This thesis describes the derivation of restriction endonuclease maps for HSV-1and HSV2 DNA; a study of the homologous regions between the genomes of HSV-1,HSV-2,EHV-1and PRV, and the effect of homology upon recombination between HSV-1 and HSV2; nucleotide sequences of the L-S joint regions in HSV-1 andHSV-2 DNA; and analysis of two HSV-1/HSV-2 intertypic recombinants which fail to invert normally in one or both segments of the genome. Restriction endonuclease maps were already available of HSV-1 DNA for XbaI, HindIII, EcoRI, BgIII and HpaI, and of HSV-2 DNA for these enzymes and KpnI. Maps of HSV-1DNA for KpnI, BamHI, XhoI and PvuII and of HSV-2DNA for BamHI, were determined using the techniques of simultaneous digestion with two endonucleases, recleavage of isolated restriction fragments, and blot hybridisation. Previously reported size heterogeneity at the L terminus and L-S joint of the two genomes was confirmed. The results suggested that HSV-2 DNA is 1-2 x10(6) larger in molecular weight in the S segment than HSV-1 DNA. HSV-1 and HSV-2 DNA share approximately 50% homology, and PRV DNA possesses not greater than 8% homology with HSV-1 DNA. Hybridisation of 32 P-labelled recombinant plasmids containing HSV-1 or HSV-2 DNA inserts to blot strips of restriction endonuclease digests of HSV-1 or HSV-2 DNA inserts to blot strips of restriction endonuclease digsts of HSV-1 or HSV-2 DNA showed that the two genomes are collinear, within the resolution attained in these experiments. Hybridisation of 32 P-labellea HSV-1 or HSV-2 DNA to similar blot strips allowed seven regions of the genome to be identified which are more homologous than neighbouring regions. EHV-1 and PRV DNA also showed greater homology to these regions of the HSV genome than to others, but no significant homology between HSV DNA and HCMV DNA was detected. Homologous regions probably reflect greater conversation of the structures of polypeptides encoded by them. Five good candidates for conserved HSV-1 polypeptides are major DNA- binding protein, the major capsid protein, the DNA polymerase, and two immediate- early polypeptides, Vmw IE 175 amd Vmw IE 136'(143). Hybridisation of cloned HSV DNA fragments to blot strips of EHV-1 or PRV restriction endonuclease digests showed that homologous regions in the L segment of the EHV-1 genome are collinear with the L segment of the HSV-1 genome in the IL arrangement. Homologous regions between HSV and PRV DNA where shown not be arranged in a simple collinear fashion. The genome structures were analysed of more than a hundred HSV-1/HSV-2 intertypic recombinans produced by marker rescue of HSV-1 tsD with HSV-2 restriction fragments spanning the L-S joint. The recombinants possessed crossovers preferentially in homologous regions. At least two of the genome arrangements (P and IL) recombined, a result which disproves the earlier proposition that only one of these two arrangements is able to take part in the generation of viable recombinants.

Herpes simplex virus type 1 (HSV-1) Immediate-early protein ICP0 is important for regulating the balance between lytic and latent infections. The RING finger domain of ICP0 acts as an E3 ubiquitin ligase, binding to E2-ubiquitin conjugating enzymes and target proteins, promoting their polyubiquitination and subsequent degradation. ICP0 localises to cellular nuclear sub-structures known as ND10 at early stages of infection, and the RING finger domain induces the degradation and dispersal of ND10 proteins, which form part of the cell's intrinsic antiviral defence mechanism. The RING finger domain of ICP0 consists of an alpha-helix, and this and the loop regions are involved in interactions with the E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2E1. Previous work using an ICP0-inducible cell line system found a mutation (N151D) in ICP0's alpha-helix, which allowed complementation of an ICP0-null mutant virus plaque formation but caused a substantial defect in the induction of reactivation of quiescent HSV-1. This raised the possibility that the mechanisms controlling lytic infection and reactivation may be separable. The main focus of this study was to investigate the N151D mutation and other mutations located within the alpha-helix (including K144E and K144E/N151D) using an inducible ICP0 expression cell system and virus infection studies. The phenotypes of these alpha-helix mutants were characterised to investigate if complementation/lytic infection and reactivation involve differential activities of ICP0. Additionally, these alpha-helix mutants were analysed for their in vitro E3 ligase ability and ability to interact with components of the ubiquitin pathway, focusing on E2 ubiquitin-conjugating enzymes. The results from this study using the inducible ICP0 cell system confirmed previous results. The alpha-helix mutants had a greater defect during reactivation than complementing the plaque forming defect of an ICP0-null mutant virus, and this was more noticeable for the K144E mutant. The virus infection studies showed a greater correlation between the effects of the mutations on the degradation of ND10 proteins, plaque formation and replication at low multiplicity of infection. The defect of the K144E mutation was more profound than N151D in all the assays, and the activity of a double mutant including mutations at both K144 and N151 (KE/ND) was reduced to levels comparable with the RING finger deletion mutant. Infection with mutants K144E and KE/ND greatly reduced the efficiency of reactivation of quiescent HSV-1 even at multiplicities of infection where their lytic infection was not severely affected, whereas N151D showed an intermediate phenotype. Furthermore, this study showed that ICP0 has the potential to interact with multiple E2 ubiquitin-conjugating enzymes, and the alpha-helix mutations may affect these interactions. Further investigation will be required to examine the roles that these E2 ubiquitin-conjugating enzymes play during HSV-1 infection. The data in this study indicate that there is no strong evidence to suggest that ICP0 utilises differential activities of its RING finger to mediate reactivation and the stimulation of lytic infection, despite the likely dissimilar nature of the viral chromatin structure in the two situations. These findings, however, provide an insight into the biological importance of the RING finger alpha-helix of ICP0 during the course HSV-1 infection and especially during reactivation from quiescence. In addition, motifs present within ICP0 that may be involved in interactions with other cellular proteins were analysed. These included the PPEYPTAP motif present within retroviral Gag proteins, the SIAH-1 interaction motif and residue T67 (which are all involved in interactions with cellular E3 ubiquitin ligases), and the CoREST binding region. Furthermore, a region of homology between alphaherpesvirus ICP0 proteins downstream of the RING finger domain, which had not previously been investigated in detail, was studied. These studies indicated that the region downstream of the RING finger domain (residues 211-222) may contribute to ICP0's activity, but no major role was detected for the other motifs studied.

Viral respiratory infections are associated with substantial mortality, morbidity, and a vast economic and healthcare burden. The diagnosis of such infections has been revolutionised by the introduction of molecular methods such as RT-PCR. This has resulted in high levels of sensitivity and specificity along with a rapid turnaround time in comparison to previous methods. As a product of this success, the diagnosis of respiratory infections makes up a large proportion of the workload in most diagnostic laboratories. The development of next generation sequencing (NGS) may be the next revolution in the field of virus diagnostics. This allows a metagenomic approach to specimen processing whereby target independent sequencing of all genetic material is carried out. The research presented in this thesis initially sought to examine if NGS would be feasible in the field of respiratory virus diagnostics. The aim was to apply NGS to clinical specimens in parallel with the current diagnostic RT-PCR assays employed by the West of Scotland Specialist Virology Centre (WoSSVC) to determine if NGS could give the same level of results as RT-PCR and whether the sequence information generated in the process could be used in further characterising the detected pathogens. Further to this, the NGS method was then applied to the detection of norovirus from faecal specimens to demonstrate the utility in other areas of viral diagnostics. The results show that multiple viral pathogens can be detected from clinical specimens without specific virus targeting. The method was less sensitive than RT-PCR but sequence data generated during the process was utilised viral detection, subtyping and phylogenetic analysis. We also demonstrated that a single workflow could be applied to multiple specimen types in the detection of RNA viral pathogens.

Picornaviruses are important causes of human illness and it is necessary to understand more about how these viruses function. Human parechoviruses (HPeV) are common pathogens and studies have shown that 95% of people become infected with HPeV at a very early age, usually with symptoms such as mild diarrhoea and fever. However, one virus type HPeV3, is implicated in much more serious cases of neonatal disease and so it is important to understand HPeVs to increase the opportunity to develop drugs or vaccines against the infection. The HPeV1 genome encodes a single polyprotein that is cleaved into structural and non-structural proteins. Analysis of one region of the genome (encoding the polymerase, 3Dpol) shows that some codons are perfectly conserved, suggesting functions in addition to protein coding. This region seems to fold into an RNA secondary structure made up of three stem-loops and a tertiary structure “kissing” interaction. The structure was validated by comparing all the available HPeV sequences and found to be highly conserved. To investigate if the structure has a role in RNA stability, an EGFP fluorescent assay was used. Sequences containing the structure was added to the 3’ UTR of the EGFP gene. A mutant with 21 mutations which completely destroys the RNA structure was also used. A FACS-based method was used to measure expression levels of EGFP. The results showed that there was a significant reduction in fluorescence from the mutant construct. The effect of the structure was also investigated in infected cells and in cells exposed to different stresses which could mimic virus infection. The results suggest that the structure can positively affect RNA stability/translation. Further investigation on other possible roles such as RNA replication and translation should be performed to improve the understanding of the biology of the structure in HPeVs and a Renilla Luciferase reporter gene system was assembled to facilitate the studies in the future.

Picornaviuses replicate in the cytoplasm, but there is growing evidence that the cell nucleus is affected by infection e.g. transcription factor cleavage, relocation of nuclear proteins and alteration to nucleo-cytoplasmic shuttling. It was previously observed that Parechovirus genus members affect the distribution of the nuclear paraspeckle protein PS PC-1, which is an RNA-binding protein involved in splicing and RNA export. To investigate if this is a general feature of picornaviruses infection, coxsackie virus A9 (CAV-9) was studied. This is a typical member of the large and most medically-important picornavirus genera Enterovirus, which is genetically divergent from Parechovirus. Using an EGFP-PSPC-1 fusion, we found that infection changes the distribution of PS PC-1 from nuclear paraspeckles to cytoplasmic granules that do not seem to correspond to known cytoplasmic foci of RNA-binding proteins e.g. stress granules and P-bodies. They also do not correspond to CAV-9 replication complexes. Two other paraspeckle proteins (PSF and NONO) colocalise with PSPC-1 in these structures. The effect does not seem to be due to cleavage of these proteins by virus proteases, phosphorylation at two sites known to be involved in PSF translocation or sumoylation. It is dependent on part of PSF, between amino acids 452-606, which is also needed for paraspeckle localization and which is involved in key interactions between PSF, PSPC-1 and NONO. There are few reports on the significance of paraspeckle proteins in virus infection. Our results suggest that we have identified a novel cellular compartment, or a structure induced by virus infection. If this is proved to be required by the virus, then it could be a potential drug target for the development of a new class of antiviral agents against this important group of viruses.

Human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immunodeficiency syndrome (AIDS), a leading cause of death worldwide infecting millions of people each year. Despite intensive research in vaccine development, therapies against HIV-1 infection are not curative and the huge genetic variability of HIV-1 challenges drug development. Current animal models for HIV-1 research present important limitations, impairing the progress of in vivo approaches. Macaques require CD8+ depletion or large portions of the genome to be replaced by sequences derived from simian immunodeficiency viruses to progress to AIDS, and the maintenance cost is high. Mice are a cheaper alternative, but need to be 'humanized' and breeding is not possible and knockout experiments are difficult. The development of an HIV-1 clone able to replicate in mice is a challenging proposal. The lack of human co-factors in mice impedes function of the HIV-1 accessory proteins Tat and Rev, hampering HIV-1 replication. The Tat and Rev function can be replaced by constitutive/chimeric promoters, codon-optimized genes and the constitutive transport element (CTE), generating a novel HIV-1 clone able to replicate in mice without disrupting the amino acid sequence of the virus. By minimally manipulating the genomic 'identity' of the virus, we propose the generation of an HIV-1 clone able to replicate in mice to assist in antiviral drug development. My results have determined that murine NIH 3T3 cells are able to generate pseudotyped HIV-1 particles, but they are not infectious. Codon-optimized HIV-1 constructs are efficiently made in human HEK-293T cells in a Tat and Rev independent manner and capable of packaging a competent genome in trans. CSGW (an HIV-1 vector genome) efficiently generates infectious particles in the absence of Tat and Rev in human cells when 4 copies of the CTE are placed preceding the 3’LTR. HIV-1 replication competent genomes lacking tat expression and encoding different promoters are functional during the first cycle of replication when Tat is added in trans. Finally, we developed a replication competent HIV-1 clone lacking tat and rev genes and encoding 4CTEs that could be a future candidate for HIV research. My results shown that the development of an HIV-1 Tat-Rev independent clone could become a candidate for HIV research in a near future, but further investigations are necessary before proposing our model as an alternative yet.

The bacterium Vibrio cholerae is the causative agent of the acute diarrhoeal disease cholera. V. cholerae is naturally found in aquatic environments but can switch lifestyles to cause disease in humans. The lifestyle switch requires modulation of genetic systems. Much of the regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, I show that the global transcription regulator, cAMP receptor protein (CRP), plays an integral role in the regulatory network that controls lifestyle switching. I have identified two sites for CRP in the intergenic region between rtxHCA and rtxBDE, a locus which encodes the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin and toxin transport system respectively. Using a combination of genetics, biochemistry and in vivo animal studies, I have determined a CRP dependent regulation of gene expression for toxin transport in response to host infection. This work shows that rtxHCA is constitutively expressed and not subject to regulation by CRP whist CRP acts as a repressor of rtxBDE transcription. Examination of further CRP targeted genes reveals similar behaviour upon host colonisation. These findings suggest that toxin export occurs in nutritionally rich environments, where the MARTX toxin can exert cytopathic and cytotoxic effects on host cells.

The family Hantaviridae, of the Bunyavirales order, contains many important human pathogens of which Puumala virus (PUUV) is the most widely distributed member in Europe. It causes nephropathia epidemica, a milder form of haemorrhagic fever with renal syndrome and mortality rates of up to 1% have been reported. They are enveloped viruses, with a tripartite single-stranded negative sense RNA genome, that replicate solely in the cell cytoplasm. Several factors have been proposed to play a role in hantavirus pathogenicity, including regulation of innate immune responses, cell signalling and enhancement of endothelial cell permeability. The work presented in this thesis describes biological and molecular characterisation of the mechanisms behind a hantavirus infection. Transcriptome analysis was a valuable tool that allowed the investigation of the broader picture of the effect of PUUV on the host cell. 549 and as many as 7,283 genes were differentially expressed at 24 and 48 hours post infection, respectively, in PUUV-infected cells, revealing extensive transcriptional change. By 48 hours normal cellular function appeared severely disrupted. Most genes involved in mitochondrial functioning were down-regulated, suggesting a reduced cellular energy level. Dysregulation of an important signalling hub such as mitochondria might have a more global impact on cellular functions, consistent with findings in this study. Intrinsic apoptosis pathway, which is mediated by mitochondria, appeared inhibited. Whereas, death receptor signalling was activated. Pathways associated with actin formation, organisation and signalling also appeared inhibited. Members of Rho family of GTPases, which are key regulators of actin dynamics, were down-regulated overall. Furthermore, integrin signalling, which mediates Rho GTPase activity, was also inhibited. Immunofluorescence studies revealed marked morphological changes in mitochondria and substantial remodelling of the actin cytoskeleton. Further analysis revealed a direct interaction between PUUV N protein and anillin, a scaffolding protein that mediates formation and organisation of actin filaments, suggesting a potential novel mechanism behind actin cytoskeleton reorganisation. Biological interferon (IFN) assays enabled the identification of two IFN antagonists encoded by PUUV, the cytoplasmic tail of the Gn glycoprotein and the non-structural protein, NSs. The Gn tail inhibited type I IFN induction at the level of TRAF3-TBK, in agreement with previous studies on other hantaviruses. Whereas, NSs was found to block IFN induction downstream of IRF3, suggesting it was able to disrupt transcription or translation. Utilising immunofluorescence and chromatin immunoprecipitation methods, it was found that PUUV NSs possessed a potential mechanism to inhibit transcription by blocking serine 2 phosphorylation at the C-terminal domain of RNA polymerase II in a similar manner to the previously described Bunyamwera virus NSs. The data presented in this thesis illustrates the broad range of mechanisms employed by PUUV to alter cell function to aid virus replication and subvert innate immune responses.

Microbial lab-based evolution is a technique to study evolutionary theory. It is a method which can provide insights into the ability of a microbe to adapt to a biological process such as low pH. To investigate pathways that could lead to an acid resistant phenotype in E. coli, we evolved six independent lines or populations of E. coli K-12 MG1655 by iterative growth and dilution experiments for approximately 740 generations at pH 4.5. Clones isolated from evolved populations were significantly fitter than the ancestor at pH 4.5. Five of the six evolved strains had acquired an identical mutation in rpoA, and mutations in cytR in addition to other mutations. PCR analysis of the fossil record of the evolved populations showed that the arcA mutations always arose first followed by the rpoA mutations. Investigating the genetic basis of adaptation showed that the mutations in arcA were loss of function in nature and conferred caused an intermediate increase in fitness. Transcriptional analysis showed a global change in their transcriptional signatures with significant upregulation of the arcA regulon. Our study showed that loss of function of ArcA caused an increase in the RpoS activity of the acid evolved strains leading to a general stress resistant phenotype.

The work presented in this thesis is concerned with the characterisation of the UL32 gene of herpes simplex virus type 1 (HSV-1). UL32 encodes an essential 596 amino acid cysteine-rich, zinc-binding protein that is highly conserved throughout the herpesviruses. The UL32 protein is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Conservation is highest at the C-terminus and three CxxC motifs are present in almost all known herpesvirus UL32 sequences The UL32 antibodies available in the laboratory at the beginning of the project were incapable of detecting small amounts of UL32 protein and so new rabbit antisera were created. Soluble extracts from insect cells infected with a UL32-expressing baculovirus (AcUL32) were fractionated by anion exchange chromatography and the UL32-containing fractions used to immunise rabbits. The resultant antisera successfully recognised UL32 from transfected, HSV-1 infected and baculovirus infected cells on western blots, and UL32 in transfected cells by immunofluorescence. I performed random mutagenesis of the UL32 gene in an effort to examine structure-function relationships within this protein, and generated a panel of 37 mutants containing 5 amino acid insertions at distinct positions. The abilities of these mutants to complement the DNA packaging and growth defects of a virus lacking a functional copy of UL32 (the null mutant hr64) were examined and 15 of the mutants retained functionality in both assays. A complete correlation was found between the ability of mutants to support growth and DNA packaging, suggesting that the key functions of UL32 are confined to the DNA packaging pathway. There was also good correlation with the degree of amino acid conservation within UL32, with most of the mutants which abolished functionality being located in the highly conserved regions, and the functional mutants in less conserved regions. A number of site-specific mutants were also created, in which the paired cysteine residues were replaced with serines (i.e. CxxC to SxxS). Mutation of the first and third cysteine pairs (from the N-terminus) completely abrogated growth and packaging, whereas significant functionality was maintained following mutagenesis of the central pair. Finally, removal of the C-terminal 4 amino acids also resulted in generation of a non-functional protein. Generation of an HA-tagged UL32 construct and the introduction of this into HSV-1 allowed the localisation of UL32 in infected cells to be studied. In contrast to previous reports, I detected UL32 predominantly in the nuclei of infected cells, co-localising with ICP8 in replication compartments. DNA packaging has previously been shown to occur within the replication compartments and a number of the other packaging proteins also localise to these sites. It was previously reported that UL32 played a role in the localisation of capsids to the replication compartments. However, work presented in this thesis shows this not to be the case, and that capsid proteins VP5 and VP19C were correctly localised in replication compartments during infections with the UL32 mutant hr64. I found no evidence of UL32 interaction with the UL6, UL25 or UL17 DNA packaging proteins in HSV-1 infected or transfected cells, or using immunoprecipitation from baculovirus-expressed cells. Immunofluorescence studies of co-transfected cells showed that UL15 could direct the partial re-localisation of UL32 from the cytoplasm to the nucleus. The addition of the other terminase subunits UL28 and UL33 caused the complete re-localisation of UL32 to the nucleus, suggesting that UL32 might interact with the terminase complex. Fifteen of the insertional mutants were completely re-localised to the nucleus in the presence of UL15, UL33 and UL28, with eleven further mutants showing an intermediate phenotype of partial nuclear localisation. The ability to at least partially co-localise with the terminase complex appeared necessary for the ability of the mutants to support virus growth and DNA packaging, suggesting that this interaction may be essential for the function of UL32. However, no interaction could be demonstrated between UL32 and any of the individual terminase subunits using immunoprecipitation from insect cells. A series of experiments was undertaken to further characterise the UL32 protein. A new UL32 mutant virus (Δ32EP) was generated by insertion of a kanamycin resistance cassette in place of a large portion of the UL32 gene. This mutant had an indistinguishable phenotype from hr64, confirming that the main function of UL32 is within DNA packaging. The functional conservation between HSV-1 UL32 and the homologues from HCMV and VZV was examined, but neither protein could support the growth of Δ32EP. DNA fragments from replicated concatemeric DNA from Δ32EP infected cells behaved similarly to wt HSV-1 fragments in PFGE, suggesting that UL32 is not involved in the resolution of branched structures within the genome prior to packaging. UL32 had previously been reported to bind zinc, and this was confirmed using a zinc-release colourimetric assay. The amount of zinc bound to soluble baculovirus-expressed UL32 was quantified, showing that UL32 bound zinc in a 1:1 molar ratio. UL32 does not share all of the characteristics of a zinc finger motif, but the results of the mutagenesis experiments suggest that the outer CxxC/CxxxC motifs may be important for zinc binding. Because of its zinc-binding properties and potential interaction with the terminase complex, the DNA binding properties of UL32 were also investigated. It was found that UL32 did not bind to dsDNA containing either the minimal packaging sequence (Uc-DR1-Ub) or an unrelated non-HSV-1 sequence using an electrophoretic mobility shift assay (EMSA).