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Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.

Arboviruses are transmitted to vertebrates by arthropod vectors such as mosquitoes or ticks. The replication of Semliki Forest virus (SFV) (Togaviridae; Alphavirus) in vertebrate cells is well established and triggers cell death. SFV infection of Aedes albopictus mosquito cells was characterised. Virus growth curves were compared in three cell lines. Infection of U4.4 cells was persistent and did not affect growth of the culture. In contrast, infection of C6/36 and C7-10 cells resulted in a static culture with no cell division and no cell death. The response of U4.4 cells was characterised in greater detail using viruses containing fluorescent or luciferase markers within the replicase or structural open reading frame of the virus genome. Activation of the STAT/IMD pathway prior to SFV infection significantly reduced virus driven luciferase expression and virus production. Activation of the Toll pathway prior to SFV infection had no effect. However, activation of Toll in addition to STAT/IMD had a cumulative effect on luciferase expression and virus production. viRNAs were characterised by Illumina Solexa sequencing. Two percent of the small RNA species found in virus infected cells were derived from virus RNA. These were predominantly 21 nt long and mapped along the entire SFV genome and genome complementary RNAs. Generation of these viRNAs was not random. Some areas produced high frequencies and others no or very few; hot and cold spots respectively. There were no correlations between viRNA frequency and base pairing or secondary structures predictions. Cold spot-derived viRNAs were more effective than hot-spot viRNAs in inhibiting virus replication. Similar results were observed in Aedes aegypti-derived cells. Attempts were made to investigate the source of these viRNAs using a virus containing an IRES element which had been reported to prevent virus replication in insect cells but which did not efficiently do so in this study. A virus containing the RNAi inhibitor p19 was characterised and shown to increase virus production. Techniques for infecting mosquitoes via a blood meal feed were established. No infection was observed with virus replicon particles carrying a fluorescent marker gene. Infection was established using virus containing p19.

Fast-evolving RNA viruses are a leading cause of morbidity and mortality among human and animal populations, contributing significantly to both global health and economic burden. The advent and revolution of high-throughput sequencing has empowered phylogenetic analyses with increasing amounts of temporally and spatially sampled viral data. Moreover, the parallel advancement in molecular evolution and phylogenetic methods has provided investigators with a unique opportunity to gain detailed insight into the evolutionary and epidemiological dynamics of emerging viral pathogens. Using state-of-the-art statistical approaches, this thesis addresses some of the important but controversial questions in viral emergence. Chapter 2 introduces a new framework to quantify and investigate reassortment events in influenza A viruses. By developing a computationally efficient algorithm to calculate the largest common subtree for a pair of tree sets, which are estimated from diffe rent parts of the genome for the same taxa set, the level of phylogenetic incongruency due to reassortment can be appropriately ascertained. Chapters 3, 4 and 5 investigate the evolutionary origins of three diff erent viruses: the novel emergence and cross-species transmission of SARSCoV, the genesis and dissemination of the unique HCV circulating recombinant form, and the ancient divergence of all influenza viruses, respectively. Moreover, Chapter 4 presents an improved statistical framework, which provides more precise evolutionary estimates, by utilizing the hierarchical bayes approach to investigate recombination events in emerging RNA viruses. The last empirical study, presented in Chapter 6, applies the recently developed Bayesian phylogeography models to a large viral sequence dataset sampled from southern Viet Nam to examine the fine-scale spatiotemporal dynamics of endemic dengue in Southeast Asia. The work presented here reflects both the advancements made in sequencing technology and statistical phylogenetics, along with some of the challenges that remain in studying the emergence of fast-evolving RNA viruses. This thesis proposes new and improved solutions to these evolutionary problems, such as incorporating non-vertical evolution (i.e. homologous recombination and reassortment) into the phylodynamic framework, with the aim of facilitating future investigations of emerging viral diseases.

Epstein-Barr virus (EBV) is linked to the development of several human malignancies. Epstein-Barr Nuclear Antigen 2 (EBNA 2) is required for the immortalisation and continued proliferation of EBV-infected B-cells. EBNA 2 is a transcriptional regulator of both viral and cellular genes. The viral C promoter (Cp), regulated by EBNA 2, drives transcription of an ~120 kb pre-mRNA that is differentially spliced to generate messages encoding all the other EBNAs required for immortalisation. To study the regulation of Cp-transcript elongation, we used a pair of EBV-infected cell-lines to compare the transcriptional complexes associated with the Cp transcriptional unit and two shorter EBNA 2-regulated viral genes, LMP1 and LMP2A. Interestingly, we found an accumulation of RNA Polymerase II (Pol II) in association with the pausing factors DSIF and NELF at Cp, which were absent at the LMP gene locus. Further experiments revealed that C promoter sequences have a much higher propensity to occlude nucleosome formation, promoting TBP recruitment and Pol II accumulation. We also found highlevel recruitment of the Pol II C-terminal domain (CTD) kinase, pTEFb at Cp, increased Pol II Serine 2 CTD phosphorylation and retention at promoter-distal regions. Furthermore pTEFb recruitment at Cp was facilitated by association with the bromodomain protein Brd4 and Pol II pausing. By sustaining a nucleosome-free region and recruiting high levels of pTEFb, Cp elongation appears highly adapted to ensure production of the long EBNA-encoding transcript required to establish and maintain B-cell immortalisation. In additional studies we examined the association of methylated forms of EBNA 2 with viral and cellular genes. EBNA 2 is modified by asymmetric (aDMA) or symmetric (sDMA) arginine di-methylation in the arginine-glycine repeat region. We found that aDMA-modified EBNA 2 preferentially bound promoters to regulate gene expression, implicating this modification as a key regulator of EBNA 2 activity.

Coxsackievirus B5 (CBV5) is a positive sense, single-stranded RNA virus belonging to the Enterovirus genus of the Picornaviridae family. It can cause many serious diseases, including viral myocarditis (which can lead on to dilated cardiomyopathy), aseptic meningitis, and pancreatitis. The structure and cell cycle of CBV5 is typical of a picornavirus. Viral RNA is detected by Toll-like receptors (TLRs) and retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs). The RLR family, consisting of RIG-I, MDA5 (melanoma differentiation-associated gene 5), and LGP2, are pattern recognition receptors that detect a range of different viruses. RIG-I and MDA5 are homologous cytoplasmic proteins containing an N-terminal region with two caspase activation and recruitment domains (CARDs), a central SF2 type DExD/H-box RNA helicase domain, and a C-terminal repressor domain (RD). Once a viral ligand has been detected and bound by RIG-I and MDA5, both signal downstream through their CARDs to activate IRF3/7 and NF-κB indirectly, via the protein intermediate IPS-1 (IFN-β promoter stimulator 1), and initiate an immune response. RIG-I and MDA5 contribute to antiviral signalling in different ways depending on the virus involved. MDA5 has been shown to be critical for Picornaviridae detection, whilst RIG-I can detect a wide variety of different viruses and pathogen associated molecular patterns. Results presented here show the expression levels of both are upregulated in response to CBV5 infection in human cardiac cells, with MDA5 expression levels being slightly greater than RIG-I. However, in Huh cells, RIG-I expression levels are greater than those of MDA5, indicating that it plays a role in CBV5 sensing. The presence of both phospho-IκB (corresponding to NF-κB activation) and IRF3 is detected in both cardiac cells and Huh cells in response to CBV5, and IFN-β production is also greatly upregulated. RIG-I and MDA5 colocalise with the adaptor protein IPS-1 in response to CBV5 infection, again indicating the synergistic response by the two RLRs, and both RLRs form homodimers in the cytoplasm. Overall, this suggest that both MDA5 and RIG-I act synergistically to detect CBV5 and initiate a downstream immune response, although MDA5 appears to be the marginally stronger sensor.

Human noroviruses of the Caliciviridae family are the major cause of acute gastroenteritis in the developed world, but little is known about their molecular mechanisms of translation and replication. Since human noroviruses are not cultivatable in vitro, most molecular studies have therefore been based on the cultivatable murine norovirus (MNV) or feline calicivirus from the Vesivirus genera. Calicivirus translation is dependent on the viral VPg protein covalently linked to the 5’ end of viral RNA and removal of the VPg from calicivirus RNA results in loss of infectivity. VPg functions as a ‘cap substitute’ by interacting with eIF4E to recruit translation initiation factors (eIFs). Given that VPg plays a critical role in the virus life cycle, it is potentially a good anti-viral target. As an initial attempt to identify ways of targeting VPg as an anti-norovirus therapy, we used phage display to identify peptides with an affinity for VPg. Despite repeated attempts however, we failed to generate a peptide that had clear specificity for the calicivirus VPg. Since VPg has ability to bind the cap binding protein eIF4E, m7-GTP Sepharose affinity chromatography was used to isolate eIF4E and the associated VPg. Similar to the analysis of the MNV VPg-translation initiation factor complex, this method successfully isolated not only the human norovirus VPg, but also isolated the entire eIF4F complex and PABP from a cell based-norovirus replicon system. Human norovirus and MNV VPg proteins share 54% amino acid identity and MNV has been proposed as a good model for human norovirus. To begin structural and functional analysis of the human norovirus VPg in the context of an authentic viral life cycle, we introduced the human norovirus VPg into an infectious clone of MNV, to generate a chimera containing all the MNV non-structural and structural proteins but with the human norovirus VPg. Whilst the insertion of the human norovirus VPg into the MNV genome did not affect polyprotein processing, we were unable to recover infectious viral particles. Since the requirement for other cellular factors in the VPg-dependent translation is not fully understood, we used “tandem affinity purification” to further define the components of the initiation factor complex associated with VPg. Our data demonstrated that VPg associates with both canonical initiation factors and possibly non-canonical initiation factors. Further investigation suggested that the scaffold protein eIF4G, which bridges the interaction between PABP and eIF4E, binds directly to VPg via its central domain. To determine the function of eIF4G in the norovirus life cycle, we have silenced eIF4G isoforms in 293T cells using specific siRNAs and transfected them with infectious VPg-linked viral RNA. The siRNA-mediated knockdown of either eIF4G isoform resulted in a 9 fold reduction in virus titre, as well as reduction of viral RNA and protein synthesis, confirming that eIF4G has a functional role in norovirus life cycle. Furthermore, it is not known if the eIF4G-eIF4E interaction is not limiting for MNV translation. Thus, we altered the availability of eIF4E to bind eIF4G by overexpressing wild type or non-phosphorylatable mutant forms of the eIF4E binding protein 1 (4E-BP1) in cells. Overexpression of either the wild type 4E-BP1 or the non-phosphorylatable 4E-BP1 mutant in MNV infected cells showed no significant change in viral RNA, virus titre or protein expression suggesting that the eIF4G-eIF4E interaction in a VPg-translation initiation complex is not limiting for MNV translation. In addition to studying the interaction between VPg and the eIFs, we also examined the effect of mutations in VPg on virus viability and ability to bind the eIF4F complex. A number of mutants were generated in MNV infectious clone to identify residues that play a role in calicivirus translation. The mutational analysis of MNV VPg indicated the C-terminus of VPg is essential for the association of VPg with the components of eIF4F complex and virus viability. This work provides new insights on the interaction between noroviruses and the host cell, as well as the novel mechanisms that viruses use to synthesis their proteins.

Meq is a viral c-Jun analog and member of the AP-1 transcription factor family that acts as the primary oncoprotein encoded by Marek’s disease virus. Previous studies have shown that Meq interacts with a variety of proteins as part of its pivotal function in the development of Marek’s disease virus-induced lymphoma. The primary focus of this study was to identify the global interactome of the Meq oncoprotein. This was initially carried out by review of the Meq sequence and the subsequent identification of BATF and BATF3 as potential analogous partners. Interactions with novel proteins were also predicted based on the nature of charged interactions that mediate a leucine zipper dimerisation event. Furthermore, the Meq protein was used as bait in a yeast two-hybrid screen to produce a large data set of both known and novel interacting proteins. Interactions with JunB, JunD and CtBP1 were confirmed along with a set of novel proteins including Par-4, ATF3, RACK1, N4BP1 and Pin1. The chicken Par-4 and ATF3 genes were cloned and expressed to confirm co-localisation with Meq in the cell nucleus while further biochemical investigations for each interaction were carried out by co-immunoprecipitation. The Par-4 and ATF3 proteins have been shown to play a variety of key roles in cellular mechanisms such as apoptosis, cell cycle regulation and transformation but we failed to see expression of these proteins in either normal or transformed lymphocytes. However, Par-4 and ATF3 are expressed in the feather follicle epithelium of infected birds providing the potential for a role during lytic infection in the skin. It is clear that more work is required in order to explore this hypothesis further but this thesis describes a number of novel interactions made by Meq and in doing so we have contributed to the greater understanding of Marek’s disease virology.

Human adenoviruses (Ads) are DNA viruses believed to hold potential as gene therapy vectors. However, Ad vectors often express residual late structural proteins, which stimulate immune responses resulting in rapid clearance of the vector. Therefore a greater understanding of the mechanisms controlling Ad late gene expression is required. During the late phase of adenovirus 5 (Ad5) infection it was previously shown that a nuclear compartment involved in RNA metabolism known as the Cajal body (CB) is disassembled from 1-6 punctate domains per cell into numerous microfoci. Furthermore, the marker protein of CBs, p80 coilin, was suggested to play a role in the expression of Ad late phase proteins. However, the exact function of coilin in Ad late protein expression is unknown. The aim of this investigation was to determine the roles of coilin and additional CB proteins during Ad infection. Immunofluorescence microscopy was utilised to investigate the redistribution of key CB proteins following Ad5 infection of A549 cells. Whilst coilin was redistributed from CBs into microfoci, another CB protein, termed survival of motor neuron (SMN), was redistributed from CBs into the nucleoplasm. To assess the roles of coilin and SMN during Ad5 infection, coilin was depleted in A549 cells by RNA interference (RNAi). Cells were infected with Ad5 and the virus yield, Ad proteins levels and Ad mRNA levels were assessed. Depletion of coilin reduced the virus yield and decreased the synthesis of Ad early, intermediate and late phase proteins. Although Ad mRNA expression was mostly unaffected, nuclear export of Ad mRNAs was abrogated in coilin-depleted cells. This indicated that coilin plays a role in Ad mRNA transport. Similar to coilin, SMN depletion significantly reduced the virus yield. However, in contrast to coilin, SMN depletion resulted in significant decreases in the levels of Ad capsid proteins, whilst non-structural proteins were either increased or unaffected. SMN depletion was found to reduce early gene transcription and altered alternative splicing patterns of Ad mRNAs. This suggested that SMN plays a role in Ad transcription and mRNA splicing. This investigation uncovered the involvement of two CB proteins in two very distinct roles during Ad infection. This is the first report suggesting a role for CBs in mRNA export. Further study is now required to identify the exact function of coilin in Ad mRNA export. Furthermore, investigation of a role for coilin in cellular mRNA export and mRNA export during infection with other viruses is also warranted. Although SMN was known to play a role in cellular mRNA splicing, this is the first report indicating that SMN may be involved in splicing of virus mRNAs. Additional work is required to define the precise function of SMN in transcription and mRNA splicing during Ad infection and during infection with other virus species.

Retroviruses must integrate their genome into the host DNA as a necessary step of their replication cycle. Normally, retroviruses integrate into somatic cells and are transmitted, from infected to uninfected hosts, as “exogenous” retroviruses. On rare occasions, they can infect germ line cells and become part of the host genome as “endogenous” retroviruses (ERVs), which are transmitted vertically to the offspring and inherited as Mendelian genes. During evolution, most ERVs have accumulated mutations that rendered them defective and unable to produce infectious viral particles. Some ERVs, however, have maintained intact open reading frames for some of their genes, and have been co-opted by the host as they fulfil important biological functions. Sheep betaretroviruses represent a unique model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) co-exists with the highly related endogenous JSRVs (enJSRVs). The sheep genome harbours at least twenty-seven enJSRV loci and, most likely, the process of endogenization is still occurring. During evolution, one of these enJSRV loci, enJS56A1, has acquired a defective and transdominant Gag polyprotein that blocks the late replication steps of related retroviruses, by a mechanism known as JSRV late restriction (JLR). Interestingly, enJSRV-26, a provirus that integrated in the sheep germ line less than two hundred years ago, possesses the unique ability to escape JLR. In this thesis, the molecular basis of JLR escape was investigated. The main determinant of JLR escape was identified in the signal peptide of enJSRV-26 envelope protein (SP26). A single amino acid substitution in SP26 was found to be responsible for altering its intracellular localization as well as its function as a post-transcriptional regulator of viral gene expression. Interestingly, interference assays demonstrated that enJSRV-26 relies on the presence of the functional signal peptide of enJS56A1 envelope protein (SP56) in order to escape JLR. In addition, the ratio between enJSRV-26 and enJS56A1 Gag polyproteins was found to be critical to elude JLR. Finally, sequence analyses revealed that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus, reinforcing the hypothesis that this locus has provided an evolutionary advantage to the host. This study unveils critical aspects of JLR that were previously unknown, and provides new insights on the molecular mechanisms governing the interplay between endogenous and exogenous sheep betaretroviruses.

There is a delicate evolutionary balance between viruses and their hosts. The host has evolved the intrinsic, innate and adaptive immunity to fight viral infections. However, viruses have acquired several counteracting measures to evade host defences. Ovine Betaretroviruses, including the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) and the highly related endogenous enJSRVs are a unique model system to investigate virus-host interaction over long evolutionary periods. Sheep have co-opted some defective enJSRV loci to (i) counteract infection by exogenous viruses and likely (ii) to cope with the continuous retroviral invasion of their genome. In addition, various genes of the innate and intrinsic immunity of the host have evolved to block viral replication. The work presented in this thesis focuses on the ovine bone marrow stromal cell antigen 2 (Bst-2)/ tetherin, a recently identified cellular restriction factor with a broad antiviral activity, and its interaction with sheep Betaretroviruses. In sheep, the BST-2 gene is duplicated into two paralogs termed oBST-2A and -2B. Studies presented in this thesis show that oBST-2B possesses several biological properties distinct from the paralog oBST-2A and from all the other BST-2 orthologs. oBST-2A prevents the release of JSRV/enJSRV viral particles by ‘tethering’ them at the cell membrane similarly to what observed by human BST-2. On the other hand, oBST-2B, does not reach the cell membrane but remains within the Golgi stacks and the trans-Golgi network. Several lines of evidence obtained in this thesis suggest that oBST-2B reduces significantly Env incorporation into viral particles. Therefore, oBST-2B possesses a unique antiviral activity that complements the classical tethering restriction provided by oBST-2A.

The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.

Bacteriophage T5 is a bacterial virus known to have a remarkably high replication rate. It is a double-stranded DNA virus and encodes many of the proteins needed for its own replication. During replication, the viral double-stranded genomic DNA has to be separated by enzymes called helicases, which are motor proteins that utilize chemical energy from ATP to move along and unwind nucleic acid duplexes. Until now, no helicase has been characterized in bacteriophage T5. A bioinformatic analysis on the T5 replication gene cluster showed that several early gene products (D2, D6 and D10), which possess key helicase signature sequences (motifs), may be T5 helicases. This is the first report to investigate helicases of bacteriophage T5 and the study focused on bioinformatic and biochemical characterization of these three potential helicases. Here, D2 and D10 were identified to be two novel T5 helicases, showing helicase activity in vitro as well as having some unique properties previously uncharacterised in other helicases. However, D6 did not show ATPase activity under the condition employed and a further investigation on characteristics of D6 is required. Except for a Walker A motif, no other common conserved motifs related to helicase activity were identified in the D2 protein sequence. However, D2 was found to have a rare bipolar helicase activity giving it the ability to unwind partial duplex DNA with either a 5' or a 3' ssDNA tail (ss-dsDNA). This indicates D2 may possess some unconventional motifs relevant to its helicase activity. The extent of 5'→3' or 3'→5' unwinding activity of D2 was revealed to be dependent on 5' or 3' tail length. Interestingly, D2 displayed biased polarity preference with its 3'→5' unwinding activity being several fold greater than its 5'→3' unwinding activity when the substrates have identical tail length. Differential inhibition of the bipolar helicase activities by high NaCl concentration was also observed. The 5'→3' unwinding activity was more sensitive to inhibition by high NaCl concentration than the 3'→5' unwinding activity. The D10 protein can unwind branched DNA substrates, including forks, Y-junctions and Holliday junctions, which resemble DNA replication, recombination and repair intermediates. Furthermore, D10 was shown to catalyze branch migration of the Holliday junction substrate. Intriguingly, the ability of D10 to unwind the Y-junction substrate was found to be structure-dependent and sequence-dependent. Also, the unwinding activity can be affected by the strand discontinuity of the substrate. All the findings in this study contribute to a new insight into functional properties of helicases.

A significant number of RNA viruses assemble their protein containers and genomic material simultaneously. Here the implications of this protein-RNA co-assembly are investigated using an extended version of a model first proposed by Adam Zlotnick in 1994 (Zlotnick, 1994). The inspirations for this extended model are the cases of bacteriophage MS2 and the STMV virus, viruses that have been well characterised experimentally. Example pathways of RNA virus assembly have been enumerated and kinetic simulations have been run on these networks. The results show the most likely pathways of virus assembly and the concentrations of the intermediates. This work will also demonstrate how kinetic traps may be avoided when proteins are able to bind RNA during assembly. Additionally modelled are DNA cages, which are three-dimensional shapes made from double-helical DNA molecules. Such cages have been seen within viruses but may also be constructed artificially. This model has been used to produce energetically optimised designs for icosidodecahedron-shaped DNA cages.

The structure and symmetry of viruses has been the subject of study since Crick and Watson in 1956, and there have been several complementary theories describing different aspects of the geometry of these complicated entities. Included here is a unified theory that relates the structure and sizes of the different viral components, from the capsomeres to the packaging of the genomic material, providing, through a set of structural constraints on viral structures, a new classification scheme for viral structures. Moreover, aspects of this theory also apply to fullerene structures in chemistry, showing that this symmetry principle is deeper than just biological in nature.

The last two decades have seen a major increase in the use of cryo-eleclron microscopy for virus reconstruction. Icosahedral virus reconstruction is particularly successful partly because the high symmetry of the structure can provide a guide to orient images via . for example. common lines. In this thesis. we introduce two improvements to t he icosahedral virus reconstruction methodology. Firstly. we propose a systematic probability-based orientation method to increase the accuracy of orientation. It will be shown that. relying on the statistical properties rather than the magnitudes of common-line residuals. the accuracy! can be improved substantially. Viruses such as adenovirus can be reconstructed straight- away without any model-based orientation refinement. Secondly_ we introduce a novel approach to identify icosahedral druses so that a) efficient icosahedral virus reconstruction methods can be used correctly on , viruses to achieve higher resolution" b) biologists ca better understand the structure and mechanism 0 1" ,viruses" This is done by decomposing common- line residuals into pair residuals and modelling them by normal mixture distributions. An innovative Markov chain Monte Carlo method. partition sampler. is design to efficiently estimate the number of components in the normal mixture distribution an hence help to identify the icosahedral viruses. The methodology ha'-e been tested on fixed viruses. Among these adenovirus. PRD1 and SFV are successfully identified as icosahedra viruses. and MMTV and HIV as non-icosahedral viruses.

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

Enigmatic amphibian declines are strongly associated with infectious diseases, including chytridiomycosis, caused by the fungus 'Batrachochytrium dendrobatidis' (Bd), and ranaviral disease induced by ranaviruses. A series of data analyses, field sampling, laboratory experimentation and molecular techniques were utilized to test the hypothesis that the international live animal trade is contributing to the spread of these amphibian pathogens. Regions sampled included specific locations in North America, South America and Asia. The magnitude of the live importation of amphibians into the United States (U.S.) was calculated in the millions of animals per year. Batrachochytrium dendrobatidis and ranavirus infections were identified in live food animals in: (a) U.S. wet markets (in 'Lithobates catesbeianus', the North American bullfrog); (b) frog farms in Taiwan (in 'L. catesbeianus'); and (c) wet markets in Taiwan (in 'Rana tigrina', the Chinese bullfrog). 'Batrachochytrium dendrobatidis' infections were also identified in 'L. catesbeianus' farmed in Brazil and in 8 species of amphibians in the U.S. pet trade (in captive bred and directly imported animals). Detection of Bd in individuals upon initial entry into the U.S. demonstrated definitively the trans-continental movement of this potentially lethal amphibian pathogen. Laboratory experimentation illustrated transmission of Bd from a highly traded carrier host ('L. catesbeianus') to frogs of the same species, and to a known susceptible frog species ('Litoria caerulea', White's tree-frog). Molecular data linked U.S. market frogs and their associated ranaviral infections to origins in Asia and South America. Genotyping of pure, cultured isolates of Bd from farmed 'L. catesbeianus' in Brazil showed that they were most similar to isolates from native amphibians in Latin America when compared to a global dataset, providing evidence for transmission between wild and farmed amphibians. Analysis of isolates from market 'L. catesbeianus' led to the discovery of a novel genotype of Bd in Ypsilanti, Michigan that appeared to have a disparate lineage compared with the previously identified panzootic genotypes. DNA sequence analysis from the Ypsilanti frog indicated its origin in Brazil, where the novel genotype was also discovered in native Brazilian frog populations. The demonstration of amphibian pathogens on frog farms, in live wet markets and at border crossings, the evidence of inter- and intra-species transmission, and the existence of a novel genotype in traded anurans, all indicate that international transport of amphibians and their pathogens could produce not only mixing of pathogen genotypes, but the inadvertent spread of strains of unknown virulence that could negatively impact amphibian health. In 2008, data from research contained in this thesis were instrumental in the decision to list both Bd and ranaviral disease in the World Organization for Animal Health Aquatic Animal Health Code, providing guidelines (such as quarantine procedures) to limit the spread of these pathogens through the trade. The evidence provided in this work re-enforces the need for urgent action to minimize the potential spread of amphibian diseases through international trade routes, for the benefit of amphibian populations throughout the world.

Recombination is an important biological process in a diverse range of viruses, particularly those with single-stranded ribonucleic acid (RNA) genomes including the enteroviruses. Mutations caused by the error-prone RNA-dependent RNA polymerase (RdRp) and the vast population size of these virus populations are evolutionary mechanisms that generate genetic diversity – this allows viruses to survive under changing environmental pressures (e.g. adaptive host immunity). Ribonucleic acid recombination has been identified as another contributing mechanism involved in diversification, by removing interfering or lethal mutations from a virus genome, and by establishing new viruses. Virus RNA recombination is well documented and is identified in several virus families including picornaviruses. However, recombination is a rare event and the study of the molecular mechanisms behind virus recombination is complicated by our ability to isolate and analyse recombinants from a mixed virus population including the parental viruses. The objectives of this study were to firstly devise a method for generating populations of natural recombinant viruses, and secondly, to study the molecular processes that determine where and when recombination occurred in the enteroviral genome. During this project, an in vitro system was developed to allow the recovery of recombinant enteroviruses in the absence of their parental viruses. Two virus RNA molecules containing “lesions” rendering them unable to generate viable virus on their own were co-transfected into mouse L929 cells. The method required two parental virus RNA molecules to be present in a single cell to produce a viable recombinant virus. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and sequencing analysis confirmed the recombinant nature of progeny virus genomes. Experimental data confirmed the effectiveness of the method and provided evidence that recombination occurs in at least two phases. Initial template switching, referring to the transfer of RdRp from one RNA template to another mid-replication, occurred apparently indiscriminately and with the addition of extra virus and non-virus sequence at the junction sites. In the second phase, any additional sequence was lost during subsequent rounds of replication and selection. The approach was expanded to incorporate viruses from different enterovirus species to investigate intra- and interspecies recombination events.

Human and bovine respiratory syncytial viruses (HRSV and BRSV), along with pneumonia virus of mice (PVM) are the members of the genus Pneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae. Although both HRSV and BRSV have been associated with important diseases in human and livestock, there is no clearcut description of the molecular aspects of their pathogenesis. For HRSV, the lack of a suitable study model is one of the main reasons hampering the study of aspects of pathogenesis of the virus. HRSV infects a wide range of animal models, however most of the common laboratory animal models are not sufficiently permissive to study the infectivity of the virus. PVM naturally infects mice and causes a disease indistinguishable from that of HRSV in humans. Two strains of PVM have been described: strain 15 (Warwick) which is not pathogenic and strain J3666 which is highly pathogenic. The main difference between these two strains lies in the organisation of the gene encoding the attachment (G) glycoprotein. The G gene in strain J3666 has two ORFs. The larger second ORF codes for the G glycoprotein and is located downstream of the first ORF which has no known function. The strain 15 G gene also contains two ORFs but in this case both the first and main ORFs overlap each other. The aim of the project was to investigate the molecular basis for pathogenesis of PVM as a model for pneumoviruses. As a first step, the pathogenesis of PVM strain J3666 was revaluated and the effect of consecutive tissue culture passages on the pathogenicity of the virus was examined. It was shown that consecutive passages of PVM strain J3666 caused attenuation of the virus. To investigate the possible mutations causing the attenuation the majority of the virus genome was sequenced from three passage stocks where the transition from pathogenic to non-pathogenic occurred. No differences in the genome sequences for the three passage stocks were found. However, sequence analysis of individual clones of the SH and G genes of the viruses showed evidence that the stocks contained a mixed population of sequences. A robust reverse genetics system was established to rescue recombinant PVM from cDNA using co-transfection of plasmids coding for the ribonucleoprotein complex of the virus (N, L, M2-1 and P proteins) and a cDNA copy of the virus genome cloned under the control of the bacteriophage T7 RNA polymerase. Using this system, four viruses differing in their G gene organisation were generated and used to infect mice to study the effect of mutations on pathogenicity. It was shown that the viruses with the G gene of strain 15 (Warwick) lacking the first ORF manifest a modest increase in their pathogenicity compared to the non-pathogenic PVM strain 15(Warwick) parent. The recombinant viruses containing the G gene organisation of strain J3666 showed the highest level of pathogenicity. The reverse genetics system was used to study the role of the first ORF in G glycoprotein expression. Using a dicistronic minigenome construct, the effect of the presence or absence of the first ORF in both the strain 15 and strain J3666 G gene organisation was studied. It was shown that the presence of the first ORF of the G gene in the strain 15 (Warwick) suppressed the expression of the G protein, while the first ORF in the strain J3666 did not have any significant effect on G protein expression.

Epstein-Barr virus (EBV), a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this B-cell growth transformation programme depends on the activation of a viral promoter, Wp, present in each tandemly arrayed BamHI W repeat of the EBV genome. In order to examine the role of the BamHI W region in B cell infection and growth transformation, we constructed a series of recombinant EBVs carrying different numbers of BamHI W repeats and carried out B cell infection experiments. We concluded that EBV requires at least 2 copies of BamHI W repeats to be able to activate transcription and transformation in resting B cells in vitro. At least 5 copies of BamHI W were required for optimal transcription and transformation; while increasing the number beyond 5 copies had no further effect. Experiments to try to rescue the impaired virus indicated that the expression of sufficient levels of EBNA-LP and EBNA2 from Wp are the key determinants of virus-driven B cell transformation. We believe that EBV has evolved to contain multiple copies of BamHI W repeats to ensure high levels of Wp-initiated transcripts immediately post infection.

Rotaviruses are a leading cause of acute gastroenteritis in infants and young children worldwide and possess a genome of 11 double-stranded (ds) RNA segments. Early morphogenesis of RV particles and viral RNA replication occur in cytoplasmic inclusion bodies called ‘viroplasms’, of which the viral non-structural proteins NSP2 and NSP5 are essential components. Using confocal microscopy (CM), we demonstrated association of viroplasms with lipids and lipid droplet (LD)-associated proteins (perilipin A, ADRP). LD-associated proteins were found to co-localise with viroplasm-like structures (VLS) in cells co-transfected with NSP2- and NSP5- expressing plasmids in the absence of rotavirus infection, as well as viroplasms containing NSP5-EGFP. Close spatial proximity between NSP5-EGFP and perilipin A was demonstrated by Fluorescence Resonance Energy Transfer (FRET). Time course CM studies showed increasing recruitment of perilipin A to viroplasms during the progression of rotavirus infection. Separation of RV-infected cell extracts on iodixanol gradients demonstrated co-localisation of rotavirus dsRNA, NSP5 and perilipin A in low density fractions. Chemical compounds interfering with LD formation or homeostasis (isoproterenol + IBMX, triacsin C, C75) decreased the number and size of viroplasms in rotavirus-infected cells, dsRNA replication and the production of infectious progeny virus, whilst significantly protecting cells from cytopathicity caused by rotavirus infection. These data contribute to a novel understanding of the role of LDs in rotavirus replication. Using a cell line stably expressing NSP5-EGFP, the dynamics of viroplasm formation in relation to dsRNA replication and production of infectious virus was investigated.

Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. The sigma virus (DMelSV) is currently the only natural hostspecific pathogen to be described in Drosophila melanogaster. In this thesis I have examined; the diversity and evolution of sigma viruses in Drosophila, their transmission and population dynamics, and their ability to host shift. I have described six new rhabdoviruses in five Drosophila species — D. affinis, D. obscura, D. tristis, D. immigrans and D. ananassae — and one in a member of the Muscidae, Muscina stabulans (Chapters two and four). These viruses have been tentatively named as DAffSV, DObsSV, DTriSV, DImmSV, DAnaSV and MStaSV respectively. I sequenced the complete genomes of DObsSV and DMelSV, the L gene from DAffSV and partial L gene sequences from the other viruses. Using this new sequence data I created a phylogeny of the rhabdoviruses (Chapter two). The sigma viruses form a distinct clade which is closely related to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they may deserve to be recognised as a new genus. Furthermore, this analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing me to reconstruct the major transitions that have occurred during the evolution of the family. This data suggests that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. In Chapter three I examined whether the new sigma viruses in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for DMelSV, both males and females can transmit these viruses to their offspring. Males transmit lower viral titres through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. I then examined natural populations of D. obscura in the UK; 39% of flies were infected and the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data I estimate that the virus has swept across the UK within the last ~11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on lab estimates of transmission rates, I show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations, at rates consistent with those measured in the field. Therefore, as predicted by the simulations, the virus has undergone an extremely rapid and recent increase in population size. In Chapter four I investigated for the first time whether vertically transmitted viruses undergo host shifts or cospeciate with their hosts. Using a phylogenetic approach I show that sigma viruses have switched between hosts during their evolutionary history. These results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts. In Chapter five I examined the ability of three Drosophila sigma viruses to persist and replicate in 51 hosts sampled across the Drosophilidae phylogeny. I used a phylogenetic mixed model to account for the non-independence of host taxa due to common ancestry, which additionally allows integration over the uncertainty in the host phylogeny. In two out of the three viruses there was a negative correlation between viral titre and genetic distance from the natural host. Additionally the host phylogeny explains an extremely high proportion of the variation (after considering genetic distance from the natural host) in the ability of these viruses to replicate in novel hosts (>0.8 for all viruses). There were strong phylogenetic correlations between all the viruses (>0.65 for all pairs), suggesting a given species’ level of resistance to one virus is strongly correlated with its resistance to other viruses. This suggests the host phylogeny, and genetic distance from the natural host, may be important in determining viruses ability to host switch. This work has aimed to address fundamental questions relating to host-parasite coevolution and pathogen emergence. The data presented suggests that sigma viruses are likely to be widespread vertically transmitted insect viruses, which have dynamic interactions with their hosts. These viruses appear to have switched between hosts during their evolutionary history and it is likely the host phylogeny is a determinant of such host shifts.

Coronaviruses have extremely large RNA genomes and use complex regulatory mechanisms to replicate and express their genomes. This involves the expression of two large polyproteins, pp1a and pp1ab, that are autoproteolytically processed to produce nonstructural proteins (nsp) 1 to 16. In this study, enzymatic activities associated with Human coronavirus 229E (HCoV-229E) nsp3 were characterised. First, N-terminal processing of HCoV-229E nsp3 by viral papain-like proteases (PLP) 1 and 2 was investigated. The two proteases were previously shown to have partially overlapping substrate specificities and functionally replace each other at specific sites. Using polyclonal antisera, nsp3 and nsp4 were now detected as proteins of 175 and 43 kDa, respectively, in virus-infected cells. Immunofluorescence assays revealed a punctate perinuclear staining pattern, suggesting that nsp3 and nsp4 associate with viral replication complexes. Experiments using recombinant vaccinia viruses expressing wild-type or mutant forms of the HCoV-229E pp1a/pp1ab nsp1-to-nsp4 region revealed that PLP2 is responsible for processing at the predicted nsp3|nsp4 cleavage site while PLP1 does not cleave at this site. The data provide new insight into HCoV-229E PLP substrate specificities and polyprotein processing pathways. Second, a conserved nsp3 domain called ADRP (ADP-ribose -1”-phosphatase) was investigated using a reverse genetics approach. To better understand the role of the ADRP in viral replication, a set of HCoV-229E ADRP mutants was generated, including viruses with a (i) mutation in the putative ADRP substrate-binding site (N1357A), (ii) deletion of the entire ADRP domain and (iii) substitution of the ADRP domain with that of HCoV-NL63. Characterisation of ADRP mutants revealed defects in RNA synthesis and viral growth in cell culture. Sequence analysis of viable clones following serial passage in cell culture identified a number of second-site mutations, possibly contributing to restoration of viral fitness and suggesting important (though nonessential) functions of the ADRP domain in coronavirus replication in cell culture.

Riboswitches are cis-acting gene regulatory RNAs, which function without involvement of proteins. They have been implicated as drug targets and are attractive systems for the study of RNA-ligand binding and RNA folding. The purine riboswitch was used as a model system for RNA-ligand docking. Published binding data was successfully reproduced in silico and compounds predicted to bind the riboswitch in a virtual screening were tested experimentally. Structural data confirming the predicted binding mode for several cases was obtained. The problems encountered were not specific to RNA-ligand docking but known from the far more explored field of protein-ligand docking.The SAM-I riboswitch was also subjected to virtual ligand screening. This receptor is a system of greater complexity than the purine riboswitch and consequently posed a harder challenge to the docking protocol. After initial validation of the docking setup based on previously published data, a set of compounds selected from the in-house database of commercially available compounds was screened. One compound identfied in silico was cofirmed to bind experimentally.The k-turn motif found in the SAM-I riboswitch was investigated with respect to its folding. The k-turn motif was found to be foldable in context of the SAMI riboswitch as well as in isolation as was expected. Furthermore, mutations disrupting key interactions within the k-turn motif were found to be prohibitive of k-turn folding in isolation as well as in context of the riboswitch, leading to a loss of ligand binding. Interestingly, two sequences were identfied which fold in context of the riboswitch but do not fold in isolation. This confirms the contribution of tertiary interactions to k-turn folding. This conclusion was backed up with structural data is a system of greater complexity than the purine riboswitch and consequently posed a harder challenge to the docking protocol. After initial validation of the to its folding. The k-turn motif was found to be foldable in context of the SAMI riboswitch as well as in isolation as was expected. Furthermore, mutations disrupting key interactions within the k-turn motif were found to be prohibitive of k-turn folding in isolation as well as in context of the riboswitch, leading to a loss of ligand binding. Interestingly, two sequences were identi ed which fold in context of the riboswitch but do not fold in isolation. This con rms the contribution of tertiary interactions to k-turn folding. This conclusion was backed

Foot-and-Mouth Disease Virus (FMDV) induces rearrangements of host-cell membranes to generate vesicles that are believed to provide platforms for formation of the viral replication complex. The cellular origin of these vesicles and the properties that make them favourable for replication are poorly understood. For some Picornaviruses these vesicles are thought to derive from membranes of the secretory pathway. In this thesis, I have investigated a role for membranes of the secretory pathway in FMDV infection. Key cellular proteins involved in regulating the flow of membranes through the secretory pathway between the ER and Golgi were inhibited using expression of dominant-negative (dn) proteins and small interfering RNA (siRNA) and the effect on FMDV infection determined. Inhibition of ER export using a drug (H89) or Sar1 (the GTPase required for COPII transport vesicle formation at ER exit sites) reduced FMDV infection. In contrast, stabilisation of COPII coats, or inhibition of Arf1 or Rab proteins, that are involved in the secretory pathway after the formation of COPII vesicles, had little or no inhibitory effect on infection. Interestingly inhibition of Arf1, Rab1 or Rab2 enhanced infection. In contrast, Arf1 reduced infection by bovine enterovirus which is inhibited by Brefeldin-A, and therefore likely to be dependent on Arf1 for replication. These results show that Sar1 and/or COPII vesicle formation is necessary for FMDV infection and that inhibiting the formation of COPI coats is in some way advantageous to FMDV infection. These results suggest that FMDV targets COPII vesicles membranes before the COPII/COPI exchange and facilitates FMDV infection and that COPI components are not required.

VACV gene C16L encodes a 37-kDa protein that is highly conserved in orthopoxviruses and functions as an immunomodulator. Intranasal infection of mice with a virus lacking C16L (vΔC16) induced less weight loss, fewer signs of illness and increased infiltration of leukocytes to the lungs compared with wild-type virus. To understand C16’s mechanism of action, tandem affinity purification and mass spectrometry were used to identify C16 binding partners. This revealed that Ku70, Ku80 and PHD2 interact with C16 in cells. Ku70 and Ku80 constitute the Ku heterodimer, a well characterised DNA repair complex. MEFs lacking Ku, or the other component of the DNA-dependent protein kinase (DNA-PK) complex, the catalytic subunit of DNA-PK (DNA-PKcs), were shown to be deficient in the upregulation of IRF-3-dependent genes such as Cxcl10, Il6 and Ifnb in response to transfection of DNA, but not poly (I:C). Furthermore, following infection of MEFs with VACV strain MVA the activation of Cxcl10 or Il6 transcription was dependent on DNA-PK. Therefore, DNA-PK is a DNA sensor capable of detecting poxvirus DNA and activating IRF-3-dependent innate immunity. C16 inhibited the binding of Ku to DNA, and therefore inhibited DNA-mediated induction of Cxcl10 and Il-6 in MEFs. The role of C16 in vivo was also examined: infection with vΔC16 led to increased production of Cxcl10 and Il-6 following intranasal infection of mice compared with wild-type virus. C16 is therefore an inhibitor of DNA-PK-mediated DNA sensing and innate immune activation. C16 was also shown to bind to PHD2, an enzyme involved in regulation of hypoxic signalling. VACV was found to activate the transcription of hypoxia-related genes, and C16 expression in cells was also capable of doing this. The role of hypoxic signalling in VACV infection remains poorly understood.

The aim of this research was to assess the incidence of dsRNA mycoviruses in the opportunistic human pathogenic fungus Aspergillus fumigatus, where previously no dsRNA viruses had been reported and to investigate the effects of any dsRNAs on the growth and pathogenicity of the fungus. Thus far 366 isolates (clinical and environmental) have been screened, 24 of which posses dsRNA elements. Successful efforts were made to completely characterise the two dsRNA segments of the isolate 88, partitivirus to obtain novel sequence information. Fungal viruses or mycoviruses are widespread and they usually infect their hosts persistently without any detectable phenotypic effects. They have been however linked with both hypovirulence and hypervirulence but are normally cryptic. To obtain information on the effect of the dsRNAs on their respective hosts, efforts were made to ‘cure’ isolate 88 of its dsRNA infection by cycloheximide treatment. However, following cycloheximide treatment, a sensitive reverse transcription polymerase chain reaction (RT-PCR) amplification assay showed that the dsRNA elements, whilst being reduced in amount, were not eliminated completely and that high levels of cycloheximide also interfered with spore production, pigmentation and overall growth of the isolate. In further experiments attempts were made to mobilise the dsRNAs from 4 isolates viz. A-56, A-54, A-78 and isolate 88 into isolate Af-273y, which is hygromycin resistant and yellow in colour, by hyphal tip fusion, protoplast fusion and protoplast transfection with purified virus. Protoplast fusion and viral transfection experiments were successful for some isolates, as assessed by the RT-PCR assay and small scale extractions of nucleic acids. Subsequently comparative growth experiments by radial growth assay and mycelial weight measurements between isolate Af-273y and Af-273y transfected with isolate 88 partitivirus in essentially the same genetic background were performed. These experiments showed that the partitivirus infection resulted in a sectored phenotype and significantly lowered the growth of the fungus. All efforts to initiate the molecular characterisation of uncharacterised dsRNA elements found in isolates A-54, A-78 and isolate-66 have thus far proven unsuccessful but a new approach (cDNA library construction) is proposed for the characterisation of these dsRNAs.

Acanthamoeba causes Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) in humans and in its cystic form is resistant to extreme environmental conditions. Both human pathogenic water-borne viruses and free-living protozoa share the same aquatic environment. This study set out to test the ability of both Acanthamoeba and Tetrahymena to internalise and protect enteric viruses; coxsackievirus (B3, B5), poliovirus (PV) and rotavirus (RV) following co-culture. Viral uptake was assessed by infection of cultured mammalian cells, by indirect immunofluorescence (IF), and by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that none of the free suspended viruses were internalised in Acanthamoeba or Tetrahymena. However, both coxsackievirus B3N and rotavirus Wa could be detected within Acanthamoeba by IF and confirmed by RT-PCR when the amoebae were co-cultured (fed) with virally infected mammalian cells. The co-cultured amoeba was allowed to encyst but following this procedure no viruses were detected either by cell culture or RT-PCR. In a second series of experiments, the efficacy of solar disinfection (SODIS) against viruses either alone or when co-cultured with Acanthamoeba was assessed. SODIS reduced the viral infectivity by over 3log10 after 1 h for CVB3N and over 2log10 for PV after 2 h. Repeating these experiments in the presence of riboflavin, a 6log10 reduction was observed for CVB3N after 1 h of light exposure and 6log10 after 6 h for all other viruses tested. The results suggest that Acanthamoeba does not internalise or protect viruses in suspension. However, if a virus is located with an infected mammalian cell then it may be internalised; a new potential mechanism for virus dissemination in the environment. Secondly, solar disinfection is an effective treatment method for water contaminated with viruses which is further enhanced by the addition of riboflavin. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.

Hepatitis C virus (HCV) readily establishes a chronic infection and it is currently thought to infect 2-3% of the global population. Treatment for HCV places a severe burden on patients, leading to a premature treatment termination in approximately 20% of individuals, and has poor efficacy against the predominant genotype. A greater understanding of the virus lifecycle and mechanisms of persistence will provide valuable information in the continuing development of direct-acting antiviral compounds. HCV encodes 10 proteins which are translated as a single polyprotein. Non-structural protein 2 (NS2) is a small, hydrophobic, trans-membrane protein, yet the precise number and position of its trans-membrane domains remain unclear. NS2 is required for virion morphogenesis but it is not required for replication of the viral genome and it does not form part of the mature virion. NS2 has been shown to interact with other viral proteins, potentially via intra-membrane contacts. Determining the topology of NS2 is therefore vital to our understanding of how NS2 interacts with the other viral proteins and functions within the virus lifecycle. The interactions between NS2 and membranes and the viral glycoprotein E2 were investigated by truncation analysis and fusion with reporter proteins. Glycosylation analysis demonstrated that the N- and C-termini of NS2 are oriented to the luminal and cytosolic faces of the ER membrane, respectively. Truncation of NS2 at residue 70 oriented a C-terminal reporter fusion to the ER lumen consistent with the formation of a luminal loop. This is the first biochemical evidence that NS2 assumes a 3 TMD topology. Co-immunoprecipitation studies with E2 and eGFP-tagged truncation of NS2 revealed that NS2 forms multiple interactions with E2 and that these interactions are dependent upon NS2 targeting to membranes but likely independent of NS2 topology. A model of NS2 topology is presented.

Introduction: Multiple sclerosis (MS) is a progressive, neurological condition in which balance difficulties and falls are commonly reported. Measuring balance is important for assessment and to measure outcome of intervention and measures used should have known levels of reliability and validity. This research aimed to identify the measures used by physiotherapists for balance, walking and gait, and to review psychometric testing of commonly used measures for people with MS. Research tested psychometric properties of the measures among patients with MS. Method: A survey of physiotherapists identified widespread outcome measurement, that reliability and validity of measures was important and that the Berg balance scale (BBS) and 10-metre walking test (10MWT) were commonly used measures. A literature review highlighted limited evidence of psychometric testing of these measures among people with MS. The short form BBS (sfBBS) had not been tested at all. The BBS and sfBBS were tested for reliability and validity among people with MS. The 10MWT and objective force-plate data were also tested as measures of balance. Results: The national survey of physiotherapists yielded an encouraging 77.5% return rate and showed that therapists valued measurement. The patient-based study recruited a smaller sample than desired leading to caution in interpretation. The sfBBS was strongly related to the BBS and both had acceptable levels of intra-rater reliability. Inter-rater reliability was affected by interpretation of scoring guidelines and the sfBBS had significant measurement error. The 10MWT, especially at maximal pace, may be used as an estimator of balance ability. Conclusion: There must be clear instructions for scoring the BBS particularly between raters. Caution is required in generalising from a small sample size however analyses demonstrated acceptable levels of intra-rater reliability in MS. The sfBBS and 10MWT, as balance estimates, may save time for patients and clinicians and reduce the burden on patients.

Matrix proteins play a central role in the assembly of enveloped pleomorphic viruses. In order to study matrix protein function during assembly, cryo-electron microscopy and tomography studies were carried out on two virus families. Studies of coronavirus virus-like particles and virions revealed that the matrix protein M forms a two-dimensional network of endodornain-Iinked homodimers at the membrane. Structural differences were detected between M on membranes with rigid positive curvature and M on flaccid membranes with variable curvature. Studies of filamentous and round influenza A virus revealed structural and functional differences in the homodimeric matrix protein Ml that were associated with differences in local membrane curvature. The conformational change happened more commonly on filamentous particles than on spherical. In both virus families matrix proteins were found to control membrane bending and rigidity through a process that appears to involve ribonucleoprotein-dependent conformational changes. Both influenza Ml and coronavirus M lost rigid membrane bending activity upon exposure to pH 5.0, demonstrating that the interactions that are proposed to lead to budding can be reversed under conditions that simulate the endosomal environment. Together, these observations help to explain how matrix proteins are able to produce sufficient force to drive the budding process through homotypic and heterotypic intramolecular interactions, and how the structural changes that drive egress can be reversed to facilitate entry.

The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.

The Bunyaviridae family is one of the largest among RNA viruses, comprising more than 350 serologically distinct viruses. The family is classified into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Orthobunyaviruses, nairoviruses and phleboviruses are maintained in nature by a propagative cycle involving blood-feeding arthropods and susceptible vertebrate hosts. Like most arthropod-borne viruses, bunyavirus replication causes little damage to the vector, whereas infection of the mammalian host may lead to death. This situation is mimicked in the laboratory: in cultured mosquito cells no cytopathology is observed and a persistent infection is established, whereas in cultured mammalian cells orthobunyavirus infection is lytic and leads to cell death. Bunyaviruses encode four common structural proteins: an RNA-dependent RNA polymerase, two glycoproteins (Gc and Gn), and a nucleoprotein N. Some viruses also code for nonstructural proteins called NSm and NSs. The NSs protein of the prototype bunyavirus, Bunyamwera virus, seems to be one of the factors responsible for the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of bunyaviruses in cultured mosquito cell lines other than Aedes albopictus C6/36 cells. Here, I compared the replication of Bunyamwera virus in two additional Aedes albopictus cell clones, C7-10 and U4.4, and two Aedes aegypti cell clones, Ae and A20, and investigated the impact of virus replication on cell function. In addition, whereas the vertebrate innate immune response to arbovirus infection is well studied, relatively little is known about mosquitoes’ reaction to these infections. I investigated the immune responses of the different mosquito cells to Bunyamwera virus infection, in particular antimicrobial signaling pathways (Toll and IMD) and RNA interference (RNAi). The data obtained in U4.4 cells suggest that NSs plays an important role in the infection of mosquitoes. Moreover infection of U4.4 cells more closely resembles infection in Ae and A20 cells and live Aedes aegypti mosquitoes. My data showed that the investigated cell lines have various properties, and therefore they can be used to study different aspects of mosquito-virus interactions.

Unfortunately, in the last few years a large-scale colony loss called Colony Collapse Disorder syndrome reduced the overall number of hives in different countries. However, the cause of this is still not clear and for this reason the Department for Environment, Food and Rural affairs (DEFRA) funded this research in order to gain a better knowledge of honey bee virus epidemiology and taxonomy in England and Wales. A National level sampling plan was designed to be statistically representative of the honey bee population present in England and Wales. In order to detect viruses at low prevalence level, a new viral RNA extraction method based on virus precipitation was developed and the viral RNAs obtained were screened for eight honey bee viruses using Real Time rt-PCR. Once information about the prevalence and the distribution of the eight honey bee viruses was obtained, a further characterisation of Deformed wing virus DWV and Black queen cell virus BQCV was performed using classic rt-PCR coupled with Sanger sequencing, and phylogenetic trees were obtained using bioinformatic tools. The viruses, grouped according to their nucleotide similarity, were reported on a geographically referenced map in order to highlight the distribution of the variants found in England and Wales. The results obtained in this thesis have resulted in a better knowledge of the epidemiology and taxonomy of honey bee viruses, determination of the full sequence of Slow paralysis virus SBPV for the first time, and a new virus RNA extraction method that can be exploited in other research fields.

Caliciviruses are single-stranded positive-sense RNA viruses. The viral genome is polyadenylated at the 3' end and 7-8 kb in length. The 5' end of the genome does not possess a cap structure to direct translation of the mRNA. Instead, a viral protein VPg is present which has been demonstrated to act as a novel proteinaceous ‘cap-substitute’ to direct the translation of both the genomic and subgenomic RNAs. Initial work has shown that eIF4A is required for the translation of feline calicivirus (FCV) RNA. The aim of this study was to carry out further analysis of the initiation factors of the eIF4F cap binding complex (eIF4A, eIF4G, eIF4E and poly A binding protein-PABP) and their requirement translation of feline calicivirus (FCV) mRNA. We have developed a system to knock down eIF4AI expression in Crandell Rees Feline Kidney (CRFK) cells using siRNA, and have shown that this leads to an inhibition of virus translation and replication. We have also shown that eIF4AI binds to the FCV RNA and have determined the minimal binding site of eIF4AI on FCV RNA. As a second parameter of our study, we have also demonstrated the knock down of PABP expression in CRFK cells using siRNA and showed that this knockdown also leads to inhibition of FCV translation and replication. Finally we have shown that knockdown of eIF4GI in Human Embryonic kidney (HEK-293) cells using shRNA leads to inhibition of FCV protein synthesis and replication. On the basis of this research, we have proposed a model of the interactions of the FCV RNA with components of the eIF4F complex i.e., eIF4A, eIF4G and PABP. Furthermore, our model also suggests a possible mechanism for the switch from translation to replication of viral RNA late in infection.

NSP4, encoded by rotavirus genome segment 10 has been shown to be a transmembrane, endoplasmic reticulum (ER) specific N-linked glycoprotein. Consistent with its localization to the ER membrane, NSP4 was first shown to have a role in the morphogenesis of the infectious virion. The protein has also been reported to have cytotoxic activity when applied extracellularly to cells. Consequently it has been earmarked as an enterotoxin being secreted from virus-infected cells to cause early cellular pathology in the gut. The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions. Site-directed mutagenesis was used to block N-linked glycosylation at both of the known glycosylation sites near the amino terminus of NSP4. This revealed that the NSP4 induced formation of the cytoplasmic extrusions was dependent on the protein’s ability to become fully glycosylated. The cytoplasmic extrusions seen in cells expressing glycosylated NSP4 were also evident in virus-infected cells. Using real-time confocal microscopy a dynamic elongation of the cytoplasmic extrusions with a growth speed of 2 μm/min was observed in virus-infected cells. The cytoplasmic extrusions were found to contain β-tubulin and F-actin. Inhibiting their polymerization prevented the formation of the extrusions from virus-infected cells. Functional studies using Cell Tracker dyes showed that the cytoplasmic extrusions could disseminate vesicles from virus-infected cells onto the plasma membrane surface of uninfected cells. The vesicles were then found in the interior of the uninfected cells. Mono-specific antibody to NSP4 revealed the presence of the protein in the vesicles suggesting that the cytoplasmic extrusions facilitated the direct cell-cell spread of NSP4. The effect of NSP4 expression on the microtubular network of cells was analysed. It was found that NSP4 de-polymerized the microtubular network from the centre of cells and promoted the assembly of microtubules at the periphery of the cells in a glycosylation independent manner. Similar de-polymerization and re-assembly of the microtubules was observed in the virus-infected cells. Interestingly in the presence of nocodazole, tubular structures containing tubulin and viral proteins excluding NSP4 were found in virus-infected cells. A YFP-PCA assay was established to screen for cellular partners of NSP4. The functionality and the sensitivity of the assay were examined, but only two false positive colonies were isolated in the first screening. In conclusion, the function of glycosylated and unglycosylated NSP4 was examined with the former possessing the ability to promote the formation of the cytoplasmic extrusions from cells and both being capable of disrupting the microtubular network indicating that two forms of NSP4 play different roles in NSP4 function. The cytoplasmic extrusions seen in our studies may be relevant to rotavirus infection and pathogenesis.

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.

Erwinia carotovora subsp. atroseptica (Eca) mutants resistant to a variety of phages were isolated and characterized, identifying lipopolysaccharide (LPS) as a frequently used phage receptor. Various strains deficient in steps in LPS assembly were isolated. One mutation mapped to horizontally acquired island (HAI) 5. Although lateral transmission of this locus to Eca has enhanced virulence in potato, it has also rendered Eca sensitive to phage infection. The genome sequence of Eca identified 17 HAIs, including 2 prophages. Genomic deletion of the prophages resulted in decreased motility, the ability of these strains to cause soft rot of potato was diminished. One phage newly isolated on Eca, φTE, was sequenced. Only 20% of the ORFs could be assigned a function. Characterization of this phage demonstrated that infection depended on the presence of a flagellum. This phage was able to mediate generalized transduction, indicating a potential use in functional genomic analysis. The generalized transducing phage that infects Serratia sp. ATCC39006 (φ0T8) was also shown to be flagellatropic following mutational analysis of the host. Further characterization of this phage identified an alternative and unexpected host, Pantoea agglomerans. It was possible to move a plasmid-encoded antibiotic resistance determinant between the two bacterial genera with high efficiency by φ0T8-mediated transduction. This φ0T8-sensitive strain of P. agglomerans has antifungal capacity, and is therefore of potential application as a biocontrol agent. The antifungal activity was investigated. P. agglomerans was found to produce an N-acyl homoserine lactone signalling molecule that is typically used in quorum sensing. The potential impact of quorum sensing on antifungal production was investigated via mutagenesis.

Using fluorescent antibody virus neutralisation tests, optimised for each virus, I have measured the neutralisation titres of rabbit sera against a global panel of divergent lyssaviruses. Those neutralisation titres were then used to generate antigenic maps, allowing visualisation of antigenic distances to within 1.3 two-fold dilutions in titre. Antigenic distances were compared with glycoprotein ectodomain sequence data, of which 20/30 sequences were obtained by direct sequencing as part of this study. These comparisons revealed clinically important differences between genetic and antigenic relationships. Vaccine strains and wild-type genotype 1 rabies viruses were antigenically distinct, and three of the Eurasian bat viruses were antigenically similar to known genotypes. To evaluate the validity of extrapolating animal model data to other species, I have compared the serological responses of mice, dogs, monkeys, foxes and raccoon dogs using antigenic cartography. The responses of rabbits, mice, monkeys and dogs correspond but orally vaccinated foxes and racoon dogs do not. Passive immunoglobulin treatment is a crucial part of rabies post exposure treatment, and here I have also shown that it is possible to predict the titre of polyclonal passive immunoglobulin treatments to divergent lyssaviruses. Together the results of this study show a measurable spectrum of antigenic distances between lyssaviruses that can be used for rational design of active and passive preventative treatments.

The murine cytomegalovirus (MCMV) immediate early 1 (IE1) protein has been described as a trans-activator of viral and host gene expression. However, the precise role that IE1 plays in the viral life cycle, and in particular its effect on the host immune response is not known. This thesis investigates the functional relationship of the IE1 protein and the immune response induced after infection. By using an ie1-deletion mutant MCMV (MCMVdie1) it was demonstrated that, early after infection, tumor necrosis factor (tnf ) gene activation and protein production was significantly induced in infected-primary macrophages (M ) to a much greater extent than its wild type counterpart. In addition, preliminary studies on the signalling pathways activated upon infection were carried out in order to gain information about the pathways that might be involved in MCMVinduced modulation of tnf activation. Initial observations on the MAPK family members Erk1/2, p38 and JNK did not revealed any differential activation in the absence of IE1. However, due to a number of limitations, it was not possible to draw any firm conclusions from this study. Investigation of the role of IE1 in the in vivo production of TNF were also performed in both susceptible (BALB/c) and resistant (C57Bl/6) mice. These experiments confirmed the attenuated phenotype of MCMVdie1 in vivo, whereby the mutant strain grew to much lower titers than wild type. When cytokine production was assessed in relation to PFU levels a significant production of TNF after infection is observed in different organs of both mice strains. This raises the question whether IE1 contributes to MCMV modulation of TNF production in the natural host. Although, because it is still unclear whether the phenotype of MCMVdie1 in vivo is due to a defect in the virus or the result of a immune response, it was not possible to conclude unequivocally that IE1 is responsible for dampening this cytokine response. This thesis also tested whether the attenuated replication of MCMVdie1 in vivo was due to the increased TNF production induced after infection. An initial investigation in tnf depleted mice revealed that the MCMVdie1 growth phenotype is not due to TNF response. Overall, this study has provided insight into a potential immune modulatory function by MCMV associated with IE1 protein and the regulation of TNF in vivo and in vitro.

Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.

Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.

Marine vibrio MV-1 is a magnetotactic bacterium capable of aligning its cell in response to the Earth’s magnetic field. This ability is due to the presence of chainlike structures comprising magnetosomes, magnetite particles enclosed in a lipid membrane with associated proteins. Strain MV-1 differs from other, bettercharacterized strains of magnetotactic bacteria as the cells produce higher amounts of biomagnetite per litre of culture and its magnetosomes are unique in shape. This study investigates the presence and organisation of a gene cluster termed a “magnetosome island” within the genome of MV-1. In other magnetotactic bacteria this genomic region has been shown to contain many of the genes associated with magnetosome formation but has not been previously investigated for MV-1. One of the conserved fragments of this region was amplified using degenerate primers followed by extension of the known sequence using inverse PCR based technique and constructing plasmid libraries. Sequencing of the genome of strain MV-1 was accomplished as a part of this study. Significant work was done on comparison of the sequence quality obtained from SOLEXA, 454 and Sanger sequencing technologies. A number of obtained contigs were joined manually and the resulting sequence was automatically annotated using RAST. The obtained genome sequence of 3.6 Mb with a G+C content of 54.3 % was preliminarily analysed and used to search for magnetosome related genes. This study also analysed proteins associated with the magnetosomes of strain MV-1 using MALDI-TOF, LC-MS and Orbitrap mass spectrometry. These approaches allowed the identification of a number of proteins in the isolated magnetosome membrane fraction. Some of these proteins have very low similarity with other characterized proteins (either in magnetotactic bacteria or in other organisms). Another significant point is that genes that code for proteins such as MamR, MamK and MmsF were found to be present in several homologous copies within the “magnetosome island” of MV-1. Interestingly, this study shows that all homologous copies of these proteins were identified in the magnetosome membrane fraction. Generation of knock-out mutants of several specific genes from the “magnetosome island” of strain MV-1 was attempted; constructs were made based on suicide plasmids carrying the cre-lox or I-SceI systems. Despite altering numerous experimental conditions it was not possible to obtain conclusive evidence of the isolation of MV-1 transconjugants containing the integrated constructs. In order to investigate the cell localization of the magnetosome associated protein CAV30779.1, an enhanced green fluorescent protein (EGFP) fusion based construct was generated and transferred into MV-1 cells. The EGFP fluorescent protein fusions within the cells were detected by microscopy. This study reveals novel information about magnetosome formation in marine vibrio MV-1. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of the complex process of magnetosome formation in bacteria.