This thesis reports an investigation of the role of Vaccinia virus (VACV) gene K7R in evasion of innate immunity. The K7 protein is an inhibitor of intracellular signalling pathways leading to the activation of the innate immune response and was shown to act by inhibiting TBK1/IKK-ε - mediated IRF signalling by targeting DDX3, a cellular RNA helicase (Schroder et al., 2008). To characterise K7 further and study its interaction with DDX3, K7 in complex with fragments of DDX3 was expressed in bacterial cells with the aim of obtaining co-crystals. The role of K7R in the context of VACV infection was also studied. Previous in vivo work using a recombinant virus lacking K7R had shown that K7 contributes to virulence. However, virulence was retained when a mutation of the initiating methionine codon abrogated K7 protein expression while preserving the nucleic acid sequence. It has been shown here that this is likely to be due to the presence of viral miRNA(s) encoded from the K7R sequence. Computational analysis identified putative pre-miRNAs within K7R and mutated versions of the K7R ORF which contained the predicted pre-miRNA sequences but did not make protein were still able to inhibit the IFN-β, NF-κB and ISRE-dependent intracellular signalling in vitro. Several of these mutated genes were introduced into the K7R deletion VACV and their virulence will be examined in a mouse model of infection. The presence of a miRNA also needs to be formally demonstrated using northern blot and/or qPCR analysis. Finally, there is a strong interest in using attenuated VACV strains such as Modified Vaccinia Ankara (MVA) as vaccine vectors against various pathogens. To investigate whether the removal of immunomodulators from MVA, such as the orthologue of K7R, can make the virus more immunogenic, a deletion mutant and its revertant counterpart were made and characterised in vitro.

Meq is a viral c-Jun analog and member of the AP-1 transcription factor family that acts as the primary oncoprotein encoded by Marek’s disease virus. Previous studies have shown that Meq interacts with a variety of proteins as part of its pivotal function in the development of Marek’s disease virus-induced lymphoma. The primary focus of this study was to identify the global interactome of the Meq oncoprotein. This was initially carried out by review of the Meq sequence and the subsequent identification of BATF and BATF3 as potential analogous partners. Interactions with novel proteins were also predicted based on the nature of charged interactions that mediate a leucine zipper dimerisation event. Furthermore, the Meq protein was used as bait in a yeast two-hybrid screen to produce a large data set of both known and novel interacting proteins. Interactions with JunB, JunD and CtBP1 were confirmed along with a set of novel proteins including Par-4, ATF3, RACK1, N4BP1 and Pin1. The chicken Par-4 and ATF3 genes were cloned and expressed to confirm co-localisation with Meq in the cell nucleus while further biochemical investigations for each interaction were carried out by co-immunoprecipitation. The Par-4 and ATF3 proteins have been shown to play a variety of key roles in cellular mechanisms such as apoptosis, cell cycle regulation and transformation but we failed to see expression of these proteins in either normal or transformed lymphocytes. However, Par-4 and ATF3 are expressed in the feather follicle epithelium of infected birds providing the potential for a role during lytic infection in the skin. It is clear that more work is required in order to explore this hypothesis further but this thesis describes a number of novel interactions made by Meq and in doing so we have contributed to the greater understanding of Marek’s disease virology.

Foot-and-mouth disease virus (FMDV) is a member of the picornavirus family of non-enveloped, positive-sense, single-stranded RNA viruses. Picornavirus assembly involves the multimerisation of a capsid subunit (P1 or P1-2A) into pentameric structures, which further assemble into intact capsids containing the viral RNA genome. The capsid subunit is co-translationally myristoylated and proteolytically cleaved by a viral protease (3Cpro) to initiate the assembly cascade. A cell-free assay was developed to analyse the requirement for these processes in pentamer assembly. Pentamer assembly was found to be dependent on myristoylation. In these assays, two 3Cpro recognition sites in the P1 protein could be cleaved independently and were both required for the efficient formation of pentamers. In addition, a system was developed for the production of large quantities of purified recombinant capsid precursor that could be used for future structural studies. Existing studies have shown that molecular chaperones such as heat-shock protein 90 (Hsp90) are required for capsid assembly of other picornaviruses. Pharmacological inhibition of Hsp90 reduced growth of FMDV in cell culture and prevented pentamer assembly in the cell-free assay. Hsp90 was not required for processing, which contrasted with existing models for this part of the picornavirus life cycle. Upon RNA encapsidation a maturation cleavage occurs on the inside of the capsid generating the structural protein VP4. Upon virus entry into cells, capsids disassemble into pentamers that no longer contain VP4. Pentamers from the disassembly pathway are therefore thought to have different properties than pentamers found on the assembly pathway. A maturation-like cleavage event was engineered into recombinant pentamers. Cleavage of this site altered pentamer sedimentation from that expected of assembly pentamers (14S) to that expected of disassembly pentamers (12S). This confirmed that loss of VP4 from pentamers controlled the switch in the properties of these pentamers.

VACV gene C16L encodes a 37-kDa protein that is highly conserved in orthopoxviruses and functions as an immunomodulator. Intranasal infection of mice with a virus lacking C16L (vΔC16) induced less weight loss, fewer signs of illness and increased infiltration of leukocytes to the lungs compared with wild-type virus. To understand C16’s mechanism of action, tandem affinity purification and mass spectrometry were used to identify C16 binding partners. This revealed that Ku70, Ku80 and PHD2 interact with C16 in cells. Ku70 and Ku80 constitute the Ku heterodimer, a well characterised DNA repair complex. MEFs lacking Ku, or the other component of the DNA-dependent protein kinase (DNA-PK) complex, the catalytic subunit of DNA-PK (DNA-PKcs), were shown to be deficient in the upregulation of IRF-3-dependent genes such as Cxcl10, Il6 and Ifnb in response to transfection of DNA, but not poly (I:C). Furthermore, following infection of MEFs with VACV strain MVA the activation of Cxcl10 or Il6 transcription was dependent on DNA-PK. Therefore, DNA-PK is a DNA sensor capable of detecting poxvirus DNA and activating IRF-3-dependent innate immunity. C16 inhibited the binding of Ku to DNA, and therefore inhibited DNA-mediated induction of Cxcl10 and Il-6 in MEFs. The role of C16 in vivo was also examined: infection with vΔC16 led to increased production of Cxcl10 and Il-6 following intranasal infection of mice compared with wild-type virus. C16 is therefore an inhibitor of DNA-PK-mediated DNA sensing and innate immune activation. C16 was also shown to bind to PHD2, an enzyme involved in regulation of hypoxic signalling. VACV was found to activate the transcription of hypoxia-related genes, and C16 expression in cells was also capable of doing this. The role of hypoxic signalling in VACV infection remains poorly understood.

Influenza B viruses encode two small proteins, NB and BM2. BM2 is translated from segment 7 mRNAs by a mechanism involving sequence complementarity to the 18S ribosomal subunit. The importance of these complementary sequences was tested in the context of infectious virus using a reverse genetic approach. A series of mutations were introduced into this region of segment 7. Recombinant viruses with disrupted 18S complementarity displayed deficiency in BM2 expression in infected cells. The BM2 protein is essential for virus replication because its ion channel activity is required during virion entry to the cell. There is also evidence that the cytoplasmic tail of BM2 is involved in viral assembly. A series of amino acid truncations and substitutions in the BM2 cytoplasmic tail were engineered. Recombinant viruses that lacked more than 5 residues at the carboxyl terminus of the protein were not recovered and key residues in the region -5 to -10 were identified. The influenza B virus RNA segment 6 encodes the neuraminidase protein, NA, as well as NB. NB is a 100 amino acid transmembrane protein with a glycosylated ectodomain. NB is conserved in all natural influenza B virus isolates. Influenza B viruses that lack the NB protein can replicate in cell culture to wild-type levels, However, the deletant viruses showed attenuated growth in complex airway cultures derived from humans and ferrets. In vivo, infected ferrets excreted infectious virus in the nasal wash one day later than for viruses that encode NB. Alterations in the expression of the NA protein were not responsible for the attenuated phenotype shown by NB deletant viruses. The role of the host cell ESCRT pathway or of the interferon-induced tetherin protein in assembly and release of influenza viruses was assessed. No evidence was found for either host pathway in the replication of influenza viruses.

Foot-and-Mouth Disease Virus (FMDV) induces rearrangements of host-cell membranes to generate vesicles that are believed to provide platforms for formation of the viral replication complex. The cellular origin of these vesicles and the properties that make them favourable for replication are poorly understood. For some Picornaviruses these vesicles are thought to derive from membranes of the secretory pathway. In this thesis, I have investigated a role for membranes of the secretory pathway in FMDV infection. Key cellular proteins involved in regulating the flow of membranes through the secretory pathway between the ER and Golgi were inhibited using expression of dominant-negative (dn) proteins and small interfering RNA (siRNA) and the effect on FMDV infection determined. Inhibition of ER export using a drug (H89) or Sar1 (the GTPase required for COPII transport vesicle formation at ER exit sites) reduced FMDV infection. In contrast, stabilisation of COPII coats, or inhibition of Arf1 or Rab proteins, that are involved in the secretory pathway after the formation of COPII vesicles, had little or no inhibitory effect on infection. Interestingly inhibition of Arf1, Rab1 or Rab2 enhanced infection. In contrast, Arf1 reduced infection by bovine enterovirus which is inhibited by Brefeldin-A, and therefore likely to be dependent on Arf1 for replication. These results show that Sar1 and/or COPII vesicle formation is necessary for FMDV infection and that inhibiting the formation of COPI coats is in some way advantageous to FMDV infection. These results suggest that FMDV targets COPII vesicles membranes before the COPII/COPI exchange and facilitates FMDV infection and that COPI components are not required.

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