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.

No description available.

No description available.

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.

Hodgkin lymphoma (HL) is one of the most common haematological malignancies in the Western world. Although HL responds favourably to cytotoxic therapy in the majority of cases, late side-effects such as secondary malignancies and cardiovascular disease are becoming of great concern, particularly in younger patients. The current challenges are to maintain treatment efficacy whilst reducing side-effects and to develop biomarkers to predict response to treatment(s). Protein degradation pathways in HL cells have remained largely unstudied in this malignancy and represent an opportunity for more targeted therapy. This thesis describes the activities of protein degradation pathways in HL-derived cell lines and their sensitivities to inhibition. The results suggest that the use of proteasome and HDAC6 inhibitors, alone or in combination, may be of clinical benefit in the treatment of HL in the future. The presence of p62 was used to monitor protein handling stress; however, its diverse expression patterns in HL-derived cell lines and in paraffin-embedded HL biopsy material preclude its use as an informative biomarker in HL. p62 was found to traffic between the cytoplasm and nucleus in HL-derived cell lines and its association with a DNA damage marker in both cellular compartments implicate it as a chaperone for the cytoplasmic degradation of nuclear proteins. A previously unreported nuclear expression of the lysosomal enzyme cathepsin B in HL-derived cell lines was identified, and this may have implications for the aberrant transcriptional profile of these cells given the regulatory activities of other members of this family of cysteine proteases. The non-lymphoid origin of the HD-MyZ cell line, a putative HL-derived cell line, was corroborated by phenotypic differences in protein handling pathways in comparison to accepted HL-derived cell lines. The activities and insensitivity to inhibition of protein handling pathways in this cell line suggest that it will be an interesting model for studying alternative protein degradation pathways. The presence of a number of small cell populations with cancer stem cell (CSC)-like characteristics was confirmed in HL-derived cell lines and, although these populations require further characterisation, this has implications for HL patients who suffer relapse.

Background: There are a large number of viruses spread by mosquitoes, many of which cause debilitating, often fatal, neurological disease such as acute encephalitis. In this study we have used two different neurotropic viruses: Semliki Forest virus (SFV), and West Nile virus (WNV), both of which can cause severe panencephalitis in the mouse. The influx of leukocytes into the infected tissues is mediated by chemokines and is believed to be important for virus clearance. To date, we have only limited insights into the precise nature of chemokine involvement, and an improved understanding of these important axes provides a new target for the development of novel therapies. Hypothesis: Based on previous studies investigating the role of chemokines during neuroinflammation it was hypothesised that chemokines and other cytokines are highly upregulated during viral encephalitis, and the blockade of selected chemokine receptors would lead to altered disease outcome. It was also hypothesised that chemokine receptors would present plausible targets for the treatment of viral encephalitis. Results: To test these hypotheses, the chemokine expression pattern and the kinetics of chemokine mediated leukocyte recruitment during viral encephalitis were analysed in unprecedented detail by TaqMan low density array, and flow cytometry, respectively, and key chemokine receptor were identified as therapeutic targets. Both SFV and WNV exhibited a similar pattern of chemokine upregulation, although WNV induced significantly higher fold expression. The key chemokines upregulated were CCL2, 3, 5, 7, CXCL9 and CXCL10. The upregulation of chemokines coincided with leukocyte influx into the CNS. After identifying the key chemokines upregulated during viral encephalitis, next a selected panel of chemokine receptor antagonists was utilized to evaluate the hierarchy and relative importance of distinct chemokine receptors for CNS leukocyte influx, viral clearance, neuropathogenesis and host survival. We identified the CXCR3 axis as being the key instigator of CNS inflammation in response to alphavirus infection, placing it at the top of a hierarchal cascade that is followed by CCR2 and CCR5. Critically, inhibition of both CXCR3 and CCR2 simultaneously, significantly improved host survival to otherwise lethal encephalitis. Conclusion: These data suggest that chemokine receptors represent plausible therapeutic targets for viral encephalitis.

In this PhD project we show for the first time that novel dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with L-chirality represent promising antiviral candidates for use against pox and measles viruses. We suggest a mechanism of action based on a cellular target. Our lead compound (Cf2642, with side chain C9H18–O–C5H11) is active against vaccinia virus (a surrogate poxvirus for smallpox) and measles virus, with IC50 concentrations of 0.19 and 7.5 µM, respectively. This is a 60-fold enhancement over cidofovir (viral DNA polymerase inhibitor; IC50 of 11.5 µM against VACV). A structure activity relationship was established, which was similar for both viruses, indicating a common and specific mechanism of action. Cf2642 does not inhibit HSV-1/2, influenza, adeno or yellow fever viruses. The mechanism of action for the ddBCNAs has been investigated and, though not defined, has been narrowed down. Based on our observations of drug activity in cell lines derived from various sources, we have suggested a cellular target for the ddBCNAs, most likely cellular membrane compartments or the proteins located therein. Though inhibition of vaccinia is observed within two hours of infection, we have shown that the ddBCNAs are unlikely to be entry inhibitors. Acidification of the extracellular medium was observed but, whilst it may be linked to the mechanism of action, this is not the cause of the antiviral effects. With a possible cellular target, toxicity was carefully evaluated. We have not observed significant cytotoxicity in any of our cell models. Antivirals active against cellular targets are less subject to viral resistance, which may develop rapidly with virus-targeting drugs. This could be critical since, there are currently no effective measles antiviral drugs available on the market, and resistance to measles RNA polymerase inhibitors and the potential antipoxviral drug cidofovir has already been described.