The major structural protein of the retrovirus HIV-1 is called Gag and is expressed as a 55 kDa poly-protein with six contiguous domains. These are labelled from the N-terminus as MA, CA, SP1, NC, SP2 and P6. There are two distinct assembly steps in the lifecycle of HIV-1, termed immature and mature assembly, both of which are essential to the production of infectious viral progeny and are as such potential targets of therapeutic intervention. The immature assembly step involves self-association of, typically, 1000- 2500 copies of Gag in a nucleic acid-dependent manner, resulting in formation of a spherical capsid immediately below the host cell membrane. The resulting immature, noninfectious virions are released from the cell and the viral protease, PR, hydrolyses Gag into its component domains. MA remains at the membrane and NC remains in complex with the genome, whilst CA reassembles as a mature capsid with a conical shape and 5,7- Fullerene geometry. In the immature and mature state, CA forms a lattice in which N-CA is arranged as hexamers linked to one another by C-CA dimerisation, but the exact interfaces and CA conformations are different between the two states. In this thesis, experiments are described which seek to establish how a single protein, CA, can form two distinct lattices, and what the role of NC-nucleic acid interactions are in immature assembly. Several Gag mutants are studied using a combination of NMR spectroscopy, fluorescence spectroscopy, electron microscopy and in vitro virus capsid assembly assays. It is shown that the NC domain does not intrinsically effect any modulation of the C-CA domain at the level of the first intermediates in the assembly pathways, and that nucleic acid is required to link two Gag molecules together in order to promote immature assembly.

The viral infected cell protein 0 (ICP0) is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. Following its entry to the nucleus, HSV-1 genomes become associated with nuclear sub-structures known as nuclear domain 10 (ND10). Individual ND10 proteins, including the major constituent protein promyelocytic leukaemia protein (PML), have been reported to repress viral gene expression and confer intrinsic antiviral resistance against viral infection, a mechanism that is counteracted by ICP0. ICP0 has been shown to target specific cellular proteins for proteasome-mediated degradation through the E3 ubiquitin ligase activity conferred by its N-terminal C3HC4 RING finger domain. The RING finger domain of ICP0 has been reported to induce poly-ubiquitin chain formation in vitro in the presence of two E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2E1. However, the residues required for this interaction remain unknown. The primary purpose of this study was to map the interaction interface between the RING finger domain of ICP0 and its cognate E2 ubiquitin-conjugating enzymes and to investigate the importance of these interactions with regard to ICP0’s function during HSV-1 infection. In this study, site-directed mutagenesis was used to mutate specific ICP0 RING finger residues corresponding to residues on other RING finger proteins previously reported to be required for interaction with their respective cognate E2 ubiquitin-conjugating enzymes. Using yeast two-hybrid analysis, we demonstrate that point mutations at specific residues within loop-1 and -2 of the RING finger domain inhibited ICP0’s ability to interact with either UBE2D1 or UBE2E1. These mutations impaired or abolished ICP0's E3 ubiquitin ligase activity in vitro, inhibited its ability to conjugate ubiquitin and induce the degradation of PML in cell culture model systems. Furthermore, RING finger mutants that were unable to interact with either UBE2D1 or UBE2E1 also failed to complement the plaque formation efficiency (PFE) defect of an ICP0 null mutant virus and induce transcription from quiescent HSV-1 genomes. Molecular homology modelling of the ICP0 RING finger domain interaction interface with UBE2D1 based upon the X-ray crystallography structure of the Casitas B-lineage lymphoma proto-oncogene (c-Cbl)-UBE2L3 complex demonstrated that these residues form a potential contact interface with UBE2D1. These findings provide unique insight into the biological importance of ICP0’s ability to interact with components of the host-cell ubiquitin pathway for the efficient initiation of viral lytic infection and the reactivation of viral genomes from latency, two fundamentally important aspects in the life cycle and replication of HSV-1.

The Bunyamwera group of arthropod-borne viruses is one or the 13 serological groups that make up the genus, Bunyavirus of the family Bunyaviridae. There are 19 serologically distinct viruses in the Bunyamwern group and these have been subdivided into five, on the basis of greater antigenic similarity, as follows; the Bunyamwera Complex, the Wyeomyia Complex, Main Drain virus, Kairi virus and Guarda virus. The Bunyarnwera Complex contains 12 of these 19 viruses and the three viruses used in the genetic studies reported in this thesis represent the span of antigenic variation in the complex, The bunyaviruses , in general, are negitive-strand RNA viruses with three unique single-stranded genome segments (L,M and S) of total molecular weight, 4-6x106 daltons. There are four virus structural proteins two of which are nonglycosylated and are designated L (large) and N (nucleocapsid). The other two, G1 and G2, are glycosylated proteins located in the viral envelope. This thesis consists of a description of genetic interaction between three members of the Bunyamwera Complex and demonstrates that recombination by reassert-ment of genome segments occurs in the Bunyamwera Complex. The three viruses studied, Batai virus, Bunyamwera virus and Maguari virus, originated from three different continents - Asia, Africa and South America respectively - with no recognised overlap in their distribution.

A number of bacteriophages were isolated on the "soft rot" phytopathogens Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Several of these phages were used to obtain phage resistant mutants of SCRI1043, in order to investigate the role of the bacterial cell surface in virulence. While a number of phenotypic properties relating to pathogenicity and virulence of this strain have already been uncovered, little is known about the role of the cell surface in virulence. It was hoped that the use of phages would allow selection of mutants altered in both cell surface and virulence. Two phage resistant mutants, A5/22 and A5/8, exhibited reduced virulence when inoculated into potato plants, and were investigated further. Both mutants showed pleiotropic phenotypes. As well as reduced virulence and phage resistance, these mutants showed a number of other phenotypic alterations including, a reduction in the production of plant cell wall degrading enzymes, increased sensitivity to surface active agents, alterations in lipopolysaccharide and outer membrane protein profiles and reduced motility. A5/22 also exhibited bacteriostasis in the presence of galactose. Mutant A5/22 was more severely affected in its virulence than A5/8, which reflected in its greater deviation from the wild type phenotype. While no one phenotypic alteration could be directly associated with the reduced virulence of either mutant, a combination of several phenotypes may have been responsible. The phages isolated in this study were the first reported for these strains of Erwinia, and were therefore characterised under a number of criteria. All phages were grouped on the basis of structural morphology, restriction endonuclease digestion and host range. This is the first detailed characterisation of phages for Erwinia carotovora subsp. atroseptica. All isolated phages were tested for generalised transduction, a method of molecular genetic analysis so far unavailable to Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Two phages, ØKP and ØMl, were capable of generalised transduction in SCRI193 and SCRI1043 respectively. Both these phages were characterised and transducing frequencies improved. ØMl is the first transducing phage reported for Erwinia carotovora subsp. atroseptica and ØKP is only the second for Erwinia carotovora subsp. carotovora. Both phages are now being used extensively in the laboratory.

Several Invertebrate cell lines were examined for the presence of retrovirus particles. When cells of a Drosophila melanogaster cell line were disrupted and analysed on sucrose density gradients, a subcellular fraction with a density of 1.22 g/ml was found to possess endogenous DNA polymerase activity and could catalyse polymerization of deoxynucleotide triphosphates in response to added template primers. The latter activity had the cation and template, primer responses expected for reverse transcriptase. A high molecular weight polyadenylic acid-containing RNA was also purified from this fraction and could be dissociated by heat treatment into 30 to 35S and smaller species. Electron microscopy revealed the presence of toroidal forms reminiscent of intracytoplasmic A-type retrovirus particles within the Drosophila cells. Similar forms were found associated with the subcellular fraction of 1.22 g/ml. Drosophila cells could be induced by halogenated pyrimidines to release a polymerase activity capable of utilizing the template, primer poly(rA).oligo(dT). Pellets were prepared from induced cell culture fluids and analysed on sucrose gradients. Two peaks of poly(rA).oligo(dT) utilization were obtained of density 1.14 and 3,.20 g/ml. Radiolabelled RNA was prepared from these fractions. Radiolabelled material from both 1.14 and 1.20 g/ml density fractions sedimented at a position corresponding to 60-70S molecular weight RNA. Those high molecular weight RNAs could be dissociated by heat treatment into 30-35S species. Infection of insect cells with known Avian and Murine retroviruses was carried out. No positive results could be obtained by using polymerase activity as a measure of replication. By using Southern blot analysis, it was found that Drosophila cells could acquire exogenous viral sequences from XLV (KMSV). A number of cell lines were treated with the induced agent from Drosophila cells in an attempt to demonstrate infectivity. This could not be detected by the use of poly (rA) .oligo (dT) utilization as a measure of replication. These results are discussed and conclusions are drawn.

The invasion of novel host species by pathogens is one of the primary causes of pandemics and emerging diseases. Such host shifts are difficult to anticipate, in part because we lack an understanding of the barriers that prevent maintenance of pathogens by multiple species. Rabies virus provides an ideal model system in which to study these barriers, as well as the mechanisms used by host-shifting pathogens to overcome them. This virus appears to able to infect all mammals, but paradoxically is maintained in species-specific transmission cycles. These maintenance hosts are almost exclusively found within the Carnivora (carnivores) and Chiroptera (bats), and were established by numerous host shifts within and between these taxonomic orders. However, it remains unclear which – if any – adaptations are required when switching to a new host species, nor is it known why the virus is incapable of utilising multiple host species despite evidence of relatively frequent spill-overs between maintenance host species. This thesis describes investigations of the determinants of rabies virus host shifts across multiple scales of organisation, which aim to understand the host-specialisation of this virus. A meta-analysis of within- and cross-species infection studies shows evidence for a reduction in effective dose as the body temperature difference between the original and inoculated species increases. This affects the duration of the incubation period, suggesting adaptation of infectivity may be involved. Crucially, previous studies have linked temperature differences to infectivity differences in cell culture, and to the process of cell entry in particular. These data further show evidence for phylogenetic clustering of incubation period durations, which may be linked to clustering of sensitivity among inoculated host species. This in turn may help explain why only some carnivore and bat species are maintenance hosts of rabies virus. At the molecular level, whole genome sequencing of samples from a system in which rabies virus has made an unusual host shift to bovids suggests involvement of a single amino acid change in the acetylcholine receptor-binding site of the glycoprotein. Phylogenetic analyses involving these and other sequences shows evidence for distinct maintenance cycles in three host species in the same region, despite numerous spill-over infections between species. The involvement of the glycoprotein in this host shift further points to a role for infectivity changes in host adaptation. At the cellular level, the bovid-associated viruses show increased infectivity to a bovid cell line compared to canid-associated viruses. The bovid-associated viruses also show decreased infectivity to a canid cell line representing the original host species. Combined, these results point to a new model for the host adaptation of rabies virus. In this model, the infectivity of viruses is decreased at temperatures to which they are not adapted. This allows the emergence of genotypes with increased infectivity in the novel host species, which can be achieved through relatively minor genomic alterations. Because these adaptations are detrimental in the original host, they may combine with ecological differences between hosts to create the single-species maintenance cycles observed. Identifying the mechanisms constraining maintenance host range will allow better predictions of which host shifts are likely to be successful. In particular, the effect of body temperature difference found here is shown to explain a large part of the previously observed effect of phylogenetic distance between host species in reducing the frequency of rabies virus host shifts. The phylogenetic clustering of host sensitivity meanwhile, may explain how host shifts between very divergent hosts are possible.

The human immunodeficiency virus Rev protein is required for the cytoplasmic accumulation and probably the translational utilisation of mRNAs encoding the late viral structural proteins. Rev function is mediated by direct binding of the protein to a c/5-acting RNA sequence, the Rev-responsive element (RRE), carried by these mRNAs. Human adenovirus type 5 (Ad5) also encodes a protein, E1B 55K, required for the cytoplasmic accumulation of late viral mRNAs. Rev and E1B SSK both regulate gene expression post-transcriptionally and directly or indirectly act on the mRNA transport pathway. To explore the potential functional analogy between these two regulatory proteins, the components of the Rev/RRE system were introduced into AdS. By inserting the RRE into Ad5 late region L3 (expression of which is normally dependent on E1B 55K), and a rev expression cassette into early region E1A, a system was set up where the action of Rev could be directly compared with that of E1B 55K. A series of six recombinant adenoviruses was constructed which, together with two viruses already available, contained the rev gene and/or the RRE and/or the Ad5 E1B SSK gene in all possible combinations. Expression of functional Rev from the appropriate recombinants was confirmed by a CAT reporter gene assay. The eight viruses were used to study the effects of Rev and the RRE on the expression of Ad5 late RNAs and proteins. It was shown that the Rev/RRE system can detectably increase the cytoplasmic accumulation of certain Ad5 mRNAs in the absence of E1B 55K. Surprisingly, these included some mRNAs in which the RRE, although present in the primary transcripts, was removed from the mature species. A mechanism was proposed in which Rev/RRE action in this system may commit an RNA to a pathway of facilitated nuclear export before excision of the RRE during processing.

It has been reported that the phenomenon of biased hypermutation, associated with measles virus recovered from brain tissue, can be reproduced experimentally by propagation of measles virus in cultured neuroblastoma cells. The purpose of the research described in this thesis was to evaluate this claim and establish whether biased hypermutation could be induced in measles virus from an acute infection (ADD-MV), or in an RNA virus not implicated in neural disease such as human respiratory syncytial virus (huRS virus). Accordingly, an isolate of ADD-MV and huRS virus were passaged 10 times in two neuroblastoma cell lines (SK-N-SH and IMR-32), in human lung fibroblasts (MRC-5) and in monkey kidney epithelial (Vero) cells. The M gene sequence of huRS virus passaged 10 times in each cell line was compared with the M gene sequence of virus passaged once in the same cells. No base change was observed in the M gene of huRS virus as a result of passage in these cell lines. A 750 base pair fragment of the M gene of ADD-MV was sequenced. Comparison of the sequence obtained from virus passaged 10 times in Vero cells with that from virus passaged once did not reveal any base changes. The M gene sequence of virus passaged 10 times in SK-N-SH cells contained one base change, at position 222, a uracil to cytosine transition. Two base changes were observed in the M gene of virus passaged 10 times in MRC-5 cells, one at position 222, as described above, and one at position 217, a cytosine to guanine base change. No changes were observed in virus passaged 10 times in IMR-32 cells when compared to that passaged once. These data indicate that neither ADD-MV, nor huRS virus, underwent enhanced mutational events during propagation in cells of neural origin, contrary to observations reported previously by Wong, et al. (1989), which suggested that propagation in neuroblastoma cells induced biased hypermutation. To determine if biased hypermutation occurred at a frequency too low to be detected by cycle sequencing, the M gene of ADD-MV passaged 9 times in IMR-32 cells, generated by PCR, was cloned into the bacteriophage vector M13. Twenty-four clones were sequenced in full. In total 21 base changes were observed, however, no more than 5 changes were seen in any one clone, and there was no directional bias in the mutations observed. However, each clone contained a large deletion at the 3' region of the gene, ranging from 1008 to 622 bases. Thirteen of the clones also contained a 56 base insertion. This insertion was shown to have high identity with human mitochondrial transfer RNA (tRNA). The M gene of Yamagata-1 (SSPE) virus resembles that of acute measles, but contains a number of additional mutations, mainly uracil to cytosine transitions. Propagation of this virus in neuroblastoma cells resulted in additional mutations not observed in virus propagated in Vero cells (Wong et al., 1989). Yamagata-1 virus was passaged in the two neuroblastoma cell lines (SK-N-SH and IMR-32), both with and without propagation in human lung fibroblast cells prior to passage in the neuroblastoma cells. Three M gene clones from passage numbers one and five in both cell lines used for this experiment, were sequenced. Analysis of the M gene sequence of Yamagata-1 virus passaged in each cell line revealed that the virus stock contained a heterogeneous population of M gene mRNA's. Both mutated sequences and sequences identical to the M gene of ADD-MV were obtained. Comparisons of the M gene sequence from virus passaged 5 times in neuroblastoma cells with that passaged once revealed no biased hypermutational events. However, the sequence data determined from two clones appeared to be a chimera consisting of both ADD-MV M gene sequence and Yamagata-1 virus M gene sequence. The recombination point is at position 457, a G-A mutation which is the only base change common to both ADD-MV M gene and Yamagata-1 M gene sequence.

This work was undertaken to molecularly clone the Nirand strain of the polycythaemia inducing Friend spleen focus forming virus (F-SFFVp) together with its replication competent helper virus. Friend murine leukaemia virus (F-MuLV). To this aim viral extrachromosomal DNA molecules (of both linear and circular nature corresponding to both of these viruses) which could be induced in Friend cell lines were molecularly characterised with respect to their quantitative increase during differentiation and subcellular location. One cell line, F4-6 in which considerable amounts of these extrachromosomal DNAs could be detected was utilised for the large scale production and purification of both SFFVp and F-MuLV extrachromosomal DNA molecules. Restriction enzyme analysis of such molecules in combination with Southern blotting enabled the construction of primary restriction enzyme maps which allowed the selection of a molecular cloning strategy. Both of these viruses were subsequently molecularly cloned and were subsequently shown to be biologically active after transfection into recipient cells. A further aspect of this work was to construct a biologically active SFFVp containing a dominant selectable marker gene. A selectable construct was generated by joining different regions of the genome of the myeloproliferative sarcoma virus (MPSV) and SFFVp. A construct with the U3 region from the long terminal repeat (LTR) of SFFVp and the envelope gene region (gp55) of SFFVp (designated neo2 SFFVp) was found to be fully active as a selectable retroviral vector with identical biological properties to the wild type SFFVp neo2 SFFVp induced erythroid differentiation in vivo, with infected cells no longer requiring erythropoietin for differentiation. Furthermore neo2 SFFVp infected spleen cells could be used to generate immortal Friend leukaemia cells which were selectable with geneticin (neo2). A further construct, neo2 SFFV-M, which had a U3 region originating from NPSV was able to cause erythropoietin independent erythroid differentiation. However as compared to neo2 SFFVp and indeed the wild type SFFVp, neo2 SFFV-M was able to induce the erythroproliferative disease with a different kinetics. All attempts to isolate transformed Friend cells with this construct failed.

Mammalian reoviruses have been used as a model to study the control of host cell protein synthesis following infection. The reovirus genome is composed of 10 segments of double stranded RNA each of which, with one exception, encodes a single protein. The three serotypes of the virus show marked differences in their effects on host cell protein synthesis following viral infection. Serotypes 2 and 3 give pronounced inhibition and serotype 1 has little effect. Genetic studies using intertypic reassortants have mapped this property to the S4 gene of the virus which encodes the major outer capsid polypeptide σ3. The S4 gene of serotypes 1 and 3 have been sequenced and show 96 % homology at the amino acid level. Full length cDNA clones of the S4 gene of serotypes 1 and 3 (S4-1 and S4-3) were used to develop in vitro and in vivo assay systems to study the mechanism underlying the differential effects on host cell protein synthesis of the corresponding σ3 proteins. The ability of σ3 to inhibit host cell protein synthesis was investigated in two in vitro translation systems: the rabbit reticulocyte lysate system and S-10 cell extracts prepared from uninfected and type 1 and type 3 infected L-cells. Σ3 had no effect on either the translation of B-actin when used as a marker for eukaryotic protein synthesis, or endogenous protein synthesis in either of these systems. In vivo assay systems made use of the HIVLTR/tat inducible reporter system to drive σ3 expression. The effects of σ3 on the expression of two reporter genes, chloramphenicol acetyltransferase (CAT) and B-galactosidase were investigated in transient in vivo assay systems. Σ3 expressed from S4-3 cDNA caused a significant stimulation of reporter gene expression. In contrast σ3 from S4-1 cDNA had no effect on gene expression, suggesting a domain of S4-3 was responsible for this stimulation. In an attempt to domain map this property two hybrids were constructed, one containing the S' 800 base pairs of S4-1 and the 3' 400 base pairs of S4-3 (HY-1), and the other, HY-3, the converse of this. Σ3 synthesised from HY-1 stimulated reporter gene expression whereas that from HY-3 had no effect. Σ3 was readily detected in cells transiently expressing either the parental type 3 S4 cDNA or the HY-1 hybrid by immunoprecipitation and immunofluorescence. By contrast only very small amounts of oi were detected when either the S4 cDNA from type 1 or the HY-3 were used. Pulse-chase experiments indicated that the σ3 from type 1 virus is less stable than the type 3 protein and that the domain responsible for this stability is in the 3' terminus of the S4 gene. This domain is also that shown by others to be responsible for the ability of σ3 to bind dsRNA.