The N-terminal part comprises a predicted and a structurally reso

The N-terminal part comprises a predicted and a structurally resolved amphipathic ��-helix, designated AH1 and AH2, respectively. AH2 comprises amino acids 42 to 66 and has been shown to play an important role in HCV RNA replication (14). Intriguingly, it has the potential to traverse the phospholipid bilayer as a selleckchem 17-AAG transmembrane segment, likely upon oligomerization (14). Oligomerization of membrane proteins represents a potential mechanism to induce membrane curvature and vesicle formation (44, 50). A previous study involving chemical cross-linking provided evidence for the oligomerization of NS4B (49). However, interactions of membrane proteins are inherently difficult to study. Therefore, we aimed to validate and extend these observations by using a different experimental strategy.

In this study, we explored fluorescence resonance energy transfer (FRET) to investigate the determinants for oligomerization of NS4B. FRET is based on the transfer of energy from a fluorescent donor protein (e.g., cyan fluorescent protein [CFP]) to an acceptor protein (e.g., yellow fluorescent protein [YFP]). It has been employed successfully to investigate interactions of membrane proteins, e.g., the G-protein-coupled receptors (5) and nodavirus replicase protein A (7). In acceptor photobleaching FRET, photobleaching of the acceptor results in increased donor emission when the distance between the two is <10 nm, i.e., when the two proteins or protein segments fused to the fluorophores physically interact (5).

Thus, acceptor photobleaching FRET offers the unique opportunity to investigate protein-protein interactions at a defined subcellular location within the membrane environment of intact cells. By the use of FRET and confirmatory coimmunoprecipitation analyses, we found that HCV NS4B oligomerizes through several conserved determinants involving homotypic and heterotypic interactions. Amphipathic ��-helix AH2 was identified as a major determinant for the oligomerization of NS4B. Furthermore, mutations in NS4B that affected oligomerization disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. MATERIALS AND METHODS Cell lines and reagents. U-2 OS human osteosarcoma (40) and Huh-7 human hepatocellular carcinoma (38) cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum.

The U-2 OS-derived, tetracycline-regulated cell lines UHCVcon-57.3 and UHCVcon-AH2mut, expressing the entire polyprotein derived from the HCV H77 consensus clone and harboring wild-type NS4B and NS4B with alanine substitutions of the 6 fully conserved aromatic residues in AH2 (AH2mut), respectively, have been described Brefeldin_A previously (14, 42). Transfections were performed by calcium phosphate precipitation (3).

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