Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

Abstract

Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.Hepatitis C virus (HCV) has a single-stranded, positive-sense RNA genome, which encodes a polyprotein. The polyprotein is processed into at least 10 individual proteins (Core-E1-E2-p7-NS2-NS3-NS4A-NS3-NS4A-NS4B-NS5A-NS5B). Core-E1-E2-p7 proteins are structural proteins and are cleaved by cellular proteases. NS2 to NS5B are nonstructural proteins, which are cleaved by viral proteases (NS2 and NS3) (4). By using a subgenomic-replicon system (21), our laboratory has shown that HCV RNA replication occurs on a specialized membrane microdomain, or lipid raft (2, 30). NS4B and the cellular protein hVAP-33 (human homologue of the VAMP-associated 33-kDa protein) are the two main players in initiating the formation of the RNA replication complex (14). Other nonstructural proteins of HCV also reside in lipid rafts (2, 30); furthermore, a subpopulation of core proteins has been detected in detergent-resistant membranes (24). Therefore, this specialized micromembrane domain could be important in the HCV replication cycle.NS4B protein is a relatively hydrophobic membrane protein with a protein size of 27 kDa. According to a topology study (22), NS4B has four transmembrane segments, with the N and C termini being located on the cytoplasmic side. The amino acid composition of the cytoplasmic N terminus is very hydrophobic; thus, a fifth transmembrane domain has been proposed in this region (22). However, it is still unknown whether the flipping of the fifth transmembrane domain indeed occurs. A putative amphipathic helix on amino acid residues 1 to 26 of the NS4B N terminus is crucial for virus replication (13). In addition, a nucleotide-binding motif located between the second and third transmembrane domains of NS4B (residues 129 to 135 of NS4B) has also been identified (12).Although the exact function of NS4B in viral replication is still unclear, it is striking that the expression of NS4B alone or in the context of HCV polyprotein induces a specialized membranous web (11), which has been postulated to be the HCV RNA ...

Hepatitis c virus and host cell lipids: An intimate connection

“…107 Since NS4B oligomerization is required for the formation of the MW and a functional replication complex, 53 NS4B palmitoylation could also be involved in HCV RNA replication. 107 However, further studies will be needed to confirm the role of NS4B palmitoylation in the context of an HCV infection.…”

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

“…Our in silico and in vitro data are in agreement with previous publications, reporting polymerization of different NS4B molecules. 22 It cannot be excluded yet that NS4B molecules may also form multimers. Furthermore, there may be uncertainty as to exclude a yet unidentified additional binding partner, mediating the NS4B-NS4B interaction observed in this study.…”

“…22 Thereby, the main polymerization determinants were mapped within the aminoterminal cytosolic domain of NS4B. 22 Furthermore, the aminoterminal part of NS4B was shown to be involved in physical interaction with the cellular ER-stress response element CREB-RP/ATF6ß.…”

“…22 Thereby, the main polymerization determinants were mapped within the aminoterminal cytosolic domain of NS4B. 22 Furthermore, the aminoterminal part of NS4B was shown to be involved in physical interaction with the cellular ER-stress response element CREB-RP/ATF6ß. 23 However, neither the molecular basis for specific protein-protein interactions nor the biological relevance of this finding is known.…”

Characterization of hepatitis C virus NS3 modifications in the context of replication

“…Together, these 10 proteins coordinate the complete life cycle of HCV (Moradpour et al, 2007). For some of these proteins, it is known that they have a dual function involved in more than one process of the virus life cycle, requiring precise regulation (Kim et al, 2004;Lindenbach et al, 2007;Tellinghuisen et al, 2008;Yu et al, 2006). …”