Compartmentalization of intracellular organelles is one of the key features representing eukaryotes. Each organelle has a unique structure composed of different membrane lipids and serves a distinct function to uphold cellular homeostasis. All known positive-strand RNA viruses exploit intracellular membranes to build their own membrane compartments, so-called replication organelles (ROs). ROs have been recognized as an essential platform for efficient viral RNA genome replication and have been implicated as a physical barrier against sensors of the innate immune system during replication. Interactions with host factors are crucial for the virus to induce the endomembrane reorganization and the subsequent RO formation. This thesis uncovers underlying mechanisms of virus-host interactions over the replication process, especially during the formation of ROs by enteroviruses of the family Picornaviridae. For enteroviruses, viral protein 3A and the cellular lipid kinase PI4KB, among other viral and cellular factors, have been suggested to be the key factors that drive this process. Findings described in this thesis provide mechanistic insights and a deeper understanding of the functions of viral proteins and specific lipids during enterovirus replication.