No description available.

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope proteins, E1 and E2, are heavily glycosylated. They interact with host cell receptors and provide a target for host immune recognition. The host virus interactions determine the pathogenesis and outcome of HCV infection. L-ficolin is a soluble pattern recognition molecule of importance in innate immune defence against microorganisms. It activates the lectin complement pathway upon binding to carbohydrate recognition patterns on microorganisms. It was hypothesised that L-ficolin could interact with HCV glycoproteins. Both recombinant and serum derived L-ficolin were investigated for binding to the envelope glycoprotein E1E2 of HCV. Specific, dose-dependent binding of L-ficolin to HCV glycoprotein E1E2 was observed. The interaction between L-ficolin and HCV particles in infected sera was also demonstrated. Binding of L-ficolin to HCV pseudoparticles expressing E1E2 glycoproteins resulted in neutralisation of virus infectivity. The serum L-ficolin level was significantly higher in patients with mild HCV liver fibrosis compared to patients with severe HCV liver fibrosis. These results suggest a potential protective effect of L-ficolin, as an innate immune defence, against HCV infection. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the levels of anti-E1E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.

Retroviruses must integrate their genome into the host DNA as a necessary step of their replication cycle. Normally, retroviruses integrate into somatic cells and are transmitted, from infected to uninfected hosts, as “exogenous” retroviruses. On rare occasions, they can infect germ line cells and become part of the host genome as “endogenous” retroviruses (ERVs), which are transmitted vertically to the offspring and inherited as Mendelian genes. During evolution, most ERVs have accumulated mutations that rendered them defective and unable to produce infectious viral particles. Some ERVs, however, have maintained intact open reading frames for some of their genes, and have been co-opted by the host as they fulfil important biological functions. Sheep betaretroviruses represent a unique model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) co-exists with the highly related endogenous JSRVs (enJSRVs). The sheep genome harbours at least twenty-seven enJSRV loci and, most likely, the process of endogenization is still occurring. During evolution, one of these enJSRV loci, enJS56A1, has acquired a defective and transdominant Gag polyprotein that blocks the late replication steps of related retroviruses, by a mechanism known as JSRV late restriction (JLR). Interestingly, enJSRV-26, a provirus that integrated in the sheep germ line less than two hundred years ago, possesses the unique ability to escape JLR. In this thesis, the molecular basis of JLR escape was investigated. The main determinant of JLR escape was identified in the signal peptide of enJSRV-26 envelope protein (SP26). A single amino acid substitution in SP26 was found to be responsible for altering its intracellular localization as well as its function as a post-transcriptional regulator of viral gene expression. Interestingly, interference assays demonstrated that enJSRV-26 relies on the presence of the functional signal peptide of enJS56A1 envelope protein (SP56) in order to escape JLR. In addition, the ratio between enJSRV-26 and enJS56A1 Gag polyproteins was found to be critical to elude JLR. Finally, sequence analyses revealed that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus, reinforcing the hypothesis that this locus has provided an evolutionary advantage to the host. This study unveils critical aspects of JLR that were previously unknown, and provides new insights on the molecular mechanisms governing the interplay between endogenous and exogenous sheep betaretroviruses.

No description available.

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.

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.

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.

Recombination is an important biological process in a diverse range of viruses, particularly those with single-stranded ribonucleic acid (RNA) genomes including the enteroviruses. Mutations caused by the error-prone RNA-dependent RNA polymerase (RdRp) and the vast population size of these virus populations are evolutionary mechanisms that generate genetic diversity – this allows viruses to survive under changing environmental pressures (e.g. adaptive host immunity). Ribonucleic acid recombination has been identified as another contributing mechanism involved in diversification, by removing interfering or lethal mutations from a virus genome, and by establishing new viruses. Virus RNA recombination is well documented and is identified in several virus families including picornaviruses. However, recombination is a rare event and the study of the molecular mechanisms behind virus recombination is complicated by our ability to isolate and analyse recombinants from a mixed virus population including the parental viruses. The objectives of this study were to firstly devise a method for generating populations of natural recombinant viruses, and secondly, to study the molecular processes that determine where and when recombination occurred in the enteroviral genome. During this project, an in vitro system was developed to allow the recovery of recombinant enteroviruses in the absence of their parental viruses. Two virus RNA molecules containing “lesions” rendering them unable to generate viable virus on their own were co-transfected into mouse L929 cells. The method required two parental virus RNA molecules to be present in a single cell to produce a viable recombinant virus. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and sequencing analysis confirmed the recombinant nature of progeny virus genomes. Experimental data confirmed the effectiveness of the method and provided evidence that recombination occurs in at least two phases. Initial template switching, referring to the transfer of RdRp from one RNA template to another mid-replication, occurred apparently indiscriminately and with the addition of extra virus and non-virus sequence at the junction sites. In the second phase, any additional sequence was lost during subsequent rounds of replication and selection. The approach was expanded to incorporate viruses from different enterovirus species to investigate intra- and interspecies recombination events.

Despite being closely related viruses with similar replication cycles, HIV-2 replicates more slowly than HIV-1 and produces fewer particles, resulting in a lower plasma viral load. Expression of the major structural gene, gag, from HIV-1 and HIV-2 proviruses was compared to investigate whether this could play a role in the difference in particle production observed between HIV-1 and HIV-2 infection. Using quantitative RT-PCR, significantly less full-length HIV-2 gag mRNA was found to be transcribed from its provirus than for HIV-1. Sub-cellular fractionation allowed us to determine HIV-1/2 gag mRNA levels in the nucleus and cytoplasm throughout a time course. RNA export of HIV-2 gag mRNA was shown to be slower than for HIV-1 gag mRNA. HIV-2 full-length gag RNA was shown to be translated much less efficiently than HIV-1 in a range of cell lines. Both HIV-1 and HIV-2 Gag have been proposed to be translated by internal ribosome entry. Shutting down capdependent translation (by poliovirus-mediated eIF4G cleavage) significantly reduced translation from both HIV-1/2 gag RNAs, with no evidence of compensatory IRES activity. This suggests that cap-dependent translation is the predominant mechanism for translation of both HIV-1 and HIV-2 RNA. Additional work explored HIV RNA-protein interactions by UV cross-linking experiments using cellular proteins. Several proteins differentially binding to HIV-1/2 5’ UTR RNAs were identified and, in particular, a 45 kDa protein binding only to the HIV-1 5’ UTR. Attempts were made to characterise the proteins binding with different affinities to HIV-1 and HIV-2 RNAs. Confocal microscopy was used to visualise HIV-1/2 Gag expression within the cell. Both HIV-1 and HIV-2 Gag expression was shown to be reduced when siRNA was used to inhibit the cellular clathrin adaptor protein AP-1. In conclusion, HIV-2 Gag gene expression was found to be less efficient than HIV-1 at the level of transcription, RNA export and translation. Future work will continue to investigate the mechanisms behind these differences.

Cyanophages, viruses that infect cyanobacteria, are known to be abundant throughout the world’s oceans. They are important because of the ecological significance of their hosts which are prominent primary producers. In the natural environment cyanobacteria undergo light-dark cycles, which might be expected to exert significant effects on the way in which cyanophages reproduce. The results in this study show how light plays an important role in cyanophage adsorption to the host cell using a model system consisting of cyanophage S-PM2 and Synechococcus sp. WH7803. An initial investigation of the role of light on phage adsorption revealed a striking light-dependence. In the dark, the phage S-PM2 was virtually not capable of adsorbing to WH7803, but adsorption resumed as soon as the light was switched on. This light-dependent phage adsorption was not just limited to the phage S-PM2, four out of nine other cyanophages showed the same effect. The host photosynthetic activity and light/dark cycles were demonstrated not to influence phage adsorption. The presence of the photosynthetic reaction centre gene psbA in cyanophage genomes was not associated with the light-dependent phage adsorption. No photoreceptor was detected from the phage S-PM2 particle. A phage-resistant mutant that S-PM2 can’t adsorb to WH7803 was isolated. A putative multicopper oxidase was found to be absent from the outer membrane fraction of the mutant. This outer membrane fraction in the wild type showed a moderate phage neutralisation activity (up to ~ 30%). To test whether the putative multicopper oxidase was the S-PM2 receptor, a recombinant WH7803 strain was constructed by inactivating the putative multicopper oxidase gene. As S-PM2 can still adsorb to the knockout mutant as efficiently as to the wild type, it suggests that the multicopper oxidase is not the phage receptor and that loss of the putative multicopper oxidase is probably a pleiotropic consequence of the loss of the S-PM2 receptor or other components, such as lipopolysaccharide, that is needed for a successful S-PM2 adsorption.