The work carried out in this thesis comprises two main sections: i) characterisation of the role of ICP34.5 and ORF P in HSV-1 replication; and ii) identification of cellular and viral proteins which interact with ORF P. A complication in the analysis of the role of ICP34.5 in the HSV-1 lifecycle is the presence of overlapping antisense genes, ORF O/P, which are also deleted in ICP34.5 negative mutants. In 1991, MacLean, A. et al, isolated 1716, a HSV-1 17+ ICP34.5/ORF O/P deletion mutant. This mutant has been demonstrated to have specific characteristics both in vitro and in vivo. To attribute characteristics which were originally attributed solely to ICP34.5 to each of the genes, a number of HSV-1 recombinant viruses that express ICP34.5 and ORF O/P independently were constructed, purified and characterised. In all recombinant viruses an expression cassette was inserted into the 1716 UL43/UL43.5 locus containing the gene of interest under the control of the HSV-1 gD promoter and b galactosidase in the opposite orientation. Recombinants 1622 and 1625 express ICP34.5 and in both 1624 and 1624.5 ORF P is inserted in UL43 and UL43.5, respectively. Additionally, in 1625 ORF P is inserted in the non essential US5 gene. Western blotting analysis of 1622 and 1625 infected BHK cells demonstrated at least an eight-fold increase in ICP34.5 levels compared to wild type 17+. In the recombinants 1624, 1624.5 and 1625 no ORF P was detected, suggesting that these recombinants express low levels of ORF P, as the rabbit polyclonal sera used exhibited problems of detection with low amounts of ORF P from 17+ but not from the overproducing ts mutant in ICP4, tsK. Also, it is possible that the recombinants do not express ORF P. As we were not able to detect ORF P from our recombinant viruses, we proceeded to look for its RNA. A band with the size expected for ORF P RNA of about 700 bp was detected in 1624, 1624.5 and 1625. The 700 bp ICP34.5 transcript was also detected in 1622.

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline nuclease. Although the UL12 gene is not absolutely essential for replication, UL12 null mutants produce 100-1000 fold less viable virus than wt HSV-1. It has been suggested that the alkaline nuclease functions to remove branched structures from high molecular weight concatemeric DNA prior to its cleavage into monomeric genomes that are packaged into viral capsids. Failure to remove the branches results in unstable packaging of DNA into capsids which fail to egress from the nucleus. This thesis describes detailed characterisation of the HSV-1 mutant, ambUL12 (Patel et al., 1996) which fails to express the alkaline nuclease due to the insertion of an amber stop codon into the UL12 open reading frame. The ability of ambUL12 to replicate and package both viral genomic DNA and amplicons (plasmids containing the HSV-1 origin of replication and DNA encapsidation signal) was examined. In contrast to results obtained with other alkaline nuclease mutants, which replicate and package DNA with close to wt HSV-1 efficiency (Shao et al., 1993; Martinez et al., 1996b), ambUL12 displayed a 3-5 fold drop in replication and a 15-20 fold drop in packaging of genomic DNA. Similar reductions were observed in the replication and packaging of amplicon DNA. The replication and packaging of amplicons by ambUL12 in these transient assays could be partially complemented when UL12 was supplied in trans. Close inspection of the DNA molecules synthesised during transient assays demonstrated that amplicon replication intermediates are complex high molecular weight concatemers that undergo intermolecular recombination, analogous to viral DNA replication intermediates.

ICP27 is an HSV-1 immediate early protein required for the switch from early to late gene expression. ICP27 binds viral and cellular RNAs, and recently a yeast three-hybrid (Y3-H) analysis has identified an array of viral RNA sequences that interact with ICP27. Presented here are analyses of functional assays using a selection of the Y3-H identified RNA sequences inserted into the 5’ untranslated region (UTR) of a chloramphenicol acetyl transferase (CAT) reporter plasmid. A set of plasmids was transfected into baby hamster kidney (BHK) cells and CAT assays carried out to analyse the effects of the sequences on gene expression. Results indicated that expression was increased when ICP27-binding sequences were present even though no viral proteins were present. Comparison of sequences revealed that no common activation code or RNA structure was present that could be responsible for the increase in CAT gene expression. The levels of expression were further determined in the presence of wild type (wt), ICP27-null or ICP27 mutant HSV-1 infection to investigate whether ICP27 had any affect on CAT expression when ICP27-binding sequences were present. Interestingly, enhanced expression was observed during wt HSV-1-infection when ICP27-binding sequences were present, whereas little to no enhancement was observed during ICP27-null or mutant virus infections. However, a higher fold increase in CAT gene expression was observed during a HSV-1 infection when ICP27-binding sequences were not present. This indicated that an inhibitory effect on CAT expression observed during wt HSV-1 infection when ICP27-binding sequences were present was ICP27-depdnent. As a control, a noncoding, protein binding regulatory HPV RNA sequence was inserted into the CAT reporter plasmid, transfected into BHK cells and then infected with wt HSV-1, ICP27-null or ICP27 mutant viruses. Surprisingly, CAT expression was increased, albeit to only a limited extent, indicating that the previously observed increase in gene expression was not HSV-1 sequence specific. However, upon transfection of plasmids with the HPV control sequence inserted in the reverse orientation and a subsequent infection of cells no increase in CAT expression was observed. Analysis of the control sequence in the reverse orientation identified a shortage in G residues, which led to the construction of CAT reporter plasmids containing homopolymer sequences to inserted into the 5’UTR. A series of transfections and subsequent mock, wt HSV-1 or ICP27-null virus infections were carried out using this set of constructs. CAT assay analysis revealed an increase in CAT expression, to levels similar to those observed when the HSV-1 sequences were present, when poly(G) homopolymers were used as inserts during wt HSV-1 infection, whereas poly(A), (C) and (T) gave low levels of expression.

Herpes simplex virus (HSV) is a neurovirulent virus that in the course of natural infection of man predominantly infects sensory neurons. The aim of this project was to develop a safe, nonvirulent HSV, capable of expressing exogenous genes which altered the binding characteristics of the virus so that tropism was directed predominantly to motor nerves. It was envisaged that these viruses could then act as prototypes for gene therapy vectors targeted to the treatment of motor nerve diseases. To achieve this, two mutant viruses were created, RFa and RFb. These contained deletions of the main HSV glycoprotein involved in cellular binding (glycoprotein C). Gene fusions were created of truncated portions of gC (amino acids 377-511(RFa) and amino acids 477-511 (RFb)) to E. coli heat-labile enterotoxin B-subunit (LTB). The gene fusions were inserted in the RL1 gene thereby abolishing expression of the virulence factor ICP34.5. LTB is a ligand which binds to several gangliosides, including GM1 and GM2 which are motor neuron markers. It was hoped that by deletion of the main viral protein involved in adsorption to cells and replacing it with an LTB-containing fusion protein, the tropism of the mutant viruses could be altered to promote an increase in motor neuron infection. RFb was constructed. RFa constructed but could not be purified to homogeneity. This was thought to be due to poor adsorption/penetration or cell-to-cell spread, brought about by expression of the LTB fusion protein. RFb was analysed to determine the effect of expression of the novel LTB fusion protein within the context of the HSV genome. Western blot analysis using antibodies directed against LTB failed to detect expression of the LTB-gC fusion protein. In vitro replication studies showed that the RFb was non-virulent as demonstrated by its inability to replicate in growth arrested 3T6 cells, a phenotype characteristic of HSV which fails to produce ICP34.5. However no marked difference in virus replication kinetics was seen between RFb and wild type HSV (17+) on two motor neuron-like cell lines (NSC-19 and NSC-34).

Nuclear domain 10 (ND10) are punctate subnuclear structures that exist in most cell types. It has been suggested that ND10 structures serve as the site for initial viral genome deposition and some viral proteins, such as the ICP0 protein of HSV-1, have been shown to colocalise to, and subsequently induce the degradation of, proteins present in ND10. However the exact function of ND10 during HSV-1 infection is still unknown. In this study, the potential role of ND10 in HSV-1 infection was investigated by assessing the efficiency of viral IE and E gene expression or viral replication in cultured cells in which the expression of ND10 proteins was increased by IFN treatment or transient transfection methods. Furthermore, this study objective was also approached by using a human neuron precursor cell line (NT2) that naturally lacks one ND10 component, Sp100, and was found to have abnormal ND10 characteristics. The infectivity of HSV-1 in NT2 and differentiated hNT neuronal-like cells was investigated and the function of ICP0 in these cells was studied in more detail. Treatment with IFN- increased the number and size of ND10, whereas heat-stress caused dispersal of ND10 proteins, although this did not appear to affect HSV-1 infection. Pre-treatment with IFN- slightly inhibited HSV-1 gene expression, however it was not clear whether this effect was mediated by the ND10 proteins that are up-regulated by IFN. Examination of cells transiently transfected with plasmids expressing ND10 proteins (either PML, Sp100, SUMO-1 or Ubc9) demonstrated that the expression of viral IE and L genes was not affected by high expression levels of ND10 proteins. However, decreased viral gene expression was observed at an early time of infection in cells co-transfected with three plasmids expressing PML, Sp100, and Ubc9 or with four plasmids expressing PML, Sp100, Ubc9, and SUMO-1. Notably, in cells transfected with multiple plasmids, the exogenous ND10 proteins were sequestered in ND10 to a greater extent than in cells singly transfected. These observations suggested that over-expression of ND10 proteins, properly located in ND10, confers a marginal resistance to HSV-1 infection at an early time of infection, but this effect was less clear at later times of infection.

Transformed cells expressing BPV-1 E5 or BPV-4 E5 retain major histocompatibility class I (MHC I) molecules in the Golgi apparatus and cause the inhibition of transport of the complex to the cell surface. This effect can be ascribed to a failure of acidification of the Golgi apparatus. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Results from this thesis show that: a) Transcription inhibition can be alleviated by interferon treatment and the degradation of MHC I heavy chain (HC) can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC I to the cell surface is irreversible. b) E5 is capable of physically interacting with heavy chain. Together with the inhibition of the vacuolar (ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and heavy chain is likely to be responsible for retention of MHC I in the Golgi apparatus. c) C-terminus deletion mutants of E5 are incapable of either down-regulating surface MHC class I or interacting with heavy chain, establishing that the C-terminus domain of E5 is important in the inhibition of MHC I. d) Other two PV E5 proteins, BPV-1 E5 and HPV-16 E5 are able to interact physically with heavy chains from different alleles. BPV-1 E5 interacts with two equine classical class I heavy chains (EqB2 and EqB4) in vitro and the HPV-16 E5 interacts with classical HLA-A1, A2 and B8 in vitro and with HLA-A3 in vivo. e) Like HPV-16 E5, BPV-4 E5 is incapable of down-regulating certain non-classical MHC genes. These observations lead to a deeper understanding of the general down-regulation of MHC class I by the E5 proteins. Lack of surface classical MHC class I and presence of non-classical MHC class I in infected cells expressing E5 would allow evasion of cytotoxic T lymphocyte and NK killing and thus establishment of viral infection.

The UL33 gene of herpes simplex virus type 1 (HSV-1) encodes a 130 amino acid (aa) protein that is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Several lines of evidence have suggested that UL33, along with the UL15 and UL28 gene products, forms part of a terminase enzyme responsible for catalysing this process. This thesis describes the creation and characterisation of a number of UL33 insertion mutants in an effort to examine structure-function relationships within this protein and gain further insights into its function. Sixteen distinct mutants, encoding polypeptides with 5 aa insertions located at 14 separate positions throughout the protein, were generated. The abilities of these mutants to complement the DNA packaging and growth defects of viruses lacking functional copies of UL33 (the null mutant dlUL33 and the temperature sensitive mutant ts1233) were examined. Nine of the mutants were defective in both assays, and the capacity of all 16 mutants to support DNA packaging correlated precisely with their ability to complement virus growth. Regions of UL33 sensitive to insertion displayed a high degree of sequence conservation with UL33 homologues of other herpesviruses. In agreement with previous reports, a direct interaction between UL33 and UL28 was demonstrated in immunofluorescence and immunoprecipitation assays. Although all sixteen mutants appeared to interact with UL28 in co-immunoprecipitation experiments, four of the insertion mutants were defective in co-localisation with UL28 in immunofluorescence assays. Interestingly, of these four mutants, three supported DNA packaging to wt levels. Similar experiments confirmed that UL33 interacts directly with UL15, and immunofluorescence assays indicated that none of the mutants was impaired in this interaction. Novel interactions were also demonstrated between UL33 and the HSV-1 DNA packaging proteins UL6 and UL25. UL6 forms a portal vertex through which DNA is inserted into capsids, whilst UL25 is thought to play a structural role in stabilising capsids upon addition of DNA and is required only during the latter stages of encapsidation. All sixteen UL33 mutants were again able to interact with both partners in immunofluorescence assays. Of the remaining HSV-1 proteins necessary for genome encapsidation, neither UL17 nor UL32 interacted with UL33. Immunofluorescence studies of virally infected cells revealed that UL15 was necessary for the localisation of the remaining terminase components (UL28 and UL33) to nuclear sites of viral DNA replication, where packaging occurs. This is consistent with a model originally proposed by Yang et al. (J. Virol. 81:6419-6433, 2007), who suggested that a nuclear localisation signal within UL15 was necessary for the nuclear import of the terminase complex. Similar experiments revealed that, in the absence of UL6, none of the terminase components localised to replication compartments (RCs), suggesting that UL6 might be required for retaining the terminase at sites of DNA packaging. Together, the data presented in this thesis are consistent with UL33 forming part of the HSV-1 terminase via its interactions with UL15 and UL28. It is also possible that UL33 contributes to the transient interaction of terminase with the portal protein, UL6, during packaging. Although the interaction between UL33 and UL25 warrants further examination, it could be relevant to the mechanism by which UL25 is recruited to capsids and functions at the late stages of the head-filling process. Surprisingly, no clear evidence was obtained that any of the 16 mutants was defective in interactions with UL6, UL15, UL25 or UL28. It is therefore not yet possible to conclude whether the observed interactions of UL33 with these four proteins are essential for viral DNA packaging. By the same token, the reason(s) why nine of the 16 mutants are defective in DNA packaging remains unclear, but does not appear to be associated with their ability to form known protein-protein interactions or to localise to sites of DNA packaging. The development of cell free systems and biochemical assays will be an important step in further characterising these proteins.

Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae and infects approximately 170 million people worldwide. Unlike other known RNA viruses, HCV causes a persistent infection in the majority of infected people and can lead to cirrhosis of the liver and hepatocellular carcinoma. For these reasons, HCV is rightly classified as a major human pathogen. HCV core protein is believed to form, by analogy with other members of the Flaviviridae family, the nucleocapsid of the virus. As well as this, core has been shown to modulate many cellular processes via interactions with numerous host-cell proteins. One such protein shown to interact with HCV core is the DEAD-box RNA helicase DDX3. In cells expressing either HCV core alone, or as part of the full length HCV polyprotein, DDX3 is redistributed from its normal diffuse cytoplasmic localisation to lipid droplets where it colocalises with core. The cellular function of DDX3 is still unknown although it has been suggested to be involved in processes such as splicing, translation and RNA transport. The aim of this study was to investigate the role of DDX3 in the life cycle of HCV. This was aided by the recent discovery of a fully infectious HCV genotype 2a clone (strain JFH-1), allowing previously inaccessible aspects of the virus life cycle to be studied such as particle assembly and release. A library of HCV core mutants (residues 1-59 only) was produced by error-prone PCR and subsequently expressed in bacteria and analysed for their ability to bind bacterially expressed DDX3 using a rapid, high throughput ELISA screen. Six HCV core residues, conserved throughout all genotypes, were identified as being critical for interaction with DDX3. These residues were confirmed as being critical for the interaction by transfection of mutant core (together with E1 and E2 to ensure correct processing of core) into Huh7 cells. None of the 6 mutant core proteins were able to redistribute cellular DDX3. In order to study the effects of abolishing the core-DDX3 interaction in terms of a fully infectious HCV life cycle, the 6 critical residues were individually mutated to alanine in the cell culture infectious strain JFH-1 genome. All 6 mutant JFH-1 RNAs were capable of replication and being translated. Further investigation however, suggested that replication rate of mutant JFH-1 RNA was >50-fold lower than that of wild type JFH-1 RNA replication. Mutant core proteins colocalised with the lipid droplet marker ADRP, indicating correct subcellular localisation of the viral protein. Western-immunoblot analysis of mutant cores also confirmed that core proteins of same molecular weight to that of wild type core were produced, suggesting mutant cores were correctly processed. Of the 6 mutant JFH-1 clones analysed, 5 of them were capable of secreting infectious HCV particles that could subsequently infect naïve Huh7 cells, as detected by immunofluorescence and RT-PCR. However, one mutant, in which residue 33 of core had been changed from glycine to alanine, was initially unable to produce infectious particles. Upon passaging of cells electroporated with this mutant, infectious particles were eventually produced. The production of infectious particles consistently coincided with the presence of a second mutation in the surrounding area of the originally mutated residue 33. However, JFH-1 RNA containing both the mutation at residue 33 and the second identified mutation nearby, was unable to produce infectious particles upon electroporation, suggesting another lesion elsewhere in the HCV genome may also be required in order to overcome the effect of mutating residue 33. A recent report has indicated that DDX3 may be a nucleo-cytoplasmic shuttling protein, utilising the CRM1 export pathway. To confirm this, DDX3 localisation was analysed in the presence of the CRM1 inhibitor leptomycin B (LMB). In the absence of LMB, DDX3 was seen to have a diffuse cytoplasmic localisation while a small proportion was also seen in the nucleus. In the presence of LMB however, a build-up of DDX3 was seen in the nucleus, confirming that DDX3 uses the CRM1 pathway to shuttle from the nucleus to the cytoplasm. The results of this study indicate that the interaction of the cellular DEAD-box helicase DDX3 with core protein is not essential for the life cycle of HCV. It has been shown here however, that the replication rates of mutant HCV RNA are lower than that of wild type, suggesting that DDX3 may enhance either replication itself, or translation (which in turn provides the machinery required for viral RNA replication). Investigating this possibility is the subject of our future work. The identification of glycine 33 of core protein as being essential for production of infectious virus particles (without abolishing replication) will provide the basis for further studies on the production of infectious particles and the role that core protein plays in this process. The panel of JFH-1 core mutants will also be useful in studying the core-DDX3 interaction in a much wider context involving the role of DDX3 in normal cells. This study has uncovered important details regarding the interaction between core and DDX3 and, together with the reagents produced throughout this investigation, should enable further successful study into the role of DDX3 in the life cycle of HCV.

In order to successfully invade a new species, lentiviruses must overcome restriction factors, dominant blocks to replication, and be able to make use of available host factors such as entry receptors for replication. Human immunodeficiency virus (HIV-1) infection is blocked at a post-entry stage by rhesus macaque TRIM5α, an effect that is enhanced by host factor cyclophilin A (CypA), but largely evades restriction by the human TRIM5α variant. HIV-1 replication is also inhibited by simian APOBEC3G but is able to evade restriction by human APOBEC3G by inducing its degradation through expression of accessory protein Vif. Viral entry and tissue tropism are determined by an interaction of the viral Env glycoprotein with a cell surface receptor and a seven transmembrane domain co-receptor. The feline immunodeficiency virus (FIV) infects diverse felid species including the African lion, where infection has likely been endemic since at least the late Pleistocene, and the domestic cat, a more recent host. For domestic cat strains of FIV (FIV-Fca), entry is mediated by host proteins CD134 and CXCR4, but the identity of receptors in non-domestic strains of FIV is unknown. This thesis demonstrates that two strains of FIV isolated from lions (FIV-Ple subtypes B and E) differ in their receptor tropism and that subtype E shares entry receptors with FIV-Fca. The findings suggest that alternative receptor usage is a strategy employed by FIV in this species and has implications for the disputed pathology and tissue tropism of infection in African lions. Next, we tested the hypothesis that species which have harboured lentiviral infection for a long time are better able to prevent viral replication than recent hosts. Whilst we found that TRIM5α is non-functional in all felid species tested, evidence of potent APOBEC3 activity was found and, in lion cells, potently restricts production of infectious FIV. Moreover, lion primary T-cells prevent replication of diverse FIV strains and restrict primate lentiviruses at post-entry stages, suggesting that co-evolution with lentiviruses has driven the selection of broad-ranging restriction factors. Structural and biophysical analyses suggest that whilst FIV’s interaction with CypA appears to be conserved in affinity with HIV-1, the interaction does not appear to be crucial for replication and, in the absence of restriction by TRIM5α, the role of the capsid-CypA interaction is discussed. Overall the study explains the permissivity of domestic cat cells to retroviral infection and identifies FIV infection of lions as an example of host adaptation driven by current lentiviral infection.

With respect to proposed novel ORFs identified by in silico analysis, in most cases, we found no evidence for transcript expression. Of those that gave positive hybridisation signals, most might be explained by overlapping transcripts from genes in the same region and coding in the same direction. Other novel ORFs lie within regions of the genome now considered to be non-coding, but where transcripts have previously been reported, while the remainder may represent genuine coding ORFs. The lack of signal for previously described ORFs that are now considered non-protein coding confirms their status as discounted genes. In order to test the microarray system, the temporal expression kinetics of selected virus genes were investigated by alternative methods including; real-time PCR, and northern blots to check the identity of specific transcripts, and where antibodies were available, western immunoblotting to confirm the expression kinetics of specific proteins. The expression kinetics obtained for specific genes both differentially and non-differentially expressed by these various methods were entirely consistent with those obtained for the same genes with the HCMV microarray. It was concluded that the Merlin microarray system was a valid and reliable research tool for the investigation of HCMV gene expression.