Seasonal and pandemic Influenza virus infections cause about three to five million cases of severe illness and about 250,000 to 500,000 deaths world-wide annually according to the WHO. Although investigated intensively, Influenza virus pathogenesis is still not very well understood and hard to predict. Influenza A viruses contain a segmented, single-(-) stranded RNA genome encoding at least 10 different proteins and are highly diverse due to hypermutation and reassortment. In previous work, 56 viral genes from six different influenza A virus isolates had been cloned and genome-wide screened for virus-host protein interactions using yeast-two hybrid technology and several human and chicken cDNA libraries, leading to the identification of 127 high-confidence cellular interactors of which 40 have also been identified by RNA interference in other studies. In this thesis, two of the cellular interactors identified which both bound to the viral multifunctional protein NS1, TRBP and PACT, were further investigated with regard to their role in virus life cycle. These two proteins are known to be involved in miRNA silencing and PKR regulation. Both interactions between NS1 and TRBP and NS1 and PACT were confirmed by co-immunoprecipitation, and both TRBP and PACT co-localized with NS1 in a cytosolic compartment. NS1 was also found to be present in the RISC complex in pull-down assays with the RISC core component Ago2. In functional assays, NS1 dose-dependently inhibited RNA silencing. Although no differences in TRBP-binding between NS1 proteins of various different influenza strains could be detected in direct mating Y2H assays, they varied with regard to their inhibitory activity on RNA silencing. TRBP and PACT alone were unable to restore NS1-induced inhibition of RNA silencing activity, however both together restored RNA silencing. Moreover, the siRNA knockdown of PACT abolished the association of NS1 with Ago2, and NS1 competitively inhibited the binding of TRBP and PACT to Ago2. The depletion of either TRBP or PACT led to an inhibition of influenza virus replication. The depletion of TRBP also lifted cellular IFNβ level without infection. However, the knockdown of TRBP but not PACT blocked IFNβ production and increased cell viability post infection. These results indicate that NS1 inhibits the binding of PACT and TRBP to the RISC complex and thereby inhibits miRNA-induced gene silencing. The hypothesis that TRBP supports influenza replication potentially by regulating PKR regulation and IFNβ induction requires further investigation. In conclusion, this study provides evidence for the complexity of virus-host interactions and the dual role of viral proteins in activating both positive and negative regulatory cellular mechanisms.

Regulation of gene expression is a complex process in mammalian cells with many levels of control. In recent years non-coding RNAs in the form of microRNAs (miRNA) have surfaced as important regulators of protein coding genes, with biologically important roles in development, differentiation and cell growth. In this dissertation the complex interactions between miRNAs and mRNAs in ovarian cancer are investigated using a combination of computational and experimental techniques. In vitro studies and current models predict that increases in levels of miRNA should result in corresponding decreases in the levels of targeted mRNAs due to miRNA induced degradation. Profiling the global miRNA and mRNA expression patterns in epithelial ovarian cancer cells from patients and surface epithelial cells from normal ovaries reveal only ~11% of predicted targets of miRNAs are inversely correlated in vivo. In an effort to dissect the mechanisms behind these unexpected observations single miRNA transfection experiments are carried out followed by gene expression profiling. Analysis of genes altered following these transfections reveal majority of the altered genes are not direct targets of the miRNAs. Network analysis however suggests that miRNAs may target "hub genes" to cause altered expression in downstream transcripts. Pathway enrichment analysis of altered genes demonstrates miRNAs may regulate specific pathways rather than causing random off-target effects. Finally investigation of miRNA regulation reveals miRNAs may also affect the levels of other miRNAs, which may indirectly affect more genes downstream. Together these results provide a detailed view of the mechanisms employed by miRNAs to regulate the expression of hundreds of genes in ovarian cancer cells.