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Phenotypic mapping of the rubella virus genome Lund, Karen Diane 1999

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PHENOTYPIC MAPPING OF THE RUBELLA VIRUS GENOME by Karen Diane Lund M.Sc. University of Saskatchewan, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine We accept this thesis as conforming to the required standard: The University of British Columbia November, 1997 © Karen Diane Lund, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P^TH o k o a--/ *--lfifjo&& To&Y f/eD'C/'V The University of British Columbia Vancouver, Canada DE-6 (2788) April 23, 1998 Re: D o c t o r a l T h e s i s of K a r e n D i a n e L u n d Dear Dr. Granot The material contained in the doctoral thesis of Karen Diane Lund is pending patent by the University of British Columbia. This material has been disclosed to the University and discussions in this regard have been Ongoing since December, 1997 with the Industry Liaison Office. For this reason we would ask for permission to restrict access to the contents of the thesis for a period of twelve months from March 25, 1998 (completion of defense). Candidate (Karen Diane Lund) Supervisor (Dr. J.K. Chantler) A B S T R A C T An infectious clone of the Cendehill vaccine strain of rubella virus (RV) has been constructed, as well as two chimeric clones containing cDNAs from Cendehill and Therien (wt+) strains. These clones were used to map the regions of the genome responsible for restriction of the Cendehill strain in human synovial cells (SC). Attenuating mutations have been mapped to two of the nonstructural gene regions. Substitution of Cendehill cDNA representing nts 2803 - 5355 into the Therien infectious clone (pROBO302) resulted in a decreased yield of progeny following electroporation of RNA transcripts into SC. This region contained five mutations, at nts 2829, 3060, 3164, 3528 and 4530, in the COOH-third of P150. Substitution of the Cendehill sequence representing nts 1 - 2803 contributed to a further restriction of the progeny in SC. This region contained two mutations at nts 37 and 55, within the 5' stem-loop structure. The further observation that Cendehill bound equally well to SC and the permissive Vero cell line indicated that restriction was not at the level of receptor binding, a function of the envelope proteins. Moreover, substitution of the Cendehill structural gene region into pROBO302 had no effect on replication of the progeny of transfection suggesting that the structural genes were not involved in restriction. In addition it was found that transfection of RV RNA resulted in progeny with an altered phenotype relative to the parental strain. Phenotypic alteration remained stable after six consecutive passages in Vero cells and occurred regardless of. the cell type being transfected, the transfection reagent or whether replicative intermediates or purified genomic RNAs were used. Phenotypic properties that were altered were: plaque morphology, growth rate, temperature sensitivity and cell tropism. I l l T A B L E O F C O N T E N T S A b s t r a c t ii L is t of F i g u r e s vii L is t of T a b l e s jx, A b b r e v i a t i o n s x A c k n o w l e d g e m e n t s xi I N T R O D U C T I O N 1 V I R U S S T R U C T U R E 2 G E N O M E 2 N O N S T R U C T U R A L P R O T E I N S . 7 S T R U C T U R A L P R O T E I N S 8 LIFE C Y C L E 11 Attachment and Entry 11 Replication 13 Assembly 15 M E D I C A L O V E R V I E W 16 R A T I O N A L E A N D O B J E C T I V E S 21 M A T E R I A L S A N D M E T H O D S 24 1. Suppliers 24 2. Protocols- 24 2.1 Cells, Viruses and Bacteria 24 2.2 Cell and Virus Culture 24 2.3 Primary Culture of Synovial Cells 25 2.4 Plaque Titration of Virus 26 2.5 Plaque Purification of Virus : 26 i v 2.6 Virus Concentration 27 2.7 Comparative Binding Assay 27 2.8 Transfection 28 Electroporation 28 1. BHK 28 2. Synovial Cells 28 3. E.coli 29 Lipofectamine 29 DEAE Dextran 29 2.9 Plasmid Isolation and Purification 30 Large-Scale Plasmid Preparation 30 Small-Scale Plasmid Preparation 31 2.10 Ligation 31 2.11 Northern/Southern Blot 32 Probe Preparation 32 2.12 Infectious Clone Construction 33 Isolation of Viral RNA 33 Reverse Transcription 33 Thermal Cycling Amplification 35 pCL1921 35 pCLPC 35 pROBO302 37 pROC3 38 pROC3M 38 pJCND 39 Construct Screening 41 2.13 Virus Growth Assay 41 2.14 Intracellular versus Extracellular Virus Growth 42 Infection 42 Electroporation 42 2.15 Western Blot, Immunoprecipitation and SDS PAGE 42 2.16 Growth at 39°C 44 2.17 Virus Decay . 44 2.18 Co-infection 44 2.19 Sequencing 45 Manual 45 Automated 45 R E S U L T S A N D D I S C U S S I O N 46 1. R E S T R I C T I O N IN J O I N T 46 1.1 Infection of Synovial Cells 46 1.2 Comparative Binding 47 1.3 Electroporation 49 Summary - Restriction in Joint 50 2. C O N S T R U C T I O N O F T H E T H E R I E N / C E N D E H I L L C H I M E R A S A N D C E N D E H I L L I N F E C T I O U S C L O N E 52 2.1 First Strand Priming and cDNA Synthesis 52 2.2 Chimeras and Infectious Clone 54 Summary - Infectious Clone Construction 57 3. P H E N O T Y P I C P R O P E R T I E S O F R O B O 3 0 2 A N D T H E R I E N / C E N D E H I L L C H I M E R I C S T R A I N S 59 3.1 Phenotypic Alteration Following Transfection 59 3.1.1 Intracellular versus Virion RNA 60 3.1.2 Transfection Reagent 60 3.1.3 Cellular Environment 60 3.1.4 Plaque-Purification 61 3.1.5 Other Strains 62 3.1.6 Thermal Cycling Amplification 62 3.1.7 Western Blot and Immunoprecipitation 62 3.1.8 Electroporation of Nucleocapsid 65 3.1.9 Co-infection 65 3.2 Growth in Synovial Cells 68 3.3 Binding to Synovial Cells 3.4 Electroporation of Synovial Cells 3.5 Plaque Morphology 3.6 Viral Growth Curves 3.7 Growth in Raji Cells 3.8 Growth at 39°C 3.9 Virus Decay 3.10 Western Blot Summary - Phenotypic Properties 4. S E Q U E N C E A N A L Y S I S 4.1 Nonstructural Gene Region 4.1.1 5'SL 4.1.2 P150 4.1.3 P90 4.2 Subgenome 4.2.1 NTR 4.2.2 Capsid 4.2.3 E2 4.2.4 E1 4.2.5 3'SL Summary - Sequence Analysis C O N C L U S I O N R E F E R E N C E S A P P E N D I C E S Appendix A Appendix B Appendix C . Nonstructural Mutations . Structural Mutations . Solutions LIST O F F I G U R E S Int roduct ion Fig. 1 Genomic Organisation of Togaviridae 3 Fig.2 Putative Stem-Loop Structures of Rubella Virus RNAs 5 Fig.3 Nonstructural and Structural Protein Production in Rubella Virus 9 Fig.4 A. Rubella and RAA 19 B. Growth of RV in Joint 19 Fig.5 Growth of Rubella Virus in Vero and Synovial Cells 20 Mater ia ls a n d M e t h o d s Fig.6 Cendehill (and RV) cDNAs 34 Fig.7 pROBO302 37 Fig.8 Cloning Stragegy for Cendehill/Therien Chimeras and Cendehill Infectious Clone 40 R e s u l t s a n d D i s c u s s i o n Fig.9 Titration of RV in Infected SC 47 Fig.10 Synthesis of Cendehill First Strand cDNA 53 Fig.11 Cendehill/Therien Chimeras and Cendehill Infectious Clone 55 Fig.12 A. Western Blot of Transfected and Nontransfected RV Strains 63 B. Immunoprecipitation of Parental RV Strains 63 Fig. 13 Titration of Chimeric Viruses in Infected SC 69 Fig. 14 Plaque Morphology of Rubella Virus Strains 74 Fig.15 Growth in Vero Cells - 7.5 hr PI 78 Fig.16 Growth in Vero Cells - 25 hr PI 79 Fig. 17 Decay of RV Titres at 37°C 83 Fig.18 A. Nonstructural Proteins - Amino Acids Alterations in Cendehill Strain 90 B. 5'Stem-Loop Mutations 90 V I 1 1 Fig. 19 Structural Proteins - Amino Acid Alterations in Cendehill Strain 94 Fig.20 Nonstructural Gene Region - Sequence Comparison 101 Fig.21 Subgenome - Sequence Comparison 117 i x LIST O F T A B L E S Mater ia ls a n d M e t h o d s Table 1 Oligonucleotide Primers 36 R e s u l t s a n d D i s c u s s i o n Table 2 Comparative Binding to SC and Vero Cells (i) 48 Table 3 Electroporation of Viral RNA into SC 50 Table 4 Comparative Binding to SC and Vero Cells (ii) 71 Table 5 Electroporation of Chimeric Viral RNA into SC 72 Table 6 Growth of RV Strains in Vero Cells 77 Table 7 Growth of RV Strains in Raji Cells 81 Table 8 Temperature Sensitivity of RV Strains 82 1. A B B R E V I A T I O N S BHK baby hamster kidney cDNA copy DNA CPE cytopathic effect ddH 2 0 distilled & deionised water DEPC diethylpyrocarbonate DMEM Dulbecco's minimal essential mediu DMSO dimethylsulfoxide DTT dithiothreitol EDTA ethylenediamine tetraacetic acid FBS fetal bovine serum HBSS Hank's buffered salt solution HIFBS heat-inactivated fetal bovine serum KOAc potassium acetate LB Luria broth MOI multiplicity of infection O/N overnight PBS phosphate buffered saline PI post-infection RV rubella virus SDS sodium dodecyl sulphate SC synovial cells TAE Tris/acetate/EDTA buffer TE Tris/EDTA buffer Tris tris(hydroxymethyl)aminomethane TBS tris buffered saline Vero African green monkey kidney xi A C K N O W L E D G E M E N T S I would like to thank Dr. J.K. Chantler for her excellent supervision and friendship over the years we have worked together. I would also like to thank my Supervisory Committee; Dr. S. Gillam, Dr. D. Theilmann, Dr. I Sadowski and Dr. G. Krystal, for their rigorous and thoughtful reviews and unfailing support. Dr. Konstantine Pougatchev was responsible construction of the pROBO302 infectious clone, which enabled the mapping studies. The technical assistance of Tracy Evans and the Nucleic Acid and Protein Sequencing unit, at the University of British Columbia, and Ho-Chun Wei is gratefully acknowledged. Moral support was provided in generous quantities by Ellie Stadnick, Jaskamal Girn, Assal Sadeghi, Jessica Boname, Lorraine Lewis, Alison Cox, Dave Leggett and Georgia Tai. Kwee Downie deserves special thanks for formatting of the final manuscript. Finally I would like to thank my parents; Ann and Rainier Lund, who supplied the confidence to pursue any goals and tackle all obstacles. This project was supported by a studentship from the Arthritis Society of Canada. 1 I N T R O D U C T I O N Rubella virus, the etiologic agent of "German measles," belongs to the family Togaviridae and is the only member of the genus Rubivirus. Natural infection is common in childhood causing a systemic illness characterised by a short-lived maculopapular rash and mild fever (Young & Ramsay, 1963). The disease is generally benign and infection is often asymptomatic. Although the symptoms of acute rubella have been recognised since the 1800's, it was not until 1941 that Gregg made the association between rubella infection in pregnancy and subsequent fetal abnormalities (Gregg, 1941). It is the teratogenic potential of rubella that brought the virus to the forefront of public health interests and provided the impetus for isolation of the virus and subsequent vaccine development (Parkman & Buescher, 1962; Weller & Neva, 1962). Humans are believed to be the only natural host for the virus . Rubella replicates poorly, if at all, in animals, although it propagates well in most common animal tissue cultures (Wolinsky, 1990). This is in contrast to the other member of the Togaviridae, the genus Alphavirus, containing at least 26 species which cause some human disease but replicate primarily in animals and depend on transmission by an arthropod vector (Strauss & Strauss, 1994). Sindbis and Semliki Forest are the most well characterised alphaviruses. A number of different wild isolates and several attenuated vaccine strains have been studied in some detail (Bosma et al, 1996; Chantler et al, 1993). Current evidence suggests that these strains are, on average, 97-99% genetically identical (Dominguez et al, 1990; Clarke et al, 1987; Pugachev et al, 1997b), indicating that 2 rubella is an unusually stable RNA virus. The Therien strain is the best characterised of the wild strains and was the first to be cloned and sequenced (Wang et al, 1994). V I R U S S T R U C T U R E The rubiviruses and alphaviruses have been grouped together based on a number of shared characteristics. Most notably, they have a single-stranded, positive polarity RNA genome which is arranged with the structural gene region at the 3' terminus and the nonstructural coding region at the 5' end (Fig. 1). The genomic RNA is associated with oligomerised units of the capsid protein to form a 30 nm electron-dense icosahedral core (Alain et al, 1987; Bardeletti et al, 1975). This is, in turn, surrounded by a host-derived lipid envelope in which are inserted the two viral proteins E1 and E2 (Chong & Gillam, 1971). G E N O M E The positive polarity RNA genome is 9762 nts and is capped at the 5' terminus and polyadenylated at the 3' end, similarly to most eucaryotic mRNAs, enabling it to function directly as a template for protein translation. The approximate G/C content of the genomic RNA is 70% which is the highest recorded for any RNA virus (Dominguez et al, 1990). Such high G/C content is associated with the formation of stable G-C paired secondary structures which provide stability to the genome but certainly creates difficulties in experimental handling of the material as it impedes enzymatic manipulations. G e n o m i c O r g a n i s a t i o n of Togav i r i dae SIN H A P — I MTR I H PRO I ? I P O I [—\ I I II I— A n nsP1 "nsP2 / nsP3 nsP4 C E3 E2 6K E1 PAPHlJ i iMWRlJ ? P R O I H P O I 1—I I I I — A n P150 P90 C E2 E1 RUB Fig.l Oganisation of the genomes of Sindbis virus (SIN) and rubella virus (RUB). Sindbis functional domains and regions of rubella with corresponding homology in the nonstructural genes are highlighted with stippling. These are: methyltransferase (MTR), helicase (H), protease (PRO), unidentified essential function of nsP3 (?) and polymerase (POL). The genomes are 5'-methy| capped and 3'-polyadenylated. (Adapted from Strauss and Strauss, 1994.) Rubella has two long open reading frames (ORFs) encompassing the structural and nonstructural gene regions (Fig.1). The first RV sequence published reported that the nonstructural ORF overlapped the structural ORF (Dominguez et al, 1990); however, further analysis has shown this to be incorrect (Pugachev et al, 1997b). A sequencing error resulted in the omission of GC at nt 6262 of the original sequence. Inclusion of this pair of nts shows that the two ORFs are separated by a short nontranslated region. The nonstructural region comprises two-thirds of the genome nearest the 5' end and is believed to code for enzymes involved in viral replication. It is translated into a >200 kD polyprotein which is cleaved into two nonstructural proteins; P150 and P90 (Marr et al, 1994). The structural ORF comprises the remaining third of the genome nearest the 3' end. This region is separately transcribed from the 4 negative-strand template as a 3327 nt subgenomic RNA which is translated into a 110 kD polyprotein (Hemphill et al, 1988). This is subsequently cleaved to yield the three structural proteins: the unglycosylated capsid protein C, and E1 and E2 the glycosylated envelope proteins (Oker-Blom et al, 1983). This system of independent transcription of the structural gene region enables the structural proteins to be made in greater amounts than the nonstructural proteins. Each of the ORFs is preceded by a short nontranslated region which is presumed to be involved in initiation of transcription and/or translation (Fig.2). The first 65 nts of the 5' end of the genomic RNA is capable of forming a stem-loop (SL) structure/s which has been implicated in efficient translation of the nonstructural proteins and positive strand replication (from the negative strand intermediate) (Pugachev & Frey, 1997; Pogue et al, 1993; Nakhasi et al, 1991). Work by Nakhasi's group has shown that the 5'(+) SL, which is expected to be involved in translation, binds several host proteins, one of which has been identified as the La host protein antigen (Pogue et al, 1993; Pogue et al, 1996). Although the role of La in RV replication remains to be clarified, it is a known RNA-binding protein and has been shown to complex with the RNAs of several other viruses, including Sindbis (Meerovitch et al, 1993; Svitkin et al, 1994; Keene et al, 1987). In the alphaviruses, however, La binds to the negative strand complement of the 5'(+) terminus, alternatively called the 3'(-) SL, which is expected to be involved in transcription, suggesting that the role of La in these two genera might be different. The RV 3'(-) SL has been shown to bind other host proteins which also bind to the homologous region of alphaviruses (Nakhasi et al, 1991). 5 Puta t i ve S t e m - L o o p S t r u c t u r e s of R u b e l l a V i r u s R N A s + strand genomic RNA 3'(+) S L y - 3 5'(+) S L SG(+) S L - strand replicative intermediate 3'(-) S L SG(-) S L 5'(-) S L + strand subgenomic RNA J l 3'(+) S L IT 3 SG(+) S L Fig.2 The location of the three major stem-loop (SL) structures predicted for the rubella genome. The 5'(+) SL is presumed to be involved in the initiation of translation from the genomic RNA. Its complement, the 3'(-SL) may be involved in initiating transcription of new + strand genome. The subgenomic, SG(+) SL may have a role in transcription of the subgenomic RNA and/or translation of the structural proteins. The 3'(+) SL is thought to be involved in initiation of - strand transcription. The same region also contains three AUGs (Fig.2, Fig:20); one beginning at nt 3, one at nt 41 and one at nt 57. The second AUG, beginning at nt 4 1 , is the one utilised by the virus for translation of the nonstructural long ORF. This long ORF is terminated by three TAA stop codons; the first beginning at nt 6389. The first and third AUG are in a different reading frame from 4 1 AUG and are quickly terminated by stop codons at nts 54 and 90 respectively. Deletion studies have suggested that the Therien strain remains viable in the absence of the first initiation codon (Pugachev & Frey, 1997). Pogue et al (1993) have shown that translation can be initiated using the third AUG; ' ' • 6 however, it remains to be determined whether either of these short ORFs is used during virus production. These studies also showed that the 5'(+) SL was "required for efficient translation of the cholesterol acyltransferase (CAT) gene in cDNA constructs. Pugachev and Frey (1997) have shown that mutations in the 5' SL often resulted in reduced protein synthesis, but appeared to have little effect on RNA production. Collectively these results suggest that the major role of the 5'(+) SL structure is in translational enhancement. A 78 nt nontranslated region preceeds the ORF of the subgenomic RNA which can also form a SL structure. This region is presumed to be involved in synthesis of the subgenome and/or regulation of translation of the structural proteins. Within this region RV has 58% homology with the analogous region of Sindbis virus which acts as a promoter for transcription of the subgenomic RNA (Levis et al, 1990). However, the RV sequences, unlike Sindbis, were not capable of initiating transcription when placed in front of the CAT gene in vitro (Hertz & Huang, 1992). Finally, there is a short stretch of nucleotides at the 3' terminus, following the final stop codon of the structural gene ORF, which contains a predicted stable 3'(+) SL structure, Nakhasi's group showed that cells transfected with constructs containing the CAT gene followed by the 3'(+) SL synthesised negative strand RNA. Their experiments demonstrated that RNA extracted from these cells could act as a template to extend primers specific for the negative strand of CAT. They also showed that the primers were only extended in extracts from RV-infected cells which suggests that the RV replicative enzymes were required (Nakhasi et al, 1994). In a somewhat surprising observation, they also demonstrated that translation of the CAT gene by the 5'(+) SL was enhanced approximately 10 fold by the addition of the 3'(+) SL to the distal end of 7 the construct (Pogue et al, 1993). This raises the interesting possibility that these two ends of the genome interact during translation. Such an interaction could occur in cis or, possibly between the genome and the subgenome. The 3'(+) SL has also been shown to bind host proteins, one of which has been identified as calreticulin (Nakhasi et al, 1994; Atreya et al, 1995). The role this protein plays is still unknown. It is a Ca 2 +-binding protein found in quantity in the lumen of the endoplasmic reticulum in muscle cells, where it has been implicated in the regulation of C a 2 + flux for contraction (Michalak et al, 1991). However, perhaps of more interest in this system is the observation that small amounts of calreticulin appear to be associated with small cytoplasmic RNAs, although its function in this context is also undetermined as yet (McCauliff et al, 1990). A protein with the same molecular weight has also been reported to bind to the 3'(-) SL. If this protein is also calreticulin, it suggests that the protein may be involved in the initiation of synthesis of both the plus and minus-strand RV RNAs. N O N S T R U C T U R A L P R O T E I N S The RV nonstructural proteins are made in very low quantities making them extremely difficult to detect against the high background of host protein synthesis. Consequently they were not studied until the completion of a cDNA construct containing this gene region (Wang et al, 1994). The nonstructural long ORF is translated into a >200 kD polyprotein which undergoes a single cleavage to yield two products (Fig.3). P150, nearest the 5' terminus, is 1300 amino acids in length and encodes the putative methyltransferase region and viral protease functions (Marr et al, 1994; Chen et al, 1996; Forng & Frey, 1995; Dominguez et al, 1990). The protease operates via a 8 catalytic cysteine at residue 1151 and is required for the cleavage of the nonstructural polyprotein (Chen et al, 1996). P150 also contains a region with sequence homology to the alphavirus protein NSP3. Although the function of this protein has not been elucidated it is required for alphavirus replication (Hahn et al, 1989). It is interesting to note that the order of the NSP3 and protease domains is reversed between rubella and the alphaviruses (Fig. 1). p90 is 905 amino acids long and has regions with homology to global helicase and replicase motifs. Gros and Wengler (1996) have confirmed NTPase activity associated with this region, one of the functions representative of helicase. RV contains a GDD tripeptide at amino acids 1965-1967 which has been associated with RNA polymerase activity and which forms the anchor of a region of homology with a global replicase consensus sequence (Kamer & Argos, 1984). S T R U C T U R A L P R O T E I N S The 24S subgenomic RNA is translated into a 110kD precursor polyprotein (Fig.3). This precursor is not processed following in vitro translation in the absence of microsomes indicating that it does not contain an autoproteolytic function as is the case for alphaviruses (reviewed in Strauss & Strauss, 1990; Clarke et al, 1987). Instead, the structural proteins appear to be cleaved by host signal peptidase during co-translational insertion into the lumen of the rough endoplasmic reticulum (RER). Translocation of E2, and cleavage of E2 from C, requires a signal peptide sequence contained in the carboxy-terminus of C. Similarly, translocation of E1, and cleavage of E1 from E2, requires a signal sequence located at the carboxy-terminal of E2. (Hobman & Gillam, 1989; Marreta l , 1991; Hobman eta l , 1988; Suomalainen eta l , 1990) The 9 N o n s t r u c t u r a l a n d S t r u c t u r a l P r o t e i n P r o d u c t i o n in R u b e l l a V i r u s MTR PRO H P O L P150 t P90 viral protease MTR PRO T H P O L 5' 200 kD polyprotein translation t • + strand genomic RNA strand replicative intermediate RNA transcription / subgenomic RNA translation Fig. 3 Schematic showing protein production for the Therien strain of RV. The nonstructural proteins are translated directly from the genome, while the structural proteins are translated from subgenomic RNA.. (MTR = methyltransferase, ? = region homologous to Sindbis virus NSP3, PRO = protease, H - helicase, POL = polymerase, C - capsid, E1/E2 = envelope glycoprotein 1 & 2, Y = N-linked glycosylation site.) 110 kD polyprotein E2 E1 ^ host protease E2 E1 ^ host glycosidase J Y J Y _ E2 E1 5' 10 immature structural proteins have molecular weights of approximately 33kD, 30kD and 53kD for C, E2 and E1 respectively (Oker-Blom et al, 1983). In the case of the Therien strain, sequence analysis has shown that E1 contains three potential glycosylation sites and E2 contains four, all of which appear to be utilised (Bowden & Westaway, 1985; Hobman et al, 1991; Vidgren et al, 1987). Following translocation into the RER, E2 and E1 each acquire N-linked sugar moities and are transported to the Golgi apparatus as integral membrane proteins. In Cos cells the majority of the envelope proteins remain localised in the Golgi, only a minor fraction being carried further to the plasma membrane (Hobman et al, 1993; Hobman & Gillam, 1989; Hobman et al, 1990). Although it remains cytoplasmic, the capsid protein has also been found to co-localise with the Golgi (Hobman et al, 1990; Qiu et al, 1994). A loose association of C with the ER has been shown to be mediated by the signal peptide retained at the COOH-terminus (Suomalainen et al, 1990). This may facilitate development of a stronger interaction between C and the cytoplasmic tail of E1, a connection which appears to be necessary for virion formation (Hobman et al, 1994). E1 forms heterodimers with E2 shortly after translocation into the RER and requires co-expression of E2 in order to move past the Golgi and reach the plasma membrane, while E2 is capable of being transported alone (Hobman et al, 1993). The capsid protein is not processed beyond the initial cleavage event but is found, in the virion, almost exclusively as 66kD homodimers. After glycosylation E1 has a molecular weight of approximately 58kD. Mature E2 exists in a variety of forms, presumably due to variable levels of glycosylation, the predominant species being approximately 47kD and 42kD (Oker-Blom et al, 1983). The virion envelope proteins are also thought to exist primarily in hetero- or homodimeric forms. 11 L IFE C Y C L E A t t a c h m e n t a n d Ent ry Rubella is believed to enter the cell using a receptor-mediated endocytotic pathway, although no receptor has yet been identified. RV has been found to replicate in virtually all tissues of fetuses infected in utero, which suggests that the receptor is common to most cell types (Monif et al, 1965). The fact that the virus is much more contained if the infection is acquired post partum is perhaps more likely due to the influence of the immune system or alterations in cellular factors as differentiation progresses, than to a loss of the receptor on cells in later life. This speculation is supported by the observation that, although humans are believed to be the only reservoir for RV, it also grows very well in cell cultures from many animals, including mice, rabbits, hamsters, ducks, dogs and primates. We may infer from this that the RV receptor is widespread and well conserved across many species boundaries, suggesting that it is constitutively expressed on most cells. Mastromarino et al (1989; 1990) suggested the receptor was a lipid based on studies showing that several phospoljpids and glycolipids were able to neutralise RV. However these studies did not include controls to eliminate the possibility that inhibition was ocurring through simple steric hindrance. Since no confirmatory work has been published it may be that these effects were found to be nonspecific. Other experiments using immunological methods have thus far failed to reveal the cellular receptor for RV (Nath et al, 1989). The mammalian receptor for Sindbis virus was identified by testing a panel of monoclonal antibodies (MAbs), raised against BHK (baby hamster kidney) cells, for their ability to inhibit virus binding. The MAb which reduced binding by 80% was found 12 to be specific for the laminin receptor (Wang et al, 1992). Although this seems to be the major receptor in mammalian cells, Sindbis is also able to use other receptors. In chicken cells it has been found to use a second receptor which is not inhibited by MAb to laminin, but is blocked by an anti-idiotypic antibody which recognises a completely different protein (Wang et al, 1991). In addition, two strains of Sindbis virus which bound equally well to BHK cells were shown to have very different binding affinities for neuronal cells. These two strains varied by one amino acid in the E2 structural protein (Tucker & Griffin, 1991). These results show that one strain of an alphavirus can use more than one receptor and, that different strains can use different receptors. This flexibility has no doubt been instrumental in allowing the alphaviruses access to such a broad host range. The majority of RV binding appears to occur relatively rapidly. Using plaque titration, Vaheri et al showed that a 1 hr adsorption period, followed by overlaying the cells with agarose, produced 80% of the plaques found after 3 hrs of adsorption (Vaheri et al, 1967). Unpublished studies in our laboratory support these findings and further showed that shortening the adsorption period to 30 min resulted in a significant reduction of plaque development. Interestingly however, in most cell types only a fraction of the cells appear to be susceptible to infection at any given time, making it impossible to attain synchronous infections of RV. This has been demonstrated using infectious center assays in which a post-adsorption monolayer, infected at 5 pfu/cell, was disrupted and the serially diluted cells were plaque titrated. Only 10% of BHK21 cells were found to be infected, and up to 50% of Vero cells (Sedwick & Sokol, 1970; Hemphill et al, 1988). During later rounds of replication the remainder of the monolayer will eventually support virus replication. Studies using 13 synchronised cell cultures have shown that this is not a simple cell-cycling phenomenon (Chantler, 1979). Inside the endosome the transition to acidic pH is thought to trigger dissociation of the RV core and release of the viral RNA into the cytoplasm. Exposure to pH of 6.0 or lower elicits a fusogenic activity which has been found to reside in E1 and which is thought to facilitate fusion of the viral envelope with the endosomal membrane (Katow & Sigura, 1988; Gillam S, personal communication, 1997). It has also been shown that exposure to low pH causes an increase in the solubility of the C protein (Mauracher et al, 1991). This presumably provides a mechanism for uncoating of the RNA concommitant with its release into the cytoplasm. Although conclusive experiments to show that RV uses this pathway for entry have not been performed, receptor-mediated endocytosis followed by low-pH induced fusion via the envelope glycoproteins is the preferred mechanism for most enveloped animal viruses including the alphaviruses (Kielian & Helenius, 1986; reviewed in: Wiley, 1986). .,. V . R e p l i c a t i o n Once free in the cytoplasm, translation of the positive polarity parental RNA allows synthesis of the viral nonstructural proteins. Subsequently replication occurs through a negative-strand intermediate which acts as a template for the generation of: multiple copies of new genomic and subgenomic RNAs which can be used for further rounds of translation or, in the case of the full-length plus-strand, for encapsidation. The control systems for RV replication have not yet been studied. In the alphaviruses experimental evidence suggests that a complex containing the uncleaved 14 nonstructural proteins is instrumental in initiation of the negative strand intermediate. Later accumulation of the nonstructural proteins results in cleavage of the polyprotein by the viral protease, which acts in trans. The cleaved complex then switches from negative to positive strand synthesis (Shirako & Strauss, 1994; Lemm et al, 1994). Current work by Yao et al, (1997) indicates that the RV nonstructural protease also functions in trans and it is tempting to extrapolate a similar regulatory mechanism. In most virus systems infection is accompanied by inhibition of host protein synthesis. In the case of the alphaviruses this can begin as early as 3 hr after infection, beyond which time most of the translation in the cell is viral (reviewed in: Strauss & Strauss, 1994). One function of the viral glycoproteins appears to be involved in disruption of the Na+/K+ gradient in the cell; a phenomenon which happens at the same time as inhibition of host protein synthesis (Ulug & Bose, 1985; Ulug et al, 1984). The altered ionic balance may decrease the affinity of cellular initiation factors for host mRNA and confer an advantage on the alphaviral subgenomic mRNA which has been shown to bind more tightly to initiation factors like cap binding protein and e1 F-4B (Berben-Bloemheuvel et al, 1992). In RV-infected cells however, inhibition of host protein synthesis is minimal although a similar preference for viral translation occurs in hypertonic medium (Chantler, 1979). The lack of ability to switch off cellular translation may be one of the reasons rubella grows so poorly relative to many other viruses. While the alphaviruses routinely reach titres in cultured cells of 10 9 pfu/ml (10 3 pfu/cell), RV rarely exceeds 10 7 pfu/ml (30 pfu/cell) (Strauss & Strauss, 1986; Bardeletti et al, 1979; Vaheri et al, 1967). 15 A s s e m b l y Following synthesis of sufficient quantities of mature genomic RNA and processed structural proteins, the assembly of progeny virions begins. New virions are reported to assemble either at the cytoplasmic surface of vacuoles, the Golgi apparatus or, in some cell lines, at the inner surface of the plasma membrane . The virus particles then exit the cell primarily by budding at cytoplasmic membranes and subsequent exocytosis, or less frequently by budding directly from the plasma membrane (Von Bonsdorf & Vaheri, 1969; Bardeletti et al, 1979; Payment et al, 1975). Its apparent preference for budding at cytoplasmic membranes distinguishes rubella from the alphaviruses which bud only from the plasma membrane (reviewed in: Strauss & Strauss, 1985). Although the first progeny of a wild infection may be detected outside the cell by 12 hr the highest titres generally occur around 36-48 hr, sometime after the peak of RNA synthesis at 26-30 hr (Vaheri et al, 1965; Maes et al, 1966; Sedwick & Sokol, 1970; Hemphill et al, 1988). Rubella cytopathology may not be apparent until 3-5 days after inoculation in tissue culture and in many cases may not be visible at all. This is particularly true when the infective dosage is low (<1 pfu/cell) or if the strain has become attenuated through serial passages in culture. The factors which limit viral cell damage are probably those which allow rubella to establish a persistent infection in most of the cell lines tested (Rawls et al, 1968; Stanwick et al, 1974). This is in marked contrast to the alphaviruses which can release progeny as early as 3-4 hr post-infection and are characterised by a rapid, aggressive lytic infectious cycle with gross cytopathology by 6-8 hr(Sawicki et al, 1981). 16 M E D I C A L O V E R V I E W Rubella virus (RV) is the causative agent of "German measles", a common mild disease of childhood which is endemic worldwide. The major complication of rubella infection occurs during early pregnancy when transplacental passage of the virus results in the various manifestations of congenital rubella syndrome (CRS) including blindness, deafness, cardiac abnormalities and mental retardation (Wesselhoeft,:1947). The seriousness of CRS was underscored by the estimated 30,000 children born with rubella-associated defects in the USA alone following an epidemic in 1963.-4 (Cooper, 1975). ; ^ ' r.< The first vaccines, HPV77.DE5 and HPV77.DK12, were derived by serial passage of the wild M33 strain (Parkman et al, 1966; Hilleman et al, 1969; Parkman & Meyer, 1969). These strains were licensed but were withdrawn from medical use due to an unacceptably high risk of adverse reactions, particularly joint problems (Spruance et al, 1972; Thompson et al, 1973). The Cendehill vaccine strain was produced by serial passage of a wild isolate in rabbit kidney cells at reduced temperatures (32°C) (Peetermans & Huygelen, 1967) and was licensed for use both in Europe and North America. Although it had a very low association with adverse reactions it was recently replaced , in Europe, with the North American vaccine strain, RA27/3 which is reported to have superior immunogenicity (Best, 1991; Best et al, 1974, Plotkin & Baser, 1985). RA27/3 has been very effective in eliminating the incidence of congenital T u b e l l a syndrome but continues to be associated with acute and late onset neurologic and joint symptoms (Tingle et al, 1985). . 17 Rubella has been circumstantially linked to "diabetes in children with congenital rubella although this has not been studied sufficiently to know what role the virus plays in the disease. Rubella panencephalitis is a very severe late-onset consequence of infection. It is usually seen during adolescence in children with congenital rubella but can follow a natural infection. It is always fatal but has not been well studied as it is also very rare. Finally, rubella infection is believed to be the trigger for a wide range of rheumatic manifestations including rubella-associated arthritis (RAA) (Chantler et al, 1981, Chantler et al, 1982, Chantler et al, 1985) and fibromyalgia, and may also have a role in juvenile rheumatoid arthritis. The association of rubella with acute, transient joint manifestations was first described by Sir William Osier in 1905 and has since been widely documented (Osier, 1905; Heggie & Robbins, 1969; Cooper et al, 1969; Wolinsky, 1990). Rubella-associated arthritis (RAA) is usually short-lived and resolves completely; however, a number of patients go on to develop chronic and/or recurrent pauci- or polyarticular symptoms which can persist for many years (Chantler et al, 1981; Chantler et al, 1982; Tingle et al, 1986). It is a non-degenerative inflammatory disease and is usually treated with drugs rather than invasive techniques. It occurs predominantly in adult women which suggests a genetic or hormonal contributing factor. RAA is thought to be immune-mediated since the presentation of symptoms usually occurs 2-3 weeks after infection or vaccination, coinciding with induction of the immune response rather than the acute stage of viral infection. (Chantler et al, 1982). In addition to sex (F>M), other predisposing factors are age (>15) and a familial history of rheumatic or autoimmune disease (Ford et al, 1988). .18 Apart from its temporal relation to rubella infection the strongest diagnostic evidence for viral involvement in RAA is the observation that the frequency and severity of symptoms can be correlated with the infecting strain of rubella, wild strains being the most aggressive, and vaccine strains the least. A study performed by Dr. Aubrey t ingle (Tingle et al, 1986), estimated that up to 30% of women experienced recurrent arthropathy following infection with a wild strain of rubella and approximately 4% following vaccination with the RA27/3 strain (Fig.4.A). The pathologic mechanism of rubella-associated arthritis (RAA) is not presently known. Rubella is known to persist at low levels in peripheral blood lymphocytes and has also been isolated from synovial mononuclear cells both during the acute and chronic stages of disease (Ogra et al, 1975, Chantler et al, 1981, Chantler et al, 1982, Tingle et al, 1985, Chantler et al, 1993). Viral persistence in the joint and deposition of circulating immune complexes of virus and antibody in the joint have both been proposed as possible initiators of RAA (Phillips, 1986). There is presently no animal model for RAA; however, the frequency and intensity of clinical symptoms correlates directly with the ability of the infecting strain to propagate in human synovial organ culture in vitro (Fig.4B)(Miki & Chantler, 1992). Although rubella strains are genetically around 98% homologous they display striking phenotypic variation (Chantler et al, 1993). Wild strains, such as Therien, which have the highest association with arthritic sequelae (30%), commonly replicate to titres of 10 6 - 10 7 pfu/ml in human joint organ culture, comparable to yields from the most permissive cell lines for rubella (Fig 5.A). The vaccine strain RA27/3, which is associated with dramatically lower levels of recurrent arthritis (4%), is severely restricted in these cultures and does not attain titres greater than 10 2 pfu/ml. However RA27/3, 19 A. Rubella and RAA O 5 a. 100 80 60 40 20 Cend RA27 DE5 DK12 Wild Rubella Virus Strain B. Growth of RV in Joint 6 3 4 a. D) O Cend RA27 Therien Rubella Virus Strain Fig.4.A. The relative association of several strains of RV with acute and recurrent arthritis is shown using data from a study by Tingle et al (1986). The reports were collected from 1969-1985. The RV strains examined were: wild (mixture), HPV77/DK12 (vaccine), HPV77DE5 (vaccine), RA27/3 (vaccine) and Cendehill (vaccine). B. The ability of Therien (wild), RA27/3 (vaccine) and Cendehill (vaccine) to grow in human joint organ culture; constructed using data from Miki and Chantler, 1992 . as well as Therien, was found to persist in joint culture for the 3.5 months duration of the study. No replication was detected in these tissues using the former European vaccine strain, Cendehill, which is reported to have a very low association with acute arthritis and none with late-onset joint manifestations (Best et al, 1974). These results lend strength to the hypothesis that recurrent RAA might be triggered by reactivation of rubella virus which has established a persistent infection in the joint. In summary, the primary medical problem associated with rubella virus infection is congenital rubella syndrome. Although rubella teratogenicity has been largely abolished in North America and the European community in the wake of an aggressive campaign of vaccination, it continues to have devastating effects in many other nations with less well-developed health programs. More recently in the western world the medical focus of rubella research has been directed toward secondary complications of virus infection;including rubella-associated arthritis (RAA) and diabetes. Growth of Rubella Virus Strains in Vero and Synovial Cells 2 3 4 Days Post-Infection B S y n o v i a l C e l l s 0 I - 1 0 1 2 3 4 5 6 7 8 Days Post-Infection Fig.5. A. The growth of RV strains in Vero cells, one of the most permissive cell lines for the virus. B. Growth of RV strains in primary cultures of dissociated synovial cells. Figures were constructed using data from Miki and Chantler, 1992. 21 R A T I O N A L E The studies proposed are based on two separate lines of investigation which have been developed over the last few years. An infectious clone containing cDNA for the Therien strain of RV was recently constructed by Dr. T.K. Frey (Georgia State University, Atlanta, Ga.) (Dominguez et al, 1990; Wang et al, 1994; Pugachev et al, 1997a; 1997b). The use of infectious clones to map properties of RNA viruses was pioneered by Racaniello and Baltimore (1981) with poliovirus, another positive-stranded RNA virus. Following the conversion of poliovirus RNA to a complementary cDNA, these investigators then incorporated the viral sequences, as dsDNA, into plasmid vectors which allow molecular manipulations which were not readily available for RNA. They were then able to alter the viral genome in a controlled manner and observe the effect/s by transfecting the plasmid-derived transcripts into mammalian cells, giving rise to progeny in a manner similar to that of the parental virus. Since this early work with poliovirus, infectious clone technology has been used to map properties like cell tropism, temperature sensitivity and attenuation in a large number of other RNA viruses including some of the alphaviruses (Bouchard et al, 1995; Polo & Johnston, 1990). This opened up the possibility of undertaking genetic manipulation of the RV genome in order to be able to map phenotypic characteristics to specific genes. A second line of experiments carried out in our laboratory showed that the ability of an RV strain to replicate in human joint culture paralleled its association with joint complications. Increased frequency and severity of joint symptoms were correlated with increased virus titres in vitro, wild strains being the most aggressive (Miki & Chantler, 1992). The attenuated Cendehill vaccine strain was not able to grow in human joint organ culture and was not associated with arthritic sequelae. A similar 22 growth trend was observed in cultures of dissociated synovial cells (SC). In these primary cultures Cendehill appeared to be able to replicate at a very low level but was still highly restricted, never reaching titres greater than 10 2 pfu/ml. This is at least 100 fold lower than RA27/3 in this system and 10 3-10 4 fold lower than the Therien strain (Fig 5.B). Therefore, although restriction of Cendehill was less stringent in dissociated SC, these cells should still provide a valid model for growth in joint and they have the advantage of being easier to manipulate and providing greater consistency for comparative experimentation than primary organ cultures. Moreover, in the absence of an animal model system, the properties which allow growth in human synovial cells might provide a satisfactory model to study the arthritogenicity of rubella virus. The rationale of the work reported was therefore based on the fact that a phenotypic and genetic comparison of the highly attenuated Cendehill strain with a wild strain which grows well in joint and is linked to a relatively high incidence of subsequent arthritis could provide information about the pathogenesis of RAA. Although the individual wild strains included in the RAA studies have not been typed, the virus of choice for such a comparative study was naturally the well-characterised Therien strain, which grows to high titre in joint tissue (Miki et al, 1992) and for which an infectious clone was available. This strain was to be compared with the non-arthrotropic Cendehill strain. The current study was, therefore, designed to examine the ability of each of these strains to grow in human joint cell culture under a variety of conditions in order to address which stage(s) of the infectious cycle was involved in the growth restriction of the vaccine strain. In conjunction with this, chimeric strains comprising part-Therien and part Cendehill were to be produced and the phenotypic properties of 23 these compared with the parental strains in order to map properties of interest on the genome. Finally by completing construction of an infectious clone of the Cendehill strain and comparing its sequence with the published wt+ sequences of Therien and M33 strains, we hoped to identify nucleotide changes in Cendehill associated with attenuating features including joint cell growth restriction. 24 M A T E R I A L S A N D M E T H O D S 1. S u p p l i e r s ABI (Applied Biosystems (Canada) Inc., Mississauga, Ontario, Canada American Can Co., Greenwich, Connecticut, USA American Type Culture Collection, Rockville, Maryland, USA BioRad Laboratories, Hercules, California, USA Boehringer Mannheim (Canada), Laval, Quebec, Canada GibcoBRL, Life Technologies, Gaithersburg, Maryland, USA ICN (ICN Pharmaceuticals Canada Ltd., Montreal, Quebec, Canada Millipore Corp. (Waters Chromatography), Marlborough, Massachusetts, USA Qiagen Inc., Chatsworth, California, USA Sigma Chemical Co., St. Louis, Missouri, USA USB (United States Biochemical Corp.), Cleveland, Ohio, USA Vector Laboratories, Burlingame, California, USA 2. P R O T O C O L S 2.1 C e l l s , V i r u s e s a n d B a c t e r i a The Therien strain of rubella virus is derived from a wild strain isolated in the United States by A. Schluederberg (Oker-Blom et al, 1983). The Cendehill vaccine strain was originally obtained from Rohm Pharma. Vero (African green monkey kidney) and BHK (baby hamster kidney) cells were obtained from the American Type Culture Collection. Primary cultures of fetal human synovial cells were prepared from autopsy material. WM1100 electrocompetent Escherichia coli cells were purchased from Bio Rad. 2.2 C e l l a n d V i r u s C u l t u r e Vero cells were cultured in Medium 199 (M199 - GibcoBRL) supplemented with 10% fetal bovine serum (FBS). BHK cells were grown in DMEM-F12 supplemented with 10% FBS. Primary human synovial cells were cultured in RPMI-1640 supplemented with 10% FBS, 1 % gentamicin. 25 In all cell lines, virus was cultured by washing the monolayer with PBS and allowing inoculating virus to adsorb for 2 hr @ 35°C. Following adsorption the virus inoculum was removed and the cells were washed and overlayed with medium, as described above, but containing 5% HIFBS, 1 % gentamicin. To prepare stock virus, this medium was replaced at 2 days PI, or at the first visible sign of cytopathology and the fresh supernatant collected after 24 hr, clarified by low speed centrifugation, and stored in aliquots at -70°C. In the case of strain Therien R the virus supernatant was collected 18 hr PI, since a gross cytopathic effect (CPE) was observed in almost 100% of the cells at this time. All cell cultures (infected or uninfected) were grown in humidified incubators at 35°C in an atmosphere of 5% C O 2 . 2.3 P r i m a r y C u l t u r e of S y n o v i a l C e l l s Human fetal knee joints were obtained from Vancouver General Hospital after autopsy. Joints were stored in transport medium at 4°C for a maximum of 6 hours. Synovial cell (SC) cultures were prepared as previously described (Miki & Chantler, 1992). Sections extending approximately 2 mm to either side of the joint were excised following removal of the surrounding tissue and kneecap. The sections were minced into 1 mm pieces and cultured overnight in RPMI 1640, 10% FBS, 1 % gentamicin. The matrix was disrupted by incubation in 1 % collagenase (Boehringer Mannheim) for 4 hr @ 37°C, 5% C 0 2 . Cell material was pelleted at 3,000 x g (1000 RPM, IEC Centra) for 10 min and the collagenase mixture was removed by aspiration. Cells were dissociated by 3 x 15 min treatments in 0.25% trypsin, 0.2% EDTA. Following each digest the enzyme mix was transferred to 4 volumes of RPMI 1640, 10% FBS to inactivate the trypsin. The cell suspension was mixed and allowed to settle briefly to 26 remove large, visible debris. Single cells were collected by pelleting as described above and were .cultured in RPMI 1640, 10% FBS, 1 % gentamicin (GibcoBRL). (Note: Aspiration of medium from the pellet results in approximately 3 fold greater recovery than removal by decanting, as the pellet is fragile.) SCs were passaged by trypsinisation once a week when necessary and were not used later than the fourth passage. 2.4 P l a q u e T i t ra t ion of V i r u s Virus titers were determined by plaque assay as described by Fogel & Plotkin (1969). Serial ten fold dilutions of virus were prepared in M199, 5% FBS. One ml of each dilution was inoculated onto subconfluent Vero cell monolayers in 35 x 10 mm petri.dishes and "allowed to adsorb for 2 hr. The inoculum was removed by aspiration and the monolayers were overlaid with 2 ml/dish of M199, 5% FBS, 0.5% agarose (SeaKem ME, w/v in ddH 2 0) . Six days following inoculation the monolayers were examined for visible plaques or foci and were overlaid with 2 ml of M199/agarose containing neutral red dye (1:8000 w/v) and observed for clearings after 24 hours. 2.5 P l a q u e P u r i f i c a t i o n of V i r u s Virus was titrated as described and observed 5 days post-inoculation for visible clearings in the monolayer. Cores of agarose overlying individual foci were removed by aspiration using a 1.0 ml Gilson micropipettor with a disposable tip which had been severed to give an inner bore of approximately 2mm. The agarose plugs were ejected into petri dishes (60 x 15 mm) containing subconfluent Vero cell monolayers in 3 ml of virus propagation medium. After 4 days the cells were trypsinised and split 1:2. 27 Supernatants from these cells were used to prepare virus stocks; final working stocks were harvested and frozen in aliquots at 3 passages after inoculation of the agarose plug. 2.6 V i r u s C o n c e n t r a t i o n a) Virus supernatants were collected and pelleted by ultracentrifugation for 4 hr at 80,000 x g (18,000rpm, Beckman L870 M, SW27 rotor), 4°C. b) Virus supernatants were collected and layered onto 25% sucrose/PBS. Virions were pelleted through the sucrose cushion by ultracentrifugation for 2 hr at 150,000 x g (25,000rpm), 4°C. After centrifugation the layer above the cushion was removed by aspiration, and the remainder poured away. Tubes were drained by inversion for 5 min to minimise the amount of sucrose retained. 2.7 C o m p a r a t i v e B i n d i n g A s s a y Stocks of Therien, Therien R, Cendehill and CendehillR, grown in Vero cells, were labelled overnight with 30uCi/ml Trans 3 5S-cysteine/methionine (ICN) in RPMI 1640 (Sigma), 5% HIFBS deficient in cysteine and methionine. Virions were purified by pelleting through 25% sucrose, then resuspended in M199, 5% HIFBS. Controls were prepared using supernatant from uninfected Vero cells which was labelled and pelleted through sucrose in an identical manner. The stocks were adjusted to pH 7.2 by the addition of 0.1 volumes of 500mM Hepes (Sigma) buffer. Lightly confluent monolayers of either Vero or SC cells in 1.9 cm 2 wells (24-well plates) were inoculated in triplicate with 80ul of each stock and allowed to equilibrate at 5% C 0 2 for 5 min, then sealed with Parafilm (American) and incubated a further hour at 25°C with gentle agitation. The 28 cells were washed by filling each well twice with ice-cold PBS, then solubilised in 0.5 ml of 0.5% SDS/H 2 0. Each aliquot was suspended in 9 volumes of Cytoscint (ICN) and counted by liquid scintillation using a Beckman LS6000IC counter. An average value for each set of triplicates was used to determine the amount of SC/Vero binding. 2.8 T r a n s f e c t i o n Electroporation: Cells were electroporated following the general procedure outlined in Liljestrom et al (1991). A Bio Rad Gene Pulsar™ with Pulse Controller was used to shock the cells, using Bio Rad electoporation cuvettes with a 0.2mm electrode gap. 1. BHK: Subconfluent monolayers of BHK cells were trypsinised and washed, first with ice cold DMEM-F12, 5% HIFBS to inactivate the trypsin, then with ice cold PBS. The cells were resuspended in ice cold PBS to a density of 1 x 10 7 cells/ml. The cells were mixed with RNA in 1.5 ml Eppendorf tubes on ice. The mixture was transferred to pre-cooled electroporation cuvettes (0.45ml/cuvette) and subjected to two pulses at 1.5kV, 25uFD with the pulse controller set at infinity (time constant approximately 0.7msec). Following the pulse the cells were allowed to recover at room temperature for 10 minutes prior to the addition of 1ml of DMEM-F12, 5% HIFBS. The cell/medium mixture was then added to 60 x 35 mm petri dishes containing 4ml of the same medium. Twenty-four hours after electroporation the medium was replaced, following a gentle wash with PBS. >. 2. Synovial Cells: Synovial cells were trypsinised and pulsed in the same general manner as described for BHK cells but using a pulse of 0.75kV. Following 29 electroporation and recovery the cells were mixed with 1ml of RPMI 1640, 5% HIFBS and then added to petri dishes containing 3ml of the same medium and 1ml of conditioned medium (medium harvested from SC cultures after 4 days growth). Medium replacement and washing were carried out 48 hours after electroporation. 3. E. coli: Electrocompetent WM1100 cells were electroporated using a modification of the method of Dower et al (1988) according to the procedure outlined in the Bio Rad product description. Plasmid DNA (1-5 ul suspended in ddH 2 0) was mixed with 40 ul of WM1100 cells on ice. The mixture was pulsed once using 0.1 cm cuvettes, 1.8kV, 25uF, with the pulse controller set at 200 (time constant approximately 4.5 msec). One ml of SOC medium was added immediately following the pulse and the cells were incubated for 1 hr at 37°C prior to being spread on LB plates. Lipofectamine: RNA was diluted in 100 ul of M199 without serum or antibiotics. This was mixed with 10 ul of Lipofectamine™ which had been similarly diluted and allowed to incubate 30 min at room temperature. This mixture was then added to 0.8 ml of the same medium, layered onto 80% confluent Vero cells in 60 x 15 mm petri dishes and allowed to incubate 6 hr at 37°C. After addition of 3.0 ml of virus propagating medium (without antibiotic) the cells were incubated a further 18 hr, at which time the medium was replaced. After 4 days samples of the supernatant were removed for plaque titration. DEAE Dextran: RNA was diluted in 1.0 ml M199 containing 0.25 mg/ml DEAE dextran (Sigma) and 0.1 M Tris-HCI (pH 7.5) and incubated with 80% confluent Vero cells for 18 hr at 37°C. The cells were rinsed with tris-buffered saline (TBS) before and after treatment with transfection medium with TBS. After removal of the dextran mixture the cells were treated with 10% DMSO in TBS for 3 min at RT, then washed 3 x 30 with TBS and incubated a further 4 days in virus propagating medium prior to sampling for plaque titration. 2.9 P l a s m i d Iso la t ion a n d P u r i f i c a t i o n Large-Scale Plasmid Preparation: Plasmids were isolated by alkaline lysis (Birnboim & Doly, 1979) and purified by polyethylene, glycol precipitation Sambrook et al, 1989). All centrifugation steps were carried out using a Sorvall centrifuge at 4°C, unless otherwise noted. Bacterial cultures containing plasmids were inoculated into 250 ml of 2 X yeast/tryptone (2 X YT) broth containing the appropriate antibiotic for selection and incubated overnight at 37°C with shaking. Cultures were harvested by pelleting for 10 min at 10,000 x g (6000 RPM, Sorval centrifuge, GSA rotor). The bacterial pellets were resuspended in 5 ml of Solution 1 containing 5mg/ml lysozyme and incubated at 25°C for 10 min. 10 ml of Solution 2, containing 0.2N sodium hydroxide and 1 % (w:v) sodium dodecyl sulfate was added and the mixture was incubated at 4°C for 10 min. Following lysis, the preparation was neutralised by the addition of 7.5 ml of Solution 3 and incubated for 20 min at 4°C. Genomic DNA and cell debris was removed by centrifugation at 25,000 x g (10,000 RPM), 20 min, 4°C. Nucleic acid was collected from the supernatant by precipitation with 0.6 volumes of isopropanol and centrifugation at 25,000 x g (10,000 RPM), 10 min, 4°C. The pellets were transferred to 30 ml Corex centrifuge tubes and washed with ice-cold 70% ethanol. After washing, the pellets were solubilised by gentle agitation at 25°C in 3 ml tris-EDTA (TE) buffer. The solution was incubated for 10 min at 4°C after addition of one volume of 5M lithium chloride and centrifugation at 20,000 x g (10,000 RPM, SS-34 31 rotor) for 10 min. Nucleic acid was precipitated from the supernatant with an equal volume of isopropanol and washed with ice-cold 70% ethanol as described. Samples , which were not to be used for transcription were treated with 1900U of RNase T1 (1 ul, GibcoBRL) for 30 min at 25°C to digest low molecular weight RNA. Plasmid DNA was precipitated by incubation for 10 min at 25°C with an equal volume of 13% polyethylene glycol 8000, 1.6M sodium chloride and pelleted by centrifugation at 8,000 x g (10,000 RPM, Eppendorf Microfuge) for 10 min. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) to remove protein contaminants. The plasmid DNA was precipitated a final time by addition of 0.1 volumes of 3M sodium acetate and 2 volumes of 100% ethanol. Precipitates were generally visible immediately and were collected by centrifugation at 25,000 x g (13,000 RPM, Eppendorf Microfuge) for 10 min. Plasmid preparations were resuspended in 0.1 x TE buffer for storage and quantitated by visualisation on agarose gels and by spectrophotometry. A ratio of absorbance at 260nm/280nm of 1.8 or higher was considered acceptable for RNase-treated samples. Small-Scale Plasmid Preparation: Several mis of bacterial culture were grown overnight in capped test tubes with agitation. Cells were pelleted from 1.0 ml aliquots of culture fluid in Eppendorf centrifuge tubes at 25,000 X g (13,000 RPM, Eppendorf Microfuge) for 20-30 sec. The cells were resuspended in 100 ul of Solution 1, followed by addition of 200 ul of Solution 2 and 150 ul of Solution 3. The mixtures were incubated on ice for 4.0 min and centrifuged for 5 min as described to remove cell debris. Isopropanol (v/v) was used to precipitate the plasmid DNA which was washed with 70% ethanol and resuspended in 20 ul of ddH 2 0. 32 2.10 L i g a t i o n Ligations were carried out at 15°C for 20 hr using T4 DNA ligase; 1 U/ug (BRL). The 5 X ligase buffer supplied by the manufacturer contained: 250mM Tris-HCI (pH 7.6), 50mM MgCI 2 l 5mM ATP, 5mM DTT, 25% (w/v) polyethylene glycol-8000. 2.11 N o r t h e r n / S o u t h e r n B lo t RNA samples were prepared in 53% DMSO, 17% glyoxal and 10mM N a P 0 4 pH 6.5. After heating for 10 min at 55°C each sample received 0.1 volumes of RNA loading buffer and was electrophoresed (65V) through 1 % agarose in 10mM N a P 0 4 in recirculating 10mM NaP0 4 buffer. All buffers were prepared using diethylpyrocarbonate (DEPC)-treated H 2 0 . Following electrophoresis 20 X SSC was used to effect overnight capillary transfer of the RNA to a Magnagraph nylon membrane (Micron Separations Inc.) by standard procedures. The membranes were baked at 80°C for 2 hr and incubated with prehybridisation buffer for at least 2 hr at 65°C with rocking. 25ul of 3 2P-labelled probe was diluted 1:1 with formamide and added to the prehybridisation mix and the membranes were incubated a further 18hr. The membranes were washed twice with 1 x SSC, 0 . 1 % SDS, then once with 0.1 x SSC, 0 . 1 % SDS for 30 min each (65°C). After drying the hybridised membranes were exposed to X-ray film overnight. Probe Preparation: 3 2P-labelled probe was prepared by nick translation using DNase 1 and DNA Pol 1 with cDNA from the Therien infectious clone, pROBO302, as template. The probe was labelled with 2uM alpha 3 2 P . The reaction was allowed to proceed for 1 hr at 15°C. 33 2.12 I n fec t ious C l o n e C o n s t r u c t i o n RNA isolated from mature virions was used as a template for reverse transcription, primed with specific oligonucleotides, to generate a complementary first strand of DNA. Double-stranded cDNA was made by thermal cycling amplification (Minicycler™, MJ Research) of the first strands using specific primers. To confirm insertion of the correct fragments, the sequence of each infectious clone was compared over several regions with that of Cendehill cDNA which was sequenced directly following reverse transcription and amplification. Isolation of Viral RNA: Cendehill virions were obtained by pelleting virus supernatant for 4 hr @ 18000 rpm as described above. Virions were solubilised in 20-100 ul of 1 % SDS and viral RNA was isolated by extraction with 1-2 ml of acidified phenol/guanidinium isothiocyanate (Chomczynski & Sacchi, 1987) using Trizol™ (GibcoBRL) according to the manufacturer's instructions. RNA was precipitated from the aqueous phase by the addition of isopropyl alcohol (1:1) and washed with 75% ethanol diluted in DEPC-treated H 2 0 . prior to drying and resuspension in 20-50 ul DEPC-treated ddH 20. Reverse Transcription: Specific primers complementary to the published sequence of the Therien strain (Dominguez et al, 1990) were-used to initiate the first strand of DNA synthesis. The primers used were #16, 38 and 125 (Fig.6/Table 1). A mixture of primers and viral RNA in ddH 2 0 (total volume 11 ul) was heated for 3 min @ 90°C. RNA was then transcribed using 200U of Superscript II (Life Technologies) with the addition of 1 % DMSO, for 1 hr @ 42°C. To increase the amount of first strand DNA, after 1 hr the mixture was re-heated to 90°C for 3 min, allowed to cool to 42°C and incubated a further hour with the addition of 200U of fresh enzyme. The standard reaction mixture contained 10mM dithiothreitol and 1mM dNTPs. The volume was brought to 10Oul by addition of TE buffer and heated to 90°C to inactivate the reverse transcriptase. Enzyme, primers and excess nucleotides were removed by extraction of the mixture with 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1 ,v:v:v), followed by precipitation at -20°C in 0.3M sodium acetate and 66% ethanol. Precipitation was facilitated by the addition of 20ug of glycogen (Boehringer Mannheim). C e n d e h i l l (and RV) c D N A s 36 1 37 1 Hindlll, SP6 F2 Pvul Nhel Bgl l 125 36 2 38 2 _ EcoRI F1 Fig.6. The cDNAs used to construct the Therien/Cendehill chimeric viruses and the Cendehill infectious clone are shown underneath a schematic of RV genome. The location of the restriction sites used for construction is given by dotted lines. Primers used to generate each cDNA are noted at the termini. The cDNAs made from TherienR and M33R for sequence analysis are designated by 1 and 2, respectively. 35 Thermal Cycling Amplification: After generation of the first strand of DNA by reverse transcription, the products were amplified using specific primers (Fig.6/Table 1) and repeated cycles of incubation with Deep Vent™ (NEB) thermostable polymerase with 3'-5' proof-reading exonuclease activity. The standard reaction mixture contained 400uM dNTP, 2mM MgS0 4 , 0.5uM primer and 1U of polymerase. After cycling unincorporated dNTP, proteins and oil were removed using a Qlquick Spin Column (Qiagen). The products were resuspended in ddH 20. If removal of primers was required the cDNA was further purified by agarose gel electrophoresis. pCL1921: pCL1921 is a plasmid vector with spectinomycin resistance constructed by Lerner and Inouye (1990) but which contains the polycloning site of pUC19 (Yanisch-Perron et al, 1985) in place of the polylinker region of pGB2 (Churchward et al, 1984). pCLPC: pCLPC is a plasmid vector constructed by inserting a polycloning site containing restriction sites found in Therien strain cDNA, into the pCL1921 vector to facilitate subcloning of rubella virus cDNAs. The oligonucleotide 5'-GAT CAC CGG TAC GCG TAG ATC TAT CGA TCA ATT GGC GCG CCG CTA GCG-3', was constructed, as well as the largely complementary oligonucleotide 5'-GAT CCG CTA GCG GCG CGC CAA TTG ATC GAT AG A TCT ACG CGT ACC GGT-3'. The two were annealed to produce a dsDNA containing restriction sites for Agel, Mlul Bgl II, Clal, Muni, AscI, Nhel and overhanging ends compatible with BamHI-digested material (one of the terminal BamHI sites was destroyed by altering the terminal sequence from GATCC to GATCA). This polycloning segment was ligated with pCL1921 which had been digested with BamHI. The resulting plasmid was sequenced manually using the 36 dideoxy termination method, and analysed by restriction enzyme digestion and agarose gel electrophoresis to confirm insertion of the polycloning site in the orientation: Hindlll, Pstl, Sal I, Xbal, BamHI, Nhel, AscI, Muni, Clal, Bql II, Mlul. Aqel. Smal, Kpnl, Sad, EcoRI. Ol igonuc leot ide Pr imers RV 3' end complement F1 5'-CGCGAATTCTTTTTTTTTTTTTTTTTTTTCTATACAGCAACAGGTGC-3' EcoRI — > . RV 5'start F2 5'-TCGAAGCTTATTTAGGTGACACTATAGCAATGGAAGCTATCGGACCTCGCTTAGG-3' Hindi 11 SP6 9 5TGCAGCGTTCGACGCAAACG-3' 2133-2153 10 5'-TCCGAGTGCCGTTGCGATC-3' 2243-2262 16 5'-GCGTTCTTGATGTCGATATCGCG-3' 4410-4431 18 5'-CTCACTGATGTCTACACGCAGATG-3' 5281-5763 36 1 2 5'-CGCTGTTCGCCCTTGCTAGCT-3' 8676-8697 371 5'-ACTGCTGATTGCCGGTGTAAT-3' 8880-8901 RV 3' end complement 382 5'-GGAATTCCACTAGTTTTTTTTTTTCTATACAGCAAC-3' 46 5'-CAACCACCTCGGGAATGC-3' 3241-3260 125 5'-TAGTCTTCGGCGCTTGG-3' 5747-5763 251 5'-TTTGCCAACGCCACGGC-3' 2603-2618 Table.1 Oligonucleotides used to prime first strand synthesis and thermal cycling amplification are shown with their nucleotide location in the RV genome. The primers used to amplify TherienR are designated by 1, those used to amplify M33R are designated by 2 (Sec.3.1.6). All primers, including those complementary to the plus-strand equivalent, are shown in the 5'-3' orientation. 37 pROBO302: The first Therien clone used in our experiments was pROBO102 which was constructed by Wang et al (1994) using cDNAs generated by reverse transcription and amplified with Taq™ polymerase. This clone was found to be infectious at a frequency of only 4 pfu/10 ug of RNA transcript, which indicates that infectivity was arising as a result of sporadic mutation of pROBO102, and that the clone itself likely contained deleterious mutations and was not infectious. This clone was subsequently repaired by Pugachev et al (1997) by replacing most of the Therien cDNA with corresponding fragments made using the proof-reading polymerase, ExTak™ (Takara Shuzo Co., Ltd). This new clone, pROBO302, was infectious at 10 4 pfu/ug and was used in all experiments to prepare the Cendehill/Therien chimeric viruses. A diagramatic representation of pROBO302 is given in Fig.7. pROBO302 is constructed with a HindllI restriction site and an SP6 RNA polymerase promoter upstream of the negative-strand complement of the 5'-terminus of the RV genome. An EcoRl restriction site was added to the 3' terminus to facilitate cloning into the low-copy vector pCL1921. 2246 2803 Pvul Nhel l _ / HinDl 11 13935 Fig.7 The Therien infectious clone, pROBO302, was constructed using the plasmid pCL1921 as a propagation vector. pCL1921 is replicated at approximately 5 copies/cell and carries resistance genes for spectinomycin. An SP6 RNA polymerase promoter was inserted to allow transcription of full-length, genome (+)-sense RNA. • PCL1921 IT! Therien cDNA EcoRl 9782 38 pROC3: (Fig. 11 .A) Oligonucleotide 38 was used to prime reverse transcription of Cendehill RNA (described above.). The first strand was then amplified by thermal cycling using oligonucleotide 18 as the forward, and F1 as the reverse primers. F1 contained an T 2o tract and additional nucleotides for an EcoRI restriction site downstream of the viral sequence. The buffer was adjusted to 4mM Mg 2 + . Cycling conditions were: 98°C - 30 sec; 60°C - 30 sec; 75°C - 30 sec; 80°C - 4 min, 10 sec (34 cycles). The final product was a 4478 bp cDNA homologous to the Cendehill genomic RNA from nt 5281 to the 3' end of the Cendehill genomic RNA and also contained an EcoRI site downstream of the terminal poly A sequence to facilitate cloning. The fragment was digested with EcoRI and Bglll (nt 5355) and ligated into pROBO302 which had been similarly digested. The products were transformed into WM1100 cells to yield an infectious clone in which the Therien cDNA had been replaced by the analogous Cendehill sequence from nt 5355 to the 3' end. pROC3M: (Fig. 11 .B) Oligonucleotide 125 was used to prime reverse transcription. The first strand was then amplified by thermal cycling using oligonucleotide 251 as the forward, and 125 as the reverse primers. The buffer was adjusted to 3mM Mg 2 + . Cycling conditions were: 98°C - 30 sec; 52°C - 30 sec, 72°C -2.5 min (34 cycles). The product was a 3160 bp cDNA homologous to nt 2603 - 5765 of the Cendehill genomic RNA which was digested with Nhel (nt 2803) and Bgl II (nt 5355). Since the yield was low (approx 100ng prior to gel purification) the digested cDNA was ligated into the cloning vector pSUPC and transformed into WM100 cells by 39 electroporation. A mixture of cDNA from 4 clones, pSUM (1-4), with appropriate molecular weights was digested with Nhel and Bgl II for insertion into pROC3. Since pROC3 contains 2 sites for Nhe I (nt 2803 and nt 8690), an appropriate deletion was created by digesting completely with Bgl II then partially with Nhel to obtain the desired 11448 bp vector. These 2 fragments were ligated and electroporated into WM1100 cells to yield an infectious clone in which the Therien cDNA had been replaced by the analogous Cendehill sequence from nt 2805 to the 3' end. pJCND: (Fig. 11 .C) Oligonucleotide 16 was used to prime reverse transcription. Two fragments were amplified separately from the product of the first strand reaction. Oligonucleotides F2 (forward) and 10 (reverse) were used to amplify a fragment from the 5' end to nt 2262. A Hindlll restriction, and SP6 RNA polymerase promoter sequence were included in primer F2 upstream of the viral sequences. The buffer was adjusted to 3mM Mg 2 + . Cycling conditions were: 98°C - 30 sec, 50°C - 1 0 sec, 75°C -30 sec, 80°C - 2 min (34 cycles). Oligonucleotides 9 (forward) and 46 (reverse) were used to amplify a second fragment from nt 2133 - 3260. The cycling conditions were: 98°C - 30 sec, 54°C - 30 sec, 78°C -1.5 min (34 cycles). These two cDNAs were restricted with Pvul, which cuts at nt 2246 (and 9475). Fragment 9 - 46 was dephosphorylated to inhibit self-annealing and the two were ligated overnight at 15°C using approximately 1.5 ug of each (2 fold molar excess of 9 - 4 6 ) . Following ligation the mixture was digested with Hindlll and Nhel and purified from the gel. The fragment was ligated overnight at 15°C into pCLPC which had been digested with Hindlll and Nhel and gel purified to remove the intervening segment of the polycloning site. Following transformation by electroporation into WM1100 cells an appropriate clone, pCL5', was selected by screening using digestion with restriction enzymes. 40 Cloning Strategy for Cendehil l /Therien Chimeras and Cendehi l l Infectious C lone Cendehill RNA Bgl11/EcoR1 I' ds-cDNA C 1 Nhe1/Bgl11 2803 5355 Pvul I I 1 2246 I I 2246 3260 Hindi 11/Nhe 1 2803 • 5355 9 7 6 2 / pCL1921 pROBO302 Therien cDNA 1 pROC3 2 pROC3M ' i 3 pJCND 'j,:,:,,,1 L Fig.8 Cendehill RNA is converted by reverse transcription and thermal cycling amplification into ds-cDI The cDNAs are cut using using appropriate restriction enzymes and inserted sequentially into the analc region of the Therien infectious clone (pROBO302) which have been similarly restricted. 41 Since pR0C3M contains two Nhel sites (nt 2803 and nt 8690), to prepare for insertion of the 5'-terminal Cendehill cDNA, it was first digested with Hindlll and Bglll to create a deletion from nt 1 - 5357 of the pROC3M sequence. The clone carrying the deletion was then ligated 4 hr at 22°C with the fragment 2803-5357 isolated from a second pROC3M digest using Nhel and Bgl II to produce a version of pROC3M carrying a deletion from nt 1 - 2803. The ligation mixture was digested with Hindlll to reduce self-annealing. The desired 2827 bp product was ligated overnight at 15°C with the Hindlll - Nhel fragment purified from pCL5' and transformed into WM 1100 cells by electroporation to yield the Cendehill infectious clone pJCND in which all of the Therien cDNA had been replaced by corresponding Cendehill sequences. Construct Screening: Each potential infectious clone was screened for infectivity. Smail-scale plasmid preparations were precipitated with an equal volume of 5M potassium acetate. Nucleic acid was precipitated from the supernatant by addition of an equal volume of isopropanol, washed with 70% ethanol and resuspended in 20ul of ddH 2 0. The partially purified preparations were linearised by restriction digestion with EcoRl at the 3' terminus of the viral sequence. Positive-polarity viral RNA was generated by transcription from the SP6 promoter and the products were transfected into BHK21 cells by electroporation. After 2 days the supernatants were transferred,to Vero cells and samples removed for plaque titration 4 days later. 2.13 V i r u s G r o w t h A s s a y Subconfluent Vero cells in 9.5 cm 2 dishes were inoculated with 4 x 10 5 pfu of virus (approx. 0.1 pfu/cell). After 1 hr adsorption at 35°C, the cells were washed twice with 2 x 5 ml PBS and supplemented with 5 ml fresh propagating medium. At various 42 times 500 ul aliquots were removed (and replaced) and stored at -70°C pending plaque titration. Results were compiled from 2 separate experiments with staggered sampling points; one beginning at 4 hr P1 and one beginning at 12 hr P1. Zero time samples were taken immediately following the post-adsorption washes from both experiments for baseline determination. 2.14 Int race l luar v e r s u s E x t r a c e l l u l a r V i r u s G r o w t h Infection: Subconfluent cells were infected as described for the Virus Growth Assay. At 4 days PI the medium was harvested and stored at -70°C. The cells were washed with 2 x 5 ml of PBS. One ml of fresh virus propagating medium was layered onto the cells and the petri-dishes were sealed and frozen in a horizontal position at -70°C to fracture the cells allowing release of intracellular virus. Prior to plaque titration the frozen cells were centrifuged for 2 min at 25,000 x g (13,000 rpm, Eppendorf Microfuge) to remove debris. Electroporation: Following electroporation of viral or plasmid-derived RNA, SCs were propagated for 6 days. The cells and supernatants were harvested as described above for plaque titration. 2.15 W e s t e r n B l o t , I m m u n o p r e c i p i t a t i o n a n d S D S P a g e Virus samples were partially purified by sedimentation of supernatants from infected cells (about 20 ml) through 25% sucrose as described above. The pellets were resuspended in solubilisation buffer containing 2% SDS, 5% beta-mercaptoethanol and heated at 100°C for 3 min. The proteins were separated by electrophoresis through 43 discontinuous 10% polyacrylamide gels containing 0.1%SDS at 32mA for 2.5 hr (Laemmli, 1970). Proteins were transferred to Immobilon PVDF membrane (Millipore) using a sodium carbonate buffer system containing 20% methanol (Dunn, 1986). Transfer was carried out at 50mA for 30 min, followed by 300mA for 3 hr in a water-cooled apparatus. PVDF membranes were blocked with 3% gelatin (40°C) and incubated with a 1 :-100 -1:250 dilution of antiserum to different RV strains(dilutions varied with antibody preparations) for 1.hr at RT with gentle agitation. The bound-antibodies were detected using the Vectastain ABC kit (Vector Laboratories) with 4-chloro-1-naphthol as substrate. The immobilised proteins were visualised using polyclonal antiserum to Therien or Cendehill strains. The antisera (Therien, M33, HPV77, Cendehill) were produced in rabbits by inoculation of virus preparations recovered by sedimentation from supernatants of virus-infected Vero cells. Two injections were given at 1 month intervals and the rabbits were bled 10 days later for collection of antiserum. The antibody fraction was precipitated with saturated NH4(SO)4 and the IgG fraction collected after column filtration using DEAE Sepharose (Pharmacia). Immunoprecipitation experiments were carried out using supernatant virus which had been labelled O/N with 3 5S-cys/met (25 uCi/ml) and concentrated by pelleting from 10% polyethylene glycol (PEG) 3500 for 10 min at 10,000 x g (6000 RPM, Sorvall centrifuge, GSA rotor). Labelled virions were resuspended in 1.0 ml of virus propagating medium (10% PEG) and re-pelleted in an Eppendorf microfuge to facilitate removal of PEG. The virions were then resuspended in 1.0 ml 20mM Tris, 150 mM NaCl, 1 % NP40 (pH 7.6. Labelled virus was incubated with polyclonal rabbit aniserum 44 to RV for 1 hr at 4°C. Immune complexes were precipitated by incubation with Pansorbin (Calbiochem) for 30 min at 4°C. The precipitate was collected by pelleting for 3 min at 25,000 x g (13,000 RPM, Eppendorf microcentrifuge). Precipitates were washed 3 times in 1.0 ml of resuspension buffer, solubilised in SDS PAGE sample buffer and separated by PAGE as described above. Gels were dried and exposed to X-ray film for approximately 1 week at-70°C. 2.16 G r o w t h at 39°C The ability of the virus to grow at elevated temperatures was examined using the procedure outlined above for plaque titration of virus, but with the following changes: 1) The adsorption period was reduced to 1 hr. 2) Immediately following addition of the agarose overlay the cells were transferred to 39°C. 2.17 V i r u s D e c a y Virus stocks, in virus propagation medium, were adjusted to pH 7.2 by addition of 50 ul 0.5M Hepes buffer (Sigma) and were diluted to approximately 10 5 pfu/ml. The aliquots were placed at 35°C, 5% C 0 2 and sampled periodically to determine the concentration of viable virus by plaque titration. Zero time values were established using samples taken 10 min after C 0 2 equilibration and were normalised to 1 x 105 pfu/ml. 2.18 C o - i n f e c t i o n Subconfluent Vero cells were infected with 1 pfu/cell of Therien. After 24 or 48 hrs the cells were washed and inoculated with 0.1 pfu/cell of Therien R. The cultures 45 were monitored 24 hrs later for development of CPE. 2.19 S e q u e n c i n g Manual: Manual sequencing was carried out using dideoxynucleotide-termination reagents supplied with the Sequenase DNA Sequencing Kit (USB). 3 5 S -dATP was used to label the products for 5 min prior to addition of the termination mixture. After a further 5 min incubation the reactions were stopped and the products were separated by electrophoresis through an 8% polyacrylamide gel containing 8M urea. Dried gels were analysed following exposure to X-ray film overnight. Automated: Automated sequencing was carried out by the University of British Columbia, Nucleic Acid and Protein Sequencing Unit (Biotechnology Laboratory). Amplitaq Dye Terminator Cycle Sequencing (ABI) reagents were used and fluorescent products were analysed spectrophotometrically. 46 R E S U L T S & D I S C U S S I O N 1. R E S T R I C T I O N O F V I R A L R E P L I C A T I O N IN J O I N T While wt+ strains of RV readily infect human joint tissue, the vaccine strains RA27/3 and Cendehill are highly growth-restricted (Miki & Chantler, 1992). The level at which such restriction might occur can be arbitrarily separated into three broad categories. 1) The first is viral attachment and entry. If the binding regions of the structural proteins of one strain are sufficiently different it may not be able to recognise and bind to the cellular receptor. Alternatively, once bound it may not be able to interact with other cellular components required to complete the process of entry. 2) The second is the ability of the virus to replicate following release of the genome into the host cytoplasm. This category can involve many events including replication of the genetic material, production of nonstructural and structural proteins and assembly of the nucleocapsid. 3) The third category is the ability of the assembled virus particle to exit the cell. Since rubella is believed to bud from internal membranes or the plasma membrane (Bardeletti et al 1979; Payment et al, 1975) impaired exit might stem from altered targeting signals for intracellular transport of viral glycoproteins. 1.1. In fect ion of S y n o v i a l C e l l s (SC) To confirm previous results on the relative permissiveness of synovial cells to different RV strains, SC cultures were infected with Cendehill, Therien, and M33 strains, and washed rigorously following the adsorption period. After four days the levels of intracellular and supernatant virus were assayed (Fig.9). A baseline of 5 x 10 2 pfu/ml of virus remaining after removal of the inoculum was determined by titration of medium 47 Ti t ra t ion of R V Infect ion in S C Cend Ther M33 Day 4 Post Infection Fig.9 Titration of Cendehill (vaccine), Therien (wt+) and M33 (wt+) after infection in synovial cells. The range for two experiments is shown by error bars. Intracellular virus was released by one freeze-thaw cycle. The level of virus remaining in the supernatant immediately following removal of the inoculum (and 3 x washes in PBS) was used as a baseline for non-specific residual virus (ie. titres below this level were not assumed to constitute evidence of replication). | j extracellular | | intracellular sampled immediately after the adsorption period. In confirmation of previous results, Therien and M33 strains both replicated well and produced high titres both inside and outside the cells while Cendehill strain appeared to be severely restricted in its replication (Miki & Chantler, 1992). Titres above baseline were not detected in either the cellular or supernatant fraction and no cytopathology was seen. 1.2. C o m p a r a t i v e B i n d i n g In order to determine whether the block to Cendehill strain replication in SC occurs due to impaired receptor interaction, we examined the binding of 3 5S-labelled virus to SC. Since virus preparations vary in such factors as the particle/pfu ratio and 48 intensity of 3 5S-labelling, the ability of each strain to bind to SC was quantitated relative to its ability to bind to Vero cells which are permissive to replication of all the strains. The raw data were normalised to compensate for nonspecific binding of 3 5S-labelled cell proteins in each cell type (the lowest level of virus binding was at least 2 fold higher than control in each case). The results of four separate experiments are shown in Table 2. The average variability of replicates within an experiment was 7% for Vero cells and 10% for SC. Since the Therien strain replicates to high titres in both cell types, we had expected comparable binding to Vero and SC. Surprisingly, we found that the level of Therien strain binding to SC was approximately half that of Vero. In contrast, the Cendehill strain bound almost equally well to both cell types. In order to confirm the unexpected result for the Therien strain, a second stock was prepared and the binding in both cell types was compared in conjunction with the earlier stock preparations. The results were the same for each preparation. Since the binding of the Cendehill strain to both Vero and SC was nearly equivalent it seemed Table.2 35S-labelled, sucrose-purified RV was examined for the ability to bind to SC and Vero cells. The results are presented as a proportion of the binding to SC. A second preparation of Therien stock was used in two of the experiments denoted by *. The range for each series of experiments is shown in brackets. Comparative Binding to SC and Vero Cells (i) SC/Vero Average Cendehill 0.86 0.83 0.79 1.10 Therien 0.47 0.52 0.48* 0.43* 0 . 9 0 (+/- 0.2) 0 . 4 8 (+/- 0.04) 49 unlikely that its growth restriction in SC occurred at the level of receptor recognition and binding. Moreover as Therien grows well in both cell types, the 48% reduction in binding to SC does not appear to interfere with its ability to establish a productive infection. 1.3 E l e c t r o p o r a t i o n The comparative binding studies described above suggested that the restriction of Cendehill in SC was not a result of impaired attachment. To determine whether a block occurred later in the replicative cycle, RNA from Cendehill and Therien was electroporated into synovial cells (0.5 ug/5 x 10 6 cells) to examine the ability of the strains to replicate following entry and release of viral nucleic acid. Northern dot blot quantitation was carried out to ensure that approximately equivalent quantities of input RNA were used. The cells were harvested 5 days post-electroporation and assayed for the presence of intra- and extracellular virus. The results are presented in Table 3. Although no intracellular virus was found in the cells electroporated with the Cendehill strain RNA, 10-20 pfu/ml was found in the supernatant indicating that replication was occurring although at a severely restricted level. This is in marked contrast with Therien strain RNA which produced relatively high titres both intra- and extracellularly. In a second experiment where the supernatant of electroporated SCs was used to infect Vero cells to amplify any virus present, the Cendehill yield at 4 days (Vero) remained low at approximately 10 3 pfu/cell, whereas the companion Therien culture yielded around 5 x 10 6 pfu/cell (not shown). 50 E l e c t r o p o r a t i o n of V i ra l R N A into S C Table 3 SC were examined 5 days post-electroporation for intracellular and extracellular virus production. Cells were suspended in 1.0 ml of medium and subjected to freeze-thaw to release virus. pfu/ml RV Strain Cells Medium Cendehill Therien 4.0 x 105 10-20 4.0 x 104 Summary - Growth Restriction in Joint These experiments suggest that the block to replication of Cendehill in SC does not occur at the level of receptor binding or of viral exit from the cells. Although we have not directly examined the ability of the virus to successfully enter and uncoat following receptor binding, the observation that Cendehill is not able to replicate following electroporation of genomic RNA into synovial cells (while Therien is), gives weight to the idea that Cendehill restriction occurs subsequent to attachment and entry. Moreover as Cendehill grows relatively well in Vero cells, these results implicate the nonstructural proteins in restriction, since it is the processes of viral replication and translation which are believed to be most sensitive to cell-specific host factors. Future experiments could examine the rate of amplification of viral RNA following electroporation into SC to determine whether replication of the genome is hampered. Given the problems we have encountered in immunological identification of the Cendehill strain (R&D, Sec.3.1.7) it would likely prove difficult to monitor the kinetics of structural protein translation. These experiments indicate that extremely low levels of Cendehill replication occur after infection which are not readily distinguishable above background. In fact, earlier studies by Miki and Chantler (1992) showed that although replication of 51 Cendehill was completely restricted in joint organ culture, minimal replication of Cendehill in dissociated fetal synovial cells was likely occuring (Fig.5.B.); the low levels of virus found following electroporation of Cendehill into SC support this idea. 52 2. C O N S T R U C T I O N O F T H E T H E R I E N / C E N D E H I L L C H I M E R A S A N D C E N D E H I L L I N F E C T I O U S C L O N E In order to localise the genetic sequences involved in the restriction of Cendehill in joint, two chimeric viruses were constructed by replacing portions of the Therien cDNA of pROBO302 (Fig.7) with corresponding regions of Cendehill cDNA. Finally a full-length infectious clone of Cendehill was prepared (Fig.8). 2.1. F i r s t S t r a n d P r i m i n g a n d c D N A S y n t h e s i s First strand synthesis was carried out using 3 specific primers designed to be complementary to the published sequence of the Therien strain (Dominguez et al, 1990). Fig.10.A & B shows the results of a nucleic acid blot of the first strands synthesised using primers 38, 125 and 16 and a schematic of their location. The genomic and subgenomic RNAs of RV are used as markers for 9762 and 3327 bases respectively. Fig.6 (M&M, Sec.4.12) provides a diagramatic representation of the strategy used for thermal cycling amplification and the sequences of the oligonucleotide primers is given in Table 1 (M&M, Sec 4.12). Synthesis from primer 16 produced one predominant species that migrated slightly above the subgenomic RNA. This suggested that the first strand might be extended as far as the 5' terminus; which would result in a 4228 nt cDNA. This was confirmed using thermal cycling amplification with primers F2 and 10 which produced a 2257 bp product homologous to the 5' end of the genome. Approximately 10 7 pfu were required to synthesise this amplified dsDNA. A similar quantity of starting material was used to synthesise a second 1127 bp dsDNA with primers 9 and 46 from the same first-strand mix. 53 Fig.lO.A. First-strand cDNA reactions primed with oligonucleotides 16, 38 and 125 were separated on agarose gels and hybridized with 32P-labelled probe made by nick-translation using whole cDNA for the Therien strain as template. The RV genomic (9762) and subgenomic (3327) RNAs are used as size markers. Species which are thought to be required to carry out subsequent thermal cycling amplification for construction of chimeric viruses are marked by •<. Panel a: 24 hr exposure Panel b: 72 hr exposure B. The starting nts of primer 16, 38 and 125 are shown schematically. Synthesis of first strand from primer 38 resulted in a range of products including a single major band running slightly below the 3327 nt subgenomic mRNA. Longer exposures revealed a smear of material extending from around 4500 nt to the bottom of the gel. The very low quantities of these larger species is likely the reason that 5 x 10 8 pfu of virus was required as starting material for cDNA synthesis in order to amplify the 4478 nt product homologous to the 3' end, using primers 18 and F1. Shorter fragments near the 3' end could be amplified using much less starting material (10 7 pfu). It is possible that the major product represents termination due to secondary structures near the start of the RV structural protein coding region. Priming of first strand synthesis with oligonucleotide 125 yielded one major product and several minor products near the 3327 nt subgenomic marker. None of Synthesis of Cendehill First Strand cDNA 9762" genome 3327-subgenome B . 16 125 38_ 4228 5763 " 4 - 9762 54 these was made in abundance and in order to amplify a 3160 bp cDNA, RNA from about 5 x 10 8 pfu of virus was needed in the first strand reaction. This fragment, made with primers 251 and 125, never yielded in excess of about 300 ng, whereas each of the other cDNAs yielded about 1.0 ug/reaction. First-strand cDNAs were amplified using Deep Vent™, an extremely thermostable DNA polymerase isolated from Pyrococcus sp.. This enzyme contains a 3'-5' proofreading exonuclease function which allows the extension of long amplified fragments with relatively few errors. Comparing the sequences of cloned cDNAs to the direct products of amplification mixes, we found no differences in the 3'-terminal 1000 nts. Although our results have not been subjected to statistical analysis, preliminary observations suggest that the error rate for Deep Vent was less than 1 per thousand nts. Its high thermostability enabled efficient extended DNA synthesis at temperatures up to 80°C, an advantage in dealing with high G/C nucleic acids like RV RNA. As indicated by the manufacturer, we found that the proofreading enzymatic activity necessitated using considerably more starting material than is required when using polymerases without exonuclease function. This difficulty is likely further compounded by the high G/C content of RV which would be expected to hinder the progress of polymerase as a result of secondary/tertiary structures in the nucleic acid. 2.2. C h i m e r a s a n d In fect ious C l o n e Two infectious Therien/Cendehill chimeras were constructed and finally, an infectious clone containing the complete cDNA for Cendehill strain. Details of the cloning strategy are provided in Materials and Methods (M&M, Sec.4.12.) 55 p R 0 C 3 (ROBO /Cendehil l 3') contains the nonstructural genes of Therien strain (to nt 5355) and the remaining 4405 nt, including the structural genes, from Cendehill strain (Fig. 11 .A). There were relatively few difficulties in construction of pROC3. Preparation of pROBO302 for insertion of the 3' end fragment utilised Bgl II and EcoRl restriction sites which are unique and could therefore be carried out to completion and entailed no problems in separation of the digestion products. The 3' cDNA insert was made in fairly high quantities which ensured ample concentrations of both components of the ligation reaction. In addition, the cohesive ends each had overhangs of 4 bp which facilitated ligation. RNA produced from the infectious clone pROC3 produced approximately 10 6 pfu/ml of progeny after three serial passages in Vero cells. The progeny were designated as strain ROC3. Cendeh i l l / The r ien C h i m e r a s a n d C e n d e h i l l In fect ious C l o n e Therien Nonstructural f Therien Structural ' I V~ Bglll I - I Cendehill Structural • • I Nhel ^ I I Cendehill Structural • I v ^ Cendehill Nonstructural I Cendehill Structural I pROBO302 [ A. pROC3 C B. pROC3M L C. pJCND [ Fig.11 The regions of Therien cDNA which have been replaced by analogous regions from Cendehill strain in pROC3 (A), pROC3M (B), and pJCND (C) are shown schematically. pR0C3M (ROBO/Cendehill 37Midsection) contains Therien cDNA from 1-2803 bp and Cendehill cDNA from 2803-9762 bp (Fig.11 .B.). In order to construct pROC3M, the nt 2803-5355 (Nhel/Bgl II) cDNA was subcloned into pCLPC prior to cloning into pROC3. Initial quantities of the fragment primed with oligonucleotides 125 and 251 were never greater than 100 ng. Following restriction digestion and gel purification the total amounts were estimated at 50 ng. Given the problems with efficiency described above, the fragment was subcloned in order to amplify the working quantity and ensure successful cloning. cDNA from four subclones were combined for the final ligation into pROC3 to increase the probability of obtaining a viable clone without deleterious mutations. The infectious clone pROC3M produced approximately 5 x 10 5 pfu/ml of progeny after three serial passages in Vero cells. The progeny were designated as strain ROC3M. For the construction of the Cendehill infectious clone, p J C N D (J Chantler CeNDehill), the two fragments, 1-2262 and 2136-3263, were digested with Pvul (nt 2246), ligated and gel purified to create the 1-3263 bp product corresponding to the 5' terminus of Cendehill (Fig.11 .C) . Digestion with Pvul produces a two bp overhang and ligation at this site was very inefficient. Consequently only very small amounts of this product were recovered following restriction digestion (with Hindlll and Nhel) and gel purification. For this reason a subcloning strategy similar to that used for pROC3M was employed to amplify the 1-2803 bp product for insertion into pROC3M (note: RV carries a second Pvul site at nt 9475). The infectious clone pJCND produced approximately 10 6 pfu/ml of progeny after three serial passages in Vero cells. The progeny were designated as strain JCND. 57 Summary - Clone Construction Two Therien/Cendehill chimeric viruses, and a full-length infectious clone of Cendehill were made by sequential substitution of Cendehill cDNA into the Therien infectious clone, pROBO302. The plasmid constructs were first screened by restriction digestion for correct size and restriction fragment patterns. These candidates were then linearised at the 3'-terminus with EcoRI and used to generate RNA transcripts from an SP6 polymerase promoter located upstream of the 5' start of the genomic sequence. The transcripts were electroporated into BHK cells, which were co-cultivated with Vero cells after 2 days to amplify any virus produced. In order to ascertain that only viable virus was selected, plaque titration was carried out on the supernatants 5 days after Vero/BHK co-culture. This ensured that we would not be identifying defective particles within the transfected cells which were capable of causing CPE but were not able to initiate an independent infection. Earlier attempts using non-proofreading thermostable enzymes to achieve infectivity from RV cDNA constructs of Therien and M33 were largely unsuccessful (Wang et al, 1994; Giliam S, personal communication, 1997). This is presumably a result of the higher error rate in DNA synthesis generated by these enzymes in comparison to those with proofreading capability. The use of proofreading enzymes like Deep Vent™ (New England Biolabs) used in our study, and Ex-Taq™ (TaKaRa, Shuzo Co.) which was used to construct pROBO302, has inherent difficulties associated with degradation of the starting material. However, the final products have fewer errors introduced making them the enzymes of choice in systems like this that require high fidelity. Progeny of the infectious clones were designated ROC3, ROC3M 58 and JCND and were found to be infectious in Vero cells at levels similar to the progeny of pROBO302 (ROBO302) and wild Therien strain. The sequence of Cendehill cDNAs taken directly from thermal cycling amplification mixes was compared with the sequence of the selected clones to confirm that the correct fragments had been inserted. Further details of the sequencing procedures for these clones are provided in: R&D, Sec.4. After production of the cDNAs the two main problems encountered in construction of the Therien/Cendehill infectious chimeras and, finally, the infectious clone of Cendehill strain were: a) the apparent toxicity of some rubella sequences for the host bacteria and, b) the relative inefficiency of transformation when using large plasmids. In early experiments using the high copy vector pCMV, no viable rubella clones were recovered. This was assumed to be due to the toxicity of some of the viral sequences since viable clones were recovered when the vector pCL1921 was used to carry the viral cDNA. pCL1921 is a low copy plasmid (approx. 5 copies/cell), used for the construction of pROBO302 (Pugachev et al, 1997a), which facilitates the cloning of cDNAs which may be toxic to bacteria when replicated to high copy numbers (Lerner & Inouye, 1990). In addition our observations suggested that the 14 kb plasmids of the RV infectious clones were transfected into bacteria at least 100 fold less efficiently than 4 kb control plasmids, using heat shock. In our hands, electroporation of large plasmids into bacteria was roughly 10 fold more efficient than heat shock and was used to maximise the number of transformants. 59 3. P H E N O T Y P I C P R O P E R T I E S O F C E N D E H I L L , T H E R I E N , R O B O 3 0 2 A N D C E N D E H I L L / T H E R I E N C H I M E R I C S T R A I N S 3.1. P h e n o t y p i c A l t e r a t i o n F o l l o w i n g T r a n s f e c t i o n Initially experiments to test the infectivity of the pROBO302 infectious clone, provided by Dr. T.K. Frey, were carried out using Lipofectamine to transfect transcribed RNA into Vero cells. When the supernatants of these transfected cells were assayed for virus progeny by plaque titration it was immediately apparent that the morphology of the plaques derived from the infectious clone was markedly different from that of Therien. Therien produces pinpoint microfocal plaques which are not visible on staining with neutral red, while pROBO302 produced larger turbid plaques which were unstained in neutral red similar to those commonly found for attenuated strains like RA27/3 and Cendehill (Sato et al, 1979, Fogel and Plotkin, 1969) (plaque morphologies are described in detail in: R&D, Sec.3.5/Fig. 14). Since the plaque morphologies of the Therien infectious clone (pROBO302) and the Therien wt + strain were so markedly different, we transfected RNA extracted from Therien to determine whether the change was a result of the transfection protocol, or whether pROBO302 contained mutations which altered its plaque phenotype. Surprisingly, the progeny of Therien transfected RNA also demonstrated an altered plaque morphology in the form of large clear plaques which were visible in neutral red-stained monolayers. The observation that the altered phenotype was stable during 6 subsequent passages of the progeny in Vero cells by normal infection (not transfection) procedures suggested that the process of transfection might be altering the genotype of the progeny virions. A transition of this nature has not previously been reported in any 60 other virus system. We therefore wanted to test the effects of a variety of parameters on the phenomenon of phenotypic alteration following transfection. 3.1.1. Intracellular versus Virion RNA: The initial experiments were performed using viral RNA extracted from cells and may have been due to anomalous transcription from double-stranded RNA replicative intermediates or co-transfection of several incomplete viral RNAs. To test this idea, RNA was extracted from viral pellets of the clarified supernatants of infected cells prior to visible cytopathology to ensure collection of mature virions containing single-stranded genomic RNA. The progeny from transfection of virion RNA also produced large clear plaques in neutral red-stained monolayers, rather than wild type focal plaques. 3.1.2. Transfection Reagent The initial transfections were performed using the cationic lipid reagent, Lipofectamine. We examined the possibility that alterations to the input RNA by the transfection reagent resulted in anomalous transcription by introducing Therien virion RNA into BHK cells using: i) DEAE/dextran and, ii) electroporation which adds no exogenous reagents. In each case the progeny demonstrated the altered phenotype. 3.1.3. Cellular Environment Host factors have been shown to play a role in viral replication and translation in several virus systems including alphaviruses (Kuhn et al, 1992, Meerovitch et al, 1993, Svitkin et al, 1994). To determine the effect of the cellular environment on the the phenotype of viral progeny following the process of transfection, 61 Therien virion RNA was transfected into BHK, and later synovial, cells using electroporation. The progeny all displayed the same altered plaque phenotype that had been observed in virus from transfected Vero cells. 3.7.4. Plaque-Purification: Since our Therien stock was occasionally observed to produce clear or turbid plaques (0.001%) it was possible that the process of transfection was causing preferential selection of a rare subspecies of viral RNA. Therien virus stock which contained no clear-plaque variants was prepared by plaque-purification of a single microfocus. This was grown into high-titre stock in 3 subsequent passages in Vero cells. RNA from supernatant virus, harvested 3 days post-infection, was electroporated into BHK cells which were co-cultivated with Vero cells two days later. The progeny of this transfection also produced clear plaques, although larger than those found with the non-plaque-purified Therien stock. The transfection-derived virions also produced cytopathology more rapidly and aggressively than observed previously. The plaque-purified parental strain also appeared to produce more intense CPE than our mixed population, although not as aggressively as the transfection-derived stock, suggesting we had isolated a variant which was more virulent in tissue culture. The progeny of transfection of the plaque-purified Therien RNA was serially passaged three times in Vero cells to prepare a working stock. Aliquots of this stock were subjected to a further three serial passages without observable changes in the altered phenotype. This stock was designated T h e r i e n R and used in all of the following assays. 62 3.1.5. Other Strains: To determine whether the anomalous phenotype observed after transfection of Therien RNA was unique to that strain we obtained virion RNA from the M33 strain which was independently purified in Dr. Shirley Gillam's laboratory. M33 RNA was electroporated into BHK cells and the plaques examined on Vero cells. The wild M33 strain produces microfocal plaques similar to Therien, but the progeny following electroporation also gave turbid/clear plaques in neutral-red stained monolayers indicating that the phenomenon was not unique to the Therien strain. Progeny of transfected M33 RNA were designated M 3 3 R . 3.1.6. Thermal Cycling Amplification: To confirm the identity of the progeny of transfection, RNA was extracted from Therien R and M33 R virions and used as template for reverse transcription of cDNA and subsequent thermal cycling amplification with primers specific to the published sequence of RV (Dominguez et al, 1990). M&M,Sec.4.12.(Fig.6/Table 1) shows the strategy for synthesis of these cDNAs and lists the oligonucleotides used to generate them. Therien R strain cDNA was amplified from nt 8671 -8896. M33 R strain cDNA was amplified from nt 8671 -9762. Sequencing confirmed that the amplified fragments were derived from RV. 3.1.7. Western Blot and Immunoprecipitation: In order to determine whether the antigenicity of the stocks derived from transfection differed from that of the parental strains, analysis of viral protein by Western blot was performed using virus that had been partially purified by sedimentation through 25% sucrose. The immobilised proteins were probed using polyclonal antibodies to Therien strain (Fig.12.A.). No 63 Western Blot of Transfected and Nontransfected RV Strains MW MK C C R T T R 302 ROC3 Fig. 12.A . Immobilised proteins (nonreduced) were probed with rabbit polyclonal antiserum to Therien strain. MW=molecular weight marker; MK=mock; C=Cendehill (vaccine); CR=Cendehill from transfection; T=Therien (wild isolate); TR=Therien from transfection; 302=stock from transfection of pROBO302 RNA; ROC3=stock from transfection of pROC3 RNA. Positions of E1 and E2 monomers are marked. Immunoprecipitation of Parental RV Strains MK T RA C 1B2 M33 Fig.12. B Autoradiogram of 35S-labelled mature virions (reduced) immunoprecipitated using polyclonal antiserum to Therien strain. MK=mock; T=Therien wt+; RA=RA27/3 (vaccine); C=Cendehill (vaccine); 1B2=wt+; M33=wt+. Positions of El , E2 and C monomers are marked. 64 significant differences in the antigenic pattern were observed between the parental and transfection-derived stocks. E1 migrated as a single band near 58kD, E2 as a series of bands between 42 and 47kD. In addition, numerous other higher MW bands were observed due to the tendancy of the RV structural proteins to form homo- and hetero-dimers (Hobman et al, 1993). The capsid protein is expected to migrate as a 66kD homodimer under nonreducing conditions and is probably masked by nonspecific material migrating in that region (which is also found in the mock-infected lane). The staining pattern for Cendehill was extremely weak. Since the assay of viable virus from the sucrose pellets of Therien, M33 and Cendehill was similar (+/- 0.4) this phenomenon was not felt to be due to insufficient input of Cendehill virus in the sample. Further, increasing the concentration of the Cendehill proteins 20 fold did not result in any enhancement of the protein pattern and increases beyond that level led to visible distortion of the gel lanes. Moreover we were not able to improve the Cendehill pattern using polyclonal antiserum to M33, HPV77 or Cendehill strains (not shown). One explanation for these findings may be that Cendehill is inherently less stable than the wt + strains and its structural proteins undergo conformational alterations during the blotting procedure which prevent recognition by the antisera . Although a carbonate buffer was used for blotting in order to facilitate renaturation of the proteins, transfer to a solid support necessitates considerable denaturation of the proteins which can result in a loss of antigenicity. Our antisera were raised against native virions, and since the majority of RV epitopes are reported to be conformationally defined (Terry et al, 1988; Miller et al, 1997) this process might eliminate a large fraction of the antigenic recognition. To circumvent this process, infected cells were labelled with 3 5S-cystine and methionine and viral proteins were 65 extracted from polyethylene glycol-precipitated supernatant virions with buffer containing non-ionic detergent to remove the proteins from the envelope with minimal conformational disruption. The extracts were immunoprecipitated with the same antisera used previously and the immunoprecipitated proteins were then separated using SDS PAGE and visualised by autoradiography (Fig.12.B.). This relatively gentle procedure allowed visualisation of a weak band in the E2 region and a light smear co-migrating roughly with E1 for RA27/3 and Cendehill. The E1 band in Cendehill appeared to have a slightly lower MW than was seen in the other strains and probably reflects the loss of one N-linked glycosylation site (discussed further in R&D, Sec.4.2.4). RA27/3 produces a stronger pattern than Cendehill on western blots (Chantler et al, 1993). The difficulty in visualising the Cendehill proteins using immunological methods may relate to its low antigenicity, a property which contributed to its replacement with RA27/3 as the vaccine of choice in Europe (Best, 1991). This idea is supported by the observation that polyclonal antiserum to the Cendehill strain gave poor visualisation for all the strains (not shown). It might be possible to improve detection of dissociated Cendehill proteins using antisera against denatured viral proteins. 3.1.8. Electroporation of Nucleocapsid: We speculated that the capsid protein might be required as a chaperone to present the infecting RNA in a specific conformation, and that in the absence of capsid, secondary structures might form leading to mis-replication of the parental strand resulting in stably altered progeny following transfection. To "test this hypothesis we removed the viral envelope from plaque-purified Therien strain by solubilisation in 1 % Triton X-114 (Maurachaer et al, 1991) and 66 isolated; nucleocapsids by pelleting through 25% sucrose. The nucleocapsids were electroporated into BHK cells. Two days post-electroporation, supernatant from these cells was passaged onto subconfluent Vero cell monolayers. Cytopathology was observed after 24 hours that appeared to be identical to that of the rapidly-growing phenotype recovered after electroporation of Therien RNA (TherienR). The plaque morphology of the virus recovered was indistinguishable from the Therien R phenotype. Although it seems unlikely that RNA would remain bound to nucleocapsids under the conditions of detergent extraction, the core preparations were treated with RNase and re-pelleted through 25% sucrose. The RNase-treated nucleocapsids were electroporated as before, however, no cytopathology was observed and no plaques were found when the supernatant was titrated. These results seem to confirm the observations of Hovi (1972) who reported that encapsidated RNA from either rubella or alphavirus was sensitive to digestion with RNase, suggesting either that the capsid is permeable, or that the RNA may be partially exposed on the capsid surface. We were not able to demonstrate that electroporation of encapsidated Therien RNA gave rise to progeny with parental characteristics. However, since it is believed that rubella virus infects via an endocytic pathway and may undergo pH-dependant uncoating (Mauracher et al, 1991), it is possible that conformational changes to the capsid protein occur within the endosome which are required for correct presentation of the RNA. Electroporation of nucleocapsids directly into the cytoplasm may eliminate entry-associated morphological changes. Conformational changes to the capsid during the entry process are known to occur in poliovirus, another positive strand RNA virus. In the case of the non-enveloped polio virus, this event occurs upon attachment of the virus capsid to the cellular receptor 67 and results in enhanced sensitivity to protease and an increased affinity for liposomal binding (Fricks & Hogle, 1990; Gomez et al, 1993). The morphological changes presumably facilitate entry and allow destabilisation of the core so that the nucleic acid can be released. In another RNA virus system, the HIV core protein NCp7 has been found to affect the synthesis and stability of proviral DNA (Berthoux et al, 1997). Our experiments have not ruled out an interactive role for the C protein of RV in initial events of the replicative cycle which results in the altered phenotype of progeny. Another possibility is that high levels of RNA present in the cell (up to 10 genome copies/cell assuming 100% entry) following electroporation, relative to infection, may in some way mediate this effect. However, experiments using 0.1 genome copies/cell of starting material for the electroporation failed to yield virus). 3.1.9. Co-infection: Apart from a possible chaperoning role for the C protein there was a possibility that another, as yet undetected, protein or small nucleic acid was carried into the cell with the virion and was required for "normal" transcription. To test this hypothesis we infected Vero cells with 5 pfu/cell of parental Therien. The infection was allowed to proceed for 24 hr before infection with 0.5 pfu/cell of the Therien R strain and the cells were monitored for the appearance of the rapid CPE with highly rounded cells characteristic of infection with TherienR (R&D, Sec.3.1.4.). Our rationale was that any additional factors carried by the wild strain would be present in the cells at the time of release of Therien R nucleic acid and might, therefore, result in a "reversion" to the wild phenotype. However, 24 hr after superinfection with Therien R the cells exhibited approximately 90% TherienR-like CPE, suggesting that such a form of phenotypic rescue was not occurring. ,1. 68 Since Vero cell monolayers will not initially be infected at 100% (Sedwick & Sokol, 1970; Hemphill et al, 1988), we repeated the experiment and extended the Therien strain infection to 48 hr PI before addition of the second strain. Previous studies in our laboratory, using immunohistochemical staining methods, have shown that a minimum of 70% of the monolayer should be infected under similar conditions (Chantler and Lund, unpublished observations). Superinfection with the Therien R strain still resulted in 90% TherienR-like CPE after 24 hr. Only large clear plaques characteristic of Therien R were found, indicating; a) Therien R was capable of superinfecting Therien-infected cells and, b) products of Therien wt+ infection were not able to facilitate a reversion of the Therien R phenotype to microfocal (wt+) plaques. In light of these results it became clear that it would be necessary to include progeny from transfection of each parental strain as controls in all studies examining phenotypic properties of the infectious Therien/Cendehill chimeras since these studies depend on progeny of transfected RNA transcribed from plasmids. Therefore, in addition to TherienR and M33R, a stock of CendehillR strain was also produced by electroporation of Cendehill virion RNA. 3.2. G r o w t h in S y n o v i a l C e l l s The experiment shown in Fig.9 (R&D, Sec.1), confirmed earlier findings that synovial cells restricted the growth of Cendehill but were very permissive for the wt+ Therien strain. In order to determine the region of the RV genome responsible for growth in SC, cells were i n fec ted with ROC3, ROC3M and ROBO302 strains. We expected that ROBO302 would be able to grow in these cells but that one of the chimeric viruses would not (or both, if the genetic changes involved in Cendehill growth 69 restriction were distributed in several areas of the genome). Therien R, CendehillR, M33 and M33 R strains were included as controls and both intracellular and extracellular virus production were examined. The results are presented in Fig.13. As expected CendehillR, like the parent strain, appeared to be completely unable to infect these cells and showed no titres above the baseline of non-specific adsorption T i t ra t ion of C h i m e r i c V i r u s e s In Infected S C 7 " 6 - I 4 -log pfu/nSl 3 — baseline 1 — f X I \ | extracellular | | intracellular I F JX i Cend CendR Ther TherR M33 M33R ROC3 302 Day 4 Post Infection Fig.13 Titration of the Therien/Cendehill chimeric viruses (ROC3), compared with wild strains (Therien, M33), vaccine (Cendehill), transfected controls (TherienR, CendehillR, M33R) and progeny of the Therien infectious clone (ROBO302) after infection in synovial cells. The range for two experiments is shown by error bars. Intracellular virus was released by one freeze-thaw cycle. The level of virus remaining in the supernatant immediately following removal of the inoculum (and 3 x washes in PBS) was used as a baseline for non-specific residual virus (ie. titres below this level were not assumed to constitute evidence of replication). 70 of input virus (5 x 10 2 pfu/ml). The Therien R control also showed similar growth characteristics to its parent wild strain, showing high intracellular titres. However, as in the case of directly electroporated Therien RNA (R&D, Sec.1.3, tab le 3.) we repeatedly found reduced levels in the supernatant suggesting that transfection afters the progeny in a manner which may result in impaired viral egress, th i s is in contrast to the parental strain which produced considerably higher extracellular titres. We were surprised to find that ROBO302 was also unable to infect SC. th i s indicated that the cDNA used to prepare ROBO302 was either prepared from a variant of our therien strain, or that it had accumulated mutations which had caused an alteration in phenotype. t h e chimeric virus ROC3 was also restricted in SC, a logical finding since neither ROBO302 nor Cendehill, from which it was constructed, could replicate in these cells. In light of these findings we did not carry out this experiment using ROC3M or JCND. Another unexpected finding was that M33R was also restricted in joint cells producing a maximum titre in the supernatant of only 1.1 x 10 3 pfu/ml and even lower intracellular titres marginally above baseline, th i s result provided further evidence that transfection of viral RNA could result in a stably altered phenotype, since the wt+ parental M33 strain replicated to titres of 1.0 x 10 7 pfu/ml of supernatant virus and 2.9 x 10 5 pfu/ml inside the cells, comparable to the titres observed for ther ien. Samples were also taken at 7 days PI to ensure that intracellular replication was not delayed for some strains. No increase was observed beyond that found at 4 days PI indicating that none of the strains was producing a late-onset burst of replication. 71 3.3. B i n d i n g to S y n o v i a l C e l l s Therien was shown to bind to synovial cells with roughly half the efficiency of Vero cells (Table 2). A similar phenomenon was observed for CendehillR and TherienR (Table 4). In light of the observation that Therien R, like Therien, is able to grow relatively well in SC (R&D, Sec.3.2, (Fig.13), these results provide further support for the idea that the restriction of Cendehill is likely due to factors other than impaired attachment, since Cendehill bound equally well to both SC and Vero. 3.4. E l e c t r o p o r a t i o n of S y n o v i a l C e l l s Previous results suggested that growth restriction of Cendehill in joint cells might be a result of events occurring subsequent to viral entry (R&D, Sec.1), therefore the ability of the infectious clones to grow in SC if the entry step was bypassed was tested. C o m p a r a t i v e B i n d i n g to S C a n d V e r o Ce l ls ( i i ) Cend C e n d R Ther T h e r R SC/Vero 0.86 0.41 0.47 0.38 0.83 0.39 0.52 0.42 0.79 0.46 0.48* 0.29 1.10 0.41 0.43* 0.37 Average 0.90 (+/- 0.2) 0.42 (+/- 0.04) 0.48 (+/- 0.04) 0.37 (+/- 0.O8) Table 4 35S4abelled, sucrose-purified RV was examined for the ability to bind to SC and Vero cells. The results are presented as a proportion of the binding to SC. Therien and Cendehill values have been incorporated from Table 2 (R&D, Sec.1.1.2). A second preparation of Therien stock was used in two of the experiments denoted by *. The range for each series of experiments is shown in brackets. 72 Transcripts from pROC3, pROC3M, pJCND and pROBO302, as well as Cendehill and Therien RNA, were e l e c t r o p o r a t e d into SC and the cells and supernatants were harvested after 5 days for virus titration (Table 5). Transcripts were quantitated by ethidium bromide staining in agarose gels. Intracellular and extracellular titres of ROC3 were found to be equivalent to those of ROBO302. This shows that substitution of the Cendehill structural genes into ROBO302 was not having a restrictive effect on replication. In contrast, the titres of ROC3M showed a 10 fold reduction both inside and outside the cells. Thus it appears that mutations in the region of 2803 - 5355 play a role in growth restriction of Cendehill in joint. The observation that ROC3M titres, although lower than ROC3 and ROBO302 strains, were still 10 fold higher than those found after electroporation of Cendehill RNA, suggested that other mutations within the 5' terminus might contribute further to restriction of replication in SC. E l e c t r o p o r a t i o n of C h i m e r i c V i ra l R N A into S C pfu/ml RV Strain Cells Medium Table 5 SCs were examined 5 days post-electroporation for intracellular and extracellular virus production. Cells were suspended in 1.0 ml of medium and subjected to freeze-thaw to release virus. Values for Cendehill and Therien have been incorporated from Table 3. (R&D, Sec.1.3) Cendehill Therien pROC3 pROC3M pJCND1 pJCND2 pJCND3 pROBO302 4.0 x 105 1.6 x 102 40 1.6 x 102 ND ND ND 10-20 4.0 x 104 2.5 x 103 2.4 x 102 1.9 x10 3 9x 101 73 Transcripts were prepared from 3 clones of pJCND and electroporated into SC. In two cases no virus was recovered from the supernatant, although 1 clone yielded 9 x 10 1 pfu/ml. Since all 3 replicated equally well following electroporation into BHK cells and subsequent passage into Vero cells (10 4 pfu/ml after 4 days), these results suggest that mutations in the 5' terminus, from nt 1 - 2803 also contribute to restriction of Cendehill in SC. Future studies to examine the sequence of these three clones in this region will provide information about the mutations involved in growth restriction. It was immediately apparent in these studies that the ROBO302 strain was impaired in its ability to replicate in SC in a manner that was at least partially unrelated to changes in the attachment/entry process. Intracellular titres after electroporation did not exceed 5 x 10 2 pfu/ml, indicating that if minimal replication had occurred during infection of SC with ROBO302 (R&D, Sec.3.2, Fig. 13) it would not have been detectable above the baseline of reversible viral adsorption. The supernatant titres after electroporation were also low, but at 1.9 x 10 3 pfu/ml would be just above the baseline for the infection assays. The disparity in the results for Therien RNA and pROBO302 transcripts is another indiction that the genomes of these two strains must not be identical. 3.5. P l a q u e M o r p h o l o g y Therien and M33 strains produce pinpoint microfoci: small accumulations of cellular material above the monolayer which are visible microscopically by about 4 days post-overlay, and by eye as small opaque dots approximately 0.5mm in diameter after about 4-6 days. The microfoci are not visible after staining with neutral red, which suggests that the cells surrounding the foci are viable since neutral red is actively taken P l a q u e M o r p h o l o g y of R u b e l l a V i r u s S t r a i n s c = Cendehill C R = CendehillR T - Therien T R - Therien R M33 = M33 M33R = M33R ROC3 = ROC3 302 = ROBO302 JCND = JCND MK = mock Fig.14.A Morphology of RV plaques in Vero cells stained with neutral red 6 days after inoculation. Progeny of transfection (CendehillR, TherienR, M33R), chimeras (ROC3, ROC3M), the Cendehill infectious clone (JCND) and the Therien infectious clone (ROBO302) all produce clearings while wt+ (Therien, M33) strains produce microfoci which cannot be visualised with neutral red. Fig.14.B Morphology of RV microfoci in Vero cells stained with methylene blue 6 days after inoculation. Cendehill does not produce microfoci. 75 up by living cells. They can be stained as dark dots on a lighter background of cells using 0 . 1 % methylene blue if great care is taken not to dislodge the foci during removal of the agarose overlay (Fig.14.B). Microfocal plaques are also observed for other wild strains, such as Thomas and have been described by other researchers (Kouri et al, 1974; Miki & Chantler, 1992). Many of these strains produce turbid plaque (described below) variants but these occur at a frequency of less than 0.001% in the population. The Therien R strain, the progeny of transfection of RNA from plaque-purified Therien (R&D, Sec.3.1.4), produced large plaques visible by eye, first as turbid foci, within two days. These foci expanded rapidly, forming large clearings (approx. 5mm) by the third day, and encompassing the entire monolayer after 4-5 days. The Cendehill strain produces turbid plaques which are visible on staining with neutral red, and sometimes by eye without staining, about 4-6 days after inoculation. Turbidity is generally a hallmark of mild cytopathology and lack of cell lysis. Microscopically these plaques appear as a series of very small holes within a relatively intact monolayer; collectively producing a turbid focus which reaches 2-3 mm at its maximum size around day 6. In many cases Cendehill plaques have a characteristic "bullseye" appearance after staining, with an unstained circle surrounding a stained center. This has been interpreted as demonstrating recovery of.the central cells, a phenomenon which presumably is due to the relatively mild cytopathology induced by this strain (Sato et al, 1979). CendehillR, the progeny of transfection of RNA extracted from the Cendehill strain, produced turbid plaques that were indistinguishable from the parent strain. As described in R&D, Sec.3.1.5, M33R produced turbid plaques which were visible in neutral red. These plaques were similar to those observed for Cendehill and CendehillR 76 strains but tended to be slightly larger and more clear, although microscopic examination confirmed that they did not achieve the complete clearance of the monolayer that was seen with Therien R. All of the viruses from infectious clones; ROC3, ROC3M, JCND and ROBO302, produced plaques which were morphologically indistinguishable from those of Cendehill. Each of the chimeric viruses generally produced plaques which became visible about 1 day sooner than those of Cendehill and CendehillR strains. With the exception of Therien R, all of the other strains achieved a maximum plaque size 5-6 days after inoculation; these do not increase up to 12 days. 3.6. V i ra l G r o w t h C u r v e s In view of the unusually rapid cytopathology induced in Vero cells by Therien R, we examined the growth of some of the stocks derived from transfection relative to their parental strains. Vero cells were infected with Therien, Therien R, Cendehill, CendehillR, ROC3, ROC3M and ROBO302 strains. The cells were washed rigorously following the adsorption period and the supernatant was sampled periodically and titrated to determine the amount of virus present (Table 6). A baseline of 5 x 10 2 pfu/ml was determined by titration of medium sampled immediately after the adsorption period (0 hr); this titre represented the amount of input virus reversibly adsorbed onto the outside of the cells which could be dissociated into the medium. The earliest titre above baseline was produced by Therien R at 8 hr PI at approximately 10 4 pfu/ml suggesting that the initial virus is released somewhat earlier, between 4-8 hr. At this time about 2% of the cells appeared to be more retractile and slightly rounded (Fig. 15 ). At 24 hr approximately 50% of the monolayer showed CPE 77 Growth of RV Strains in Vero Ce l ls pfu/ml RV Strain 4hr 8hr 12hr 16hr 20hr 24hr Cend CendR Ther TherR ROC3 R0C3M ROBO302 8.9 x 103 1.5x 103 1.3x 105 2.7 x 104 1.8 x 105 8.9 x 102 2.3 x 103 2.5 x 103 6.7 x 102 ND 1.2 x 103 3.7 x 103 ND ND 5.7 x 103 5.5 x 104 5.1 x 104 5.3 x 105 6.7 x 103 1.2'x 104 1.0x 104 Table 6 Titres of RV strains at various times after infection. A baseline titre of 5 x 102 pfu/mlwas determined by titrating the medium immediately following adsorption (and washing) to determine the amount of non-specific virus adsorbed to the outside of the cells. Titres below this level were not recorded. ND = not done (Fig.16) with many of the cells detached, and the titres had risen to around 5 x 10 5 pfu/ml. In separate experiments using an MOI of 1 pfu/cell complete detachment of the monolayer was observed within 18 hr (not shown). The first progeny Therien titres were observed at 12 hr PI. At 1.5 x 10 3 pfu/ml, only slightly above baseline, this is presumably close to the earliest release of virus. The titre increased gradually to about 5 x 10 4 pfu/ml at 24 hr PI. These results are in agreement with reports for other wild strains (Chantler et al, 1993; Miki & Chantler, 1992; Vaheri et al, 1965; Oker-Blom, 1984). Cytopathology was not visible until around 80 hr at which time it appeared abruptly in about 60% of the monolayer (not shown). Cendehill virus emerged from the eclipse phase approximately 12 hrs later than Therien, between 20 and 24 hours. The eclipse phase of CendehillR was slightly shorter than the parent strain, between 16 and 20 hr PI, and titres of about 10 4 were reached by 24 hr PI. No CPE was observed up to 96 hr for either Cendehill or CendehillR strains. Growth in Vero Cells - 7.5 hr PI Therien Cendehill Fig.15 The growth of parental strains (Cendehill, Therien) compared with the progeny of electroporated RNA from the same viruses (CendehillR, TherienR). TherienR CPE was estimated at 2%; no CPE was observed for any of the other strains. Photographs were taken using phase-contrast microscopy (40X). Growth in Vero Cells - 25 hr PI s Fig. 16 The growth of parental strains (Cendehill, Therien) compared with the progeny of electroporated RNA from the same viruses (CendehillR, TherienR). TherienR CPE was estimated at 60%; approximately 2% CPE was observed for the other strains. Photographs were taken using phase-contrast microscopy (40X). 80 ROC3, ROC3M and ROBO302 strains had intermediate growth patterns compared with CendehillR and Therien, with phases of eclipse between 16 - 20 hr PI. These strain reached titres of about 10 4 pfu/ml by 24 hr. 3.7. G r o w t h in Raj i C e l l s The growth of RV strains in lymphoid cells is of interest because it could have a bearing on the ability of the virus to disseminate throughout the body as a passenger of these cells. Chantler et al (1993) reported that wild strains grew well in cultures of mixed peripheral blood mononuclear cells (PBMC), B and T cell lines and macrophage cell lines. In contrast, RA27/3 and Cendehill strains were severely restricted in PBMC. Although further studies were not performed with Cendehill, RA27/3 was shown to be incapable of replicating in B cell lines (Raji, Cess), and replicated only slightly in a macrophage line (U937). Raji cells were infected with Therien, Therien R, Cendehill, CendehillR, M33, M33 R , ROC3, RQC3M and ROBO302 strains for a 1 hr adsorption period and were washed by pelleting three times in PBS. The supernatant was harvested after 5 days for plaque titration (Table 7). As previously shown the two wild strains, Therien and M33 grew well in these cells, producing titres up to 10 5 pfu/ml. Therien R also gave high yields with a titre of 10 6 pfu/ml. As expected from previous results, Cendehill and CendehillR showed no growth in these cells. We had hoped to gain some insight into the location of the genes involved in B cell tropism by examining the growth of the various chimeras. However, neither M33R , ROC3, ROC3M nor ROBO302 showed any ability to grow in Raji cells. This is a similar pattern to what we observed upon infection of synovial cells with these strains (R&D, Sec.1.1). Again M33R , the progeny of 81 G r o w t h of R V S t r a i n s in Raj i C e l l s pfu/ml RV Strain 0 Hr 5 Days Cendehill Cendehi l l R Therien Ther ien R M33 M 3 3 R R O C 3 R O C 3 M ROBO302 3.9 x 10 2 2.7 x 10 2 2.7 x 10 2 4.8 x 10 2 1.5 x 10 2 2.4 x 10 2 3.0 x 10 1 3.5 x 10 2 4.5 X 10 2 1.4 x 10 5 4.1 x 10 6 2.4 x 10 5 Table 7 Growth of RV strains in the Raji B-cell line. Zero hr titres were taken immediately after adsorption and subsequent washing. All titres were recorded. electroporated M33 RNA, lost the ability of the parent to grow in this cell line. The finding that pROBO302 was unable to grow in Raji cells, in contrast to Therien R, meant that we were unable to determine the genes involved in B cell restriction using this infectious clone. 3.8. G r o w t h at 39°C The optimum growth temperature for rubella virus is 35°C. Previous studies had shown that Therien and M33 were capable of replicating in Vero cells at 39°C, while the vaccine strains, Cendehill and RA27/3, were not able to form plaques at the elevated temperature (Chantler et al, 1993). This sensitivity is likely related to the serial passaging of these viruses at reduced temperatures as part of the process of attenuation of the vaccine strains. Therien, Therien R, Cendehill, Cendehillp, M33, M33 R , ROBO302, ROC3 and 82 T e m p e r a t u r e S e n s i t i v i t y of R V S t r a i n s Cend C e n d R Ther T h e r R M33 M33 R R 0 C 3 R 0 C 3 M R O B O 35°C + + + + + + + + + 39°C - - + + + -Table 8 Viruses were asorbed to Vero cells for 1 hr at 35°C, then transferred to 39°C for 6 days. Wt+ plaques (Therien, M33) were observed without stain; all other strains were stained with neutral red at 5 days PI. Strains with positive growth at 39°C all had titres > 104 pfu/ml. ROC3M were assayed for their ability to form plaques after transfer to 39°C (Table 8). As previously observed (Chantler et al, 1993), both wild strains (Therien and M33) produced focal plaques at 39° similar to those found at 35°C, while Cendehill did not form plaques. Like the parental strain, Therien R was also able to grow at 39°C and formed plaques comparable to those seen at the lower temperature (large clearings after neutral red staining). In contrast, M33R did not form plaques at 39°C, showing that this strain had become temperature-sensitive relative to the parent. The fact that M33R became temperature sensitive, while Therien R did not, may reflect an enhancement of a pre-existing property of the parent strain. Chantler et al (1993) showed in a comparison of four wild strains, that some grew equally well at 35°C and 39°C (eg. Thomas strain) while others had reduced titres at the higher temperature. Of these M33 showed the greatest sensitivity with a 10 3 pfu/ml reduction of virus yield at 39°C, compared to Therien which was reduced by only 10 1 pfu/ml. Neither ROBO302, ROC3 nor ROC3M was able to form plaques at the higher temperature. Since stocks from transfected Therien RNA (TherienR) grew well, the inability of ROBO302 to grow at 39°C further suggests that the cDNA used was from variant of Therien strain, or that it had accumulated mutations during construction resulting in a different phenotype. Since ROBO302 and Cendehill strains were both temperature sensitive, the inability of the chimeras to plaque at 39°C was expected (JCND was not tested). 3 . 9 . V i r u s D e c a y Aliquots of Therien, Therien R, Cendehill, Cendehillp, ROC3, ROC3M and pROBO302 were suspended in virus propagating medium and incubated at 37°C to examine the stability of the strains at 35°C (Fig. 17). The strains were found to segregate into two groups, apparently based on the composition of their structural D e c a y of R V T i t res at 3 7 °C 6 5 E 4 Fig.17 RV stocks were suspended in virus propagation medium (in the absence of cells) and incubated at 37°C. Samples were removed periodically to determine the number of viable particles. 3 O 3 2 — PFCC3 — pFCECBE 0 28 51 74 Hours Post Infection 84 genes. Cendehill, CendehillR and R0C3 strains, which contain the structural gene region of Cendehill, all decayed more rapidly than the strains which contained wild-type structural genes. After three days Cendehill, CendehillR and pROC3 strains had all decayed from 10 5 to approximately 10 2 pfu/ml. This pattern of decay is in agreement with the findings of Miki & Chantler, 1992. In contrast, each of the strains with wild-type structural genes retained a level of viability at least 10 fold higher. These results suggest that alterations to the structural genes have rendered Cendehill less stable than Therien and M33 strains. 3.10. W e s t e r n B l o t The antigenic profiles of ROC3 and ROBO302 were compared on western blots to those of the parental strains (Cendehill, Therien) (R&D, Sec.3.1.7; Fig.12.A). The progeny of pROBO302 had an identical immunological staining pattern to parental Therien strain, whereas the progeny of pROC3 showed very poor staining in common with the parental, Cendehill structural proteins. These results indicated that the structural genes of these infectious clones were being expressed in a way that was antigenically similar to the parental strains. ROC3M and JCND strains were not examined as they contain the same Cendehill structural gene region as ROC3. Since the antisera used in these experiments were raised against mature virus recovered from infected cellular supernatants, they were expected to recognise the structural proteins only. Summary - Phenotypic Properties Electroporation of the chimeric viruses into SC demonstrated that the 5' 85 terminus, f r o m nt 1 - 2803, a n d the r e g i o n f r o m nt 2803 - 5355 of C e n d e h i l l w e r e e a c h contr ibut ing t o the g r o w t h restrict ion of C e n d e h i l l in S C . In contrast, substitution of the Cendehill structural genes for those of Therien in the pROC3 construct, had no effect on virus production after electroporation, relative to the ROBO302 control. These results are in agreement with those of the earlier binding and electroporation studies using Therien and Cendehill RNA (R&D, Sec.1.2/1.3) which suggested that the nonstructural genes might be involved in growth restriction. The inclusion of Therien R as a control in these experiments demonstrated that the Therien infectious clone, pROBO302, is not well-suited for genetic mapping of growth restriction of Cendehill in joint. Like the Cendehill strain, the progeny of ROBO302 were also stringently restricted in their ability to infect SC. Since Therien R replicated reasonably well in this situation, the growth restriction of ROBO302 was not a result of changes induced during the transfection process, and likely reflects either deleterious alterations in the Therien sequence introduced during construction of the clone, or that the cDNA was derived from a variant strain of Therien. This conclusion was supported by the further observations that ROBO302 had lost the ability to grow at 39°C and to grow in Raji cells. Since Therien R replicates in each of these systems, construction of another infectious clone using our plaque-purified parent of Therien R should enable more detailed study of phenotypic alterations between these two strains. However, since ROBO302 was able to replicate slightly following electroporation into SC, we were able to use differences between this clone, the ROBO/Cendehill chimeric clones (pROC3 and pROC3M), and the Cendehill infectious clone (pJCND) to map the growth restriction of Cendehill in joint. Future studies using a revised Therien infectious clone will be required to ascertain that other regions of the Cendehill genome 86 are not also involved in SC restriction. The experiments also show that transfection of RV RNA can give rise to virus with an altered phenotype which is stable on passage in Vero cells. Phenotypic alteration appears to develop independently of the virus ;strain, cell type and mode of transfection. The fact that the phenotype remains stable after numerous passages strongly suggests that the alteration has occurred at the genetic level, possibly due to anomalous transcription from the free genomic RNA. One mechanism for such an alteration might be a requirement for the virus structural protein/s acting as RNA chaperones to maintain a specific secondary structure for correct transcription. It would be interesting to compare the sequence of the progeny of transfection with that of the parent strains to determine where these changes take place. Other possibilities include alterations in the nonstructural gene region. The progeny of transfected viral RNA (TherienR and CendehillR strains) were found.to have accelerated rates of growth relative to their respective parental strains. In each case the eclipse phase was reduced by approximately. 4 hr. One possibility is that the rates of replication are increased as a result of alterations to the viral enzymes. However, Nakhasi's demonstration that translation from the nonstructural ORF was enhanced by the presence of the 3' SL also suggests a mechanism for growth rate alterations mediated through this region (Pogue et al, 1993). The Cendehill strain generally produces relatively little CPE and this property was mirrored in CendehillR. However Therien R produced spectacularly aggressive CPE relative to the parental strain with complete detachment of the monolayer by 18 hr. This extreme rapidity of CPE was not observed in earlier experiments using a mixed population of the Therien strain and probably represents an unusual subspecies 87 selected during plaque purification. Future experiments comparing Therien R and less aggressive variants of the Therien strain should provide valuable insights into the mechanisms of RV virulence. In addition to restricted cell tropism, the growth and decay experiments point to other levels of attenuation for Cendehill relative to Therien. The growth rate of Cendehill, as indicated by the,length of the eclipse phase, was at least 8 hr longer than Therien. Delayed growth may provide surrounding cells with more time to become resistant to infection through production of cytokines like interferon, thus limiting the spread of virus. This effect would be amplified by the restriction of Cendehill in lymphoid cell lines which could prevent its transport by these cells within the body. The optimum temperature for growth of most RV strains is 35°C, rather than 37°C (Vaheri, 1967). As rubella infection is associated with a mild fever the reduction in growth above 37°C suggests that any elevation of body temperature is likely to significantly restrict viral growth. Finally, the increased instability of Cendehill at 37°C probably reduces the level of dissemination via circulatory viremia. 88 4. S E Q U E N C E A N A L Y S I S In order to determine the genomic changes which might account for the phenotypic alterations we observed in our experiments, the sequence of Cendehill was compared with that of the three other strains for which the complete sequence has been determined: i) Therien (Dominguez et al, 1990), M33 (Clarke et al, 1987, and RA27/3 (Pugachev et al, 1997b). It should be noted that omissions in the original Therien sequence result in nucleotide numbering which differs slightly from the one presented here. These changes include insertion of an arginine codon at nt 1268 (CGC) and GC at nt 6262 (Pugachev et al, 1997b). A consensus M33 sequence is used for comparison with Cendehill (Clarke et al, 1987; Zheng et al, 1989; Pugachev et al, 1997b) Approximately 5000 nucleotides of the genome were sequenced directly from amplified reverse transcription mixes, the remainder was derived from Cendehill plasmid cDNAs. Where significant unique changes were found in the plasmids, these were confirmed using direct cDNA amplification mixes and reverse polarity primers. pROBO302 was also sequenced over various regions to confirm that changes were not due to anomalies in the sequencing reaction or pre-existing mutations in the Therien infectious clone. In no case did we find that nucleotide changes specific to Cendehill, relative to the other strains, were present in pROBO302. A comparison of the 5' nontranslated and nonstructural gene regions of Cendehill and RA27/3 vaccine strains, with Therien and M33 wt+ strains is shown in Fig.21, while Fig.22 shows a comparison of the subgenomic RNA, containing the structural gene region, of the same strains. 89 4 . 1 . N o n s t r u c t u r a l G e n e R e g i o n Cendehill contains 81 substituted bases in the 6426 nts of the nonstructural region relative to Therien strain, a frequency of 1.3% (compared to 2% in the subgenome). The majority of these result in silent mutations leaving eighteen amino acid substitutions (Fig.20; Appendix A). Thirteen of the altered residues are also found in the M33 wt+ strain and most will not be discussed as they are unlikely to contribute to the phenotypes under examination since M33 produces microfocal plaques, grows well in joint cells, in B cells and at 39°C. Fig.18.A shows the relative locations of these mutations in the Cendehill nonstructural proteins. 4.1.1. 5'SL Cendehill contains two mutations within the putative 5' stem loop (SL); a T to C transition at nt 37 and an A to G at nt 55 (Fig. 18.B). The mutation at nt 37 occurs in the terminal loop and does not affect the structure. The G at position 55 causes an extra mis-pairing, resulting in an increase in the size of the first bulge in the stem of the potential SL structures. These mutations could result in altered binding of host factors associated with translation 5'(+) SL or positive strand replication 3'(-) SL. Interestingly the mutation at nt 55 results in the deletion of a stop codon for the first ORF of the virus. This ORF is defined by the first AUG, which begins at nt 3 (Fig.21). The first AUG has the potential to synthesise a 16 amino acid protein in the Therien strain. Elimination of the stop at nt 54-56 would allow this protein to be extended to 28 amino acids in length, generating a protein of approximately 3kD. Although it appears that this first ORF is not essential for viral replication (Pugachev & Frey, 1997), 9 0 Nonst ruc tu ra l P r o t e i n s - A m i n o A c i d A l te ra t i ons in C e n d e h i l l S t ra in P150 P90 U I U I — D T " tnnnr innnr c>Y N>GU A>v Y > H T>l i Cendehill D Cendehill + M33 Fig.18.A. Amino acid substitutions in Cendehill, compared to Therien strain are shown by: D . The altered amino acids are noted for those mutations which are specific to Cendehill (ie. not found in M33 also). P r e d i c t e d 5 'SL S t ruc tu res for Ther ien a n d C e n d e h i l l 5' 3 7 1 ' I I i \ » — c — o _ t > _ 55 AUG, Therien r \ / o O \ T - C A 3 7 \ o 5' \ r x« / \ I T — P - - 0 — C _ p ' \ • , _ o — O 55 AUG., Cendehill Fig.18.B. 5'SL structures predicted by the program RNA DRAW, for the first 65 nts of Therien and Cendehill. The Cendehill mutation at nt 55 increases the size of the bulge in the stem structure. The mutation at nt 37 does not affect the structure of the terminal loop. 91 read-through in this area might interfere with normal protein synthesis or with synthesis from the third AUG. Alternatively, it is possible that a small protein is produced that, although not essential, could act to enhance virus replication or translation in some way. Another possible effect of mutations in the 5' stem-loop is interference with initiation of positive strand RNA synthesis from the negative strand replicative intermediate (since this structure becomes part of the 3', or initiating, end of the intermediate). Alterations to the analogous structure in Sindbis virus, had deleterious effects on viral replication (Niesters & Strauss, 1990). A similar phenomenon may occur with rubella, although preliminary evidence suggests that viral protein synthesis is affected by alterations in the 5' SL to a much greater degree than RNA synthesis (Pugachev & Frey, 1997). Using the pROBO302 infectious clone, Pugachev and Frey (1997) have shown that a variety of single point mutations within the SL loop are tolerated by the virus. Many of these changes had an effect on plaque morphology and some caused a small reduction in virus titre. However none of these effects could be correlated with specific perturbations in the SL structure. Tolerance for substitutions within the SL was supported by the report of 5 separate alterations found variously in the 5' terminal sequences of 6 other strains of RV (Johnstone et al, 1996). 4.1.2. P150 Beginning at A U G 4 1 , P150 comprises the first 1300 codons from the 5'-terminus of the genome. One change was found in Cendehill at nt 358, within the 347-375 region reported to be essential for binding of genomic RNA to capsid protein (Liu et al, 1996). No changes in this binding domain were found in M33, RA27/3 or any of the 92 6 other strains examined by Johnstone et al (1996). The first amino acid change unique to Cendehill in the nonstructural gene region is found at residue 929 where a G to A substitution results in replacement of cysteine with tyrosine. This occurs within the region of homology with the alphavirus NSP3 (Dominguez et al, 1990). Removal of the -SH-containing cysteine might alter intra/inter-molecular bonding and consequently the structure of the mature protein. A second change from asparagine to glycine is found at residue 1006. This mutation may also be included in the same domain or may be part of the beginning of the protease domain; the 5' extent of this domain has not yet been determined. Another substitution at residue 1041 substitutes tyrosine for histidine. Although both have bulky aromatic side groups, histidine is positively charged which could interfere with intra/inter chain bonding. Proximity to the catalytic region suggests that this change is included in the protease domain. The fourth alteration results in replacement of alanine with valine at position 1162. Although this mutation is placed within eleven amino acids of the catalytic cysteine (1151) of the protease (Chen eta l , 1996), given the highly conservative nature of the switch it may not have a significant effect on proteolytic function. p150 was found to contain a total of 43 substituted amino acids collectively in M33, RA27/3 and Cendehill, relative to Therien. This is an overall frequency of 3.3% which suggests that a higher degree of variability is tolerated in this protein. Strauss & Strauss (1994) reports that while one of the two NSP3 domains in the alphaviruses is highly conserved, the other is not. Fifteen (about 1/3) of the collective changes in p150 occur within 90 amino acids N-terminal to the NSP3-equivalent motif which may reflect a similar tolerance for variability. In the case of the alphaviruses the variable region of 93 NSP3 is C-terminal to the conserved domain. Since we observed high variability on tfote N-terminal side, it is interesting to speculate whether such a reversal might be related to the reversed order of the NSP3 and protease domains between rubella and the alphaviruses (Fig.1). 4.1.3. P90 P90 contains two putative functional domains. Homology to a global helicase motif has been identified between amino acids 1300 - 1600, near the amino terminus of the protein, and homology to a replicase domain has been identified between residues 1871 and 1971 (Dominguez et al, 1990). Cendehill has only one unique change within P90, located at amino acid 1496, in which replacement of C with T at nt 4527 resulted in a change from a polar threonine to a nonpolar isoleucine. The amino acid sequence of P90 is highly conserved and Cendehill, RA27/3 and M33 collectively contain only 6 substituted amino acids in the 905 amino acids of the protein (0.7%). 4.2. Subgenome Cendehill contains 67 substitution mutations relative to Therien in the subgenome (Fig.21). Two of these occur in the non-translated region upstream of the translational start site and two occur following the stop codons for the structural proteins. Of the remaining 62 substitutions within the coding region, 45 occur as the third base of a cbdon and do not affect the amino acid composition. 4.2.1. NTR Two changes were found in the 78 nucleotides upstream of the polyprotein start site, a region presumed to be involved in initiation of translation (Frey & 94 Marr, 1988), however both of these were also found in the wt+ M33. There are 16 substitutions in the 1062 amino acids (1.4%) of the Cendehill structural genes relative to Therien (Fig. 19/Fig.21). Ten of these are also found in the M33 (wt+) strain. The remaining 6 mutations appear to be unique to Cendehill, not being found in Therien, M33, HPV77 and RA27/3 (Zheng et al, 1989; Clarke et al, 1989; Pugachev et al, 1997b). Two mutations appear in the capsid protein (C), one in E2 and four in E1. The amino acids are numbered from the start of the polyprotein in the following discussion. S t r u c t u r a l P r o t e i n s - A m i n o A c i d A l t e r a t i o n s in C e n d e h i l l S t r a i n S>P T>G E2 Y Y Y Y A>V E1 N>D L>M < I I U H>L A>T Fig.19. Amino acid substituions in Cendehill, compared to Therien strain are shown by: D. The altered amino acids are noted for those Cendehill mutations which are specific to Cendehill (ie. not found in M33 also). Glycosylation sites are marked by: Y. Mutations which affect Cendehill + M33 glycosylation sites are marked by: * . 95 4.2.2. Capsid Of the 2 changes in C, one occurs at amino acid 34. Substitution of C for T results in replacement of polar serine with nonpolar proline. Addition of proline will introduce a sharp bend into the primary structure of the protein. This mutation is particularly interesting since it occurs within a stretch of 12 amino acids (28-56) which have been identified as the sequence with the highest specificity for binding genomic RNA and believed, therefore, to be important in encapsidation (Liu et al, 1996). A strong conformational change in the center of this region would almost certainly alter its RNA-binding affinity. Serine 34 of the C protein has also been identified as one of several phosphorylated residues, although the function of this modification is not yet understood (S Gillam personal communication, 1997). The second change in C converts amino acid 87 from threonine to glycine resulting from an AC to GG substitution at nts 6770 and 6771. This switch removes the potentially reactive hydroxyl group and the bulk of the threonine side-chain. Interestingly, the M33 strain also contains the mutation at nt 6771 (but not 6770) creating a serine codon; a much more conservative change which retains the hydroxyl group. Neither of the altered residues in C falls within the reported major antigenic domains for natural infection, although at least one monoclonal antibody was found to recognise a region between amino acids 64 and 97 (Wolinsky et al, 1991). 4.2.3. E2 The single change in E2 specific to Cendehill occurs at amino acid number 306 of the polyprotein, six amino acids from the NH2-terminus. Substitution of T for C results in replacement of alanine with valine. This falls within the first twenty-six residues of E2, a region which has been identified as a neutralising epitope of E2 (O'Brien, 1989) using a monoclonal antibody produced by Green & Dorsett (1986). 96 Although this is very conservative change, since both have small non-polar side chains, the poor antigenicity of Cendehill E2 suggests that it may have a negative effect on antibody recognition (R&D, Sec.3.1.7). The difficulty in stimulating production of monoclonal antibodies against E2 is a further argument for the relatively low antigenic potential of this molecule (Best, 1991; Plotkin & Buser, 1985), at least in animal systems. A threonine to isoleucine change at amino acid 112 in E2 eliminates one of the four, apparently filled, N-linked glycosylation sites in E2 (Bowden & Westaway, 1985; Zheng et al, 1989; Qiu et al, 1992). Although Zheng et al (1989) reported that this site was retained in the M33 strain, the M33 sequence of Clarke et al (1987) indicates that the same mutation is present. It therefore seems unlikely that loss of the carbohydrate moiety in E2 contributes significantly to the phenotypic alterations of Cendehill. 4.2.4. E1 As is the case for the mutation in E2, the four alterations in E1 all occur in the region of the protein which is extruded into the lumen of the ER; destined to be exposed on the surface of the mature virion. Substitution of G for A, and T for C at nts 8786 and 8788 respectively, alters uncharged asparagine to charged aspartic acid at position 177 in Cendehill. This change may be particularly significant because it disrupts one of the three Asn-X-Thr/Ser N-linked glycosylation sequences found in Therien E1, all of which appear to be utilised (Bowden & Westaway, 1985; Hobman et al, 1991). In conjunction with the mutation to the glycosylation site in E2, this means an overall loss of 2 glycosylation sites in Cendehill relative to Therien. The second unique change is found 26 amino acids.downstream at position 785, where a leucine is converted to a methionine by replacement of C with A. Both of these 97 are nonpolar and have side chains of similar length so this substitution may not alter the structure of the protein excessively. The remaining two substitutions also occur relatively close together, at amino acids 890 and 915. The first is a switch from charged histidine to nonpolar leucine (A to T); the second is an alteration of nonpolar alanine to polar threonine alteration (T to C). All of these changes could affect protein structure and/or stability. No altered amino acids were found in Cendehill E1 in the region associated with hemagglutination and neutralisation functions (from amino acids 796-867; or 214-285 numbering from the start of E1) (Terry et al, 1988; Chaye et al, 1992; Wolinsky et al, 1991; 1993). Nor were any unique changes found in the C, E2 or E1 regions mapped as dominant epitopes of the cell-mediated immune system (McCarthy et al, 1993; Ou et al, 1992a, b, c, 1994; llonen et al, 1992). However, it appears certain that the substitutions found in Cendehill must have a profound effect on its recognition by the humoral immune system since immunoprecipitation and western blot studies using a variety of RV polyclonal antisera (R&D, Sec.3.1.7) have shown that Cendehill is poorly antigenic. 4.2.5. 3'SL No unique mutations were found in Cendehill within the 58 nts of the putative 3'SL, which is believed to play a role in the initiation of negative strand \ . synthesis. Summary - Sequence Analysis Within the first 2803 nts of Cendehill are 8 base substitutions, all of which are silent. Only the mutations at nts 37 and 55 would result in a change of the structure of 98 a putative regulatory sequence, the 5' stem-loop, which has been shown to interact with host factors (Fig.18.B) (Pogue et al, 1993). The mutation at nt 37 should not alter the structure of the terminal loop but might interfere with sequence-specific recognition. Mutation 55 causes an increase in the size of the first bulge in the stem of this structure. Nakhasi's group reported that mutations which increased the size of this bulge resulted in enhanced binding of the La antigen to this structure (Pogue et al, 1993; Nakhasi et al, 1991). It would be useful to determine the effect of increases in La-binding on replication and translation. Since Pugachev and Frey (1997) reported that the main consequence of mutations within this region appeared to be reductions in nonstructural protein production, it is tempting to speculate that these might be attenuating mutations for Cendehill, resulting in decreased levels of translation. In addition, mutation 55 eliminates a stop codon for the first short ORF, allowing potential read-through of a slightly longer peptide. Although this ORF is not required for replication in Vero cells, it is conserved in all of the strains examined to date (Pugachev & Frey, 1997; Johnstone et al, 1996) and may have some role in tropism or attenuation. M u t a t i o n s at n ts 37 a n d 55 in the 5 ' SL are the re fo re the m o s t l i ke ly c a n d i d a t e s fo r the i n c r e a s e d r e s t r i c t i o n of p J C N D re lat ive to pROC3M t r a n s c r i p t s f o l l o w i n g e l e c t r o p o r a t i o n into S C . Mutations which result in 5 amino acid substitutions unique to Cendehill strain were found between nts 2828 - 4531 in the nonstructural gene region (Fig.18). Three of these mutations involve nonconservative amino acid replacements in the NSP3(Sindbis)-homology/protease domain. One additional nonconservative change occurs farther downstream, in the helicase domain. These unique changes fall within the Cendehill cDNA fragment used to construct pROC3M, which showed decreased 99 ability to replicate in synovial cells relative to pROC3 containing the structural gene region alone. T h e m u t a t i o n s at nt 2829, 3060, 3164 a n d 4530, w h i c h a re r e s p o n s i b l e fo r 4 n o n c o n s e r v a t i v e a m i n o a c i d c h a n g e s , a re the m o s t l i ke ly c a n d i d a t e s fo r r e s t r i c t i n g y i e l d s f r o m p R O C 3 M t r a n s c r i p t s f o l l o w i n g e l e c t r o p o r a t i o n into S C . Cendehill was found to have a mutation both at amino acid 34 of the C protein, the primary region involved in binding to the genomic RNA, and in nt 358 of the genomic RNA, the region of the genome which is bound (Liu et al, 1996). Although other sequences of the C protein are also capable of binding RNA to some degree, a drastic alteration of the primary binding site might reduce the yield of encapsidated virus or the specificity of genomic binding, resulting in a higher proportion of DI particles or inappropriately packaged RNAs. Encapsidation of inappropriate RNAs has been reported for Sindbis capsids in the absence of genome (Wengler et al, 1982; 1984). This is likely also the case for RV since virus-like-particles, which are formed when cells are transfected with the structural genes only, have electron-dense cores suggesting that they are packaging RNA (Qiu et al, 1992). These mutations might result in a lower particle/pfu for Cendehill than for wt-i- strains, thus contributing to attenuation. Another way in which mutations in the capsid protein might contribute to attenuation is through destabilisation of the core structure. As discussed in R&D, Sec.3.9, Cendehill is less stable than wt-i- virus strains. Both of the nonconservative mutations in C, but particularly the substitution of proline for serine (at amino acid 34) with its accompanying change to the architecture of the protein, would be good candidates for destabilisation of the core. 100 Instability of the virus particle might also be influenced by mutations in the envelope proteins. Nonconservative amino acid changes might alter the conformation of the proteins. The loss, in Cendehill, of 2 glycosylation sites might also contribute to instability of the virus particle and/or receptor-binding. In eucaryotic cells the surface glyco-groups are believed to act as a physical barrier to protect the cell, in addition to having a role in adhesion (Alberts et al, 1994). Our studies did not find any involvement of the structural proteins in restriction of Cendehill in synovial cells. However, since the Therien infectious clone, pROBO302, was also considerably restricted in these cells (in contrast to transfected Therien RNA) it remains possible that mutations in the structural genes also play a role in this phenomenon. Poor recognition of Cendehill on western blots and in immunoprecipitates (R&D, Sec 3.1.7) indicates that it is not recognised well by antibodies to native (undenatured) virus. These results support earlier reports that Cendehill was a poor immunogen, which ultimately resulted in its replacement by RA27/3 as the European vaccine strain (Best, 1991). However, recognition of Cendehill by the cell-mediated immune system, which relies on linear peptide antigens, should not be impaired. 101 Fig. 20 Nonstructural Sequence Comparison The sequences of Cendehill, RA27/3 (Pugachev et al, 1997b) and M33 (Clarke et al. 1987) were compared to Therien strain (Domingues et al, 1990; Pugachev et al, 1997b). Since much of the original Therien sequence was based on pROBO102 which was known to contain errors the Therien sequence was adjusted where sequencing of pROBO302 resulted in a base change which was common to all of the other strains sequenced (Pugachev et al, 1997). These adjustments are marked with an asterisk (*). Where each strain is homologous with Therien no sequence is entered. Base changes are noted at the appropriate position beneath the Therien sequence with -(signifying no change) to clarify their position within a codon. Amino acids which are altered in Cendehill strain are noted below the nucleotide substitution in the sequence. Amino acid substitutions that are specific to Cendehill strain are in bold type. The symbol ">" denotes "change to" (eg. A>T signifies an amino acid change from A to T.) Domains of homology with other organisms are § § J | | § § and marked with above the Therien sequence. The symbol.... signifies an unsequenced region. Nucleotide numbering is listed on the left side of the page; amino acid numbering on the right. All amino acids are numbered from the start of the polyprotein. TH = Therien RA =RA27/3 M33 =M33 JCND = Cendehill 102 R U B E L L A V I R U S NONSTRUCTURAL RNA • stem-loop l > p200 T h C A A § § G A A G C T A T C G G A C C T C G C T T A G G A C T C C C A T T C C C § 1 | | G A G A A A 3 M33 : RA g t - g -J C N D c 50 stop JL stop 3 T H C T C C T A G p B p l A G G T T C T T GCC C C C GGT GGG C C T T A T A A C T T A ACC G T C GGC 20 M33 RA J C N D - - g 101 T H A G T TGG .GTA AGA GAC C A C G T C CGA T C A A T T G T C GAG GGC GCG TGG GAA G T G 37 M 3 3 — c RA — c J C N D - - t - - C 152 T H CGC GAT G T T G T T A C C G C T GCC C A A A A G CGG GCC A T C G T A GCC GTG A T A 53 M33 RA t — J C N D 200 methyltransf erase TH C C C AGA C C T G T G T T C A C G CAG* A T G JjCAG G T C A G T ^ G A T CAC C C A GCA C T C CAC 70 M33 ' * """"** RA J C N D 251 T H GCA A T T T C G C G G \ T A T A C C CGC CGC C A T TGG A T C GAG TGG GGC C C T A A A GAA 87 M33 RA J C N D 302 T H GCC C T A C A C G T C C T C A T C GAC C C A AGC C C G GGC C T G C T C CGC GAG G T C 103 M33 RA J C N D 350 T H G C T CGC G T T GAG CGC CGC T G G G T C G C A C T G T G C C T C CAC AGG ACG GCA CGC 120 M33 RA J C N D — C 401 T H A A A C T C GCC A C C GCC C T G GCC GAG A C G GCC AGC GAG GCG TGG CAC G C T GAC 137 M33 RA J C N D 103 5 0 2 T H T A C G T G T G C GCG C T G C G T GGC G C A C C G AGC GGC C C C T T C T A C G T C C A C 153 M33 RA J C N D 5 0 0 T H C C T GAG GAC G T C C C G C A C GGC GGT CGC GCC G T G GCG GAC AGA TGC T T G C T C 1 7 0 M33 - - C - - t RA J C N D - - C 5 5 1 T H T A C T A C A C A C C C A T G C A G A T G T G C GAG C T G A T G C G T A C C A T T GAC GCC A C C 1 8 7 M33 - - C RA J C N D — C 6 0 2 T H C T G C T C G T G GCG G T T GAC T T G T G G C C G G T C GCC C T T GCG GCC C A C G T C 2 0 3 M33 t — RA - - C J C N D t — L > L 6 5 0 T H GGC GAC GAC T G G GAC GAC C T G GGC A T T GCC T G G C A T C T C GAC C A T GAC GGC 2 2 0 M33 - - t RA J C N D — t 7 0 1 T H GGT TGC C C C GCC GAT TGC CGC GGA GCC GGC G C T GGG C C C A C G C C C GGC T A C 2 3 7 M 3 3 — t RA J C N D — t - - t 7 5 2 T H A C C CGC C C C TGC A C C A C A CGC A T C T A C C A A G T C C T G C C G GAC A C C GCC 2 5 3 M33 RA J C N D 8 0 0 T h CAC C C C GGG CGC C T C T A C CGG T G C GGG C C C CGC C T G T G G A C G CGC GAT TGC 2 7 0 M33 - - c RA J C N D 8 5 1 T h GCC G T G GCC GAA C T C T C A TGG GAG G T T GCC C A A C A C TGC GGG CAC CAG GCG 2 8 7 M33 RA J C N D 104 902 T H C G C G T G C G C G C C G T G C G A T G C A C C C T C C C T A T C C G C C A C G T G C G C A G C 303 M33 R A — g JCND 950 T H C T C C A A C C C A G C G C G C G G G T C C G A C T C C C G G A C C T C G T C C A T C T C G C C G A G 320 M33 ~ t R A JCND - - a 1001 T H G T G G G C C G G T G G C G G T G G T T C A G C C T C C C C C G C C C C G T G T T C C A G C G C A T G 337 M33 t - - - - t R A JCND 1052 T H C T G T C C T A C T G C A A G A C C C T G A G C C C C G A C G C G T A C T A C A G C G A G C G C 353 M33 - - g — t R A JCND 1100 * T H G T G T T C A A G T T C A A G A A C G C C C T G A G C C A C A G C A T C A C G C T C G C G G G C A A T 370 M33 R A JCND 1151 T H G T G C T G C A A G A G G G G T G G A A G G G C A C G T G C G C C G A G G A A G A C G C G C T G T G C 387 M33 - - a R A JCND — a 1202 T H G C A T A C G T A G C C T T C C G C G C G T G G C A G T C T A A C G C C A G G T T G G C G G G G A T T 403 M33 — g - - g c — R A JCND 1253 R T H A T G A A A GGC GCG A A G CGC T G C GCC GCC GAC T C T T T G AGC G T G GCC GGC TGG 420 M33 a — — c RA a - -JCND 1304 * T H C T G GAC A C C A T T TGG GAC GCC A T T AAG CGG T T C T T C GGT AGC GTG C C C C T C 437 M33 - - c RA - g - — c JCND 105 1 3 5 5 T H GCC GAG CGC A T G GAG GAG T G G GAA C A G GAC GCC GCG GTC GCC GCC T T C 4 5 3 M33 RA J C N D 1 4 0 3 T H GAC CGC GGC C C C C T C GAG GAC GGC GGG CGC C A C T T G GAC A C C G T G C A A C C C 4 7 0 M33 - - a RA J C N D 1 4 5 4 T H C C A A A A T C G C C G C C C CGC C C T GAG A T C GCC GCG A C C TGG A T C G T C C A C G C A 4 8 7 M33 RA J C N D 1 5 0 5 * T H GCC AGC GCA GAC CGC C A T T G C GCG T G C G C T C C C CGC TGC GAC GTC C C G 5 0 3 M33 - - c - - a RA - - t J C N D 1 5 5 3 T H CGC GAA C G T C C T T C C GCG C C C GCC GGC C C G C C G G A T GAC GAG GCG C T C A T C 520 M33 — c - - t - - t RA J C N D — t 1 6 0 4 T H C C G C C G T G G C T G T T C GCC GAG CGC C G T GCC C T C CGC TGC CGC GAG T G G GAT 537 M33 RA - a -J C N D 1 6 5 5 T H T T C GAG G C T C T C CGC GCG CGC GCC G A T A C G GCG GCC GCG C C C GCC C C G ' 553 M33 RA - t -J C N D 1 7 0 3 * T h C T G G C T C C A CGC C C C GCG CGG T A C C C C A C C G T G C T C T A C CGC CAC C C C GCC 5 7 0 M33 — t RA - - t J C N D — t 1 7 5 4 T H C A C C A C G G C , C C G T G G C T C A C C C T T GAC GAG C C G GGC GAG G C T GAC GCG GCC 5 8 7 M33 - - t ? ; : v - g - - - c RA - - t J C N D — t 106 1805 T H C T G G T C T T A T G C GAC C C A C T T GGC C A G C C G C T C CGG GGC C C T GAA CGC 603 M33 RA C--JCND 1853 T H C A C T T C GCC GCC GGC GCG C A T A T G T G C GCG C A G GCG CGG GGG C T C CAG G C T 620 M33 - a -RA JCND 1904 T H T T T G T C C G T G T C G T G C C T C C A C C C GAG CGC C C C T G G GCC GAC GGG GGC GCC 637 M33 --C — t RA JCND — t 1955 T H A G A GCG T G G GCG A A G T T C T T C CGC GGC T G C GCC T G G GCG C A G CGC T T G 653 M33 RA JCND 2003 T H C T C GGC GAG C C A G C A G T T A T G C A C M33 — g --c RA JCND --g - - c 2054 T H C A G C T G A T C G C A C T G G C T T T G CGC M33 a — — c RA JCND — c 2105 T H G C A C T C T C G G T G C G T GAC C T G C C C M33 RA JCND C T C C C A T A C A C C GAT GGC GAC G T G C C A 670 A C G C T G GCC C A A CAG GGG GCC GCC T T G 687 GGG GGT G C A GCG T T C GAC GCA A A C 703 C-- a — — g — t — t 2153 T H GCG G T C A C C GCC GCC G T G CGC G C T GGC C C C CGC C A G T C C GCG GCC GCG T C A 720 M33 g — c t - a — RA g - - , a — JCND g — c t - a — R>G S>L A>T 2204 T H C C G C C A C C C GGC GAC C C C C C G C C G C C G CGC CGC G C A CGG CGA T C G C A A CGG 737 M33 RA t - - t - -JCND 107 2255 T H C A C T C G GAC G C T CGC GGC A C T C C G C C C C C C GCG C C T GCG CGC GAC C C G 753 M33 --C - t -RA --C J C N D --C - t -A>V 2303 T H C C G C C G C C C G C C C C C A G C C C G C C C G C G C C A C C C C G C G C T G G T GAC C C G G T C 770 M33 --c -gc cag - t g RA --g J C N D --g 2354 T H C C T C C C A T T C C C GCG GGG C C G GCG G A T CGC GCG C G T GAC GCC GAG C T G GAG 787 M33 - c - a-- -a- c--RA - c - t - -J C N D - c - -a-I>T G>E 2405 T H G T C GCC T G C GAG CCG AGC GGC C C C C C C A C G T C A A C C AGG GCA GAC C C A 803 M33 - t - -a- --a -a-RA -a- --a -a-J C N D -a- --a -a- --g C>Y R>K 2453 T H GAC A G C G A C A T C G T T GAA A G T T A C G C C C G C G C C - G C C GGA C C C ^ G T G C A C C T C 82 0 M33 " "* " "' - - t RA J C N D - - t 2504 NSP3 : T H C G A - G T C CGC G A C A T C A T G * GAC C C A C C G C C C G G C ^ T G C AAG , G T C r G T G G T C "AAC 837 M33 ** " ' ' - - t *" "~ —t ' RA J C N D — t — t 2555 : T H GCC GCC A A C j G A G ' G G G C T A T C T G G C C . G G G - . T C T GGC G T G T G C G G T , G C C A T C 853 M33 *' ' * " t-g • * ' ""--c --g RA --g J C N D --g --C 2603 • T h TTT-^GCC A A C GCC A C G G C G ; < G C C C T C - G C T G C A A A C r i f G C , CGG CGC CTC" GCC C C A 870 M33 • • • ' ' ' "** g--~ " " . " RA J C N D g — . N>D 2654 NSP3 T H T G C "CCC A C C '.GGC GAG GCA'^GTG G C G A C A / C C C GGC C A C GGC T G C ' G G G T A C A C C 887 M33 - t - ' * --g * "" " K * RA J C N D --g 108 2705 j^Z ~~Z, 1 TH ® f ATC^^AC^GCC GTC •GCG CCG CGG CGT^CCT CGG. GAtfSCCC.! G C l p l l 903 M33 --a " " ' """" R A JCND 2753 — -_ M33 R A JCND 2804 ^ NSP3 T H C T A 3 G C C - ; G C C [GCG C G T C G G T G G G C G T G T G T C ' G C G T G C C C C ~ C T C > C T C T * G G C < G C T 937 M33 c-c ~* '*'""* R A C — JCND - a -c>y 2855 T H G G C G T C T A C G G C T G G T C T G C T G G G G A G T C C C T C C G A G C C G C G C T C G C G 953 M33 " — t R A JCND — t 2903 T H G C T A C G C G C A C C G A G C C C G T C G A G C G C G T G A G C C T G C A C A T C T G C C A C C C C 970 M33 g — - c -R A - c - — t JCND g--T > A 2954 T H G A C C G C G C C A C G C T G A C G C A C G C C T C C G T G C T C G T C G G C G C G G G G C T C G C T 987 M33 - - t R A JCND 3005 T H G C C A G G C G C G T C A G T C C T C C T C C G A C C G A G C C C C T C G C A T C T T G C C C C 1003 M33 R A JCND 3053 T H G C C G G T G A C C C G G G C C G A C C G G C T C A G C G C A G C G C G T C G C C C C C A G C G A C C 1020 M33 R A JCND - g -N>G 3104 T H C C C C T T G G G G A T G C C A C C G C G C C C G A G C C C C G C G G A T G C C A G G G G T G C G A A 1037 M33 — c --c R A JCND 109 3155 T H C T C T G C CGG T A C A C G CGC G T C A C C A A T GAC CGC GCC T A T G T C A A C C T G 1053 M33 , t — RA J C N D C-- --C Y>H 3203 T H TGG C T C GAG CGC GAC CGC GGC GCC A C C AGC TGG GCC A T G CGC A T T C C C GAG 1070 M33 — g RA J C N D — g 3254 T H G T G G T T G T C T A C GGG C C G GAG C A C C T C GCC A C G C A T T T T C C A T T A A A C C A C 1087 M33 - - t - - c RA J C N D c — L > L 3305 T H T A C AGT G T G C T C A A G C C C GCG GAG G T C AGG C C C C C G CGA GGC A T G T G C 1103 M33 RA J C N D 3353 * T H GGG A G T GAC A T G TGG CGC T G C CGC GGC T G G CAG GGC A T G C C G C A G G T G CGG 1120 M33 RA g - -J C N D 3404 T H T G C A C C C C C T C C A A C G C T C A C GCC GCC C T G TGC CGC A C A GGC GTG C C C C C T 1137 M33 RA J C N D 3455 C 1151 T H CGG GCG AGC A C G C G A GGC GGC G A G . C T A GAC C C A A A C A C C | | | § T G G C T C 1153 M33 - t -RA - t -J C N D - t -A>V 3503 T h CGC GCC GCC GCC A A C G T T GCG C A G G C T GCG CGC GCC T G C GGC GCC T A C A C G 1170 M33 RA J C N D - t -A > V 3554 T H AGT GCC GGG TGC C C C A A G T G C GCC T A C GGC CGC GCC C T G AGC GAA GCC CGC 1187 M33 — t RA - g -J C N D 110 3 6 0 5 T H A C T C A T GAG GAC T T C GCC GCG C T G A G C C A G CGG T G G AGC GCG AGC C A C 1203 M33 t - -RA a--J C N D 3 6 5 3 T H GCC GAT GCC T C C C C T GAC GGC A C C GGA GAT C C C C T C GAC C C C C T G A T G GAG 1 2 2 0 M 3 3 — c RA t — J C N D --C 3 7 0 4 T H A C C G T G GGA T G C GCC T G T T C G CGC G T G T G G G T C GGC T C C GAG C A T GAG GCC 1 2 3 7 M33 --a --c RA --C J C N D — a — a 3 7 5 5 * T H C C G C C C GAC C A C C T C C T G G T G T C C C T C C A C C G T GCC C C A A A T GGT C C G 1 2 5 3 M33 — C RA J C N D — C 3 8 0 3 T H TGG GGC G T A G T G C T C GAG G T G C G T GCG CGC C C C GAG GGG GGC A A C C C C A C C 1270 M33 — c RA J C N D 3 8 5 4 H 1272 T H GGC C A C T T C G T C T G C GCG G T C GGC GGC GGC C C A C G C C G C G T C T C G G A C CGC 1 2 8 7 M33 RA J C N D > p 9 0 3 9 0 5 G 1300 T H C C C CAC C T C TGG C T T GCG G T C C C C C T G T C T CGG GGC | p l GGC A C C T G T 1303 M33 - - t --C --C - - t RA - - t — c J C N D — t — c --C - - t 3 9 5 3 ' T H GCC GCG A C C GAC GAG GGG C T G GCC C A G GCG T A C T A C GAC GAC C T C GAG G T G 1 3 2 0 M33 — c RA J C N D 4 0 0 4 T H CGC CGC C T C GGG G A T GAC GCC A T G GCC CGG GCG GCC C T C G C A T C A G T C C A A 1 3 3 7 M33 — c a — RA J C N D 111 4055 - - - - h e l i c a s e - - - - -T H CGC C C T CGC A A A GGC C C T T A C A A T ^ ^ r ^ ^ ^ ^ ^ P | ^ ^ ^ T 4 w 0 © e 5 G C A ' 1353 M33 --C --c *** RA JCND 4103 — -T H GGC G C T GGC A A G A C T A C C "CGC A T C C T C G C T G C C , T T C 'ACG CGC GAA GAC C T T 1370 M3 3 --C " " " --C RA — c JCND --C --c 4154 T H T A C G T C T G C C C C A C C A A T GCG C T C C T G C A C GAG A T C C A G GCC A A A C T C CGC 1387 M33 RA JCND 4205 T H GCG CGC G A T A T C GAC A T C A A G A A C GCC GCC A C C T A C GAG CGC CGG C T G 1403 M33 gc-RA --g gc-JCND gc-R>A 4 2 5 3 h e l i c a s e zi~~ T H A C G A A A C C G C T C GCC GCC T A C CGC ^ G ^ g ^ p % f A C f ^ f C ^ G A ' T ,GAG, G C G T T C A C T 1420 M33 *** RA JCND — a 4304 ' T H C T C "GGC G G C G A G T A C T G C * G C G " T T C G T T R G C C A G C C A A A C C A C C ' G C G " G A G G T G 1437 M3 3 R A JCND 4355 h e l i c a s e T H A T C ; T G C G T C G G T G A T C G G G A C C A G " ' T G C ' G G C C C A C A C - T A C G C C A A T A A C 1453 M33 R A JCND 4403 * T H TGC CGC A C C C C C G T C C C T GAC CGC T G G C C T A C C i ^ p i f p i i f t CGC CAC A C T 1470 M33 "'"*"" "*~ RA JCND 4454 T H T G G CGC T T C C C C GAC T G C T G G GCG GCC CGC C T G CGC GCG GGG C T C GAT T A T 1487 M33 RA JCND 112 4505 T H G A C A T C G A G G G C G A G C G C A C C G G C A C C T T C G C C T G C A A C C T T T G G G A C 1503 M33 R A JCND - t -T>I 4553 T H G G C C G C C A G G T C G A C C T T C A C C T C G C C T T C T C G C G C G A A A C C G T G C G C C G C 1520 M33 R A JCND 4604 h e l i c a s e T H C T T C A C G A G G C T G G C A T A ' C G C 1 G C A ^ - T A C A C C " G ' T G ^ C G C G A G " G C C ^ C A G *GGT A T G 1537 M33 " "' * " ' ~ " • *" ** R A JCND AGC jGTC\J3GC ACC *<3<5C -JTGC ATC CAT.; GTA GGC AGA GAC GGC ACG GAC GTT 1553 — g --c __g __g - h e l i c a s e ~~Z~ GCC CTG GCG CTG ACA CGC GAC CTC GCC ATC GTC^AGC CTG ACC CGG~ GCC TCC 1570 t — L > L 4754 • TH GAC GCA CTC TAC 'CTC't'CAC GAG CTC GAG GAC GGC TCA CTG CGC GCT GCG GGG 1587 M33 --c"~ ' _ t_ RA JCND — C 4805 TH CTC AGC GCG TTC CTC GAC GCC GGG GCA CTG GCG GAG CTC AAG GAG GTT 1603 M33 - - t RA JCND - - t --a 4853 TH CCC GCT GGC ATT GAC CGC GTT GTC GCC GTC GAG CAG GCA CCA CCA CCG TTG 1620 M33 --c RA JCND — c 4904 TH CCG CCC GCC GAC GGC ATC CCC GAG GCC CAA GAC GTG CCG CCC TTC TGC CCC 1637 M33 RA JCND 113 4955 T H C G C A C T C T G G A G G A G C T C G T C T T C G G C C G T G C C G G C C A C C C C C A T T A C 1653 M33 --a — g R A JCND — a 5003 T H G C G G A C C T C A A C C G C G T G A C T G A G M 3 3 R A JCND G G C G A A C G A G A A G T G C G G T A C A T G C G C 1670 - - t — t — t 5054 T H A T C T C G C G T C A C C T G C T C A A C A A G A A T C A C A C C G A G A T G C C C G G A A C G G A A 1687 M33 R A JCND 5105 T H C G C G T T C T C A G T G C C G T T T G C G C C G T G C G G C G C T A C C G C G C G G G C G A G G A T 1704 M33 R A JCND 5156 T H G G G T C G A C C C T C C G C A C T G C T G T G G C C C G C C A G C A C C C G C G C C C T T T T C G C 1721 M33 ~ c R A -JCND — C 5207 T H C A G A T C C C A C C C C C G C G C G T C A C T G C T G G G G T C G C C C A G G A G T G G C G C A T G 173 8 M33 — t — t R A JCND 5258 T H A C G T A C T T G C G G G A A C G G A T C G A C C T C A C T G A T G T C T A C A C G C A G A T G 1754 M 3 3 R A — c JCND 5306 T H G G C G T G G C C G C G C G G G A G C T C A C C G A C C G C T A C G C G C G C C G C T A T C C T G A G 1771 M33 a — R A JCND 5357 T H A T C T T C G C C G G C A T G T G T A C C G C C C A G A G C C T G A G C G T C C C C G C C T T C C T C 1788 M33 — t --c R A JCND - - t 114 5408 T H A A A GCC A C C T T G A A G T G C G T A G A C GCC GCC C T C G G C C C C A G G G A C A C C 1804 M33 --g --a R A J C N D 5456 T H G A G GAC T G C C A C G C C G C T C A G GGG A A A G C C G G C C T T GAG A T C C G G G C G T G G 1821 M33 — t --a R A ~ t J C N D - - t --a 5507 T H G C C A A G G A G T G G G T T C A G G T T A T G T C C C C G C A T T T C C G C G C G A T C C A G A A G 1838 M33 --C — t R A J C N D — c 5558 T H A T C A T C A T G C G C GCC T T G CGC C C G C A A T T C C T T G T G GCC G C T GGC C A T 1854 M33 — t C--R A J C N D - - t 5606 ~~JZ. T H A C G G A G C C C G A G G T C G A T G C G T G G T G G C A G G C C C A T T A C A C C A C C A A C §Xg§ 1871 M33 — c --c - - t R A - - t J C N D — c - - t <• 5657 r e p l i c a s e ~ T H A T C G A G G T C / G A C T T C A C T ^ G A G T T C G A C A T G , . ' A A c f C A G A C C ' C T C - ^ G C T 'ACT C G G 1888 M33 ' * " * " * * " " ' "--V R A J C N D 5708 T H GAC G T C G A G C T C GAG A T T AGC GCC G C T C T C T T G G G C C T C C C T T G C G C C 1904 M33 — c R A J C N D 5756 T H G A A GAC T A C CGC GCG C T C CGC GCC GGC AGC T A C T G C A C C C T G CGC G A A C T G 1921 M33 — t — t R A J C N D - - t - - t 5807 ^ - - r e p l i c a s e T H G G C T C C - A C T G A G A C C GGC T G C * G A G C G C A C A <AGC~7GGC GAG C C C GCC" A C G C T G 1938 M33 - " ' *• ~--t" " --a R A J C N D - - t 115 5 8 5 8 TH CTG -CAC'S^iC-»AC€'^CXi>G^j<3CC• ATG TGC ATG ' G C C ' A T G ^ G C ATG GTC CCC 1 9 5 4 M33 — t RA J C N D ~ t 5 9 0 6 G _ - D - D ~--_ZZZ TH AAA <3GC *<5^ i^^i^^^^SSSPs ' ^TS i ^ ^ - ^ G Q^T»^©s!^SJ^^™fi®7J^K3?'®; 1 9 7 1 M33 * - - c RA —t J C N D — C 5 9 5 7 TH CTC CCC GAG GGC GCG CGC AGC GCG GCA CTC AAG TGG ACC CCC GCC GAG GTG 1 9 8 8 M33 - a t RA - a t J C N D — t 6 0 0 8 TH GGC TTG TTT GGC TTC CAC ATC CCG GTG AAG CAC GTG AGC ACC CCT ACC 2 0 0 4 M33 - - c - - a —t - - a RA • —c —t J C N D — c - - t — a —t — a 6 0 5 6 TH CCC AGC TTC TGC GGG CAC GTC GGC ACC GCG GCC GGC CTC TTC CAT GAT GTC 2 0 2 1 M33 RA J C N D 6 1 0 7 TH ATG CAC CAG GCG ATC AAG GTG CTT TGC CGC CGT TTC GAC CCA GAC GTG CTT 2 0 3 8 M33 - - a —c. RA J C N D — a —c 6 1 5 8 TH GAA GAA CAG CAG GTG GCC CTC CTC GAC CGC CTC CGG GGG GTC TAC GCG 2 0 5 4 M33 RA J C N D 6 2 0 6 Th GCT CTG CCT GAC ACC GTT GCC GCC AAT GCT GCG TAC TAC GAC TAC AGC GCG 2 0 7 1 M33 —t RA J C N D - - t 6 2 5 7 TH GAG CGC GTC CTC GCT ATC GTG CGC GAA CTT ACC GCG TAC GCG CGG GGG CGC 2 0 8 8 M33 RA J C N D 116 6308 T H GGC C T C GAC C A C C C G GCC A C C A T C GGC GCG C T C GAG GAG A T T CAG A C C 2104 M33 — t RA JCND 6356 Stop 2 T H C C C T A C GCG CGC GCC A A T C T C C A C GAC GCC GAC T A A CGC C C C T G T A C G T G G 2115 M33 ~ t c — RA — t JCND — t c — 6407 stop 2 stop 2 > subgenome s t a r t s T H GGC C T T T A A T C T T A C C T A C T C T A A C C A GGT C A T C A C C C A C C G T T G T T T CGC M33 c - t RA JCND c — 117 Fig.21 Subgenomic Sequence Comparison The sequences of Cendehill, RA27/3 (Pugachev et al, 1997b) and M33 (Clarke et al. 1987) were compared to Therien strain (Domingues et al, 1990; Pugachev et al, 1997b). Where each strain is homologous with Therien no sequence is entered. Base changes are noted at the appropriate position beneath the Therien sequence with - (signifying no change) to clarify their position within a codon. Amino acids which are altered in Cendehill strain are noted below the nucleotide substitution in the sequence. Amino acid substitutions that are specific to Cendehill strain are in bold type. The symbol ">" denotes "change to" (eg. A>T signifies an amino acid change from A to T.) Known or putative structural domains are | K I S B and marked with , or ////// above the Therien sequence. Potential glycosylation sites are noted below the sequences and are l!§|llt a n c l designated "CHO." The symbol..... signifies an unsequenced region. Nucleotide numbering is listed on the left side of the page; amino acid numbering on the right. All amino acids are numbered from the start of the polyprotein. TH = Therien RA = RA27/3 M33 = M33 JCND = Cendehill 118 SUBGENOMIC SEQUENCE COMPARISON 6407 stop stop > subgemome s t a r t s T H GGC C T T T A A T C T T A C C T A C T C T A A C C A G G T C A T C A C C C A C C G T T G T T M33 c - t RA JCND C--6451 T H T C G C C G C A T C TGGTGGGTAC C C A A C T T T T G C C A T T C G G G A GAGCCCCAGG G T G C C C G A M33 c c RA JCND C c 6500 > C s t a r t T H A T G G C T T C T A C T A C C C C C A T C A C C A T G GAG GAC C T C C A G A A G GCC 15 M33 --C — t RA JCND — C - - t 6557 T H C T C G A G GCA C A A T C C CGC GCC C T G CGC GCG GAA C T C GCC GCC GGC GCC T C G 32 M33 — g -gt RA a — JCND 6608 genomic RNA b i n d i n g T H C A G T C G CGC CGG C C G CGG C C G C C G CGA C A G CGC GAC T C C AGC A C C T C C 48 M33 RA JCND c — S>P 6656 T H GGA G A T GAC T C C GGC C G T GAC T C C GGA GGG C C C CGC CGC CGC CGC GGC A A C 65 M33 RA JCND 6707 T H CGG GGC C G T GGC C A G CGC AGG GAC T G G T C C AGG GCC C C G C C C C C C C C G GAG 82 M33 -a- — c --a RA JCND -a- --a R>K 6758 T H GAG CGG C A A GAA A C T CGC T C C CAG A C T C C G GCC C C G AAG C C A T C G CGG 98 M33 -g-RA JCND g g - --a T>G 119 6806 T H GCG C C G C C A C A A C A G C C T C A A C C C C C G CGC A T G C A A A C C GGG C G T GGG GGC 115 M33 — t RA . - ~ t J C N D — t 6857 T H T C T GCC C C G CGC C C C GAG C T G GGG C C A C C G A C C A A C C C G T T C C A A GCA GCC 132 M33 — t —g — g RA J C N D — t — g --g 6808 T H G T G G C G C G T GGC C T G C G C C C G C C T C T C C A C G A C C C T G A C A C C GAG G C A 148 M33 - - t RA J C N D --C — t ~ t 6956 T H C C C A C C GAG GCC T G C G T G A C C T C G T G G C T T T G G AGC GAG GGC GAA GGC GCG 165 M33 - - t --a RA --a J C N D — a 7007 T H G T C T T T T A C CGC G T C GAC C T G C A T T T C A C C A A C C T G GGC A C C C C C C C A C T C 182 M33 —c — t - t -RA J C N D — C 7058 T H GAC GAG GAC GGC CGC TGG GAC C C T GCG C T C A T G T A C A A C C C T TGC GGG 198 M33 RA J C N D 7106 T H C C C GAG C C G C C C G C T C A C G T C G T C CGC GCG T A C A A T C A A C C T GCC GGC GAC 215 M33 — t --C RA J C N D — t — t --C 7157 T H G T C AGG GGC G T T T G G GGT A A A GGC GAG CGC A C C T A C GCC GAG C A G GAC T T C 232 M33 c — --a - - t RA — t - - t J C N D - - t 7208 T H CGC G T C GGC GGC A C G CGC T G G C A C CGA C T G C T G CGC A T G C C A G T G CGC 248 ' M33 RA J C N D --C 120 7256 T H GGC C T C GAC GGC GAC AGC GCC C C G C T T C C C C C C C A C ACC A C C GAG CGC A T T 265 M33 -eg — t RA J C N D 7307 T H GAG A C C CGC T C G GCG CGC C A T C C T T G G CGC A T C CGC T T C GGT GCC C C C ® | § 282 M 3 3 — RA J C N D 7358 E2 s i g n a l — T H GCC " T T C C W ^ C g ^ S ^ ^ T C T T C T g T C j p e ^ C G ^ G ^ 298 M33 — c g — "~ """""" RA g — J C N D — c g~-7406 >E2 s t a r t T H CGC GCC GGG C T C CAG C C C CGC G C T G A T A T G GCG GCA C C T C C T A C G C T G C C G 315 M33 --c - t - - c - --a RA J C N D - t - --c - c -A>V L>P 7457 T H C A G C C C C C C T G T GCG C A C GGG C A G C A T T A C GGC C A C CAC CAC C A T C A G C T G 332 M33 C - - - - t — t RA C - -J C N D C — - - t O R 7508 T H C C G T T C C T C GGG CAC GAC GGC C A T C A T GGC GGC A C C T T G CGC G T C GGC 348 M33 - - C RA J C N D - - C 7556 T H C A G C A T T A C CGA A A C GCC AGC GAC G T G C T G C C C GGC CAC TGG C T C C A A GGC 365 M33 c — RA c — J C N D c — Y>H . l l f f C H O ' 7607 T H GGC TGG GGT TGC T A C A A C C T G A G C . G A C T G G C A C C A G GGC A C T C A T G T C T G T 382 M33R RA J C N D CHO 7658 T H C A T A C C AAG C A C A T G GAC T T C TGG T G T G T G GAG CAC GAC CGA C C G C C G 398 M33 — c RA - - C - - t J C N D --c 121 7706 T H C C C GCG A C C C C G A C G C C T C T C A C C A C C GCG GCG A A C T C C A C G A C C GCC GCC 415 M33 t — - a - - t t g - -RA J C N D - - t t OfSIliili 7757 T H A C C C C C GCC A C T GCG C C G GCC C C C T G C C A C GCC GGC C T C A A T GAC AGC T G C 432 M33 C — RA J C N D * \ CHO 7808 T H GGC GGC T T C T T G T C T GGG T G C GGG C C G A T G CGC C T G CGC C A C GGC G C T 448 M33 RA J C N D 7856 T H GAC A C C CGG TGC GGT CGG T T G A T C T G C GGG C T G T C C A C C A C C GCC C A G T A C 465 M33 RA — t J C N D 7907 T H C C G C C T A C C CGG T T T GGC T G C G C T A T G CGG T G G GGC C T T C C C C C C T G G GAA 482 M33 - - C — c RA J C N D - - C 7958 T H C T G G T C G T C C T T A C C GCC CGC C C C GAA GAC GGC T G G A C T T G C CGC GGC 498 M33 a - t — t — t RA J C N D - - t 8006 T H G T G C C C GCC C A T C C A GGC GCC CGC T G C C C C GAA C T G G T G AGC C C C A T G GGA 515 M33 — t a — RA - - C a - -J C N D — t a - -A>T 8057 T H CGC GCG A C T TGC T C C C C A G C C T C G GCC C T C T G G C T C GCC A C A GCG A A C GCG 532 M33 — t RA J C N D 8108 E2 t r a n s m e m b r a n e T H CTG T C T C T T G A T ^ ^ p P < ^ P ^ ^ ^ ^ § g ^ | g B ^ p ^ € * G - • C T G , . G T C C C G * T G G 548 M33 — c — g t — - - t " t - - "• RA J C N D - - c — g — t 122 8156 / / / / / E 2 cytoplasmic///////// T H G T C C T G A T A T T T A T G G T G T G C CGC CGC GCC T G T CGC CGC CGC GGC GCC GCC 565 M33 RA --c a--JCND 8207 - - - E l signal T H GCC GCC C T C A C C GCG G T C G T C C T G CAG GGG T A C A A C C C C C C C GCC T A T GGC 582 M33 --a RA JCND — a 8258 > E l s t a r t T H GAG GAG G C T T T C A C C T A C C T C T G C A C T G C A C C G GGG T G C GCC A C T C A A 598 M33 RA JCND 8306 ? T H G C A C C T G T C C C C G T G C G C C T C G C T GGC G T C C G T T T T G A G T C C A A G A T T G T G 615 M33 a-- --c --c --C - - t RA --C JCND a-- --c --c A>T 8357 T H GAC GGC GGC TGC T T T GCC C C A T G G GAC C T C GAG GCC A C T GGA GCC T G C A T T 632 M33 --C ~ t RA JCND --C 8408 T H T G C GAG A T C C C C A C T G A T G T C T C G TGC GAG GGC T T G GGG GCC TGG G T A 648 M33 - - t RA • JCND 8456 T H C C C GCA GCC C C T TGC GCG CGC A T C TGG A A T GGC A C A C A G CGC GCG T G C A C C 665 M33 a--RA a — JCND a--V>T . ". *"CH©.-*/ 8507 T H T T C TGG G C T G T C A A C GCC T A C T C C T C T GGC GGG T A C GCG C A G C T G GCC T C T 682 M33 RA JCND 8558 T H T A C T T C A A C C C T GGC GGC A G C T A C T A C A A G C A G T A C C A C C C T A C C GCG 698 M33 — c RA JCND --c 123 T H T G C GAG G T T GAA C C T GCC T T C GGA C A C A G C GAC GCG GCC T G C TGG GGC T T C 715 M33 RA JCND T H C C C A C C GAC A C C G T G A T G AGC G T G T T C GCC C T T G C T AGC T A C GTC C A G C A C 732 M33 — C RA JCND 8708 T h C C T C A C A A G A C C G T C CGG G T C A A G T T C C A T A C A GAG A C C AGG A C C G T C 748 M33 RA — t - ~ - t JCND ~ t ~ t T h T G G C A A C T C T C C G T T GCC GGC G T G T C G TGC A A C G T C A C C A C T GAA C A C C C G 765 M33 - - a RA — t JCND - - a £ T _ t N>D CHO T H T T C T G C A A C A C G C C G C A C GGA C A A C T C GAG G T C CAG G T C C C G C C C GAC C C C 782 M33 " _ t RA JCND _ _ t T H GGG GAC C T G G T T GAG T A C A T T A T G A A T T A C A C C GGC A A T CAG CAG T C C 798 M33 - - C — c - - a RA - - C C - - , FCND a— — a JCND a— _ _ a L>M , CHO 8906 T H CGG TGG GGC C T C GGG A G C C C G A A T T G C C A C GGC C C C GAT TGG GCC T C C C C G 815 M33 ~ t — t RA — t JCND — C — t — t 8957 T H G T T T G C C A A CGC C A T T C C C C T GAC M33 - - c RA JCND T G C T C G CGG C T T GTG GGG GCC A C G C C A 832 — t 9008 T H GAG CGC C C C CGG C T G CGC C T G G T C GAC GCC GAC GAC C C C C T G C T G CGC 848 M33 — t - ~ t RA - - t JCND - - t 124 9056 T H A C T GCC C C T GGA C C C GGC GAG G T G T G G G T C A C G C C T G T C A T A GGC T C T C A G 865 M33 — c - - g RA - - g JCND - - g 9107 T H GCG CGC A A G T G C GGA C T C C A C A T A CGC G C T GGA C C G T A C GGC C A T G C T A C C 882 M33 RA JCND 9158 T H G T C GAA A T G C C C GAG T G G A T C C A C GCC C A C A C C A C C AGC GAC C C C T G G 898 M33 — t RA JCND - t - - - t H>L 9206 T H C A T C C A C C G GGC C C C T T G GGG C T G A A G T T C A A G A C A G T T C G C C C G G T G G C C 915 M33 - - c RA — c J C N D — c a — A>T 9257 T h C T G C C A CGC A C G T T A GCG C C A C C C M33 g — — t RA JCND g ~ T>A CGC A A T G T G C G T GTG A C C GGG T G C T A C 932 - - c — t 9308 T H CAG TGC GGT A C C C C C GCG C T G G T G GAA GGC C T T GCC C C C GGG GGA GGC 948 M33 - - a — g RA — g JCND — a — g 9356 T H A A T T G C C A T C T C A C C G T C A A T GGC GAG GAC C T C GGC GCC G T C C C C C C T GGG 965 M33 - - C — t g - - t - -RA - - t t — JCND - - c g — t — L>V V>F 9407 T H A A G T T C G T C A C C GCC GCC C T C C T C A A C A C C C C C C C G C C C T A C C A A G T C AGC 982 M33 — t RA JCND 125 9458 T H T G C GGG GGC GAG AGC G A T CGC GCG A C C G C G CGG G T C A T C GAC C C C GCC 998 M33 - g - - - t RA - g - - - t JCND - g - - - t T>S 9506 T H GCG C A A T C G T T T A C C GGC G T G G T G T A T GGC A C A C A C A C C A C T G C T G T G T C G 1015 M33 RA JCND 9557 - •--T h G A G A C C CGG C A G A C C T G G G C G G A G T G G G C T G C T G C C C A T - T G G T G G C A G C T C 1032 M33 - - t RA JCND .—t 9608 E l transmembrane T h A C T , C T G GGC G C C A T T T G C G C C C T C C C A C T C G C T GGC " T T A C T C G C T T G C 1048 M33 RA - t -JCND stem-loop — 9656 / / / / / / / / / / / / / E l c y t o p l a s m i c / / / / / / / / / / / / / / / / / / / / / / / / stop T h T G T G C C A A A T G C T T G T A C T A C T T G CGC GGC G C T A T A G C G C C T CGC T A G T G G M33 —a — g c RA - - g JCND - - g 9707 Stop T h G C C C C C G C G C G A A A C C C G C A C T A G G C C A C T A G A T C C C C G C A C C T G T T G C T M33 c t RA c t JCND C t t . 9757 T h G T A T A G RA g JCND . . . . . . 126 CONCLUSIONS Attenuating mutations in Cendehill virus have been mapped to two of the nonstructional gene regions, the 5'SL and the COOH-terminal third of P150. The idea that more than one locus contributes to a phenotypic property is well documented in virology. For example, two mutations in E1 and one in E2 of Sindbis virus have been found to participate in attenuation of neurovirulence. Alteration of E1 or E2 alone produces a reduction in neurovirulence, but the two combined yield a greater degree of attenuation (Polo and Johnston, 1990). The polymerase protein of the P1/Sabiri strain of poliovirus contains three amino acid changes which are required to produce temperature sensitivity. Neither of these mutations alone is sufficient to achieve a sensitive phenotype (Bouchard et al, 1995). Conversely, the same phenotypic property can be generated by completely different mutations. Two separate determinants of neurotropism were found to reside in either the 5' nontranslated region or in the E2 glycoprotein of Sindbis virus (Dubuisson et al, 1997). These studies indicate that one phenotypic property might be generated through a variety of mutations. That this occurs in RV is suggested by our observations that Cendehill and RA27/3 did not contain any mutations in common, that were not also found in the wt+ strain, M33 (Fig.20/21). This means that shared phenotypic traits between the two vaccine strains, such as temperature sensitivity and growth restriction in B-cell lines, have been generated by different mutational events. These observations also highlight the advantage of using mapping studies in which blocks of analogous cDNA containing multiple mutations are replaced, in addition to single site-specific changes. 127' Mutations at nt 37 and 55, within the 5' stem-loop structure, have been identified as candidates for partial restriction of Cendehill in joint. These mutations could affect the rate of translation of the nonstructural polyprotein. The mutation at nt 55 might also disrupt the production of a small peptide product of the first short open reading frame (ORF). An additional 4 nonconservative amino acid substitutions between nts 2828 and 4531 in the nonstructural genes appear likely to contribute further to the restricted phenotype. One of these occurs in a region with homology to the essential NSP3 protein of Sindbis virus, two near the protease domain, and one in the viral helicase domain. However, the functional consequences of such mutations may reach outside of the proteins in which they are found. In the alphaviruses the RNA polymerase activity resides in a separate protein, NSP4, but appears to require the helicase and all of the other nonstructural proteins, as well as host factors, to form an active replication complex (Barton et al, 1988; Barton et al, 1991). In addition, it is postulated that a complex of all of the alphavirus nonstructural proteins may be required to regulate the synthesis of positive and negative strand genomic RNAs (Strauss and Strauss, 1994). Instead of the four nonstructural proteins found in the alphaviruses, in RV the helicase and polymerase are linked on P90, while the methyltransferase, protease and NSP3-homologous domains are combined in P150. By analogy with the alphaviruses, these domains likely interact both within and between the two RV nonstructural proteins and a change in the helicase domain, for example, might affect the function of a putative replicase complex. These observations underscore the need for biochemical characterisation, as well as genetic location, when determining the mechanisms which underlie phenotypic diversity. 128 Binding of Cendehill strain to synovial cells did not appear to be impaired. Moreover, substitution of the structural gene region of Cendehill into the Therien infectious clone, pROBO302, did not have any effect on the replication of progeny in synovial cells, suggesting that these genes and control regions do not play a role in joint. However, since the progeny of pROBO302 (ROBO302) are also considerably restricted in synovial cells, involvement of the structural genes cannot be ruled out. Restriction of RQBO302 in SC was not due to alterations as a result of the transfection process, since the progeny of electroporated Therien RNA (TherienR) replicated well in these cells. Rather, pROBO302 may have been constructed using cDNA from a phenotypic variant of our Therien strain. Pugachev et al (1997) report that both the f-Therien and w-Therien strains which were used to construct pROBO302 had clear or turbid plaque morphologies, respectively. Neither of these strains were reported as generating microfocal plaques which we and other researchers have found as the predominant plaque morphology for Therien and other wt+ strains (Kouri et al, 1974; Miki & Chantler, 1992; Chantler et al, 1993). These observations illustrate the importance of correlating phenotype with sequence for each strain under study. A survey of the literature shows considerable diversity in the sequences obtained for the same reported strains (Bosma et al 1996; Johnstone et al,1996; , Domingues et al; Pugachev et al 1997b; Lund & Chantler current study). In order to study joint restriction in more detail another infectious clone would need to be constructed to replace pROBO302, using cDNA from our Therien strain, and shown to have the correct phenotype in joint tissue. The observation that transfection of RV RNA can produce progeny with an altered phenotype which is stable upon serial passage is previously unreported in the 129 literature. If such a phenomenon is also able to occur in the body this suggests a new mechanism for the generation of viral diversity. Although a rare event, Koch (1973) reported that isolated poliovirus RNA was able to infect Hela cells in isotonic medium containing no additives. These studies emphasise the importance of including transfection-derived controls in studies contrasting the properties of infectious clones (which must undergo transfection to produce virus) with parental strains. Sequence analysis has shown that the collective frequency of amino acid substitution for three strains (Cendehill, RA27/3, M33) relative to Therien is 3.3% for P150 and 0.7% for P90, suggesting that the helicase/replicase domains are highly conserved. There appears to be a mutational "hot spot" between nts 2100 - 2450, immediately upstream from the region with homology to the Sindbis virus NSP3 domain. The frequency of substitution within these 117 amino acids is 15%. Pugachev et al (1997) noted that this region bears sequence homology to a "proline hinge" motif which is also found in hepatitis E and beet yellow necrotic vein viruses (Koonin et al, 1992). ; Given the relative conservation of the rest of the genome it seems unlikely that such a high frequency of mutation has arisen because this region is non-essential and tolerates high random variability. It is interesting to consider that diversity may have evolved in this segment of the polyprotein to facilitate interactions with a host factor/s which would vary between cell types. In the structural genes the collective frequency of amino acid substitution is 2.6% for C, 5.3% for E2 and 2.2% for E1. We would expect that E1 would be the more conserved of the two envelope proteins since it is reported to carry the major neutralisation and hemagglutination domain, signifying its role in receptor-binding. In 130 addition E1 is believed to carry the fusion properties of the virus which participate in viral entry (Gillam S, personal communication, 1997). Although Cendehill strain has no reported association with recurrent joint complications, it was also considered to generate insufficient humoral immunity and was recently replaced as the European vaccine by RA27/3. Although vaccination with RA27/3 results in satisfactory immunity it has been linked to a low level of recurrent or chronic arthritis. Since the genetic changes which restrict the growth of Cendehill in synovial cells have been localised to the nonstructural gene region, it should be possible to engineer a vaccine by recombination between the Cendehill and RA27/3 strains which retains the antigenicity of RA27/3, but is incapable of growth in joint. 131 R E F E R E N C E S Alain R, Nadon F, Seguin C, Payment P, Trudel M (1987) Rapid virus subunit visualisation by direct sedimentation of samples on electron microscope grids. 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Those mutations which are specific to Cendehill strain are marked by * and b o l d type; others are in common with the M33 strain. Mutations which are specific to Cendehill and which result in amino acid changes are shaded. The symbol ">" denotes "changed to" (eg. A>C = A changed to C) (ntr = nontranslated region) NT# NT AA 37* T>C (nt 55* A>G 118* O T 124 A>C 358* T>C 502 T>C 586 T>C 602 O T 655 O T 718* O T 721 O T 1000* G>A 1186 G>A 1573 O T 1717 O T 1762 O T 1942 O T 2014 A>G 2020 T>C 2071 T>C 2152 O T 2183 O G R>G 2189 T>C S>L 2190 O T Ii 2198 O A A>T 2266 T>C 2292 O T A>V 2341 T>G 2361 T>C I>T 2370 O A O E 2412 G>A O Y 2416 O A 2442 O A R>K 2449* A>G 2494 O T 2533 O T 2545* O T 2572 A>G 2584 T>C 2633 A>G N>D 2671 A>G r) Appendix A cont. NT# N T A A 2829* G>A O Y 2 8 8 7 O T 2 9 1 2 A > G T > A 3060* A>G N>G 3164* T>C Y>H 3193* T>C 3 2 3 8 O G 3296* T>C L>L 3 4 5 9 O T A > V 3528* O T A>V 3 6 8 2 T>C 3 7 3 0 G>A 3748* T>A 3 7 9 3 .' A > G 3 9 1 3 C > T 3 9 1 9 T>C 3 9 4 3 T>C 3 9 4 9 C > T 4 1 1 7 ' T>C 4 1 5 3 T>C 4 2 4 7 C>G R > A 4 2 4 8 G>C II 4276* O A 4530* O T T>I 4681* A>G 4 6 8 7 -. A > G 4712* O T L>L 4 7 5 9 A > C 4825* O T 4840* O A 4 8 5 8 T > C 4 9 6 3 G>A 5 0 4 7 • C > T . 5 2 0 3 T > C 53-62- C > T 5497* O T 5 5 0 3 G>A 5 4 2 7 T > C ' 5 5 6 Q C > T 5 6 2 3 T > C 5 6 3 8 C > T 5 7 8 8 C > T 5 7 9 4 C > T 5848* O T 5 8 9 0 C > T . 5 9 3 8 T > C 5 9 7 7 O T 6 0 1 6 T > C 6 0 2 8 * O T 6 0 3 1 . O A 6 0 4 0 O T 6 0 5 2 T > A Appendix A cont. NT# NT A A 6118 G>A 6148 A>C 6244 O T 6385 O T 6398 T>C 6 4 1 9 * T>C ( n t r ) ( n t r ) 146 A P P E N D I X B S t r u c t u r a l G e n e R e g i o n M u t a t i o n s The mutations found in Cendehill relative to Therien within the structural gene region is shown. Those mutations which are specific to Cendehill strain are marked by * and b o l d type; others are in common with the M33 strain. Mutations which are specific to Cendehill and which result in amino acid changes are shaded. The symbol ">" denotes "changed to" (eg. A>C = A changed to C) (ntr = nontranslated region; g = glycosylation site) NT# NT AA 647 6 A>C ( n t r ) 6480 T>C ( n t r ) 6520 T>C 6547 O T 6611* T>C S>P 6 7 2 ? O A R>K 6756 O A 6770* A>G T>G 6771* O G II 6856* O T 6871 O T 6883 A>G 6901 A>G 6922* G>C 6937 O T 6946* O T 6979 O A 7012 T>C 7108 O T 7117* O T 7141 T>C 7204 O T 7234* A>C 7366 T>C 7385 A>G T>A 7428* O T A>V 7444 T>C 7452 T>C L>P 7466 T>C O R 7489 O T 7534 T>C 7562 T>C Y>H 7660 T>C 7746 . O T T > I ( g ) 7747 O T " (g) 7945 • T>C 7966 O T 8023 O T 8024 O A A>T 8119 T>C 8125 O G Appendix B cont. NT# NT A A 813 7 O T 8 2 2 1 G>A 83 0 6 G>A A>T 8338 T>C 8353* T>C 8407* T>C 8459 G>A V>T 8599 T>C 8732 O T 8744 O T 8770 T>A 8786* A>G N>D(g) 8788* O T " (g) •QQSf'- C>T 8864* O A L>M 8897 O A 8929* A>T 8932 O T 8935 O T 9013 O T 9 0 67 ' A>G 9180* A>T H>L 9193* O T 9208 T>C 9254* G>A A>T 92 66 A>G T>A 9299* G>T 9346 O A 9355 O G 9358 T>C 9386 O G L>V 9395 O T V>F 9483 O G T>S 9496 O T 9592 O T 9697 , T>G 9730 O C ( n t r ) 9740 O T ( n t r ) 9741* O T (ntr) A p p e n d i x C S o l u t i o n s Antibiotics Collagenase Buffer: HBSS+ Hybridisation Buffer (Northern) Nick Translation Buffer Northern Gel Buffer (50 X) spectinomycin 50 ug/ml chloramphenicol 35 ampicillin 50 1 % collagenase (els II) 0.1 M Tris (pH 7.5) 0.1MCaCI 2 5.4mM KCL 0.44mM K H 2 P 0 4 137mMNaCI 0.33mM N a 2 H P 0 4 5.5mM D-glucose 1.26mM CaCI 2 0.81 mM M g S 0 4 5 x S S C 1mM EDTA 1 % SDS 200 ug/ml denatured salmon sperm 50mM Tris-HCI (pH 7.5) 10mM MgCI 2 0.1 mM DTT 2mM (dATP, dTTP, dGTP) 2uM alpha 3 2 P-dCTP 2.5 U DNA Pol 1 0.025 ng DNase 1 0.5 ug DNA template ddH 2 0 to 25 ul 500mM N a H 2 P 0 4 500mM N a 2 H P 0 4 pH 6.5 PBS (pH 7.0) 150mM NaCl 7.5mM N a 2 H P 0 4 2.5mM N a H 2 P 0 4 149 RNA Sample Buffer RNA Loading Buffer SOC Broth Solution 1 16 ul DMSO 3 ul 10mM N a P 0 4 (pH 6.5) 5 ul 6M glyoxal (5 ul RNA in DdH 20) 50% glycerol 0.4% bromophenol blue 10mM N a P 0 4 (pH 6.5) 2% tryptone 0.5% yeast extract 10mM NaCl 2.5mM KCI 10mM MgCI 2 10mM M g S 0 4 20mM glucose 50mM glucose 25mM Tris-CI (pH 8.0) 10mM EDTA Solution 2 0.2M NaOH 1%SDS Solution 3 5M KOAc 300 ml glacial acetic acid 57.5 ml ddH 2 0 142.5 ml SSC 0.15 M NaCl 0.015M Na-citrate pH 7.0 TAE TBS 40mM Tris-acetate 2mM EDTA pH 8.5 25mM Tris-HCI (pH 7.4) 140mM NaCl 3mM KCI TE Buffer Transport Medium: 10mM Tris-CI (pH 7.5) 1'mM EDTA (pH 8.0) HBSS+ 300U/ml penicillin 300ug/ml streptomycin 0;75ug/ml amphotericin Washing Buffers (Northern) 2 X YT Broth 1) 2 x SSC, 0 . 1 % SDS 2) 1 x SSC, 0 . 1 % SDS 3) 0.1 xSSC, 0 . 1 % SDS yeast extract 10 g/l tryptone 20 g NaCl 10 g 


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