Investigating the importance of
co-expressed rotavirus proteins in the
development of a selection-free rotavirus
reverse genetics system
JF Wentzel
20134045
Thesis submitted for the degree Philosophiae Doctor in
Biochemistry at the Potchefstroom Campus of the North-West
University
Promoter:
Prof AA van Dijk
Co-promoter:
Dr HG O’Neill
ii
Ek wou so graag ‘n liggie sien… -Totius
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ACKNOWLEDGEMENTS
I am indebted to many individuals who gave their time, expertise, support, assistance and prayers to make this epic, and at times painful, adventure possible. To each and every one who contributed in any way, my most heartfelt appreciation. I wish to express my sincere gratitude, appreciation and thanks to the following people and institutions whose assistances made the completion of this thesis possible:
My supervisor, Prof. Alberdina A. van Dijk, for all the encouragement, guidance and moral support.
My co-supervisor, Dr. Hester G. O’Neill, for her valuable assistance and giving me the opportunity to take on this ambitious study.
Prof. Christiaan A. Potgieter for his interest in this study, sound scientific advices and providing me with cultured cells and plasmids.
Prof. Ulrich Desselberger for many invaluable discussions, ideas and encouragements throughout this study.
Dr. L. Yuan for providing the information on the passage history of four rotavirus Wa variants.
Angelique Lewies for her unwavering support, encouragement and understanding throughout all the ups and downs of this study. For project and personal finances I thank the European Foundation Initiative for Neglected Tropical Diseases, the National Research Foundation, the Poliomyelitis Research Foundation and the North-West University Potchefstroom Campus.
The unconditional love, support, trust and encouragement of my family and friends especially my mother, sister and brother.
Finally, my Heavenly Father who blessed me with wonderful opportunities and determination to complete this challenging study with endurance.
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TABLE OF CONTENTS
Acknowledgements iii
Table of contents iv
List of Figures x
List of Tables xii
Abbreviations xiv
Summary and Keywords xvii
Opsomming en Sleutel woorde xix
Publications associated with this study xxi
Conference presentations xxiii
1. CHAPTER 1: INTRODUCTION 1
1.1 Background and problem identification 1
1.2 Hypothesis 3
1.3 Aims and objectives 3
1.3.1 Aim of this study 3
1.3.2 Specific objectives of this study 4
1.4 Structure of thesis 4
1.5 Methodology and experimental procedures 6
2. CHAPTER 2: LITERATURE OVERVIEW 9
2.1 Introduction 9
2.2 Historical look at gastroenteritis and rotavirus 10
2.3 Rotavirus genome and protein structure 11
2.4 Rotavirus particle structure 14
2.5 Rotavirus evolution and classification 16
2.6 Replication cycle of rotavirus 22
2.6.1 Rotavirus attachment to host cell 22
2.6.2 Rotavirus cell penetration and uncoating 23
2.6.3 Transcription of viral mRNA 27
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2.6.5 Genomic RNA Replication and Packaging 29
2.6.6 Rotavirus Assembly 30
2.6.7 Virus release 31
2.7 Rotavirus pathogenesis 31
2.8 Immune response to rotavirus infections 32
2.8.1 Innate response to rotavirus infections 32
2.8.2 Acquired immunity 33
2.9 Rotavirus vaccines 39
2.10 Viral reverse genetic systems 40
2.11 dsRNA and rotavirus reverse genetic systems 41
3. CHAPTER 3: CONSENSUS SEQUENCE DETERMINATION AND ELUCIDATION OF 45 THE EVOLUTIONARY HISTORY OF A ROTAVIRUS Wa VARIANT REVEAL A
CLOSE RELATIONSHIP TO VARIOUS Wa VARIANTS DERIVED FROM THE ORIGINAL Wa STRAIN
3.1 Introduction 45
3.2 Materials and Methods 46
3.2.1 Rotavirus and cell culture propagation 46
3.2.2 Sequence-independent cDNA synthesis and genome
Amplification 46
3.2.3 Sequence and phylogenetic analyses 47
3.2.4 Molecular clock analyses and evolutionary rate
Estimations 49
3.3 Results and Discussion 50
3.3.1 Sequence data analysis and comparison to similar rotavirus
strains in GenBank 50
3.3.2 Description of rotavirus Wa variants and molecular clock
Analyses 56
3.3.3 Substitution rates and evolutionary pressures analyses 62
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4. CHAPTER 4: OPTIMISATION OF TRANSFECTION CONDITIONS FOR MA104, 66 COS-7, BSR and HEK293H CULTURED CELLS USING eGFP
4.1 Introduction 66
4.2 Materials and Methods 68
4.2.1 Evaluation of chemical based transfection reagents 68
4.2.1.1 Cultured cells and eGFP encoding plasmids 68
4.2.1.2 Transfection procedures of the different chemical
transfection reagents 68
4.2.1.3 In vitro transcription, transfection and expression
determination of rotavirus SA11 transcripts 70
4.2.1.4 In vitro transcription of the rotavirus SA11 genome
segments using double-layered particles 71
4.2.1.5 Transfection and expression determination of
rotavirus SA11 transcripts 71
4.2.2 Evaluating the effectiveness of electroporation in four
different mammalian cells using eGFP 72
4.3 Results and discussion 73
4.3.1 Determination of the best transfection reagent to DNA ratio 73
4.3.2 Evaluation of the effect of different transfection reagents
on cell viability 75
4.3.3 Optimisation of transfection methods and determination of the most effective transfection reagent for each
cultured cell type 76
4.3.4 Determining the four different transfection reagents
ability to successfully transfect rotavirus transcripts 79
4.3.5 Determining the efficacy of eGFP incorporation
with electroporation in four different mammalian cells 81
4.4 Summary 82
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ROTAVIRUS PROTEINS IN THE DEVELOPMENT OF A ROTAVIRUS TRANSCRIPT-BASED REVERSE GENETIC SYSTEM
5.1 Introduction 84
5.2 Materials and Methods 85
5.2.1 MA104 Codon-optimised rotavirus SA11 expression
Plasmids 85
5.2.2 Description of Wa and SA11-based expression plasmids 87
5.2.3 Transformation of ABLE C competent cells and
plasmid amplification 88
5.2.4 Plasmid extraction and purification 89
5.2.5 Transfection optimisation of rotavirus plasmids in
mammalian cell cultures 90
5.2.6 Preparation of transcriptionally active rotavirus SA11
double-layered particles 91
5.2.7 In vitro transcription of the rotavirus SA11 genome
segments using double-layered particle 91
5.2.8 Transfection of rotavirus SA11 transcripts on a variety
of cell lines 92
5.3 Results and Discussion 93
5.3.1 Optimisation of transfection of rotavirus expression plasmids conditions and expression of the viral
proteins they encode 93
5.3.2 Standardisation of transfection conditions for rotavirus
Transcripts 101
5.3.3 Transfection of DLP-derived rotavirus SA11 transcripts into a variety of mammalian cells in an attempt to
recover viable virus 105
5.3.4 Transfection of DLP-derived rotavirus SA11 transcripts in the presence of pre-expressed plasmid derived
rotavirus proteins 107
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6. CHAPTER 6: INVESTIGATING A ROTAVIRUS TRANSCRIPT SYSTEM AS A 117 POSSIBLE REVERSE GENETIC APPROACH
6.1 Introduction 117
6.2 Materials and Methods 119
6.2.1 Cells, plasmids and transcripts 119
6.2.2 In vitro transcription of GLYAT and RVFV mRNA 120
6.2.3 Transfection of HEK 293H cells and quantitative RT-PCR 120
6.2.4 Detection of the expression of RIG-I and MDA5 by flow
Cytometry 122
6.2.5 Determining the effect pre-expressed plasmid derived rotavirus proteins on the interferon response elicited by
rotavirus transcripts 122
6.2.6 Statistical analysis of qRT-PCR data 123
6.3 Results and Discussion 124
6.3.1 Comparison of the effect of rotavirus transcripts and plasmids encoding for rotavirus proteins on HEK 293H,
MA104 BSR and COS-7 cells 124
6.3.2 The interferon response of HEK 293H cells to rotavirus transcripts and plasmids containing rotavirus genome
segments 125
6.3.2.1 Cytokine response to rotavirus SA11 infection 126
6.3.2.2 Cytokine response to rotavirus SA11 transcripts 133
6.3.2.3 Cytokine response to rotavirus Wa encoding
plasmids 134
6.3.2.4 Cytokine response to rotavirus SA11 encoding
plasmids 136
6.3.3 The interferon response of HEK 293H cells to in vitro
derived transcripts of GLYAT and RVFV 137
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rotavirus transcripts in HEK 293H cells 139
6.4 Summary 146
7. CHAPTER 7: Closing remarks and future prospects 148
8. REFERENCES 153 9. APPENDICES APPENDIX A 175 APPENDIX B 190 APPENDIX C 198 APPENDIX D 199 APPENDIX E 203 APPENDIX F 207 APPENDIX G ` 222
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LIST OF FIGURES
CHAPTER 2 Page no.
Figure 2.1: Diagrammatic illustration of the genome and proteins organization of rotavirus SA11.
14
Figure 2.2: The structural architecture of rotavirus. 15
Figure 2.3: Phylogenetic relationship of rotavirus serogroups A – H based on all 11 genome segments.
19
Figure 2.4: Schematic illustration of the replication cycle of rotavirus. 25
Figure 2.5: Schematic summary of the innate immune response of a mammalian host cell to a rotavirus infection.
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CHAPTER 3
Figure 3.1: Nucleotide alignments of the WaCS and closely related Wa variants and rotavirus reference strains.
52
Figure 3.2: Amino acid alignments and predicted secondary structure of viral proteins exhibiting novel or noteworthy amino acid changes.
54
Figure 3.3: Schematic diagram summarising the known passage history of the rotavirus Wa variants originating from the 1974 infant human rotavirus Wa stool sample isolate.
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Figure 3.4: Combined maximum clade credibility (MCC) tree of all 11 genome segments of the 17 rotavirus sequences analysed using the Bayesian MCMC framework.
60
Figure 3.5: Comparison between genome segment 5, 7 and 8 of the WaCS and related variants exhibiting nucleotide repeats.
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CHAPTER 4
Figure 4.1: Comparison of eGFP expression in cultured cells transfected with different ratios of transfection reagents to eGFP DNA .
74
Figure 4.2: The influence of the four different transfection reagents on cell viability.
76
Figure: 4.3: Efficacy of four different transfection reagents for transfecting and expressing eGFP containing plasmids in mammalian cells.
78
Figure 4.4: Efficacy of four different transfection reagents for
transfecting rotavirus SA11 transcripts in mammalian cell
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Figure 4.5: Four different cell lines were electroporated in the presence of 1 μg eGFP containing plasmid DNA
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CHAPTER 5
Figure 5.1: Comparison between a normal T7 promoter sequence and the expression T7 promoter sequence used in the rotavirus Wa and SA11 constructs and the resulting rotavirus
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terminal sequences.
Figure 5.2: An overview of the general rotavirus insert design and plasmid composition
86
Figure 5.3: Rotavirus SA11 multiple genome segment insert plasmid composition
87
Figure 5.4: Plasmid map of phCMVDream 88
Figure 5.5: Comparison of the expression of rotavirus VP6 in cultured cell lines after transfection with SA11 and Wa expression
plasmids.
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Figure 5.6: The effect of different rFPV infection times and number of plasmid transfections on the percentage of MA104 cells expressing plasmid derived rotavirus SA11 VP6
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Figure 5.7: Comparison of the expression of plasmid derived rotavirus SA11 VP6 in MA104 cells infected with rFPV at different time.
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Figure 5.8: Immunostaining of MA104 cells expressing VP6 after transfection optimisation.
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Figure 5.9: Immunological detection of plasmid derived rotavirus protein expression in HEK 293H cells.
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Figure 5.10: Immunostaining of rotavirus SA11 VP6 of MA104 cells after the transfection of rotavirus SA11 VP6 containing plasmid DNA and DLP derived SA11 VP6 transcripts.
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Figure 5.11: Immunological detection of rotavirus protein expression from transcript.
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Figure 5.12: Comparison of expression of VP6 and NSP2 between the CMV controlled MA014 codon-optimised rotavirus SA11 plasmids and the T7 controlled rotavirus SA11 plasmids.
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CHAPTER 6
Figure 6.1: The log2 relative quantities of cytokine-encoding mRNA expression induced in HEK 293H cells by rotavirus SA11 transcripts and plasmids encoding rotavirus Wa and SA11 containing plasmids.
128
Figure 6.2: Comparison of the relative quantities of the expression of various cytokines induced in HEK 293H cells by in vitro derived GLYAT-, RVFV- and rotavirus transcripts.
138
Figure 6.3: The log2 relative quantities of cytokine-encoding mRNA expression induced in HEK 293H cells 24 hours after transfection of rotavirus SA11 transcripts.
140
Figure 6.4: Comparison of flow cytometry detected expression of RIG-I and MDA5 induced in HEK 293H cells by rotavirus transcripts and plasmids.
142
Figure 6.5: Innate immune response to rotavirus infection and likely strategies of rotavirus to subvert this immune response.
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LIST OF TABLES
Page no. CHAPTER 1
Table 1.1 Specific methods applied in this study. 7
CHAPTER 2
Table 2.1: The 11 rotavirus genome segments, encoded viral proteins and their functions based on the human rotavirus Wa.
13
Table 2.2: dsRNA viruses (Group III) classification. 18
CHAPTER 3
Table 3.1: GenBank accession numbers of rotavirus strains used in phylogenetic analysis and pairwise comparisons.
48
Table 3.2: Summary of the WaCS data determined with 454® pyrosequencing
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Table 3.3: Summary of the nucleotide substitution rates and possible sites under diversifying selection of the genome segments of the Wa rotavirus lineages.
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CHAPTER 4
Table 4.1: Electroporation parameters for the different mammalian cell lines.
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CHAPTER 5
Table 5.1: The effect of transfecting plasmids containing individually rotavirus Wa genome segments on MA104, COS-7, BSR and HEK 293H cells over a 120 hour period.
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Table 5.2: Evaluation of cell death following transfection with SA11 transcripts and level of VP6 expression detected.
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Table 5.3: Summary of transfection experiments in different tissue cultured cells with rotavirus SA11 DLP- derived transcripts.
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Table 5.4: MA104 cells transfected with different combinations of rotavirus SA11 DLP-derived transcripts and rotavirus Wa genome segment encoding plasmids.
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Table 5.5: MA104 cells transfected with different combinations of rotavirus SA11 DLP-derived transcripts and rotavirus SA11 genome segment encoding plasmids.
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Table 5.6: MA104 cells transfected with different combinations of rotavirus SA11 DLP-derived transcripts and rotavirus SA11 codon-optimised genome segment encoding plasmids.
112
CHAPTER 6
Table 6.1: Mechanisms of different viruses to evade the innate immune response of the host cell.
118
Table 6.2: Comparison of cell death following the transfection of rotavirus SA11 transcripts and rotavirus encoding plasmids.
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Table 6.3: Cytokine-encoding mRNA expression/suppression induced in HEK 293H cells following rotavirus infection and transfection of rotavirus SA11 transcripts and plasmids encoding rotavirus Wa and SA11 containing plasmids.
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ABBREVIATIONS
µg: Micro gram µl: Micro litre o C: Degrees Celsius aa: Amino acidATP: Adenosine triphosphate BCA: Bicinchoninic acid bp: Base pairs
cfu: Colony forming units CPE: Cytopathic effect CsCl: Caesium chloride Da: Dalton
ddH2O Double-distilled water DNA: Deoxyribonucleic acid DLP: Double-layered particle DPI: Days post infection
dsRNA: Double-stranded ribonucleic acid EB: Elution buffer
EDTA: Ethylene-diamine-tetra-acetic acid ELISA: Enzyme-linked immunosorbent assays EM: Electron microscope
ER: Endoplasmic reticulum FBS: Foetal bovine serum
FDA: Food and Drug Administration GSK: GlaxoSmithKline
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HPI: Hours post infection H: Hour
HSC70: Heat-shock cognate 70 proteins IgA: Immunoglobulin A
IgG: Immunoglobulin B IgM: Immunoglobulin M LB: Lysogeny broth kDa: Kilo Dalton
MA104: African green monkey kidney cell line MOI: Multiplicity of infection
ml: Millilitre
NIH: National Institutes of Health NTPase: Nucleosidetriphosphatase NWU: North-West University NSP: Non-structural protein OD: Optical density
ORF: Open reading frame
PAGE: Polyacrylamide gel electrophoresis PBS: Phosphate-buffered saline
PCR: Polymerase chain reaction pfu: Plaque forming units PLC: Phospholipase C
RdRp: RNA-dependent RNA polymerase RE: Restriction endonuclease
RNA: Ribonucleic acid
RPM: Revolutions per minute
RT-PCR: Real time polymerase chain reaction siRNA: Small interfering ribonucleic acid
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SOC: Super optimal broth with catabolite repression ssRNA: Single-stranded ribonucleic acid
SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis siRNA: Small interfering RNA
SLP: Single-layered particle TLP: Triple-layered particle TOI: Time of infection
UTR: Untranslated terminal region V: Volts
VP: Structural viral protein WHO: World Health Organisation
SUMMARY
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Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues.
This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence.
The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system.
Summary
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Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined co-expression systems were able to produce a viable rotavirus.
Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus.
Keywords:
rotavirus; transcript-based reverse genetics system; sequence-independent genome amplification; rotavirus Wa and SA11 strains; phylogenetic analysis; nucleotide substitution rate analysis; next-generation 454® pyrosequencing; consensus sequence determination; plasmid derived expression of rotavirus proteins; transfection optimisation; rotavirus transcript translation; innate immune response; interferon suppression; role of viroplasms
OPSOMMING
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Tru-genetika is ʼn innoverende molekulêre biologie gereedskap wat wetenskaplikes in staat stel om virale genome op cDNA vlak te manipuleer ten einde spesifieke mutante of kunsmatige virusse te genereer. Die tru-genetika sisteem vir die influensa virus is een van die beste illustrasies van die potensiële voordele van hierdie tegnologie. Hierdie tru-genetika sisteem vorm die basis vir die vermoë om die influensa vaksien variante gereeld aan te pas, om tred te hou met die nuutste seisoenale uitbrake. Tans, is daar ook tru- genetika sisteme beskikbaar vir talle RNA virusse. Seleksie-vrye tru-genetika sisteme is al ontwikkel vir lede van die Reoviridae familie, insluitende Afrika perde siekte virus, bloutongvirus en orthoreovirusse. Hierdie deurslaggewende tegnologie het al tot die generering van waardevolle bewyse gelei, met betrekking tot die replisering en patogenese van hierdie virusse. Ongelukkig was die ekstrapolering van die plasmied gebaseerde- sowel as die transkrip gebaseerde tru-genetika sisteme, nog onsuksesvol vir rotavirus. Die ontwikkeling van ʼn seleksie-vrye tru-genetika sisteem vir rotavirus sal ʼn belangrike bydrae lewer tot die sistematiese ondersoek van aspekte in die rotavirus lewensiklus waaroor daar nog onvoldoende kennis is. Sodanige begrip sal verder aanleiding gee tot die ontwikkeling van beter vaksines.
In hierdie studie word die belangrikheid van mede-uitgedrukte rotavirus proteïene in die ontwikkeling van ʼn seleksie-vrye rotavirus tru-genetika sisteem ondersoek. Die konsensus volgordes van die rotavirus Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) en SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) variante is ingespan om rotavirus uitdrukkingsplasmiede te ontwerp. Die konsensus nukleotied volgordes van die menslike rotavirus Wa variant was bepaal deur volgorde onafhanklike cDNA sintese en vermeerdering in kombinasie met volgende-generasie 454® pyrosequencing tegnologie. ʼn Totaal van 4 ongekende nukleotied veranderinge, wat ook lei tot aminosuur veranderinge, was opgemerk in genoom segment 7 (NSP3), genoom segment 9 (VP7) en genoom segment 10 (NSP4). In silico analises het aangedui dat geen van die opgemerkte nukleotied veranderinge, en gevolglike aminosuur veranderinge, enige merkwaardige effek op die virale struktuur het nie. Evolusionêre analises het daarop gedui dat die WaCS variant, waarvan die volgorde bepaal was, baie nou verwant is aan die ParWa en VirWa variante wat afkomstig is van die oorspronklike 1974 Wa isolaat. Ten spyte van reeks passerings in beide diere en selkulture, kom die rotavirus Wa genoom stabiel voor. As dit in ag geneem word dat die huidige verwysingsvolgorde vir rotavirus Wa saamgestel is uit verskeie Wa variante se volgordes, mag die WaCS volgorde moontlik ʼn meer gepaste verwysingsvolgorde wees.
Die rotavirus Wa en SA11 variante is geselekteer vir plasmied gebaseerde uitdrukking van rotavirus proteïene, onder die beheer van ʼn T7 promoter volgorde, omdat beide virusse goeie groei in MA104 selle toon, sowel as die toeganklikheid van hul konsensus volgordes. Die T7 RNA polimerase is verskaf deur ʼn rekombinante voëlgriepvirus. Na ekstensiewe transfeksie optimalisering op ʼn verskeidenheid soogdier sellyne, was MA104 selle die mees geskikte vir die uitdrukking van rotavirus proteïene vanuit plasmiede. Die uitdrukking van
Opsomming
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rotavirus Wa en SA11 VP1, VP6, NSP2 en NSP5 kon bevestig word deur immunokleuring van MA104 en HEK 293H selle. ʼn Alternatiewe benadering, wat die kodon geoptimaliseerde uitdrukking van die rotavirus SA11 repliseringskompleks in MA104 selle behels, is ook getoets. Hierdie stel plasmiede is onder beheer van ʼn CMV promotor en is onafhanklik van die voëlgriepvirus. Al drie plasmied uitrukkingstelle was ontwerp om in kombinasie met die transkrip gebaseerde tru-genetika sisteem te werk, wat die moontlikheid vir ʼn suksesvolle rotavirus tru-genetika sisteem te verbeter.
Rotavirus transkripte is voorberei deur van aktiewe rotavirus SA11 dubbellaag partikels (DLPs) gebruik te maak. MA104 en HEK 293H selle was die mees geskikte vir die uitdrukking van rotavirus transkripte, alhoewel die uitdrukking van rotavirus VP6 ook in HEK 293H, BSR en COS-7 gedemonstreer kon word deur immunokleuring. Addisioneel, kon die uitdrukking van VP1, NSP2 en NSP5 bevestig word deur immunofluorosensie in MA104 en HEK 293H selle. Hierdie is die eerste bewys van translasie van rotavirus transkripte in selkulture. ʼn Opvallende seldood patroon is opgemerk binne 24 uur in reaksie op die transfeksie van rotavirus transkripte. Hierdie seldood patroon hou nie verband met normale sitopatiese effekte (CPE) nie en geen lewensvatbare virusse kon herwin word nie. In ʼn poging om transkrip- en plasmiedsisteme te kombineer is ʼn tweeledige transfeksie strategie gevolg waar plasmiede, wat vir rotavirus proteïene kodeer, eerste getransfekteer word, gevolg deur rotavirus transkripte, 12 ure later. Die kodon geoptimaliseerde plasmiedstel was ontwerp op grond van die hipotese dat die uitdrukking van ʼn DLP-repliserings kompleks (VP1, VP2, VP3 en VP6) die replisering en verpakking van transkripte sou bystaan. As die kodon geoptimaliseerde plasmiede eerste getransfekteer is, het dit ooglopende vertraging in massa seldood, wat veroorsaak word deur transkripte, tot gevolg gehad. Geen van die mede-uitdrukkingsisteme was daartoe in staat om lewensvatbare rotavirus te lewer nie.
Laastens is die nie-spesifieke, aangebore immuunreaksie wat deur die transfeksie van rotavirus transkripte en plasmied uitgedrukte rotavirus proteïen veroorsaak word, ondersoek. Kwantitatiewe RT-PCR (qRT-PCR) eksperimente het daarop gedui dat rotavirus transkripte hoë vlakke van uitdrukking van die sitokines IFN- α1, IFN-1β, IFN-λ1 en CXCL10 induseer. Die uitdrukking van sekere rotavirus proteïene vanaf plasmiedes (VP3, VP7 en NSP5/6) het spesifieke aangebore immuunreaksies gestimuleer, terwyl ander virale proteïene (VP1, VP2, VP4 en NSP1) weer die vermoë het om sekere aangebore immuunreaksies betekenisvol te onderdruk. In die lig van hierdie onderdrukkingstendense, is die vermoë van spesifieke en/of kombinasies van rotavirus proteïene, om die aangebore immuunreaksies wat deur rotavirus transkripte uitgelok word, te onderdruk, ook ondersoek. qRT-PCR resultate het aangedui dat selle wat vooraf getransfekteer was met plamiede wat kodeer vir NSP1, NSP2 of ʼn kombinasie van NSP2 en NSP5, betekenisvol die uitdrukking van spesifieke sitokines onderdruk wat deur rotavirus transkripte geïnduseer word. Hierdie bevindinge dui daarop dat daar moontlik addisionele virale meganismes is om die aangebore immuunreaksies te onderdruk, as slegs die degradering van interferon regulerende faktore deur NSP1. Die onderdrukking van die sterk aangebore immuunreaksie wat rotavirus transkripte tot gevolg het, mag uiters belangrik wees in die veldtog om die
Opsomming
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repliseringsiklus van hierdie virus beter te verstaan en uiteindelik ʼn seleksie-vrye tru-genetika sisteem vir rotavirus te ontwikkel.
Sleutel woorde:
Rotavirus; transkrip-gebaseerde tru-genetika sisteem; volgorde onafhanklike cDNA sintese en vermeerdering; rotavirus Wa en SA11 variante; filogenetiese analise; nukleotied substitusie tempo analise; 454® pyrosequencing tegnologie; konsensus volgorde bepaling; plasmied afkomstige uitdrukking van rotavirus proteïene; transfeksie optimalisering; rotavirus transkrip translasie; aangebore immuunreaksie; interferon onderdrukking; rol van viroplasmas
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PUBLICATIONS ASSOCIATED WITH THIS STUDY
Published article:
Wentzel, J.F., Yuan, L., Rao, S., van Dijk, A.A. and O'Neill, H.G. 2013. Consensus sequence determination and elucidation of the evolutionary history of a rotavirus Wa variant reveal a close relationship to various Wa variants derived from the original Wa strain. Infection, Genetics and Evolution. 20. 276-283
Submitted manuscript:
J. F. Wentzel, L. H. du Plessis, L. Mlera, H.G. O’Neill and A. A. van Dijk. 2014. Rotavirus non-structural proteins NSP1, NSP2 and NSP5 suppress several innate immune responses in cells transfected with rotavirus (+)single-stranded RNAs. Submitted to Journal of General Virology. VIR/2014/061101
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CONFERENCE CONTRIBUTIONS
Poster presentations:
Wentzel, J.F., van Dijk, A.A. and O’Neill H.G. 2011. DETERMINATION OF THE ROTAVIRUS Wa CONSENSUS SEQUENCE BY SEQUENCE INDEPENDENT cDNA SYNTHESIS AND AMPLIFICATION COMBINED WITH 454 PYROSEQUENCING. Virology Africa Congress. Cape Town, South Africa
Wentzel, J.F., Yuan, L., Roa, S., van Dijk, A.A. and O’Neill H.G. 2013. CONSENSUS SEQUENCE DETERMINATION AND ELUCIDATION OF THE EVOLUTIONARY HISTORY OF A ROTAVIRUS Wa VARIANT DERIVED FROM THE ORIGINAL Wa ISOLATE. 5th European Rotavirus Biology Meeting, Valencia, Spain
Wentzel, J.F., du Plessis L. H., Mlera, L. van Dijk, A.A. and O’Neill H.G. 2013.SUPPRESSING THE INTERFERON RESPONSE ELICITED BY ROTAVIRUS TRANSCRIPTS USING PLASMID DERIVED ROTAVIRUS NON-STRUCTURAL PROTEINS NSP1, NSP2 and NSP5/6. 5th European Rotavirus Biology Meeting, Valencia, Spain