| In eukaryotes, cell membranes are important in separating cell into various compartments for various biological functions. To replicate inside cell, viruses have developed various strategies to penetrate the membrane boundaries or to utilize the membrane system for replication. In this dissertation, we will see how two RNA viruses, Hepatitis C Virus (HCV) and Coronavirus, involve cellular membranes.; HCV is an enveloped positive-stranded RNA virus. Similar to other RNA viruses, HCV replication also induces membrane alteration (membranous web) which could be an important replication platform for RNA replication in cell. HCV nonstructural protein 4B (NS4B) is involved in this membrane alteration, and is also crucial for maintaining the replication complex in detergent-resistant membrane, suggesting a structural role of NS4B in replication. First part of this dissertation (Chapter 2) focuses on the characterization of NS4B protein in order to reveal the mechanism by which NS4B could execute its functions. Mainly, the lipid modification and polymerization activity of NS4B were identified. NS4B palmitoylation is on cysteine 257 residue and Cysteine 261 at the C-terminal end of NS4B. The lipid modification of NS4B is involved in protein stability, protein interaction and replication site targeting. Also, 2-bromopalmitate, the palmitoylation inhibitor, could inhibit HCV RNA replication, suggesting that NS4B lipid modification could be important for RNA replication. The polymerization activity of NS4B on the N-terminal cytoplasmic region was in a scattered pattern. The NS4B palmitoylation also contributes to the polymerization activity. The lipid modification and polymerization activity could be two important properties for NS4B to induce specialized structure and to maintain the virus RNA replication in detergent-resistant membrane.; Along with the NS4B study, several NS4B-interacting proteins were identified by mass spectrometry. Two of these NS4B-interacting candidates, Rab1B and FALDH, are included in this dissertation (in Chapter 2 and 3, respectively). Rab1B belongs to the Rab GTPase family and has GTPase activity and geranylgeranyl modification at its C-terminal end. Rab1B is involved in the directing of vesicle transport between ER to Golgi. Our results suggest that Rab1B could specifically interact with NS4B in single protein expression or in the context of subgenomic replicon. Also, Rab1B subcellular localization was changed in the presence of NS4B protein, and Rab1B is partially colocalized with NS5A in replicon cell. (Abstract shortened by UMI.)... |
