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.

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.

Vaccinia virus (VACV) strain Western Reserve gene A40R encodes a type II membrane glycoprotein with a C-type lectin-like domain at the C terminus. The A40 protein is not incorporated into virions, is nonessential for virus replication in cell culture and does not affect virus virulence in a murine intranasal model of infection. However, A40 does affect the outcome of infection in an intradermal infection model in which the virus lacking gene A40R produced smaller lesions and alterations in the host immune response. A40 has amino acid similarity to C-type lectins, such as NKG2A and DC-SIGN. This observation together with its location on the infected cell surface and its ability to bind to the surface of cells of the immune system is consistent with A40 functioning as an immunomodulator. It is possible that A40 might function by mimicking native host lectins or by modulating recognition of VACV-infected cells by cells of the immune system. To investigate the mechanism by which A40 affects the outcome of infection in vivo, a cloning and experimental strategy was devised to search for its ligand(s) and try to determine its structure in collaboration with protein crystallographers. To achieve these goals, the recombinant A40 protein has been produced in E. coli, mammalian cells, and from insect cells infected with recombinant baculoviruses. Bacterially expressed recombinant A40 was used to generate an antibody specific to A40 and this was then purified, characterised and used to further the characterisation of A40. The potential for A40 to interact with a ligand on the surface of another cell was investigated by a cell binding assay using recombinant A40 protein. This protein was produced in a mammalian system and was found to bind to the surface of immune cells but not to epithelial cells. In cytotoxicity assays, the absence or over-expression of A40 was found to modulate the ability of NK cells to kill VACV infected cells.

This thesis describes the genetic analysis of the heterogeneity of HGV/GBV-C and the characterization of the terminal regions of the viral genome. The sequence diversity across the HGV/GBV-C genome was significantly lower than that observed with HCV isolates. Comparative analysis of twenty-seven complete genome HGV/GBV-C sequences indicated the presence of four phylogenetic groups and this study demonstrated that these groupings could be reproduced by analysis of the 5'-untranslated region (5'-UTR) and of various sub-fragments. At the same time, the analysis of the 5'-UTR variability indicated the existence of group-specific polymorphisms, many of which are covariant and consistent with the proposed secondary structure of this region. An important difference between the polyproteins of HGV/GBV-C and HCV is the absence of a putative HGV/GBC-C core protein which is usually encoded at the 5'-end of the genome of flaviviruses. The buoyant density of HGV/GBV-C particles in human plasma was estimated to be between 1.07-1.12 g/ml, much lower than that of the other members of <I>Flaviviridae </I>family, except HCV. No HGV/GBV-C RNA was detected in fractions with densities higher than 1.17 g/ml which is expected for virus particles in immune complexes or in fractions with densities higher than 1.21 g/ml, the density range of HCV nucleocapsids. These biophysical properties correlate with the absence of a core-like protein in the genome of HGV/GBV-C isolates from different phylogenetic groups. The absence of the HGV/GBV-C nucleocapsid was also revealed by the sequence analysis data since no conserved open reading frame capable of encoding a core-like protein was identified. Generally, the untranslated regions at the 5' and 3' termini of a RNA virus genome contain regulatory elements important for viral RNA replication, transcription, translation and viral packaging. A comprehensive comparison and analysis of the primary sequence and secondary structure of the 3'-UTR of different HGV/GBV-C isolates allowed the construction of a common secondary structure model for this region and the identification of structural elements that may be involved in viral replication.

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.

Type I interferon is a cytokine induced upon virus infection that generates an antiviral state in neighbouring cells. Historically, research into avian interferon has lagged behind that into the mammalian system and hence reagents and assays are poorly developed. The avipoxvirus Fowlpox virus is naturally host restricted to avian cells. Modified Vaccinia virus Ankara (MVA) is an attenuated Vaccinia virus derived by serial passage in chicken embryonic fibroblasts (CEF), so that it too is now host restricted. In CEF pretreated with chicken interferon (ChIFN), plaque formation by Fowlpox virus (FP9 strain) was approximately 500-times less inhibited than MVA. A lack of bioinformatic clues within the FP9 genome sequence justified a broad-scale screen to identify any molecular determinants that contributed to this interferon resistance. The 266 kbp FP9 genome was divided into 61 overlapping fragments, each approximately 8 kbp long, to include each unique open reading frame (ORF) intact in at least one fragment. Splice-overlap-extension PCR protocols were optimised to assemble each genomic fragment with a guanine phosphoribosyl transferase (GPT) gene under the control of a poxvirus promoter, between two MVA flanks. These linear constructs were transfected into cells co-infected with MVA, for homologous recombination to insert the FP9 fragment and GPT cassette into the MVA genome. GPT-positive virus was enriched by culture with mycophenolic acid. In total, 65 recombinant viruses were recovered, representing 85% of the FP9 ORFs. Fourteen fragments were independently duplicated to control for mutations that may have been introduced during PCR. Recombinant viruses were screened for an increased ability to plaque in ChIFN-treated cells identifying 2 FP9 fragments, Fowlpox Interferon Resistance locus (FIR) 1 and FIR2. Further recombinant MVA viruses each containing a single gene from FIR1 were constructed and assessed for interferon resistance using the same techniques, identifying FP9.014 as contributing to FP9 ChIFN resistance.

This thesis concerns gene 176R from camelpox virus (CMLV) that encodes a protein known as viral schlafen (v-slfn). v-slfn has an N-terminal domain related to the p26 protein from baculovirus and a C-terminal domain related to mammalian schlafen proteins. A full length v-slfn is expressed by all sequenced orthopoxviruses except vaccinia virus (VACV) and variola virus. The baculovirus p26 proteins are poorly characterised, with no known function. In contrast, murine schlafen (m-slfn) proteins are upregulated in response to infection and the promoter for m-slfn2 has NF-κB and AP-1 binding sites. The prototypic slfn, m-slfn1, halts cellular proliferation by inhibition of cyclin D1 expression in vitro and both m-slfn1 and m-slfn8 reduce thymocyte proliferation in vivo. v-slfn is a predominantly cytoplasmic protein of 57 kDa that is expressed both early and late during CMLV infection. Expression of v-slfn reverses the growth arrest resulting from m-slfn1 expression, and this is a result of a reversal of the inhibition of cyclin D1 expression. This effect can be seen following overexpression of various transcription factors that upregulate cyclin D1 expression. Recombinant VACV expressing enhanced levels of v-slfn replicated and spread at a comparable rate to control viruses in vitro, but was less virulent than controls in the intranasal model of infection in vivo. A group of viruses based on VACV WR were constructed, which lack the gene fragments (B2R and B3R) corresponding to CMLV 176R. The undisrupted sequence for 176R was also re-inserted at this locus, resulting in a virus that expresses v-slfn from its natural promoter. In vitro characterisation showed no differences in replication or spread when compared to controls. Thus, v-slfn is an orthologue of mammalian slfn proteins, and may exert its effect by reversing their inhibition of cellular proliferation.

Vaccinia virus (VACV) encodes multiple proteins which modulate NF-[kappa]B activation to evade the host immune response. One example is B14, a virulence factor that reduces NF-[kappa]B activation via interaction with IKK [Beta]. B14 has orthologues in many VACV strains, including modified virus Ankara (MVA), which lacks many of the immunomodulators present in other VACV strains. Here, the MVA counterpart of B14, protein 183, was characterised. 183R was both removed from MVA and inserted into the B14R locus of a VACV strain Western Reserve (WR) lacking B14R, but in each case there was no change of phenotype. Protein 183 shares 95 % amino acid identity with B14 but, unlike B14, was unstable in eukaryotic cells, unless protein degradation was inhibited. Protein 183 did not inhibit NF-[kappa] B activation in response to cytokine stimulation, as does B14, nor did it restore the virulence phenotype of WR lacking B14R back to wild type. Therefore, the mutations incurred by 183 during the derivation of MVA have rendered the protein non-functional. However, other MVA immunomodulators remain to be characterised and this thesis describes a bacterial artificial chromosome (BAC) system that may facilitate the improvement of MVA in its role as a vaccine vector. To further characterise VACV modulation of NF-[kappa] B, a VACV WR strain was constructed lacking B14 and another VACV modulator of NF-[kappa]B, WR A49. Initial characterisation showed no change with this mutant virus in replication or spread. The effect of the B14 on NF-[kappa]B was further characterised by studying the B14-IKK [Beta] interaction using IKK[Beta] and B14 mutants. Residues 300 to 480 of IKK [Beta] were shown to be required for the interaction and a mutant encompassing this region co-purified to a degree with B14 when expressed in E. coli. In addition, mutagenesis showed that B14 residue F130 is required for the interaction of the two proteins.

Epstein-Barr virus (EBV) establishes a latent infection in the human host. In order to produce infectious virus particles EBV must reactivate from latency and enter its lytic cycle. BZLF1 is the immediate early gene in EBV that mediates the switch between latency and the lytic cycle. The BZLF1 gene is under the control of the Zp promoter. Reactivation from latency is studied in EBV positive Akata cell lines where EBV can be reactivated by crosslinking the B-cell receptor (BCR) using antibodies to mimic antigen binding. This system uses stably transfected reporter plasmids to study Zp regulation. Mutagenesis identified additional regions of the promoter that contribute to regulation and the ZID MEF2 binding site was demonstrated to be functionally important during the initial stages of Zp activation. XBP-1 splicing, previously implicated in Zp reactivation, was found to occur rapidly in this system in response to BCR crosslinking and parallels the transient induction of Zp. Chromatin remodelling also plays an important role in Zp regulation. An inducible BZLF1 expression system, independent of BCR signalling, was developed in Akata cells that accurately mimics BZLF1 activity and provides a novel approach to study repression at Zp. The BZLF1 protein is related to the bZIP family of transcription factors. BZLF1 contains a bZIP motif in which C-terminal residues fold back against a zipper region that forms an a-helical coiled-coil. The 208SSENDRLR215 sequence in the zipper region is conserved between BZLF1 and C/EBP. Point mutagenesis in this sequence revealed the importance of individual residues for transactivation and progression to DNA replication. The restoration of BZLF1 DNA replication activity by complementation of two deleterious mutations (S208E and D236K) indicated that the interaction of the C-terminal tail and the core zipper region is required for DNA replication, identifying a functional role for this structural feature unique to BZLF1.

The high mutation rate of influenza virus results in antigenic drift, meaning that each of the three components in the trivalent influenza vaccine are updated regularly so that the vaccine antigen closely matches the predominant or emerging strain. The production of influenza vaccines from the chosen seed has relied on embryonated chickens eggs for more than 40 years. Recent technological advances have resulted in the evaluation of several cell lines as alternative substrates for influenza vaccine production. Reverse genetics of influenza viruses allows the creation of viruses at will from cDNA. However, licensed cell lines have so far proved unpermissive for virus rescue and permissive lines remain largely unlicensed. A reverse genetics system for the production of influenza vaccines in PER.C6 cells has been developed and optimised. Many recent clinical isolates do not grow in eggs and hence require reassortment with a high growth, egg permissive backbone. Adventitious agents aside, cell lines able to support growth of clinical isolates could be used directly included in the vaccine, without the need for reassortment. However, this could lead to year on year variation in the quality and characteristics of the vaccine. Particularly pertinent, is the variation in the amount of IFN a clinical isolate can induce, which could severely limit yields. Yields and effects of IFN for a panel of high and low inducing viruses are investigated in a number of potential vaccine cell lines. The mechanisms behind virus IFN induction have been investigated. Using a panel of high and low inducing viruses the differences in growth and viral protein expressions, and activation of PRR has been analysed. The IFN response in PER.C6 cells has been characterised. Through an improved understanding of the mechanisms of IFN induction and inhibition, antagonists could be introduced into the vaccine cell line to improve yields.