Kaposi's sarcoma-associated herpesvirus (KSHV) is the 8th human herpesvirus discovered in 1994. After primary infection, KSHV establishes latency and, in the context of immunosuppression, has been associated with specific malignancies: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Seroepidemiological surveys suggest that KSHV is not a ubiquitous virus and several transmission routes and risk factors must exist to explain its global distribution. The increased risk of KSHV-associated cancers in human immunodeficiency virus (HIV)-infected individuals, and the decrease of KS incidence after the introduction of highly active anti-retroviral therapy (HAART) in 1997 suggest that cellular immunity plays an important role in controlling KSHV. In this thesis, seroepidemiological studies were conducted in African, Middle Eastern, Mediterranean European and South American countries to determine the infection rate, and to determine risk factors and possible routes of KSHV transmission. A new quantitative real-time PCR method was developed to detect KSHV in clinical samples. This assay has diagnostic and prognostic implications for the management of KSHV-associated diseases. Anti-viral and immunological responses were measured and analysed, and cellular immune responses to KSHV were demonstrated in a cohort of HIV infected individuals undergoing HAART treatment. The interaction between KSHV and HIV was evaluated and the impact of HAART on KSHV immune reconstitution was investigated.

Gammaherpesviruses are associated with a number of diseases including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) constitutes the most amenable animal model for this family of pathogens. However experimental characterisation of gammaherpesvirus gene expression, at either the protein or RNA level, lags behind that of other, better-studied alpha and beta herpesviruses. A differential display system and 2 independent array systems have been developed, with the aim of characterising MHV-68 gene expression profiles and providing experimental supplement to a genome that is chiefly annotated by homology. Furthermore, the 3 technologies are compared in terms of their strengths and weaknesses, with respect to these goals. The MHV-68 transcriptome is presented through a lytic infection in vitro, both as quantitative measures of transcript abundance, and as part of a hierarchical cluster analysis. Functional predictions have been made for various genes based on co-expression with better characterised genes. Each gene has also been categorised as being expressed with kinetics by blocking de novo protein synthesis and viral DNA replication. This fundamental characterisation furthers the development of this model and provides an experimental basis for continued investigation of gammaherpesvirus pathology.

Human herpesvirus type 7 (HHV-7), with its high prevalence and apparently non pathogenic nature, is believed to be a virus very well adapted to humans. It is therefore useful to study the transcription patterns of the virus and the host to understand the life cycle and pathogenesis (or the lack of it) of HHV-7 infection. In order to obtain the global pattern of host and viral gene expression, we have made a DNA microarray containing probes that target all 86 predicted HHV-7 open reading frames and approximately 1000 cellular genes. This is the first description of an integrated host- pathogen microarray for HHV-7. The microarrays were used to follow the gene expression patterns over a 72-hour time course infection of a CD4+ T-cell line (Sup-Tl) by HHV-7 in vitro, which revealed an overall increase of HHV-7 transcripts over time, as well as the coordinated transcription profiles for groups of viral genes with similar functions. HHV-7 genes predicted to be immediate-early transcription factors were the first genes to be expressed, followed by genes involved in viral DNA replication, and at later stages, the genes encoding structural proteins and proteins of packaging functions were expressed. Temporal classification was assigned for 68 HHV-7 ORFs, of which a selection was confirmed by RT-PCR in the presence of metabolic inhibitors of de novo protein synthesis and viral DNA replication. Both gene expression profiling and inhibitor treatment demonstrated that the lytic gene expression of HHV-7 was temporally regulated, consistent with the classification of viral gene expression into immediate-early, early and late transcripts in other herpesviruses.

The latency-associated nuclear antigen (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a multi-function protein involved in maintenance of the viral episome and has been shown to interact with several proteins including p53 and pRB. It is likely that the multifunctional role of LANA1 exceeds these observations therefore the work in this thesis aimed to study LANA1 at both the transcription and protein-protein interaction level. I developed a lentiviral system for the infection of primary endothelial cells with LANA1, to analyse changes in gene expression profiles by gene expression microarrays. While the system was successfully developed, no significant changes in gene expression could be attributed to LANA1. Subsequently, using the yeast two-hybrid system and large-scale immunoaffinity purification coupled to mass spectrometry, I identified 26 novel binding partners of the KSHV LANA1 complex. The majority of these proteins have functions in splicing or mRNA processing and further analysis lead to the discovery of predominant protein domains. Several of the identified proteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, A2/B1, D and I, constitute members of the human H-complex. hnRNP A1, A2/B1 and D are also implicated in telomere biogenesis. I show that LANA1 binds UP1, the proteolytic derivative of hnRNP A1, which modulates telomere elongation and maintenance. Furthermore, I show an in vivo interaction between LANA1 and human telomerase reverse transcriptase (hTERT) and the ability of LANA1 to recover telomerase activity from cell lysates. These findings suggest a function for LANA1 in the maintenance of telomeres and may have important implications for the role of LANA1 in KSHV-related tumours. I propose models for the role of these complexes in splicing and telomere biogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) was discovered in 1994. KSHV is associated with at least three types of human cancer; KS, an endothelial tumour, and two lymphoproliferative disorders, primary effusion lymphoma (PEL) and a variant of multicentric Castleman's disease (MCD). In KSHV, the genes expressed in latency have been implicated in cell transformation. One of a cluster of three latency-associated genes, that regulate proliferation and apoptosis, encodes a viral FLICE inhibitory protein (vFLIP) in open reading frame 71 (ORF71). Two roles have been proposed for vFLIP; when expressed in heterologous cells it both blocks Fas-mediated apoptosis and activates the NF-KB pathway by interaction with IKB kinase (IKK). Given the two contrasting roles assigned to vFLIP, the aim of this study was to determine the function of vFLIP in KSHV-infected cells. vFLIP was therefore immunoprecipitated from PEL cells and four associated proteins were identified by mass spectrometry: IKK components and the chaperone, Hsp90. Using gel filtration, a single population of vFLIP in the cytoplasm of PEL cells co-eluted and co-precipitated with an activated IKK complex. An inhibitor of Hsp90, geldanamycin, inhibited vFLIP-induced IKK activity and killed PEL cells, inferring that vFLIP activation of NF-KB contributes to PEL survival. In a yeast-two-hybrid screen, our collaborators identified IKK as an interacting partner of vFLIP. Fragments of IKK were expressed in mammalian cells and bacteria, and the central portion of IKK? (amino acids 150-272) was identified as the vFLIP binding region. Finally, it is suggested that vFLIP activates the alternative pathway of NF-KB activation, leading to processing of p100 and generation of p52. This process is phosphorylation dependent and results in nuclear translocation of RelB and p52. A possible mechanism of action is suggested by the physical interaction between p100/p52 and vFLIP. These data strongly support an important role for vFLIP in NF-KB activation. This may be crucial for cell transformation by KSHV, for survival of infected cells and for the maintenance of latency.

No description available.

HCMV infection is usually asymptomatic in immunocompetent hosts, but serious disease can occur in immunocompromised individuals and in congenitally infected newborns. The importance of virus fitness determinations became evident in 1976, when it was proposed that the infecting strain of HCMV is important for clinical outcome, along with the intensity and duration of viral replication. HCMV strains exhibit different levels of virulence in vivo, depending on their passage history in cell culture. High and low passage HCMV strains exhibit tropism differences in vitro, suggesting that different tissue tropism may occur in vivo. In addition, approximately 13kb of novel DNA sequences located near the right edge of the unique long component of the genome has been identified in Toledo and clinical strains. This region (UL/b') encodes several open reading frames, which are missing from the high passage laboratory-adapted variants of Towne and AD 169 and are thought to play important roles in pathogenesis. One of the aims of this thesis was to determine the replication dynamics of different HCMV strains in vitro as well as compare their ability to bind to cells and mediate cell-to-cell spread of infection using pair-wise competition experiments in cell culture. AD 169 was shown to replicate better than Toledo in fibroblasts. Furthermore, assessment of the replication of Toledo in a different cell type, HUVEC, indicated that the virus replicated to higher levels in fibroblasts. Towne was found to bind to HEL cells with higher affinity compared to AD 169. The results showed phenotypic differences between high and low passaged HCMV variants and also illustrated that fitness differences between them are variable and highly dependent upon the status of the virus inoculum. To begin to understand the complex relationship between tissue tropism, virulence and HCMV genome composition, a DNA microarray approach was developed to examine host and HCMV gene expression during the productive infection of two distinct cell lines, fibroblasts and endothelial cells. The results showed that genes wifrojn tye fPSIPp were expressed in a cell type-specific fashion. Ip the context of host cell gene expression, cell type-specific host gene transcriptional changes were observed, reflecting different viral modulation of distinct cell type environments. The results provided potential insight into the function of genes encoded in the UL/b' region. Of particular note was that transcriptional changes frequently occurred in genes associated with pathways involved in the pathology of HCMV in the human host.

To substantiate the hypothesis that multiple infection by human herpesvirus 8 (HHV-8) and by cytomegalovirus (CMV) commonly occurs, intraperson nucleotide variation in DNA amplified from hypervariable subgenomic domains of HHV-8 and CMV was evaluated in a group of people living in Malawi, where both viruses circulate hyperendemically. Mouth rinses, throat gargles, palatal exfoliates and blood were sampled from 89 people (age range: 4 m to 45 y). In 24 people (27%), HHV-8 DNA could be amplified in >1 sample 9 (38%) were seropositive for human immunodeficiency virus-1 and 7 (29%) exhibited Kaposi's sarcoma. Sequence variation was sought in DNA segments derived from: the domain in open reading frame (ORF) 73 that encodes latent nuclear antigen the Bam330 segment of ORF 26 and the VI region of ORF Kl. Restriction fragment-length polymorphism analysis, nucleotide sequencing, PCR cloning and denaturing gel gradient electrophoresis were applied to study the sequence diversity of these segments. Significant intraperson/inter- and intra-sample sequence polymorphisms could be found in 15 people (62.5%). Variation in urine-derived VI sequences could, in addition, be evaluated: in 5 people, the sequences were monotypic, and in 2, urinary and oral sequences were genotypically different. Variation in hypervariable domains in the gO and gN regions of CMV was studied in urine and saliva samples of 77 people. In 41 individuals (53%), DNA could be amplified from at least one domain, and, in 14 (18%), from both domains. In 4 individuals (5%), urinary and oral sequences were genotypically different. The extent of inter- and intra-person variation in the 2 CMV domains studied was significantly less than inKWl of HHV-8. Multiple infection by HHV-8 and by CMV is common. It would be important to determine if such infection reflects coinfection occurring during initial transmission or superinfection, as the latter implies that vaccination might be ineffective to prevent and control the spread of the viruses.

HSV1 vectors have previously been produced with the ability to direct long-term expression of a transgene within the nervous system. These viruses have an arrangement of the latency promoter and enhancer regions LAP1 and LATP2 such that LATP2 exerts long-term expression onto LAP1 and an exogenous promoter at the same time, in a back-to-back fashion. To characterise the LATP2 region, two series of vectors were produced containing deletion mutations of the region placed in different orientations to LAP1 within the context of the original vectors. The vectors were tested in vitro and in vivo in the PNS and a potentially repressive region within LATP2 was identified. The enhancer activity of the region was also localised to a defined area. As the HSV1 genome is associated with histones and modification of these is a method of transcriptional control, histone modification could be one mechanism that the virus uses to keep the LAT region active during latency. This was investigated by examining the acetylation of histones associated with the LAT region, including LATP2, at lytic and latent timepoints in an in vitro system by ChIP assay. These studies found that although no significant differences in acetylation at different loci of LATP2 was found, the LAT regulatory region generally appears to be more associated with hyperacetylated histones during latency than non-LAT promoters and that this increased acetylation is conferred onto an exogenous promoter when placed within the LAT region. The findings in this thesis should provide insight into the functioning of the LAT region and may allow the development of improved HSV1 vectors for gene therapy in the nervous system.

No description available.