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.

There are many RNA virus pathogens of humans including influenza, parainfluenza, enteroviruses, parechoviruses, coronaviruses, pneumoviruses, and metapneumoviruses. These commonly invade and infect the respiratory and gastrointestinal tracts, giving rise to acute and chronic respiratory tract infections, and some may also reach the central nervous system (CNS) either via haematogenous or neural routes resulting in a variety of clinical presentations, (e.g. meningitis, encephalitis) which may lead to severe irreparable damage such as poliomyelitis especially in young children. Early correct diagnosis of viral infections is indispensable in order to prevent potential outbreaks which threaten the public health worldwide and might lead to high morbidity and some with significant mortality. Several laboratory techniques such as virus isolation, direct visualization of viral particles, detection of viral antigens and/or nucleic acids, and detection of host immune response (e.g. anti-viral antibodies) to infection, are available for diagnosis, but may have significant drawbacks such as time-inefficiency, cost and certain individual limitations. Microarray technology offers one way to overcome some of these limitations. In this study, a microarray chip containing 7967 oligonucleotides (probes) covering the whole genomes of human enteroviruses, rhinoviruses, respiratory syncytial viruses, metapneumoviruses and influenza viruses was designed and constructed using both OligoArray and Agilent eArray software, to allow simultaneous detection of any of the above viruses present in any clinical specimen. This virochip was tested against positive controls and clinical samples known to contain RNA nucleic acids of these viruses. Viral RNA was reverse transcribed, and amplified. Considerable effort was expended in trying to optimise a multiple displacement amplification (MDA) protocol for whole genome amplification, and in addition, long-range PCR was also utilised. Amplification products were fragmented, labelled and loaded into a hybridization reaction with the designed viral probes printed on the virochip. The results revealed (i) a number of technical problems associated with MDA; (ii) that some probes either failed to recognise their intended targets, or produced cross-reactive signals with non-intended targets; (iii) that many of the designed probes hybridized to their relevant viral nucleic acids and generated hybridization signals of high fluorescent intensity offering an opportunity to develop this probe array in order to be used for the identification of a wide variety of virus species up to the serogroup level or beyond (if required) specifically those causing CNS and respiratory tract infections.

Numerous endogenous retroviruses (ERVs) are found in all mammalian genomes and represent retroviruses which have, by chance, integrated into the germline and are transmitted vertically from parents to offspring. In many non-human primates these insertions have not been well-studied. ERVs provide a snapshot of the retroviruses a host has been exposed to during its evolutionary history, including retroviruses which are no longer circulating. Accurate annotation and characterisation of ERV regions is an important step in interpreting the huge amount of genetic information available for increasing numbers of organisms. This project represents an extensive study into the diversity of ERVs in the genomes of primates and related ERVs in rodents, lagomorphs and tree shrews. The focus is on groups of ERVs for which previous analyses are patchy or outdated, particularly in terms of their evolutionary history and possible transmission routes. A pipeline has been developed to comprehensively and rapidly screen genomes for ERVs and phylogenetic analysis has been performed in order to characterise these ERVs. Laboratory study was used to complement the bioinformatics analysis. Almost 200,000 ERV fragments, many of which have not previously been characterised, were identified. A novel endogenous member of the lentivirus genus of retroviruses, which are rarely found in an endogenous form, was identified in the bushbaby Galago moholi. This ERV may represent an ancient ancestor of modern human immunodeficiency virus (HIV). Another retrovirus, gibbon ape leukaemia virus, previously thought to be a common pathogen in gibbons, was found to not exist in contemporary gibbons and a route through which a single cross species transmission event may have resulted in all known cases of this disease worldwide was identified. Endogenous epsilonretroviruses, usually considered to be viruses of fish and amphibians, were identified in all screened species of primates.

Hantaviruses are a group of rodent-borne viruses. Seoul hantavirus is considered to be the only global hantavirus, although reported human cases outside of Asia are rare. Human infection occurs when breathing in aerosols of excreta from infected rodents. Hantaviruses have been listed as a major impacting factor leading to a rise in acute kidney injury (AKI) throughout the Western world. In the UK, historically, there was evidence for human and animal exposure to hantaviruses demonstrated by the detection of specific antibodies and classic renal disease, however it is only during this study that existence of a UK hantavirus in wild rodent populations has been proven. Since 2012, several cases of acute AKI due to hantavirus infection in the UK have been confirmed. Two cases were from Yorkshire and had documented exposure to wild rats. Wild rodents were trapped from the farm belonging to one of the patient’s and a strain of Seoul virus, named Humber virus, was isolated from rats. Subsequent cases of AKI were in people with exposure to specially-bred pet fancy rats. Rats from one private breeding colony were tested and a second highly similar Seoul virus, named Cherwell virus, was described. Evidence for Cherwell virus was demonstrated in a human sample with genetic data of the virus recoverable from a serum sample. It was 100% identical to the pet rat strain, thus confirming SEOV as the causative agent of the patient’s AKI. These findings have implications for public health as Seoul virus is capable of causing moderate-severe human disease. The overarching aim of the thesis was to confirm hantaviruses cause human infection in the UK and raise clinical awareness; this was achieved.

Turnip mosaic virus (TuMV, family Potyviridae, genus Potyvirus) has the widest host range amongst potyviruses. Globally it was said to be the second most important virus infecting field vegetables. Brassica juncea (Oriental mustard, family Brassicaceae), is an amphidiploid plant species with the genome AABB, comprising the genomes of the two diploid species, Brassica rapa (AA) and Brassica nigra (BB). It is widely grown and has various uses including as a leaf, stem, or root vegetable, oilseed crop, forage crop, condiment and biofumigant. Most B. juncea cultivars are very susceptible to TuMV, resulting in severe losses. Research on TuMV resistance and the mapping and identification of natural resistance genes would be very useful in order to speed up breeding resistant crops through marker-assisted selection. Sources of resistance to TuMV have been identified in B. juncea. The specificity of the resistances has been determined. A B. juncea DH line for which there is genomic information has been challenged with TuMV and found to be susceptible. This line has been used as a susceptible parent in crosses with resistant plants derived from different sources to develop segregating populations for mapping the resistance gene(s). Two BC1 populations (222 plants and 205 plants) and one F2 population (159 plants) have been phenotyped and segregation ratios were not significantly different from a Mendelian model based on the action of two recessive genes. Parental lines and selected plants in the two BC1 populations have been analysed by SNPs genotyping using the Illumina Infinium Chip. Genetic linkage maps have been constructed and QTLs have been mapped. Additionally, attempts are being made to identify a dominant TuMV resistance gene present in both Brassica napus and B. rapa. Inter-specific crosses have been made in order to introgress this gene into B. juncea. Resynthesised B. juncea plants possessing this dominant resistance have been produced through embryo rescue and polyploidy induction of F1 plants from crosses between resistant B. rapa and susceptible B. nigra plants. BC2 plants have also been developed by crossing B. rapa and B. napus plants possessing the dominant TuMV resistance with a susceptible B. juncea plant line.

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.

Picornaviruses are non-enveloped, positive sense single-stranded RNA viruses which cause many diseases ranging from slight illness to fatal meningitis and encephalitis. There are no vaccines against most picornaviruses so drugs need to be developed. Human parechoviruses (HPeVs) are being increasingly recognised as important human pathogens, but are genetically diverse from other picornaviruses. We therefore need to understand the details of virus replication to improve the opportunities to develop antiviral drugs. Viruses often rearrange the ER, ERGIC and Golgi to give a new membrane structures involved in viral replication and/or assembly. To improve our understanding of HPeV replication, we first studied the secretory pathway compartments and we found in HPeV-infected cells that the Golgi was rearranged and became more concentrated near to the replication complex. ER seemed to disappear almost completely. In terms of HPeV non-structural proteins, 2C had a major effect on the compartments and also co-localised and aggregated lipid droplets. Many viruses such as hepatitis C virus (HCV) and Dengue virus recruit lipid droplets to replication complexes. We found that lipid droplets became larger in HPeV-infected cells, but do not co-localise with replication complexes. To investigate the interaction between 2C and lipid droplets we made several 2C mutants. Mutation of NTPase domain of 2C did not change the interaction with lipid droplets. Instead we found that other domains, including a novel amphipathic helix are important. The results suggest that lipid droplets play a role in HPeV replication and so we investigated the effect of drugs which target lipid droplet formation and lipid homeostasis on HPeV replication. We demonstrated that drugs which target the enzyme DGAT1, which is involved in lipid droplet formation, have a potent effect on HPeV replication. Our results suggest that blocking lipid droplet formation is could be an important strategy for the treatment of HPeV infection.

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.

Morphological and hydrolytic enzyme cytochemical investigations have been applied to three normal and transformed cell systems in order to relate changes in hydrolytic enzyme activity to changes in ultrastructural organisation occurring when normal cells in vitro are transformed by tumour virus. On transformation changes in cell shape, growth and social behaviour are detected with the light microscope. At the ultrastructural level the major changes are in the extent of the GERL (Golgi, endoplasmic reticulum, lysosome) systems and are likely to be in response to altered rates of entry of metabolites through the cell membrane caused by transformation-dependent modifications of the cell surface. Five hydrolytic enzymes are demonstrated by light microscopic enzyme cytochemistryi mainly by azo-dye methods. Differences in levels of activity are difficult to assess visually but microdensitometry shows that transformed 3T3 cells have higher acid phosphatase activity than normal cells. This is confirmed by biochemical assays which also show that, unlike acid phosphatase, glucosaminidase has higher activity in normal cells. Biochemistry additionally provides a means of studying the effect of glutaraldehyde fixation on enzyme activity. Azo-dye techniques are found to be unsuitable for use in electron microscopic enzyme cytochemistry since dye is lost from cultured cells during processing and end product-like deposits appear in secondary lysosomes of control material. This latter phenomenon is investigated in other tissues. Metal-salt methods are, therefore, utilised for the demonstration of three enzymes, activity being largely confined to Golgi elements and lysosomes. Some activity is localised at the cell surface and this lends support to the possibility of surface modification through a process of sublethal autolysis by lysosomal enzymes. Examination of GERL morphology and enzyme activity gives an insight into the functioning of the system and provides evidence of possible mechanisms of cellular autophagy.

Promyelocytic Leukaemia proteins (PML), the core component of PML nuclear bodies (NB), are implicated in many critical cellular functions. One of those is confronting virus infection: DNA and RNA viruses target PML-NB and interact with PML proteins and this is thought to facilitate efficient replication by interfering with their antiviral functions. One of those viruses is human adenovirus type 5 (Ad5). Its early E4Orf3 protein interacts specifically with PML isoform II (PML-II), disrupting PML-NB into track-like structures to inhibit PML- antiviral responses. PML-II has been shown to be a positive regulator of type I interferon responses, as are typically induced by virus infection, through regulating the transcription factors that control this innate immune response. Given this role of PML-II and its interaction with Ad5 E4Orf3, the first question that addressed here is how does PML-II affect the progress of Ad5 infection under normal conditions? PML-NBs are also involved in cellular stress responses, being disrupted by heat shock and other stresses. Thermo-tolerance due to mild heat conditioning protects cells from more aggressive stresses such as higher temperatures through triggering the heat shock response, which helps in re-folding the affected cellular proteins. Interferon and other cellular cytokines are also induced in response to heat shock, suggesting a mechanism whereby heat stress might affect infection, possibly dependent on PML-NBs. The second question addressed here was whether cells became more resistant to infection by pre- stressing them and if so would PML-II have a role in that resistance? To address these questions, PML-II-depleted cells were first engineered by lentiviral vector delivery of specific shRNA. Physical and functional knock-down was confirmed by measuring mRNA levels for PML-II and for genes normally induced by NF-kB activation. The biology of Ad5 infection in these cells was then assessed in comparison with control cells. Ad5 gene expression displayed substantial increases with the transient or permanent depletion of PML-II in HeLa and MRC5 cells. This effect was particularly marked for late gene expression; hexon mRNA and all other late proteins showed substantial increase in PML-II depleted cells compared to several distinct control cell lines. This increase reached up to 100% with PML-II removal, quantified by flow cytometry. Virus yield also showed a 3-fold increase in PML-II depleted cells. This effect was only partly due to the impaired interferon pathway in these cells, which would be expected to augment Ad5 gene expression. The more significant factor in that increase was the overexpression of HSP70 chaperone, a specific member of the heat shock protein family, in PML-II depleted cells. siRNA-mediated HSP70 reduction caused a drastic decrease in Ad5 gene expression in PML-II Kd cells to more or less the same level seen in control cells . This effect was not NF-kB-dependent but HSP70 depletion did increase expression of interferon response genes such as ISG56. Pre-exposure of cells to mild heat stress made them significantly more resistant to Ad5 infection: such treatment reduced viral gene expression, in particular hexon and other late mRNA and protein expression, in HeLa cells and MRC5 cells. Ad5 genome replication was also reduced in pre-stressed cells compared to the control conditions. Consistent with previous studies of more extreme heat stress, mild heat stress affected the morphology of PML-NB. It also affected expression of PML protein and specifically showed an increase in PML-II mRNA expression after 3 hours of exposure to 40 ̊C compared to the control conditions. In light of the enhanced infection seen when PML-II was depleted, this increased expression of PML-II following heat stress might play a role in the reduced infection efficiency in such cells. Indeed, PML-II removal reversed the negative effect of the mild heat stress on Ad5 infection: such cells showed more late gene expression after heat stress than control cells.