MOLECULAR BIOLOGY OF THE FAMILY REOVIRIDAE

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Positive-sense ssRNA viruses

In the case of positive-strand RNA viruses, the full -length genomic RNA functions as mRNA, directing the production of some or all viral proteins necessary for the initiation of virus propagation, and as template for viral RNA replication, making these viruses highly amenable to genetic engineering.
Advances in molecular techniques have enabled direct genetic manipulation of positive-strand RNA viruses, through the use of cDNA intermediates to produce biologically active RNA molecules . Thus, infectious positive-strand RNA viruses can be generated from cloned cDNAs, by transfecting plasmids (or RNA transcribed from plasmids) containing the viral genome directly into cells, as was first demonstrated with Poliovirus (PV; Racaniello & Baltimore 1981). Due to their generally smaller genome sizes compared to DNA viruses, whole RNA virus genomes can be cloned as cDNA and manipulated at will.
This approach has been successfully achieved for multiple small and medium sized positive-strand RNA viruses (see Boyer & Haenni 1994), greatly enhancing the potential of investigations. Indeed, they can facilitate studies of viruses that are present only in low titres in infected cells or whose isolation is problematic. For instance, the development of a reverse genetics system for caliciviruses (Sosnovtsev & Green 1995) was anticipated to assist in the identification of the molecular basis for the strong host- and tissue-specific restrictions of many members of the Caliciviridae and to lead to the development of recombinant DNA-based systems for the non-cultivatable caliciviruses.
Clearly, the synthesis and cloning of full-length cDNA of larger positive ssRNA viral genomes, with correct termini, and the instability of these clones in bacteria, can be troublesome (reviewed in Boyer & Haenni1994). However, remarkable success has been achieved in studies using alphaviruses ·such as Sindbis virus (Rice et al. 1987) and Semliki forest virus (Liljestrom et al. 1991). cDNA-derived RNAs of these positive-strand RNA viruses were used to efficiently rescue infectious viruses, thus allowing extensive analyses of promoter elements of the viral RNAs as well as structure-function studies of the viral proteins. Furthermore, these viruses have shown excellent potential for expressing large quantities of heterologous proteins via recombinant constructs.

CHAPTER 1: LITERATURE SURVEY
1. REVERSE GENETICS
1.1 DNA viruses
1.2 RNA viruses
1.2.1 Retroviruses
1.2.2 Single-strand RNA viruses
Positive-sense ssRNA viruses
Negative-sense ssRNA viruses
1.2.3 Double-strand RNA viruses
2. MOLECULAR BIOLOGY OF THE FAMILY REOVIRIDAE
2.1 Infection cycle 1
2.2 Development of reverse genetic systems for the Reoviridae
2.2.1 Transcription
2.2.2 Assortment
2.2.3 Replication
2.2.4 Assembly
3. COI\JCLUSION AND AIMS
CHAPTER 2: AHSV eDNA SYNTHESIS AND CLONING
1. INTRODUCTION
2. MATERIALS AI\JD METHODS
2.1 Cells and viruses
2.2 Isolation and purification of viral dsRNA
2.3 Oligonucleotide ligation
2.4 Determination of efficiency of oligonucleotide ligation
2.5 dsRNA size fractionation and purification
2 .6 cDNA synthesis
2.7 Size separation and purification of cDNA
2.8 Annealing of cDNA
2.9 G/C-tailed cloning of cDNA
2.10 PCR amplification of cDNA
2.11 Cloning of cDNA
2.12 Northern blotting of dsRNA
2.13 Sequencing of plasmid DNA
2.14 Labelling of probes
2.15 Geneclean™ purification of DNA
2.16 In vitro translation
2.17 Molecular biological manipulation of DNA
3. RESULTS
3.1 Efficiency of oligonucleotide ligation to AHSV dsRNA
3.2 Poly(dA)-oligonucleotide ligation strategy for cloning of AHSV genome segments
3.2.1 Poly(dA)-oligonucleotide ligation
3.2.2 cDNA synthesis
3.2.3 cDNA amplification and cloning
3.2.4 Clone analysis
4. DISCUSSION
CHAPTER 3: SEQUENCING OF GENOME SEGMENT 1 OF AHSV-9
1. INTRODUCTION
2. MATERIALS AND IVIETHODS
2.1 Cloning and construction of the AHSV-9 VP1 gene
2.2 Sequencing of the AHSV-9 VP1 gene
2 .3 Analysis of the AHSV-9 VP1 gene and gene product sequences
3. RESULTS
3.1 Cloning and sequence determination of AHSV-9 genome segment 1
3.2 Sequence analysis of AHSV-9 genome segment 1 and its encoded protein
3.3 Observation w ith reference to colony morphology and insert orientation
4 . DISCUSSION
CHAPTER 4: EXPRESSION ANALYSIS OF VP1 OF AHSV-9
1. INTRODUCTION
2 . MATERIALS AND METHODS
2 .1 In vivo polymerase assays
2.1.1 Cells and viruses
2 .1.2 Vectors and plasm ids
2.1 .3 Plasmid constructions
2.1.4 Sequencing
2 .1.5 In vitro transcription and translation
2.1 .6 In vivo protein expression, labelling and analysis
2.1 .7 RNA polymerase assay
2.1.8 Northern hybridisation
2 .2 Baculovirus protein expression
2.2.1 Cells
2.2 .2 Plasmid constructions
2 .2 .3 Transposition and preparation of recombinant bacmid DNA
2.2.4 Transfection
2.2.5 Baculovirus infection
2.2.6 Expressed protein analysis
2 .2.7 Virus titration and plaque purification
2.2.8 Protein labelling
2.2 .9 VP1 solubility assays
2 .3 Bacterial expression
2.3.1 Cells
2.3.2 Vectors
2.3 .3 Plasmid constructions
2.3.4 Expression
2.3.5 Expression analysis
3. RESULTS
3.1 vTF7-3 driven in vivo gene transcription and expression
3.1 .1 Vector constructs
3 .1 .2 In vitro transcription and translation
3 .1.3 In vivo expression
3.1 .4 In vivo polymerase assay
3.2 Baculovirus expression of AHSV VP1
3.2 .1 Construction of recombinant baculoviruses
3 .2 .2 VP1 expression
3.2.3 VP1 solubility assays
3.3 Bacterial expression of AHSV VP1
4. DISCUSSION
4.1 vTF7-3 driven in vivo gene transcription and expression
4 .2 Baculovirus expression
4 .3 Bacterial expression
CHAPTER 5: CONCLUDING REMARKS
REFERENCES
PUBLICATIONS