INDUCTION OF APOPTOSIS BY AFRICAN HORSE SICKNESS VIRUS IN MAMMALIAN CELLS

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GENERAL INTRODUCTION

Just under a 120 years ago, in 1892, Dimitry Ivanovsky demonstrated that the sap of leaves infected with tobacco mosaic disease remained infectious, even after filtration through a Chamberland filter capable of retaining bacteria. In 1898, Beijerinck showed that the tobacco mosaic agent could pass through the fine filter of an agar plug, that it required growing cells (unlike most bacteria) and that it survived drying but not boiling. In the same year, Loeffler and Frosch showed that the agent causing foot-and-mouth disease of cattle could not be removed by filtration. Not only did these reports point to the existence of disease agents smaller than any known before, but they are considered to be the first reports to establish the existence of viruses. Indeed, the term filterable agent was the name first used to describe these organisms well before the term viruses were specifically applied to them (Levy et al., 1994). Today, hundreds of viruses are known and many of them are of agricultural and medical importance. Amongst these is African horse sickness virus (AHSV), the causative agent of African horse sickness (AHS).
This disease is one of the most lethal diseases of equids and is characterized by clinical signs that develop as a consequence of damage to the circulatory and respiratory systems, thus giving rise to serious effusion and haemorrhage in various organs and tissues (Mellor and Hamblin, 2004). Since the first demonstration by Clem et al. (1991) that apoptotic cell death plays a major role in viral disease mechanisms, it is now recognized that many animal viruses are capable of inducing apoptosis in infected cells (Clarke and Tyler, 2009) and that apoptosis contributes significantly to heir pathogenesis (O’Donnell et al., 2005; Umeshappa et al., 2010).
Much of the pioneering research on AHS was performed by Sir Arnold Theiler during the early 20th century. In 1900, he demonstrated the filterability of the pathogen through Berkefield and Chamberland filters, thereby indicating that the pathogen was indeed a virus. Theiler’s research lso indicated that there existed immunologically distinct strains of the AHS agent, since immunity acquired against one strain did not always protect the horse when challenged by a heterologous strain. In 1903, Theiler and Pitchford-Watkins established that AHSV may be transmitted by biting insects and, in 1921, Theiler reported the first detailed descriptions of the clinical signs and lesions produced by infections with AHSV (reviewed in Coetzer and Erasmus, 1994). During the late 1960s and 1970s, several studies were undertaken aimed at characterizing the structure and morphology of AHSV (Verwoerd and Huismans, 1969; Oellerman et al., 197 Bremer, 1976). With the advent of gene cloning, genetic engineering and protein expression technologies, much progress has been made regarding structure-function relationships of different AHSV genes and encoded gene products (Uitenweerde et al., 1995; Maree and Huismans, 1997; van Niekerk et al., 2001; de Waal and Huismans, 2005). Despite this progress, much still remains to be learned, amongst other, regarding the role of individual AHSV proteins within the context of infected host cells, the interaction of individual viral proteins with host cellular proteins, as well as viral proteins and cellular mechanisms that contribute to the molecular basis of AHS disease and pathogenesis. During the last decade, the phenomenon of RNA interference (RNAi), a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) initiates specific cleavage of cytoplasmic mRNA (Fire et al., 1998), has emerged as a powerful genetic tool whereby some of these types of questions may be addressed.
The review will summarize the current information concerning AHSV and will highlight the role of individual viral proteins in the infectious cycle of the virus. This will be followed b discussions of RNAi and its development as a tool for heterologous gene silencing in mammalian cells, as well as signalling pathways involved in virus-induced apoptosis in mammalian cells.

CHAPTER ONE: LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
1.2 AFRICAN HORSE SICKNESS (AHS
1.3 AFRICAN HORSE SICKNESS VIRUS (AHSV)
1.3.1 Taxonomic classification
1.3.2 Virion structure
1.3.3 Viral genome
1.3.4 AHSV proteins
1.3.4.1 Nonstructural proteins
1.3.4.2 Core proteins
1.3.4.3 Outer capsid proteins
1.4 ORBIVIRUS REPLICATION AND MORPHOGENESIS
1.5 RNA INTERFERENCE (RNAi)
1.5.1 The mechanism of RNAi
1.5.2 Developing RNAi for use in mammalian cells
1.5.2.1 siRNA design, synthesis and delivery
1.5.2.2 Plasmid- and viral vector-expressed shRNAs
1.5.2.3 Specificity of siRNA
1.5.3 Application of RNAi to viruses with a segmented dsRNA genome
1.6 APOPTOSIS
1.6.1 Caspases
1.6.2 Caspase signaling pathways
1.6.2.1 The intrinsic pathway
1.6.2.2 The extrinsic pathway
1.6.3 Regulation of apoptosis and caspase activation
1.6.4 Viruses and apoptosis
1.7 AIMS OF THIS INVESTIGATION
CHAPTER TWO: SILENCING OF AFRICAN HORSE SICKNESS VIRUS VP5 GENE EXPRESSION BY SHORT HAIRPIN RNA AND SMALL INTERFERING RNA IN MAMMALIAN CELLS
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Bacterial strains and plasmids
2.2.2 Cell culture and viruses
2.2.3 DNA oligonucleotides for shRNA construction
2.2.4 Construction of recombinant pENTR™/H1/TO vectors
2.2.4.1 Preparation of double-stranded DNA oligonucleotides
2.2.4.2 Cloning of double-stranded DNA oligonucleotides
2.2.4.3 Plasmid DNA extraction and quantification
2.2.4.4 Nucleotide sequencing
2.2.5 Short hairpin RNA (shRNA)-mediated silencing of AHSV-9 VP5 gene expression in Vero cells
2.2.5.1 Generation of stably transfected Vero cell lines
2.2.5.2 Viral challenge assay
2.2.6 Small interfering RNA (siRNA)-mediated silencing of AHSV-9 VP5 gene expression in BHK-21 cells
2.2.6.1 siRNAs
2.2.6.2 Viral challenge assay
2.2.7 Quantitative real-time polymerase chain reaction (real-time PCR)
2.2.7.1 Oligonucleotides
2.2.7.2 RNA extraction
2.2.7.3 cDNA synthesis
2.2.7.4 Control PCR reactions
2.2.7.5 Quantitative real-time PCR
2.2.7.6 Data analysis
2.3 RESULTS
2.3.1 Characterization of the β2-microglobulin (β2-MG) gene as an appropriate reference gene for quantitative real-time PCR
2.3.2 Short hairpin RNA (shRNA)-mediated silencing of AHSV-9 VP5 gene expression in stable Vero cell lines
2.3.2.1 Construction of recombinant pENTR™/H1/TO RNAi entry vectors
2.3.2.2 shRNA-mediated gene silencing of AHSV-9 VP5 gene expression in Vero cells
2.3.3 siRNA-mediated silencing of AHSV-9 VP5 gene expression in BHK-21 cells
2.4 DISCUSSION
CHAPTER THREE: EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF THE AFRICAN HORSE SICKNESS VP5 PROTEIN
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Bacterial strains and plasmids
3.2.2 DNA amplification
3.2.2.1 Oligonucleotides
3.2.2.2 Polymerase chain reaction (PCR)
3.2.3 Agarose gel electrophoresis
3.2.4 Recovery of DNA fragments from agarose gels
3.2.5 Cloning of DNA fragments into plasmid vectors
3.2.5.1 Ligation of DNA fragments to vector DNA
3.2.5.2 Preparation of competent cells
3.2.5.3 Transformation of competent cells
3.2.5.4 Plasmid DNA extraction
3.2.5.5 Restriction endonuclease digestions
3.2.6 Nucleotide sequencing and sequence analysis
3.2.7 Plasmid constructs
3.2.8 Generation of recombinant baculoviruses
3.2.8.1 Cells and culture conditions
3.2.8.2 Co-transfection of Sf-9 cells
3.2.8.3 Plaque assays
3.2.8.4 Preparation of large-scale virus stocks
3.2.9 Analysis of recombinant baculovirus-expressed proteins
3.2.9.1 Expression of recombinant fusion proteins
3.2.9.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
3.2.9.3 Immunoblot analysis
3.2.10 Cytotoxicity assays
3.2.10.1 Determination of the optimal cell concentration
3.2.10.2 Cytotoxicity of baculovirus-expressed VP5 proteins
3.2.10.3 Cytotoxicity of synthetic VP5 peptides
3.3 RESULTS
3.3.1 Secondary structure analysis of AHSV-9 VP5
3.3.2 Construction of recombinant baculoviruses expressing full-length and truncated VP5 proteins
3.3.3 Characterization of VP5 proteins synthesized in recombinant baculovirusinfected Sf-9 cells
3.3.4 Effect of baculovirus-expressed full-length and truncated VP5 proteins on plasma membrane permeability of Sf-9 cells
3.3.5 Effect of synthetic VP5 peptides on plasma membrane permeability of Sf-9 cells
3.4 DISCUSSION
CHAPTER FOUR: INDUCTION OF APOPTOSIS BY AFRICAN HORSE SICKNESS VIRUS IN MAMMALIAN CELLS
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.2.1 Cells and viruses
4.2.2 Analyses of AHSV-infected BHK-21 and KC cells
4.2.2.1 Preparation of AHSV-infected cell lysates
4.2.2.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
4.2.2.3 Immunoblot analysis
4.2.3 Microscopy
4.2.4 DNA fragmentation analysis
4.2.5 Quantification of apoptosis
4.2.6 Caspase-3 activation assays
4.2.7 Detection of mitochondrial membrane depolarization
4.2.7.1 Flow cytometry
4.2.7.2 Confocal laser scanning microscopy of AHSV-infected BHK-21 cells
4.3 RESULTS
4.3.1 Microscopic examination of AHSV-infected BHK-21 and KC cells
4.3.2 DNA fragmentation analysis in AHSV-infected BHK-21 and KC cells
4.3.3 Caspase-3 activation in AHSV-infected BHK-21 cells
4.3.4 Mitochondrial membrane depolarization in AHSV-infected BHK-21 cells
4.4 DISCUSSION
CHAPTER FIVE: CONCLUDING REMARKS
PUBLICATIONS AND CONGRESS CONTRIBUTIONS
REFERENCES