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Analysis of the structural proteins of rubella virus Berkowitz, Cheryl Anne 1988

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ANALYSIS OF THE STRUCTURAL PROTEINS OF RUBELLA VIRUS  By  CHERYL ANNE BERKOWITZ B.Sc.  (Microbiology), University of Alberta, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1988 Cheryl Anne Berkowitz, 1988  In presenting degree  this  thesis  in partial  fulfilment  at the University of British Columbia,  of the requirements I agree  for an advanced  that the Library shall make it  freely available for reference and study. I further agree that permission copying  of this  department  or  thesis by  for scholarly  his  publication of this thesis  or  It  is  by the head  understood  that  for financial gain shall not be allowed without  po.4-l>o  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  may be granted  her representatives.  permission.  Department of  purposes  for extensive of my  copying  or  my written  ii ABSTRACT  Complications of rubella virus infection, including congenital rubella syndrome and the association of rubella virus with joint inflammation, emphasize the need for continued research on rubella virus.  The finding that the association of rubella virus infection with  joint manifestations i s more pronounced with wild strains than with vaccine strains suggested the possibility of strain variation.  Several different techniques have been employed in order to compare six rubella virus strains and identify variations in their structural proteins.  Differences in biological activities were detected, including  the extent of virus production and the ability of various c e l l types to support replication of rubella virus (tissue tropism).  However, the  strains were shown to have remarkably similar electrophoretic patterns. Variation appeared to result from alterations in glycosylation.  Efforts  to isolate the protein components of the two envelope glycoproteins were unsuccessful, and i t was therefore not possible to localize variation to either the protein or the carbohydrate components.  Future work  employing more sensitive methods for examination of fine molecular structure and the correlation of these structures with biological activity w i l l further our understanding of the pathogenesis of rubella virus infection.  iii TABLE OF CONTENTS  INTRODUCTION I.  Rubella Virus - Molecular Biology  pl~3  II.  Structural Organization of Virion  P3~4  III.  Sequences of Rubella Virus Structural Proteins  p4-7  IV.  Monoclonal Antibody Studies  p7~8  V.  Rubella-Associated Arthritis  p8-ll  VI.  Correlation of Viral Protein Structure  VII.  with Pathogenicity  pll-16  Glycoproteins - Introduction  pl6-17  VIII. Glycoprotein Structure  pl8-21  IX.  Viral Glycoproteins  p21-22  X.  Rubella Virus Glycoprotein Structure  p22-24  PURPOSE OF THIS STUDY  p25-26  MATERIALS I.  Abbreviations  p27  II.  Materials  p28  III. Solutions  P29-30  IV.  P31-32  Gel Solutions  METHODS I.  Virus Stock Preparation  p33  II.  Plaque Assay  p33  iv III.  Precipitation with Polyethylene Glycol (PEG)  p34  IV.  Immunoprecipitation  p34  V.  One-Dimensional SDS-PAGE  P34-35  VI.  Two-Dimensional PAGE  P35~36  VII.  Western Blotting  P36-37  A. Transfer  p36  B. Detection  p37  VIII. Digestion by Hydrogen Fluoride IX.  Staphylococcus aureus V8 Protease Digestion  p37~38 P38-39  RESULTS I.  One-Dimensional SDS-PAGE 1. Introduction  p tO-53 1  p40  2. Choice of Primary Antibody for Immunodetection p40-4l 3. One-Dimensional SDS-PAGE  p4l-47  4. Time Course of the Production of Rubella Virus Structural Proteins  p^7-48  5. Detection with Monoclonal Versus Polyclonal Antisera II.  p48-53  Two-Dimensional Gel Electrophoresis  P53 6l  1. Introduction  p53  2. Results and Discussion  P56-61  III. Tissue Tropism 1.  Introduction  _  p6l-67 p6l-62  2. Permissiveness of Cess and U937 Cells to Different Rubella Virus Strains  p62-67  V IV.  Rubella Virus Glycoprotein Analysis  p67~8l  1. Deglycosylation - Introduction  p67~70  2. Chemical Deglycosylation - Hydrogen Fluoride  p70~71  3. Enzymatic Deglycosylation - Endoglycosidase F p71~79 k. Discussion of Digestion of Rubella Virus Glycoprotein by Endoglycosidase F V.  p79-8l  Peptide Mapping  p8l-89  1. Introduction  p8l-83  2. Results and Discussion  p83 91 _  SUMMARY AND CONCLUSION  P92-95  BIBLIOGRAPHY  P96-113  vi LIST OF TABLES  Table  I: Replication of Rubella Virus in Different Cell Lines  p63  Table II: Plaque Titration of Rubella Virus in Vero, Cess and U937  Cells  P  64  vii  LIST OF FIGURES  Fig.  1. Structures of the Oligosaccharide Chains of Asparagine' linked Glycoproteins  Fig.  pl9  2. Detection of Rubella Virus Structural Proteins By Three Polyclonal Rabbit Antisera  Fig.  3- Strain Comparison of Rubella Virus Structural Proteins  Fig.  4. Comparative Cytopathic Effect of Rubella Virus Infection in Vero Cells (72 hours post-infection)  Fig.  5- Time Course of Production of RV Structural Proteins  Fig.  6. Comparative Detection of RV El Glycoprotein With  p42 p^5  p^9 p51  Polyclonal and Monoclonal Antisera Fig.  p54  7- Strain Comparison of Rubella Virus Structural Proteins By Two-Dimensional Gel Electrophoresis  Fig.  8. Strain Comparison of Rubella Virus Structural Proteins (Cess Cells)  Fig.  p57  p65  9- Strain Comparison of Rubella Virus Structural Proteins , (U937 Cells)  Fig. 10. Immunoprecipitation of the El Glycoprotein  P  68  p74  Fig. 11. Comparison of Methods for Isolation of the El Glycoprotein  p77  Fig. 12. Rubella Virus Structural Proteins: S. aureus V8 Protease Digestion  p84  viii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, Dr. J.K. Chantler, for the opportunity to carry out this project in her laboratory, and for her generous support and guidance throughout the course of this study. I would also like to thank the members of my supervisory committee, Drs. J.B. Hudson, G. Krystal, W.R. McMaster, P.E. Reid and G. Weeks for their guidance and helpful suggestions. My thanks also go to Ms. Karen Lund, Ms. Georgia Tai and Mr. Malcolm Barth for helpful,discussions. I would also like to express my profound gratitude for the neverending support of my parents and family.  1  INTRODUCTION  I. Rubella virus - Molecular Biology  Rubella virus (RV) i s a small enveloped virus i n the family Togaviridae and i s the sole member of the genus Rubivirus.  Electron  microscopy shows i t to be a roughly spherical particle, 50-70 nm i n diameter with a 30 nm electron-dense core.  This particle i s surrounded  by a lipid-containing envelope, acquired by budding through the host c e l l membrane, into which virus-specific glycoproteins have been inserted (59.151,154).  The virus has a single-stranded, positive-sense  (infectious) RNA genome of about 10 000 nucleotides (47,159) or about 3.5 x 10 daltons (89,154). 6  The genomic RNA sediments at 40S and  contains both a 5' methyl cap structure and a 3' Poly(A) tract (107). A subgenomic capped and polyadenylated mRNA species, which hybridizes by Northern blot to the 3* portion of the 40S genomic RNA, also exists i n infected cells (107).  This subgenomic RNA of approximately 3500  nucleotides (47,159) i s translated into a 110 000 molecular weight precursor polyprotein (pi10) which i s co-translationally cleaved into the three structural proteins of rubella, the envelope proteins El and E2, and the core protein C. The gene order of these three proteins on the polycistronic subgenomic mRNA species has been shown to be NH -C-E22  E1-C00H (105).  A review of early reports on rubella virus, a l l dating from the  2 1970s, revealed disagreement in the number and size of rubella virus proteins, with different investigators describing from three to twelve virus-specific proteins produced during infection (15,89,15^.156).  This  lack of consistent data on the number of structural proteins and their molecular weights may in part have been due to difficulty in producing high v i r a l titres and purifying intact rubella virions.  Furthermore,  while most of the early investigators studied preparations of purified virus, one report (15)  was a study of intracellular virus.  Since  t  infection by rubella virus does not inhibit cellular metabolism (15.59.15^). i t i s probable that the true number of v i r a l proteins was masked by host proteins. More recently, consensus has been reached that the virus consists of three structural proteins, the C or core protein, which i s an 1  unglycosylated protein of about 33k daltons, and two glycosylated envelope proteins, El and E2.  El has a molecular weight of about 58k  daltons, while E2 has been variously reported as a series of two (76,107) or three (163)  closely related glycoprotein species with  molecular weights of about 42-48k daltons.  Peptide maps generated by  cyanogen bromide cleavage of E2 species appeared to differ by only a single peptide (161), while other peptide mapping furnished similar maps, but different isoelectric points (65).  The consequent suggestion  that the same polypeptide exists in more than one charged form i s supported by tryptic peptide analyses (105) sequencing (76),  and by partial amino acid  which showed the E2 species to have identical carboxyl-  terminal structures. Together with the fact that the coding capacity of  3  the subgenomic 2 \S RNA i s sufficient for only one E2 polypeptide, these l  reports, confirm that the E2 species have an identical apoprotein moiety and that the various forms are the result of heterogeneous processing.  I I . S t r u c t u r a l Organization of V i r i o n  Waxham and Wolinsky (162)  have modelled the structural organization  of the proteins in the rubella virion from results of gel electrophoresis under non-reducing conditions. Results generated by twodimensional SDS-PAGE showed that the El glycoprotein exists in three distinct species, in a monomeric form (El), as a disulphide-bonded El-El dimer with a relative molecular weight of 105k daltons and as a 95k dalton disulphide-bonded E1-E2 heterodimer.  Non-reduced E2 was shown to  occur almost exclusively in the E1-E2 heterodimeric form, and C was reported to exist only in a dimeric form of 78k daltons, associated with the v i r a l genome.  The structure of the rubella virion appears to be unique in the Togaviridae family, as the relative proportions of E l , E2 and C are not equal (162).  The model therefore describes the central core of the  virion as an association of multiple dimeric units of the C protein with the genomic RNA to form an icosahedral structure (35.162).  This capsid  structure then acquires an envelope into which repeating hexamers, each composed of five El molecules and one E2 molecule, are inserted. It was proposed that one E1-E2 heterodimer i s inserted in this hexamer, while the remaining El molecules remain as monomers or interact to form  4 i  homodimers. That the antigenic domain on E2 i s generally inaccessible to recognition by antibodies may thus be explained by i t s position i n the E1-E2 heterodimer of the hexamer (162).  Furthermore, the variable  accessibility of the multiple antigenic domains of E l may be the result of El configurational changes which depend upon i t s three potential interactions (El, El-El, and E1-E2).  I I I . Sequences o f Rubella Virus S t r u c t u r a l Proteins  The amino acid sequences of the E l , E2 and C proteins of the wild type M33 strain of rubella virus (23) and El (47,159), E2 (159) and C (l4l) of the Therien wild type strain together provide details of a unique togavirus.  The E l and E2 glycoprotein genes are both preceded by a series of approximately twenty uncharged, mainly hydrophobic, amino acids (23,47,159), typical of signal sequences which direct the translocation of proteins through the endoplasmic reticular membrane.  At the carboxy  termini of both E l and E2 l i e similar sequences of hydrophobic amino acids which probably function as trans-membrane domains and anchor the glycoproteins to the l i p i d bilayer. Additionally, there are two long stretches of uncharged amino acids i n the coding region for E l (47) and three long hydrophobic regions i n the E2 coding region (23).  Both the  El and E2 sequences are unusually G/C-rich, with a high number of proline and cysteine residues (23,159)-  The sizes of E l and E2, as  calculated from the amino acid sequences, are i n good agreement with  5 those determined by gel electrophoresis for the unglycosylated forms (present in tunicamycin-treated cells) (47,159)•  The deduced amino acid  sequence for El indicates the presence of three potential sites for relinked glycosylation (23,47,159), while E2 has been reported as having either three (23)  or four (159)  potential glycosylation sites.  The  large shift in the mobility of E2 synthesized in the presence of tunicamycin suggests that a l l (three or four) potential glycosylation sites are f i l l e d (159). but whether a l l three potential sites in El are f i l l e d i s s t i l l uncertain.  The difference between the molecular weights  of the glycosylated and unglycosylated forms of El i s only 5000 daltons (159), although gel f i l t r a t i o n studies by Frey et a l . (47)  showed that  at least two of the sites are f u l l .  The nucleotide sequences of the C protein gene show i t to be unusually rich in C (41.6%) and G (31.2%) residues, but poor in A (15.4%) and U (11.8%) (141).  Codon usage i s non-random, with C and G  residues preferentially found in the third position of a l l codons except those for glutamine (l4l).  Regions with long stretches of up to 35% G  or 45% C residues are found throughout the gene, with highest G + C content (B0%) in the amino-terminal  third.  The C protein i s strongly  hydrophilic as well as rich in proline (l4.1#) and arginine (14.4%) residues ( l 4 l ) .  Clusters of both amino acids are concentrated in the  amino-terminal  third of the protein (l4l), and this positively-charged  region may be involved in binding the protein to the negatively-charged 40S genomic RNA in the v i r a l nucleocapsid  (23).  6 The amino acid sequences for E2 as determined by Clarke et a l . (23) and Vidgren et a l . (159) show good agreement for about half the sequence.  The disagreement i n the remaining half i s likely the result  of single nucleotide changes which could lead to a switch i n frameshift. These changes may reflect strain differences, but could also result from the d i f f i c u l t y i n sequencing this region because of i t s high G + C content.  The amino acid sequence of El as determined by Vidgren et a l . (159) differs from that determined by Frey et a l . (47) i n only three positions, a l l due to single base differences which may have accrued from errors i n sequencing or from mutations i n the Therien strain of rubella virus acquired during cultivation.  Comparison of the sequences  of the M33 strain C gene (23) and the Therien strain C gene (l4l) reveals five single amino acid changes that probably reflect strain differences.  Three nucleotides at various positions i n the Therien  sequence appear to be absent i n the M33 sequence, resulting i n a different amino acid sequence because of frameshift alterations. However, this may be an artefact that illustrates the difficulty of sequencing the C gene because of i t s high G + C content.  A comparison of the nucleotide and amino acid sequences of the rubella virus E l , E2, and C proteins reveals no homology with other alphaviruses, including Semliki Forest virus and Sindbis virus (l4l). The overall base composition of the coding regions, with their high G + C content, i s very different from the alphaviruses, and i s unique among  7  RNA viruses. The unusual codon usage, which differs markedly from that generally observed i n human cells, may affect the rate of translation by contributing to a shortage of specific isoacceptor tRNA species (141). This may indeed also explain the relatively slow production of rubella virus particles i n c e l l culture.  IV. Monoclonal Antibody Studies  Monoclonal antibodies (mAbs) have been used to isolate regions of the structural polypeptides of rubella virus that bear on the infectivity of the virus.  Once identified, these specific regions may  be amino acid sequenced, an essential step toward producing a synthetic, non-replicating subunit vaccine for rubella virus.  Numerous recent studies have attempted to define epitopes important for infectivity.  Most of the mAbs produced to date by various  investigators recognize E l , which implies an immunodominant role for this protein as an antigen, with at least three distinct epitopes having been defined on E l (52,68,69,153,163).  A l l of the studies have located  the haemagglutination site i n E l , and neutralizing epitopes have been located i n E l as well as E2 (35,52,68,69,152,161,163).  As well as defining functional epitopes, monoclonal antibodies against E2 have also been used to immunoprecipitate a precursor to the E2 glycoprotein from infected cells (163).  This precursor i s smaller  and lacks the molecular weight heterogeneity of E2 found i n the virus,  8  further supporting the suggestion that post-translational modifications, including association with E l , account for the multiple forms of E2 present in mature rubella virus and reported by so many investigators.  Although several reference strains of rubella virus exist, i t appears that there i s only one serotype (35,151)•  However, early  morphologic studies demonstrated that rubella virus strains could be differentiated by their ability to form large or small plaques in rabbit kidney (RK) c e l l culture (43) plaque formation.  and by differences in the kinetics of  Strain differences have also been detected by  neutralization tests, which revealed differences in rate constants for neutralization of six rubella virus isolates (51)although Ho-Terry and co-workers (67)  Furthermore,  found no difference between the  electrophoretic patterns of a wild-type rubella virus strain and the RA27/3 vaccine strain by polyacrylamide gel electrophoresis, the strains could be distinguished by competitive radioimmunoassays.  These results  suggested the existence of strain-specific antigens, a view supported by the detection of strain-specific epitopes (35)  on the E2 glycoprotein of  a l l tested strains.  V. Rubella-Associated  Arthritis  Naturally-acquired  rubella i s a common childhood illness that  manifests i t s e l f as a generally mild and self-limiting infection characterized by fever, coryza and malaise followed by an atypical rash consisting of round, slightly raised, discrete macules (151).  Its  9 c l i n i c a l importance, however, l i e s , f i r s t l y , i n i t s ability to cause severe birth defects (congenital rubella syndrome) i f a pregnant woman is infected particularly i n her f i r s t trimester of pregnancy and, secondly, i n i t s association with joint inflammation. i  Arthritis associated with rubella virus infection was recognized as early as 1906 (109) and acute joint inflammation has since then been recognized as a common complication of both natural infection and rubella vaccination.  Although normally transient and without sequelae,  and generally with complete resolution of symptoms within two to three years, recurrent inflammation has, i n a small number of cases, been reported for as long as five to fifteen years (17,18,137,147,148). In a l l these cases, continuing involvement of the virus i s suggested by the presence of rubella virus in peripheral blood lymphocytes and synovial fluid.  Rubella-associated arthritis (RAA) may also progress to a  condition resembling rheumatoid arthritis, characterized by RF (rheumatoid factor)-positivity (91,9*0 . However, i n most instances, the inflammation i s resolved before permanent joint damage results.  Although RAA has been described in patients of a l l ages, development of arthritis seems most often to be associated with adolescent and adult females.  The incidence of joint inflammation also  appears to be more frequent after natural infection than after immunization with the currently licensed vaccine strains HPV77/DE5 and RA27/3: a recent study (147) thus found that 52% of women who contracted natural rubella subsequently developed acute arthritis, as compared with  10 only 14% of immunized women. It has also been observed that the different vaccine strains (Cendehill, HPV77/DE5 and RA27/3, etc.) vary in their ability to produce joint manifestations.  Isolation of rubella virus from joints affected by inflammation has been reported after both natural infection (60) and immunization (104,166).  In a large number of cases, rubella virus has also been  isolated from peripheral blood lymphocytes of patients with rubellaassociated arthritis (18, Chantler, unpublished).  This suggests the  possibility that the virus persists at sites other than joints, and that the inflammatory response i s the result of v i r a l reactivation which produces immune complexes that accumulate i n the joints (45).  This  hypothesis i s supported by an ability to detect rubella-specific immune complexes for up to eight months following rubella vaccination, with an associated higher incidence of joint symptoms ( I 5 8 ) .  Immune complexes  have also been implicated i n acute rubella-associated arthritis, as suggested, f i r s t l y , by a temporal association of the appearance of antirubella !virus antibodies with the onset of joint symptoms (25,104) and, secondly, by the higher levels of circulating immune complexes i n symptomatic patients ( I 5 8 ) .  However, association of elevated levels of  immune complexes with development of acute arthritis after rubella immunization was not confirmed by one study (133).  Rubella virus has also been advanced as a cause of chronic inflammatory arthritis of unknown etiology (29,54,57)-  The evidence for  this i s twofold: f i r s t l y , that rubella virus can persist i n humans for  11 long periods in congenital rubella syndrome, and secondly, the recognized frequency of arthritis associated with acute rubella virus infection and the prolonged occurrence of rubella arthritis in some patients.  Several reports have described rubella arthritis  to classical rheumatoid arthritis (91.94).  progressing  Other investigators have  isolated rubella virus from several cases of rheumatoid arthritis with no known association with recent rubella infection (16,44), and study (20)  one  has offered evidence for implicating rubella virus as an  etiological agent in approximately 30% of juvenile chronic arthropathies.  It has also been suggested that rubella virus may be the  i cause of a small proportion  (<10#) of adult rheumatoid arthropathies.  i VI. C o r r e l a t i o n of V i r a l Protein Structure with  Pathogenicity  Mutations accumulate spontaneously and very rapidly in the genomes of RNA viruses, and consequently make i t d i f f i c u l t to correlate identified gene alterations with specific changes in biological properties (79)•  Studies of numerous v i r a l systems have shown that  changes in pathogenicity are commonly the result of alterations in v i r a l protein structure.  Both tissue tropism and virulence have been found to  be altered by minor changes in the amino acid sequence of certain v i r a l polypeptides,  particularly envelope glycoproteins  (82).  pathogenicity i s generally under multigene control (4l),  Although even a single  amino acid substitution can, in some systems, render a virus avirulent. A correlation between specific nucleotide substitutions and altered v i r a l virulence has been suggested for numerous viruses, including  12 rabies virus, polio virus and reovirus type 3-  In rabies the pathogenicity of different rabies virus strains for  i  adult mice depends upon the presence of a specific antigenic determinant i  on the v i r a l glycoprotein, and the ability to use neutralizing monoclonal antibodies for selecting nonpathogenic mutants of rabies virus makes i t reasonable to assume that the pathogenic properties of the virus are indeed determined by the glycoprotein. (33)  Dietzschold et a l .  analyzed tryptic peptides of the glycoproteins of the pathogenic  parent virus and nonpathogenic variants, and then amino acid sequenced a specific tryptic peptide variant.  This work established that the change  in pathogenicity coincided with an amino acid substitution at position 333 of the glycoprotein molecule, resulting in the replacement of an arginine residue with either isoleucine or glutamine (33) or glycine (130).  or glutamine  While amino acid substitutions were also detected at  other positions in the glycoprotein, these changes affected only neutralization, not pathogenicity.  Thus, an arginine at position 333  appears,to be essential for retention of pathogenicity.  It has been  suggested that the replacement of this arginine residue by another amino acid may cause a significant change in glycoprotein conformation. Sequence analysis of poliovirus vaccine strains has yielded a similar correlation between specific nucleotide substitutions and altered v i r a l virulence.  Five base substitutions in the genome of  poliovirus type 3 (P3/Leon/37) may be responsible for the attenuated phenotype of the Sabin vaccine strain (138), while reversion of the  ! i  13 Sabin type 3 poliovirus vaccine strain to a neurovirulent phenotype i s consistently associated with a point mutation i n the 5' noncoding region of the v i r a l genome (39)•  Work with reassortment mutants of influenza virus (which has a segmented genome) has identified the v i r a l haemagglutinin as one of the major determinants of v i r a l virulence.  Although i t appears that an  i-  optimal gene constellation, rather than a single virus gene, i s responsible for the pathogenicity of influenza virus (8), infectivity of the influenza virus i s directly related to the presence of the haemagglutinin i n i t s cleaved form.  The haemagglutinin (HA) i s post-  translationally modified by cleavage at a connecting peptide region into two subunits, HA1 and HA2, and this cleavage i s a prerequisite for v i r a l infectivity.  Furthermore, HA cleavage can be correlated with strain  virulence and virus production i n tissue culture (31).  Another study  (78), a comparison of a virulent and an avirulent strain, showed that while the HA of the avirulent strain was cleaved only i n the presence of 1  trypsin, the virulent strain HA precursor was also cleaved into HA1 and HA2 when trypsin was absent.  However, the amino acid sequences of both  strains were indistinguishable through the connecting peptide region, and difference i n the susceptibility of the HA to cleavage i s therefore not directly attributable to the sequence of this peptide. While the apparent molecular weight of the HA1 subunit from the avirulent strain was higher than that of the virulent strain, the molecular weights of the haemagglutinins were indistinguishable when the viruses were grown in the presence of tunicamycin.  Since monoclonal antibodies indicated  14 that at least one epitope on the HA differed between virulent and avirulent strains, and there were no deletions or insertions i n the amino acid sequences, i t was thought possible that the difference in molecular weight was due to the loss of a carbohydrate side chain i n the virulent strain.  But further examination of the amino acid sequences  detected only one amino acid change in the virulent strain that could affect a glycosylation site in the area of the connecting peptide.  This  loss of a carbohydrate moiety may permit access of an enzyme that recognizes the basic amino acid sequences and results i n cleavage activation of the haemagglutinin i n the virulent virus strain.  The revelation that protease activation results i n exposure of new terminal amino acid sequences explains i n part the role of cleavage i n determining v i r a l virulence as well as tissue tropisms.  Since the  rubella virus structural proteins are specified by a polycistronic message, relative sensitivity to cleavage of the pllO precursor of different strains could determine virulence i n this system also.  Differences exist not only i n the susceptibility of the haemagglutinin to cleavage and pathogenicity between different subtypes of influenza virus, but even within one subtype ( 8 ) . These differences exist i n the structures of the glycoproteins,  as indicated by variation  in the molecular weights (electrophoretic mobilities) of the haemagglutinin and the neuraminidase, and in the presence of carbohydrate moieties.  For example, the acquisition of virulence by one  strain may, as already noted earlier, be associated with a point  15 mutation which results i n the loss of a glycosylation site (31)-  In  avian influenza and Newcastle disease viruses, the susceptibility of the v i r a l glycoproteins  to proteolytic cleavage (which determines  pathogenicity) i s determined primarily by the structure of the glycoproteins  (8,100).  In Sendai virus as well, the fusion protein F  must be cleaved into i t s F  1  and F  2  subunits to become active.  Although  conversion of the F protein from i t s inactive to i t s active form may be accomplished i n vitro by trypsin, trypsin sensitivity can be changed by a single amino acid substitution at the cleavage site of F. The acquisition of trypsin resistance has been traced to a mutation resulting i n the replacement of arginine by an isoleucine residue at the cleavage site (trypsin cleaves on the carboxy terminal side of arginine and lysine residues) (73)•  Viral particle variation has also been implicated i n the establishment of persistent infections.  For example, the periodic  nature of equine infectious anemia has been attributed to sequential production and release of novel antigenic strains which temporarily escape host immune surveillance (97)•  The results from this study  suggest that structural variation of the EIAV glycoproteins occurs during persistent infection and remains stable during replication i n c e l l culture.  The variation can be identified by altered migration  rates i n SDS-PAGE as well as by generation of distinctive peptide maps, and may be due either to carbohydrate heterogeneity or to alterations occurring i n the amino acid sequence.  A more recent study of EIAV was  directed toward antigenic and biochemical characterization during  16 persistent infection (119)-  The EIAV isolates could be distinguished  antigenically by neutralization assays and Western blot analysis. Again, virion glycoproteins displayed different electrophoretic mobilities upon SDS-PAGE. Further work, involving tryptic peptide and glycopeptide maps of each virus isolate, revealed biochemical alterations in both the amino acid sequence and glycosylation patterns of the virion surface glycoproteins gp90 and gp45, but no structural variation was observed in the internal v i r a l proteins.  Oligonucleotide  mapping showed structural variation at the level of the v i r a l genome and indicated that point mutations were probably responsible for this variation.  Since virulence i s likely to be determined by a variety of factors that contribute independently  to the capability of a virus to cause  illness in an infected host, characterization of the molecular determinants of v i r a l virulence i s important in understanding the biology of virus infections.  VII. Glycoproteins - Introduction  Many biologically important proteins contain constituents attached to the polypeptide chain.  These range from small molecules, such as  sulphates and phosphates, to complex structures, including oligosaccharides and nucleic acids.  Among these constituents - and  perhaps the most important - are the oligosaccharide side chains which, when covalently attached to peptide backbones, characterize the  17 glycoproteins.  Glycoproteins are found in bacteria, viruses, fungi,  plants and higher animals and are involved in many cellular functions. These include c e l l - c e l l interaction, adhesion of cells to substratum, enzymic activity, histocompatibility and, i n the case of enveloped viruses, the attachment of the virus to i t s host via specific receptor recognition and the elicitation of a specific antiviral response.  Viral glycoproteins are synthesized by an orderly process that involves the insertion of nascent polypeptide chains into the endoplasmic reticulum membrane, processing of amino-terminal peptides and glycosylation of the nascent chains.  The maturation and  incorporation of the glycoproteins into mature progeny virus involves the migration of the glycoprotein from the endoplasmic reticulum to the smooth membrane, the Golgi apparatus and finally to the plasma membrane. Viral glycoproteins may be found i n several distinct states. An unglycosylated polypeptide exists i n the c e l l only when inhibitors of glycosylation are present.  A partially glycosylated polypeptide may be  found intracellularly as an intermediate product; but by the time the glycoprotein reaches the c e l l surface, i t i s fully glycosylated. The fully glycosylated form i s associated with or anchored to the c e l l membrane through a hydrophobic region, and i s incorporated into virus particles as part of the envelope.  In general, the v i r a l genomes are  too small to specify glycosyl transferases, and glycosylation of the v i r a l glycoprotein therefore involves the glycosylating mechanisms of the host c e l l .  18 VIII. GLYCOPROTEIN STRUCTURE  The linkage between the oligosaccharide and the protein i s a glycosidic linkage that results from a condensation reaction between an amino acid side chain on the protein and the anomeric carbon on the f i r s t residue of the oligosaccharide (80).  In glycoproteins of higher  organisms, carbohydrate linkages to protein may be either N-glycosidic, in which the carbohydrate i s linked to the amido nitrogen of asparagine, or O-glycosidic, with the carbohydrate linked to the hydroxyl oxygen of serine, threonine or, rarely, hydroxylysine.  0- and N-glycosyl classes  of carbohydrates differ not only in the type of sugar-protein linkage, but also in monosaccharide composition, mode of biosynthesis and resistance to inhibitors of glycosylation.  N-glycosides are more common  than 0-glycosides in glycoproteins of higher organisms, but a single glycoprotein may have multiple chains, some of which are 0-glycosides and some N-glycosides.  The carbohydrates in N-glycosides are generally  somewhat longer than in 0-glycosides (7 25 -  residues versus  2-10  residues).  Asparagine-linked oligosaccharides (N-glycosides) on glycoproteins share a common precursor and thus share a common "core" sequence: Ma  1  + 3(Ma  1 +  6)MB1-" 4 G N B 1 " * 4GN-Asn  glucosamine) (Figure 1 ) . attached to the  a  1 , 3 " and  (M:D-mannoses, GN:N-acetyl-D-  Sequence differences occur in the sugars a&  l , 6 - l i n k e d mannose residues of the core.  There are three major types of asparagine-linked oligosaccharides: high mannose, complex and hybrid oligosaccharides (Figure 1 ) .  In high  19  FIGURE 1: STRUCTURES OF THE OLIGOSACCHARIDE CHAINS OF ASPARAGINE-LINKED GLYCOPROTEINS  The core structure and modifications of the oligosaccharide chain of asparagine-linked glycoproteins are diagrammed.  20  STRUCTURES OF THE OLIGOSACCHARIDE CHAINS OF ASPARAGINE-LINKED GLYCOPROTEINS  Ma1 - 3 ( M « 1 -6)M/31 — 4GN/31 — 4GN-Asn or M  . "M—GN —GN—Asn M  M  M—GN —GN —Asn  X—GN—M  Core Structure  (biantennary hybrid) M M  M  \ M—GN —GN —Asn  /  y  M  \  M M  M— G N —GN —Asn  /  (high-mannose, M5)  X—GN—M  / X —GN (triantennary hybrid)  M — M M —M  M —GN —GN —Asn  M M  M — M— M (high-mannose, M9)  GN — M — G N — G N — A s n X—GN—M  M  M —GN —GN —Asn  M — M — M'  /  (bisected hybrid) X—GN—M  (high-mannose, M8)  \  GN — M — G N — G N — A s n  /  X — GN — M  X—GN—M  / X — GN — M  M — G N —GN —Asn  (bisected complex)  (biantennary complex) X—GN—M ""M — G N — G N —Asn  X—GN—M  /  /  X —GN (triantennary complex)  ABBREVIATIONS: F: fucose M: mannose X: variable o l i g o s a c c h a r i d e GN: N-acetylglucosamine Asn: asparagine •: attachment p o i n t f o r optional branching residues  X —GN  \ X—GN—M  ~M — G N — G N — A s n  X—GN—M / X —GN (tetraantennary complex)  (From Boehringer Mannheim Biochemica Info)  21 mannose oligosaccharides, a l l the residues attached to the core are mannose residues and number five to nine (including the three i n the core) i n most mammalian glycoproteins.  Complex oligosaccharides are  characterized by having N-acetylglucosamine (GN) attached to both the a1,3~  and al,6-linked mannose residues i n the core.  In the hybrid type  of asparagine-linked oligosaccharide, the l , 6 - l i n k e d core mannose has a  only mannose residues attached to i t while the a i , 3 l i n k e d core mannose -  has one or more GN-initiated branches attached to i t .  IX. VIRAL GLYCOPROTEINS  Two types of oligosaccharide moieties may be found i n v i r a l glycoproteins.  These are the high mannose type, which contains N-  acetylglucosamine  and mannose, and the complex type which consists of  galactose, fucose and s i a l i c acid i n addition to mannose and Nacetylglucosamine.  Sialic acid i s usually the terminal sugar of the  oligosaccharide side chain i n the complex moiety (48).  Both the high  mannose and complex types have been found to be present i n the glycoproteins of toga-, myxo- and retroviruses. Hybrid structures have yet to be identified i n viral glycoproteins.  Although N-glycosides are more common than O-glycosides i n glycoproteins, there i s evidence from studies of virus and host c e l l systems that both types of linkages may exist i n the same glycoproteins. While O-linked oligosaccharides are predominant i n many c e l l surface glycoproteins such as glycophorin, and i n secreted glycoproteins such as  22 submaxillary mucins, numerous other cellular glycoproteins, including fetuin, glycophorin and immunoglobulins contain both asparagine- (N-) and serine- or threonine- (0-) linked oligosaccharides (62). Glycoproteins with 0-linked or 0- and N-linked oligosaccharides have been found i n vaccinia virus, coronaviruses, herpes simplex virus and respiratory syncytial virus (RSV) (53)•  Vaccinia virus haemagglutinin,  a structural glycoprotein, has both 0- and N-linked oligosaccharide chains (62), with N-linked oligosaccharides predominating, but 0-linked chains participating i n the expression of biological activity (132). The El glycoprotein of mouse hepatitis virus, a coronavirus, seems to be the f i r s t identified v i r a l structural glycoprotein that contains only 0linked oligosaccharides (62).  Other studies have also suggested the  occurrence of 0-linked, as well as previously identified N-linked, oligosaccharides i n the glycoproteins of herpes simplex virus type I (108).  X. RUBELLA VIRUS GLYCOPROTEIN STRUCTURE  Preliminary analysis of the composition and structure of rubella virus glycoproteins has yielded several features.  The f i r s t studies  reported that, since the glycoproteins could be labelled with tritiated sodium borohydride after oxidation with galactose oxidase, galactose must be the terminal carbohydrate moiety (149).  Other carbohydrate  components were identified as N-acetyl-D-glucosamine and mannose. Neuraminidase treatment indicated the apparent absence of s i a l i c acid residues i n the terminal oligosaccharides of both El and E2  23 (neuraminidase cleaves terminal s i a l i c acid residues) (10,149)•  Another  study showed that while E l and the E2 species E2a and E2b were a l l glycosylated, only El and E2b were efficiently labelled with ^Hmannose, thus underscoring the possibility that the difference i n migration between E2a and E2b i s due to differences i n glycosylation (106).  Treatment with endoglycosidase H, which cleaves N-linked high mannose or hybrid structures, had no effect on the mobilities of E2a and E2b (106), and thereby suggests that their glycan moieties are not of the high mannose type. excluded.  The presence of complex glycans, however, i s not  The difference i n mobility between E2a and E2b can therefore  be attributed to both the number and structure of the glycan moieties. The same study reported that the major peptide migrating at the position of E2b apparently contained N-linked glycans since i t could not be detected i n tunicamycin-treated infected cells.  In i t s place, a smaller  protein, representing the unglycosylated form of E2a or E2b or both, was found.  More recently, i t was shown (9) that, i n infected cells, the counterparts of E l and E2 migrated as sharp bands, indicating that the heterogeneity i n virion E2 i s a function of virus maturation.  Further  analysis indicated that while El comprises predominantly simple or high mannose oligosaccharide side chains, the composition of E2 i s more heterogeneous, with the higher molecular weight species being relatively low i n mannose and high i n galactose (the complex glycan pattern), and  2k the lower molecular weight components high i n mannose and low i n galactose (the simple pattern).  Another report by the same investigators (10)  included the analysis  of GP59 and G P 4 3 , the intracellular counterparts to El and E2 respectively, after digestion with endoglycosidase H.  It appeared that  while the intracellular species GP59 (El) and GP43 (E2) were sensitive to endo H, the El and E2 present i n extracellular virus contained both sensitive and resistant species. Although this finding i s not in agreement with previous work by Oker-Blom et a l . (106), who found that endo H had no effect, the methods of analysis differed, with the former focussing on digestion of individual glycopeptides and the latter, likely a less sensitive method, on the digestion of whole virions.  25 PURPOSE OF THIS STUDY  Rubella virus infection remains a serious problem twenty years after the development of a vaccine.  Congenital rubella syndrome i s  s t i l l seen too often, even in highly developed countries, and the involvement of rubella virus i n transient and chronic joint pain and rheumatoid arthritis following natural infection and immunization has only begun to be studied.  The development of a vaccine of high efficacy  and few complications thus remains a priority, but must be preceded by in depth study of the molecular characteristics of the virus.  Numerous reports have noted that the incidence and severity of joint manifestations varied in dependence on the different rubella virus strains.  Several other biological differences have also been reported,  including variation in neutralization kinetics, haemagglutinating activity and, from this study, tissue tropism, which may be defined by the ability of the virus to enter specific c e l l types and establish a productive infection.  Tissue tropism may be characterized by the extent  of virus production and the time, post-infection, at which maximum cytopathic effect i s observed, and plays an important role i n the course of infection.  The molecular basis for these biological variations i s s t i l l poorly understood.  The variations may be the result of mutations i n the  nucleotide sequence which lead to alterations i n the amino acid sequence.  The biological variation may also be the result of variable  26  processing, with differences in the carbohydrate moieties of the glycoproteins possibly resulting in conformational variation and differential presentation of antigenic epitopes.  Precedence for both  nucleotide sequence mutations and processing differences resulting in biological variation exists in other v i r a l systems.  It i s perhaps most  likely that rubella virus strain differences result from a combination of types of variation.  With these facts in mind, preliminary studies were made in efforts to correlate the incidence of joint symptoms caused by wild type and vaccine strains of rubella virus with tissue tropisms in vitro.  Further  to these experiments, specific rubella virus variants, chosen on the basis of differences in biological activity were analyzed by polyacrylamide gel electrophoresis for alterations in structural protein make-up and by peptide analysis for alterations in the molecular structure of the v i r a l structural proteins.  Although non-structural  proteins can also play a role in determining virulence by directly or indirectly influencing v i r a l replication, these proteins have yet to be fully characterized and therefore studies were concentrated on the structural proteins.  It was expected that these studies would enable  correlation of biological differences among rubella virus strains with specific molecular alterations, a correlation that has been accomplished in other v i r a l systems.  27 MATERIALS  I. ABBREVIATIONS  1. APS:  ammonium persulphate  2. BSA:  bovine serum albumin  3. DOC:  deoxycholic acid  4. EDTA:  ethylenediamine tetraacetic acid  5. FBS:  foetal bovine serum  6. HIFBS: heat-inactivated  foetal bovine serum  7. NP40: Nonidet P40 8 . PAGE: polyacrylamide gel electrophoresis 9. PBS:  phosphate-buffered saline  10. PBS+:  phosphate-buffered saline with calcium and magnesium  11. PEG:  polyethylene glycol  12. PMSF:  phenylmethyl  13- SDS:  sodium dodecyl sulphate  14. TEMED:  sulphonylfluoride  N.N.N',N*-tetramethylethylenediamine  15. TPS:  Tween, phosphate, saline buffer  16. Tris:  t r i s (hydroxymethyl) aminomethane  28 II.  MATERIALS  1. Cells Vero (African green monkey kidney), Raji, Cess, and U937 cells were obtained from the American Type Culture Collection (ATCC). A l l cells were maintained as continuous cultures. 2. Virus Stocks (Original) M33 HPV77/DE5 RA27/3 Thomas Therien 1B2  ATCC-wild type Meruvax I - Merck, Sharp and Dohme 1979 Meruvax II - Merck, Sharp and Dohme 1982 Congenital rubella syndrome isolate 1978 adapted i n Finland 1984 plaque-purified patient isolate 1981  3- Electrophoresis chemicals A l l chemicals for one- and two-dimensional gel electrophoresis were purchased from Bio-Rad, except SDS (BDH). 4. Enzymes Staphylococcus aureus V8 protease: purchased from Miles Laboratories Ltd. Endoglycosidase F (Endo F): purchased from Genzyme 5. Miscellaneous materials Hydrogen fluoride was generously donated by Dr. Dutton, Department of Chemistry, UBC. Nitrocellulose was purchased from Bio-Rad and Schleicher & Schuell. Film for autoradiography: Kodak X-Omat RP x-ray film Staphylococcus aureus protein A: Calbiochem Protein A - Sepharose: Pharmacia  29 III. SOLUTIONS (not described in METHODS section)  1. TNE (pH 7.4) - 0.01 M Tris 0.1 M NaCl 1 mM EDTA 2. TPS (pH 7.6) - 10 mM sodium phosphate 0.9$ NaCl 0.05$ Tween-20 3. PBS (pH 7-0) - 0.15 M NaCl 7.5 mM Na HP0 2.5 mM NaH P0 .H 0 2  4  2  4  4. PBS+ (pH 7.3) - 0.14 M NaCl 2.7 mM KC1 8 mM Na HP0 1.5 mM KH P0 0.9 mM CaCl 2  2  4  2  4  2  5. SDS-PAGE sample buffer - 0.0625 M Tris-HCl (pH 6.8) 2% SDS 10% glycerol 5% B-mercaptoethanol 0.001% bromphenol blue 6. Staphylococcus aureus V8 protease buffer - 0.0625 M Tris-HCl (pH 6.8) 10% glycerol 0.1% SDS 0.001% bromphenol blue 7. Endoglycosidase F reaction buffer - 0.1 M sodium phosphate (pH 6.1) 50 mM EDTA 1% NP40 0.1% SDS 1% B-mercaptoethanol 8. Neuraminidase reaction buffer - 0.1 M Na acetate (pH 5-0) 1 mM Tris-HCl (pH 7.6) 2 mM PMSF 9. SDS-PAGE running buffer (pH 8.3) - 0.025 M Tris 0.1% SDS 0.192 M glycine 10. Western Blot Transfer Buffer I (pH 8.3) - 0.013 M Tris 0.096 M glycine 20% methanol  30 11. Western Blot Transfer Buffer II (pH 9.9) - 10 mM NaHC0 3 mM Na C0 20% methanol 3  2  3  12. Gel fixative - 25% methanol 7% acetic acid 2% glycerol 13. Culture medium (Vero cells) - 199 + 10% FBS + 1% antibiotic/antimycotic (Penicillin 10 000 U/ml Streptomycin 10 000 ug/ml Amphotericin-B 25 pg/ml) 14. Culture medium (Raji cells) - RPMI + 15% FBS + 1% antibiotic/antimycotic 15. Culture medium (Cess and U937 cells) - RPMI + 10% FBS + 1% antibiotic/antimycotic 16. Culture medium (post-infection) Vero cells - 199 + 5% HIFBS + 1% antibiotic/antimycotic Raji, Cess, U937 cells - RPMI + 5% HIFBS + 1% antibiotic/antimycotic  31 IV. GEL SOLUTIONS A. One-Dimensional SDS-PAGE gels 1. non-gradient separating gel (10% acrylamide) 10% acrylamide ( 0.8 bis:30 acryl.) 0.375 M Tris-HCl (pH 8.6) 0.1% SDS 0.1% TEMED 0.033# APS 2. gradient separating gel (7-5$ to 20% acrylamide) stock I - 7^5# acrylamide (0.3 bis:60 acryl.) 0.558 M Tris-HCl (pH 8.6) 0.1% SDS 0.1% TEMED 0.01% APS stock II - 20% acrylamide 0.558 M Tris-HCl (pH 8.6) 0.1% SDS 0.1% TEMED 0.006% APS ratio stock I:stock II 1.4:1 3. stacking gel - 4.6% acrylamide (0.3 bis:60 acryl.) 0.128 M Tris-Hcl (pH 6.8) 0.1% SDS 0.1% TEMED 0.07% APS B. Two-Dimensional PAGE gels (with IEF gels) 1. Sample buffer - 9.5 M urea 2% NP40 2% ampholines 5% B-mercaptoethanol 2. Sample overlay - 8 M urea 1% ampholines 3. Anode electrode solution - 0.01 M H-jPO^ 4. Cathode electrode solution - 0.02 M NaOH 5. SDS equilibration buffer (pH 6.8) - 10% glycerol 5% p-mercaptoethanol 2.3% SDS 0.0625 M Tris  Isoelectric Focussing Tube gels - k% acrylamide (1.62 bis 28.38 acryl.) 2% NP40 2% ampholine (4:1 pH 5-7:pH 3.5-10) 0.01% APS 0.07% TEMED 9-2 M urea  33 METHODS  I. Virus Stock Preparation  Virus stocks were prepared by infecting Vero c e l l monolayers or Raji, Cess, or U937 c e l l suspensions at an MOI of between 1 and 10 for an adsorption period of k hours, after which the inoculum was removed and replaced with fresh medium. Supernatants were collected when cytopathic effect (CPE) was observed, at approximately 3 to 5 days postinfection.  These were centrifuged at 3000 rpm for 15 minutes (in an IEC  Centra~7R centrifuge) to remove cellular debris, and titrated by plaque assay.  II. Plaque Assay  Serial ten-fold dilutions (10"  1  to 10"  6  or 10" ) of virus stocks 8  were prepared i n 199 medium + 2% HIFBS and were used to infect Vero c e l l monolayers for a k hour adsorption period.  The inoculum was aspirated  and the monolayers were overlaid with 0.5% agarose i n 2 x 199 medium and incubated at 35°C.  Plaques were counted after 10 to 14 days.  Modification for RA/27 strain: The monolayers were overlaid a second time 3 days after the f i r s t overlay with 0.5$ agarose i n 2 x 199 medium containing neutral red.  34 III. Precipitation with Polyethylene glycol (PEG)  Supernatants from virus-infected c e l l cultures were clarified by centrifugation at 3000 rpm for 15 minutes i n an IEC Centra~7R centrifuge.  An equal volume of 20% (w/v) PEG (M.W. 3350) i n PBS was  added to each sample.  Samples were incubated on ice for 2 hours and the  precipitates were collected by centrifugation at 10 000 rpm for 10 minutes i n a Sorvall centrifuge.  IV. Immunoprecipitation  Virus particles, precipitated with PEG from supernatant medium, or c e l l pellets, were taken up i n 1 ml 1% NP40 (V/V) i n TNE, vortexed and incubated on ice for 30 minutes.  Cell debris was pelleted i n an  Eppendorf centrifuge for 3 to 5 minutes.  Rabbit antiserum (50 ul) was  added to each supernatant and the samples were vortexed and incubated on ice for 2 hours (or overnight at 4°C).  S_^ aureus protein A (100 ul) was  added to a l l samples, which were vortexed again and incubated on ice for 2 hours.  Immune complexes were pelleted by centrifugation and washed  twice i n 1% NP40 ( V / V ) , 0.5% DOC (w/v), 0.1% (w/v) SDS i n TNE (pellets were dispersed by sonication).  The pellets were taken up i n sample  buffer prior to gel electrophoresis.  V.  One-Dimensional SDS-PAGE  SDS-PAGE was carried out by the method of Laemmli ( 8 6 ) . Gradient  35 separating gels or single-concentration 10% acrylamide gels were poured, allowed to polymerize and overlayed with a stacking gel i n which a template was embedded.  Samples (supernatants precipitated with PEG or  immunoprecipitates) were taken up in SDS sample buffer, boiled for 3 minutes, centrifuged briefly and applied to the sample wells. Gels were run at 32 mA per gel until the bromphenol blue marker dye reached the bottom of the gel. Gels were then either fixed and dried under vacuum for autoradiography or electroblotted onto nitrocellulose paper for immunodetection.  VI.  Two-Dimensional PAGE  Two-dimensional PAGE gels were carried out by the method of O'Farrell (103).  In the f i r s t dimension, isoelectric focussing (IEF)  gels were poured in glass tubes to a height of 12.5 cm.  The tubes had  been prepared by pretreating them i n a dichromate cleaning solution overnight, rinsing them i n dH 0 and thereafter washing them again 2  overnight i n ethanol saturated with potassium hydroxide (KOH).  After  one more rinsing, they were l e f t to dry and capped at one end with 3 or 4 layers of Parafilm.  The tube gels were overlaid with 20 ul of 8 M urea, followed by dR* 0. 2  Polymerization was allowed to take place for 30 to 60 minutes,  after which the Parafilm plugs were removed and the tube gels loaded into the gel tank containing anode buffer.  The i n i t i a l overlay was  sequentially replaced with 10 u l of sample buffer, 10 u l of sample  36 overlay and, finally, the cathode electrode solution. The tube gels were pre-electrophoresed for 15 minutes at 200 v, 30 minutes at 300 v and 30 minutes at kOO v.  Overlay buffers were removed and the samples,  dissolved in sample buffer, layered on top of the gels.  Samples were  sequentially overlaid with 10 ul sample buffer, 10 ul sample overlay and, finally, cathode electrode solution. Gels were run for 18 hours at 400 v, removed from the tubes, equilibrated in SDS equilibration buffer for 2 hours at room temperature and stored at -20°C until required.  A single-concentration 10% SDS-PAGE gel was used for electrophoresis in the second dimension.  The tube gels were embedded in 1%  agarose (in SDS equilibration buffer) applied to the top of the stacking gels, and the slab gels were run at 32 mA per gel until the bromphenol blue marker dye reached the bottom of the gel.  The slab gels were then  fixed, dried and autoradiographed.  VII.  Western Blotting  A.  Transfer Following electrophoresis, the gels were sandwiched between  sheets of nitrocellulose paper that had previously been wetted in blotting buffer, and placed in a Hoeffer TE42 transfer apparatus. Transfer was carried out, with cooling, at 50 mA overnight, followed by 2 to 4 hours at 300 mA, or, after a brief equilibration period minutes at 50 mA),  for k hours at 300 mA.  (30  Following transfer, the blots  were washed for 2 x 10 minutes in TPS and air-dried.  37 B.  Detection The nitrocellulose sheets were blocked in warm (40°C) 3%  gelatin in TPS for 20 minutes (to reduce background) and washed i n TPS for 10 minutes.  The sheets were then incubated for 1 hour at room  temperature (r.t.) while being agitated with a primary rabbit polyclonal antibody of anti-rubella antiserum diluted 1:200 in TPS, and were then washed twice i n TPS for 10 minutes.  The antiserum had been previously  prepared by column-purification on DEAE-Sephacel columns and adsorbing the serum against Vero cells to reduce non-specific binding. Secondary incubation with biotinylated goat anti-rabbit antiserum (Vectastain), diluted 1:200 in TPS, was carried out for 1 hour at r.t. with agitation. Again, the sheets were washed twice for 10 minutes in TPS prior to colour development using biotinylated horseradish peroxidase complexed to avidin (Vectastain) and prepared i n TPS.  The sheets were incubated  with this complex for 30 minutes at r.t. with agitation. followed by a 5 minute wash in PBS.  This was  Colour development was accomplished  by immersing the sheets i n a solution of 0.015% H 0 2  2  and  3 mM 4-  chloro-l-naphthol (dissolved in methanol) in PBS at r.t. with agitation for 5 to 15 minutes.  The sheets were then rinsed i n dR*0 and air-dried. 2  In some cases, the samples were radioactively labelled, and the sheets also autoradiographed either before or after the immunological detection of antigens.  VIII.  Digestion by Hydrogen Fluoride  Rubella virus samples were prepared from infected Vero c e l l  38 supernatants precipitated with PEG. 0.5 ml PBS and lyophilized.  The precipitates were taken up i n  Duplicate samples were treated with  hydrogen fluoride (HF) under both mild (1 hour, 0°C) and harsh (3 hours, 23°C) conditions. Samples were then either taken up i n dH 0 and 2  lyophilized again before solubilization i n SDS sample buffer, or taken up directly i n sample buffer.  Thereafter they were loaded on to 10%  polyacrylamide mini gels, which were electrophoresed at 32 mA per gel. The gels were then either stained with Coomassie blue stain and dried under vacuum or were blotted onto nitrocellulose paper.  The  nitrocellulose sheets were developed as previously described and then stained briefly i n amido black stain.  IX. Staphylococcus aureus V8 Protease Digestion  One-dimensional  gradient gels were poured and run as usual.  Individual sample lanes were cut out and equilibrated for 30 minutes, without stirring, i n 0.125 M Tris-HCl, pH 6.8 containing 0.1% SDS. Previously prepared gradient gels were overlaid with the same buffer, and the gel strips placed horizontally through the buffer 10 to 15 mm above the separating gel. The buffer was then aspirated and replaced with stacking gel, overlaid with 0.1% SDS.  After polymerization of the  gels, the electrode compartments were f i l l e d with SDS running buffer, and the gels overlaid with V*8 protease buffer with or without V8 protease.  The protease was employed at a concentration of 8 ng per  square millimeter of slot surface, or 2.5 ug/ml. Electrophoresis was carried out at 32 mA per gel until the bromphenol blue marker dye  39 approached the bottom of the stacking gel, at which point the current was turned off for 30 minutes to allow for digestion to take place. Following electrophoresis, the gels were either fixed and dried for autoradiography or electroblotted onto nitrocellulose paper for immunodetection.  40  RESULTS  I. ONE-DIMENSIONAL SDS-PAGE  1. Introduction  The simplest and most common method for characterizing v i r a l proteins i s one-dimensional SDS-PAGE, which separates proteins by molecular weight (size).  This method can be used in preliminary studies  to determine the molecular weights of proteins of interest and can also indicate major differences in specific proteins of several strains of a single virus.  In conjunction with Western blotting and immunological  detection procedures, SDS-PAGE can thus offer much information  and  answer many questions about basic protein structure.  2. Choice of Primary Antibody for Immunodectection  Rabbit polyclonal antisera had been prepared against many of the rubella virus strains examined in this study, but despite previous reports of the cross-reactivity of anti-rubella virus antisera, there was s t i l l a possibility that one antiserum might cross-react more strongly than any of the other antisera.  Identical rubella virus protein samples were electrophoresed in triplicate, transferred to nitrocellulose sheets and developed with antisera prepared against three different strains. Results are shown in  Hi Figure 2, and reflect the high degree of cross-reactivity among antisera.  In each panel, the indicated antiserum detected the three  structural protein groups of the three major virus strains (Therien, HPV77/DE5 and M33)- Even so, i t appears that antiserum prepared against the HPV77/DE5 strain most strongly detects the v i r a l proteins of the strains examined (panel 2). This antiserum also detects the numerous degradation products i n each sample lane, which disappeared i n other experiments when greater care was taken to prevent digestion by proteases.  High molecular weight species (HMWS) probably representing  El or E2 dimers or polymers were also clearly detected, but decreased i n intensity or disappeared altogether i n other experiments when higher concentrations of p-mercaptoethanol were used.  As a result of this  experiment, rabbit anti-HPV77/DE5 antiserum was used as the primary antibody for immunodetection i n the majority of further experiments.  3. One-Dimensional SDS-PAGE  With the optimal antiserum for detection determined, a basic comparison of the structural proteins of six rubella virus strains was made.  Earlier studies had determined that rubella virus infection of  Vero cells produced satisfactory v i r a l titres i n the supernatant and preliminary strain comparisons were therefore made using this c e l l type. This c e l l line was also found to be satisfactory for studies of intracellular proteins and thus could be used for both kinds of studies while the use of lymphoblastoid c e l l lines was limited to studies of extracellular proteins.  42  FIGURE 2: DETECTION OF RUBELLA VIRUS STRUCTURAL PROTEINS BY THREE POLYCLONAL RABBIT ANTISERA  Vero c e l l cultures (10 x 10 cells) were infected with rubella 6  virus stock preparations.  Supernatants from 72 to 96 hours post-  infection were collected and precipitated with 10% PEG. The resulting pellets were resuspended i n SDS sample buffer (60 ul) and the samples (15 ul) electrophoresed and transferred to nitrocellulose paper for immunodetection.  The E l , E2 and C polypeptides are indicated. Also of note are the high molecular weight species (HMWS) and the species appearing between the El and E2 regions, both of which are greatly decreased by the addition of higher concentrations of B-mercaptoethanol, suggesting that these species are disulphide-linked polymers.  Other minor bands (a  "laddering" effect) are believed to result from sample degradation.  43  D E T E C T I O N  O F T H R E E  Kd  fffi*  R U B E L L A  V I R U S  P O L Y C L O N A L  H i i i i  S T R U C T U R A L R A B B I T  P R O T E I N S  B Y  A N T I S E R A  $ i i  0  130  HMWS  75 50  )E2  )C  39 27 17  rabbit anti-Therien  rabbit anti-HPV77/DE5  rabbit  anti-M33  44  Figure 3 illustrates the great similarity of the protein patterns produced by the Therien, HPV77/DE5 and 1B2 strains.  The E l glyco-  protein i s detected as one major and several minor bands just above the band representing IgG.  The latter i s commonly seen i n Western blots of  immunoprecipitated protein samples and results from recognition of the rabbit antiserum used i n the immunoprecipitation  protocol by the goat  anti-rabbit secondary antibody i n the immunodetection system. E2 appears as a series of three or four bands (labelled), and the C protein appears very faintly (lanes E and F).  The inconsistent presence of the  C, or core, protein on Western blots may possibly be attributed to a loss of antigenicity upon transfer to nitrocellulose sheets since i t appears consistently by autoradiography.  Also, since a change i n  transfer buffer, from a Tris-glycine (150) to a carbonate (36) buffer system which returns proteins to a more native form after electrophoresis, resulted i n stronger and more consistent detection of C, this explanation i s deemed the most satisfactory.  The species  migrating below the Ig band, but above the E2 region may represent altered conformational forms of E2 and are not consistently observed.  Figure 3 also shows that the patterns of proteins of the Thomas, M33 and RA27/3 strains differ from the protein patterns of the other three strains.  The Thomas strain, although producing a pattern for the  E2 glycoprotein species identical to those of the f i r s t three strains, gives a different pattern for E l , with two major E l species migrating at the same rate and another E l species migrating more quickly than i n the other strains.  This band i s not clearly observed i n Figure 3, but has  45  FIGURE 3: STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS  Vero c e l l cultures (10 x 10^ cells) were infected with rubella virus stock preparations.  Cells were collected at 72 hours post-  infection, washed in PBS and resuspended in SDS sample buffer (100 ul) containing 46 ul B-mercaptoethanol (2x normal volume).  Samples (10 pi)  were electrophoresed and the resulting gel transferred to nitrocellulose paper.  The blot was developed with rabbit anti-Therien  antiserum as the primary antibody.  The E l , E2 and C species are indicated. Of interest are the high molecular weight species and displaced band in the E2 region of the M33 strain (arrows).  Also of note are the species appearing between the El  and E2 regions which are present inconsistently on Western blots and may represent altered conformational forms of E2.  STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS  ABC  D  E  F  G  47 been noticed as a characteristic of this strain (see Figure 6).  The M33 strain shows the most marked differences, with the presence of large quantities of a high molecular weight species (arrow), most likely a dimer of E l and E2. While this species was also detected i n the protein patterns of the other strains (Figure 2), and could be greatly reduced by the addition of higher concentrations of a reducing agent, the M33 high molecular weight species appeared more resistant to reducing agents. strains.  The E2 region i s also different from that of the other  Examination of the E2 group reveals that the fastest  migrating, and therefore the smallest, species i s displaced relative to the smallest species i n the other strains (arrow).  The RA27/3 strain did not produce any pattern, a failure that reflects the much lower virus titres (10-100 fold) obtained by infection with this strain than with the other strains.  PAGE analysis using  highly concentrated RA27/3 supernatant virus resulted i n detection of RA27/3 proteins, and thereby suggests that detection problems i n this study are solely a reflection of low protein concentration.  4 . Time Course of the Production of Rubella Virus Structural Proteins  An experiment designed to explore the course of infection and production of rubella virus proteins was carried out i n Vero cells. Cell supernatants were collected and the culture medium replaced every 24 hours until maximum cytopathic effect (CPE) was observed, about 4 to  48  5 days post-infection.  Parallel infected c e l l monolayers were also  photographed at 24 hour intervals.  Figure 4 i s a comparison of the CPE  produced by five rubella virus strains at 72 hours post-infection.  The  CPEs produced by the M33. Therien and 1B2 strains are the most marked when compared with the control, while that produced by HPV77/DE5 i s less intense, but s t i l l obvious.  Infection by the RA27/3 strain, however,  appears to leave the c e l l monolayer intact, even up to five days postinfection.  This result agrees with the protein analysis in Figure 3 and  is perhaps not unexpected, since plaque titration data have shown infection with R A 2 7 / 3 to produce lower virus titres than any of the other strains.  Infection with this strain i s thus less productive, and  host cells are able to survive intact for longer periods.  A time course of the appearance of structural proteins i s shown in Figure 5.  Patterns for the E2 region of a l l the strains except RA27/3  are distinct 48 hours after infection, while the El patterns are not clearly detectable until much later, about 96 to 120 hours (4 to 5 days).  In the RA27/3 sample, a single band, which may represent the C  protein, appears faintly at 96 hours and, by 120 hours, has become quite strong.  This band i s often the only protein species detected in  unconcentrated samples of RA27/3-  The detection of protein patterns  correlates well with the appearance of cytopathic effects.  5 - Detection with Monoclonal Versus Polyclonal Antisera  Since a monoclonal antibody directed against an El epitope was  49  FIGURE 4: COMPARATIVE CYTOPATHIC EFFECT OF RUBELLA VIRUS INFECTION IN VERO CELLS (72 HOURS POST-INFECTION)  Vero c e l l cultures (5 x 10^ cells) were infected with rubella virus stock preparations at an MOI of 1 to 10.  Photographs of cytopathic  effect (CPE) were taken at 24 hour intervals.  HPV77/DE5  1B2  51  FIGURE 5: TIME COURSE OF PRODUCTION OF RV STRUCTURAL PROTEINS  Vero c e l l cultures (10 x IO cells) were infected with rubella 6  virus stock preparations and supernatants collected and replaced every 24 hours.  Supernatants were precipitated with 10% PEG and resulting  pellets resuspended i n SDS sample buffer (100 u l ) . Samples (5 ul) were electrophoresed, transferred to nitrocellulose paper and developed with rabbit anti-HPV77/DE5 antiserum as the primary antibody.  52  TIME COURSE OF PRODUCTION OF RV STRUCTURAL PROTEINS  48 h p . i .  72 h p.  .  )  A B C  A  B  C  D E F G  D  E  F  G  H I  H  J  K  L  M  N  I J K L A A N  E 2  53 available, the proteins detected by the monoclonal and polyclonal antisera were compared.  Figure 6 (lanes H - N ) shows the pattern produced  by development with rabbit anti-HPV77/DE5 polyclonal antiserum,  and  lanes A-G the pattern detected by a monoclonal antibody against M33 E l . While the polyclonal pattern i s that consistently seen, the detection of a low molecular weight band in the M33 sample by the monoclonal antibody is an interesting finding.  More specifically, the El patterns of the  Therien, HPV77/DE5 and 1B2 strains show El to migrate with an approximate molecular weight of 68k daltons, while the Thomas El migrates much faster (this band i s barely detectable in Figure 6), the M33 El migrates to the E2 region.  and  This detection of a low molecular  weight species by the monoclonal antibody represents the most striking strain difference yet observed among rubella virus strains and i s most likely explained by the epitope to which the monoclonal antibody reacts only being accessible in the unglycosylated form of El in this particular strain.  The detection of the Thomas El species at a  molecular weight of approximately 58k daltons may be explained by the same mechanism, although in this case, an underglycosylated form, rather than an unglycosylated one, i s recognized.  II. TWO-DIMENSIONAL GEL ELECTROPHORESIS  1. Introduction  Although a number of biological differences, including plaque formation, haemagglutination  activity and antigenicity, have been  54  FIGURE 6: COMPARATIVE DETECTION OF RV El GLYCOPROTEIN WITH POLYCLONAL AND MONOCLONAL ANTISERA  Vero c e l l cultures (10 x IO cells) were infected with rubella 6  virus stock preparations and supernatants collected and replaced every 24 hours.  Supernatants were precipitated with 10% PEG and the resulting  pellets resuspended i n SDS sample buffer (100 u l ) .  Samples (5 ul) were  electrophoresed, transferred to nitrocellulose paper.  Sample lanes A  through G were developed with the monoclonal antibody, 2B5 (directed against the M33 El) and lanes H through N, with rabbit anti-HPV77/DE5 antiserum.  55  C O M P A R A T I V E W I T H  D E T E C T I O N  P O L Y C L O N A L  A N D  O F  R V  E l  G L Y C O P R O T E I N  M O N O C L O N A L  A N T I S E R A  Kd  130 75  50  »•=  = §  )«  39  >C  27 17  A  B  C  D  E  120 h p.i.  F  G  J  K  96 h p.i.  L  M  N  56  detected among rubella virus strains, analysis of the structural proteins of rubella virus by one-dimensional (1-D) SDS-PAGE yielded differences i n only three (M33, Thomas, RA27/3) of the six strains examined.  Since the extent of the biological differences indicated the  likelihood of further, perhaps more subtle, molecular variation among the virus strains, the method of two-dimensional (2-D) gel analysis was used to explore this possibility.  In the O'Farrell (103) two-dimensional gel system, proteins are separated i n the f i r s t dimension on the basis of charge by isoelectric focussing.  Thereafter, the proteins are separated i n the second  dimension on the basis of molecular weight (size), as i n one-dimensional SDS-PAGE.  This system allows for the resolution of two or more distinct  proteins which may have the same mobility (molecular weight) under reducing conditions.  2 . Results and Discussion  As seen i n Figure 7. the protein patterns generated by twodimensional gel electrophoresis of six rubella virus strains are remarkably similar.  The most obvious difference i s the lack of a clear  pattern for the RA27/3 vaccine strain.  As previously mentioned, this  strain grows to much lower titres (10 to 100-fold less) i n c e l l culture than the other strains, and supernatants from RA27/3 infected cells _  therefore contain substantially less v i r a l protein for analysis than i s found for any other rubella virus strain.  Until more concentrated  57  FIGURE 7: STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS  Vero c e l l cultures (10 x 1 0 cells) were infected with stock virus 6  preparations and labelled overnight three to four days post-infection with 50 uCi/ml 3 S-methionine following a two hour starvation period i n 5  1/10 methionine-containing medium.  Supernatants (5 ml/sample) were  collected, precipitated with 10% PEG and immunoprecipitated with rabbit anti-HPV77/DE5 antiserum (50 pi).  Immunoprecipitates were resuspended  in 2D-PAGE sample buffer (45 pi) and the samples (20 pi) loaded onto tube gels for electrophoresis i n the f i r s t dimension.  The pH gradient formed i n the f i r s t dimension was pH 5-2 to 7-0.  Gels were autoradiographed for four weeks at 4°C.  Legend: One-Dimensional Gel A Control B Therien C HPV77/DE5 D M33 E RA27/3  F 1B2 G Thomas  STRAIN  COMPARISON OF R U B E L L A VIRUS STRUCTURAL PROTEINS BY TWO-DIMENSIONAL CEL ELECTROPHORESIS —  2D  I  CONTROL  IEF  m  THERIEN  59  STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS 2D THOMAS  I  •  »  I  C  D  i  I•  m  y y  >  6  60  protein samples of the RA27/3 strain can be obtained, comparison of this pattern with those of the other strains i s impossible.  The most significant difference among the strains examined i s seen by comparison of the M33 wild type strain with any of the other strains, most notably Therien and 1B2. As indicated by the arrow in Figure 7c, the M33 pattern shows a single displaced spot i n the E2 region relative to those i n the Therien and 1B2 patterns.  This may be the same  displaced protein species detected by 1-D SDS-PAGE. The identity of the protein remains undetermined, but i t may be the M33 E l protein which was shown to co-migrate with the E2 species i n 1-D SDS-PAGE. This could be substantiated by using the techniques of Western blotting and immunodetection with a monoclonal antibody directed against E l . Electrophoretic conditions for transfer of the proteins from a 2-D gel onto nitrocellulose paper and conditions for development may, however, need to be altered, since Western blots developed with even polyclonal antisera failed to reveal a l l protein spots detected by autoradiography (data not shown).  Because two-dimensional SDS-PAGE affords greater  resolution of proteins than one-dimensional electrophoresis, each resolved spot probably contains less protein.  In part, this was  suggested by the fact that samples had to be radiolabelled to a higher specific activity than for one-dimensional SDS-PAGE.  Two-dimensional SDS-PAGE thus failed to detect any differences i n the structural proteins of rubella virus that could not be detected by one-dimensional SDS-PAGE. For routine examination of the structural  61 proteins of new rubella virus strains, i t was therefore decided to reject this technique i n favour of the faster, less complicated and less labour-intensive  technique of one-dimensional SDS-PAGE. This decision  was also prompted by the d i f f i c u l t y of interpreting two-dimensional SDSPAGE patterns.  III. TISSUE TROPISM  1.  Introduction  Biological differences among strains of a virus can manifest themselves i n many ways, including variation i n the kinetics of neutralization, haemagglutinating activity and tissue tropism.  Earlier  experiments i n this laboratory had revealed several differences i n biological activity among the strains, including differences in plaque formation, haemagglutination and tissue tropism (particularly i n chondrocytes).  As well, previous studies on the relative permissiveness  of various reticular c e l l types to infection by rubella virus showed variation.  The Therien, HPV77/DE5, 1B2, Thomas and M33 strains a l l gave  comparable titres i n mixed peripheral blood mononuclear cells and Raji cells (Epstein-Barr virus-transformed human B-cells), while the RA27/3 strain replicated very poorly or at undetectable levels (Table I) (Chantler, unpublished).  These results have great bearing for the  potential development of persistent infections, as i n congenital rubella syndrome and rubella-associated  arthritis.  62 Table II shows the results of plaque assays carried out with virusinfected Vero, Cess (EBV-transformed human B-cell line) and U937 (human monocyte-like c e l l line) cells.  Again, although the f i r s t five rubella  virus strains replicated satisfactorily i n a l l the c e l l lines tested, the RA27/3 strain replicated poorly i n both Vero and U937 cells and appears not to replicate at a l l in Cess cells.  2.  Permissiveness of Cess and U 9 3 7 Cells to Different Rubella Virus  Strains  Further information on the degree of expression of rubella virus proteins by lymphoblastoid c e l l lines was obtained by one-dimensional SDS-PAGE of both intracellular and supernatant virus.  Very l i t t l e  protein was found intracellularly (data not shown) and thus while both intra- and extracellular proteins from infection of Vero cells could be analyzed, only supernatant v i r a l proteins produced by infection of these c e l l lines were examined. Protein samples obtained from Cess c e l l infection gave patterns similar to those obtained from infected Vero cells (Figure 8 ) .  As before in other work, no R A 2 7 / 3 pattern could be  detected, but i n this case, rather than low production of virus particles relative to the other strains, the evidence from plaque assays (previously described) suggests that RA27/3 cannot replicate in this c e l l line.  The M33 strain again gave the most strikingly different  protein pattern, with i t s high molecular weight dimer and displaced band in the E2 region. In this c e l l type, however, the displaced band i s not the smallest species, but rather, the second smallest, with a faster  63  TABLE I: REPLICATION OF RUBELLA VIRUS IN DIFFERENT CELL LINES  Cell Line Virus Strain  Mixed PBMC  B-Cell Line Raji  T-Cell Line CCRF-CEM  1B2  +++  +++  ++  Therien  +++  +++  ++  Thomas  +++  ++  NT  M33  ++  ++  NT  HPV77/DE5  +++  +++  ++  RA27/3  +/-  -  NT  NT - Not Tested (From J.K. Chantler, unpublished)  64  TABLE II: PLAQUE TITRATION OF RUBELLA VIRUS IN VERO, CESS AND U937 CELLS  Cell Line U937 (pfu/ml)  Virus Strain  Vero (pfu/ml)  Cess (pfu/ml)  1B2  3.8  X  107  4.4  x  10  8  8.5  X  107  Therien  2.5  X  107  4.2  x  10  8  2.7  X  10  Thomas  1.4  X  107  3-5 x 10  8  5-3  X  107  M33  1.2  X  107  3.1  x  10  4.9  X  107  HPV77/DE5  1.7  X  10  8  5.1  x  10  1.6  X  10  8  RA27/3  1.3  X  10  6  l.l  X  10  6  8  8  <10  2  8  65  FIGURE 8: STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS (CESS CELLS)  Cess c e l l cultures (5 x 10° cells) were infected with rubella virus stock preparations.  Cell supernatants were collected at 72 hours post-  infection and the v i r a l proteins precipitated with 10%  PEG.  Precipitates were resuspended in SDS sample buffer (100 ul) and 5 ul samples used for electrophoresis.  Of particular note are the high molecular weight species and the displaced E2 band in the M33 strain.  Also of interest i s the low  molecular weight band in the E2 region of a l l the strains which appears below the displaced M33 band.  The other minor bands ("laddering"  effect) are believed to result from sample degradation.  o  J  3  Q  3  8  V  1  (ST133 SS30) SNI310cJd "lVrJlllOPcJlS  snyiA  \m3ana  JO  Niosiavdwoo  Nivais  67  migrating species, running just below the M33 displaced band, being detected i n a l l the v i r a l strains.  El species appear only very faintly  in the M33 and Thomas strains and, since the E2 bands appear i n approximately equimolar concentrations with the E2 bands of the other strains, suggest that recognition of this protein by the antiserum used for detection i s somehow impeded, possibly as a result of glycosylation differences which have altered or obscured the epitopes recognized by the antiserum.  Rubella virus infection of U937 cells generated the same patterns as those seen i n Cess cells (Figure 9)• But like RA27/3 infection of Vero cells, and unlike that of Cess cells, the lack of pattern may be attributed to lower production of virus particles than by other rubella virus strains.  Again, the pattern produced by the M33 strain i s the  most different, with i t s faint E l species and i t s displaced band i n the E2 region, the latter a characteristic of this strain i n a l l c e l l types examined to date.  IV. RUBELLA VIRUS GLYCOPROTEIN ANALYSIS  1. Deglycosylation - Introduction  In the study of glycoproteins, i t i s often advantageous to be able to isolate the polypeptide backbone free of attached glycan moieties. This facilitates the analysis of either component or, indeed, the identification of the type of linkage involved.  Deglycosylation may be  68  FIGURE 9: STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS (U937 CELLS)  U937 c e l l cultures (5 x IO stock preparations.  6  cells) were infected with rubella virus  Cell supernatants were collected at 12 hours post-  infection and the v i r a l proteins precipitated with 10%  PEG.  Precipitates were resuspended in SDS sample buffer (100 ul) and 5 ul samples used for electrophoresis.  Of note are the high molecular weight species and the displaced E2 band i n the M33 strain.  Also of interest i s the low molecular weight  species appearing below the displaced M33 band, also seen in Figure 8.  STRAIN COMPARISON OF RUBELLA VIRUS STRUCTURAL PROTEINS (U937 CELLS) Jo  lb  4?  70 accomplished by either chemical or enzymatic methods.  2. Chemical Deglycosylation - Hydrogen Fluoride  Highly glycosylated proteins are well known to be d i f f i c u l t to purify.  Microheterogeneity  due to varying degrees of glycosylation  contributes to the difficulty, making i t especially hard to establish the presence of one unique peptide species.  However, i t i s possible to  achieve satisfactory deglycosylation by means of hydrogen fluoride (HF), which w i l l cleave glycosidic linkages, and thereby remove microheterogeneity  as well as reduce the molecular weight of the  (glyco)protein.  Exposure of glycoproteins to anhydrous hydrogen fluoride at 0°C w i l l cleave a l l the linkages of neutral and acidic sugar moieties within one hour while leaving the peptide bonds and glycopeptide linkages of amino sugar moieties intact (99)-  Harsher conditions, of three hours at  23°C w i l l cleave the 0-glycosidic linkages of amino sugars, but leave peptide bonds and N-glycosidic linkages intact.  And since treatment of  glycoproteins with HF results i n l i t t l e or no degradation of the sugars themselves, quantitative recovery i s possible.  Rubella virus glycoproteins were treated with hydrogen fluoride i n an attempt to further demonstrate the type of glycosidic linkages i n these glycoproteins.  Unfortunately, the results obtained by this  technique were uninterpretable, because the positive and negative  71 control proteins, as well as the rubella virus test proteins, were completely hydrolyzed by the acid (data not shown). This may have been due to a large excess of HF, since the addition of the HF to the reaction v i a l was inexact at best, and the system employed had been developed for large samples of glycoprotein.  Since generation of  accurate data depends on digestion with hydrogen fluoride under anhydrous conditions, i t i s also possible that despite a l l precautions, some water entered the system.  Overall, then, this system failed to  furnish any further information on the structure of rubella virus glycoproteins.  3. Enzymatic Deglycosylation - Endoglycosidase F  The use of enzymes as means for deglycosylating glycoproteins eliminates the hazards associated with highly toxic and corrosive chemicals.  Another obvious advantage of enzymatic deglycosylation i s  the specificity of cleavage which may be achieved. exoglycosidases  For example,  w i l l only act on terminal residues, while  endoglycosidases may be active at sites within the glycoprotein structure.  Furthermore, endoglycosidases have been identified that  operate only on specific types of structures or at specific cleavage sites.  For this study of rubella virus glycoprotein deglycosylation, endoglycosidase F (Endo F) was chosen.  This enzyme i s active on N-  linked glycosides, the type believed to be present in rubella virus from  72 studies with monensin and tunicamycin.  It cleaves high mannose,  biantennary hybrid or biantennary complex structures, but w i l l not cleave bisected hybrid structures, triantennary complex or tetraantennary complex structures (Figure 1, pl9)•  Preliminary experiments on the digestion by Endo F of a crude preparation of rubella virus proteins precipitated with PEG were unsuccessful (with no decrease in the molecular weights of the glycoproteins being detected).  Since this preparation contains large  amounts of protein (including protein in the foetal bovine serum of the culture medium), this failure was attributed to the possibility that the small amount of enzyme added to the reaction mixture was being overwhelmed by substrate.  It was therefore thought essential to develop  a methodology by which individual rubella virus glycoproteins could be isolated.  A f i r s t attempt at isolation of a rubella virus glycoprotein involved careful disruption of the virus particles and selective precipitation of the El glycoprotein with a monoclonal antibody.  This  was carried out by resuspending PEG-precipitated HPV"77/DE5-infected c e l l supernatants in an SDS/Tris buffer, boiling the samples for 90 seconds and then centrifuging to recover the pellet.  NP40 (1 ml) was added to  each of two sample pellets to dilute out residual SDS, while the remaining two pellets were resuspended in a 1:1 mixture of NP40 and SDS/Tris buffer to retain a high SDS concentration, thus preventing reassociation of El and E2 subunits into dimers.  A l l four pellets were  73  then immune-precipitated with a monoclonal antibody, 2B5 (directed against M33 E l ) , followed by goat anti-mouse IgG and, finally, by S. aureus protein A containing 5% bovine serum albumin (BSA). shows the results of this experiment.  Figure 10  Despite the differences i n  i n i t i a l sample treatment, each lane on the nitrocellulose sheet shows successful isolation of the rubella virus E l glycoprotein.  However, a disadvantage of this method lies i n the immunoprecipitation of a complex of glycoprotein, monoclonal antibody and S.aureus protein A which forms a dense pellet that can only be dispersed by sonication.  Because of the large quantity of protein i n the pellet,  not a l l of i t v i r a l , conditions were deemed unfavourable for digestion by enzymes.  It was therefore felt that this method of isolating  glycoproteins was not ideal, and that treatment with Endo F of immune complexes precipitated with S. aureus protein A was not feasible.  An attempt to precipitate the glycoprotein/monoclonal antibody immune complex by a centrifugation step, thereby avoiding the addition of S. aureus protein A altogether, was made. However, this also failed, likely a result of the formation of an immune complex of insufficient size to be precipitated i n this manner.  A modification of the f i r s t method, with protein A-sepharose replacing S. aureus protein A, was attempted.  Protein A-sepharose i s a  highly purified form of S. aureus protein A and has the advantage of producing less solid pellets upon centrifugation.  As before, HPV77/DE5-  74  FIGURE 10: IMMUNOPRECIPITATION OF THE El GLYCOPROTEIN  Vero c e l l cultures were infected with HPV77/DE5 and the supernatants collected at 48 hours post-infection followed by precipitation with 10% PEG. Resulting pellets were differentially treated prior to immunoprecipitation with the monoclonal antibody 2B5 (75 ul), goat anti-mouse Ig (10 ul) and S. aureus protein A + 5% BSA (100 u l ) .  Sample Pretreatments: A HPV77/DE5: SDS concentration kept high B HPV77/DE5: SDS concentration kept high C HPV77/DE5: SDS diluted out D HPV77/DE5: SDS diluted out E Control:  SDS concentration kept high  F Control:  SDS diluted out  IMMUNOPRECIPITATION OF THE E l GLYCOPROTEIN  A  B  C  D  E  F  76 infected c e l l supernatants were precipitated with PEG, the resultant pellets resuspended i n SDS sample buffer, boiled and immunoprecipitated as previously described, except that protein A-sepharose was used to precipitate the immune complexes.  The results of this experiment are  shown in Figure 11, and although not as striking as those shown i n Figure 10, demonstrate once again, that successful isolation of El was achieved.  The method for isolating the rubella virus El glycoprotein using protein A-sepharose was used i n an experiment designed to test digestion of E l by Endo F as well as by neuraminidase. Purified E l was digested by Endo F at concentrations of 0.5 and 2 units (enzyme from NEN) for 1 hour at 37°C.  Identical samples were digested by neuraminidase at a  concentration of 0.01 units for 4 hours at 37°C.  Neuraminidase was  considered a negative control i n these experiments as this enzyme functions to cleave terminal s i a l i c acid residues, which do not exist in the rubella virus glycoproteins.  Neither enzyme treatment produced any  effect on the mobility, and thus the molecular weight, of the El glycoprotein (data not shown).  Since the ideal method of isolating rubella virus glycoproteins would eliminate the need for introduction of additional protein species into the system, one other procedure was investigated.  Rubella virus  proteins were electrophoresed on 10% acrylamide "mini" gels on which stained molecular weight markers were also run.  Gel slices  corresponding to the regions on the gel to which the rubella virus  77  FIGURE 11: COMPARISON OF METHODS FOR ISOLATION OF THE El GLYCOPROTEIN  Immunoprecipitation protocol: A HPV77/DE5: monoclonal antibody 2B5 (75 ul) goat anti-mouse Ig (10 ul) protein A-sepharose (150 ul) B Control:  monoclonal antibody 2B5 (75 ul) goat anti-mouse Ig (10 ul) protein A-sepharose (150 ul)  C HPV77/DE5: monoclonal antibody 2B5 (75 ul) goat anti-mouse Ig (10 ul) S. aureus protein A + 5% BSA (100 ul) D Control:  monoclonal antibody 2B5 (75 ul) goat anti-mouse Ig (10 ul) S. aureus protein A + 5% BSA (100 ul)  E HPV77/DE5: rabbit anti-HPV77/DE5 antiserum ( 4 0 ul) protein Asepharose (150 ul) F Control:  rabbit anti-HPV77/DE5 antiserum ( 4 0 yl) protein Asepharose (150 yl)  G HPV77/DE5: anti-HPV77/DE5 ( 4 0 yl) (100 yl)  S. aureus protein A + 5% BSA  anti-HPV77/DE5 ( 4 0 yl) (100 yl)  S. aureus protein A + 5% BSA  H Control:  I HPV77/DE5: goat anti-mouse Ig (10 yl) protein A-sepharose (150 ul) J Control:  goat anti-mouse Ig (10 yl) protein A-sepharose (150 yl)  Lanes A and C show successful isolation of the E l glycoprotein (arrow).  Secondary bands i n lanes A through D represent Ig species; the  smaller Ig band i n lanes A and B i s precipitated with protein Asepharose and the larger band i n lanes C and D i s precipitated with S. aureus protein A + 5% BSA.  COMPARISON OF METHODS FOR ISOLATION OF THE El GLYCOPROTEIN  A  B  C  D  E  F  G  H  I  J  79 glycoproteins were expected to migrate were excised and extruded through 18 gauge needles.  Samples were boiled i n Endo F reaction buffer for 90  seconds prior to the addition of enzyme at a final concentration of 0.2 milliunits/ml (enzyme from Genzyme).  Samples were incubated overnight  at 37°C, and both the gel slurry and i t s eluate were electrophoresed on gradient acrylamide gels as previously described.  Neither preparation  proved to be satisfactory as the gels appeared to electrophorese inconsistently and the resulting Western blots could not be interpreted (results not shown).  4. Discussion of Digestion of Rubella virus Glycoproteins by Endoglycosidase F  More work must evidently be done with endoglycosidases i f meaningful insight into the composition and structure of rubella virus glycoproteins i s to be gained.  The f i r s t step i n such work must be the  development of a means for isolation of individual glycoproteins.  One  possible method which has very recently shown potential involves immunoprecipitation  of specific glycoproteins by monoclonal antibodies,  PAGE separation and ultimate recovery of each glycoprotein group by preparative gel electrophoresis followed by electroelution.  Success with digestion of rubella virus glycoproteins by endoglycosidases appears to be d i f f i c u l t to achieve.  This may be due to the  structure of the glycan moieties, as yet unstudied.  It i s possible that  the N-glycosides present i n rubella virus glycoproteins consist of  80  structures of such high complexity that access to the cleavage site by the enzyme used i n this study (Endo F) i s prevented.  I f so, an enzyme  with a different specificity might be employed, or, f a i l i n g that, alterations i n the complexity of the structures involved might be made. These could include reduction of glycoproteins to glycopeptides, or reduction of the complexity of the oligosaccharide chain by preliminary digestion with specific exoglycosidases.  The latter method may result  in conversion of a substrate sterically unfavourable for digestion by endoglycosidases into a more favourable form.  Employment of a mixture  of enzymes of varying specificities may also serve to deglycosylate the protein of interest by sequentially removing branch structures.  During the course of this study, another enzyme, often a contaminant i n preparations of Endo F, became available i n purified form from several biotechnology  companies (Boehringer Mannheim, Genzyme).  This enzyme, glycopeptidase F (GPase F), has a broader range of activity than Endo F.  It i s active on N-glycosides and cleaves most high  mannose, hybrid and complex oligosaccharide structures, but w i l l not cleave oligosaccharides attached to an N- or C-terminal asparagine residue, nor w i l l i t cleave 0-linked oligosaccharides.  It may thus be a  more useful enzyme for isolation of polypeptide backbones free of attached glycan groups.  Like Endo F, i t i s sensitive to SDS, and i t i s  therefore necessary to dilute the SDS as well as to include a non-ionic detergent activity.  (NP^O) i n the incubation buffer i n order to retain enzymic  81  Both Endo F and GPase F may be found to be more active on glycopeptides than glycoproteins.  I f so, the use of proteases with  narrow specificities should be considered for the production of glycopeptides, especially prior to digestion with GPase F, because a non-specific protease will produce many small fragments.  This would  increase the likelihood of an asparagine residue, to which oligosaccharide groups are attached, being the N- or C-terminus of a fragment, a structure on which GPase F i s not active.  Although technical complications s t i l l exist, deglycosylation of the rubella virus glycoproteins remains as a promising research tool for studying the effects of carbohydrate moieties on biological function.  V. PEPTIDE MAPPING  1. Introduction  Peptide mapping involves the reduction of proteins to peptides by either chemical or enzymatic means and characterization of the resulting peptides by a technique such as electrophoresis.  Since i t i s not  possible to identify relationships between proteins on the basis of electrophoresis alone, the technique may be used as means for assessing whether proteins of similar size are the product of a single gene, but have undergone differential processing.  For example, proteins may vary  in mobility as a result of slight chemical modifications or posttranslational cleavage events.  82 Both chemical and enzymatic cleavage methods produce specific cleavages at given amino acids.  Chemical methods result i n complete  cleavage at a small number of specific amino acids, and thus tend to produce larger peptides (50-100 residues) than those furnished by enzymatic cleavages (5 20 residues). _  Both types of cleavages may be  controlled by a variety of factors which include pH, temperature, protein to enzyme ratio (enzymatic cleavage), reaction period and protein conformation.  The choice of a proteolytic enzyme depends greatly on the amino acid composition of the protein i n question.  One proteolytic enzyme of  high specificity which has been found to be particularly useful for peptide analysis i s Staphylococcus aureus V8 protease, which cleaves at the carboxy-terminal side of glutamic acid residues i n a bicarbonate buffer (pH 7.8) or acetate buffer (pH 4.0), and at aspartic and glutamic acid residues i n a phosphate buffer (pH 7.8)  (70).  This protease was  employed i n a modification (146) of the Cleveland technique (24) for the digestion of proteins i n two-dimensional SDS-PAGE, and that modification has become popular for the comparative analysis of proteins because of i t s relative simplicity and the fact that i t generates a pattern of peptide bands that i s highly reproducible and characteristic of both the protein and the enzyme used.  The resolving power of the method depends  largely on the molecular weight distributions, relative amounts and protease susceptibilities of the individual proteins i n the sample. Limitations of the method l i e primarily i n i t s inability to resolve the numerous small peptides generated by extensive digestion.  Resolution of  83  very complex samples may be further limited by d i f f i c u l t i e s in identifying a l l peptides generated by digestion of a given protein and by the possibility that two peptides, although generated from different proteins, have the same mobility in the second electrophoretic dimension.  2 . Results and Discussion  As already noted earlier, Western blots developed with a monoclonal antibody directed against an epitope of El indicated that the rubella virus strains examined in this study have El glycoproteins of varying mobilities.  More specifically, in the M33 strain, the protein band  detected by the monoclonal antibody (El) was found to have an approximate molecular weight of 48k daltons, and thus co-migrated with the E 2 species of a l l the rubella virus strains.  This suggests that  proteins in this molecular weight range varied not only in the extent of processing, specifically glycosylation, but actually represented different proteins.  two  To examine this possibility, digestion of the  rubella virus proteins by V8 protease was used.  As seen in Figure 1 2 , digestion patterns were obtained for the E 2 species of a l l the rubella virus strains, except  RA27/3  (pattern not  shown), but no patterns for digestion of El could be detected.  The best  result was that obtained by digestion of the Thomas strain (D), which produced several fragments from the two spots corresponding to the two most clearly labelled E 2 species in the control figure (C). Although  84  FIGURE 12: RUBELLA VIRUS STRUCTURAL PROTEINS: S. AUREUS V8 PROTEASE DIGESTION  Vero c e l l cultures (10 x 10 cells) were infected with rubella b  virus stock preparations. At the f i r s t indication of CPE (M33 and RA27/3- 48 hours post-infection, a l l other strains: six days postinfection), the cells were starved for two hours i n 1/10 methioninecontaining medium and labelled overnight with 50 uCi/ml S-translabel. 35  The labelled supernatants (5 ml/sample) were precipitated with 10% PEG, the resulting pellets resuspended i n 1% NP40 i n TNE containing 2mM PMSF (a protease inhibitor) and immunoprecipitated with rabbit anti-HPV77/DE5 antiserum (50 ul).  Immunoprecipitates were resuspended i n SDS sample  buffer (60 ul) and 10 u l samples were electrophoresed through the f i r s t dimension gel. Dried gels were autoradiographed for four weeks at 4°C.  85  RUBELLA VIRUS STRUCTURAL PROTEINS: STAPHYLOCOCCUS AUREUS V8 PROTEASE DIGESTION 10-—. 2 0  + C O N T R O L- V I  |  C O N T I O l + V I  86  RUBELLA VIRUS STRUCTURAL PROTEINS: STAPHYLOCOCCUS AUREUS V8 PROTEASE DIGESTION  HPVT7/DII-VI  HPV77/OIS+VI  >  F 11J-VI  i  RUBELLA VIRUS STRUCTURAL PROTEINS: STAPHYLOCOCCUS AUREUS V8 PROTEASE DIGESTION + THEHIEN-Vl  ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ THtRICN+VI  88  many of the fragments are indistinct, i t i s clear that at least one of the bands appears i n both lanes, indicating that they are probably identical.  If, however, the two parental E2 spots are different forms  of the same species, i t would be expected that more of the digestion pattern would correlate.  Digestion patterns for the other rubella virus  strains examined show distinct bands for only one E2 species (F,H,J,L). In each case, two fragments are detected from one of the two major E2 species corresponding to those in the controls (E,G,I,K), but cannot be compared with fragments resulting from digestion of the other E2 species.  Published sequences of the E2 glycoprotein show that there are thirteen potential sites for cleavage by V8 protease, and i f a l l the sites are recognized, digestion by the enzyme would therefore generate fourteen peptide fragments.  However, one of the potential cleavage  sites lies within a potential glycosylation site and two other cleavage sites occur immediately carboxy-terminal to two other potential glycosylation sites.  If these glycosylation sites are indeed f i l l e d ,  the V8 protease may be incapable of cleaving the protein because of conformational inhibition or steric hindrance by the glycosidic groups. Thus, theoretically indicated generation of fourteen peptides by V8 may not be achievable in practice.  The digestion patterns presented here must also be considered in light of the detection system used.  I n i t i a l attempts to Western blot  the final gels and immunodetect the peptides generated by digestion with  89  V8 protease failed.  Although there was some evidence of digestion of  the proteins, only one or, occasionally, two peptides were visualized (data not shown), and useful comparison of digestion patterns was therefore impossible.  The most likely explanation for this inability to  detect the peptide fragments immunologically lies in the need to retain intact antigenic epitopes.  It i s probable that generation of fragments  was accompanied by destruction of epitopes recognized by the antisera, and the majority of the fragments consequently remained undetected.  Autoradiography  was next tested as a means for detecting peptides.  Protein samples labelled with either S-methionine or S-translabel 35  35  (labelled methionine and cysteine residues) were digested by V8 protease and electrophoresed.  Results with S-Met-labelled samples were 35  unsatisfactory, with few peptides being detected. 35  However, digestion of  S-translabelled proteins was much more successful. This may be  explained by once again examining the amino acid sequence for E2. Comparison of mole percentages of amino acid residues present in the E2 protein as reported by Clarke et a l . (23)  and Kalkkinen et a l .  (76)  shows consistency in methionine (2.3-2.5%) and cysteine (5.0-5.3%) residues; and when these residues are identified within the sequence, i t can be seen that labelling with S-Met alone w i l l show up a maximum of 35  five out of a possible fourteen generated fragments, while translabel w i l l label ten fragments.  35  S-  In either case, several potential  fragments may go undetected i f this method of detection i s used.  The situation i s even more d i f f i c u l t for the El glycoprotein.  90 There are thirty-four potential V8 cleavage sites i n this protein and none exist within or adjacent to any of the three potential glycosylation sites.  Thirty-five El peptide fragments may thus be  generated by V8 protease digestion.  Again, i f mole percentages of  methionine (0.6-0.7) and cysteine (4.9 5.0) residues are considered, and _  these residues located within the sequence, 35g_Met alone w i l l only label a maximum of three of the possible thirty-five fragments, but S 35  translabel could label a possible eighteen fragments.  Even i f some of  the labelled peptide fragments of El were sufficiently hot to be visualized by autoradiography, i t must also be borne i n mind that i f thirty-five fragments are indeed generated by V8 protease digestion, some of the individual fragments would be small enough to migrate completely through the acrylamide gel. In this work a 7.5 to 20% gradient gel was used.  This acrylamide concentration may not be optimum  for recovery of possible small E l fragments, and future work with this technique should therefore include some experimentation with different gel formulations and acrylamide concentrations.  If interpretable results are to be obtained from protease digestion, some means of visualization that could detect a l l the fragments generated by digestion will have to be developed; for only when a l l fragments are visualized, would i t be possible to gain information about the identity of similarly-sized proteins. Possible solutions to this problem include radioactively labelling tyrosine residues with  1 2 5  I , but since tyrosine residues do not occur i n each  possible fragment, this will also leave some fragments undetected.  The  91 best solution appears to be one which completely avoids the need for protein-labelling.  Silver staining of the second dimensional gel might  be a satisfactory technique for visualization.  This i s a sensitive  technique and may be completed i n a day, eliminating the long wait for results when autoradiography i s employed.  It does, however, have  drawbacks, including the high cost of the reagents and difficulty in reducing background staining, although this latter complication may be minimized by experimentation to find the right conditions for staining.  92 SUMMARY AND CONCLUSION  Major variation i n the biological activities of several rubella virus reference strains has been detected within the last decade. Differences in haemagglutinating activity, neutralization kinetics, association with joint inflammation and, from this study, tissue tropism a l l play important roles in determining the course of rubella virus infection, including which c e l l types will be infected, how much progeny virus i s produced and how quickly infection w i l l spread, as well as the occurrence of complications and the establishment of persistent infections.  The variety and extent of this biological variation among  strains suggests the existence of underlying differences in molecular structure.  Mutations i n the nucleotide sequences could result i n amino  acid substitutions, resulting, i n turn, i n conformational changes or i n additions or deletions of side chain structures.  These changes can  alter the ability of the virus to enter a particular c e l l type (tissue tropism) and set up a productive infection.  Changes i n side chain  structures, namely carbohydrate moieties, can also result i n alterations of virus activity, especially i f receptors on host cells no longer recognize the structures.  Thus, biological activity can be vastly  altered by changes in molecular structures.  An attempt was made i n this study to examine six rubella virus strains for gross differences i n structural protein composition. Onedimensional SDS-PAGE analysis revealed limited strain variation, with three strains (Therien, HPV77/DE5, 1B2) producing apparently identical  93  protein patterns and obvious differences in proteins appearing i n only the M33. Thomas and RA27/3 strains. Since the many differences i n biological functions detected among these strains cannot be accounted for solely by this variation in protein pattern, other means designed to detect more subtle strain differences were employed.  Two-dimensional  electrophoresis was one such method, since i t i s capable of resolving proteins of the same molecular weight (size) by isoelectric focussing. However, this technique failed to provide any additional evidence of strain variation due to gross protein differences.  Since an important question yet to be answered about rubella virus structure involves the localization of strain differences to either the protein core structure or the carbohydrate side chain moieties, attempts were also made to strip the rubella virus glycoproteins of their side chains.  I f this had been successful, proof of whether the strain  variation resulted from genetic mutation or post-translational modification (as suggested by the presence of several forms of each glycoprotein) may have been provided.  Unfortunately,  despite several  attempts at deglycosylation, the location of strain differences remains unknown.  Tissue tropism i s another characteristic that has been found to vary among rubella virus strains. While basic patterns of the structural proteins were observed i n studies of fibroblastic (Vero) and lymphoblastoid (Cess and U937) c e l l lines infected with five of the six rubella virus strains examined, the sixth strain (RA27/3) appeared to  94 selectively replicate in only two of the three c e l l lines, and even in these c e l l lines, did not replicate to the same extent as the other virus strains. Furthermore, an additional E2 species running just below the displaced M33 E2 band was found in U937 and Cess cells, but not in Vero cells, suggesting that processing (glycosylation) may be different between fibroblastic and lymphoid cells.  An experiment to compare the patterns of structural proteins detected by a monoclonal antibody directed against the El protein in comparison with the polyclonal anti-rubella virus antiserum resulted in the observation that, in the M33 strain, the El band detected by the monoclonal antibody migrated to the E2 region.  It was therefore likely  that the different proteins migrating in this region were not solely the result of differential processing of a single species, but also included a second protein species.  The  aureus V8 protease digestion  experiments were designed to examine this possibility, and the preliminary evidence obtained suggests that this i s indeed the case, with only one peptide generated being detected in both major constituents of the E2 region.  It i s apparent from this study that the variation in biological activity of the six rubella virus strains examined results from subtle variations in the molecular structure of the virus and not gross alterations in polypeptide structure of the structural proteins.  While  i t remains a possibility that the variation may result from alterations in the non-structural proteins as well, this remains speculation only  95  until more characterization of these proteins has been done.  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