The Anelloviruses Torque Teno virus (TTV) and TTV-like minivirus (TLMV) are small, circular DNA viruses which infect humans and non-human primates. They are highly prevalent in the general population; however infection is without any apparent pathology. Both viruses are extremely heterogeneous, especially for DNA viruses, and the role of the immune system in controlling the infection has yet to be established. Initial experiments involved establishing prevalence figures for TTV and TLMV as well as SENV D and H, subtypes of TTV implicated in potential transfusion transmitted non A-E hepatitis, in Scottish blood donors. 88% of serum samples were PCR positive for either TTV, TLMV or a heterogeneous mixture of both viruses. The presence of SENV D and H was determined by Southern blot and revealed 0.5% of samples tested were infected with SENV D, 10.9% with SENV H and 1% with both SENV D and SENV H. We compared the titre of both TTV and TLMV in the bone marrow and spleen from 3 groups: HIV negative individuals, HIV positive individuals and HIV positive individuals who had progressed to AIDS, leading to immunosuppression. The AIDS group had higher tires, which were statistically significant compared with both the HIV positive and negative groups for both bone marrow and spleen. TTV/TLMV tires in HIV positive and transplant patients were compared to individuals infected with viruses not known to cause immunosuppression (HCV and HBV) and healthy blood donors. Both the immunosuppressed groups of individuals had titres of TTV/TLMV in serum higher than the other three groups. The suggestion that farm animals were infected with TTV similar to human TTV led to an investigation of TTV/TLMV homologues infecting non-human primates and farm animals. None of the farm animals were shown to be PCR positive. Sequence analysis of the primate samples determined they were infected with viruses which were genetically distinct from human TTV and TLMV.

The existence of functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognised. In this thesis several computational methods to detect RNA secondary structure in the coding regions of hepatitis C virus (HCV), hepatitis G virus (HGV)/GB virus C (GBV-C) and related viruses have been used. These include thermodynamic prediction of folding free energies (FFEs), evolutionary conservation of minimum energy structures between virus genotypes, suppression of synonymous variability and analysis of covariant and semi covariant substitutions in thermodynamically favoured structures. Each of the predictive methods provided evidence for conserved RNA secondary structure in the core and NS5B encoding regions of HCV and throughout the entire coding region HGV/GBV-C. Positions in the HCV genome with predicted RNA structure localises precisely to regions of marked suppression of variability at synonymous sites, indicating that RNA structure constrains sequence change at what are generally regarded as phenotypically neutral sites. Combining these methods, the computational data obtained in this thesis demonstrates the existence of at least ten conserved stem loop structures within the NS5B encoding region and three in that coding for the core protein in the coding region of HCV. Analysis of the NS5B encoding region and 3’ untranslated region (3’UTR) of HGV/GBV-C indicates an even greater degree of RNA secondary structure. Remarkably, it appears from analysis of FFEs that extensive RNA secondary structure may exist along the entire length of both HCV and HGV/GBV-C genomes, a finding with considerable implications for future functional studies. The existence of predicted RNA structures in the HCV genome was determined using controlled nuclease mapping of RNA transcripts from the core and NS5B regions under conditions which retained potential long-range RNA interactions. The pattern of cleavage sites of nucleases specific for single and double stranded RNA provided strong experimental support for structures previously predicted in this study.

The clinically relevant gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), are the subject of intensive research as both are associated with fatal neoplastic disorders. Due to the species specificity and inefficient replication in vitro of EBV and KSHV, several animal models have been proposed to allow analysis of viral pathogenicity in vivo. Murine gammaherpesvirus-68 (MHV-68) represents a tractable animal model of gammaherpesvirus pathogenesis as it is able to infect numerous murid rodent species, including laboratory mice, and replicate productively in a wide range of cell types in vitro, permitting efficient manipulation of the viral genome. MHV-76, a related gammaherpesvirus, is a deletion- mutant of MHV-68 and lacks 4 MHV-68- specific genes (M1-M4) and 8 viral tRNA-like sequences at the 5'-end of the genome. These genes are implicated in latency and/or immune evasion. Consequently, MHV-76 is attenuated during productive infection and the early stages of splenic latency, with respect to MHV-68. The aim of the project was the characterisation of the M4 gene of MHV-68. There is little data describing the properties of the M4 gene product; sequence analysis predicted an open- reading frame of 1376bp in length, encoding a -45kDa product. Thus far, M4 expression has been detected during lytic infection, but not during latent infection. Recently, it has been demonstrated that M4 is expressed as an immediate -early /early transcript during lytic replication of MHV -68 in vitro. To elucidate the contribution M4 makes to in vivo pathogenesis, a novel MHV-76 mutant (MHV-76inM4), in which the region of MHV-68 coding for M4 and accompanying putative promoter elements was inserted into the 5'-region of the MHV-76 genome, was created. A revertant virus was subsequently generated (MHV-76.Rev) which restored the 5'- region of the MHV- 76inM4 genome to that of MHV-76. Genomic rearrangements were confirmed by Southern analysis and sequencing. The growth of MHV- 76inM4 in vitro was indistinguishable from that of MHV-76 and MHV-68. However, viral titres from MHV-76inM4- infected BALB/c mice were significantly increased with respect to MHV -76 at early times in the lung, suggesting an important role for M4 during productive infection. Additionally, at days 17 and 21 post- infection, there was a significant elevation in latent viral load in splenocytes of MHV-76inM4- infected mice compared to MHV-76, as measured by ex vivo reactivation assay and real-time PCR. Like MHV -76, MHV-76inM4 displays no evidence of overt splenomegaly, characteristic of MHV -68 infection at this time. M4 expression in vivo was detectable by RT-PCR during productive infection in the lung and during the establishment of latency in the spleen, but in general was not detectable during long-term latency. The data demonstrate that M4 modulates both productive and latent infection, and suggest that M4 has a role in subversion of the innate immune response.

In this study, the gene encoding the GM-CSF inhibitory factor (GIF) was isolated and mapped to the right terminal quarter of the orf virus genome. The orf virus GIF cDNA was expressed as a secreted protein in Chinese hamster ovarian cells as detected by GM-CSF inhibition ELISA. Recombinant GIF was purified by ovine GM-CSF affinity chromatography and gel filtration. Sequence analysis of the 20 N-terminal amino acids was performed on the purified GIF. The GIF gene encodes a 28 kDa protein that exhibits 32% amino acid sequence similarity to the predicted sequence of the A41L gene product encoded by vaccinia virus. Although the vaccinia virus A41L protein has sequence similarity to the T1 secreted chemokine-binding proteins of leporipoxviruses, its function is not known. GIF did not share any homology with any cytokine receptor molecule identified to date. In contrast to other parapoxvirus immunomodulatory proteins that are products of early viral genes, GIF was found to be the product of an intermediate/late viral gene of orf virus infected cells. GIF formed homodimers and homotetramers in solution and bound ovine GM-GSF with a Kd of 369 pM. In addition GIF bound ovine IL-2 with a Kd of 1.04 nM. Although orf virus infects humans, GIF did not bind human GM-CSF or IL-2. GIF was shown to inhibit the binding of ovine GM-CSF labelled with ¹²⁵I to its receptor on isolated sheep neutrophils and it inhibits the haematopoietic activity of ovine GM-CSF in a soft agar bone marrow colony assay. GIF also inhibited the binding of ovine IL-2 labelled with ¹²⁵I to CD4⁺ T cells and inhibited the stimulatory activity of ovine IL-2 in a T cell proliferation assay. This inhibitory activity was neutralised by a rabbit antiserum raised against purified GIF. GIF was produced in vivo during orf virus reinfection. GIF was detected in skin, localised to the area of orf virus infected cells and in afferent lymph draining the skin site of infection. The presence of GIF, 3-7 days after virus infection was associated with reduced levels of GM-CSF in the lymph plasma and the period of maximum viral replication in the skin.

The structure and symmetry of viruses has been the subject of study since Crick and Watson in 1956, and there have been several complementary theories describing different aspects of the geometry of these complicated entities. Included here is a unified theory that relates the structure and sizes of the different viral components, from the capsomeres to the packaging of the genomic material, providing, through a set of structural constraints on viral structures, a new classification scheme for viral structures. Moreover, aspects of this theory also apply to fullerene structures in chemistry, showing that this symmetry principle is deeper than just biological in nature.

A significant number of RNA viruses assemble their protein containers and genomic material simultaneously. Here the implications of this protein-RNA co-assembly are investigated using an extended version of a model first proposed by Adam Zlotnick in 1994 (Zlotnick, 1994). The inspirations for this extended model are the cases of bacteriophage MS2 and the STMV virus, viruses that have been well characterised experimentally. Example pathways of RNA virus assembly have been enumerated and kinetic simulations have been run on these networks. The results show the most likely pathways of virus assembly and the concentrations of the intermediates. This work will also demonstrate how kinetic traps may be avoided when proteins are able to bind RNA during assembly. Additionally modelled are DNA cages, which are three-dimensional shapes made from double-helical DNA molecules. Such cages have been seen within viruses but may also be constructed artificially. This model has been used to produce energetically optimised designs for icosidodecahedron-shaped DNA cages.

In tailed bacteriophages and evolutionarily related herpes viruses, the portal protein is a central component of the DNA packaging molecular motor, which translocates viral genomic DNA into a preformed procapsid. The motor is the most powerful molecular machine discovered in nature, generating forces reaching ~50 pN and translocating DNA with a speed of several hundred bp/sec using ATP as an energy source. The oligomeric portal protein ring is situated at a unique vertex of the procapsid forming a conduit for DNA entry and exit. Although the three-dimensional structure has already been determined for portal proteins from bacteriophages P22, SPP1 and phi29, several important questions about the role of individual protein segments in DNA translocation and their interaction with other components of the motor remain unanswered. Structural information on portal proteins from other bacteriophages, like T4 for which a wealth of biochemical information is already available, will help to answer at least some of these questions. The portal protein of bacteriophage SPP1 (gp6) can form circular oligomers containing 12 or 13 subunits. It is found as a 12-subunit oligomer when incorporated into the viral capsid and as a 13-subunit assembly in its isolated form. The X-ray structure of the SPP1 portal protein is available only for the isolated 13-subunit assembly of the N365K mutant form. Because this mutation results in a reduction in the length of packaged DNA, determining the structure of the wild type portal protein would shed light on the mechanism of DNA translocation. Elucidation of the mechanism of DNA packaging depends also on the availability of accurate structural information on the SPP1 portal protein in its 12-subunit biologically active state. Such structural knowledge would be particularly useful in future, for designing a stable molecular machine that can function in vitro. In this thesis, experiments were designed to promote the formation of the dodecameric gp6: viz fusing gp6 with TRAP protein that forms a stable circular dodecamer as well as the co-expression of gp6 with the SPP1 scaffolding protein gp11. The protein targets were cloned, expressed and purified, and the oligomeric state of gp6 was characterised by a combination of biochemical, biophysical and structural approaches. The structure of the wild type gp6 was solved at 2.8 Å resolution, revealing a 13-fold symmetrical molecule. The protein’s fold is the same as for the N365K mutant, with most significant conformational differences observed in the tunnel loop and in segments of the clip and crown domains. Comparison with the structure of N365K mutant reveals significant differences in subunit-subunit interactions formed by tunnel loops, including different hydrogen bonding and van der Waals interactions. It is likely that these differences account for the different amount of packaged DNA, indicating involvement of tunnel loops in DNA packaging. The portal protein of bacteriophage CNPH82, cn3, was also successfully cloned, expressed and purified. Promising crystallisation conditions have been identified that yield crystals diffracting to 4.2 Å. Further optimisation should lead to determination of the X-ray structure of this protein in not too distant future. Self-rotation function calculations and SEC-MALLS analysis indicate that the cn3 protein forms 13-subunit assemblies, in common with the SPP1 portal protein. Foundation work has been carried out for the bacteriophage T4 portal protein, aimed at identifying suitable production and purification conditions. In addition, the full-length bacteriophage SPP1 scaffolding protein gp11 has been cloned, purified and crystallised. Degradation was observed in the full length gp11 protein and therefore a series of truncations were designed, cloned and purified aiming to improve the stability. Further studies on limited proteolysis of the full-length gp11 should lead to a stable gp11 tuncation that will form crystals with better diffraction.

Crick and Watson were the first to recognise the importance of symmetry in the structures of viral capsids. This observation was the departure point for Caspar-Klug’s theory in which the possible positions and orientations of the protein building blocks are predicted and classified in terms of T-numbers. Whilst this theory predicts the layouts of the protein containers, it provides no information on the thickness of the capsid or its surface features. The creation of icosahedrally invariant point arrays via affine extension of the icosahedral symmetry group and their mapping to viral capsids in [37] has shown that they provide geometrical constraints on viral structure that not only correlate positioning of proteins on the capsid, but also relate structural features on different radial levels including genome organisation. In this study we have extended this approach using the quasilattices embedding these point arrays. To derive further geometric constraints on virus architecture we firstly show how classifying the possible transitions between the quasilattices modelling the structure of the virus before and after the transition allows us to derive information on the most likely transition paths taken by the protein shell during the structural transformation. Next, a new algorithm matching tile sets to viral capsids has been implemented to investigate further the geometrical constraints quasilattices place on these structures over and above the point arrays in [37].

Bacteriophage T5 is a bacterial virus known to have a remarkably high replication rate. It is a double-stranded DNA virus and encodes many of the proteins needed for its own replication. During replication, the viral double-stranded genomic DNA has to be separated by enzymes called helicases, which are motor proteins that utilize chemical energy from ATP to move along and unwind nucleic acid duplexes. Until now, no helicase has been characterized in bacteriophage T5. A bioinformatic analysis on the T5 replication gene cluster showed that several early gene products (D2, D6 and D10), which possess key helicase signature sequences (motifs), may be T5 helicases. This is the first report to investigate helicases of bacteriophage T5 and the study focused on bioinformatic and biochemical characterization of these three potential helicases. Here, D2 and D10 were identified to be two novel T5 helicases, showing helicase activity in vitro as well as having some unique properties previously uncharacterised in other helicases. However, D6 did not show ATPase activity under the condition employed and a further investigation on characteristics of D6 is required. Except for a Walker A motif, no other common conserved motifs related to helicase activity were identified in the D2 protein sequence. However, D2 was found to have a rare bipolar helicase activity giving it the ability to unwind partial duplex DNA with either a 5' or a 3' ssDNA tail (ss-dsDNA). This indicates D2 may possess some unconventional motifs relevant to its helicase activity. The extent of 5'→3' or 3'→5' unwinding activity of D2 was revealed to be dependent on 5' or 3' tail length. Interestingly, D2 displayed biased polarity preference with its 3'→5' unwinding activity being several fold greater than its 5'→3' unwinding activity when the substrates have identical tail length. Differential inhibition of the bipolar helicase activities by high NaCl concentration was also observed. The 5'→3' unwinding activity was more sensitive to inhibition by high NaCl concentration than the 3'→5' unwinding activity. The D10 protein can unwind branched DNA substrates, including forks, Y-junctions and Holliday junctions, which resemble DNA replication, recombination and repair intermediates. Furthermore, D10 was shown to catalyze branch migration of the Holliday junction substrate. Intriguingly, the ability of D10 to unwind the Y-junction substrate was found to be structure-dependent and sequence-dependent. Also, the unwinding activity can be affected by the strand discontinuity of the substrate. All the findings in this study contribute to a new insight into functional properties of helicases.

Introduction: Multiple sclerosis (MS) is a progressive, neurological condition in which balance difficulties and falls are commonly reported. Measuring balance is important for assessment and to measure outcome of intervention and measures used should have known levels of reliability and validity. This research aimed to identify the measures used by physiotherapists for balance, walking and gait, and to review psychometric testing of commonly used measures for people with MS. Research tested psychometric properties of the measures among patients with MS. Method: A survey of physiotherapists identified widespread outcome measurement, that reliability and validity of measures was important and that the Berg balance scale (BBS) and 10-metre walking test (10MWT) were commonly used measures. A literature review highlighted limited evidence of psychometric testing of these measures among people with MS. The short form BBS (sfBBS) had not been tested at all. The BBS and sfBBS were tested for reliability and validity among people with MS. The 10MWT and objective force-plate data were also tested as measures of balance. Results: The national survey of physiotherapists yielded an encouraging 77.5% return rate and showed that therapists valued measurement. The patient-based study recruited a smaller sample than desired leading to caution in interpretation. The sfBBS was strongly related to the BBS and both had acceptable levels of intra-rater reliability. Inter-rater reliability was affected by interpretation of scoring guidelines and the sfBBS had significant measurement error. The 10MWT, especially at maximal pace, may be used as an estimator of balance ability. Conclusion: There must be clear instructions for scoring the BBS particularly between raters. Caution is required in generalising from a small sample size however analyses demonstrated acceptable levels of intra-rater reliability in MS. The sfBBS and 10MWT, as balance estimates, may save time for patients and clinicians and reduce the burden on patients.