Summary and Discussion for section IV 137SUMMARY AND PERSPECTIVES 140REFERENCES 145vLIST of TABLESPageTable 1. Specificity of glycosidases used in this study 6Table la. Summary of properties of monoclonal antibodies directed to El 109aTable 2. Comparison of the HAT and VN antibodies from mice immunized withvaccinia recombniants containing different RV El glycosylationmutant cDNA inserts 114Table 3. A. HA assay of deglycosylated RV virionB. Effect of deglycosylation of RV El on antibody recognition byEl-specific monoclonal antibodies 117Table 4. Immunoreactivity of the VLPs with RV-specific monoclonal antibodies 132Table 5. Comparison of antibody titres of mouse sera from mice immunized withdifferent RV antigens 135viLIST of FIGURESPageFigure 1. Processing of N-linked oligosaccharides to a representativebiantennary complex structure 3Figure 2. Topography of the genome RNA of RV 34Figure 3. General strategy for the expression and processing ofRV structural proteins 36Figure 4. Schematic representation of mammalian cell expressionvectors used in this study 54Figure 5. Schematic representation of wild-type and glycosylationmutants of RV E2 68Figure 6. Determination of the number of N-linked glycans on RV E2 70Figure 7. Expression of wild-type and glycosylation mutants of E2in COS cells 72Figure 8. Formation of aberrant disulfide bonding in E2 glycosylationmutants 73Figure 9. Western blot analysis of steady-state wild-type and mutantE2 proteins in transfected cells under reducing andnon-reducing conditions 75Figure 10. Time course for glycan processing of wild-type andmutant E2 proteins 77Figure 11. Intracellular stability of wild-type and mutant E2 proteins 78Figure 12. Indirect immunofluorescence of wild-type and mutantE2 proteins in COS cells 80Figure 13. Intracellular processing and secretion of a soluble formof wild-type and mutant E2 proteins 82Figure 14. Effect of BFA and monensin on processing of E2 87Figure 15. Glycosidase digestion of E2 from BFA- and monensin-treated cells 89Figure 16. Effect of BFA and monensin on processing and secretionof an anchor-free form of E2 92Figure 17. Effect of BFA and monensin on the proteolytic cleavageof the polyprotein precursor for RV structural proteins 94Figure 18. Indirect immunofluorescence of RV structural proteins in cellstransfected with pCMV5-24S and treated with BFA or monensin 96Figure 19. Release of virus particles in infected cells 98Figure 20. Titration of cell-associated and released virus 99Figure 21. Electron microscopic analysis of virus assembly 100Figure 22a. Schematic representation of wild-type and glycosylationmutants of RV El 107Figure 22b. Expression of El glycosylation mutants by vaccinia recombinants 108Figure 23. Immunoblot analysis of El glycosylation mutants expressedby vaccinia recombinants 110Figure 24. Immunoblot analysis of sera from mice immunized with Elvaccinia recombinants 112viiEffect of deglycosylation on the antigenicity of RV virionIndirect immunofluorescence of El glycosylation mutants ininfected CV- 1 cellsDiagrammatic representation of RV cDNAs used in theconstruction of recombinant plasmidsImrnunoblot analysis of proteins expressed intransformed BHK cellsB. Immunoblot analysis of proteins sedimented byultracentrifugationTime course of VLPs secretion from BHK-24S cellsPurification of VLPs and RV on sucrose density gradientcentrifugationElectron microscopic analysis of the VLPs in BHK-24S cellsRadioimmunoprecipitation of RV structural proteins expressedin COS cellsFigure 33. Lymphoproliferation responses of mice immunized with VLPsFigure 25.Figure 26.Figure 27.Figure 28. A.Figure 29.Figure 30.Figure 31.Figure 32.116119124126127129130134136viiiLIST of ABBREVIATIONSAP ampicillinATP adenosine triphosphateB CIP 5-bromo-5-chloro-3-indolyl phosphate(3-Me 13-mercaptoethanolBHK baby hamster kidney cell linebp base pairBSA bovine serum albuminCon A concanavalin ADHFR dihydrofolate reductaseDMEM Dulbecco’s modified Eagle’s mediumDNase deoxyribonucleaseDTT DithiothreitolEDTA ethylene diaminetetraacetic acidELISA enzyme-linked immunosorbent assayER endoplasmic reticulumFCS fetal calf serumGC Golgi complexHA hemagglutinationHAT hemagglutination inhibitionkb kilobasekDa kilodaltonLB L brothM molar concentrationmA milliampmAb monoclonal antibodyMEM minimum essential mediumMOl multiplicity of infectionNET nitro blue tetrezolium0D405 absorbance at 405 nm wavelengthPAGE polyacylamide gel electrophoresisPBS phosphate-buffered salinepfu plaque forming unitPMSF phenylmethylsulfonyl fluorideRK rabbit kidney cell lineRNase ribonucleaserpm rotation per minuteRT room temperatureRV rubella virusS Svedberg unitSDS sodium dodecyl sulfateSFV Semliki Forest virusSIV Sindbis virusixSV4O Similian virus 40TM tunicamycinTRICT tetramethyfrhodamine isothiocyanateTris trishydroxymethylaminomethaneVLP virus-like particleVN viral neutralizingVSV vesicular stomatitis virusWGA wheat germ agglutininxACKNOWLEDGEMENTSFirst, I would like to thank my supervisor, Dr. Shirley Gillam, for her advice, supportencouragement and enthusiasm during my graduate studies, and for her help both in my researchand my life.I also want to thank the other members of my supervisory committee, Drs. Caroline Astell,Peter Candido and Frank Tufaro for their helpful advice, suggestion and discussions. Specialthanks to Dr. Aubrey Tingle for taking time to provide criticism of the manuscripts.Thank you to members of Shirley’s lab, Tom, David, D.C., Helen, Helena, Nina,Zhewei for their help, and to many people around in the Research Centre for creating such awonderful working environment.Lastly to my wife for her understanding throughout.This thesis is dedicated to my parents.xi1. INTRODUCTIONThis introduction contains three subsections: an overview of biosynthesis and biological functionsof asparagine-linked (N-linked) glycosylation on viral glycoproteins, recent progress in viralglycoprotein targeting and virus assembly, and a review of rubella virus biology. The rationaleand objectives of this thesis will conclude the chapter.1.1. N-linked glycosylation of viral glycoproteinsN-linked glycosylation is one of the most common post-translational modifications of proteinsin the exocytic pathway of eukaryotic cells. Animal viruses utilize host-cell glycosylationmachinery to synthesize and process oligosaccharides attached to viral glycoproteins. Theexpression of viral antigens in cells has proven to be a useful system for studying the stepwiseevents in glycan processing and intracellular transport along the exocytic pathway. From a largenumber of viral glycoproteins studied so far, none of the carbohydrate structures identified isunique to viral glycoproteins; they are also present in a variety of other membrane and secretoryglycoproteins. However, the impact of N-linked glycosylation on conformation and subsequentlyon biological functions of viral glycoproteins varies with the protein in question.1.1.1. Structure and biosynthesis of N-linked carbohydrate on viral glycoprotein1.1.1.1. Transfer of oligosaccharide from a glycan-lipid precursorAll of the N-linked carbohydrates on viral glycoproteins is synthesized and processed by host cellenzymes following the general pathway for this class of glycans (for review see Komfeld andKornfeld, 1985). As the first step in the biosynthesis of glycoproteins, a core structure-1-(Glc3Man9GlcNAc2)is assembled on the lipid carrier dolichol phosphate in the lumen of theendoplasmic reticulum (ER). Upon the translocation of newly synthesized polypeptide into theER, the oligosaccharide is transferred, by oligosaccharide transferase, to the asparagine residuesof polypeptides at the sequence Asn-X-Ser/Thr where X can be any amino acid except proline(Fig.l) (Kornfeld and Kornfeld, 1985). The oligosaccharide is covalently attached to theasparagine residues with a N-glycosidic linkage. A survey of protein sequences has revealed thatnot all the potential N-linked glycosylation sites are glycosylated (Kornfeld and Kornfeld, 1985).A properly oriented and accessible Asn-X-Ser/Thr motif in the protein is believed to play a majorpart in determining the efficiency of glycosylation. Sequential processing of N-linked oligosaccharideOnce the oligosaccharide becomes polypeptide bound, it undergoes a series of trimming reactionsby glycan modifying enzymes in the secretory pathway (Fig. 1) (for review see Kornfeld andKornfeld, 1985). Three glucose residues are first removed in the ER by glucosidases, followedby the removal of mannose residues by ER mannosidase or by x-mannosidase I present in theGolgi cisternae. At this stage, the oligosaccharide intermediate containing 5 to 8 mannoses isrecognized by the Golgi enzyme, acetylgiucosidase. This enzyme catalyzes the addition of anacetyiglucoside residue to the free mannose linked x1-3 to the mannose residues. Afterward, twomore mannose residues are removed by Golgi mannosidase II, leaving a free mannose for thefurther addition of acetyiglucoside. After the acetylglucoside is added, the oligosaccharidebecomes biantennary and is subjected to further addition of residues of galactose, fucose or sialicacid, catalyzed by glycosyl-transferases in the trans Golgi or trans-Golgi network, completing-2-‘cDTurncamycinblocks lhesyn thesisof lhisIDoER t.1annosidaseFig.1 Processing of N-linked oligosaccharides to a representative biantennary complex structure.The scheme depicts the processing from the transfer of Glc3Man9lcNAc2from its dolicholpyrophosphoryl derivative to the nascent polypeptide chain still bound to the ribosome, followedby processing reactions in the ER and GC. Oligosaccharide processing enzymes are listed abovethe line; the reaction they catalyze is depicted below the line (except for the alternate processingreaction, 3a). The subcellular localization of processing events are dipicted by the brackets.Structures susceptible or resistant to digestion by endo H, an enzyme frequently used as adiagnostic test for processing to complex structures, are indicated. Symbols: v, glucose; 0,mannose; , N-acetylglucosamine; •, galactose; , fucose; O, sialic acid. (Moreman and Touster,1988)yEnoopIsmsc H Goc ComplexReticulum-3-the assembly of the complex type oligosaccharide. Depending on the accessibility to glycanmodifying enzymes and cell types, the extent of processing can be varied for differentglycoproteins (for review see Kienk, 1990) or for oligosaccharides at different sites on the sameprotein species (Pollack and Atkinson, 1983), and this may explain the vast diversity of glycandifferentiation in viral glycoproteins. Characterization of the structure of N-linked glycansThe extent of N-linked oligosaccharide processing can be monitored using glycosidase digestionor lectin binding assays (Montreuil et al., 1986). Glycosidases are excellent tools to elucidate theprimary structures of glycans by sequential degradation of oligosaccharides bound to thepolypeptide backbone. Basically two types of enzymes are used: exoglycosidases hydrolyseglycosidic bonds of monosaccharides in terminal non-reducing positions and may achieve astepwise degradation of the glycans; endoglycosidases hydrolyse internal glycosidic bonds. Eachendoglycosidase usually recognizes a certain type of glycosidic linkage (Montreuil et al., 1986)(Table 1). By analysing sugar moieties that are liberated from the protein backbone with paperchromatography or FPLC, the structure of N-glycans on a protein can be defined.Lectins are sugar-binding proteins or glycoproteins of non-immune origin whichagglutinate cells and/or precipitate glycoconjugates (Lis and Sharon, 1984). Lectins are powerfultools for characterizing structure of oligosaccharides on glycoproteins because they bind withhigh specificity to certain types of glycoconjugates (Montreuil et al., 1984). Practically,immobilized lectins are widely used for isolating sugar components whereas fluorescence reagentconjugated lectins are used for visualizing subcellular compartmentation.-4-1.1.2. Approaches for functional analysis of N-linked glycosylationSeveral approaches have been used to define the functional roles of N-linked carbohydrateaddition in cells. These include the use of agents that interfere with glycosylation (for review eeElbein, 1987), elimination of each N-linked glycan addition site on DNA or cDNA byoligonucleotide-directed mutagenesis and the use of glycan-processing-deficient cell lines.Tunicamycin, a nucleoside analog, is the most widely used glycosylation inhibitor (Elbein,1987). This antibiotic inhibits the transfer of G1cNAc-1-P from UDPG1cAc to dolichol phosphate(Fig. 1). Since this step is the first in the lipid-linked pathway, tunicamycin treatment results inthe synthesis of proteins that lack N-linked glycosylation (Elbein, 1987). Drugs that inhibitspecific steps in the processing pathway have become available and have been extensively usedin the study of the role of oligosaccharide processing intermediates in biological functions andtransport of glycoproteins (Elbein, 1987). Furthermore, compounds such as brefeldin A (Fujiwaraet al., 1988) and monensin (reviewed by Mollenhauer et al., 1990) which disrupt vesicularstructures of cells and thus interfere with the normal distribution of resident glycan processingenzymes, have also been widely used in analyzing the effect of aberrantly processed glycans onthe transport and function of glycoproteins.While treatment with the above mentioned drugs may lead to altered cell metabolism(Elbein, 1987; Mollenhauer et al., 1990), the elimination of glycosylation sites on coding DNAor cDNA of a protein by site-directed mutagenesis has proven to be a valuable method to analyzethe influence of carbohydrate site chain addition on glycoproteins in cells under normal growthconditions, especially when proteins with more than one N-linked glycosylation site are beingexamined. Expression and characterization of mutant viral glycoproteins with partial or-5-Table 1. Specificity of glycosidases used in this study (Montreuil et al, 1986)Endoglycosidase H (Man)—Man\z-Man—G1cNAc- 1—G1cNAc-Asn/x—ManIyActive on N-linked oligosaccharides of glycopeptides. Enzymecleaves only high mannose structures [n=2-150, x=(Man)12 y andz=H] or hybrid structures (n=2, x and/or y =NANA-Gal-G1cNAcor similar, z=H or G1cNAc).Glycopeptidase F x\w—Man\u—Man--G1cNAc--G1cNAc- 1—Asn/y—Man/zActive on N-linked oligosaccharides of glycopeptides. Enzymecleaves high mannose structures (w, x and y= one or more Manresidues, u and z=H) or hybrid structures (w and x=Man, y and/orz=NANA-Gal-G1cNAc or similar, u=H or G1cNAc) or complexstructures (y and w=NANA-Gal-G1cNAc or similar, x and z=H,NANA-Gal-G1cNAc or similar, u=H or GlcNAc).Neuraminidase NANAt2-1—X or NGNAa2-1—XEnzyme cleaves terminal sialic acid residues which are cL2,3-, a2,6-or x2,8-linked to: Gal, G1cNAc, Ga1NAc, NANA, NGNA,oligosaccharide, glycolipid or glycoprotein.-6-complete depletion of N-linked glycosylation sites have provided greater understanding of thefunction of N-glycans.A large number of cell lines have been isolated that are deficient in glycan processingenzymes. Infection of these cells with viruses and the analysis of protein expression, processingand virus production have yielded a great deal of useful information about the function ofoligosaccharide side chains on proteins (Kennedy, 1974; Schlesinger et al., 1976; Hsieh andRobbins, 1984).1.1.3. Biological functions of N-linked glycosylationN-linked glycosylation influences a number of properties of proteins, including stability,intracellular transport, biological activity and antigenicity (for review see Olden et al., 1982;Klenk, 1990). There are no general rules existing regarding the consequences of changing thenormal glycosylation pattern on a protein. Results from experiments using tunicamycin haveindicated that the requirement for glycosylation is intrinsic to a given protein (Olden et al., 1982).Furthermore, in some cases, the biological effect of N-linked glycosylation depends on theparticular site within the polypeptide chain. Role of carbohydrate in initiating protein folding and maintaining protein stabilityOne of the most important functions of N-linked glycosylation is to initiate and maintain proteinfolding into its proper configuration. N-linked sugars are added co-tianslationally to thepolypeptide chain as the consensus sequences emerge on the luminal side of the ER membrane,prior to or during folding. Inhibition of N-linked glycosylation, using tunicamycin or elimination-7-of N-linked glycosylation sites by mutagenesis, leads to misfolded proteins, such as aggregatedor disulfide cross-linked complexes (Machamer et aL, 1985; Rose and Doms, 1988; Ng et aL,1990; Vidal et aL, 1992). It is believed that the hydrophilic oligosaccharides render foldingintermediates more soluble and less likely to form irreversible aggregates (Rose and Doms,1988).The importance of individual N-linked oligosaccharide side chains depends on the proteinin question as well as the location of the glycosylation site within the protein. Elimination ofsome sites may result in little or no defect whereas others may be essential for correct foldingand stability (Ng et aL, 1990; Sodora et al., 1991; Roberts et aL, 1991; Pique et al., 1992).Elimination of individual glycosylation sites can also lead to the generation of temperature-sensitive (ts) mutants (Gallagher et al., 1988; Machamer and Rose, 1988; Ng et al., 1990) andthe effect of such elimination can be additive and strain-specific (Machamer et al., 1985;Gallagher et al., 1992).Oligosaccharides on glycoproteins are believed to provide protection against proteaseattack (reviewed by Olden et al., 1982). After treatment with glycosidases to remove glycans onglycoproteins, the polypeptide backbones become more susceptible to protease digestion (Oldenet al., 1982). On the other hand, a higher turnover rate has been observed for many glycosylationmutant proteins and for glycoproteins from cells treated with glycosylation inhibitors (Ng et al.,1990; Sodora et al., 1991; Roberts et al., 1991). The deleterious effect of elimination ofglycosylation on protein stability parallels that of protein folding (Ng et al., 1990; Sodora et al.,1991). Proteins misfolded due to inhibition of glycosylation by tunicamycin or deletion ofglycosylation sites have a low solubiity and tend to form aggregates in the ER and be degraded-8-(Ng et al., 1990; Sodora et al., 1991; Roberts et al., 1991). Role of carbohydrate in facilitating intracellular transport and processingIt is now clear that carbohydrate itself does not determine the subcellular destination of viralglycoproteins. However, glycosylation greatly enhances the movement of many glycoproteins outof the ER, although this often results through effects on protein folding. It is possible though, thatoligosaccharides could have the effect of increasing transport rates by generating affinitiestowards enzymes sequestered in the exocytic pathway.The requirement for carbohydrate in the transport of membrane and secretory proteins isnot universal and is highly protein-specific (reviewed by Olden et al., 1982). Some proteins aretransported and function normally when glycosylation is inhibited with tunicamycin, whereasothers exhibit folding defects, frequently resulting in protein aggregation in the ER or rapiddegradation (Machamer et al., 1985; Rose and Doms, 1988; Hurtley and Helenius, 1989; Ng etal., 1990). It has also been shown that the arrest of transport of misfolded viral protein in the ERcan be rescued at a lower temperature (Gallagher et al., 1988; Machamer and Rose, 1988; Ng etal., 1990), which supports the concept that retention in the ER is due to protein misfolding.Addition of supernumerary carbohydrate side chains, (e.g. for VSV G and influenza virus HA),can also have the similar effect, disrupting folding and transport (Doms et al., 1988; Gallagher,et al., 1988; Machamer and Rose, l988a). However, some degree of flexibility has been observedin the position on the polypeptide backbone at which carbohydrate chains are required (Doms etal., 1988, Machamer and Rose, 1988b). A single amino acid substitution of VSV G protein hasbeen found to eliminate the stringent carbohydrate requirement for cell surface expression of the-9-mutant VSV G protein (Pitta et al., 1989).Carbohydrates may affect in two ways, the proteolytic processing of viral precursorglycoprotein: as a general requirement for proper protein folding and subsequently transport tothe site of cleavage, or as a factor modulating protein configuration within or adjacent to thecleavage site for the access of protease. Inhibition of N-linked glycosylation inhibits theprocessing of Sindbis virus envelope protein E2 (Leavitt et al., 1977) and the Newcastle Diseasevirus F glycoprotein (Morrison et al., 1985) due to failure in transport of nonglycosylated proteinsto the Golgi complex for cleavage. Using oligonucleotide-directed mutagenesis, others have foundthe site-specific influence of glycosylation on cleavage of the CKIPenn strain of avian influenzavirus HA (Deshpande et al., 1987), Friend murine leukemia virus envelope protein (Kayman etal., 1991) and measles virus fusion protein (Alkhatib et al., 1994). Role of carbohydrate in modulating biological activities of proteinsThe envelope glycoproteins of animal viruses may be involved in the attachment of virus to thehost cell receptor, and fusion between the virus envelope and the cell membrane; they also serveas the target for host immune surveillance (discussed below). The importance of N-glycosylationof viral glycoproteins in receptor binding has been illustrated in some detail for humanimmunodeficiency virus type I (HIV- 1) envelope glycoprotein gp 120 (Fenouillete et al., 1990).Deglycosylation of HW-1 gpl2O by glucosidase digestion results in a less than 10-fold reductionof the ability to bind to CD4, the cellular receptor for HP/-i (Fenouillet et al., 1990).Deglycosylation also significantly reduces but does not abolish, HIV- 1 binding to and infectivityof CD4 cells (Fenouillete et al., 1990). Anticarbohydrate monoclonal antibodies (mAbs) can- 10 -block infection by cell-free virus as well as inhibiting syncytium formation, probably throughsteric hindrance (Hansen et al., 1990). However, nonglycosylated HIV-1 gpl2O synthesized inthe presence of tunicamycin fails to bind to CD4 (Li et al., 1993). These data suggest thatglycosylation of gp 120 is essential to create a conformational epitope to which CD4 binds, butis not directly involved in CD4-binding.In contrast, treatment of respiratory syncytial virus with N-glycanase and 0-glycosidaseunder mild conditions to remove readily accessible carbohydrate from respiratory syncytial virusglycoprotein results in a significant loss of virus infectivity (Lambert, 1988), suggesting thatcarbohydrate exerts a considerable influence on the attachment and/or penetration function of theviral glycoproteins. Similarly, enzymatic removal of N-linked oligosaccharide fromhemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 leads to achange in the interaction of HN with the host receptor, sialic acid (Gorman et al., 1991).Site-specific contribution of N-linked glycosylation on a viral protein towards theirbiological activities has been studied in several viruses employing mutagenesis approach.Mutations at particular sites in envelope glycoproteins have been found to be responsible for lossof infectivity of Friend murine leukemia virus (Kayman et al., 1991), and a decreased inductionof polykaryon formation in measles virus (Alkhatib et al., 1994) and HW-1 (Dedera et al., 1992).Finally, lack of glycosylation at a conserved site in influenza virus neuraminidase has been foundto confer strain-specific neurovirulence in mice (Li et al., 1993). Role of carbohydrate in influencing antigenic propertiesCarbohydrates on viral glycoproteins can modulate the antigenicity and immunogenicity of viral- 11 -glycoproteins, directly or indirectly. The indirect influence refers to the fact that oligosaccharidecan promote and maintain the correct folding of viral glycoproteins and thus stabilize the epitopesor facilitate epitope exposure. It is often found that synthetic peptides bearing neutralizingepitopes of viral glycoproteins or viral glycoproteins expressed from E.coli fail to induceneutralizing antibodies, suggesting that lack of glycosylation may reduce the immunogenicity ofproteins. Since the contribution of each glycosylation site within a protein possessing multipleglycosylation sites may be different (see, the site-specific effect of glycosylation on theimmunoreactivity has been characterized for a number of viral glycoproteins. Using site-directedmutagenesis to alter N-linked glycosylation sites, it has been found that one glycosylation siteon bovine herpesvirus type 1 glycoprotein gIV is important for its immunoreactivity (Tikoo eta., 1993). Mutant proteins lacking glycans at residue 102 show altered reactivity withconformation-dependent gIV-specific mAbs and also induce significantly lower neutralizingantibody responses than wild-type (Tikoo et al., 1993).On the other hand, evidence has been obtained that carbohydrates can shield antigenicsites from immune recognition by steric hindrance. Addition of carbohydrate side chains at novelsites on influenza virus hemagglutinin results in the shielding or disruption of functional epitopeson the surface of hemagglutinin (Gallagher, et al., 1988). One of the Sindbis virus neutralizationescape mutants selected with mAbs shows a codon change which results in the gain of a newglycosylation site at amino acid residue 203 of the E2 protein (Davis et al., 1987). A morecommon approach utilized in studying carbohydrate shielding of epitopes on viral glycoproteinsis the use of glycosidases to remove glycans from expressed glycoproteins or from assembledvirions, and analysis of the antibody binding activity as well as the immunogenicity of these- 12 -deglycosylated proteins or virions. Deglycosylation does not affect the binding of Rauscherleukemia virus envelope glycoproteins to neutralizing antibodies from sera; and in fact,deglycosylated virions induce a faster neutralizing antibody response than that of untreatedcontrol virus (Elder et al., 1986). The immunoreactive conformation of envelope glycoproteinsof HIV- 1 remains unaltered after deglycosylation (Ferouillet et al., 1990). However, rabbitsimmunized with these deglycosylated glycoproteins produce lower viral neutralizing (VN)antibodies that inhibit HIV- 1 infectivity or syncytium formation in infected cells (Benjouad etaL, 1992).Besides total removal of glycans from glycoproteins, partial removal of sugar moietiesfrom carbohydrate side chains or inhibition of oligosaccharide processing have also been foundto interfere with the fine conformation of domains in the polypeptides within or adjacent toglycosylation sites and to result in an altered antigenic properties. The glycoprotein gIV of bovineherpesvirus 1 expressed from recombinant baculovirus infected insect cells, which are devoid ofsialyl transferases for the addition of terminal sialic acid to the N-glycans, reacts less efficientlywith niAbs that recognize conformation-dependent epitopes, and induces lower overall orneutralizing antibody titres, than the protein from virion grown in mammalian cells (van DrunenLittel-van den Hurk, et al., 1991). In cells treated with N-methyl-1-deoxynojirimycin, an inhibitorof a-glucosidase, the normal carbohydrate trimming is inhibited. Under these conditions, SFVE2 protein expressed from SFV-infected chicken cells contains three additional glucose residuesin the oligosaccharide side chain and has a significantly changed antigenicity (Kaluza et al.,1980).Carbohydrates can serve directly as targets for recognition by the immune system.- 13 -Because of their host specificity, carbohydrate epitopes are responsible, by a mechanism ofmolecular mimicry, for cross-reactivities with host components or with other viral glycoproteinscarrying similar oligosaccharides. This has been well documented in the case of influenza virus(Klenk, 1990). Carbohydrate epitopes are rare; more frequently, the sugar moieties are part ofthe constitution of conformation-dependent epitopes on viral glycoproteins. Depletion of N-linkedglycan either by changing the consensus sequence for N-glycosylation or by total inhibition ofN-glycosylation using tunicamycin results in the reduced reactivity of bovine herpesvirus 1 gIVto mAbs that recognize conformation-dependent epitopes but not those which react to linearepitopes (Tikoo, et at, 1993). Immunization of animals with mutant protein in whicholigosaccharide side chains involving conformation-dependent epitopes are deleted, results in nosignificant difference in total antibody responses; however, the neutralizing titre of the antibodiesis much lower (Tikoo, et al., 1993).- 14 -1.2. Targeting of viral membrane glycoproteins and virus assembly1.2.1. General concepts1.2.1.1. Assembly of enveloped virusesEnveloped viruses package their genomes within a protein shell, and this nucleocapsid (or corestructure) is then enveloped by a lipid bilayer at the final step of virus maturation, the buddingprocess, during which the nucleocapsid core extrudes itself through a certain region of the cellularmembrane. The envelope of viruses is made up of a regular lipid bilayer derived from, andsimilar in structure and composition to, one of the host cell membranes. However in this process,the host membrane proteins are effectively excluded and replaced with virus-specific membraneproteins (Suomalainen et al., 1992). The mechanism of virus budding is still largely unknown.It is believed that the interaction between the nucleocapsid and viral membrane glycoproteins,in most cases, is the driving force for virus assembly. Accumulation of viral proteins within aspecific subcellular compartment may provide ground for such an interaction and may, at leastpartially, determine the site of virus maturation.In general, the unambiguous proof of virus maturation at any particular subcellular site isprovided by the demonstration of electron microscopic profiles of virus particle accumulation andbudding. Virus assembly at almost all cellular membrane structures, plasma membrane (PM),Golgi complex (GC), endoplasmic reticulum (ER) and inner nuclear membrane has been reported.By comparing the electron microscopic profiles of virus budding with data from light- andelectron-microscopic immunolocalization of the distribution of viral glycoproteins as well as thenucleocapsid, a strong correlation between the site of virus budding and glycoprotein targeting- 15 -has been found for a number of viruses. Targeting of viral envelope glycoproteinsViral proteins destined for the plasma membrane follow the general secretory pathway alsoutilized by host cell plasma membrane proteins. In fact, viral spike proteins have beeninstrumental in dissecting the various steps involved in this exocytic pathway. The organelleswithin this system include the rough and smooth ER, the cis- medial- and trans-Golgi, the transGolgi network (TGN), secretory vesicles and granules, and the PM (reviewed by Dunphy andRothman, 1985). The ER represents the point of entry for the proteins that will traverse thiscomplex organellar pathway (reviewed by Pfeffer and Rothman, 1987). Proteins synthesized onthe polysomes associated with the ER membrane, are cotranslationally inserted into the ER dueto the presence of a signal sequence (Singer et al., 1987). During and after the process oftranslocation itself, components of the ER play a variety of roles in catalyzing posttranslationalmodifications such as proteolysis and N-linked glycosylation of the extruded proteins. In additionit has become increasingly clear that molecules residing within the lumen of the ER assist in thecorrect folding of translocated polypeptides and their assembly into oligomeric complexes (Nget al., 1990; Earl et al., 1991). The attainment of a correct structure appears to be critical fortransport out of the ER to the Golgi, and may in fact be the rate-limiting step in the process (see1.1.3.1 of the introduction) (reviewed by Rose and Doms, 1988). Proteins normally targeted tothe PM exit the ER and move to the Golgi cisternae via vesicle-mediated membrane fusion. TheGC is a set of subcompartments that comprises at least three biochemically distinct units(reviewed by Dunphy and Rothman, 1985), based on the enzymes that transform N-linked- 16 -oligosaccharide chains into their mature form. As glycoproteins are transported from cis throughmedial to trans Golgi, carbohydrate modifications are carried out by resident enzymes. A largevariety of other processes such as 0-linked glycosylation, acylation, suiphation, and proteolyticcleavage/activation also occur in the GC. The majority of membrane-spanning proteins aretransported to the PM in a constitutive, unregulated fashion, whereas secretion of soluble proteinsinvolves the TGN, which is a branchpoint directing the final destination of proteins (Pfeffer andRothman, 1987).Not all the proteins that enter the secretory pathway end up at the cell surface; a numberof cellular and viral glycoproteins are retained in one of the subcellular compartments of theexocytic pathway (the ER, the GC or the TGN). The intracellular retention of glycoproteins isbelieved to be mediated by “retention signals” located in the primary structure of proteins (Lewisand Pelham, 1989; Swift and Machamer, 1991). Many ER-resident proteins bearing an aminoacid motif of Lys-Asp-Glu-Leu (KDEL) or similar sequences interact with the KDEL receptorwhich normally resides in the cis Golgi cisterae (Lewis and Petham, 1989). Ligand bindinginduces a change of conformation in the KDEL receptor and results in the retrograde movementof the receptor-ligand complex back to the ER (Lewis and Pelham, 1991). The Golgi retentionsignal has been found to be located in the transmembrane domains of proteins (Swift andMachamer, 1991) while the cytoplasmic tail has been shown to be important in retention in theTGN.In most studies of protein targeting, the criteria of cell surface expression ofglycoproteins is defined using inimunofluorescence techniques while subcellular localization ofproteins can be determined using light- and electron-microscopic immunolocalization.- 17 -Biochemical analysis such as analysis of the extent of N- or 0-linked glycosylation, organellespecific proteolytic processing and the oligomeric state of the proteins in question have also beenextensively used to study protein transport. Experimental approachesConventional electron-microscopic profiles of virus particle accumulation and budding of infectedcells at different stages during virus replication provide adequate information on the site of virusbudding. Recent developments in irnmuno-electron microscopic techniques allow the finelocalization of virus-specified proteins in virus-infected cells. This methodology has been appliedto transfected cells expressing viral protein from DNA or cDNA constructs derived from partialor intact viral genomes. Comparison of protein localization with or without virus assembly hasshed light on which viral proteins determine the site of budding.While morphological studies create a steady-state image of virus budding and virusparticle accumulation, data from biochemical analysis have been proven to be very informativeon the stepwise processes from the synthesis of viral proteins to their incorporation into virions.In such studies, two protein transport inhibitors, brefeldin A (BFA) and monensin have beenextensively utilized. Monensin, a monovalent ion-selective ionophore, facilitates thetransmembrane exchange of principally sodium ions for protons, which results in theneutralization of acidic intracellular compartments such as the trans Golgi apparatus andassociated elements, combined with a disruption of the normal functions of these compartments.Late Golgi processing events such as terminal glycosylation and proteolytic cleavages are mostsusceptible to inhibition by monensin (reviewed by Mollenhauer et al., 1990).- 18 -BFA, a fungal metabolite, is another compound which has been extensively utilized instudying ER-Golgi trafficking in recent years. At low concentrations (ijig/mi) this lipophilicmolecule retards protein secretion, apparently acting specifically on transport from the ER to theGolgi (Misumi et al., 1986). At an intermediate concentration (2.5 ig/m1) the Golgi isdisassembled, while at 10 pg/mI, transport is completely blocked and morphological changesincluding dilation of the ER as well as loss of the Golgi structure (Fujiwara et al., 1988, Misumiet al., 1986). At high concentration, BFA causes the movement of resident Golgi proteins backinto the ER (Doms et al., 1989). This inhibitory effect of BFA is reversible. It should be notedthat although these observations are applicable to most type of cells, the effect of BFA treatmentin each cell type is cell-type specific.1.2.2. Virus assembly at the PMMost enveloped animal viruses (e.g. aiphaviruses, arenaviruses, orthomyxoviruses,paramyxoviruses, rhabdoviruses, and retroviruses) acquire their envelope at the PM. Afterbudding has been completed, virus particles are released directly into the extracellular space. Morphogenesis and assemblyFor virus maturation at the PM, in most cases the pre-assembled nucleocapsid is transported tothe cell surface and interacts with viral glycoprotein(s) embedded in the PM, leading to formationand release of the virus particles. Matrix or membrane (M) proteins are involved, for someviruses, in this nucleocapsid-glycoprotein interaction. In the unusual case of lentiviruses, assemblyof the core and virion occurs simultaneously, and does not require the envelope glycoprotein.- 19 - Assembly mediated by spike-nucleocapsid interactionsAlphaviruses are enveloped RNA viruses belonging to the Togaviridae family. The virions appearas essentially spherical, 60-65 nm diameter particles . The genome, a 49S RNA molecule withpositive polarity, is encapsidated by a single species of capsid protein arranged in an icosahedralconfiguration. This nucleocapsid is enveloped by a lipid bilayer derived from the host cell plasmamembrane. Projecting from the bilayer and embedded in it are the viral encoded glycoproteinsdesignated El and E2. For alphaviruses replicating in vertebrate cells, the assembly ofnucleocapsid takes place in the cytoplasm while the budding occurs primarily at the plasmamembrane (Smith and Brown, 1977). Several ts mutants of alphaviruses have been isolated whichhave maturation defects associated with the spike proteins at the restrictive temperature and thusare defective in the release of infectious virions. In some ts mutants, the mutated spike proteinsfail to be transported to the plasma membrane at the restrictive temperature, and electronmicroscopic (EM) analysis of these cells shows no budding structure under these conditions(Brown and Smith, 1975; Saraste et al., 1980; Smith and Brown, 1977). In one Sindbis virus tsmutant (ts2O)-infected cells, the altered spikes are able to reach the cell surface, and in thesecells, the plasma membrane is seen to be lined with nucleocapsids engaged in the buddingprocess (Smith and Brown, 1977; Saraste et al., 1980). These results indicate that thenucleocapsid cannot bud from the cell without correct nucleocapsid-spike protein interaction atthe cell surface. Amino acid substitutions in the cytoplasmic tail of E2 have been shown to leadto defects in virion assembly (Gaedigk-Nitschko and Schlesinger, 1991). Recently, usingrecombinant SFV genomes lacking the nucleocapsid protein gene or, alternatively, the spikegenes, Suomalainen et al. (1992) demonstrated that virus release is strictly dependent on the- 20 -coexpression of the nucleocapsid and spike proteins and concluded that the budding ofaiphaviruses is mediated by nucleocapsid-spike interaction. Matrix or membrane (M) protein mediated nucleocapsid-glycoprotein interactionRhabdoviruses, orthomyxoviruses and paramyxoviruses are negative-stranded RNA viruses.Although there are differences in their genomic organization (segmented or nonsegmented), virionstructure (spherical or bullet-shaped) and gene expression strategy, they are similar in that virionmaturation occurs at the cell surface and is mediated by respective M proteins. Theribonucleoprotein cores are assembled in the nucleus (orthomyxoviruses) or cytoplasm (rhabdoand paramyxoviruses) and transported to a region of the plasma membrane which contains newlyinserted but randomly distributed viral glycoproteins. The M proteins bridge the gap between theribonucleoprotein core and the cytoplasmic extension of the glycoproteins, leading to theenvelopment of nucleocapsid with glycoprotein-covered plasma membrane. For vesicularstomatitis virus (VSV), a rhabdovirus, the M (matrix) protein binds to progeny nucleocapsid andthe nucleocapsid-M-protein complex migrates to the cell surface to initiate budding (Newcombet al., 1982). In paramyxoviruses, the M (membrane) proteins aggregate in the inner aspect ofthe cell surface and noncovalently associate with glycoprotein. The complex ‘captures’ the newlyarrived nucleocapsid to activate the budding process (reviewed by Dubois-Dalq et al., 1984). Spike glycoprotein transportMuch of what is known about stepwise transport of glycoproteins from the site of biosynthesisto the cell surface has been learned from the study of some viral glycoproteins: VSV G protein,- 21 -influenza HA and alphavirus p62 and El. Although the movement of these proteins from the ERto the cell surface is fast, with a t112 of less than 30 minutes, they are subjected to a number ofstructural modulations. The lumen of the ER provides an environment optimized for proteinfolding and multi-subunit assembly. The ectodomain of the polypeptide must fold correctly inorder to be transport competent (Earl et al., 1989). Protein misfolding induced by inhibition ofglycosylation (by TM or mutagenesis) results in the aggregation and cross-linking of proteins bydisulfide bonds (Hurtley et at, 1989; Machamer and Rose, 1988b). These defective proteins areretained in the ER and eventually degraded (Lippincott-Schwartz et al., 1988). Retention preventsdelivery of nonfunctional viral membrane proteins to the site of virus budding. Movement fromthe ER to GC is the rate-limiting step in the exocytic pathway for viral membrane proteins thatare targeted to the cell surface, whereas intra-Golgi transport is fast (Pfeffer and Rothman, 1987).Transport between Golgi compartments is a vesicular process during which proteins are exposedto the sequential action of N-linked glycosylation modification enzymes residing in eachsubdivision of the GC (Dunphy and Rothman, 1985). Compartmentalization is also important inthat it allows protein cleavage/activation to occur at a specific stage of maturation within theGolgi, as in the case for the conversion of alphavirus p62 to E2 and E3 (Hakimi and Atkinson,1982).2.2.3. Studies with inhibitors, BFA and monensinBFA and monensin effectively block the incorporation of the VSV G protein and El and E2proteins of SW into their respective virions as a result of the inhibition of transport of these- 22 -proteins to the plasma membrane. In the presence of BFA, the acquisition of endo H resistanceby VSV G protein and the proteolytic conversion of SIV pE2 to E2 (a Golgi-specific event) areinhibited, suggesting that the transport of these envelope proteins is arrested in the ER (Oda, etal., 1990). In monensin-treated cells, fatty acid attachment to VSV G and SIV pE2, and theposttranslational removal of mannose residues from oligosaccharides on VSV G occurs normally,whereas proteolytic cleavage of SW pE2 to E2 is inhibited, suggesting that monensin acts duringlate Golgi processing (Johnson and Schlesinger, 1980).1.2.3. Viruses assembled in the GCMembers of the Bunyavirus family are the only viruses in which budding occurs for certain inthe GC. Although there are a large number of viruses in this family, they share a similar generalstructure and site of maturation. Morphogenesis and assemblyBunyavirus particles are 90-100 nm in diameter and contain two membrane glycoproteins, Giand G2. The internal protein N associates with RNA to form the nucleocapsid. EM studies showthat virus particles mature intracellularly by budding into smooth vesicles in a perinuclear regionand the budding structure is not observed at the PM. During Uukuniemi virus infection, both Giand G2, as well as N, probably in the form of nucleocapsids, accumulate in the GC. The helicalnucleocapsids appear to line up beneath the membrane of distended Golgi vesicles. As G 1 andG2 accumulate in the GC, progressively more nucleocapsid seems to enter the GC region. Littleif any N protein is seen associated with the ER or the PM. Thus specific interactions between- 23 -nucleocapsids and membranes containing viral glycoproteins exist only in the GC (Kuismanenet aL, 1982, 1984). Targeting of Gi and G2 to the GCBased on primary structure deduced from cDNA sequences, Gi and G2 from most of the virusesin Bunyavirus family are type I membrane glycoproteins (Pettersson, et al., 1988). Theoligosaccharides on Gi and G2 are found to be heterogeneously processed, as judged by endoH digestion and analysis of terminal glycans. The presence of immature glycans may reflect thesite of maturation in the GC. In virus-infected cells or cells expressing viral protein from vacciniarecombinant virus, most of the glycoproteins accumulate in the GC and cannot be chased outfrom there. This strong retention of glycoproteins in the GC suggests that a retention signal mayreside in either Gi or G2, or both. Inhibitor studies, BFA and monensinBFA treatment does not affect the assembly of intracellular infectious virus particles of PuntoToro virus but causes a rapid and dramatic change in intracellular distribution of Gi and G2glycoproteins, from a Golgi pattern to an ER pattern (Chen et al., 1991). In contrast, budding ofbunyanvirus is inhibited by the ionophore monensin (Cash 1982; Kuismane et al., 1985, Chenet al., 1991) whereas the association of the nucleocapsid with Golgi vesicles seems to beunaffected (Kuismanen et al., 1985). This points to the possibility that the pH or the ionic milieuprevailing in the GC is critical for bunyavirus budding. Budding in the GC may thus bedependent not only on a certain concentration of glycoproteins, but also on a conformational- 24 -change of the glycoproteins induced by this milieu.1.2.4. Virus assembly in the ER, the GC or a pre-Golgi compartmentMembers of the coronaviridae family are enveloped RNA viruses that acquire their lipoproteincoats by budding at intracellular membranes (the ER, the GC, or in a compartment between thesetwo organelles). Coronaviruses have a single species of coat protein, N protein, which isassociated with the genomic RNA in the cytoplasm to form helical, loosely coiled nucleocapsidsand two membrane glycoproteins M and S. Morphogenesis and assemblyCoronavirus budding occurs at intracellular membranes between the rough ER and the Golgiapparatus. At the budding site, nucleocapsids align on the cytoplasmic side of smooth membranein the ER, or the GC where viral glycoproteins have accumulated (Massaiski et al., 1982).Budding at the cell surface has not been observed. In a detailed electronmicroscopic study ofdifferent types of cells infected with mouse hepatitis virus A59, slight differences in the sites ofvirus maturation were observed, either between the GC and the smooth perinuclear vesicles, ortubules in a pre-Golgi region (Tooze et al., 1984; Tooze and Tooze, 1985; Tooze et al., 1987).It appears that the location of coronavirus budding is at least in part determined by the host cell. M protein targeting determines the site of coronavirus maturationAmino acid sequences predicted from cDNA sequences reveal that coronavirus M glycoproteinis a type III membrane glycoprotein with multiple transmembrane domains (Mayer et al., 1988;- 25 -Wickner and Lodish, 1985). Based on the topological model and protease protection assays, itis proposed that most of the M protein is embedded in the membrane (Mayer et al., 1988). Mprotein contains either N- or 0-linked oligosaccharides depending on the strain.Immunolocalization of M protein in coronavirus-infected cells or cells transfected with a clonedcDNA has indicated that it is predominantly found in smooth membranes of the pre-Golgi region,the GC and, later in infection, also in the ER (Rottirer and Rose, 1987; Machamer and Rose,1987; Mayer, et al., 1988). Recent studies employing immuno-electronmicroscopic andrecombinant DNA techniques have shown that the M protein is targeted to the GC through a“Golgi retention signal localized in the first transmembrane domain of M protein (Machameret aL, 1990; Swift and Machamer, 1991; Machamer et al., 1993). This retention signal” issufficiently efficient to cause proteins normally targeted at the cell surface (e.g. VSV G) to beretained in the GC (Swift and Machamer, 1991).The site of virus budding is determined by the subcellular localization of M protein, basedon several lines of evidence. M protein is never transported beyond the trans Golgi to the PM,and no virus budding has been observed at the PM, although a large amount of S protein mayaccumulate at the PM (Mayer et al., 1988). In coronavirus-infected cells treated with tunicamycin,M protein and the nucleocapsid protein are normally incorporated into virions in the ER andGolgi without S protein (Holmes et al., 1981). In this respect, coronavirus M protein resemblesthe matrix or membrane proteins of paramyxovirus and rhabdovirus in function during virusassembly, although it is a glycoprotein.- 26 - Studies with monensinStudies by Niemann et al., (1982) show that monensin does not interfere with coronavirusbudding in the rough ER or Golgi, but it does inhibit virus release and fusion of infected cells.During monensin treatment, oligosaccharides on the S protein are shown to be resistant to endoH digestion but lack fucose, indicating that transport of the S protein is inhibited between thetrans Golgi and the cell surface. The M protein incorporated into virions is devoid ofcarbohydrate, implying that the transport of M protein is also inhibited by monensin.1.2.4. Closing remarksIn the case of viruses that bud at the plasma membrane, the viral glycoproteins are rapidlytransported to the cell surface via the normal exocytic pathway (Stephens and Compans, 1988).By contrast, for most of the intracellular maturing enveloped viruses, at least one of the virus-specified glycoproteins is targeted to and accumulates in the budding compartment. Examples ofsuch glycoproteins have been discussed earlier in this introductory section, e.g. coronavirus Mprotein (in a post-ER, pre-Golgi intermediate compartment, or GC) and bunyavirus Gi and G2(GC). Thus, one important factor in determining the site of budding is clearly the targeting to andaccumulation of viral glycoprotein in the compartment.Viruses incorporate functional gene products into virions. The first step in this qualitycontrol mechanism is that newly synthesized viral glycoproteins must fold into a properconformation to obtain transport competence. For proteins destined for the cell surface, this isthe rate-limiting step in the exocytic pathway, during which they undergo a series ofposttranslational modifications and become biologically functional upon reaching plasma- 27 -membrane. For proteins targeted to an intracellular compartment, however, correct protein foldingenables proteins to exit the ER and to be retained in one of the compartments. Such retentionmay be due to 1) the presence of specific amino acid motif (linear or conformational) thatconstitute a “retention signal” (as in the case of coronavirus M glycoprotein); 2) lateral interactionbetween viral membrane glycoproteins that result in the formation of large aggregates thatexclude them from transport vesicles; 3) association with macromolecules residing in asubcellular compartment via interaction with regions of the protein other than the “retentionsignal”.To date, little is known about the mechanism underlying virus budding, particularly forvirus budding at the intracellular membrane. It is understandable that an important prerequisitefor virus budding may be the need for a critical concentration of viral glycoproteins within thebudding compartment. A good explanation is that normally a particular type of virus buds onlyin one of the subcellular compartments. Conformational changes in glycoproteins along thetransport route induced by milieu (pH, ionic conditions) or posttranslational modifications (e.g.glycan processing) may facilitate the interaction of viral glycoproteins with nucleocapsids. Lastly,compartment-specific molecules may assist the budding process.- 28 -1.3. Rubella virus biology1.3.1. ClassificationRubella virus (RV) is the sole member of the genus Rubivirus in the family Togaviridae(Porterfield et al., 1978). Based on morphological criteria, Togaviruses are defined as spherical,enveloped viruses with an icosahedral nucleocapsid. The genome is composed of a singleinfectious RNA molecule. Surrounding the nucleocapsid is the host cell-derived lipid bilayercontaining viral membrane glycoproteins. Progress in the molecular characterization of virusesin the Togaviidae family has led to reclassification of these viruses on the basis of viral genomestructure, organization, and gene expression. Under the current classification, the Togaviridaefamily consists of two genera: aiphaviruses (arthropod-borne) and rubiviruses (non-arthropodborne) (Francki et al., 1991). The two well-studied viruses, Sindbis virus (SIN) and SemlikiForest virus (SFV) are included in the aiphavirus genus whereas RV is the only known memberof rubivirus genus (Francki et al., 1991).1.3.2. Clinical aspectsRV is the etiological agent of a relatively mild childhood disease known as German measles. RVinfection in humans may be asymptomatic or can induce adenopathy, malaise, low grade fever,and exanthem. The most common complication following natural RV infection is transient jointinvolvement such as polyarthralgia and arthritis.The primary medical significance of RV infection is that the virus can cross the placentaand replicate in the fetus. Infants born to women infected during the first trimester of gestation- 29 -have a high incidence of birth defects, collectively known as congenital rubella syndrome (CRS).These include heart defects, cataracts, deafness, and mental retardation. Vaccination with live,attenuated virus has been successful in reducing the incidence of CRS. However, rubella-associated arthritis and the consequence of viral persistence in vaccinees resulting from RVvaccination remain major medical concerns (Chantler et al., 1982). Furthermore, RV infectionhas been linked to some chronic diseases, including autoimmune diseases (Wolinsky, 1990).Although the highest correlation between RV infection and chronic disease is found in the CRSpopulation (Wolinsky, 1990), the association of RV persistence with arthritis (reviewed byPhillips, 1989) and multiple sclerosis (Nath and Wolinsky, 1990) has been suggested.1.3.3. Morphology and morphogenesisEarly studies employing conventional electron microscopy of RV grown in BHK-21 cellsindicated that RV virions are spherical, 60-70 nm in diameter, with a 30 nm electron dense coresurrounded by an envelope (von Bonsdorff and Vaheri, 1969). These structures have been definedas the icosahedral nucleocapsid (the dense core) (Murphy et al., 1968) and lipid-bilayer (envelopeassociated with the hemagglutination activity) (Holmes et al., 1969). The mechanism of RVassembly and budding is largely unknown. Among reports on the RV budding site, there is anapparent discrepancy. In BHK-21 and Vero cells, intracellular maturation (in Golgi or vacuoles)and budding at the plasma membrane have been observed (Bardeletti et al., 1979; Payment et al.,1975). However, studies in which cells were infected with RV at high multiplicity of infection(MOl) and analyzed at different time intervals suggest that there exits a course of progressionof RV maturation from the Golgi towards the plasma membrane (von Bonsdorff and Vaheri,- 30 -1969; Bardeletti et al., 1979).1.3.4. Nucleic acids and genome organizationThe RV genome, a single-stranded RNA molecule with a sedimentation coefficient of 40 S, isinfectious (Hovi and Vaheri, 1970). RV-irifected cells contain, in addition to the 40 S RVgenome, an RV-specific RNA molecule which sediments at 24 S (Oker-blom et al., 1984). Thissubgenomic RNA is polyadenylated, capped, and identical to the 3’ one-third of the 40 S RNA(Oker-Blom et al., 1984). It serves as a messenger RNA for the synthesis of RV structuralproteins (Oker-Blom et al., 1984). The molecular mechanism that results in the synthesis of the24 S subgenomic RNA is not clear, but presumably involves a negative-sense RNA intermediate.Sequences of the 24 S subgenomic RNA have been determined from cDNA clones for wild typeisolates (M33 and Therien strain) (Clarke et al., 1987; Frey et al., 1986) or vaccine strains (RA27/3 and HPV 77) (Nakhasi et al., 1989; Zhang et al., 1989). A 95% homology at the nucleotidelevel is found between three reported RV 24 S RNA sequences whereas little homology wasfound with that of the alphavirus subgenomic mRNA (Frey and Man, 1988).Recently cDNA clones covering the entire RV genome for the Therien (Dominguez et al.,1990) and M33 strains (Yang et al., 1993) have been constructed and sequenced. Sequence dataderived from cDNAs reveal that the RV genome is 9756 nucleotides (Therien) or 9764 (M33)in length and has a G/C content of 69.5%, the highest of any RNA virus sequence to date(Dominguez et al., 1990). Alignment of the 40 S sequence between the Therien and M33 strainsshows homologies of 97.5% and 96.6% for overall nucleotide and deduced amino acid sequences,respectively (Yang et aL, 1993). The RV genome contains two long open reading frames (ORFs),- 31 -a 5’ proximal ORF (from nucleotide 41 to nucleotide 6385) which encodes the nonstructuralproteins and a 3’ proximal ORF (from nucleotide 6506 to nucleotide 9694) which encodes thestructural proteins (Fig.2) (Dominguez et al., 1990; Yang et al., 1993). Thus, the genomicorganization of RV closely resembles that of aiphaviruses (Strauss et al., 1984).1.3.5. Non-structural proteinsThe non-structural proteins of RV are encoded by the 5’ two-thirds of its genome, and translatedas a polyprotein precursor. In RV infected cells, protein species with molecular masses of 200,150, 87, 75 and 27 kDa, in addition to the structural proteins, have been detected using humanconvalescent serum (Bowden and Westaway, 1984). Recently, RV-specific proteins withelectrophoretic mobilities corresponding to 200, 150, and 90 kDa have been expressed in cellstransfected with a recombinant plasmid (pTM3/nsRUB) containing the RV 5’ proximal ORFunder the control of the T7 polymerase promoter (Marr, et al., 1994). Antibodies raised againstbacterial fusion proteins containing regions encoded by the 5’ proximal ORF react to the 200,150 and 90 kDa proteins from the above mentioned cDNA transfected cells as well as RVinfected cells. Mutational analysis indicates that the 150 and 90 kDa proteins are the processingproducts of the 200 kDa precursor and the order within the ORF is NH2-P150-P90-COOH (Manet al., 1994, Forng and Frey, unpublished results).The biological functions of these proteins is not known. Amino acid sequences predictedfrom cDNA sequences reveal a conserved helicase motif and a replicase motif found among wellstudied positive-stranded RNA viruses. In addition, a cysteine protease activity is found to beinvolved in the processing of the nonstructural protein precursor and an important catalytic role- 32 -has been assigned to Cys1151 of the protease (Marr et al., 1994).1.3.6. Expression and processing of structural proteinsRV contains three structural proteins: a capsid protein, C (33 kDa), and two membraneglycoproteins El (57 kDa) and E2 (42-47 kDa). In RV infected cells, the structural proteins aretranslated as a polyprotein precursor, in the order, NH2-C-E2-El-COOH, with the 24 Ssubgenomic RNA serving as a template (Fig.3) (Oker-Blom et al., 1984). The polyproteinprecursor is subsequently proteolytically processed to yield three individual structural proteins.Unlike SIN and SFV, the RV capsid protein does not possess an autocatalytic serine protease-lilceactivity used to release itself from the polyprotein (Clarke et al., 1987; McDonald et al., 1990).In the absence of microsomes, in vitro translation of the 24 S subgenomic RNA produces apolyprotein precursor of 110 kDa (Oker-Blom et al., 1984; Clarke et aL, 1988).Amino acid sequences predicted from cDNAs reveals that both RV El and E2 are typeI membrane proteins (Singer et al., 1987) with their N-termini preceeded by stretches of 20 and23 hydrophobic amino acid residues, respectively (Clarke et al., 1987; Frey and Marr, 1988).These hydrophobic sequences resemble the consensus signal peptides that mediate targeting ofnascent polypeptides to the ER membrane and initiate translocation of protein into the ER lumen(reviewed by Wiley, 1986). In vitro and in vivo expression of wild-type and mutant proteinslacking the signal sequences demonstrate that the presence of the signal peptides is required fortranslocation and processing of the polyprotein precursor (Hobman et al., 1988; Hobman andGillam, 1989; Man et al., 1991). Furthermore, mutations at the cleavage sites (von Heijne, 1984)of either the E2 or El signal peptide resulted in the accumulation of uncleaved polyprotein- 33 -0.0 1.0 2.0 30 4.0 50 6.0 7.0 8.0 9.0 10.0 11.0SG ANA•H C f E2f El polyAP90-.Fig.2 Topography of the genome RNA of RV. The scale at the top of diagram is in kilobases.Untranslated sequences are denoted by black lines and open reading frames (ORFs) by openboxes. The 5’ proximal ORF encodes nonstructural proteins and 3’ proximal ORF encodesstructural proteins. The boundaries of the individual proteins processed from the precursortranslated from each ORF are denoted. Within the nonstructural protein ORF, the location ofglobal amino acid motifs indicative of replicase (R), helicase (H), and cysteine protease activity(P) as well as the small regions of homology between the deduced amino acid sequence of RVand SIV (X motif) is shown. Also shown are: positions of regions of nucleotide homologybetween RV and aiphaviruses, (open circle); subgenomic start site, (closed circle); the 3’ terminalstem-and-loop structure, (hatched circle). An expanded topography of the RV strucutral proteinORF is shown at the bottom of the diagram. Within the ORF, the positioning of the followingdomains of the structural proteins are shown: , the hydrophilic region of C which contains ahigh concentration of basic amino acids and putatively interacts with the virion RNA; , thehydrophobic signal sequences which proceed the N-termini of E2 and El; , the transmembranesequences of E2 and El; Y, potential N-linked glycosylation sites (the site marked with a Y isnot present in the HPV-77 and M33 strain); •, a putative region for 0-linked glycosylation.Below the diagram are shown the location of domains which contain epitopes recognized bymouse mAbs. (N denotes domains containing epitopes recognized by neutralizing mAbs). (Frey,1994).RUBo :x: PHP150J.w1-;.Ik -,— YY L1JMAbQjCriE2 . El- 34 -precursor (McDonald et al., 1991; Qiu et al., 1994). Therefore, it is clear that the cleavage of Eland E2 signal peptides by cellular signalase gives rise to individual RV structural proteins duringthe processing of polyprotein precursor.1.3.7. Posttranslational modification of RV structural proteins1.3.7.1. Capsid proteinThe capsid protein of RV is nonglycosylated and associates with the genomic RNA in RVinfected cells to form nucleocapsid. The cDNA sequence indicates that the C protein has amaximal size of 300 amino acid residues with high percentages of arginine and proline residues(Clarke et al., 1987; Frey and Man, 1988). Capsid protein from virions migrates as a doublet inpolyacrylamide gels (Suomalainen et al., 1990; Man et al., 1991; Maraucher et al., 1991), thedifferences in molecular weight presumably being due to the alternative sites for translationinitiation. Recently it has been shown that capsid protein is phosphorylated, although the extentand function of phosphorylation is unclear (Sanchez and Frey, 1991). After the cleavage of theE2 signal peptide during translocation, the E2 signal peptide is found attached to capsid protein,which helps the capsid protein to become membrane-associated (Suomalainen et al., 1990). TheE2-signal sequence-mediated membrane association of the C protein may be important in thetransport of the C protein and in nucleocapsid formation. E2 glycoproteinOn an SDS gel, E2 glycoprotein from virion migrates as a diffuse band with molecular weightsranging from 42 to 47 kDa. In the presence of tunicamycin, Vero cells infected with RV and- 35 -COS cells transfected with RV cDNAs produce E2 with a molecular weight of 29 to 31 kDa(Oker-Blom et al., 1983; Sanchez and Frey, 1991) and thus the carbohydrate contributes one-thirdof the molecular mass of E2. Amino acid sequence predicted from E2 cDNAs reveals a proteinof 281 residues including three potential N-linked glycosylation sites in M33 (Clarke et al., 1987)G 40 S (.—11000 b)catj-1AA(A)-—•—• ® 24 S (—3500 b)AA(A)3A translationp110NH2)— I 1CQOHprocessingcapsid envelopeIIC E2 El- 33K 30K 53K• jj glycosylation ,E2a (47K) 58KE2b (42K)Fig.3 General strategy for the expression and processing of RV structural proteins. (Oker-Blomet al., 1984).- 36 -and HPV77 (Zheng et al., 1989) strains as opposed to four in Therien (Vidgren et aT., 1987; Freyand Marr, 1988) and RA27/3 (Frey et aL, 1986) strains. In addition to N-linked glycans, E2 isknown to contain 0-linked carbohydrates (Sanchez and Frey, 1991; Lundstrom et al., 1991).Digestion with glycosidases and lectin-binding assays reveal that N-linked glycans on E2 fromvirions contain high-mannose, hybrid-type and complex-type (Putnam and Therien strains)(Bowden and Westaway, 1985; Sanchez and Frey, 1991) or only complex-type, four branchedsugars (M33 strain) (Lundström et al., 1991), with the majority of complex-type terminating ingalactose and some fraction having terminal sialic acid (Sanchez and Frey, 1991; Lundstrom etal., 1991). The heterogeneous processing of both N-linked and 0-linked glycans on E2contributes to the diffuse nature of E2 on an SDS gel.Expression of E2 in vitro and in vivo from cDNA constructs demonstrates thattranslocation of E2 into the lumen of the rough ER is mediated by a signal peptide residing inthe C-terminus of the capsid protein, and this sequence can function externally as well as in itsnative internal context (Hobman and Gillam, 1989; Marr et al., 1991; Sanchez and Frey, 1991).Following translocation, N-linked glycosylation of E2 takes place. Processing of N-linked glycanson E2 involves at least two stable intermediates, a 39 kDa high mannose-containing precursorand a 42 kDa form bearing some complex-type sugars (Hobman and Gillam, 1989; Hobman, etal., 1990). Although it has been shown that E2 contains 0-glycans, the site of 0-linkedglycosylation and the extent of processing have not been defined. So far the importance of Nand 0-linked oligosaccharides on E2 in virion assembly and infectivity is unknown.- 37 - El glycoproteinEl is the dominant surface molecule of the RV virion (Ho-Terry and Cohen, 1984; Terry et al.,1988). El migrates as a discrete band with apparent molecular weight of 57 kDa.Nonglycosylated El synthesized in tunicamycin-treated cells has a molecular weight of 53 kDa(Oker-Blom et al, 1983; Bowden and Westaway, 1984; Sanchez and Frey, 1991). Deduced aminoacid sequence shows that El is 481 amino acid residues in length with three potential N-linkedglycosylation sites (Frey et al., 1986; Clarke, et al., 1987). 0-linked oligosaccharides are notdetected in El (Lundstrom at al., 1991) whereas palmitic acid is incorporated in El (Hobman etal., 1990). There is a stretch of seven amino acids including five arginine residues (R-R-A-C-RR-R) before the putative signal peptide sequence of El and after the putative transmembraneanchor domain of E2 that may contain basic amino acid cleavage sites for endoproteases. Sincethe C-terminal amino acid sequence of E2 has not been determined, it is not known whether otherproteolytic cleavages take place during the processing of the E2E 1 precursor polyprotein at theC-terminus of E2, besides the cleavage of the El signal peptide by host signal peptidase. Arecent mutational study of this region shows that the cleavage of the E2E1 polyprotein precursoris impaired when the signal peptide cleavage site alone, or both arginine clusters are altered,whereas partial cleavage is observed in the mutants in which one of the two arginine clusters ismodified (Qiu et al., 1994). These data indicate that the arginine clusters do not function as abasic protease cleavage site, rather, they contribute to maintaining the proper configuration of thatregion for access by cellular signal peptidase.- 38 - Conformation of structural proteinsCapsid protein forms a noncovalently bound dimer soon after translation in RV-infected cellsas well as in cells infected with a vaccinia recombinant virus expressing C protein (Baron andForsell, 1991). However, covalently linked C dimers are detected only in RV-infected cells butnot in vaccinia recombinant-infected cells (Baron and Forsell, 1991). Similarly, disulfide-boundEl-El homodimers and El-E2 heterodimers are routinely observed when rubella virions aresubjected to non-reducing SDS-PAGE (Waxham and Wolinsky, 1983; Dorsett et al., 1985),whereas such glycoprotein complexes are not detected when El and E2 are expressed fromcDNA (unpublished results). It is possible that the formation of intermolecular disulfide bondsof El and E2 occur after virions are released from the infected cells and exposed to a relativelyoxidative environment in the medium. Besides intermolecular disulfide bonds, intramoleculardisulfide bonding is found in El and E2 which is important to the maintenance of properconformation for antibody binding (Green and Dorsett, 1986; Wolinsky et al., 1991), proteinstability, hemagglutination activity and infectivity (Ho-Terry and Cohen, 1981; Katow andSugiura, 1988).Intracellularly, El and E2 form noncovalently associated heterodimers in RV-infected cellsas well as when they are expressed from cloned cDNAs (Baron and Forsell, 1991; Hobman etal., 1993). The association of El and E2 increases the intracellular transport rate of E2 (Hobmanet al., 1990) and releases El from retention in a post-ER, pre-Golgi compartment (Hobman et aL,1993).- 39 -1.3.8. Intracellular localization of RV structural proteinsIn an indirect immunofluorescence study, RV glycoproteins El and E2 were shown to beconcentrated in the juxtanuclear region of both RV-infected cells and cDNA transfected cellsexpressing all three structural proteins of RV. This region represents a reticular structure whichmay span from the ER to the Golgi stacks. Low level cell surface expression of El and E2 isalso observed (Hobman et aL, 1990). The capsid protein is also localized in the Golgi-like area,presumably due to its membrane association as well as the interaction with El and/or E2(Hobman et al., 1990; Baron et al., 1992). However, when each protein is expressed separatelyfrom cloned cDNA, a different intracellular distribution pattern is observed. The capsid proteinis found in a reticular structure extending throughout the cells (Baron et al., 1992). E2 is in theER, the Golgi cisternae and the cell surface, whereas El is retained in a pre-Golgi structure(Hobman et al., 1990). Fine mapping of the El and E2 intracellular localization usingimmunogold labelling reveals that El, when expressed alone, is arrested in a novel post-ER, preGolgi compartment near the exit site of the ER (Hobman et al., 1992). Although the coexpression of El and E2 releases such retention, they are targeted to the Golgi complex, and arenot efficiently transported to the cell surface (Hobman et al., 1993).1.3.9. Biological function of RV structural proteinsThe major biological activities associated with the RV virion (structural proteins) arehemagglutination (HA) (Schmidt et al., 1968) and low-pH induced cell-cell fusion of infectedcells (Vaananen et al., 1980). There is evidence linking both HA and fusion activities to RV El.Trypsin treatment of virions under conditions which digest El with minimum damage to E2- 40 -shows that such digestion results in the loss of the HA activity (Ho-Terry and Cohen, 1981).Murine mAbs that exhibit hemagglutination inhibition (HAT) activity, are all anti-El but not antiE2 (Waxham and Wolinsky, 1983; Green and Dorsett, 1986; Chaye et al., 1992). More directevidence is that El but not E2 expressed via vaccinia virus recombinants can induce theproduction of hemagglutination inhibitory antibodies (Gillam, unpublished result).Brief exposure of RV infected cells to a pH of 6.0 or lower results in syncytiumformation, presumably mediated by the RV glycoproteins expressed at the plasma membrane(Katow and Sugira, 1988). RV virions also induce fusion of erythrocytes (Vaananen et al., 1980)and the virions gain the ability to bind liposomes (Katow and Sugiura, 1988) after incubation inacidic media. The basis of the fusogenic activity in RV is not well defined but is thought toreside in El.1.3.10. Immune responses to RV infectionNatural RV infection or RV vaccination leaves a long-lasting immunity, which is attributed tocirculating antibodies. The initial response following infection or vaccination is a transient 1gMresponse and in most cases is El specific (Partanen et al., 1985). Although other immunoglobulinclasses (IgE, IgA) are stimulated subsequently, IgG production is the dominant serologicalresponse. Persisting IgG antibodies are directed to all three structural proteins of RV, althoughthe predominant reactivity is against El (Katow and Suguira 1985; Zhang et al., 1992; Chaye etal., 1992), indicating an important role of El in inducing a protective immunity against RVinfection.IgG antibodies to RV may have HAT and viral neutralizing (VN) properties (Green and- 41 -Dorsett, 1986; Waxham and Wolinsky, 1985). There is a good correlation between the levels ofIgG to RV, and classically measured HAT titres and neutralizing antibody titres in seropositivesera (Stokes et al., 1969). It is assumed that these responses play a positive role in viral clearanceand protection (Waxham and Wolinsky, 1985a). Circulating immune complexes containing RVspecific antibody and antigen are frequently found after RV infections (Ziola et al., 1983) but inmost cases their presence has not been associated with any of the complications following RVinfection or vaccination (Singh et al., 1986).Much less is known about the importance of cellular responses to RV infection. RVspecific cellular responses have been demonstrated using lymphocyte proliferation assays andlymphocyte mediated cytotoxicity assays (Buimovici-Klein and Cooper, 1985; Vesikari andBuimovici-Klein, 1974; Ilonen and Salmi, 1986). Cell-mediated cytotoxicity has been implicatedin the pathogenicity of RV infection (Martin et al., 1989). In these studies, intact RV was usedas the antigen for the analysis of proliferation responses. Only recently, Chaye et al. (1992)demonstrated antigen-specific lymphocyte proliferative responses in peripheral blood lymphocytesusing an in vitro proliferative assay with vaccinia recombinants expressing individual RVstructural proteins. In human populations, each individual exhibits different responses to El, E2and C; however, El is the dominant antigen to which the majority of subjects developlymphocyte proliferative responses (Chaye et al., 1992).Proliferative responses to purified, intact RV are major histocompatibility antigen (HLA)restricted (Ilonen and Salmi, 1986). Similarly, Ou et al. (1992a,b,c) isolated T-cell clones againstE2 glycoprotein and C protein from RV seropositive individuals and found that HLA restrictionswere associated with HLA DR7 for E2 epitopes, and HLA DR4 for C epitopes.- 42 -1.3.11. Immunological determinants on RV structural proteinsTo characterize the antigenic determinant on RV structural proteins, panels of murine mAbs havebeen generated and biological activities of these antibodies have been analyzed. These panels aremade up primarily by El-specific antibodies with a rare number of antibodies recognizing E2 orC (Waxham and Wolinsky, 1985; Green and Dorsett, 1986; Chaye et al., 1992). In 1985,Waxham and Wolinsky (1985) mapped HA and VN activities to the El glycoprotein. Since thisstudy, much effort has been focused on delineating functional epitopes on El (Terry et al., 1988,1989; Lozzi et al., 1990; Wolinsky et al., 1991). Numerous methods to localize HA or VNepitopes have been utilized including recombinant DNA technology (Terry et al., 1989;deMazancourt and Perricaudet, 1989; Wolinsky et al., 1991), peptide analysis (Lozzi et al., 1990;Terry et al., 1988; Mitchell et al., 1992) and competitive binding assays with mAbs (Waxhamand Wolinsky, 1985a). Six independent epitopes have been identified which are thought to beimportant for viral infectivity and HA (Green and Dorsett, 1986; Waxham and Wolinsky, 1985a).Three non-overlapping linear epitopes that react with mAbs having HAl and VN activities havebeen localized to El residues 245 to 285 (Terry et al., 1988). Wolinsky et al. (1991) and Chayeet al. (1991) separately mapped a region between residues 202 to 283 of El which consists ofoverlapping epitopes recognized by mAbs with VN, HAT or VN and weak HAT activities(Wolinsky et al., 1991; Chaye et al., 1992). Using a set of nested synthetic peptides, these twogroups subsequently narrowed the epitope to a region between residues 213 and 239, or betweenresidues 214 and 240, respectively (Chaye et al., 1992; Wolinsky et al., 1993). Contrary to thoseresults with El, B cell-epitope mapping on E2 and C has been less informative, due to the lowernumber of mAbs available. Only one of the E2-specific mAbs has been found to possess VN- 43 -activity (Green and Dorsett, 1986). It was reported recently that an anti-C mAb recognizes a 52kDa f3-cell antigen, implying that the C protein may be involved in molecular mimicry leadingto initiation of an immunopathological process (Karounos et al., 1993).Protective immunity to viral infection requires activation of helper T cells specific forviral antigens. Ou et al. (1992a, b, c, 1993) identified T-cell epitopes on RV structural proteinsby screening a nested set of overlapping synthetic peptides with peripheral blood lymphocytesfrom immune donors and subsequently with T-cell lines/clones derived from the peripheral bloodlymphocytes of immune donors. They identified regions between residues 358 and 377 of El,between residues 54 and 74 of E2, and between residues 255 and 280 of C, as the relativelyimmunodominant T-cell epitopes (Ou et al., 1992a,b,c, 1993). Mitchell et al. (1993), applyingessentially the same methodology, identified immunoreactive regions on El and E2 recognizedby T-cells of normal healthy individuals. McCarthy et al. (1993) took a different approach, usingsets of comparatively short overlapping synthetic peptides containing predicted T-cell epitopemotifs of RV structural proteins within the region bearing linear B-cell epitopes defined by RVspecific mAbs (C1 to C29, CM to C97, E231 to E2105 and El202 to E13). With one exception, all ofthe synthetic peptides were able to stimulate varied but individually specific lymphoproliferativeresponses in peripheral blood mononuclear cells from 25 to 50% of a population of normal, RVimmune donors with diverse HLA backgrounds (McCarthy et al., 1993). These studies indicatethat further fine-mapping of T-cell determinants among a larger human population with HLAdiversity is necessary for future construction of an effective synthetic peptide vaccine for RV.-44-1.3.12. Project rationale and thesis objectivesViruses utilize the host cell machinery for the synthesis and processing of viral proteins. Viralproteins undergo a series of structural modulations during transport from the site of synthesis tothe site at which they are incorporated into virions, and become functionally competent. Post-translational modifications are a major focus of studies on structure/function relationship ofproteins, and among them, glycosylation has been studied exhaustively. RV contains twomembrane glycoproteins El and E2. In recent years, although studies on a) the structure ofcarbohydrates on El and E2 (Sanchez and Frey, 1991; Lundstrom et al., 1991), b) the intracelluartransport and processing of El and E2 (Hobman and Gifiam, 1989; Hobman et al., 1990; Sanchezand Frey, 1991; Baron and Forsell, 1991; Marr et al., 1991; Baron et al., 1992) and c) theanalysis of immunological determinants on El and E2 (Wolinsky et al., 1991; Chaye et al., 1992)have greatly strengthened our knowledge about RV, little is known about the functional role ofN-linked glycosylation on RV El and E2.In this study, the importance of N-linked oligosaccharides on RV El and E2 has beeninvestigated with respect to its biological functions during replication and infection. Theapproaches taken involve a combination of recombinant DNA technology and mammalian cellexpression. The thesis describes two lines of experiments. The first line of experiments is directedat the cell biology aspects of El and E2. Studies were initiated to define the role of N-linkedglycosylation on processing and transport of E2, and these experiments were extended toinvestigate the correlation between the sorting of the El and E2 glycoproteins and virus assemblyusing two protein transport inhibitors, brefeldin A and monensin. The second line of experimentsare focused on vaccine development. In view of the fact that the immunoreactivity of RV El- 45 -glycoprotein is very dependent on its native conformation, the influence of N-linked glycosylationof El on its antigenicity and immunogenicity was analyzed. The outcome of these experimentsled to the expression and characterization of the virus-like particles containing RV structuralproteins, and studies of their immunological properties. The potential application of these virus-like particles as an antigen sources for serodiagnostic assays and vaccine development will bediscussed.- 46 -2. MATERIALS AND METHODS2.1. MATERIALS and SUPPLIESDNA modifying enzymes and restriction endonucleases were purchased from Bethesda ResearchLaboratories (BRL), Promega, New England Biolabs, Boehringer Mannheim, Pharmacia andUnited States Biochemical Corporation. All enzymes were used as specified by the manufacturerunless indicated otherwise.L-[35S]-methionine (600-800 Ci/mmole) was from Du Pont Inc. Tissueculture reagents were from Gibco (Gaithersburg, MD) or Sigma (St. Louis, IL). Brefeldin A waspurchased from Boehringer Mannheim. Tunicamycin and monensin were products of Sigma.GENECLEAN (BlO 101) was obtained from Promega. Human polyclonal anti-rubella serum wasprovided by Dr. A. Tingle (B.C. Children’s Hospital, Vacouver, B.C.). Mouse monoclonalantibodies against RV El were generated in this lab. Mouse monoclonal antibodies against RVE2 and capsid protein were generously provided by Dr. J. Safford (Abbott Laboratories, Chicago,IL) or Dr. J. Wolinsky (University of Texas, Houston, TX). Fluorescein (FITC)-conjugated goatanti-mouse or anti-human IgGs were from Kirkegaard & Perry Laboratory. IThodamine (TRICT)conjugated goat anti-mouse or anti-human antibodies, and lectins were from Zymed. A TRICTconjugated rabbit anti-Golgi protein serum was prepared in the laboratory of Dr. F. Tufaro(University of British Columbia, Vancouver, B.C.). COS, CV-l, i1Cl43, BHK, Vero and RK cells,rubella virus M33 strain and vaccinia virus WR strain were obtained from the American TypeCulture Collection.- 47 -2.2. METHODS2.2.1. Propagation of bacterial strainsE.coli strains DH5o from BRL were used for the propagation of recombinant clones. DH5o cellscontaining recombinant plasmids were grown in LB medium (1% tryptone; 0.5% yeast extract;1% NaC1) containing 100 ug/ml ampicillin (AP) for selection of antibiotic resistance. For longterm storage the bacterial strain was stored in 15% glycerol at -70°C.2.2.2. Preparation of competent cells and transformationCompetent cells were prepared using a method described in Promega technical bulletin 018.Briefly, E.coli cells were grown in 20 ml of LB medium until the absorbance at 600 nm reached0.15-0.3. Cells were centrifuged at 5000 rpm in a Sorvall SS34 rotor at 4°C for 5 minutes, andthe supernatant was discarded. The bacterial pellet was resuspended in 10 ml of cold solution A(10 mM 3-[N-morpholino] propanesulfonic acid (MOPS) (pH 7.0); 10 mM RbCl), andcentrifuged as above. Cells were then resuspended in 10 ml of cold solution B (10 m’l MOPS(pH 6.5); 10 mM RbC1; 50 mM CaC12) and incubated on ice for 30 minutes. After pelleting thecells as above, cell pellets were resuspended in 1 ml of solution B plus 15% glycerol, and quickfrozen in 0.2 ml aliquots in dry ice-ethanol and stored at -70°C.For plasmid transformation, 0.2 ml of competent cells were incubated on ice with 10-50ng of plasmid DNA for 30 minutes. After a two minute heat shock at 42°C, 1 ml of LB mediumwas added to the transformation mixture and the cells were allowed to recover at 37°C for 45minutes before plating onto selective media.- 48 -2.2.3. DNA preparation and handling2.2.3.1. Mini-prep plasmid isolationColonies containing plasmids were picked into 3-5 ml of LB containing 100 ig AP per ml andthe bacteria were grown to saturation. Bacterial cells from 1.5 ml culture were pelleted for oneminute in a microfuge. The pellet was resuspended in 100 ul of 50 mM glucose; 10 mlvi EDTA;25 mM Tris-HC1 (pH 8.0) and lysed by the addition of 200 il of 0.2 N NaOH/1% SDS for 5minutes at 0°C. Chromosomal DNA and proteins were precipitated by incubating the lysismixture with 150 il of cold potassium acetate (3 M K; 5 M CH3OO, pH 4.8) at 0°C for 5minutes, followed by centrifuging in a microfuge for 5 minutes at 4°C. The supematant wasextracted with an equal volume of phenol:chloroform (1:1), and the DNA precipitated with twovolumes of ethanol at room temperature (RT) for 5 minutes. Plasmid DNA was recovered bycentrifugation in a microfuge for 5 minutes at RT, washed in 70% ethanol, dried in a Speed VacConcentrator, and resuspended in 50 il of TE (10 mM Tris-HC1, pH 8.0; 1 mM EDTA)containing 20 ag/m1 RNase A. Aliquots were used for restriction analysis or subcloning. Thismethod is essentially that described by Maniatis et al. (1982). Large scale plasmid DNA preparationsThe protocol is a procedure obtained from Promega technical bulletin 009 (developed by Dr. P.Krieg and Dr. D. Melton of Harvard University) with modifications. Cells grown in selectivemedia overnight in 100 ml cultures were pelleted by centrifugation at 5000 rpm in a Sorvall GSArotor at 4°C for 5 minutes. The supernatant was discarded and each pellet was resuspended in3 ml of 50 mlvi glucose; 10 mM EDTA; 25 mM Tris-HC1 (pH 8.0) containing 20 mg/mi- 49 -lysozyme followed by 20 minute incubation on ice. Cells were lysed by the addition of 6 ml of0.2 N NaOH/1% SDS and incubation on ice for 10 minutes. Chromosomal DNA and proteinswere precipitated with 4 ml of cold potassium acetate solution (see mini-prep procedure) on icefor 20 minutes, followed by centrifugation at 15,000 rpm in a Sorvall SS34 rotor at 4°C for 15minutes. RNase A (100 ug) was added to the cleared lysate followed by incubation at 37°C for20 minutes. The lysate was extracted twice with equal volumes of phenol:chloroform, and thenucleic acids were precipitated with one volume of isopropanol at RT for 5 minutes. Remainingnucleic acids were recovered by centrifuging at 15,000 rpm for 10 minutes at 4°C in a SS34rotor. The pellet was dried, and dissolved in 1.60 ml of sterile water. The solution wastransferred to siliconized Corex tubes and DNA was selectively precipitated by the addition of0.4 ml of 4 M NaCl and 2.0 ml 13% polyethylene glycol (PEG, MW 8,000), mixing andincubation on ice for 60 minutes. The plasmid DNA was pelleted at 10,000 rpm for 10 minutesat 4°C in a SS34 rotor, washed with 70% ethanol, dried and dissolved in TE. Restriction endonuclease digestions and DNA modificationAll restriction digestion reactions were performed according to assay conditions specified by thesuppliers.DNA fragments were ligated using T4 DNA ligase in 50 mM Tris-HC1, pH7.6; 10 mMMgC12; 1 mM ATP; 1 mM DTT; 5% (w/v) polyethylene glycol for 2 hours at RT, except forblunt-ended fragments which were ligated overnight. Reactions were diluted five-fold with TEprior to transformation (Maniatis et al., 1982).DNA fragments with 5’ overhangs were blunt-ended with E.coli DNA polymerase I Kienow- 50 -enzyme in 50 mM Tris-HC1, pH 7.2; 10 mM MgSO4;10 mM DTT; 50 mM BSA; 80 pM dNTP’sfor 30 minutes at RT. The enzyme was inactivated by heating at 70°C for 5 minutes (Maniatiset al., 1982).Fragments with 3’ protrusions were converted to flush ends using T4 DNA polymerase in33 mM Tris-acetate, pH 7.9; 666 mM potassium acetate; 10 mM magnesium acetate; 0.5 mMDTT; 100 mg/mi BSA; for 5 minutes at 37°C. Reactions were adjusted to 25 mM EDTA, andthe DNA purified by phenol:chloroform extraction and ethanol precipitation (Maniatis et al.,1982), or using GENECLEAN (BlO 101).Removal of terminal 5’ phosphates from DNA fragments with 5’ overhangs was done usingcalf intestinal alkaline phosphatase (CIP) in 50 mM Tris-HC1, pH 9.0; 1 mM MgCI2; 0.1 mMZnC12; 1 mM spermidine for two successive 30 minute incubation periods of 15 minute at 37°Cand 15 minutes at 56°C. CIP reactions were terminated by addition of 0.3% SDS andphenol:chloroform extraction followed by ethanol precipitation (Maniatis et al., 1982).Purification of DNA fragments from agarose gels or enzyme reaction mixtures wasroutinely done using GENECLEAN. Desired fragments were excised from ethidium bromidestained TAE agarose gels (see and the gel matrix was solubilized in 2-3 volumes ofsaturated sodium iodide at 55°C. DNA was removed from the agarose solutions by vortexing themixture with a suspension of glassmilk, and a brief spin in a microfuge. Contaminants werewashed away from the glass bound DNA by three successive washes with coldNaClJethanol/water solution. The DNA was eluted from the glass beads with TE or water byincubating at 55°C for 3 minutes.- 51 -2.2.4. Expression vectors2.2.4.1. pCMV5For transient expression of RV cDNA in COS cells, pCMV5 (Andersson et al., 1990)was used (Fig.4a). This vector directs transcription by the human cytomegalovirus majorimmediate early gene promoter and provides polyadenylation signal from the human grownhormone gene at the 3’ terminus of the inserted sequence. pCMV5 contains the SV4O origin ofreplication allowing replication in COS cells as well as a prokaryotic origin of replication andAP resistance gene for growth and selection in E.coli. pGS2OFor construction of vaccinia recombinants, vector pGS2O (Fig.4b) (Mackett et al., 1985)was used. This vector contains the vaccinia virus immediately early gene promoter p7.5, flankedby sequences from the vaccinia virus thymidine kinase gene. The AP resistance gene andprokaryotic replication origin allow propagation of the recombinant plasmid in E.coli. pNUTVector pNUT (Fig.4c) (Palmiter et al., 1987) was used to construct stable transformed BHKcells. RV cDNAs were cloned into this vector at the Sma I site which is flanked by the mousemetallothionein gene (mIvlT-1) promoter and the 3’ polyA sequences of the human growthhormone (hGH). The presence of the dihydroxyfolate reductase (DHFR) cDNA permits theselection of transfected BHK cells in the presence of a high concentration of methotrexate.Sequence from the human hepatitis B virus 3’ end mediates insertion of adjacent DNA sequences- 52 -into the chromosome of host cells (Nagaya et al., 1987). The pUC18 backbone and SV4O originfacilitate replication of the plasmid in E.coli and mammalian cells, respectively.2.2.5. DNA-mediated transfection2.2.5.1. Transfection of COS cellsCOS cells were transfected with plasmid DNA using a method described by Adam andRose (1985). Subconfluent monolayers of cells grown in Dulbecco modified Eagle medium(DMEM) plus 5% fetal calf serum (FCS) were washed twice with Tris-saline (25 mM Tris-HC1(pH 7.4), 140 mM NaC1, 3 mM KC1, 1 mM CaC12, 0.5 mM MgC12,0.9 mM Na2HPO4). Cellswere incubated with DEAE-dextran (Mr=5 X iO; 500 jig/mi) and plasmid DNA (4 jig/mi) inTris-saline at 37°C for 30 minutes. The DNA solution was then removed and replaced withDMEM plus 40 uM chioroquine for 3 hours at 37°C. After removal of chioroquine solution, thecells were shocked with 10% dimethylsulfoxide/DMEM for 3 minutes at RT. Finally, themonolayer was washed three times with Tris-saline and incubated at 37°C for 40 hours in DMEMplus 5% calf serum. The expression of RV proteins was analyzed using metabolic labelling orimmunoblotting (see below). Calcium-phosphate mediated DNA transfectionTransfection of CV1 cells and BHK cells with plasmid DNAs were according to Gorman et al.,(1982). CaPO4JDNA mixture was prepared by combining 10-25 jig plasmid DNA in 219 jil ofddH2O, 31 1il of 2M CaC12 and 250 jil of 2xHBSP (1.5 mM Na2HPO4;10 mM KC1; 280 mMNaCl; 12 mM glucose; 50 mM HEPES, pH 7.0). The mixture was allowed to stand for 30- 53 -CFig.4 Schematic representation of mammalian cell expression vectors used in this study. a.pCMV5. The vector backbone is pTZ18R (Pharmacia) and contains a bacteriophage fi ampicillinresistance gene (Amps). The CMV region consists of a promotor-regulatory region of the humancytomegalovirus major immediate early gene. b. pGS2O. The vector contains the promotor foran early gene coding for a 7.5 kDa polypeptide and is placed upstream from the uniquerestriction endonuclease Sma I site, flanked by vaccinia thymidine kinase (TK) DNA (thick line).c. Recombinant plasmid from pNUT vector. The backbone for this vector is pUCl8. The essentialfeatures of this vector are: DNA sequences taken from the 3’ termini of human hepatitis B virusgenome (HBV 3’); a promotor region from mouse metallothionein I gene (mMT-1); and sequencefor dihydrofolate reductase (DHFR). RV cDNAs were inserted into the Sma I site between themIvIT-1 and hGH 3’ sequences.11onSV 40onH8V3mMT-l-RV dJNAhGH 3pUC 18- 54 -minutes at RT prior to adding to the medium of cultured cells. Cells were incubated for differentperiods of time before removing the DNA mixture. The time of incubation depended on the celltype and nature of experiment.2.2.6. Construction of vaccinia recombinantsVaccinia virus recombinants expressing RV El glycosylation mutant proteins were constructedfollowing a standard procedure as described by Mackett et al., (1985). Infection/transfection procedureConfluent monolayers of CV1 cells in Minimal Essential Medium (MEM) were infected withpurified vaccinia virus (WR strain) at a ratio of 0.05 p.f.u./cell. Inoculum was removed at 2 hourspost infection (h.p.i.). Cells were washed twice with serum-free medium and 0.5 ml of DNAsuspension (CaPO4JDNA) (see was added to the cells and the cells were incubated for30 minutes at RT prior to the addition of MEM/5% FCS. Cells were scraped into the mediumat 48 h.p.i. and viruses were released by three cycles of freeze-thawing. Selection of recombinantsOne-fifth of the released viruses were layered onto monolayers of human tk 143 cells (gift fromF. Graham, McMaster University) and incubated for 1 hour at 37°C. The inoculum was removedand cells were incubated with Eagles medium containing 5% FCS and 25 .ig/ml 5-bromodeoxyuridine (BUdR). Progeny virus was harvested at 48 h.p.i., by scraping cells intomedium and then three rounds of freeze-thawing.- 55 - Plaque purification and virus titrationMonolayers of CV1 cells were infected with 0.5 ml of a ten fold serial dilution of virus andincubated at 37°C for one hour, with occasional shaking. The inocula were removed, cells werewashed with MEM once and overlaid with MEM containing 5% FCS and 1% noble agar. Cellswere stained at 36 h.p.i. with 1% agarose containing 0.1% neutral red. Clear virus plaques werevisualized after incubation for 2-3 hours. Plaques were counted and virus infectivity wascalculated as plaque forming units/mi (pfu/ml). Well isolated plaques were picked into a Pasteurpipette and virus in agarose plugs were eluted into 0.5 ml MEM and stored at -700C. Large-scale virus purificationMonolayers of CV-1 cells (in 175 cm2 flask) were infected with wild type or recombinantvaccinia viruses at a multiplity of infection (MOl) of 5 and incubated for 48 hours. Cells wereharvested and resuspended in 10 mM Tris-HC1, pH 9.0 and homogenized. The nuclear pellet wasremoved after centrifugation at 750xg for 5 minutes at 4°C. Trypsin (0.25 mg/mi) was added tothe supernatant and incubated for 30 minutes at 37°C. The supernatant was then layered on topof an equal volume of 36% sucrose in 10 mM Tris-HC1 pH 9.0 and centrifuged at 13,500 rpmin a Beckman SW27 rotor for 80 minutes at 4°C. The pellet was resuspended in 2 ml of 1 mlviTris-HC1 pH 9.0 and layered onto continuous sucrose gradients (15-40% in 1 mM Tris-HC1, pH9.0). The centrifugation was carried out at 4°C, 12,000 rpm for 45 minutes. Banded virus wascollected with a syringe through the side of the tube and stored at -70°C.- 56 -2.2.7. Metabolic labelling(i). COS cells. Labelling of COS cells was performed according to Hobman and Gillam(1989). Briefly, 40 hours post-transfection, transfected cells (in 35 mm dishes) were washed onceand incubated with methionine-deficient DMEM for 30 minutes prior to the addition of 0.5 mlmethionine-deficient DMEM containing 100 jiCi[35S]-methionine (Du Pont) and 5% FCSdialyzed against phosphate-buffered saline (PBS). Incubation with[35S]-methionine-containingmedium was for 30 minutes. Some cells were further incubated with a chase medium containing2 mM unlabelled methionine for various periods of time. Cells were washed with cold Tris-salineand lysed with 500 a1 of RIPA buffer (1% Triton X-100; 10 mlvi EDTA; 50 mM Tris-HC1, pH7.5; 1% sodium deoxycholate; 0.15 M NaCI; 0.1% SDS). After keeping on ice for 5 minutes,lysates were scraped off the plates and cleared of nuclei and debris by centrifugation at 4°C for5 minutes at 13,000 rpm. The supernatants were subjected to immunoprecipitation.(ii). Vero cells. Labelling of RV virions released from RV-infected Vero cells was carriedout as described by Clarke et al. (1987). At 24 h.p.i., infected cells (in 60 mm dishes) wereincubated with methionine-deficient medium for 30 minutes and labeled with 100 pCi [35S]-methionine for 1 hour. Cells were washed with and incubated in MEM with 2.5% FCS forvarious periods of time. Medium samples were collected and an equal volume of 20% PEG (MW8,000) in 2.5 M NaCl were added. RV particles were precipitated by centrifugation at 4°C for10 minutes at 14,000 rpm in an Eppendorf centrifuge after incubation on ice for 1 hour. Thevirus pellets were resuspended in RIPA buffer and RV-specific proteins wereimmunoprecipitated with human anti-RV serum and subjected to SDS-PAGE andautoradiography.- 57 -2.2.8. ImmunoprecipitationImmunoprecipitation of RV structural proteins from cell lysates was performed according toHobman and Gillam (1989). Human polyclonal anti-rubella serum, mouse serum or fluid asciteswere preincubated with Protein A-Sepharose (Pharmacia) for at least 4 hours at 4°C in bindingbuffer (100 mM Tris-HC1 (pH 7.4); 400 mM NaC1) with constant mixing. The antibody-coatedbeads were washed twice with binding buffer, and once in lysate buffer (25 mM Tris-HC1 (pH7.4); 100 mM NaC1; 1 mM EDTA; 1% Nonidet P-40). Cells lysates or harvested media wereadded and mixed with the antibody-coated beads for over 8 hours at 4°C with constant rotation.Beads were washed once with lysate buffer, twice with wash buffer (25mM triethanolamine; 172mM NaC1; 1% SDS; 1 mM EDTA), three times with 10 mM Tris-HC1 (pH 7.4), and once withwater. Antigen-antibody complexes were dissociated from the Protein A-Sepharose by boilingin 1 X SDS dissociation buffer (see below) for 5 minutes, vortexing and pelleting the beads bycentrifugation. Supernatants were collected and used for further analysis.2.2.9. Endogycosidase digestionThe conditions for endoglycosidase digestion were essentially those described by themanufacturer. Digestion with endoglycosidase H (endo H, Boehringer Mannheim) was carriedout in 100 mM sodium citrate buffer (pH 5.5) containing 0.15% SDS. Digestion with 0-glycosidase, endoglycosidase F/N-glycosidase F (endo F/PNGase F) and neuraminidase (all fromBoehringer Mannheim) were performed in a buffer containing 20 mM sodium phosphate pH 7.0,10 mlvi n-octylglucoside, 0.1%SDS. Immunoprecipitates to be digested with N-glycanase wereadjusted to 100 mM sodium phosphate (pH 8.6), 1% Nonidet P-40, 100 mM EDTA, 0.5% 3-- 58 -mercaptoethanol, 0.1% SDS. All incubations were normally for 8 hours.2.2.10. ImmunoblottingRV antigens were separated by SDS-PAGE and transferred to nitrocellulose filters usinga Bio-Rad Trans-Blot apparatus for 60 minutes at 240 mA in 25 mM Tris-HC1; 192 mM Glycine(pH 8.3); 20% methanol. The non-binding sites on filters were blocked by incubating for 30minutes to overnight in TBS (25 mM Tris-HC1, pH 7.4; 150 mM NaCl) containing 4% powderedskimmed milk. Membranes were then incubated with human anti-RV serum or monoclonalantibodies (at appropriate dilutions) for 1 hour, washed with TBS/0.3% Tween-20 and treatedwith goat anti-human or goat anti-mouse IgG conjugated to alkaline phosphatase (BRL) for 1hour. Blots were washed as above and developed with NBT (nitro blue tetrazolium)/BCIP (5-bromo-4-chloro-3-indoyl phosphate). All incubations were done at RT.2.2.11. Indirect immunofluorescenceTransfected COS cells grown on polylysine-coated 9 mm glass coverslips were washed threetimes with PBS, and fixed for 20 minutes at RT in 2% formaldehyde/PBS, followed by washingwith PBS. Some cells were permeabilized with 0.1%NP-40/PBS for 30 minutes prior to blockingwith 1% BSA/PBS. BSA/PBS was substituted for PBS in all dilutions and washings after thisstep. Coverslips were overlaid with diluted human serum (1:200) or murine monoclonalantibodies (1:100), incubated for 60 minutes at RT, and washed. Incubation with secondaryantibodies, fluorescein-conjugated goat anti-human or anti-mouse IgG (diluted 1:100) was for 60minutes. For double-labelling using lectin-conjugates, permeabilized cells were incubated with- 59 -wheat germ agglutinin-rhodamine conjugated (WGA-TRICT) to visualize Golgi and post-Golgistructures or concanavalin A-rhodamine conjugated (Con A-TRICT) for ER staining at 10-15jig/mi for 30 minutes at RT prior to blocking with BSA.2.2.12. Electrophoresis2.2.12.1. Separation of DNA fragmentThe buffers used in agarose gel electrophoresis were 1XTAE (40 mM Tris-acetate, pH 8.0; 1 mMEDTA) and 1XTBE (89 mM Tris; 89 mM boric acid; 2 mM EDTA; pH 8.0) for separation ofsmall fragments. The gel concentration varied from 1% to 2% agarose with 1 jig/mI ethidiumbromide for visualization. DNA samples were diluted to 8% sucrose; 20 mM EDTA (pH 8.0);0.05% bromophenol blue; 0.05% xylene cyanol and separated by electrophoresis on 10 cmsubmarine horizontal agarose gels at 75 volt. Separation of proteinProteins were separated using a discontinuous gel system described by Laemrnli (1970). Sampleswere adjusted to 62.5 mM Tris-HC1 (pH6.8); 10% glycerol; 2% SDS; 2% f3-mercaptoethanol anddenatured at 95°C for 3 minutes. Stacking gels consisted of 4% polyacrylamide, and separatinggels contained either 10% or 11% polyacrylamide. Solutions used to prepare these gels aredescribed in (below). Gels were run at constant voltage of 115-125 volts until themarkers have run to the desired position. The stacking gel was trimmed away, and the proteinswere either fixed in 10% acetic acid for 15 minutes for fluorography or transferred tonitrocellulose membrane for immunoblot analysis. Fixed gels were immersed in the fluorographic- 60 -agent Amplify (Amersham) for 15 minutes, dried under vacuum and exposed to X-ray film at -70°C. Solutions used for electrophoresis:5X Stacking gel buffer: 0.625 M Tris-HC1 (pH 6.8), 0.5% SDS5X Separating gel buffer: 1.875 M Tris-HC1 (pH 8.8). 0.5%SDS5X Gel running buffer: 0.125 M Tris-HC1; 0.96M glycine, 0.5% SDS (pH 8.3)Polyacrylamide Stock: 30% acrylamide, 0.8% N’N’-bis methylene acrylamideGels were polymerized by adding ammonium persulfate to 0.05% and TEMED(N’N’N’N’ -Tetramethylenediamine) to 0.1%.2.2.13. RV propagation, purification and titration2.2.13.1. Virus propagationVero cells were normally used to grow RV M33 strain in this study. Subconfluent cells (70%)were infected with RV at a MOl of 5-10 at 37°C for 2 hours. The inoculum was removed andcells were incubated with MEM with 2.5% FCS after washing once with the same medium. Themedium was collected at intervals of 24 hours starting at 48 h.p.i., and replaced with freshmedium after each harvesting, till 96 h.p.i. Cell debris were cleared by centrifugation at 3,000rpm for 5 minutes and virus particles were harvested from the medium by centrifugation at27,000 rpm for 2 hours. Pelleted virus was suspended in PBS and stored at -70°C.- 61 - Purification of RV or virus-like particles using sucrose density gradientsPelleted RV or virus-like particles from 35 ml tissue culture supernatant were suspended in 0.35ml TNG buffer (50 mM Tris, pH 7.5; 100 mM NaC1; 200 mM glycine) and applied onto the topof a 12 mI-sucrose gradient of 20-50% sucrose in TNG. Centrifugation was carried out using aBeckman SW41 rotor at 90,000xg for 16 hours at 15°C. Fractions (— 0.5 mi/fraction) werecollected by puncturing the bottom of the tube and the density of each fraction was determinedusing a refractometer. 100 i1 samples from alternative fractions were diluted with an equalamount of TNG buffer and subjected to centrifugation at 90,000 rpm for 20 nun, on a Tabletopcentrifuge. The pellets were resuspended with RIPA buffer. RV proteins in the pellets, and in thesample that loaded onto the gradient was analyzed by SDS-PAGE and immunoblotting (usinghuman anti-RV serum). RV structural protein-containing fractions were considered to be purifiedvirus or virus-like-particle stocks. Titration of RVThe infectivity of harvested virus or RV stock was determined using an immunochemical focusassay (Fukuda et al, 1987). Briefly, monolayers of RK cells in a 96-well plate were infected withRV in serial dilutions (10 to l0) for 2 hours at 37°C. Infected cells were washed with PBS andfixed with 3% formaldehyde in PBS for 15 minutes at 72 h.p.i.. After washing twice with PBS,endogenous peroxidase was inactivated with 0.2 ml of 0.5% H20 in absolute methanol for 15minutes at RT. The non-selective immunoglobulin binding sites of the monolayers were blockedby incubation for 1 hour at 37°C with 0.2 ml of rabbit pre-immune serum (1:200 dilution inPBS/0.5% BSA). 0.2 ml human anti-RV serum (1:200 dilution in PBS/0.5% BSA) was added and- 62 -incubated at 37°C for 60 minutes followed by three rinses with wash buffer. Cells were thenincubated with peroxidase-conjugated rabbit anti-human IgG (1:200 in PBS/0.5% BSA) for onehour at 37°C and then were rinsed three times with wash buffer. Plaques were visualized afterapplying peroxidase substrate (0.1 ml PBS containing 0.02% cold H20 and 0.5 mg/mi 3,3’diaminobezidine tetrahydrochloride) and incubating cells at RT till brown deposits developed.Plaques were counted from three wells per virus dilution and averaged. Titres of virus wereexpressed as pfu/ml.2.2.14. Electron microscopyFor routine morphology studies, cells (RV-infected or stable transformed cells) were fixed with2% glutaraldehyde, 3%paraformaldehyde in 100mM NaCacodylate buffer, pH 7.2, scraped fromthe culture dish, and pelleted in a microfuge. The cell pellets were then postfixed (one hour) in2% 0s04 in the same buffer, stained in block (2 hours) with 2% uranyl acetate. Dehydration wascarried out with a graded series of ethanol and sample blocks were embedded in Epon plasticresin using standard methods (Barteletti et al., 1979). A series of thin (250 nm) plastic sectionswere collected on Formvar-coated slot grids after cut and analyzed.2.2.15. Mice immunization2.2.15.1. Immunization with live, purified vaccinia recombinants.High-titer vaccinia recombinants expressing wild-type or glycosylation mutant RV El proteinswere purified from infected cells by centrifugation in sucrose density gradients (see Thepurified recombinant viruses were titered on CV-l cells by plaque assay ( and lxl05 pfu- 63 -of each recombinant (in PBS) were used to immunize individual mice (four in each group) byintraperitoneal (i.p.) injection. Mice were re-injected 4 additional times at 3 week intervals. Fourmice were immunized with each vaccinia recombinant and were bled for serum 10 days aftereach injection. Sera from mice immunized with the same vaccinia recombinant were pooled andused for immunological assays. Immunization with RV and virus-like particlesRV or virus-like particles were semi-purified from culture medium using centrifugation. Identicalamounts of antigens (equivalent to 256 HA units) were emulsified in Freund’ s complete adjuvantand used to immunize mice (four in each group). Mice received three additional injections ofantigens in Freund’s incomplete adjuvant at three-week intervals. Mice were bled and sera werecollected for analysis.2.2.16. Enzyme linked immunoadsorbant assay (ELISA)RV (diluted 1:600) or individual structural proteins (diluted 1:20) expressed from recombinantbaculoviruses (Gillam, unpublished results) were coated onto Immulon-2 plates (Dynatech,Chantilly, VA) in carbonate buffer [15 mM Na2CO3,35 mM NaHCO3 (pH 9.5)]. Following onehour blocking in 0.5% skim milk-PBS, the plates were incubated with monoclonal antibodies(ascites fluid or tissue culture supernatant), mouse sera or human sera diluted in 0.5% skim milk-PBS. The one hour incubation was followed by the addition of alkaline phosphatase-conjugatedgoat anti-mouse or anti-human IgG antibodies (BRL) diluted 1:3000. The plates were developedin substrate buffer [1M diethanolamine, 5 mM MgCl2,2 mg/mi p-nitro-phenylphosphate (pH 9.6)]- 64 -and read at 405 nm on a Bio-Rad microplate reader (Bio-Rad, Richmond, CA).2.2.17. Hemagglutination (HA) assay and hemagglutination inhibition (HAl) assayHA and HAT assays were performed using a heparin/manganous chloride procedure (Liebhaber,1970). RV antigens (25 tl) were serially diluted (two fold) with HSAG (25 mM HEPES, pH 6.5;140 mM NaC1; 1 mM CaC12; 1% BSA; 0.0025% gelatin) and seeded on a polyvinyl plate. Afterchilling the plate at 4°C for 15 minutes, 50 }Jl of 0.25% one day old chick erythrocyte suspensionwas added to each well. Aggregation of chick erythrocytes was developed after incubation at 4°Cfor one hour in some wells and the HA titre of the antigen was expressed as the end-point ofserial dilution at which full agglutination was observed.For HAT assay, serum samples or ascites fluid (200 }Jl) were pre-treated with 200 il ofMnC12/heparin solution (0.5 M MnC12, 2500 lU/mi Porcine heparin). Following the 15 minutesof incubation, 200 p1 of a 50% chick erythrocyte solution in HSAG was added and incubated onice for one hour. An additional 600 p1 of HSAG buffer was added and the serum/erythrocytemixture was subjected to centrifugation for 10 minutes at l,000xg and the supernatant (a dilutionof 1:8) was collected. 50 p1 of treated serum was serially diluted two-fold in polyvinyl plates and25 p1 of RV antigen containing 4 HA units were added to each well. Following one hourincubation at 4°C, 50 p1 of 0.25% one day old chick erythrocytes in HSAG was added and theplates kept at 4°C for another hour before interpreting the results. The HAT titre was expressedas the end-point of dilution at which no aggregation of erythrocytes was observed.- 65 -2.2.18. Viral neutralization assayPurified ascites fluid and sera from pre-immune or immunized mice were heated at 55°C for 20minutes to inactivate complement, diluted 1:5 in M199 medium with 2% FCS, centrifuged for10 minutes at 10,000 rpm and sterilized by filtration through a 0.22 pm pore size filter.Serial dilutions (in triplicate) of the serum were performed in M199 medium with 2%FCS to which was added equal amounts of diluted RV (2 pfulpl in M199, 2% FCS) with orwithout rabbit complement (2.5%). The virus-antibody mixture was incubated at 37°C for onehour, then 50 p1 was layered onto subconfluent RK cells in 96-well microtitre plates, mixing forone hour at 37°C. The virus-antibody mixture was removed and monolayers were layered withM199 medium containing 2.5% FCS and incubated at 37°C for 72 to 96 hours. Plaques weredetected using the immunoperoxidase method ( and the VN titre was the reciprocal ofthe dilution that demonstrated at least a 50% reduction in plaque formation compared to controlcells.2.2.19. Lymphocyte proliferation assayLymphocytes were isolated from spleens of immunized mice. For the antigen-specific response,cells (2.5x 1 o per well) were incubated in 96-well flat bottom plates with varying concentrationsof expressed antigens in triplicate. Following 7 day incubation at 37°C with antigen, the cellswere pulse labeled with[3H1-thymidine (lpCi/well) for 16 hours, harvested and washed ontoglass-fibre filters with distilled water. After the filters were air dried overnight, 3 ml ofBiodegradable Counting Scintillant (Amersham) scintillation fluid was added to determine theincorporation of[3H]-thymidine.- 66 -3. RESULTS and DISCUSSION3.1. Section I. Role of N-linked glycosylation on E2 processing and transport3.1.1. E2 cDNAsE2 cDNAs were constructed previously in this lab (Hobman and Gillam, 1990). Oligonucleotidedirected mutagenesis was employed to introduce one or two nucleotide changes in the codonsencoding asparagine or serine, resulting in a single amino acid substitution at each potentialglycosylation site. The addition of N-linked oligosaccharides was prevented by changing the AsnX-Ser consensus sequence at asparagine residues 53, 71, and 115 to Gln-X-Ser, Asn-X-Gly, andLle-X-Ser, respectively. The mutants in which consensus sequences were altered singly arereferred to as G 1, G2, and G3; the double mutant is referred to as G 12; and the triple mutant isreferred to as G123 (Fig.5). The positions are numbered sequentially from the N-terminus of E2.The wild-type and mutant E2 cDNAs were inserted into a mammalian expression vector pCMV5(Fig.4a) (Andersson et al., 1989) and used to transfect COS cells.3.1.2. Determination of functional N-linked glycosylation sites in E2N-Glycanase digestion was performed to characterize the actual number of N-linkedoligosaccharide side chains on E2. N-Glycanase hydrolyses the glucosylamine linkage of all typesof N-linked oligosaccharides on glycoproteins to give free oligosaccharides and polypeptides.Digestion of radio-labeled and immunoprecipitated wild-type E2 expressed in COS cells with aserially diluted N-glycanase generated four species with apparent molecular weights of 37, 35,33, and 31K (Fig.6). It is likely that these four species corresponded to E2 with three, two, one,- 67 -G123 I1 Sites 1,2 &3AlteredFig.5 Schematic representation of wild-type and glycosylation mutants of RV E2. The E2 proteincontains three N-linked glycosylation sites at residues 53, 71 and 115 as depicted by branchedstructures (Y). The putative transmembrane region is located near the C-terminus of E2 ([‘).The first residue of mature E2 is glycine-1 and the C-terminal residue of E2 before El is glycine281.12 3Y1 53 71 115 281I IWTGiG2G3G12Y----II 1.. -- -YY..I...- III 1Asn ‘-Gin(53)Sec ‘-Gly(73)-Asn ‘-lie(115)Sitesl&2Altered- 68 -Fig.6 Determination of the number of N-linked glycans on RV E2.[35S]-methionine labeled E2was incubated with no (lane 1); 10 mU (lane 2); 20 mU (lane 3); 50 mU (lane 4); 100 mU (lane5) and 300 mU (lane 6) N-glycanase (Boehringer Mannheim) for 10 minutes at 37°C. E2 wasseparated by SDS-PAGE and subjected to fluorography. The positions of molecular weightmarkers are shown on the left (kDa).A12345 629-- 69 -and no carbohydrate side chain(s), suggesting that wild-type of E2 glycoprotein normally hasthree N-linked oligosaccharide chains.3.1.3. Expression of E2 glycosylation mutants in COS cellsAnalysis of the expression of E2 glycosylation mutants in COS cells was carried out accordingto procedures detailed in Materials and Methods. After a 30 minutes pulse-labelling period, wild-type E2 expressed as a prominent 37 kDa glycoprotein (Fig.7a, wt). The electrophoreticmobilities of the mutant proteins increased proportionally with the number of inactivatedglycosylation sites (Fig.7a). Removal of any single glycosylation site at position 1, 2 or 3 resultedin the synthesis of a major 35 kDa glycoprotein, while the double mutant G23 and the triplemutant G 123 directed the synthesis of proteins which migrated at 33 and 31 kDa, respectively,(Fig.7a).To verify that the differences in electrophoretic mobility between wild-type and mutant E2were due to the numbers of N-linked oligosaccharide side chains attached, some transfected cellswere treated with tunicamycin. Tunicamycin at a low concentration efficiently inhibits N-linkedglycosylation without interferring with protein synthesis in cells (Elbein, 1987). In the presenceof 3 jig tunicamycin per ml, all the E2 polypeptides synthesized in cells transfected with wild-type and different glycosylation mutant cDNAs had the same molecular weight as the triplemutant, G123 (Fig.7a). Tunicamycin did not affect the apparent molecular weight of G123 fromtransfected cells (Fig.7a, G123; 7b, E2G123) nor did digestion with N-glycanase (Fig.7b,E2G123). These results further confirmed that all three potential N-linked glycosylation sites ofE2 are normally used and that the difference in molecular weight between wild-type and mutant- 70 -Fig.7 Expression of wild-type and glycosylation mutants of E2 in COS cells. (A). Transfectedcells were labeled with[35S]-methionine for 30 minutes in the presence (+Tm) or absence of 3jig/mi of tunicamycin. RV specific proteins were immunoprecipitated using human anti-RV serumand separated by 11% SDS-PAGE. (B). Some immunoprecipitated E2 proteins were treated with100 mU N-glycanase at 37°C overnight (+ glycanase) and subjected to SDS-PAGE andautoradiography. The positions of molecular weight markers are shown on the left in kDa.ft Gi G2 G3 G12 Gi23 wt- + -+ -+ -+ -+- + TmBwt E2 E2G123— + — — + — Tm—— + — — + glycanase- 71 -E2 is due to the number of carbohydrate chain attached.3.1.4. Formation of aberrant disulfide bonds in E2 glycosylation mutantsThe possible formation of aberrant disulfide bonds in E2 glycosylation mutants was examinedby pulse-chase analysis. Radio-labeled E2 proteins from transfected COS cells wereimmunoprecipitated with human anti-RV serum and separated by SDS-PAGE under reducing andnonreducing conditions (Cohen et al., 1982). Wild-type and mutant E2 proteins migrated slightlyfaster in the absence of 13-mercaptoethanol (nonreducing) than in its presence (reducing) (Fig.8),implying the existence of intramolecular disulfide bonds in E2 that have also been observed inmany other glycoproteins (Machamer and Rose, 1988a, b; Vidal, et al., 1989). The 012 proteinran as a diffuse band, and the G123 protein was not detectable on the gel under nonreducingconditions, although in the presence of f3-mercaptoethanol, bands corresponding to these mutantproteins were readily detected (Fig.8). These results suggest that the formation of aberrantdisulfide intramolecular bonds occurs causing the proteins to migrate as diffuse smears whendisulfide bonds are not disrupted.The possible formation of aberrant intermolecular disulfide bonds in E2 mutants wasfurther analyzed by immunoblotting (Towbin et al., 1979). Under reducing conditions, singleglycosylation mutants had a prominent 35 kDa and minor 33.5 kDa and 31 kDa glycoproteinspecies (Fig.9). Two species at 33 and 31.5 kDa were detected in the double mutant (Fig.9). Onlythe 31 kDa unglycosylated E2 protein was observed in the triple mutant (Fig.9). Undernonreducing conditions, the samples migrated slightly faster because of the presence ofintramolecular disulfide bonds (Fig.9). Although the majority of E2 remained as monomer,- 72 -+13-MeA-13-MeFig.8 Formation of aberrant disulfide bonding in E2 glycosylation mutants. Transfected cells werepulse-labeled with 100 jiCi[35S]-methionine for 30 minutes and chased with excess methioninefor 2 hours. RV-specific proteins were analyzed by immunoprecipitation using human anti-RVserum, separated on 11% SDS-PAGE with or without 3-mercaptoethanol and fluorographed. Thepositions of molecular weight markers are shown on the left in kDa and the arrow indicates thestart of the separating gel.r’)—00000I- 73 -wt G123 G12 G3 G2 GIA-68-43- —-29BFig.9 Western blot analysis of steady-state wild-type and mutant E2 proteins in transfected cellsunder reducing and non-reducing conditions. Transfected COS cells were lysed (40 hour posttransfection) with RIPA buffer (50 mM Tris-HC1, pH 7.5, 1% Triton X-l00, 10 mM EDTA, 0.15M NaC1, 0.1% SDS, 1% sodium deoxycholate) containing 10 mM iodoacetamide. Cytoplasmicextracts were electrophoresed on 11% reducing (A) and non-reducing (B) gels. The proteins weretransferred to cellulose nitrate membranes. Membranes were blocked in 4% milk powder in TBS(0.15 M NaC1, 0.02 M Tris-HC1, pH 7.5) and incubated with human anti-RV serum (1:200dilution). The proteins were visualized using alkaline phosphatase-conjugated anti-human IgG.The positions of molecular weight markers are shown on the right in kDa.- 74 -protein species corresponding to the position of E2 diiner, trimer and tetramer were readilyobserved (Fig.9). Deletion of any glycosylation site from E2 seemed either to abolish the bindingof antibodies to E2 or reduce the amount of monomeric forms, especially in G12 and G123(Fig.9). It is possible that these mutants proteins exist as alternatively folded structures that arenot recognized by anti-RV serum and that the antigenic sites in G12 and G123 forms aredetectable only after unfolding of these proteins by cleavage of intramolecular disulfide bonds.These finding suggest that the pattern of disulfide bonding for E2 glycosylation mutants isheterogeneous and the glycosylation may be important in preventing aberrant disulfide bondformation.3.1.5. Glycan processing and intracellular stability of E2 proteinsThe kinetics of processing and the turnover rate of the E2 mutant proteins were examined bypulse-chase experiments followed by densitometric analysis of processed proteins. After thirty-minute pulse-labelling, wild-type E2 was found predominantly in the 37 kDa form, and removalof high-mannose glycans by endo H digestion reduced the molecular size to 31 kDa (Fig.10).Approximately 25, 40 and 50% of wild-type of E2 was found to possess complex-type sugar after1-, 2- and 4-hour chase periods, respectively (Fig.10). In contrast, Gi, G2, and G3 mutantproteins containing complex-type glycans represented only 17, 14, and 10% of the total amountof each mutant protein after a two-hour chase (Fig. 10). No endo H resistance was observed forthe double mutant, G12 (Fig. 10). As the acquisition of endo H resistance is believed to beindicative of transport of glycoproteins through the medial Golgi apparatus, it is evident thatremoval of glycosyl moieties impairs the transport of E2 mutant proteins. This effect is dependent- 75 -WTGiG243-29-o 12 4——chase(hrs)endoH—R—sFig.10 Time course for glycan processing of wild-type and mutant E2 proteins. Cells were pulse-labeled with[35SJ-methionine for 30 minutes and chased for various times as indicated. Someimmunoprecipitated samples were digested with endo H for at least 8 hours (+ endo H). EndoH-resistant (R) and sensitive (S) oligosaccharide-containing proteins are indicated. The positionsof molecular weight markers are shown on the left in kDa.— 429-43-—sG3 43-29- aiG1229- ——s- 76 -on both the position and the number of glycosylation sites altered.To determine the turnover rate of wild-type and mutant E2, immunoprecipitates fromtransfected COS cells were fractionated on SDS-PAGE and quantitated by densitometric analysisof the autoradiographs (Fig. 11). Wild-type E2 was relatively stable in COS cells, with 70% ofE2 remaining after a 4 hour chase. By contrast, the mutants exhibited a higher turnover rate. Thehalf-lives (t112) for mutant proteins in the cells were: Gi, G2 and G3=3 hours; G12=2 hours; andG123=30-60 minutes. It could be that the mutant proteins were not properly folded andtransported due to an altered glycosylation pattern, and were rapidly degraded as has beenreported for some other glycoproteins (Matzuk and Boime, 1988).3.1.6. Intracellular localization of mutant E2 proteins.The subcellular localization of E2 mutant proteins was examined using indirectimmunofluorescence. Cells expressing wild-type E2 exhibited staining throughout the cytoplasmicreticulum as well as in the juxtanuclear region (Fig.12a). The single, double and tripleglycosylation mutant proteins displayed a predominantly reticular staining pattern as well asGolgi-like staining (Fig.12c,e,g). To visualize the distribution of E2 protein in the ER and Golgi,fluorescent-conjugated WGA and ConA were used as markers for the compartments, WGA hasbeen shown to label trans Golgi cisternae, associated vesicles and the cell surface (Tartakoff andVassalli, 1983) by binding to clustered terminal N-acetylneuraminic acid residues as well as Nacetylgiucosamine-containing oligosaccharide chains on glycoproteins (Virtanen et al., 1980). Costaining of transfected COS cells with human anti-RV serum and fluorescent-conjugated WGArevealed that wild-type E2 was concentrated in the Golgi region (Fig.12b), while the mutant E2- 77 -PERCENTAGE120Fig. 11 Intracellular stability of wild-type and mutant E2 proteins. Cells were pulse-labeled with[35S]-methionine for 30 minutes and chased for various times as indicated. RV-specific proteinswere immunoprecipitated using human anti-RV serum. Rates of degradation of wild-type andmutant E2 proteins were quantified by scanning densitometry of the X-ray films from three tosix independent experiments as shown in Fig.l0. Different chase times are indicated. ---i--- wild-type, ---0--- Gi, ---•--- 02, ---a--- G3,---*--- G12, ---+--- G123.10 1HOURS2 3 4- 78 -Fig.12 Indirect immunofluorescence of wild-type and mutant E2 proteins in COS cells. Cellswere permeabiized prior to the addition of rhodamine-conjugated WGA or ConA and anti-RVserum. After the cells were washed, a secondary antibody (fluorescein-conjugated goat antihuman IgG) was added. (a) wild-type, anti-RV; (b) wild-type, TRICT-WGA; (c) G2, anti-RV;(d) G2, TRICT-WGA; (e) G12, anti-RV; (f) G12, TRICT-WGA; (g) G123, anti-RV; (h) G123,TRICT-ConA.- 79 -proteins were distributed throughout the reticulum network and Golgi region (Fig.12 d,f). Astrong reticular staining, which co-localized with ConA, was observed in COS cells transfectedwith glycosylation mutants (Fig.12h). In addition, unlike wild-type E2, which has been shownto exhibit limited amount of cell surface expression (Hobman and Gillam, 1989), theglycosylation mutants had no detectable cell surface signal (data not shown). Elimination of anyof the glycosylation sites in E2 seemed to impair the intracellular transport and to block the cellsurface expression of E2.3.1.7. Secretion of an anchor-free form of wild-type and mutant E2 proteins.To analyze the transport behavior of E2 mutants in the secretory pathway, a panel of truncatedE2 glycosylation mutants were constructed, each of which had 68 amino acids deleted from thehydrophobic C-terminus (Hobman et al., 1994). The truncated form of wild-type E2 was secretedinto the culture medium as a 36-kDa endo H-resistant glycoprotein (Hobman et al., 1994). Thetruncated form of E2 single glycosylation mutants (Gi, G2 and G3) but not double (G12) andtriple (G 123) mutants were also secreted into the culture medium, although not as efficiently asthe anchorless wild-type E2 (Fig. 1 3a). In addition, the efficiency of secretion appeared to dependon the position of the deleted glycosylation site. Deletion of the glycosylation site proximal tothe C-terminus (G3) had a more profound inhibitory effect on the secretion than of the centralsite (G2) and of that proximal to the N-terminus (G1) (Fig. 13a).The intracellular forms of anchorless wild-type E2 and E2 glycosylation mutants weresensitive to endo H digestion (Fig.13b), whereas the secreted E2 was endo H-resistant (Fig.13a).Expression of the truncated triple mutant was not detected intracellularly (Fig. 13b). The 3 1-kDa- 80 -Ic wt Gi G2 G3 G12 G123—+—+—+—+—+ —+b•-. -ÄendoHGi G2 G3 G12 G123 wt—+ —+ —+ —+ —+ —+ endoHFig.13 Intracellular processing and secretion of a soluble form of wild-type and mutant E2proteins. Cells were labeled with[35S]-methionine for 30 minutes and chased for 4 hours. (a).Immunoprecipitated samples from culture media of cells transfected with anchorless wild-typeand mutant E2 cDNA constructs. (b). Intracellular forms of each anchorless E2 protein of wild-type and glycosylation mutant. Equal volumes of each sample were incubated at 37°C for at least8 hours with or without endo H and separated on 11% SDS-PAGE. The positions of themolecular weight markers are shown on the left in kDa.29- 81 -endo H-sensitive protein species found in the culture medium of the G2 transfected cells(Fig. 13a) is probably due to lysis of a portion of cells during the chase period and the intracelluarG2 mutant protein was released into the medium. Taken together, it was evident that the singleglycosylation mutants Gi, G2 and G3 were transported out of the ER through the Golgi to thecell surface and then exited the cell into the culture medium, although not as efficiently as theotherwise unaltered anchorless E2 protein.3.1.8. Summary and DiscussionThe role of N-linked glycosylation in processing and intracellular transport of RV E2glycoprotein has been studied by expressing glycosylation mutants of E2 in COS cells. In RVM33 strain, all three sites were used for the addition of N-linked oligosaccharides. Removal ofany of the glycosylation sites resulted in slower glycan processing, lower stability and aberrantdisulfide bonding of the mutant proteins, with the severity of the defect depending on the numberof deleted carbohydrate sites. The mutant proteins were translocated into the ER and transportedto GC but were not detected on the cell surface. However, the secretion of the anchor-free formof E2 into the medium was not completely blocked by the removal of any one of itsglycosylation sites. This effect was dependent on the position of the deleted glycosylation site.Protein movement from the ER to the medial Golgi apparatus has been identified as therate-limiting step in the exocytic pathway (Rose and Bergmann, 1983), as measured by theacquisition of a variety of organelle-specific post-translational modifications. Regarding theintracellular transport rate, several viral glycoproteins that have been extensively investigated fallinto two categories. The first group includes the VSV G protein (Rose and Doms, 1988) and- 82 -influenza virus HA (Gallagher et al., 1988) which move quickly along the exocytic pathway.After a 15 minutes chase period, 50% of the oligosaccharides on VSV G and 25% on influenzavirus HA acquire endo H resistance (Rose and Doms, 1988; Gallagher et al., 1988). The secondgroup includes the HIV envelope protein (Earl et al., 1991) and simian virus 5 hemagglutininneuraminidase (SV5 HN) (Ng et al., 1989), for which acquisition of endo H resistance wasobserved within 80 minutes and 60 minutes post-labelling, respectively. We found that thecarbohydrates on wild-type RV E2 were converted to complex-type sugar moieties by 1 hourpost-labelling. However, the conversion was not complete even after a 8 hours chase period (datanot shown), reflecting a slow movement of RV E2 from the ER to the Golgi apparatus.The data from this study showed that E2 contains three potential oligosaccharide additionsites and all three potential N-linked glycosylation sites were utilized (Fig.6 and 7). Inactivationof these functional sites impaired the processing as well as the intracellular stability of E2proteins (Fig. 10 and 11), the severity of the defect depending on both the number and theposition of the glycosylation site deleted. Deletion of one N-linked glycosylation site on RV E2considerably reduced the rate of transport, as determined by the fraction of proteins that acquiredendo H-resistant carbohydrates (Fig. 10). The glycosylation site proximal to the N-terminus (Gi)seems to be less important than the site proximal to the C-terminus (G3), as judged by thefraction of molecules containing endo H-resistant carbohydrates for the membrane-bound formand by the secretion ratio of their anchorless counterparts (Fig. 10 and 13). That theoligosaccharide at each glycosylation site may play a different functional role has been notedpreviously with other glycoproteins (Matzuk and Boime, 1988; Ng et al., 1990). In addition,studies on other glycoproteins using the same approach have shown that glycosylation on all the- 83 -predicted sites is not a prerequisite for folding, assembly and transport of the protein (Guan etal., 1988). It has been suggested that the contribution of each carbohydrate chain variesdepending on its location in a different conformational circumstance of a particular protein. RVE2 is rich in cysteine and undergoes intramolecular disulfide bonding (Fig.9). Inspection of theamino acid sequence of RV E2 reveals that the 62 and G3 glycosylation sites are flanked by twocysteine residues (Clarke et al., 1987). It is possible that the oligosaccharides attached to the G2and G3 sites are important in preventing improper intramolecular disulfide bond formation,whereas glycosylation at the 61 site has less effect on proper folding and transport. The diffuseor smeared appearance of non-reduced mutant G12 and G123 proteins on an immunoblot (Fig.9)probably reflects the formation of aberrant intermolecular disulfide bonds. Thus, it appears thatoligosaccharide addition is required for proper intramolecular disulfide bonding to promotecorrect folding, which in turn is essential for efficient transport (Vidal et al., 1989). Removal ofa glycosylation site leads to formation of improper intramolecular disulfide bonds and proteinmisfolding. Dramatic alteration in protein conformation and possibly aggregation could be theconsequence when glycosylation sites are inactivated. This may account for diminished antibodybinding by G12 and 6123 proteins under non-reducing conditions (Fig.9B).Proteins that are transported slowly in cells display heterogeneity in endo H resistance.For example, this has been observed for SV5 HN (Ng et al., 1989), influenza virus neuraminidase(Kunda et al., 1991) and HIV gpl2O (Earl et al., 1991). Pulse-chase experiments havedemonstrated that endo H-sensitive, partially endo H-resistant and endo H-resistant E2 formsrepresent the ER-, Golgi- and cell surface- isoforms of RV E2. Immunofluorescence oftransfected COS cells expressing E2 wild-type and mutant genes showed that the majority of the- 84 -glycosylation mutant proteins were localized in the ER (Fig. 12). A small fraction were found inthe Golgi region (Fig. 12). Transport of the E2 single glycosylation mutants into the Golgicompartment was evidenced by the presence of partially endo H-resistant bands after the chaseperiod (Fig.1O), as well as by the secretion of the C-terminal truncated form of E2 singleglycosylation mutants (Fig. 16a). Thus the transport of E2 to the Golgi apparatus appeared to besignificantly affected but not completely blocked by the absence of any one of the N-linkedoligosaccharides.The anchorless E2 single glycosylation mutants were secreted into the culture medium,although less efficiently than wild-type anchorless E2. The oligosaccharides on the secreted formsof wild-type and mutant E2 were completely endo H-resistant as is E2 from RV virion(Lundstrom et al., 1991), suggesting that carbohydrates attached to these proteins are modifiedby Golgi enzymes. This finding indicates that the soluble forms of E2 single glycosylationmutants are transported through the normal exocytic route. Inability to detect cell surfaceexpression of E2 single glycosylation mutants could be due either to the low sensitivity ofindirect immunofluorescence in our experiments, or to the fact that mutant E2 proteins werequickly and extensively internalized from the plasma membrane, as has been observed for otherglycoproteins (Ng et al., 1989).- 85 -3.2. Section II. Effect of Brefeldin A (BFA) and monensin on protein processing and virusassembly3.2.1. Processing of N-linked oligosaccharides on E2 in BFA- and monensin-treated cellsThe expression of RV E2 in pCMV5-E2 transfected COS cells was used to determine theappropriate concentrations of BFA and monensin for the study. Interestingly, BFA seemed toenhance the level of E2 protein expression (Fig. 14A), the mechanism of which is not understood.However, no difference in the glycan processing of RV E2 was found in cells treated with BFAin the range from 1 pg/ml to 12 jig/mi (Fig.14A) and thus 6 jig/mi of BFA was chosen for usein the subsequent analysis. In contrast, monensin at higher concentrations (25 pM) inhibited E2synthesis (Fig.14C), and hence 5 pM monensin was used in the subsequent experiments.Endo H, an enzyme that removes N-linked high-mannose oligosaccharide, was used tomonitor the processing of E2 glycoproteins in pulse-chase experiments. In control (untreated)cells after 30 minutes labelling, E2 accumulated as a 37 kDa protein (Fig. 14A). Digestion withendo H reduced the molecular size of the protein species to 31 kDa (Fig. 14A). By contrast, inBFA-treated cells, only the 42 kDa species was observed by the end of 30 minutes labelling(Fig.14A) and digestion with endo H decreased its molecular weight from 42 kDa to 36 kDa(Fig.14A). By 4 hour post-labelling, in control cells, about 50% of synthesized E2 was partiallyendo H-resistant (Fig.14C). Whereas BFA induced a rapid and complete conversion of glycanson E2 to endo H-resistant forms by 4 hour post-labelling (Fig.14C). In monensin-treated cells,pulse-labeled E2 protein had a similar electrophoretic mobility to that of the control cells(Fig.14B). However, after 3 hours of chase, monensin treatment gave rise to a novel E2 protein- 86 -A 0 1 3 6 12 BFA(LJg/mI)i29-B 1 5 25 0 monensin(LIM)— +— +—- +— zLctr IC 2 3 4 4 chase(h)— + — + — + — +29-ctrl__________8_ 3 chase(h)—+ — +Fig.14 Effect of BFA and monensin on processing of E2. Transfected cells were pulse-labeledwith 100 pCi[35S]-methionine for 30 minutes and incubated with excess methionine for indicatedtime in chase experiments (C, D). BFA (in pg) (A) or monensin (in pM) (B) were added to themedium at the concentrations shown on top of A and B. In chase experiments, 6 pg/mi of BFA(C) or 5 pM of monensin (D) were present. Cells were lysed with RIPA buffer after labelling(A and B) or after chase for the indicated period of time (C, D). E2 proteins wereimmunoprecipitated with human anti-RV serum, subjected to SDS-PAGE and fluorography. Halfof each immunoprecipitated sample was digested with endo H (+) for 8 hours at 37°C beforeseparating on SDS-PAGE. Molecular weight markers (in kDa) are shown on the left. E2 proteinbands containing endo H-sensitive (s) and resistant (r) sugar moieties are marked. The novel 34kDa protein species after endo H digestion is indicated by a star (*).4—+D 3—+43’- 87-band with partial endo H-resistant sugar moieties and an apparent molecular weight of 34 kDa(Fig.14D, star) instead of 36 kDa seen in control cells (Fig.14D, ctrl).3.2.2. Processing of 0-linked glycans on E2Since it has been shown that E2 contains 0-linked oligosaccharides (Lundström et aL, 1991;Sanchez and Frey, 1991), I then analyzed the extent of 0-glycosylation of E2 in BFA- andmonensin- treated cells and in cells also treated with tunicamycin. In control cells (untreated withBFA nor monensin) after a 4 hour chase period, digestion with endo F/PNGase F reduced themolecular weights of two E2 species from 42 kDa and 37 kDa to 36 kDa and 31 kDa,respectively (Fig. 15A), similar to that observed after endo H digestion (Fig. 14C). After incubationwith neuraminidase, the 37 kDa remained unchanged whereas the 42 kDa species was reducedto 40 kDa (Fig.15A). This 40 kDa species was diminished after further digestion with 0-glycosidase (Fig.15A). Deglycosylation of E2 with the combination of endo F,PNGase F,neuraminidase and 0-glycosidase resulted in a major unglycosylated 31 kDa species and a 34kDa minor band probably due to incomplete removal of 0-linked oligosaccharide (Fig. 15A).Treatment with tunicamycin abolished the N-linked glycosylation, as well as the addition of 0-linked oligosaccharide on E2, as only a 30 kDa unglycosylated E2 species was observed(Fig. 15A). The cause for the 1 kDa difference in molecular weight between deglycosylated E2(by glycosidase digestion) and unglycosylated E2 (by tunicamycin treatment) is not known.In BFA-treated cells, the 42 kDa E2 species was reduced to 36 kDa by endo F/PNGaseF digestion, to 39 kDa by neuraminidase digestion, or to 37 kDa by further incubation with 0-glycosidase (Fig.15B). Digestion with a combination of endo F/PNGase F, neuraminidase and 0-- 88 -—— ÷ •I- +— +————— ++ill’-,____- —B__Fig. 15. Glycosidase digestion of E2 from BFA- and monensin-treated cells. Transfected cellswere labeled with[35S]-methionine for 30 minutes and incubated with medium containing excessmethionine for 4 hours before lysis with RIPA buffer. E2 proteins were immunoprecipitated withhuman anti-RV serum, digested with glycosidase for at least 8 hours at 37°C and subjected toSDS-PAGE and fluorography. BFA (6 ig/ml), monensin (5 jiM) and tunicamycin (3 jig/mi) werepresent in the medium where applicable. Samples digested with endo F/PNGase F (N-gly),neuraminidase (Neu) and 0-glycosidase (0-gly) are indicated. Molecular weight markers (in kDa)are included for reference. A, untreated cells. B, cells treated with BFA. C, cells treated withmonensin. Tunicamycin was present in the medium of some cells as indicated.N—gly—+--—+ ——0-gly———++ —NeuTmA29-÷+÷29- —C Ii:29- —______- 89 -glycosidase resulted in a single protein species of 31 kDa (Fig. 15B). In cells treated with bothtunicamycin and BFA, two protein species with apparent molecular weights of 31 kDa and 35kDa were observed (Fig. 15B). Since N-linked glycosylation was inhibited by tunicamycin, the35 kDa species must contain only 0-linked glycans. Indeed, digestion with neuraminidasereduced its molecular weight to 33 kDa while further incubation with 0-glycosidase gave riseto a single protein species of 30 kDa (Fig. 15B).E2 from monensin-treated cells migrated as a broad band with molecular weightsranging from 37K to 42K (Fig.15C). The presence of 36 kDa E2 species in endo F/PNGase Ftreated samples indicated that 0-linked glycosylation occurred (Fig. 15C), in contrast to thesituation seen in some other viral glycoproteins (Ogura et al., 1991; Collin and Mottet, 1992).A distinctive protein species with molecular weight of 34 kDa was observed in endo F/PNGaseF digested samples as well as in samples digested with a combination of endo F/PNGase F,neuraminidase and 0-glycosidase (Fig. 15C). This could be an intermediate during processing of0-linked glycans that accumulated in monensin-treated cells or alternatively is due to incompleteremoval of 0-linked sugars from E2 in an altered conformation induced by monensin-treatment.In cells treated with monensin and tunicamycin, no 0-linked glycosylation occurred (Fig.15C).Taken together, these data suggested that E2 was first synthesized in the ER as a 37 kDaprotein species containing only N-linked high-mannose type of glycans, and that as it reached theGolgi cisternae, 0-linked glycosylation took place which increased the molecular weight of E2to 42 kDa. BFA treatment caused a redistribution of Golgi enzymes back into the ER andresulted in a rapid addition of 0-linked sugars on E2 in the ER, even when N-linkedglycosylation was inhibited by tunicamycin. Whereas in monensin-treated cells, the 0-- 90 -glycosylation of E2 was not abolished, but was processed aberrantly.3.2.3. Processing and secretion of an anchor-free form of E2.To study the transport of E2 along the secretory pathway in BFA and monensin treated cells, acDNA construct encoding an anchor-free form of E2 (Hobman et al., 1994) was transfected intoCOS cells, and the expressed E2 protein was analyzed. In control cells, soluble E2 was secretedfrom the transfected cells into the culture medium at a ratio of 10-17% of total E2 protein (datanot shown). The secreted E2 was found to be resistant to endo H digestion (Fig.16), indicatingthat it had been modified by glycan processing enzymes as it traversed the secretory pathway.The majority of intracellular E2 was endo H sensitive (Fig. 16). In contrast, although the internalform of soluble E2 in BFA treated cells was partially or completely resistant to endo H digestion(Fig.16), no secreted E2 was detected (Fig.16). Monensin completely inhibited the secretion ofE2 from transfected cells, whereas intracellular E2 exhibited no obvious difference from that ofcontrol cells (Fig.16). Taken together, it is evident that BFA and monensin cause alteredoligosaccharide processing during E2 transport and completely block the movement of E2 to thecell surface.3.2.4. Effect of BFA and monensin on proteolytic processing of RV structural proteinprecursorThe expression of the polyprotein precursor and the proteolytic processing of the precursor wereanalyzed by pulse-chase experiments using pCMV5-24S (Hobman et al., 1990) transfected COScells. At the end of a 30 minutes labelling, the majority of RV structural proteins were present- 91 -internal mediumctrI BFA Mon ctrl BFA MonFig.16 Effect of BFA and monensin on processing and secretion of an anchor-free form of E2.COS cells were transfected with plasmid containing cDNA encoding a secreted form of E2 andlabeled with 100 pCi[35S]-methionine for 30 minutes at 40 hour post-transfection. Afterincubation with excess methionine for 4 hours, medium samples were collected and cells werelysed. E2 proteins were immunoprecipitated with human anti-RV serum from medium as wellas cell lysates, separated on SDS-PAGE and visualized by fluorography. Half of each sample wasdigested with endo H (+). Molecular weight markers (in kDa) are shown on the left. Ctrl, E2from untreated cells; BFA, E2 from BFA-treated cells; mon, E2 from monensin-ireated cells.—+—+—+ —+ —+ —+ endoH- 92 -ChasePulseendoHEl4C, E2rFig. 17 Effect of BFA and monensin on the proteolytic cleavage of the polyprotein precursor forRV structural proteins. Cells transfected with pCMV5-24S were pulse labeled with [35S]-methionine and chased for 3 hours. RV structural proteins were recovered from cell lysates byimmunoprecipitation with human anti-RV serum and were subjected to SDS-PAGE andfluorography. The bands corresponding to RV structural proteins without endo H treatment (onthe left) and with endo H treatment (on the right) are indicated. E2r, E2 proteins that containendo H-resistant glycans; E2s, E2 that contain endo H-sensitive sugar moieties. Endo H digestionis indicated as +. Ctrl, untreated cells; BFA, BFA treated-cells; mon, monensin-treated cells.Ct r I Mon—+ctrl BFA Mona- 93 -as individual polypeptides, although some E1fE2 uncleaved precursor protein species with highermolecular weights were also observed (Fig. 17, pulse). These minor protein species were not seenafter a 3 hour chase period (Fig. 17, chase). There was no significant difference in cleavageefficiency for RV-specific proteins between the control cells and BFA or monensin treated cells,indicating that BFA and monensin did not directly affect the proteolytic processing of RVstructural protein precursor. This is consistent with our previous fmding that the cleavage ofpolyprotein precursoris carried out by cellular signal peptidases (McDonald et al., 1991; Qiu et al., 1994), whichsuggests that it is an ER-specific event and was not interrupted by the influx of resident Golgiproteins upon BFA treatment or an impaired Golgi upon monensin treatment. RV structuralproteins appears to assemble into virus-like particles in the absence of genomic RNA and arereleased from the cells (see 3.4.), which probably is the reason that there was a slight decreasein the amount of RV structural proteins in control cells during chase.3.2.5. Subceilular distribution of RV structural proteins in BFA and monensin treated cells.Indirect immunofluorescence was used to localize the RV structural proteins in cells transfectedwith pCMV5-24S. In control cells, E2 was found to be concentrated in the juxtanuclear regionand co-localized with a Golgi marker (Fig. 18a,b), while El was localized in the pen- andjuxtanuclear region corresponding to the ER and Golgi structure (Fig.18c,d). The capsid proteinwas distributed throughout the cytoplasm (Fig.18e). A limited amount of E2 and El was detectedat the cell surface (Fig. 180. In BFA treated cells, E2 and El displayed a predominant perinuclearstaining pattern which was co-localized with a Golgi marker (Fig. 18, g-k). Combined with the- 94 -Fig.l8 Indirect immunofluorescence of RV structural proteins in cells transfected with pCMV5-24S and treated with BFA or monensin. Cells were permeabilized prior to the addition ofrhodamine-conjugated anti-Golgi protein serum, mouse monoclonal antibodies or human anti-RVserum. Some samples (F, L, and R) were not permeabiized for detection of cell surfaceexpression. After cells were washed, a secondary antibody (fluorescein-conjugated goat antimouse or anti-human IgG) was added. a-f, control cells: a) anti-E2; b) same cell, anti-Golgi; c)anti-El; d) same cell, anti-Golgi; e) anti-C; f) anti-RV, cell surface. g-1, transfected cells treatedwith BFA: g) anti-E2; h) same cell, anti-Golgi; i) anti-El; j) same cell, anti-Golgi; k) anti-C; 1)anti-RV, cell surface. m-r, transfected cells treated with monensin: m) anti-E2; n) same cell, antiGolgi; o) anti-El; p) same cell, anti-Golgi; q) anti-C; r) anti-RV, cell surface.- 95 -results from pulse-chase analysis, it appeared that in transfected COS cells, BFA treatment causedthe Golgi proteins to cycle back into the ER (as shown by the perinuclear staining with the Golgimarker) and blocked the transport of El and E2 out of the ER. In the presence of monensin, aswollen Golgi morphology was observed (Fig.18n,p), along with a diffuse E2 and El distributionin the cytoplasm (Fig. 1 8m,o). Capsid protein was distributed in the cytoplasm in BFA andmonensin treated cells (Fig. l8k,q). No cell surface fluorescence was detectable in BFA andmonensin treated cells (Fig. 1 81,r).3.2.6. Effect of BFA and monensin on RV assembly and releaseRelease of radio-labeled RV particles was monitored in RV-infected Vero cells treated with BFAand monensin (see Material and Methods). In control (untreated) cells, the amount of viralstructural proteins (C. E2, El) increased with time during the chase period (Fig. 19), indicatingthat virus particles were accumulating and that viruses were steadily assembled and released fromthe cells. In BFA and monensin treated cells, no viral structural proteins were detectable until 36hours post-labelling (Fig. 19). Thus, BFA and monensin blocked RV release from the cells duringthe early stage of the chase. After a 36 hour chase period, the viral proteins detected may be dueto lysis of BFA and monensin treated cells (Fig. 19).To further address this question, a one-step growth experiment was performed with RVinfected Vero cells treated with BFA and monensin. BFA and monensin were added to themedium 8 h.p.i. and maintained for 60 hours. Cell-associated virus and virus in the medium weretitred (Fig.20). In control cells, intracellular and extracellular viruses reached the highest titre at- 96 -E2”C‘chase(h)Fig. 19 Release of virus particles from infected cells. Vero cells were infected with RV at a MOlof 10 and labeled with[35S]-methionine for 1 hour at 24 h.p.i. Cells were further incubated innormal medium for the indicated times. Virus particles were recovered from the medium bypolyethyleneglycol precipitation, resuspended in RIPA buffer and subjected to SDS-PAGE. RVstructural proteins are indicated by arrowheads. Ctrl, untreated cells; BFA BFA-treated cells;Mon, monensin-treated cells.ctrI BFA Mon1220 2836 12202836I- 97 -PFU/m Ih.p.iPFU/ml48 60h.p.S n.Qrna( E2;mcdumFig.20 Titration of cell-associated and released virus. Vero cells were infected with RV at a MOlof 10 and incubated for the indicated time. Medium samples were collected and cells weresubjected to freezing and thawing three times to release intracellular virus. Virus infectivity wastitrated on RK cells and is expressed as PFU/ml (detailed in Materials and Methods). ,intracellular virus titre. , extracellular virus titre.24 36 48 60h.p.iPFU/mIBFA10’10’10’102024 36 48 60Monensin1 0’10224 36- 98 -48 h.p.i, about 5x107 pfu/ml, with a slightly higher extracellular virus than intracellular titre(Fig.20). Titres of cell-associated viruses were much lower in BFA and monensin treated cells,about 2x105 pfu/ml (Fig.20), indicating that BFA and monensin dramatically reduced virusassembly in infected cells. Titres of extracellular virus from BFA- and monensin-treated cellsrepresented only 0.1% of the total virus (Fig.20). Thus, BFA and monensin effectively inhibitedvirus release from infected cells. Infected cells became unhealthy with prolonged monensintreatment as an aberrant cell morphology was observed. This could explain the increasedextracellular virus titre after incubation for 60 hours (Fig.20).3.2.7. Assembly of virus particles in control or BFA and monensin treated cellsRV particles in infected cells were visualized using conventional electron microscopy. Cells werefixed at 48 h.p.i., dehydrated and embedded. Thin sections were examined after staining. In RVinfected Vero cells, virions were predominantly located in vacuoles in the proximity of the Golgicisternae (Fig.21b) and in large transport vesicles (Fig.21a). Unenveloped nucleocapsids wererarely observed in the cytoplasm and no virus budding at the plasma membrane was observedafter examining all the sections. In BFA-treated cells, the GC was disassembled and resulted ina dilated ER structure (Fig.21c). The number of enveloped virus particles was dramaticallydecreased and they were predominantly located in the cytoplasm, not associated with anymembrane structure (Fig.2 ic). In monensin-treated cells, enlarged vesicles were observed(Fig.21d). No virus particles were found except that electron-dense particles, comparable in sizeto RV nucleocapsid, was observed in enlarged vesicles (Fig.21d).- 99 -Fig.21 Electron microscopic analysis of virus assembly. Monolayers of Vero cells were infectedwith RV at a MOl of 10 and some were treated with BFA or monensin. At 48 h.p.i., cells werefixed and prepared for electron microscopic analysis. A) RV-infected cells showing virusaccumulated in large vacuole, x75,000. B) RV infected-cells showing virus maturation in theproximity to the Golgi stack, x25,000. C) RV infected-cells treated with BFA. Virus particle wasfound in the cytoplasm, near a dilated ER structure. x25,000. D) RV infected cells treated withmonensin. No virions were observed. Electron dense particles were found in large vesicles.x25,000. er, endoplasmic reticulum; Gc, Golgi complex; flu, nucleus.C•‘I—-‘II. —r er .- 100 -3.2.8. Summary and DiscussionThe effect of BFA and monensin on the transport and processing of RV structural proteins aswell as virus assembly and release was examined. BFA and monensin effectively blocked the cellsurface expression of RV E2 and El membrane glycoproteins and the secretion of an anchor-freeform of E2. A dramatic change in the intracellular distribution of RV structural proteins was alsoobserved, although the proteolytic processing of RV structural protein precursor was not affected.In the presence of BFA and monensin, virus release from infected Vero cells was only 0.1% ofthe intracellular virus. Virus particles were observed predominantly in large vesicles or Golgistacks in RV-infected Vero cells but were found in the cytoplasm in BFA treated cells.Enveloped viruses require their host’s secretory pathway for virus assembly and releasefrom the cells. The rate of intracellular transport and processing of viral proteins may play animportant role in controlling the efficiency of virus maturation, particularly for viruses that areassembled at the cell surface. Mutations that impair the transport of viral proteins to theappropriate cellular compartment have been found to significantly reduce the formation ofinfectious virus particles (Haggerty et al., 1991). In the Togavirus family, RV differs from theother subgroup (alphavirus) in that it has a relatively long latency period, slow replication andlow virus yield in cultured cells (reviewed by Porterfield et al., 1978). This is inspite of the factthat these two subgroups share similar viion structure as well as strategies for viral geneexpression. The transport rate of RV glycoproteins in transfected cells is fairly low compared tothat of the envelope glycoproteins from SFV and SIV, the two prototypes of aiphaviruses. In cellstransfected with RV E2 cDNA, about 50% of E2 contains endo H-resistant sugar moieties aftera 4 hour chase period (Fig. 14), indicating that E2 moves slowly from the ER to the Golgi stack.- 101 -Immunofluorescence studies show that the majority of RV glycoproteins El and E2 areconcentrated in the ER-Golgi region. The abundance of RV glycoproteins in the GC may becrucial to the virus’ capacity to bud from intracellular membranes (Bardeletti et a, 1979).In RV virion, E2 glycoprotein exists as multiple protein species with molecular weightsranging from 42K to 47K. It has been suggested that this is due to heterogeneous glycosylation.In transfected COS cells, a 37 kDa E2 protein with endo H-sensitive sugars and a 42 kDa E2protein with partial endo H-resistant glycans were observed. The amount of the 42 kDa proteinspecies increased with time in a pulse-chase experiment. Thus, it has been proposed that the 42kDa protein represents the Golgi-form of E2 while the 37 kDa protein is the form present in theER (Hobman and Gillam, 1989) and that the partial resistance of E2 to endo H digestion reflectsthe slow transport rate of E2. However, employing both 0-glycosidase and endo F/PNGase F toremove 0- or N-linked oligosaccharides on E2 separately, I found that the 37 kDa E2 containedonly N-linked endo H-sensitive carbohydrates whereas the 42 kDa species bore both N- and 0-linked glycans (Fig.l5). The endo H -resistant sugar moieties present on the 42 kDa protein, infact, are the 0-linked glycans. These data suggest that E2 is first synthesized as a 37 kDa proteinwith only N-linked high-mannose sugars in the ER, and is transported to the Golgi where 0-linked glycosylation occurs. Thus the acquisition of 0-linked glycans can be used to monitOr thetransport of E2 to the Golgi complex. In BFA treated cells, only the 42 kDa E2 protein with bothN- and 0-linked sugars was observed (Fig. 15, 16). This probably results from the addition of 0-linked glycans to the 37 kDa protein normally residing in the ER by the Golgi 0-glycosylationenzymes brought back into the ER by BFA (Lippincott-Schwartz et al., 1989). In the presenceof BFA, the addition of 0-linked glycans to E2 took place quickly, whereas the maturation of- 102 -N-linked sugars to complex forms was slower, as judged by the fact that complete endo-Hresistant E2 was observed after a 4 hour chase. Although monensin appeared to abolish 0-glycosylation in many cases (Colins and Mottet, 1992), I found that E2 from monensin-treatedcells possessed some 0-linked glycans.The intracellular distribution of RV structural proteins was dramatically altered in cellstreated with BFA and monensin. In BFA treated cells, the Golgi complex was disassembled asthe Golgi marker was localized in the perinuclear space (Fig.18h, 18j). A dilated Golgimorphology was found in monensin treated cells (Fig. 1 8n, l8p). There was a consistent colocalization of RV membrane glycoproteins with the Golgi markers, suggesting that RV envelopeglycoproteins interact strongly with Golgi macromolecules. Recently, it has been shown thatunassembled subunits of RV El glycoprotein are arrested in a post-ER, pre-Golgi compartment(Hobman et al., 1992); however, co-expression of El and E2 could lead to the release of El fromthis retention and allow targeting to the GC (Hobman et aL, 1993). The implication of theseobservation is that folding and multimerization of RV glycoproteins is a slow, albeit importantevent necessary for transport of El and E2 out of the ER. On the other hand, although themechanism underlying retention in the Golgi is not well understood, hydrophobic domains whichmay specify residence in the Golgi stack (Swift and Machamer, 1992), are present in both El andE2 primary structure.In RV-infected Vero cells, virions were steadily assembled and released into the medium,as judged by titres of virus in the medium and in association with cells (Fig.20). Slightly moreextracellular virus than intracellular was found (Fig.20), which is consistent with earlier reports(Payment et al., 1975; Bardeletti, et al., 1979). In BFA and monensin treated cells, very few- 103 -infectious virus particles in the medium were detected (Fig.20), indicating that BFA andmonensin effectively blocked the production of extracellular virus. The absence of radio-labeledvirions in the medium of BFA and monensin treated cells (Fig.19) ruled out the possibility thatthe decreased virus titre was due to the loss of virus infectivity. Rather, it was due to a decreasein the number of virus particles during treatment A dramatic decrease in intracellular virus titrewas also observed (Fig.20). This could be due to the inhibition of virus assembly at the internalmembrane or aberrant virus assembly, which is yet to be investigated. Recently it has beenreported that BFA affected viral RNA synthesis in poliovirus infected cells (Maynell et al., 1992)due to interruption of membrane-associated poliovirus replication complex. The nature and siteof RV RNA synthesis is not well known. However, we found that the level of protein expressionin cDNA-transfected and RV-infected cells was not affected by the concentration of BFA andmonensin used (data not shown).In Vero cells, envelopment of RV nucleocapsid to form virions has been reported to takeplace both at the internal membrane or at the cell surface (Payment et al., 1975). In this study,morphological analysis was performed in RV-infected cells in the presence and absence of BFAand monensin in order to examine the correlation between viral glycoprotein targeting and thevirus budding process. At 48 h.p.i., virus particles were found to be clustered in the vacuoles nearthe Golgi stack, with others in the transport vesicles (Fig.21). The abundance of virus particlesin the post-Golgi structures suggested that the post- Golgi membrane network may be the primarysource of membrane in RV assembly. Few virus particles, not associated with any membranestructure, were found in the juxtanuclear region of cytoplasm in BFA treated cells (Fig.2 1). Novirus particles could be detected in monensin treated cells after examining all the sections- 104 -(Fig.21). The results from morphological analyses were consistent with a significant decrease invirus titre in BFA and monensin treated cells. The reduction of intracellular virus assembly inBFA or monensin treated cells could be due to the blockage of glycoprotein transport to the siteof envelopment or the disruption of vesicular structure which may be required for efficient virusassembly. Taken together, the data presented in this study suggested a correlation betweenintracellular localization of RV structural proteins and the site of RV assembly.How cell membranes become incorporated into the envelope of the virion is as yet poorlyunderstood. The subcellular location for acquiring membranes differ with viruses, reflecting thepossibility that structural or component features of particular organelles are required to facilitatesuch envelopment. Alternatively, post-translational modification(s) on viral envelope proteinsduring transport may be essential for effective interaction with the nucleocapsids. Therefore,defining the site of virus assembly can shed some light on further studies on the mechanism ofvirus assembly.- 105 -3.3. Section III. Influence of N-linked glycosylation on the antigenicity and immunogenicityof El glycoprotein3.3.1. Construction of recombinant vaccinia viruses expressing RV El glycosylation mutantsThe cDNA fragments encoding RV El glycosylation mutants (Hobman et al., 1991) weresubcloned into the.S.n I site of vaccinia virus recombination vector pGS2O (Fig.4B), whichcontains the conventional p7.5 early/late promoter (Mackett et aL, 1984). Transfection (seeMaterials and Methods 2.2.6.) and marker rescue by cell-mediated homologous recombinationwere performed as described by Mackett et al., (1985). The vaccinia recombinants containingwild-type and glycosylation mutant cDNAs were named according to the mutations as yR-El(wild-type); VR-E1G1, VR-E 102, VR-E 103 (single mutations); VR-E1G23 (double mutation)and VR-E1G123 (triple mutation) (Fig.22a). The construction of a vaccinia recombinantexpressing RV E2 has been described (Chaye et al., 1992b).3.3.2. Expression and antigenicity of El glycosylation mutants.Cells infected with the wild-type El vaccinia recombinant expressed a 57 kDa protein (Fig.22b,El), while the vaccinia recombinants containing El glycosylation mutant cDNAs expressedproteins with apparent molecular masses of 55 kDa (Fig.22b, Gi, G2, G3), 53 kDa (Fig.22b,G23) and 51 kDa (Fig.22b, 0123), similar to those observed in COS cells (Hobman et al., 1991).The differences in electrophoretic mobility between the wild-type and glycosylation mutants weredue to differences in the numbers of oligosaccharides attached to the wild-type and mutantproteins. Corresponding polypeptides were absent from uninfected cells (Fig.22b, MI) and cells- 106 -C E2 ElwtElE1G1E1G2E1G3______________________________________El G23________ ________E1G123___ _____Fig.22a. Diagrammatic representation of RV El glycosylation mutant cDNAs used in theconstruction of vaccinia recombinant viruses. The respective portions of the E2 and El genes areindicated above the constructs. The translation initiation site is contained in the region proceedingthe N-terminus of capsid near the left end of the constructs. Three N-linked glycosylation sitesat residues 76, 177, and 209 are indicated by Y and the mutagenized glycosylation sites aremarked Gl, G2, and G3. The EcoR I (E) and Hind 111(H) sites flanking the 5’ and 3’ portionof the cDNAs, respectively, are indicated. The thick horizontal lines represent coding regions andthe thin lines indicate noncoding regions. The thick vertical lines demarcate the regions of C, E2and El coding regions (Hobman et al., 1991).I I YY, I Gi YYI I G2YI V YG3 FI I V G2G3, I Gi G2G3 F- 107 -Fig.22b. Expression of El glycosylation mutants by vaccinia recombinants. CV-l cells infectedwith wild-type vaccinia virus (WT), RV El vaccinia recombinants (El, Gi, G2, G3, G23 andG123) or mock infected (MI) were labeled with[35S]-methionine for 30 minutes at 10 h.p.i. Cellswere lysed with RIPA buffer and RV El-specific proteins were immunoprecipitated with RV ElmAbs and subjected to SDS-PAGE. Molecular weight marks in kDa are shown on the right forreference.- 108 -infected with wild-type vaccinia virus (Fig.22b, WT).To characterize the immunoreactivity of the El mutant proteins, a panel of RV El-specificmAbs was used for western blot analysis under reducing and non-reducing conditions. Six RVEl-specific mAbs (21B9H, 3D5D, 3D9F, 14D1F, 16B2C and H4C52) were used in this analysis.The mAbs were characterized as having VN (21B9H) or HAT (3D9F, 3D5D, 16B2C, H4C52)activities (Chaye et al., 1992a). Similar results were obtained from all six mAbs used and Fig.23shows two typical blots using mAbs 21B9H and 3D9F. Under reducing conditions (Fig.23, +Me), all five El glycosylation mutants reacted with each of the six mAbs, suggesting that thesemAbs recognize linear epitopes on the El primary structure. The capacity of these mAbs to bindto non-glycosylated El (Fig.23, + 13-Me, G123) indicated that these epitopes were notcarbohydrate-dependent. The weak signal observed in immunoblots under reducing conditions isdue solely to the decrease in antigenicity of El and not to the quantitative difference in theantigen used. Under non-reducing conditions, the majority of the wild-type and the singleglycosylation mutants ran as monomers with a small fraction as dimers (Fig.23, - 13-Me, wt, Gl,G2 and G3), while the double mutant G23 gave a smeared appearence on the gel (Fig.23, - 13-Me,G23) and the triple mutant G123 was not detectable (Fig.23, - f3-Me, G123). One possibleexplanation for this observation is that mutant proteins G23 and 0123 form aberrantintermolecular disulfide bonds that cause them to migrate as diffuse smears on SDS-PAGE. G123was not present on the top of the separating gel. Taken together, the data presented here suggestthat denaturation of mutant proteins G23 and G123 in the absence of 13-mercaptoethanol led todecreased or abolished antibody binding activity of these proteins. Decreased antibody bindingwas also observed in native 023 and Gl23 proteins in immunoprecipitation (Fig.22b).- 109 -Table la. Summary of propeties of monoclonal antibodies directed to ElniAbs VN or HAT Epitope21B9H VN El214 to El233H4C52 HAl ND3D5D HAT ND14D1F ND ND3D9F HAl El214 to El016B2C HAl ND- 109.a21B9H309F-M.•1 —00+ B-U.-e,Iq=o E 00000Fig.23 Immunoblot analysis of El glycosylation mutants expressed by vaccinia recombinants.Cells infected with vaccinia recombinants expressing wild-type El (WT) or El glycosylationmutants (Gi, G2, G3, G23, G123) were treated with 10 mM iodoactamine and lysed with RIPAbuffer. Proteins were separated on SDS-PAGE under reducing (+ 13-Me) and non-reducing (- 13-Me) conditions, and transferred to nitrocellulose membrane. RV antigens were probed with apanel of RV El-specific mAbs. Parental vaccinia virus infected cell lysate was used as thecontrol (ctrl). Molecular weight markers (in kDa) are included for reference.- 97- 68- 43- 29 - I97 -68 -43 -— — *--—29 -- 110-Therefore glycosylation may be required for proper folding of El to allow efficient recognitionof immunological epitopes on El.3.3.3. Immunogenic properties of the expressed El glycosylation mutants.Although glycosylation did not significantly affect antibody binding, it might affect the elicitationof HAl and VN antibody responses. To address this question, vaccinia recombinant viruses werepurified and used to immunize mice. After four injections, sera were collected, pooled and testedfor their reactivities to RV El by immunoblot analysis. All the El glycosylation mutants exceptG123 were found to elicit antibodies recognizing authentic El from RV virions when RV proteinswere separated under non-reducing conditions (Fig.24,- 13-Me). However, only the antiserumfrom wild-type El immunized mice reacted with El from RV virions under reducing conditions(Fig.24, + 13-Me). These results suggest that native immunogenic determinants of Elglycosylation mutants expressed via vaccinia recombinants are predominantly conformation-dependent. This could be due to aberrant folding of mutant proteins and masking of linearepitopes on El when normal glycosylation is blocked.To further assess the production of anti-El antibodies from mice immunized with vacciniarecombinants, an ELISA was used to quantitate the El-specific antibodies of mouse sera (Table2). The antibody titre from yR-El immunized mice was found to be three times higher than thatin sera from those immunized with VR-E1G1, VR-E1G2, VR-E1G3 and VR-E1G23, and 15times higher than that in serum from mice immunized with VR-E1G123. Table 2 shows theanalysis of HAl and VN activities of antisera from mice immunized with vaccinia recombinantviruses. VN activity was observed in sera from mice immunized with VR-El, VR-EIG1, VR- 111 -— B-Me ÷ 13-Me9768-43-29-Fig.24 Immunoblot analysis of sera from mice immunized with El vaccinia recombinants.Purified RV particles were subjected to SDS-PAGE under reducing (+ (3-Me) and non-reducing(- (3-Me) conditions, and transferred to nitrocellulose. El antigens were detected with antiserafrom mice immunized with El vaccinia recombinants. The immunizing vaccinia recombinantswere indicated on the top of the gel. Wild-type vaccinia virus was used as a negative control(WT). El-specific mAbs were used as a positive control (mAb). El monomer (p), El-E2heterodimer (o) and El-El homodimer (*) were indicated. Molecular weight markers (in kDa)are shown on left for reference.• EI H ffjj- 112--E1G2 and VR-E1G3 (Table 2). Deletion of any single glycosylation site from El did not preventneutralizing antibody production, as the ratio of VN titre to ELISA titre was similar in sera frommice immunized with yR-El, VR-E1G1, VR-E1G2 and VR-E1G3 (Table 2). Thus, carbohydrateside chains on El are not directly involved in the elicitation of VN responses. HAT activities ofthese sera were examined with regard to capacity to block the binding of RV virions toerythrocytes. Sera from mice immunized with yR-El, VR-E1G2 and VR-E1G3 showed HAlactivity while that raised from VR-E1G1 did not (Table 2), suggesting that oligosaccharideattached at the Gi site is important in eliciting HAT antibody production. In contrast, no VN orHAT antibodies were detectable in the sera from mice immunized with either VR-E1G23 or VRE 1G 123, indicating that VN and HAl epitopes were not functionally active in these mutantproteins. This may be due to an altered protein conformation when most of or all of thecarbohydrate side chains are absent.3.3.4. Antigenic properties of deglycosylated RV El from RV virions.To further confirm the above conclusion, digestion of RV virions with endo F/PNGase F wasperformed under mild conditions which remove all N-linked oligosaccharides from proteinswithout disrupting protein conformation. The extent of deglycosylation and its possible effect onantigenicity were examined by immunoblotting (Fig.25). El from deglycosylated virions migratedas a 51 kDa protein [Fig.25, + 13-Me, RV(dG)], similar to the non-glycosylated El expressedfrom vaccinia recombinant VR-E1G123 (Fig.25, + 13-Me, VR-E1G123), indicating that alloligosaccharides attached to El had been removed (Fig.25, + 13-Me). Under non-reducing- 113 -Table 2. Comparison of the HAl and VN antibodies from mice immunized with vacciniarecombinants containing different RV El glycosylation mutant cDNA insertsVN titrecVirus used forimmunization ELISA titrea HAl titreb Comp.d No comp.vve <2 <l6f <2 <2VR-El 512 128 64 32VR-E1G1 128 <16 16 4VR-E1G2 128 64 16 4VR-E1G3 128 64 8 4VR-E1G23 128 <16 <2 <2VR-E1G123 32 <16 <2 <2a Expressed as the highest dilution of antibodies yielding 0D405 two times higher thanbackground.b Expressed as the highest dilution of antibodies that completely inhibits hemagglutination.Expressed as the end point of antibody dilution that completely inhibits plaque formation.d Heat-inactivated rabbit complement (2.5%) was present in diluted antibodies.e Wild-type vaccinia virus.For the technique employed in the assay, this is the lowest dilution of serum that can beachieved. Values under 16 were considered negative.- 114-+ B-Me — B-Me>> >ELFig.25 Effect of deglycosylation on the antigenicity of RV virion. Untreated RV virion (RV),deglycosylated (dG), and cell lysate from yR-El and VR-E1G123 infected CV-l cells weresubjected to SDS-PAGE under reducing (+ p-Me) and non-reducing (- f3-Me) conditions. Proteinswere transferred onto nitrocellulose and RV El-specific antigens were probed with El-specificmAbs.- 115 -conditions, El from deglycosylated virions retained mAb binding activity, whereas nonglycosylated El from vaccinia recombinant VR-E1GT23 had lost this activity (Fig.25,- 13-Me).This suggests that removal of sugar moieties after protein folding has less effect than blockingglycosylation during protein synthesis prior to folding. The observed significant decrease inantigen mass for El from deglycosylated virions (Fig.25) could be due to aggregation ofdeglycosylated virions that pelleted during sample preparation. This observation was furtherconfirmed by using deglycosylated El from RV virions as antigen in ELISA (Table 3). Usingsix RV El-specific mAbs, I found that there was no difference in activity of binding to mAbsbetween the glycosylated and deglycosylated RV virions as determined in the ELISA (Table 3).The role of carbohydrate in HA activity of El was examined with deglycosylated RVvirions. Deglycosylation of RV virions resulted in a significant decrease in the HA titre (Table3), suggesting that carbohydrate was functionally involved in hemagglutination. However, nodifference was observed between the binding of glycosylated and deglycosylated virions to chickerythrocytes when HAT mAbs were used to inhibit the binding (Table 3), suggesting that thebinding of HAl antibodies to El is carbohydrate-independent.3.3.5. Effect of glycosylation on El cell surface expression.To localize the subcellular distribution of the wild-type and mutant El proteins, CV- 1 cellsinfected with recombinant vaccinia viruses were analyzed by indirect immunofluorescence(Fig.26). In cells infected with vaccinia El recombinants (wild-type and glycosylation mutants),El proteins were found in the ER and Golgi-like region with the exception of E1G123, whichwas found to show a more profound ER staining (Fig.26, a-d). It has been shown previously that-116-Table 3. A. HA assay of the deglycosylated RV virionGlycosylated DeglycosylatedHA activitya 320 40HA inhibition assayb3D5D 2560 2560’H4C52 5120 5120aExpressed as the highest dilution of virus yielding hemagglutination.bExpress&l as the end point of antibody dilution that completely blocks the 4 HA units bindingto chick erythrocytes (8-fold more deglycosylated viruses was used, compared to untreated virus).B. Effect of deglycosylation of RV El on antibody recognition by El-specificmonoclonal antibodies.ELISA titreamAbs Glycosylated Deglycosylated21B9H 256 256H4C52 1024 10243D5D 1024 102414D1F 256 2563D9F 256 25616B2C 128 128a Expressed as the highest dilution of antibodies yielding twice higher 0D405 than background.- 117-E2 is essential for transport of El to the cell surface (Hobman et al., 1990). The effectofglycosylation on El cell surface expression was studied by infecting CV-l cells with Elvaccinia recombinant viruses (wild-type and glycosylation mutants), or with El vacciniarecombinant viruses plus RV E2 vaccinia recombinant viruses. No cell surface expression wasdetected in cells infected with vaccinia recombinants of either El wild-type or glycosylationmutants (data not shown). In cells co-infected with both E2 and El vaccinia recombinants, theinternal distribution of El antigens remained unchanged (Fig.26, e-h), while cell surfaceexpression of wild-type and single glycosylation mutant El was observed (Fig.26i, j). Cell surfacestaining was not detected in cells co-infected with either VR-E2 and VR-E1G23 or VR-E2 andVR-E 1G123 (data not shown). The data presented here suggest that the cell surface expressionof RV El requires at least two N-linked carbohydrate side chains on El proteins in addition toco-expression of RV E2.3.3.6. Summary and DiscussionThe role of N-linked glycosylation on the antigenicity and immunogenicity of El glycoproteinwas studied using vaccinia recombinants expressing El glycosylation. The expressed Elglycosylation mutant proteins were recognized by a panel of specific monoclonal antibodies inradioimmunoprecipitation, immunofluorescence and immunoblotting, indicating that carbohydrateside chains on El are not involved in the constitution of epitopes recognized by those monoclonalantibodies. This observation was further supported by the fact that there is no significant changein the antigenicity after oligosaccharides on El from virions were removed by glycosidasedigestion. All glycosylation mutants were capable of eliciting anti-RV El antibodies at different- 118 -Fig.26 Indirect immunofluorescence of El glycosylation mutants in infected CV-l cells. For cellsurface expression, cells were fixed with 3% formaldehyde and incubated with an El-specificmAb mixture followed by the incubation with FITC-conjugated goat anti-mouse IgG. Fordetection of internal antigen, cells were permeabilized with 0.1% Nonidet-P40 prior to incubationwith El mAbs. (a) VR-El; (b) VR-E1G2; (c) VR-E1G23; (d) VR-E1G123; (e) yR-El, VR-E2;(f) VR-E1G2, VR-E2; (g) VR-E1G23, VR-E2; (h) VR-E1G123, VR-E2; (i) yR-El, VR-E2,surface; (j) VR-E1G2, VR-E2, surface. For glycosylation mutants Gi and G3, a similar stainingpattern as G2 was observed (data not shown).- 119-titres. The single glycosylation mutants (Gl, G2 and G3), but not the double mutant or thetriplemutant (G123), were found to be capable of inducing VN responses. However, among thesingle glycosylation mutants, only G2 and G3 were able to induce HAT antibodies in mice.The influence of carbohydrate on the antigenicity of viral glycoproteins has beendemonstrated in a number of viruses (see introduction section It has been suggested thatoligosaccharide side chains mask adjacent polypeptide sequence and prevent the binding ofrespective antibodies. In our case, no difference in antigenicity was observed between the wild-type and mutant El proteins expressed from vaccinia recombinants, with respect to their mAbbinding activities in immunoblotting under reducing conditions (Fig.23), suggesting that there arecarbohydrate-independent epitopes in El for the six mAbs tested. Consistent with thisinterpretation, deglycosylation of RV virions with endo F/PNGase F resulted in no quantitativechange in reactivity to El-specific mAbs when analyzed by ELISA (Table 3). However, asignificant decrease was observed in HA activity of the deglycosylated RV virions (Table 3). Itis concluded that carbohydrate on El is not involved in the constitution of epitopes recognizedby mAbs but is functional in hemagglutination. However, non-glycosylated El expressed fromVR-E1G123 failed to react to any of the mAbs under non-reducing conditions, presumably dueto aberrant folding of the mutant protein when carbohydrate side chains were absent.Most viral glycoproteiris contain multiple N-linked glycosylation sites. Site-specific effectson the processing and intracellular transport of glycoproteins have been reported for SV5 HN (Nget al., 1990) and also observed for RV E2 (see 3.1. in this section). Tn this study, all the RV Elmutants were found to reside in the ER and Golgi-like region except VR-E1G123 which waslocalized predominantly in the ER region. All three single glycosylation mutants (E11, E 1G2,- 120 -E1G3) were found to be transported to the cell surface when RV E2 was present in the cells(Fig.26j), suggesting that carbohydrate at each of the three sites affects intracellular transport ofEl equally. It has been reported previously that co-expression of RV E2 is required for cellsurface expression of RV El, whereas E2 transport to the cell surface is an E 1-independent event(Hobman et al., 1990). In this study, I have shown that besides co-expression of E2, cell surfaceexpression of El also requires any two of the three N-linked oligosaccharides. Taken together,this suggests that the interaction of El and E2 is a post-translational event rather than a cotranslational process and the formation of El -E2 heterodimer is probably important to release Elfrom retention in the Golgi apparatus.I was interested in exploring the site-specific effect of glycosylation on the immunogenicityof RV El. Immunization of mice with vaccinia recombinants expressing El glycosylation mutantsshowed that removal of any one of the carbohydrates from El does not prevent single mutantsfrom inducing VN antibodies in mice, suggesting that the protective immune response is probablydirectly toward the polypeplide backbone. Similarly, neutralizing activity is also detected in rabbitserum raised against RV El peptide expressed in E.coli (Terry et al., 1989). These results areconsistent with the finding that the VN epitopes of El map to a region of the C-terminal half ofEl that does not contain glycosylation sites (Terry et al., 1988; Wolinsky et al., 1991; Chaye etal., 1992a). The failure of VR-E1G23 and VR-E1G123 to elicit neutralizing antibodies isprobably due to improper conformation of the mutant proteins. A considerably lower antibodyresponse was found in sera from mice immunized with VR-E1G1, VR-E1G2, and VR-E 103. Ofthree single mutants, VR-E1G1 did not induce HAl antibodies, indicating that oligosaccharideattached to the 01 site is either involved in hemagglutiniation directly or is critical for facilitating- 121 -hemagglutinin epitope exposure. The hemagglutinin epitopes of El have been defined as residingin the same region as neutralizing epitopes (Terry et al., 1988; Chaye et al., 1992a), acarbohydrate-free domain. However, I have shown that hemagglutination is carbohydrate-dependent (Table 3). Thus, the expression of HA epitopes for the induction of HAT antibodyproduction is dependent on the conformation of native El protein. It has been shown that an Elpeptide expressed in E.coli is recognized by HAT mAbs but fails to produce HAl antibody inrabbits (Terry et al., 1988).The results presented here indicate that although the addition of carbohydrate is not essentialfor antibody binding to El, deletion of any one of the oligosaccharide side chains from El resultsin a less immunogenic state of El, probably due to improper folding. Thus, in developing aneffective RV subunit vaccine using El, proper combinations of different epitopes in theirimmunoactive conformations must be considered.- 122 -3.4. Section IV. Expression and characterization of virus-like particles containing rubellavirus structural proteins3.4.1. Isolation of BHK cell lines expressing RV structural proteinsThree RV cDNA constructs (Fig.27) were used in the isolation of stably transformed BHK celllines. These cDNAs encode the capsid protein (C), E2E1 polyprotein precursor (E2E1) orpolyprotein precursor for all three structural proteins of RV (24S) (Clarke et al., 1987; Hobmanet at, 1990). The cDNAs were subcloned into the I site of transfer vector pNUT (Fig.4c)(Palmiter et al., 1987), under the control of the metallothionein I promoter (Fig.4c). The resultantrecombinant plasmids were transfected into BHK cells using the calcium phosphate method( (Gorman et al., 1982). Twenty-four hours after transfection, methotrexate (2.5 mM) wasadded to the culture medium and cells were incubated with this selection medium for 10 days.Methotrexate-resistant colonies were picked and screened for the integration of RV cDNAs intotheir chromosomes using the polymerase chain reaction (Saiki et al., 1988) and for the expressionof RV structural proteins using western blot analysis (Towbin et al., 1979). Isolated cell lineswere stable under normal growth conditions as they retained the capacity to express RV structuralproteins after four months of continuous culturing. Cell lines were named according to the RVcDNAs used for transfection, as BHK-C, BHK-E2E1 and BHK-24S, respectively.- 123 -RV cDNAsATG____________CCATG—__ ___________________E2E1C E2 ElATG________24SC E2 ElFig.27 Diagrammatic representation of RV cDNAs used in the construction of recombinantplasmids. Translation initiation site (ATG) from RV capsid protein was used in all constructs.The putative signal peptides and membrane anchor domams of E2 and El are indicated as or,respectively.- 124 -3.4.2. Expression of RV structural proteinsMonolayers of stably transformed cells were incubated with medium containing 30 iiM zincsulfate for 12 hours to induce the expression of RV structural proteins from the promoter. Theexpression of RV structural proteins from stably transformed cell lines was analyzed byimunoblotting using human anti-RV serum (Towbin et al., 1979). In BHK-C cells, an intracellularprotein species with molecular size of 34 kDa was observed (Fig.28A, lanes C). This protein mayrepresent the capsid protein of RV. In BHK-E2E 1 cells, protein species corresponding to the ER-and Golgi- isoforms of RV E2 (Hobman et al., 1990) and El glycoproteins were found in the celllysate but not in the medium (Fig.28A, lanes E2E1), indicating that the E2E1 polyproteinprecursor was synthesized and proteolytically processed to give rise to E2 and El proteins. InBHK-24S cells, protein species corresponding to the C, E2 and El proteins of RV were presentin the cell lysate as well as in the medium (Fig.28A, lanes 24S), suggesting that the integratedcDNA of 24S RNA was active in directing the synthesis of RV structural proteins and thesestructural proteins were released from the cells. The secretion of RV structural proteins fromBHK-24S increased with time and was linear over a period of 24 hours under ZnSO4 induction(Fig.29).3.4.3. Assembly and release of virus-like particles (VLPs) in stable BHK-24S cellsThe secretion of RV structural proteins into the medium was found to be dependent on thecoexpression of C, E2 and El, suggesting that these proteins might be assembled into subviralparticles prior to their release from the cells. To examine this possibility, medium from BHK-24Sand RV-infected cells was subjected to ultracentrifugation (350,000xg for 20 minutes), in the- 125 -Amedium lysate-OWNO w N9768-29-B 12368-leE—-Pc29-Fig.28 A. Immunoblot analysis of proteins expressed in transformed BHK cells. Monolayers ofBHK-C, BHK-E2E1 or BHK-24S cells were incubated with serum-free medium in the presenceof 30 jiM zinc sulfate for 12 hours. Culture media were collected and cell monolayers were iysedwith RIPA buffer. Samples were directly subjected to SDS-PAGE and immunoblotting. B.Immunoblot analysis of proteins sedimented by ultracentrifugation. Samples from medium ofinduced BHK-24S (Lanes 1 and 2) or RV-infected BHK cells (Lane 3) were centrifuged at90,000 rpm for 20 minutes in the absence (Lanes 2 and 3) or presence of 1% NP-40 (Lane 1).The pellets were resuspended in RIPA buffer and analyzed using SDS-PAGE andimmunoblotting. The positions of RV structural proteins are indicated by anowhead. Themolecular weight markers are included for reference.- 126 -0 0.5 1 2 4 7 10 14 IS 24 RV (hour)medium-4.—4E1;qq. )E2lysate4E1=jØJ)E2Fig.29. Time course of VLPs secretion from BHK-24S cells. Expression of RV structural proteinswas induced by the addition of ZnSO4 (30 jiM in the culture medium). Culture medium wascollected and cells were lyzed at the indicated times (hour post-induction). Samples from mediumwere subjected to 90,000 rpm centrifugation for 20 minutes and resuspended in RIPA buffer. Theresuspended pellets and the cell lysates were analyzed using SDS-PAGE and immunoblotting.The positions of RV structural proteins are indicated.- 127 -presence or absence of 1% non-ionic detergent, Nonidet P-40. Resuspended pellets were subjectedto SDS-PAGE, transferred to nitrocellulose membranes and probed with human anti-RV serum.In the absence of NP-40, C, E2 and El were detected in the pellets from BHK-24S and RVinfected cells (Fig.28B, lanes 2 and 3). In the presence of NP-40, El and E2 glycoproteinsremained in the supernatant after ultracentrifugation (not shown), although trace amounts of Cwere found in the pellet (Fig.28B, lane 1). Thus the assembled viral proteins are secreted asparticles that sediment in a gravitational field. To confirm that proteins El, E2 and C assembledinto VLPs, samples from pelleted VLPs were centrifuged for 16 hours at 90,000xg through adensity gradient from 20 to 50% sucrose. VLPs were recovered in fractions with density of 1.17-1.19 g/nil (Fig.30); similar to that of native RV virion (1.175-1.20 g/ml) (reviewed by Horzinek,1981).3.4.4. Electron-microscopic analysis of the VLPsThe morphology of the VLPs was analyzed by employing conventional electron microscopictechnique with routine Epon embedding of fixed BHK-24S cells. The VLPs found in BHK-24Scells were comparable in size to RV particles (60 nm) (Fig.3lA) and indistinguishable inappearance, with an electron dense core surrounded by an envelope (Fig.31B, C, D). Theseparticles were predominantly located within the vacuoles in the juxtanuclear region (Fig.3 lD) orcytoplasm (Fig.3 1C), which may represent the Golgi structure. Some particles were distributedin the cytoplasm (Fig.3 1B), not associated with any membrane structure. Such particles were notobserved in BHK-E2E1 or BHK-C cells (data not shown). Taken together, it is evident that VLPswere indeed assembled intracellularly prior to their release from the cells.- 128 -1 3 5 7 9 1113 15 17 19 2123 2RV—- — -4 El• E2‘N__ ___VLPIIFig.30 Purification of VLPs and RV on sucrose density gradient centrifugation. Pelleted RV orVLPs from 35 ml culture medium were resuspended in 0.35 ml TNG buffer (50 mM Tris, pH7.5;100 mM NaC1; 200 mM glycine) and applied onto the top of a 12 mi-sucrose gradient of 20-50%sucrose in TNG. Centrifugation was carried out using a Beckman SW41 rotor at 90,000xg for16 hours at 15°C. Fractions (— 0.5 mI/fraction) were collected by puncturing the bottom of thetube and the density of each fraction was determined. 100 il sample from alternative fractionswere diluted with equal amount of TNG buffer and subjected to centrifugation at 90,000 rpm for20 minutes. The pellets were resuspended with RIPA buffer. RV proteins in the pellets, and inthe samples that loaded onto the gradient (load), were analyzed by SDS-PAGE andimmunoblotting (using human anti-RV serum). Fractions are numbered from the bottom (#1) tothe top (#23) of the gradient. The positions of RV structural proteins are indicated.- 129Fig.31 Electron microscopic analysis of the VLPs in BHK-24S cells. RV-infected BHK cells (A)or induced BHK-24S cells (B, C, D) were fixed with formaldehyde/glutaraldehyde, postfixed withosmium tetroxide, ethanol dehydrated and Epon embedded. Thin sections were analyzed byelectron microscopy after staining.%_‘. 4- 130 -3.4.5. Antigenicity of the VLPs.HA activity of the VLPs was examined using a heparin/manganous chloride procedure(Liebhaber, 1970) and the HA titre was expressed as the endpoint of serial dilutions of sera atwhich erythrocyte aggregation was observed. The VLPs from BHK-24S cells displayed HAactivity of 64, while RV particles retained HA activity when diluted to 1/32. This difference isdue to the higher yield of VLPs from induced BHK-24S cells than that of RV from infectedcells.To compare the antigenicity of VLPs with that of RV, equal amounts of RV or VLPs(with respect to HA unit) were used in each assay. Table 4 shows the antibody binding activitiesof VLPs and RV in immunoblot and ELISA analysis using twelve mAbs against RV El, E2 orC. Two of the E2 mAbs showed differences in western blotting between the VLPs and RV (Table4) and VLPs displayed a higher ELISA titre with a mAb against C protein than did RV (Table4). VLPs were also used in a solid phase immunoassay to measure the IgG response in humans.With 200 human serum samples, it was found that the correlation coefficient between the VLPsand whole RV antigens was 0.96 using a non-parametric regression analysis method (data notshown). This indicated that the antigenic determinants on the VLPs resemble those of authenticRV.3.4.6. Immunogenicity of the VLPs.To evaluate the immunogenic properties of the VLPs, we immunized mice (BALB/c, four in eachgroup) with the VLPs, RV or soluble El protein expressed in transfected BHK cells (Gillam,unpublished results), respectively, with comparatively same amounts of antigens (equivalent to- 131 -Table 4. Immunoreactivity of the VLPs with RV-specific monoclonal antibodies.Western blota ELISA titrebmAb RV VLP RV VLPH15C22(C) + + 128 256H32C43(El)-- 256 25621B9H(E1) + + 256 2563D5D(E1)-- 1024 102414B1F(E1) + + 256 2563D9F(E1) + + 256 25616A1OE(El) + + 1024 1024E2-2(E2)- + 160 160E2-4(E2)- + 320 320E2-5(E2) + + 320 320E2-6(E2)-- 160 160H46C64(E2) + + 320 320a Monoclonal antibodies were used at a dilution of 1:100 for ascites fluid or 1:5 for tissue culturesupernatant.+, positive reactivity was detected.-, negative reactivity was detected.b Expressed as the highest dilution of antibodies yielding 0D405 two times higher thanbackground.- 132 -256 HA units), The presence of anti-RV antibodies in the sera of immunized mice was analyzedusing radio-immunoprecipitation. As shown in Fig.32, mice immunized with the VLPs producedantibodies against all three structural proteins of RV (Fig.32, lane 3), as did mice immunized withRV (Fig.32, lane 4). Mice immunized with El protein also developed some anti-El antibodyresponse (Fig.32, lane 2).ELISA was used to quantify the antibody titres against each of the RV structural proteins,by using individual purified RV structural proteins expressed in Spodoptera frugiperda cellsinfected with baculovirus recombinants (Gillam, unpublished data) as antigens. In the sera frommice immunized with VLPs, a significantly higher anti-C antibody titre was found, whereas anti-El and E2 antibody titres were slightly lower (Table 5). The biological functions of theseantibodies were analyzed. Sera from VLP-immunized mice displayed VN activities (Table 5) asdetermined by plaque reduction assays (Fukuda et al., 1987). HAl activities were also present inthe sera from mice immunized with the VLPs, as well as in the sera from mice immunized withRV (Table 5). These results suggested that although VLPs were less active in inducing overallanti-El and E2 antibodies compared to RV, they induced the production of both VN and HATantibodies.We have also determined cell-mediated immune responses against RV in VLP-immunizedmice in a lymphocyte proliferation assay (Chaye et al., l992a; Ou et a, 1992). Lymphocyteproliferative responses of mice were determined in vitro by direct stimulation of lymphocyteswith UV-inactivated RV or individual RV structural proteins (C, E2 and El) purified fromrecombinant baculovirus infected insect cells. Lymphocytes from VLP-immunized miceresponded strongly to UV-inactivated RV as well as to the individual RV structural proteins in- 133 -12-•• LJ.Fig.32 Radioimmunoprecipitation of RV structural proteins expressed in COS cells. COS cellswere transfected with pCMV5-24S (Honman and Gillam, 1989), labeled with[35S]-methionineand lysed. RV structural proteins were recovered from cell lysates with mouse anti-RV antibodiespre-bound to Sepharose 4B-protein A beads as previously described (Hobman and Gifiam, 1989)and separated by SDS-PAGE. Sera were from mice immunized with El protein (Lane 2), VLPs(Lane 3), RV (Lane 4) or from pre-immune mice (Lane 1). The positions of RV structuralproteins are indicated.34IE2- 134 -Table 5. Comparison of antibody titres of mouse sera from mice immunized with different RVantigens.ELISA titre to RV proteinaVNtitre” HAl titrecimmunogen C E2 ElEl <10 10 10 <2 <8RV 40 80 160 16 32VLP 320 40 80 8 16alndividual RV structural proteins (C, E2 and El) were purified from SF9 cells infected withbaculovirus recombinants expressing each RV structural protein (unpublished data).bExpressed as the reciprocal of the highest antibody dilution that show 50% reduction in plaqueformation.cExpressed as the reciprocal of the highest antibody dilution that inhibited hemagglutination.- 135 -201protein concentration (jig/mi)Fig.33 Lymphoproliferation responses of mice immunized with VLPs. Lymphocytes from VLPimmunized mice (2.5x10/well) were incubated with different concentrations of proteins El (,E2 (+) or C (*) at 37°C for 5 days before addition of[3H1-thymidine (1 jiCi/well). All assayswere performed in triplicate and results are expressed as mean values. Using UV-inactivated RV(l0 PFU/ml), lymphoproliferation responses in the presence and absence of RV antigen were23,000 and 2,000cpm respectively.CI)Ifl xl,000504030± E2El00 2 4 6 8 10 12 14 16 18- 136 -a dose-dependent manner (Fig.33).3.4.7. Summary and DiscussionNoninfectious VLPs containing three structural proteins were expressed in a BHK cell line (BHK24S) by using an inducible promoter. These VLPs were found to resemble RV virions in termsof their size, morphology and some biological activities. In immunoblotting studies, VLPs werefound to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murinemonoclonal antibodies. Immunization of mice with VLPs induced specific antibody responsesagainst RV structural proteins as well as VN and HAT antibodies. After immunization of micewith VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RVstructural proteins stimulated proliferation.The assembly of RV vision involves at least two major steps: encapsidation andenvelopment of nucleocapsid. In RV, encapsidation occur in the cytoplasm as newly synthesizedcapsid protein interacts with genomic RNA to form icosahedral nucleocapsids. The packaging ofgenomic RNA into the nucleocapsid is believed to be a specific event as the 40S genomic RNAbut not the 24S subgenomic RNA is packaged into RV vision (Oker-Blom et al., 1984). Recently,a stretch of 31 nucleotides on the 5’ end of the RV genome has been identified as responsiblefor the binding of the genomic RNA to the capsid protein in vitro (Liu et al., unpublished data).Employing reverse transcription combined with the polymerase chain reaction (Saiki et al., 1988),we failed to detect any RV-specific RNA in the pseudovirion secreted from stable BHK-24S cells(data not shown), suggesting that capsid proteins can interact with each other and form anucleocapsid-like structure. The VLPs were found to have a higher ELISA titre with a C-specificmAb (Table 4) and to elicit stronger anti-C antibody response in mice than those from RV (Table- 137 -5), implying that the VLPs contain more C protein than RV either due to its relative amount orconformational exposure in the particles. Although the pseudovirions do not contain RV-specificRNA, we cannot rule out the possibility that they might package some cellular RNAs or evenDNAs into the nucleocapsid.Incorporation of nucleocapsid into the membrane envelope to form virus particles is a yetpoorly understood event in virus assembly. We found that in BHK-C and BHK-E2E1 cells, noRV proteins were released into the medium (Fig.28A). In BHK-24S cells, all three structuralproteins were present in the medium as the result of subviral particle formation and egress(Fig.28A). These data strongly suggest that the interaction between glycoproteins and thenucleocapsid is the driving force for the assembly and release of the VLPs. This interaction hasbeen defined to occur between the cytoplasmic tail of glycoprotein El and the capsid protein.Deletion or substituton of the cytoplasmic domain of RY El abolished the delivery of the VLPsinto the medium (Hobman et al., unpublished data).Besides being a useful tool to study RV assembly, BHK-24S cells in which VLPs aresteadily assembled and released can be used as a potential source for mass production of rubellaantigens at low cost under inducing conditions. BHK-24S cells continuously produce VLPs forup to 5 days without cell lysis when 30 jiM ZnSO4 is present in the medium and up to onemonth in DME/F12 (GIBCO) medium. VLPs can be harvested daily from the medium, whichis replaced with fresh medium after harvesting. Depending on the methods used to quantitate theyields of the VLPs and RV from culture medium, the yield of VLPs was found to be two-foldhigher than RV in an HA assay, five times higher in ELISA assays using human sera, and morethan ten times higher by protein quantitation using silver staining after gel electrophoresis (data- 138 -not shown).It has been proposed that immunogenicity can be achieved by presenting antigens ona polyvalent particle structure. This concept led to the development of chimeric virus (Clarke etal., 1987; Michel et al., 1988; Li et al., 1993), virus-like particles (Griffiths et aL, 1993) orimmunostimulating complexes (Takahashi et al., 1990), in which multiple copies of antigen areintegrated in a particulate form. These particles have been found to induce both humoral and cell-mediated immune responses, including VN antibodies (Michel et al., 1988; Griffiths et al., 1993;Li et al., 1993), T helper cells or cytotoxic T lymphocytes (Takahashi et al., 1990; Griffiths etal., 1993; Li et al., 1993) in animals. In our case, the VLPs were found to be significantly moreactive than the soluble El protein in inducing antibody responses in mice, especially for theproduction of VN and HAl activity (Table 5). The VLPs also evoked cell-mediated immuneresponse to RV and RV structural proteins. This is believed to be important in providingprotective immunity against RV infection. Preliminary results have shown that CD4 T cells maybe the major effector in cell- mediated immune responses elicited by the VLPs in mice, whereasCD8 T cells may be also involved (data not shown). A study of the phenotype of the effectorcells in proliferation assays is in progress. The VLPs are composed of all three structural proteinsof RV, which make them similar to RV regarding antigen-presentation. These studies suggest thatthe noninfectious but highly immunogenic VLPs may serve as a candidate for safe vaccinedevelopment.- 139 -4. SUMMARY and PERSPECTIVESIn the Togavirus family, RV bears a striking similarity to the prototype aiphaviruses (SFV andSIV) in terms of genomic organization and strategy for gene expression. However, RV researchhas fallen far behind that of aiphaviruses due to the fact that RV has a much slower replicationkinetics and limited cytopathological effects, and there has been limited success in producinglarge numbers of monoclonal antibodies directed to RV structural proteins (C, E2). In recentyears, with the aid of recombinant DNA technology and mammalian expression systems, muchprogress has been made in studies on the expression, processing and biological functions of RVstructural proteins. In this study, I have attempted to employ various mammalian expressionsystems (COS cell transient expression, vaccinia recombinant virus, stably transformed cells andRV-infected cells), combined with recombinant DNA techniques (including site-directedmutagenesis), to explore the structure/function relationship of RV structural proteins.The role of N-linked glycosylation of RV El and E2 has been studied with respect to theirbiological function in RV maturation. RV El is the dominant surface molecule of the RV virionand the major target for human immune surveillance. The influence of N-linked glycosylationon antigenicity and immunogenicity of RV El has been investigated by expressing Elglycosylation mutant proteins via vaccinia recombinants and by analyzing the immunoreactivityof deglycosylated El protein from RV virions treated with glycosidase to remove carbohydrateson El. It appears that all three N-linked glycosylation sites on El are required to maintain anoptimal protein configuration for the exposure of epitopes determining biological functions (i.e.neutralization and hemagglutination). Deletion of any of the N-linked glycosylation sites on Elresults in reduction of antibody production and this effect seems to be additive. Carbohydrates- 140 -on El, important for the hemagglutination activity of the virion, however, are not involved in theconstitution of the epitopes recognized by monoclonal antibodies used in this study.Unlike El, the biological function of E2 is not clear except that it is believed that in RVinfected cells, E2 serves as a carrier to deliver El from a post-ER, pre-Golgi compartment to theGolgi complex. The role of N-linked glycosylation on intracellular transport and processing ofRV E2 has been analyzed by transient expression of E2 glycosylation mutant proteins in COScells. N-linked glycosylation at all three sites is essential for transport competence of E2.Deletion of any glycosylation site on E2 leads to a significant reduction in rate of glycanprocessing, lower stability and retention of mutant proteins in the ER.A conm-ion effect of deleting glycosylation sites between El and E2 is the formation ofaberrant disulfide bonds. Amino acid sequences predicted from cDNA sequences indicate thatboth El and E2 are rich in cysteine residues. The implication of this is that proper folding andcorrect disulfide bonding may be a slow process, sensitive to modulation by structural alterationor environmental influence. Iritra- and intermolecular disulfide bonds have been observed in wild-type, as well as mutant El and E2 when they are expressed separately (Fig.9, Fig.23). It appearsthat deletion of glycosylation sites adjacent to a cysteine residue (E11, E2G2 and E2G3) hasa more profound deleterious effect. Therefore, it is conceivable that the addition of carbohydratecontributes to the proper folding and subsequent correct disulfide bonding, which may be criticalfor the biological functions of El and E2.Although the role of N-linked glycosylation on the functions of El and E2 has beenstudied in some detail, the effect of glycosylation during RV replication and infection is stilllargely unknown. Deglycosylated RV virions lost hemagglutination activity (Table 3) while their- 141 -infectivity has not been determined. Recently, Dr. T.K. Frey and co-workers have succeeded inthe construction of RV genome-length cDNA clones. Transfection of cells with RNA transcribedfrom one of these clones results in the production of virus that preserved the genetic andphenotypic characteristics of the parental virus from which the cDNA clone was derived. Suchan infectious cDNA clone will facilitate the evaluation of site-specific effect of glycosylation inviral assembly and infectivity (Wang et al., 1994).Due to the fact that RV infectious cDNA was not available during this study, analternative approach was taken to elucidate the structure/function relationship of RV structuralproteins during virus assembly. Tunicamycin, BFA and monensin were used to inhibit or toinduce an altered processing and transport of RV glycoprotein in cDNA-transfected cells and RVinfected cells. Protein processing and transport as well as virus assembly and release wereanalyzed. BFA and monensin dramatically reduce the assembly of intracellular infectious viionswhile tunicamycin completely inhibit virus assembly. The effect of tunicamycin, BFA andmonensin on virus infectivity parallels that of the disruption of distribution of El and E2 in anintact Golgi complex, which points out the possibility that probably a stable association of Eland E2 with the Golgi structure may be essential for efficient assembly of RV. This is in a goodagreement with the fact that RV El and E2 are found to be targeted to the Golgi complex(Hobman et al., 1993) with a limited amount expressed at the cell surface (Hobman et al., 1990).In RV-infected cells, the released virus has a slightly higher virus titre than that of intracellularvirus (even during the early stage of virus life cycle), and is found to contain 0-linked glycansas well as complex-type of N-linked sugars on E2. The intracellular viruses has not been purifiedto homogeneity or distinguished from unassembled RV structural proteins due to technical- 142 -difficulties, therefore, the detailed oligosaccharide structures on El and E2 of intracellular virionsare not known. It is possible, however, that the difference in the status of glycan maturationbetween extra- and intracellular viruses may contribute to their infectivity. In addition, furtherstudies are necessary to define the mechanism of retention of RV glycoprotein in the Golgiapparatus, and the importance of Golgi-specific modifications (i.e. 0-linked glycosylation) onvirus assembly.Three stably transformed cell lines expressing RV structural proteins have beenconstructed. RV structural proteins are found to assemble into virus-like particles in the Golgicomplex prior to their release from the cells. The assembly of VLPs is found to be independentof genomic RNA but is strictly dependent upon co-expression of all three structural proteins. TheVLPs resemble RV virions in terms of size, buoyant density, morphological appearance andprotein composition. These observations point out the future direction in studying thestructure/function relationship and protein-protein interaction during RV assembly. Stable celllines expressing RV structural proteins in different combinations under an inducible promotor,or with molecular modifications, will be a useful system.The non-infectious VLPs have been found to bind similarly to native RV virions with RVspecific monoclonal antibodies as well as human sera. Humoral (including viral neutralizing andhemagglutination inhibition) and cell-mediated immune responses have been detected in miceimmunized with the VLPs. Therefore, the VLPs may serve as a convenient source of RV antigenfor serodiagnostic assays and a potential candidate for vaccine development. 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