Flaviviruses are found all over the world and affect and infect millions of people every year. Flavivirus infection can lead to severe clinical outcomes resulting in neuronal damages e.g. Tick-borne encephalitis virus (TBEV), or severe hemorrhagic fevers e.g. Dengue virus (DENV). In order to effectively treat infected patients and to prevent these diseases we must understand how these viruses work and how they interfere with the mammalian host. This thesis is focusing on interactions between the virus protein NS5 and human host cell proteins. The interactions presented here might be key factors for out-come of viral disease. NS5 is the largest of the non-structural proteins and is essential for the replication and the capping as it contains both RNA dependent RNA polymerase and Methyltransferase domains. We found that TBEV NS5 interacts with human PDZ domain protein Scribble, a polarization protein important e.g. in regulating membrane trafficking. We determined that the interaction depend on a novel internal motif in TBEVNS5. This interaction could be correlated to NS5s ability to interfere with the immune system as absence of Scribble prevented NS5 from blocking phosphorylation of STAT upon Interferon induction. The role of NS5 in human PDZ domain targeting was addressed further by using a PDZ array system. Both TBEVNS5 and DENVNS5 bind additional PDZ domains using the internal motif. The tight junction protein ZO-1 binds both DENVNS5 and TBEVNS5. DENVNS5 is mainly present in the nucleus and co-localize with ZO-1 in un-polarized cells. In polarized cells TBEVNS5 and ZO-1 co-localize at the plasmamembrane. Putative C-terminal PDZ binding motifs of TBEVNS5 and WNVNS5 were characterized using the PDZ array system. This detected four novel binding partners of TBEVNS5 but numerous of potential WNVNS5 binding partners. We found that TBEVNS5 co-localizes with ZO-2 in the cellular membrane. Further, we found that TBEVNS5 induce the AP-1 by a 2 fold over the control.