Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B

Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle.     

The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner

Dengue virus (DV) is a positive sense RNA virus replicating in the cytoplasm in membranous compartments that are induced by viral infection. The non-structural protein (NS) 4A is one of the least characterized DV proteins. It is highly hydrophobic with its C-terminal region (designated 2K fragment) serving as a signal sequence for the translocation of the adjacent NS4B into the endoplasmic reticulum (ER) lumen. In this report, we demonstrate that NS4A associates with membranes via 4 internal hydrophobic regions, which are all able to mediate membrane targeting of a cytosolic reporter protein. We also developed a model for the membrane topology of NS4A in which the N-terminal third of NS4A localizes to the cytoplasm, while the remaining part contains three transmembrane segments, with the C-terminal end localized in the ER lumen. Subcellular localization experiments in DV-infected cells revealed that NS4A resides primarily in ER-derived cytoplasmic dot-like structures that also contain dsRNA and other DV proteins, suggesting that NS4A is a component of the membrane-bound viral replication complex (RC). Interestingly, the individual expression of DV NS4A lacking the 2K fragment resulted in the induction of cytoplasmic membrane alterations resembling virus-induced structures, whereas expression of full-length NS4A does not induce comparable membrane alterations. Thus, proteolytic removal of the 2K peptide appears to be important for induction of membrane alterations that may harbor the viral RC. These results shed new light on the role of NS4A in the DV replication cycle and provide a model of how this protein induces membrane rearrangements and how this property may be regulated.     

An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.     

Conserved determinants for membrane association of nonstructural protein 5A from hepatitis C virus and related viruses

Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.     

丙型肝炎病毒基因组结构及功能

丙型肝炎病毒（hepatitis C virus, HCV）是单股正链的RNA 病毒,全长为9.6 kb,包括1个大的开放阅读框（ORF）和两侧的5′,3′非编码区（UTRs）.核糖体通过进入HCV 5′UTR 端的内部核糖体进入位点（IRES）,将HCV基因组翻译成1个聚蛋白前体.前体聚蛋白被宿主和病毒的蛋白酶共同切割成为若干个具有独立功能的HCV蛋白,根据功能的不同分别命名为C、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A 和NS5B,它们不但在HCV的生活史中发挥着重要的作用,也影响着宿主细胞的信号传导、凋亡及物质代谢等一系列生化过程.近年来,随着HCV体外细胞摸型的不断发展,其病毒分子生物学方面的研究取得了很大的进展.本文从基因组结构及其编码的蛋白功能等方面阐述了HCV病毒的研究进展,为致病机理的研究及抗HCV药物的开发和疫苗研制等提供理论基础.     

Topology of Endoplasmic Reticulum‐Associated Cellular and Viral Proteins Determined with Split‐GFP

The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER‐associated proteins. A 215‐amino acid fragment of GFP (S1–10) was expressed in the cytoplasm as a free protein or fused to the N‐terminus of calnexin and in the ER as an intraluminal protein or fused to the C‐terminus of calnexin. A 16‐amino acid fragment of GFP (S11) was fused to the N‐ or C‐terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N‐ and C‐termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N‐ and C‐termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.      

Genome of human hepatitis C virus (HCV): gene organization, sequence diversity, and variation

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis. HCV infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. Since the discovery of HCV in 1989, a large number of genetic analyses of HCV have been reported, and the viral genome structure has been elucidated. An enveloped virus, HCV belongs to the family Flaviviridae, whose genome consists of a positive-stranded RNA molecule of about 9.6 kilobases and encodes a large polyprotein precursor (about 3000 amino acids). This precursor protein is cleaved by the host and viral proteinase to generate at least 10 proteins: the core, envelope 1 (E1), E2, p7, nonstructural (NS) 2, NS3, NS4A, NS4B, NS5A, and NS5B. These HCV proteins not only function in viral replication but also affect a variety of cellular functions. HCV has been found to have remarkable genetic heterogeneity. To date, more than 30 HCV genotypes have been identified worldwide. Furthermore, HCV may show quasispecies distribution in an infected individual. These findings may have important implications in diagnosis, pathogenesis, treatment, and vaccine development. The hypervariable region 1 found within the envelope E2 protein was shown to be a major site for the genetic evolution of HCV after the onset of hepatitis, and might be involved in escape from the host immunesurveillance system.     

Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.     

A genetic interaction between hepatitis C virus NS4B and NS3 is important for RNA replication

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.     

Membrane topology of the hepatitis C virus NS2 protein

The hepatitis C virus (HCV) NS2 protein is a hydrophobic protein. Previous studies indicate that this protein is an integral membrane protein, which is targeted to the membrane of the endoplasmic reticulum (ER) by the signal sequence located in its preceding p7 protein. In this report, we demonstrate that the membrane association of NS2 is p7-independent and occurs co-translationally. Further deletion-mapping studies suggest the presence of two internal signal sequences in NS2. These two internal signal sequences, which are located within amino acids 839-883 and amino acids 928-960, could target the alpha-globin reporter, a cytosolic protein, to the membrane compartments in HuH7 hepatoma cells. The presence of multiple signal sequences for its membrane association suggests that NS2 has multiple transmembrane domains. The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen. Based on these results, a model for the NS2 membrane topology is presented.     

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

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 carboxyl-terminal 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 alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. 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.     

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.     

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.     

An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication

Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.     

Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft

The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.     

Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B

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 α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-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 two-thirds 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 termini of NS4B are believed to be oriented toward the cytosol, and prediction algorithms indicate the presence of four putative transmembrane segments in the central portion of the protein (21, 27, 28, 42). The cytosolic orientation of the bulk of the protein was confirmed experimentally (21), but a more refined membrane topology is so far elusive. The introduction of glycosylation acceptor sites at various positions in NS4B validated the prediction of ER luminal loops around amino acid positions 112 and 161 (27, 28). Intriguingly, the N terminus of NS4B was reported to be translocated into the ER lumen at least partially, presumably by a posttranslational mechanism (28). Interestingly, the coexpression of the other HCV proteins appears to limit this translocation (27). A recent report indicates that NS4B is palmitoylated at C-terminal residues Cys 257 and Cys 261 and forms oligomers (59).In order to define the membrane topology of NS4B we have analyzed a comprehensive panel of green fluorescent protein (GFP) fusion constructs comprising different segments of NS4B (N. Arora, V. Castet, and D. Moradpour, unpublished data). In the course of these studies, we unexpectedly found that a fusion construct comprising the N-terminal 74 amino acids (aa) of NS4B was associated with membranes, while most prediction methods located the beginning of the first transmembrane segment around aa 74 (21, 28, 42). Here, we demonstrate that an amphipathic α-helix extending from aa 42 to 66 (α-helix 42-66) in the N-terminal portion of NS4B mediates this membrane association and plays an essential role in the formation of the HCV replication complex.     

Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2

The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4 degrees C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.     

Palmitoylation and polymerization of hepatitis C virus NS4B protein

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.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.