Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic ␣-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic ␣-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.With 120 to 180 million chronically infected individuals worldwide, hepatitis C virus (HCV) infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (38). HCV contains a 9.6-kb positive-strand RNA genome that encodes a polyprotein of about 3,000 amino acids (reviewed in references 36 and 51). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 ion channel polypeptide, the NS2-3 and NS3-4A proteases, an RNA helicase located in the C-terminal twothirds of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. HCV replication takes place in a membrane-associated complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors (7,18,31,43). Determinants for membrane association of the HCV nonstructural proteins have been mapped and a likely endoplasmic reticulum (ER)-derived membrane alteration, designated the membranous web, was found to harbor the HCV replication complex (7, 18; reviewed in reference 36).NS4B is a 27-kDa integral ER membrane protein (21). The expression of NS4B alone induces the formation of the membranous web (7). Thus, an essential function of NS4B is the induction of the specific membrane alteration that serves as a scaffold for the HCV replication complex. In addition, a nucleotide-binding motif has been proposed to reside in the middle of NS4B (8), and RNA binding properties have recently been reported for NS4B (9).Both the N and the C t...

Little is known about the structure of the envelope glycoproteins of hepatitis C virus (HCV). To identify new regions essential for the function of these glycoproteins, we generated HCV pseudoparticles (HCVpp) containing HCV envelope glycoproteins, E1 and E2, from different genotypes in order to detect intergenotypic incompatibilities between these two proteins. Several genotype combinations were nonfunctional for HCV entry. Of interest, a combination of E1 from genotype 2a and E2 from genotype 1a was nonfunctional in the HCVpp system. We therefore used this nonfunctional complex and the recently described structural model of E2 to identify new functional regions in E2 by exchanging protein regions between these two genotypes. The functionality of these chimeric envelope proteins in the HCVpp system and/or the cell-cultured infectious virus (HCVcc) was analyzed. We showed that the intergenotypic variable region (IgVR), hypervariable region 2 (HVR2), and another segment in domain II play a role in E1E2 assembly. We also demonstrated intradomain interactions within domain I. Importantly, we also identified a segment (amino acids [aa] 705 to 715 [segment 705-715]) in the stem region of E2, which is essential for HCVcc entry. Circular dichroism and nuclear magnetic resonance structural analyses of the synthetic peptide E2-SC containing this segment revealed the presence of a central amphipathic helix, which likely folds upon membrane binding. Due to its location in the stem region, segment 705-715 is likely involved in the reorganization of the glycoprotein complexes taking place during the fusion process. In conclusion, our study highlights new functional and structural regions in HCV envelope glycoprotein E2.Hepatitis C virus (HCV) infects approximately 3% of the world population (72) and is currently the major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (43). A vaccine is not yet available, and the treatment fails in around 50% of the cases, depending on the virus genotype (43). Although the cloning of the HCV genome more than 20 years ago (4) allowed for a rapid analysis of the genomic organization and a biochemical characterization of its proteins (reviewed in reference 57), the lack of a cell culture system to efficiently amplify this virus has long been a major obstacle for the study of the HCV life cycle. Fortunately, in 2005, the development of a cell culture system that allowed for a relatively efficient amplification of HCV (HCVcc) was finally reported (42,71,78).HCV is an enveloped, positive-stranded RNA virus that belongs to the Flaviviridae family (41). Its genome encodes a single polyprotein of about 3,000 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases to yield at least 10 mature products (reviewed in reference 57). Cleavage of the viral polyprotein by a cellular signal peptidase gives rise to the envelope glycoproteins E1 and E2 (reviewed in reference 17). HCV envelope glycoproteins are type I transmembrane (TM) proteins containing a h...