No description available.

No description available.

No description available.

Viral infections affect millions of people worldwide and pose a major threat to human health. Therefore efforts to understand the host defences against viruses are timely and useful. There are specific receptors on the host cells such as Pattern Recognition Receptors (PRR), which are capable of sensing infectious viruses and initiate reactions collectively known as innate immune responses by detecting motifs or molecular signatures. These responses include activation of antiviral cytokines and initiation of the adaptive immune response, thus inhibiting virus replication. The main two families of PRR involved in virus recognition are the Toll like receptors and the RIG-1 like receptors (RLRs; also known as RIG-1 like proteins or RNA helicases). This study was aimed to clarify the innate immune responses and recognition pathways of Picornaviridae by the host. Picornaviridae are single-stranded RNA viruses that can infect many tissues and organs and produce a variety of symptoms and illnesses to the host. The results from this study have shown that TLRs and RLRs and more specifically TLR7, TLR8 and MDA5 are involved in the detection of Picornaviridae such as Coxsackievirus A9 (CAV-9) and Human Rhinovirus 6 (HRV6) leading to the activation of antiviral cytokines by the host cells.

Hepatitis C virus (HCV) readily establishes a chronic infection and it is currently thought to infect 2-3% of the global population. Treatment for HCV places a severe burden on patients, leading to a premature treatment termination in approximately 20% of individuals, and has poor efficacy against the predominant genotype. A greater understanding of the virus lifecycle and mechanisms of persistence will provide valuable information in the continuing development of direct-acting antiviral compounds. HCV encodes 10 proteins which are translated as a single polyprotein. Non-structural protein 2 (NS2) is a small, hydrophobic, trans-membrane protein, yet the precise number and position of its trans-membrane domains remain unclear. NS2 is required for virion morphogenesis but it is not required for replication of the viral genome and it does not form part of the mature virion. NS2 has been shown to interact with other viral proteins, potentially via intra-membrane contacts. Determining the topology of NS2 is therefore vital to our understanding of how NS2 interacts with the other viral proteins and functions within the virus lifecycle. The interactions between NS2 and membranes and the viral glycoprotein E2 were investigated by truncation analysis and fusion with reporter proteins. Glycosylation analysis demonstrated that the N- and C-termini of NS2 are oriented to the luminal and cytosolic faces of the ER membrane, respectively. Truncation of NS2 at residue 70 oriented a C-terminal reporter fusion to the ER lumen consistent with the formation of a luminal loop. This is the first biochemical evidence that NS2 assumes a 3 TMD topology. Co-immunoprecipitation studies with E2 and eGFP-tagged truncation of NS2 revealed that NS2 forms multiple interactions with E2 and that these interactions are dependent upon NS2 targeting to membranes but likely independent of NS2 topology. A model of NS2 topology is presented.

No description available.

No description available.

Human cytomegalovirus (HCMV) UL141 is a potent modulator of natural killer (NK) cell function that acts by suppressing cell surface expression of CD155, a recognised ligand for the ubiquitous NK. cell activating receptors DNAM-1 and CD96. CD155, also known as nectin-like molecule 5 (necl-5) or poliovirus receptor (PVR), is an important adhesion molecule that impacts on cell motility, proliferation and cellular signalling complexes. The focus of this study is to characterise UL141 expression, interactions and biological properties. Consistent with the role of CD 155 as a cell adhesion molecule, UL141 expression was associated with reduced cellular adhesion whether expressed in continuous cell lines or by adenovirus vector. UL14 and UL141 exhibit significant amino acid sequence homology and are members of the same HCMV gene family. Interestingly, UL14 also impaired cell adhesion. Consistent with HCMV downregulating multiple adhesion molecules, productive infection was associated with greatly reduced cell adhesion. Against this background, deletion of UL141 from the virus had no overt effect in adhesion assays. When co-expressed with C-terminal fluorescent tags, gpUL141 and CD 155 co-localise with each other and endoplasmic reticulum marker. When over-expressed CD 155 and gpUL141 co-localised in inclusion bodies along with calnexin implying gpUL141 may hold CD 155 in a partially destabilised form. gpUL141 was identified as a component of the envelope of mature HCMV virus particles, whilst CD 155 and gpUL14 were excluded. gpUL141 is predominantly an endoglycosidaseH (Endo-H)-sensitive, ER-resident glycoprotein in HCMV infected cell that is subject to post-translational modification, consistent with proteolytic cleavage, during the later stages of infection. However, the protein incorporated into virions is full length and had acquired Endo-H resistance, consistent with transit through the Golgi apparatus. As CD 155 is recognised to be a cellular receptor for poliovirus and other herpesviruses, the identification of UL141 as a virion protein may have implications for virus entry.

HCMV is the largest human virus characterised to date, encoding approximately 165 open reading frames (ORFs). Due to many years of serial passage, laboratory adapted strains of HCMV, such as strain AD 169, have lost a 13-15kb region of genome, designated UUb', compared with HCMV clinical strains (Cha et al, 1996). Loss of this 13-15kb UUb' region is correlated with decreased virulence and increased sensitivity to Natural Killer (NK) cell lysis, leading to the hypothesis that the UUb' region is harbouring one or more NK evasion functions. A comprehensive screen of the UUb * region of HCMV strain Merlin to identify novel NK evasion functions has formed the focus of this study. The UUb1 region encompasses 23 ORFs from UL128 through to UL150. In addition, UL14, a homologue of the UUb' resident ORF UL141, and UL141A, a newly identified UUb' ORF, were included in this study as potential NK evasion functions. Using the AdEasy system and the newly developed AdZ system, the generation of recombinant adenoviruses (RAds) encoding for each of the 24 UUb' ORFs, plus the UL141 homologue, UL14, has been successful and provides a complete resource for the study of these proteins. The majority of the UUb' proteins were previously uncharacterised, and the incorporation of a C-terminal Streptag II has enabled preliminary characterisation of these ORFs. Producing the bank of UUb' RAds has also enabled a functional screen of this region in order to identify novel NK evasion functions. As a result of the systematic functional NK screen of the UUb' region, two novel NK evasion functions have been identified the UL141 homologue, UL14 and the UUb' resident ORF, UL135. Their identification brings the total number of NK evasion functions encoded by HCMV up to eight: UL40, UL16, UL18, UL83 (pp65), UL141, UL142, UL14 and UL135. These provide HCMV with an impressive arsenal dedicated to evasion of NK lysis, and are further evidence for the importance of NK cells in the control of HCMV. Further analysis of the NK evasion function encoded by UL14 revealed that similar to UL141, UL14 encodes an EndoH sensitive glycoprotein and was observed to co-localise with the ER resident protein, calnexin, consistent with gpUL14 being ER- retained. The biochemical similarities of the UL141 and UL14 NK evasion ORFs may be of functional significance, indicating that gpUL14 may also be sequestering an NK activating ligand within the cell similar to gpUL141 (Tomasec et al, 2005).

Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigation