Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic ␣-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention. Hepatitis C virus (HCV)1 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). A protective vaccine does not exist to date, and therapeutic options are still limited (2, 3). HCV has been classified in the Hepacivirus genus within the Flaviviridae family which includes the classical flaviviruses, such as yellow fever virus, and the animal pestiviruses, such as bovine viral diarrhea virus (BVDV) (4). The structure and replication cycle of HCV are incompletely understood due to the low viral titers found in sera and livers of HCV-infected individuals and the lack of an efficient cell culture system or small animal model permissive for HCV infection. Nevertheless, considerable progress has been made using heterologous expression systems, functional cDNA clones, and more recently, selectable subgenomic replicons (see Refs. 5 and 6 for recent reviews).HCV contains a single-stranded RNA genome of positive polarity and ϳ9600 nucleotides (nt) length that encodes a polyprotein precursor of about 3000 amino acids (aa) (Fig. 1A). 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 protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The non-structural proteins NS2 through NS5B include the NS2-3 autoprotease and the NS3 serine protease, an RNA helicase located in the carboxyl-terminal region of NS3, the NS4A polypeptide, the NS4B ...

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxylterminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane ␣-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATPindependent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.With an estimated 170 million chronically infected individuals the hepatitis C virus (HCV) 1 is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). A protective vaccine does not exist to date, and therapeutic options are still limited (2). HCV has been classified in the Hepacivirus genus within the Flaviviridae family that includes the classical flaviviruses, such as yellow fever virus, and the animal pestiviruses (3). HCV contains a single-stranded RNA genome of positive polarity and ϳ9600 nucleotides (nt) in length that encodes a polyprotein precursor of about 3000 amino acids (aa) (see Refs. 4 and 5 for recent reviews) (Fig. 1A). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. HCV replication proceeds via synthesis of a complementary minus strand RNA using the genome as a template and the subsequent synthesis of genomic plus strand RNA from this minus strand RNA template. The key enzyme responsible for both of these steps is the RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B).The HCV RdRp has been shown to be essential for viral replication in vitro (6) and in vivo (7). It has recently been characterized both biochemically (8 -12) and with respect to its three-dimensional structure (13-15). The HCV NS5B protein contains motifs shared by all RdRps and possesses the classical fingers, palm, and thumb subdomains. As a unique feature of the HCV RdRp extensive interactions between the fingers and thumb subdomains result in a completely encircled active site. Interestingly, deletion of the highly hydrophobic ...

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated gene expression system, a novel monoclonal antibody, and in vitro transcription-translation were employed to investigate the subcellular localization and to characterize the membrane association of this viral protein. When expressed individually or in the context of the entire HCV polyprotein, NS4B was localized in the endoplasmic reticulum (ER), as shown by subcellular fractionation, immunofluorescence analyses, and double-label confocal laser scanning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred cotranslationally, presumably via engagement of the signal recognition particle by an internal signal sequence. In membrane extraction and proteinase protection assays NS4B displayed properties of a cytoplasmically oriented integral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. An efficient replication system will be essential to further define the role of NS4B in the viral life cycle.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (29). Similar to all positive-strand RNA viruses investigated thus far (reviewed in reference 2), HCV forms a membrane-associated replication complex composed of viral proteins, replicating RNA, and altered cellular membranes (9, 13). Determinants for membrane association of the HCV nonstructural proteins have been mapped (reviewed in reference 8), and a specific membrane alteration, designated the membranous web, was recently identified as the site of RNA replication in Huh-7 cells harboring subgenomic HCV replicons (13).Members of our laboratories have recently shown that the HCV RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a relatively small class of membrane proteins termed tail-anchored proteins (16, 32). Characteristic features of these proteins include (i) posttranslational membrane targeting via a hydrophobic C-terminal insertion sequence (which in the case of NS5B was mapped to the C-terminal 21 amino acid residues), (ii) integral membrane association, and (iii) a cytosolic orientation of the functional protein domain (reviewed in references 3 and 34). The prototype of this class of proteins is cytochrome b 5 . Other examples include the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and Bcl-2. NS5B represents the first polymerase within the family of tailanchored proteins.Interestingly, the deletion of its hydrophobic C-terminal...

Signal transducers and activators of transcription (STAT) 1 and STAT3 are activated by overlapping but distinct sets of cytokines. STATs are recruited to the different cytokine receptors through their Src homology (SH) 2 domains that make highly specific interactions with phosphotyrosine-docking sites on the receptors. We used a degenerate phosphopeptide library synthesized on 35-m TentaGel beads and fluorescenceactivated bead sorting to determine the sequence specificity of the peptide-binding sites of the SH2 domains of STAT1 and STAT3. The large bead library allowed not only peptide sequencing of pools of beads but also of single beads. The method was validated through surface plasmon resonance measurements of the affinities of different peptides to the STAT SH2 domains. Furthermore, when selected peptides were attached to a truncated erythropoietin receptor and stably expressed in DA3 cells, activation of STAT1 or STAT3 could be achieved by stimulation with erythropoietin. The combined analysis of pool sequencing, the individual peptide sequences, and plasmon resonance measurements allowed the definition of SH2 domain binding motifs. STAT1 preferentially binds peptides with the motif phosphotyrosine-(aspartic acid/glutamic acid)-(proline/ arginine)-(arginine/proline/glutamine), whereby a negatively charged amino acid at ؉1 excludes a proline at ؉2 and vice versa. STAT3 preferentially binds peptides with the motif phosphotyrosine-(basic or hydrophobic)-(proline or basic)-glutamine. For both STAT1 and STAT3, specific high affinity phosphopeptides were identified that can be used for the design of inhibitory molecules.The signal transducers and activators of transcription (STATs) 1 constitute a family of latent cytoplasmic transcription factors that are activated by a large number of cytokines, growth factors, and hormones. The binding of these extracellular signaling polypeptides to specific cell surface receptors typically results in receptor homo-or heterodimerization and consecutive activation of receptor-associated protein tyrosine kinases of the Jak family. Activated Jak kinases phosphorylate tyrosine residues in the intracellular domains of the receptors (1). STATs then bind with their SH2 domains to these receptordocking sites. The Jak kinases phosphorylate the STATs on a single tyrosine located carboxyl-terminal to the SH2 domain (2). The tyrosine phosphorylation of STATs is the decisive activation event, resulting in STAT dimer formation through mutual SH2 domain-phosphotyrosine interactions. STAT dimers translocate into the nucleus, bind to response elements in gene promoters, and enhance the transcription of these target genes (3-5). Seven mammalian STAT genes have been identified in three chromosomal clusters (6). The different STAT proteins are activated by distinct cytokines and growth factors, and each STAT protein activates a distinct set of target genes (5, 7). The specific coupling of the different STAT family members to cytokine receptors is crucial for the generation of diverse intracellu...