The Caliciviridae are a group of small, non-enveloped viruses with a positive sense, single stranded RNA genome. Caliciviruses include the noroviruses, responsible for winter vomiting disease, as well as several important veterinary pathogens. Feline calicivirus (FCV) is an excellent model for studying calicivirus entry, having a known protein receptor and being readily propagated in cell culture. Here we explore calicivirus entry, using FCV. Virus entry is the critical first step of infection and is therefore an important area of study. Both alpha 2-6 linked sialic acid and feline junctional adhesion molecule A (fJAM-A) have been identified as receptors for FCV. The attachment of FCV to fJAM-A, is followed by uptake via clathrin mediated endocytosis. Little is known, however, on the viral escape mechanism leading to delivery of the viral RNA into the cytoplasm. We set out to explore the nature of FCV attachment and uncoating using structural, biochemical and biophysical analyses. By cryogenic electron microscopy we have characterized the virus-receptor interaction at high-resolution. Using electron microscopy and an RNA release assay, we have investigated virion uncoating. Finally, we have explored the importance of receptor glycosylation, and oligomerisation. Our analysis has allowed us to construct an atomic model of the major capsid protein VP1. Upon binding to fJAM-A, FCV undergoes a conformational change (rotation and tilting of the capsomeres). Flexibility in the receptor decorated virion has prevented high-resolution structure analysis of the conformational change or the virus-receptor interaction. We have, however, seen that the structural changes are limited to the capsid spikes. We hypothesised that the conformational change may be a priming step that would prepare the virus for uncoating upon internalisation. We found that upon lowering the pH below 5, receptor decorated virions disassembled, supporting this hypothesis. Disassembly of the virus-receptor complex at low pH presented a tool for estimating the quantity of receptor needed to prime the capsid for uncoating. Cryo-EM studies reveal that FCV bound fJAM-A is monomeric although the receptor was found to be dimeric in solution as previously described for the human and murine homologues. Furthermore, it is hypothesised that this is the form found at tight junctions between cells. We propose that disruption of fJAM-A homodimers may be the mechanism by which induction of viral uptake by endocytosis is triggered. Finally, we have confirmed the presence of an N-linked glycosylation on fJAM-A and show that the removal of this carbohydrate moiety does not affect viral binding in vitro.

Analysis of primary sequence data revealed a subset of open reading frames that were predicted to encode HSV-I dUTPases based on five areas of local primary sequence conservation. The differences in the primary sequence organisation of these motif regions allowed the description of two distinct dUTPase classes. The class I dUTPases are encoded by a diverse range of organisms and are characterised by a trimeric arrangement with subunit protein lengths approximating 150 amino acids. The class II dUTPases are specific to the herpesviruses and are characterised by a monomeric arrangement with a protein chain length approximately double that of their class I counterparts. It has been proposed that the class II dUTPases arose by the intragenic duplication of the class I open reading frame. In this thesis the class I structures were used as a basis to investigate the HSV-1 class II dUTPase in terms of structural and evolutionary relationships. To allow a defined approach to functional analysis of the HSV-1 dUTPase a tertiary structural model was generated for the class II enzymes. Following intensive primary sequence analysis a method was devised for comparing class I and class II sequences directly. Secondary structure prediction programs were utilised to judge the basic structural similarities between the two classes allowing the proposition of several defined hypotheses. The available class I structural information was utilised in order to characterise highly conserved structural elements within the class I group. In was then possible to relate this data set to class I primary sequences and subsequently to the generation of a class II model. Various modelling techniques were used based on the constraints on the structural organisation that could achieve a functionally active monomer plus the set of hypotheses defined in the earlier work. Mutagenic analysis of the HSV-1 dUTPase was then possible using the class II model as a reference. Several targets were investigated based on predicated functionally important regions. Analysis of these mutant enzymes was performed using purified recombinant HSV-1 dUTPase expressed from the T7 E.coli expression system.

Many viruses exploit and, in some cases, promote host cell endocytic pathways for infection. These pathways include caveolar and clathrin-mediated endocytosis, as well as macropinocytosis. The entry mechanisms of many viruses are not clear cut, with more than one pathway implicated in some cases. Hepatitis C virus (HCV) is a hepatotropic virus associated with liver disease, fibrosis, cirrhosis and hepatocellular carcinoma. There are four co-receptors or “entry factors” for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI) and the tight junction proteins Claudin 1 (CLDN1) and Occludin (OCLN). Clathrin-dependent endocytosis of HCV has been demonstrated in hepatoma cell lines and has also been shown to be the route of entry for co-receptor CD81; however, other endocytic pathways have not been considered. This thesis investigates a role for caveolae in HCV entry. In addition, it has recently become apparent that the epidermal growth factor receptor (EGFR) is required for viral entry into hepatoma cells and that stimulation with EGF results in increased entry and infection. This thesis investigates the role of EGFR in the endocytosis and trafficking of HCV receptors/entry factors, with a particular focus on CD81, using live-cell and super-resolution imaging techniques.

Enterohaemorrhagic Escherichia coli (EHEC) pathogens represent major difficulties to health as they cause infections and in the gastrointestinal tracts of animals. Previous studies revealed that the locus of enterocyte effacement (LEE) pathogenicity island is the major player in triggering the attaching and effacing lesions that induce death of host cells. The first step in its expression is activation of the LEE1 promoter that controls expression of the LEE encoded regulator (Ler) that activates LEE expression. The global regulator of LEE activator (GrlA) transcription is considered to be especially important in this process. Although GrlA binds to the LEE1 promoter and activates transcription initiation in vitro, the fold of activation was only ~ 1.5 fold. The aim of this work was to discover the factors that trigger GrlA activity. Thus, in chapter 3, it is shown how bacterial attachment to host cells triggers GrlA activity to an extent not seen during planktonic growth, with up to ~20 fold activation. Data also show that the level of free unbound GrlA defines the activity of the LEE promoter by GrlA, and the previously characterised GrlR anti-GrlA protein merely serves to buffer the level of GrlA.

A capsule composed of mainly α-glucan forms the most outermost layer of cell envelope of Mycobacterium tuberculosis, the causing agent of tuberculosis. Capsule participates in modulating the immune system responses. In GlgE pathway, one of the three pathways involved in α-glucan synthesis, trehalose in converted to glucan through the activity of four enzymes, trehalose synthase, maltokinase Pep2, maltosyltransferase GlgE and branching enzyme GlgB. It has shown that M. tuberculosis TreS and Pep2 form an octameric complex together. During this study we solved the structure of the complex at 3.6 Å in M. smegmatis. The structure reveals two pairs of Pep2 monomers bind to the opposite sides of the diamond shaped TreS tetramer in a 4 + 4 complex. However, studying the stoichiometry of the complex using other techniques such as: ITC, AUC and SAXS indicates a 4 + 2 complex formation in the solution. Moreover, our data using size exclusion chromatography would suggest that this complex formation is pH dependent and favors complex formation at more acidic pHs. This study is a step forward towards better understanding of the capsule synthesis in M. tuberculosis and highlights the role of complex formation between enzymes as an effective strategy to control the metabolic pathways in mycobacteria.

Plasmids RK2 are broad host range plasmids and they carry genes for antibioticresistance. Their central control operon, encoding two global regulators KorA and KorB, is anatural example of a negatively self-regulated operon. The aim of the work described herewas to use mathematical models to better understand the function and adaptation of KorA and orB regulation, in the context of available experimental data. The deterministic models combined with statistical inference allowed for analyses of he regulation mechanism and hypothesis generation about the system dynamics. Furthermore, the extended model was applied to data from different plasmid hosts, in order to indentify processes that might cause a difference in KorB abundance between the hosts. The stochastic multi-scale models were constructed and used in order to understand evolutionary reasons for such a negative and cooperative autoregulation. The system was tested for optimizations in mRNA production, response time, fluctuation and robustness. Moreover, the effect of cooperative regulation on the response time of the switch between vegetative and conjugative transfer, was also tested. In conclusion, the studies have deepened our understanding of the model plasmid RK2, of negative and cooperative regulation more generally, and generated hypotheses suitable for experimental validations.

Human herpesvirus (HHV) –6A and HHV-6B are unique among herpesviruses in their ability to integrate into the telomeres of human chromosomes and be inherited in a Mendelian fashion. Based on current data, inherited chromosomally integrated HHV-6 (iciHHV-6) is present in 0.5-2% of the UK population. There is increasing evidence to suggest that iciHHV-6 is not a dead-end for the virus, and that HHV-6 can be excised from the genomes of iciHHV-6-positive individuals. It is hypothesised that this excision occurs from the formation of T-loops between the end of the telomere and the viral telomere like repeats (TLRs). T-loops form naturally in a cell as part of the complex that protects the end of the chromosomes; however, under certain circumstances these can be excised leading to a loss of telomeric repeats from the chromosome and the formation of circular, extra-chromosomal telomeric sequence. There is increasing evidence that excision can lead to reactivation of the virus and potentially cause disease. Our current understanding of the phenotypic associations of iciHHV-6 is based on case-reports and case-control studies of individual diseases. The work presented in this thesis set out to answer a number of questions regarding the phenotypic consequences, and genome dynamics of iciHHV-6. First, the association between both exogenously acquired HHV-6 and iciHHV-6 and classical Hodgkin lymphoma (cHL) was examined in a case-control study. Whilst exogenously acquired HHV-6 was significantly associated with cHL the virus was present at low levels in DNA extracted from cHL tumours. Virus at such low level was concluded as not having a direct role in the pathogenesis of cHL. A case control study of iciHHV-6 and cHL revealed no difference in the prevalence of iciHHV-6 amongst cases and controls, but identified a single iciHHV-6-positive individual who appeared to have four integrated viral genomes. Secondly, iciHHV-6 was examined in the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort, in a hypothesis generating study. It was revealed the iciHHV-6 was present at a prevalence of 2.7%. Further analysis showed a statistically significant difference in the prevalence of iciHHV-6 between individuals born in Scotland (2.8%) and England (1.8%). Analysis of disease phenotypes revealed potential associations between iciHHV-6 and breast cancer in an unrelated subset of the GS:SFHS cohort. Also confirmed was the recent 3 report of an association between iciHHV-6 and angina pectoris. Analysis of other variables revealed iciHHV-6-positive females had a lower average Mill Hill vocabulary test score than iciHHV-6-negative females; and that iciHHV-6-positivity was associated with participation in fewer years of education. Thirdly, the iciHHV-6 genome in an LCL generated from an iciHHV-6A-positive individual was shown to be dynamic. The gross structure of the HHV-6 genome is a unique (U) region flanked by direct repeats (DR) (DR.U.DR).Analysis using droplet digital PCR (ddPCR), on DNA extracted at various time points of the LCL culture revealed that viral sub-genomic regions were lost in a proportion of cells, which coincided with a reduction in population doublings of the culture. At the point of reduction of viral copy number, an excess of HHV-6 DRs was noted suggesting that only a portion of the viral genome had been lost in these cells. Gradually the viral copy number returned to approximately one copy per cell. It is likely this was caused by an outgrowth of cells where excision had not occurred. This in vitro model demonstrated that whilst excision of iciHHV-6 genomes is possible, it may be accompanied by a reduction in cell viability. Loss of HHV-6 genomic regions was also examined in 59 iciHHV-6-positive individuals and was infrequent. Only six showed some degree of DR loss. Further to this, inheritance of single direct repeats was observed in six individuals and two families in the GS:SFHS cohort. A novel ddPCR and mathematical model was developed to predict the iciHHV-6 genome configuration in samples where atypical U:DR ratios were observed. Through this, we hypothesise that an iciHHV-6-positive individual who had 4 U regions per cell and 5 DR regions per cell, had an integrated HHV-6A genome concatemer with the configuration (DR.U)4.DR, and hypothesised that this arose from integration of a replication intermediate. Finally, phylogenetic analysis of five regions of 26 iciHHV-6 genomes and their counterparts in exogenously acquired HHV-6 genomes was performed. There was a higher degree of divergence between iciHHV-6A and exogenous HHV-6, along with evidence of a common viral ancestor in four iciHHV-6-positive individuals. This divergence was not observed in iciHHV-6B were very little variation was observed between iciHHV-6B and exogenously acquired HHV-6B. These results shed light on the complex relationship between iciHHV-6 and the human host.

Antiviral host factors constitutively expressed to high levels can confer intrinsic immunity. Unlike, induced antiviral responses, these pre-existing intrinsic defences can protect cells against the initial stages of virus infection. However, there has been little investigation into the existence of such defences at mucosal surfaces, major portals of virus entry. Accordingly, a role for intrinsic immunity to influenza A virus (IAV) infection of the lung and respiratory tract is yet to be established. We therefore set out to investigate the hypothesis that constitutive expression of key antiviral host factors in cells of the lung and respiratory tract may confer intrinsic immunity to IAV infection. We focused on the TRIpartite Motif (TRIM) proteins, a family known to play a role in multiple aspects of host immunity to virus infection. By analysing a cell line of lung origin restrictive to IAV plaque formation, we identified constitutive expression of TRIM22, an antiviral effector and interferon stimulated gene (ISG) product reported to restrict a number of viruses as part of an innate immune response, including IAV. Analysis of open source data, respiratory tissues, and cultured cell lines, demonstrated that TRIM22 was constitutively expressed to high levels in cells of the respiratory epithelium, independently of IAV infection. By depleting TRIM22 in a cell culture model of constitutive expression, we investigated whether constitutive TRIM22 expression confers an intrinsic defence against IAV infection. We found that TRIM22 supresses IAV gene expression, inhibiting the initiating cycle of IAV replication. Reconstitution of TRIM22 expression in a cell line deficient in TRIM22 supressed the expression of foreign reporter genes in a SPRY domain-dependent manner, suggesting a potential mechanism by which TRIM22 may restrict the infection of multiple viruses. Together, our data demonstrate that TRIM22 confers intrinsic immunity to IAV infection in the respiratory tract, thereby establishing an important role for intrinsic immunity in the regulation of IAV infection.

The study achieved a better understanding of the epidemiology and ecology of Banana streak virus (BSV) in Uganda, generating information useful in designing management strategies to limit the spread of BSV. An identification key for the mealybugs on Musa in Africa was constructed. Screenhouse transmission experiments identified Dysmicoccus brevipes, Planococcus citri and a Pseudococcus sp. from bananas as mealybugs able to transmit the virus. Field observations also demonstrated that mealybugs are able to spread from plant to plant. Monitoring spread of BSV in new fields revealed that both onset and rate of spread was site specific; however, it was not clear why this was the case. The first incidence of BSV infection in Rakai was 28 months after planting (MAP), but in Ntungamo it was only 6 MAP. BSV incidence then increased to 28%, 72MAP at a rate of 0.10 new infections/infected plant/month and 43%, 28MAP at a rate of 0.23 new infections/infected plant/ month in Rakai and Ntungamo respectively. BSV spreads slowly in some locations, and is therefore probably amenable to control by phytosanitary measures. These would comprise primarily of the use of virus-free planting material and roguing infected plants. BSV spread predominantly within-field once initial infections were established. New infections were more likely to occur within 10 rows/columns from an old infection. More spread occurred to plants bordering established infected fields in Ntungamo. Spread of BSV into the new fields from the surroundings suggests need for separation of new fields from old infected fields in order to delay onset of BSV and reduce BSV incidence.

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria.