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Originally published In Press as doi:10.1074/jbc.M512697200 on January 25, 2006

J. Biol. Chem., Vol. 281, Issue 13, 8854-8863, March 31, 2006
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Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B*Formula

Sven Miller, Sandra Sparacio1, and Ralf Bartenschlager2

From the Department of Molecular Virology, The University of Heidelberg, 69120 Heidelberg, Germany

Received for publication, November 28, 2005 , and in revised form, January 25, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mosquito-transmitted Dengue virus (DV)3 is the causative agent of dengue fever, the most prevalent arthropod-borne viral illness in humans with 50-100 million individuals infected annually worldwide (1). Dengue fever is characterized by high fever, chills, body aches, and skin rash and ranges from mild, flu-like symptoms to severe forms, which are dengue hemorrhagic fever and the dengue shock syndrome. The four serotypes of DV identified so far (DV1-4) belong to the genus Flavivirus in the family Flaviviridae. Flaviviruses are small, enveloped viruses that have a single-stranded genomic RNA of positive polarity ~11 kb in length. This RNA serves as mRNA for translation of a large polyprotein (3,391 amino acids in case of DV2) at the rough endoplasmic reticulum. The viral polyprotein is co- and post-translationally processed into three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The amino termini of prM, E, NS1, and NS4B are generated upon cleavage by the host signal peptidase in the lumen of the ER, whereas processing of most of the other NS proteins and the COOH terminus of the C protein is carried out by the viral two-component protease NS2B-3 in the cytoplasm of DV-infected cells (2-4). For cleavage of the COOH terminus of NS1 an unknown ER resident protease seems to be responsible (5). A Golgi-localized furin protease mediates cleavage of prM at a late state of infection to generate the M protein (6, 7). Most, if not all NS proteins are involved in replication of the flavivirus RNA, which occurs in close association with cellular membranes in so called viral replication complexes (RCs) (8, 9). NS5 is the RNA-dependant RNA polymerase and it carries in the NH2-terminal domain a methyltransferase important for the formation of the RNA cap structure (10-13). NS3 acts as the viral serine protease, which requires the cofactor NS2B for full activity (14, 15). In addition, NS3 also comprises an RNA triphosphatase as well as an RNA helicase (15, 16). The glycoprotein NS1 plays a role in viral RNA replication, probably at an early step of viral RNA replication (17, 18). Only little is known about the functions of the small hydrophobic proteins NS2A, NS4A, and NS4B. It has been suggested that they may serve to anchor the viral replicase to cellular membranes (19). In addition, these proteins appear to inhibit the interferon-{alpha}/beta (IFN-{alpha}/beta) response of the host (20-22).

NS4B is the largest of the small hydrophobic NS proteins of the flaviviruses. In the case of DV, NS4B consists of 248 amino acids and has an apparent molecular mass of 27 kDa. Whereas NS4B of the hepatitis C virus (HCV), which has only negligible sequence identity to the flavivirus NS4B proteins, is characterized to some extent (23-26), this is not the case with flavivirus NS4B, and neither its precise membrane topology nor its role in the viral life cycle are known. It was shown that the COOH-terminal part of the NS4A protein preceding NS4B contains a signal sequence that serves to translocate NS4B into the lumen of the ER. Because of the size of 2 kDa this signal peptide is called the 2K fragment. After translocation, the 2K fragment is cleaved off the NH2 terminus of NS4B by the host signalase in the ER lumen. The signalase cleavage at the 2K-4B site requires a prior NS2B-3 proteinase-mediated cleavage at the so called 4A/2K site, which is conserved among flaviviruses, and located 23 residues NH2-terminal of the signalase site (27). NS4B of the Kunjin virus (KUNV) was shown to be located throughout the cytoplasm, in perinuclear membranes, and possibly in the nucleus of KUNV-infected cells (28). However, it was not found in partially purified RCs (29). Nevertheless, deletion studies and trans-complementation experiments of the bovine viral diarrhea virus and KUNV (30, 31) indicate an important role of this protein in viral RNA replication.

NS4B of the DV, the West Nile virus, and also the yellow fever virus was recently identified as an inhibitor of the IFN-{alpha}/beta response. Expression of DV NS4B blocks the IFN-{alpha}/beta-induced signal transduction cascade by interfering with STAT1 (transducer and activator of transcription) phosphorylation. Deletion analyses suggest that the first 125 amino acids of DV 2K-NS4B are sufficient for the inhibition of IFN-{alpha}/beta signaling, and that proper viral polyprotein processing is required for anti-IFN function (32). Likewise NS4A and, to a lesser extent NS2A, appear to block IFN signaling, and the cumulative effect of the three proteins results in robust inhibition of IFN signaling (22, 32).

To improve our understanding of DV NS4B, we characterized its subcellular localization, membrane association, and membrane topology in this study. We provide evidence that NS4B is part of the viral replication complex and established a model of how NS4B integrates into cellular membranes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Viruses—Cell monolayers of the human hepatoma cell line Huh-7 (33) and the African green monkey kidney cell line Vero were grown in Dulbecco's modified Eagle's medium (Invitrogen, Karlsruhe, Germany) supplemented with 2 mM L-glutamine, non-essential amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf-serum (Dulbecco's modified Eagle's medium complete). Huh-7/T7 cells, constitutively expressing the T7 polymerase (34), were grown under the same conditions but 5 µg/ml Zeocin (Invitrogen) was added to the medium. For the infection of Huh-7 and Vero cells the DV2 (New Guinea C strain; kindly provided by Progen, Heidelberg, Germany) was used at a multiplicity of infection of 2-4. The virus was diluted in Dulbecco's modified Eagle's medium containing 2% fetal calf serum and cells were incubated for 4 h at 37°C with occasional rocking. After 4 h the inoculum was replaced by complete Dulbecco's modified Eagle's medium, and cells were incubated for various times.

Antisera—The following primary antibodies were used in our study: rabbit polyclonal anti-calnexin, mouse monoclonal anti-protein-disulfide isomerase, and rabbit polyclonal anit-calreticulin antibodies (all obtained from Stressgen, Victoria, Canada), mouse monoclonal anti-HA tag antibody (Covance, Berkeley, CA), mouse monoclonal anti-dsRNA antibody (English & Scientific Consulting, Szirak, Hungary), mouse monoclonal anti-intermediate compartment antibody, and mouse monoclonal anti-CLIMP63 antibody (both obtained from Alexis Biochemicals, Lausen, Switzerland), mouse monoclonal anti-GalT antibody (a kind gift of Eric Berger, Zürich, Switzerland), mouse monoclonal anti-Golgin 97 antibody, and rabbit anti-GFP antibody (both obtained from Molecular Probes, Eugene, OR). The secondary antibodies used in immunofluorescence microscopy were goat antibodies conjugated to the cyanine dye Cy3 (Dianova, Hamburg, Germany). Cellular DNA was stained with 4',6'-diamidino-2-phenylindole dihydrochloride (Molecular Probes). The secondary antibodies used in Western blot analyses were a mouse and a rabbit secondary antibody coupled to the horseradish peroxidase (Sigma).

Plasmid Constructs—Standard molecular biology techniques were used for cloning (35). The structures of all plasmids were determined by nucleotide sequence and restriction analysis. The full-length DV2 NGC strain cDNA (a kind gift of Andrew Davidson, School of Medical Sciences, Bristol, UK) served as a template for polymerase chain reaction amplification (PCR) of individual DV genes. For expression of DV-glutathione S-transferase (GST) fusion proteins, DNA fragments encoding for the following amino acids (aa) of the respective DV proteins were generated by PCR and inserted into the bacterial expression vector pGEX-6P-1 (Amersham Biosciences) in-frame with the GST sequence: E, aa 2-100; NS3, aa 320-618; NS4B, aa 126-170; and NS5, aa 1-900. The PCR fragment encoding for the amino acids of the E protein was amplified with the sense primer, 5'-ATATTGTCGACTCCGTTGCATAGGAATATCAAATAGAG-3', and the antisense primer, 5'-ATAGCGGCCGCTTATCCTCTGTCCACCATGGAGTGTTTG-3', and cloned into the SalI and NotI restriction site of the plasmid pGEX-6P-1. The PCR fragments encoding for the proteins NS3 (sense primer, 5'-ATATTGGATCCAGCAGAGACCCATTC-3'; antisense primer, 5'-ATAAGATCTTTACTTTCTTCCAGCTGCA-3'), NS4B (sense primer, 5'-AGGATCCCAAGCAAAAGCAACC-3'; antisense primer, 5'-ATAAGATCTTTATTGTCCCAACTGCTTT-3'), and NS5 (sense primer, 5'-ATATGGATCCGGAACTGGCAACATA-3'; antisense primer, 5'-ATAAGATCTTTACCACAGGACTCCTGCC-3') were cloned into the BamHI and BglII sites (underlined) of pGEX-6P-1.

A modified version of the plasmid pTM-eGFP served as the basic plasmid for the creation of the pTM-NS4B-eGFP fusion constructs expressing different NS4B fragments fused to the enhanced green fluorescent protein (eGFP). To obtain pTM-eGFP the DNA encoding for eGFP was amplified by PCR from the template pEGFP-1 (Clontech) and inserted into the vector pTM1 kindly provided by B. Moss (36). pTM-eGFP was then restricted with NcoI and an annealed oligonucleotide (sense, 5'-CATGAGACCGGTGGCCGCTGC-3'; antisense, 5'-CATGGCAGCGGCCACCGGTCT-3') containing the AgeI site (underlined) was introduced. The restriction sites NcoI and AgeI were used for the insertion of PCR fragments encoding for the different NS4B regions to generate the NS4B-eGFP fusion constructs.

The pTM1-NS4B-HA expression plasmid was constructed as follows: the sequence encoding for the DV NS4B-HA fusion protein was amplified by PCR using a sense primer with the sequence 5'-ATATTCCATGGCAAACGAGATGGGTTTCCTGG-3' and an antisense primer with the sequence 5'-ATACATACCGGTTTAGGCATAATCCGGCACATCATAAGGGTACCTTCTCGTGTTGGTTGTGTTC-3', which encoded an HA tag (italics) and a stop codon (bold). The PCR fragment was digested with restriction enzymes NcoI and AgeI and inserted into the plasmid pTM1 digested with the same enzymes.

Expression of E-, NS3-, NS4B-, and NS5-GST Fusion Proteins and Generation of Antisera—The bacterial expression vector pGEX-6P-1 (Amersham Biosciences) was used for the expression of fragments or full-length versions of GST-E, NS3, NS4B, and NS5 fusion proteins. Each construct was expressed using Escherichia coli strain BL21 codon plus (DE3)-RIL (Stratagene). After induction with 1 mM isopropyl beta-D-thiogalactoside (AppliChem, Darmstadt, Germany) the cells were grown for 4-6 h at 30 °C and afterward pelleted and ruptured by sonication in phosphate-buffered saline (PBS) supplemented with protease inhibitors. Cell debris was removed by high speed centrifugation, and GST-E and GST-NS4B fusion proteins were purified from the resulting supernatant by affinity chromatography with glutathione-Sepharose 4B (Amersham Biosciences) using a batch method following the manufacturers instructions. GST-NS3 and GST-NS5 cell pellets were treated with different concentrations of urea (3-9 M), and after centrifugation, supernatants were separated by SDS-PAGE and stained with Coomassie Blue. GST fusion proteins were excised and eluted from the gel slice by using an electroelution chamber (Schleicher & Schuell) as described in the manual. Antisera were raised in New Zealand White rabbits immunized over a period of 4 months with the purified GST fusion proteins (Eurogentec, Seraing, Belgium).

For immunofluorescence studies the monospecific polyclonal antisera were purified by affinity purification as follows: lysates of DV-infected Vero or Huh-7 cells were subjected to 12% SDS-PAGE and subsequently transferred to nitrocellulose membranes. Blots on nitrocellulose membranes were stained with 0.2% Ponceau-S in 3% trichloroacetic acid and the DV-specific bands were excised, washed, and blocked for 1 h at room temperature. Subsequently, excised pieces were incubated for 1 h at room temperature with a 1:5 dilution of the DV-specific antisera in PBS containing 5% milk powder and 0.2% NaN3. Antisera solutions were removed and stored on ice prior to the next incubation cycle. Excised blot strips were washed with PBS and bound antibodies were eluted by incubation of the strips for 20 min at 52 °C in PBS containing 0.2% NaN3. Strips were incubated again with the DV antisera solutions and this procedure was repeated 10 times. Eluted antibody fractions were pooled and concentrated using Centricon Plus-20 centrifugal filter devices (Millipore, Schwalbach, Germany) as described by the manufacturer.

DNA Sequence Analysis—Nucleotide sequences of the constructs were confirmed by automated sequencing with an ABI 310 sequencer (Applied Biosystems). Big Dye version 1.1 (Applied Biosystems) was used for cycle sequencing according to the instructions of the manufacturer.

Fluorescence Microscopy—Cell monolayers were grown on glass coverslips and, following infection or transfection, fixed at given time points in 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS, incubated with primary antibodies for 1 h in PBS containing 3% bovine serum albumin, and washed 3 times for 5 min with PBS. Secondary antibody incubation was performed for 45 min in PBS containing 3% bovine serum albumin, after which the cells were washed as described above. Subsequently, cells were mounted in Mobiglow (MoBiTec, Göttingen, Germany). Fluorescence images were taken with a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) and transferred to a computer work station running LSM5 Image Browser 3.2 software (Carl Zeiss). Images were merged with Adobe Photoshop CS software.

Transfection—DNA constructs were transfected into Huh-7/T7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Membrane Flotation Assay—Cells were infected or transfected as described above. Three days after infection or 8 h after transfection, cells were collected by scraping into PBS followed by centrifugation at 1,000 x g for 5 min. Cells were resuspended in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 2 mM MgCl2), and incubated on ice for 10 min. Cells were disrupted with 15 strokes of a Dounce homogenizer, and lysates were centrifuged at 1,000 x g for 5 min to pellet nuclei, unlysed cells, and debris. Nycodenz (Axis Shield, Oslo, Norway) was added to postnuclear lysates to a final concentration of 37.5% (w/v), loaded under a 5-35% Nycodenz discontinuous gradient, and centrifuged to equilibrium in a Beckmann TLS-55 swing-bucket rotor at 100,000 x g for 20 h. After centrifugation, fractions were collected from the top to the bottom of the density gradient. Equal volumes of samples from each fraction were separated by SDS-PAGE and immunoblotted as described above. For membrane dissociation assays, postnuclear lysates were centrifuged at 20,000 x g for 10 min to pellet membranes and their associated proteins. Pellets were resuspended in PBS, 1 M NaCl, 0.1 M Na2CO3, (pH 11.5), or 0.5% Triton X-100, incubated on ice for 30 min, and fractionated in Nycodenz gradients as described above.

Western Blot Analysis—Samples were loaded onto a 12% polyacrylamide-SDS gel, and after electrophoresis proteins were transferred to nitrocellulose. Blots were blocked overnight at 4 °C in blocking solution (5% milk powder and 0.5% Tween 20 in PBS). Incubation with the primary antibody was performed in blocking solution for 1 h at room temperature. Blots were washed 3 times for 10 min in washing solution (0.5% Tween 20 in PBS), incubated for 1 h with the secondary antibody in blocking solution, and washed again as described above. Antibody-protein complexes were detected using the ECL Plus Western blotting detection system (Amersham Biosciences). Quantification of the signals was performed with a computer running Quantity One 4.5.0 software (Bio-Rad).

Proteinase K Protection Assay—Cell lysates were prepared as described under "Membrane Flotation" (see above), and centrifuged at 1,000 x g for 5 min to pellet nuclei. The postnuclear lysates were centrifuged at 20,000 x g for 10 min to pellet membranes and their associated proteins. Pellets were resuspended in PBS and treated with 50 µg/ml proteinase K (Sigma) for 1 h on ice in the presence or absence of 0.5% Triton X-100. Proteolysis was stopped by adding 10 mM phenylmethylsulfonyl fluoride (Sigma). After 15 min on ice, a preheated (95 °C) sample buffer was added and the sample was boiled for 15 min to inactivate the protease. Samples were then analyzed by SDS-PAGE and Western blot.

Protein Sequence Analysis and Transmembrane Domain Prediction—Protein sequence analyses were performed with the following computer programs: ConPredII, TMAP, TMHMM II, Tmpred, and SOSUI.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of DV-specific Antisera directed Against E, NS3, NS4B, and NS5—To unravel the role of DV NS4B in the viral life cycle, we generated rabbit polyclonal antisera monospecific for E, NS3, NS4B, and NS5. DV proteins or fragments thereof were overexpressed as GST fusion proteins in E. coli, and New Zealand White rabbits were immunized with the purified antigens (see "Experimental Procedures"). The obtained antisera were characterized by Western blot analysis of lysates of DV-infected Vero cells. All antisera reacted with proteins of the expected sizes in DV-infected but not in mock-infected cells (data not shown). These results confirmed the specificity of the antisera that were used throughout this study. Attempts to raise NS4A-specific antisera were not successful, probably because of poor immunogenicity.

Subcellular Localization of DV NS4B—We started our characterization of DV NS4B by determining its subcellular localization in DV2-infected Huh-7 cells by confocal immunofluorescence microscopy at different time points after infection (Fig. 1, A-C). The specificity of the antibody was determined by staining uninfected Huh-7 cells (Fig. 1D). In these cells no fluorescence was observed. At 14 h post-infection NS4B accumulated in the perinuclear region with a staining pattern resembling the ER (see below; Fig. 1A). At 24 h post-infection remarkable changes in the staining pattern were noted, most notably the formation of large NS4B foci distributed throughout the cytoplasm with some enrichment in the perinuclear region (Fig. 1B). At 38 h post-infection a similar pattern was found, but a considerably higher number of cytoplasmic foci had formed (Fig. 1C). In contrast to KUNV (28), we did not detect NS4B in the nucleus of DV-infected Huh-7 cells, even at very late time points after infection. The same result was found in Vero cells excluding the possibility that non-nuclear localization of DV NS4B was due to the particular cell line used (data not shown). Moreover, a similar cytoplasmic and punctuate distribution as for DV NS4B was found for NS3 (Fig. 1, E-G) arguing that these 2 proteins were part of the DV replication complex. In contrast, NS5 was found predominantly in the nucleus (Fig. 1, I-K). Only a weak, fine granular or, at later time points, reticular staining pattern was observed in the cytoplasm in agreement with previous reports (37, 38).

To explore the subcellular localization of DV NS4B in more detail we performed colocalization studies with cellular marker proteins in DV2-infected Huh-7 cells. As shown in Fig. 2 (A-F), protein-disulfide isomerase (a soluble protein resident in the ER) and cytoskeleton linking membrane protein (CLIMP-63, a marker for the reticular subdomain of the rough ER) displayed the same reticular and foci-like staining as DV NS4B. In contrast, there was no apparent colocalization of NS4B with the Golgi marker protein Golgin 97 (Fig. 2, G-I), the trans-Golgi marker protein galactosyltransferase (GalT; Fig. 2, J-L), or a marker protein for the intermediate compartment (Fig. 2, M-O). Because the observed cytoplasmic foci bore resemblance to lipid droplets, we also performed colocalization studies of DV NS4B with these structures by using Oil red O staining. However, no colocalization between DV NS4B and lipid droplets was observed (Fig. 2, P-R). From these data we concluded that NS4B accumulates at or close to the ER or ER-derived membranes in DV-infected cells.


Figure 1
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  		FIGURE 1.
Localization of DV NS4B in infected cells. Huh-7 cells were infected with DV2 (A-C, E-G, and I-K) or left uninfected (D, H, and L). At 14 (A, E, and I), 24 (B, D, F, H, J, and L), or 38 h post-infection (C, G, and K) cells were analyzed by immunofluorescence with the given primary antibodies. Slides were analyzed by confocal laser scanning microscopy.

 
NS4B Localizes to the Sites of Viral RNA Replication—Synthesis of the flavivirus positive strand RNA occurs in membrane-bound RCs. Given the localization of DV NS4B, we assumed that this protein is part of the RNA replication machinery. To address this assumption we performed dual labeling of infected Huh-7 cells (fixed 24 h post-infection) by using an antibody that recognizes double-stranded (ds) RNA. Large perinuclear foci stained with this antibody were observed in DV-infected but not in non-infected cells (compare Fig. 3, B and E). DsRNA present in these foci colocalized with calnexin (an integral protein of the ER membrane), which otherwise displayed a coarse reticular network (Fig. 3, D-F). This subcellular distribution was different from the one in non-infected cells (Fig. 3, A-C). By using the antibody directed against DV NS4B we found an extensive overlap with the dsRNA foci in the perinuclear region, whereas NS4B foci present in the cell periphery did not perfectly colocalize with dsRNA (Fig. 3, G-I). Moreover NS3, comprising helicase and protease activity as well as the structural protein E colocalized with the dsRNA foci in the perinuclear region, whereas the smaller dots in the cell periphery did not contain detectable amounts of dsRNA (Fig. 3, J-L and P-R). By using a mouse monoclonal antibody directed against the viral E protein we found the analogous colocalization pattern between E, NS3, and NS4B (supplementary Fig. 1). In contrast, no colocalization was observed between dsRNA and DV NS5 that localizes primarily to the nucleus (Fig. 3, M-O). Taken together, these data indicate that DV dsRNA is found in dot-like structures derived from the ER and that NS4B as well as NS3 and E are included in these structures that most likely represent the sites of viral RNA replication.

NS4B Is an Integral Membrane Protein and Its Membrane Association Is 2K Independent—To characterize the intracellular membrane association of DV NS4B we performed equilibrium density gradient centrifugation to examine the flotation behavior of NS4B in detergent-free lysates of DV-infected cells. Postnuclear lysates were adjusted to 37.5% Nycodenz, loaded under a 5-35% Nycodenz gradient, and centrifuged to equilibrium. Eight fractions were collected from the top of the gradient and equal volume samples of each fraction were separated by SDS-PAGE and analyzed by Western blotting. Under these conditions, membrane-associated proteins float to low density (LD; fractions 1-4), as indicated by the distribution of calnexin, an integral ER membrane protein that was used as a marker for the gradient analysis (Fig. 4C, top). Like calnexin, DV NS4B was detected almost exclusively in the LD fractions indicating its membrane association (Fig. 4A, top). To analyze the nature of NS4B membrane association in more detail, we examined its flotation after treatment of lysates with conditions that discriminate between peripheral and transmembrane association. To this end, the postnuclear lysates of DV-infected cells were centrifuged at 20,000 x g, and the pelleted membranes were subjected to different extraction methods: high salt treatment that weakens ionic interactions between peripheral proteins and membranes or other membrane proteins (39), and treatment with high pH that should release peripheral proteins by transforming microsomal structures into membrane sheets (40). Treatment with 0.5% Triton X-100 was used to disrupt membranes and to release trans-membrane proteins. Calnexin, used as a control for an integral membrane protein, remained in the LD fractions after treatment with 1 M NaCl and 0.1 M Na2CO3 (pH 11.5) but was recovered in the high density fractions after disruption of the cellular membranes with Triton X-100 (Fig. 4C). In contrast, a large amount of the peripheral protein calreticulin shifted to the high density (HD) fractions after high salt or low pH treatments. Applying the same conditions to NS4B we found that it sedimented to HD only after pre-treatment of lysates with detergent (Fig. 4A). We therefore concluded that NS4B most likely is an integral trans-membrane protein.

Previous studies indicate that the 2K fragment at the COOH terminus of yellow fever virus NS4A serves as a signal sequence for the translocation of NS4B into the lumen of the ER (27). In addition, portions of in vitro translated yellow fever virus NS4B may be capable of insertion or association with microsomal membranes even in the absence of an NH2-terminal signal sequence. To investigate whether DV NS4B can associate with or integrate into membranes in the absence of its preceding signal sequence, we performed membrane dissociation assays and Nycodenz gradient fractionation by using cells transfected with COOH-terminal HA-tagged DV NS4B. Comparable with NS4B in infected cells, all DV 4B-HA floated to LD fractions even after treatment with 1 M NaCl or 0.1 M Na2CO3 (pH 11.5) (Fig. 4B). Thus, membrane integration of DV NS4B is independent from its preceding 2K signal sequence.


Figure 2
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  		FIGURE 2.
Colocalization of DV NS4B with cellular marker proteins. Twenty-four h after infection of Huh-7 cells with DV2, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed for immunolabeling. Panels to the left depict cells stained with the NS4B-specific antiserum (A, D, G, J, M, and P) and panels in the middle depict the immunostaining of cellular markers specified in the lower left of each pictures. Merged pictures are shown on the right (C, F, I, L, O, and R). Nuclear DNA was stained with 4',6'-diamidino-2-phenylindole dihydrochloride. Slides were analyzed by confocal laser scanning microscopy.

 
NS4B Sequences Involved in Membrane Association—The data described thus far show that DV2 NS4B is an integral membrane protein and that membrane association does not depend on the 2K signal sequence. To establish a membrane topology model of NS4B we first compared predictions of trans-membrane domains (TMDs) obtained with different algorithms by using the DV2 NGC strain 2K-NS4B sequence (Fig. 5A). Three TMDs were predicted in the COOH-terminal half of NS4B at approximately the same positions by five of six programs, whereas predictions for the NH2-terminal half of the protein were much more divergent. Two potential NH2-terminal TMDs were suggested by ConPredII, SOSUI, and TMpred (possibility II; Fig. 5A, d, e, and f), whereas only one TMD is anticipated by TMpred (possibility I; Fig. 5A, c). No TMD in the NH2-terminal NS4B region was predicted by 2 other programs (Fig. 5A, a and b). Because cleavage at the 2K-4B site occurs in the ER lumen by the host signalase (27), the NH2 terminus of NS4B must be in the ER lumen, at least during cleavage. Moreover, the COOH terminus of NS4B is generated by the viral protease in the cytosol (4, 41), and therefore must be at the cytoplasmic site of the ER, at least during processing. For this reason we assumed that a topology with 2TMDsornoTMDintheNH2-terminal half of NS4B and 3 TMDs in its COOH-terminal half is the most likely possibility. To discriminate between predicted and experimentally proven TMDs, the first ones are designated potential transmembrane domains (pTMDs) throughout this report.


Figure 3
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  		FIGURE 3.
Colocalization of DV NS4B with viral dsRNA. Huh-7 cells were infected with DV2 (D-R) or left uninfected (A-C). At 24 h post-infection cells were fixed and processed for immunolabeling. Used antibodies are given in the lower left of each picture. Merged pictures are shown on the right (C, F, I, L, O, and R). Slides were analyzed by confocal laser scanning microscopy.

 
To determine the regions of NS4B mediating membrane integration, deletion mapping analyses were performed. Based on a consensus model for the topology of NS4B containing a maximum of 5 pTMDs (Fig. 5B, top), plasmids were constructed directing the expression of NS4B pTMDs fused at their COOH terminus to the eGFP (Fig. 5B). Fragments fused to eGFP were larger than the pTMDs predicted by a single program to minimize the risk of missing regions important for membrane insertion. Expression plasmids were transfected into Huh-7/T7 cells and 8 h post-transfection cells were fixed and immunostained with an anti-GFP antibody. As expected, the uniform cellular distribution of eGFP was altered by fusion with the 2K fragment to a cytoplasmic reticular staining pattern (Fig. 5C), perfectly overlapping with the ER marker calnexin (data not shown). A comparable reticular staining pattern was also observed with the proteins in which the 2K signal sequence was fused to the first pTMD (residues -23 to 56), or the first and second pTMDs (residues -23 to 93). Interestingly, this reticular staining pattern was also found for the protein in which pTMDs 1-3 without the preceding 2K sequence were fused to eGFP (residues 1-146) as well as for the proteins containing the individual pTMD 3 (residues 93-146), pTMD 4 (residues 146-190), or pTMD 5 (residues 190-248) fused to eGFP. Only the proteins including the first (residues 1-56), the second (residues 56-93), or the first and second pTMDs (residues 1-93) fused to eGFP did not display an ER-like staining pattern. These results suggest that sequences including pTMD 3, 4, and 5, but not pTMD 1 and 2, mediate membrane targeting of eGFP.


Figure 4
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  		FIGURE 4.
Characterization of the membrane association of DV NS4B. Postnuclear pellets of DV-infected Huh-7 cells (A, C, and D) and pTM-NS4B-HA-transfected Huh-7/T7 cells (B) were centrifuged at 20,000 x g for 10 min. Pellets were treated with 1 M NaCl, 0.1 M Na2CO3 (pH 11.5), 0.5% Triton X-100, or PBS and subjected to Nycodenz gradient fractionation. Fractions were collected from the top to the bottom of the tube. Fractions 1-4 represent the LD fractions, fractions 5-8 the HD fractions. Proteins in equal volumes of each fraction were separated by SDS-PAGE and Western blot analyses with antisera against DV NS4B (A), the HA tag (B), calnexin (C), or calreticulin (D) were performed. Calnexin served as a control for an integral membrane protein, calreticulin was used as a control for a peripheral membrane protein.

 
To examine the nature of the membrane association of some NS4B-eGFP fusion proteins in lysates of transfected cells, equilibrium density gradient centrifugation experiments were performed (Fig. 5D). Soluble eGFP was detected almost exclusively in the HD fractions, whereas after fusion with the 2K fragment approximately half of the fusion protein sedimented to membranous (LD) fractions. By far the highest amounts of 4B-(1-56)-eGFP and 4B-(56-93)-eGFP, corresponding to the first or second pTMD, respectively, were found in the HD fractions, indicating that these pTMDs were not sufficient to mediate membrane association of eGFP. In contrast, nearly all eGFP was detected in the membrane-containing LD fractions after fusion of eGFP with pTMD 3 (residues 93-146), pTMD 4 (residues 146-190), or pTMD 5 (residues 190-248). These data clearly support the results of the immunofluorescence studies and show that pTMDs 3, 4, and 5 contain membrane targeting sequences.

Membrane association of these fusion proteins was investigated in more detail by treatment of membrane pellets of cells transfected with the corresponding constructs with 0.1 M Na2CO3 (pH 11.5) prior to Nycodenz gradient fractionation. Western blot analysis of the corresponding samples revealed that even after this treatment the majority of eGFP fusion proteins containing the pTMDs 3, 4, or 5 was detectable in the LD fractions associated with membranes (4B-(93-146)-eGFP: 75.4%; 4B-(146-190)-eGFP: 99.9%; 4B-(190-248)-eGFP: 96.8%; data not shown), supporting the notion that pTMDs 3, 4, and 5 are integrated into intracellular membranes and can serve as internal signal sequences of DV2 NS4B.

Probing DV NS4B Topology by Protease Protection Assays—To characterize the membrane topology of DV NS4B in further detail, proteinase K protection assays were performed. To this end, we transfected Huh-7/T7 cells with constructs in which eGFP was fused with COOH-terminal truncations of NS4B after each pTMD (see Fig. 6A). Lysates of transfected cells were treated with proteinase K and Western blot analysis with a GFP-specific antibody was performed. Under these conditions the localization of the eGFP part of the corresponding fusion proteins (either cytoplasmic or luminal) depends on the ability of the preceding pTMD(s) to serve as membrane spanning fragments. In case of a luminal localization eGFP should be protected against protease digestion, whereas cytoplasmic eGFP should be degraded. As shown in Fig. 6B, eGFP fusion proteins carrying the 2K fragment or the 2K fragment together with the first pTMD (residues -23 to 56) were completely protected against protease digestion, indicating a luminal localization of the reporter. After disruption of the membranes with Triton X-100 the proteins were completely degraded. Likewise the eGFP fusion protein comprising the 2K fragment and pTMDs 1 and 2 (residues -23 to 93) was proteinase K resistant. Interestingly, this construct gave rise to 2 bands differing in their apparent molecular mass by about 2-3 kDa. Treatment of cell lysates with PNGase F revealed that the upper band corresponded to a glycosylated form (data not shown). Indeed, we identified a potential N-glycosylation site in pTMD 2 at aa position 62 of NS4B. The finding that the 2K-4B-(-23-93)-eGFP protein is completely protected against proteinase K digestion and is glycosylated indicates that the sequence comprising pTMD 2 localizes to the ER lumen. Because glycosylation was observed with all eGFP fusion proteins containing pTMD 2, this part of the protein most likely localizes to the ER lumen independent of its sequence environment. It should be pointed out that a NS4B double band was also found in DV-infected cells (data not shown), but because of production of a rather stable 2K-4B protein we could not firmly establish weather the upper band represents only 2K-4B or also glycosylated NS4B.

Unexpectedly, in the presence of Triton X-100 a smaller fragment of ~28 kDa still reacting with the GFP-specific antibody was detected with 2K-4B-(-23-93)-eGFP (see Fig. 6B). Fragments of comparable size were also observed after Triton X-100 and proteinase K treatment with the proteins containing pTMDs 1-3 (residues -23 to 146), and pTMDs 1-5 (residues -23 to 248). These bands probably represent an intrinsically protease-resistant form of eGFP.

In the case of the fusion protein containing pTMDs 1-3 (residues -23 to 146), the COOH-terminal eGFP was completely digested by the protease, indicating its cytoplasmic localization. This result suggested that pTMD 3 is the first membrane spanning segment of DV NS4B. In the case of the fusion protein comprising 2K and pTMDs 1-4 (residues -23 to 190), a protected fragment ~34 kDa in size was observed. This fragment was about 20 kDa smaller than the corresponding full-length protein and it was eliminated after the addition of Triton X-100, demonstrating that it does not represent an intrinsically protease-resistant portion of the molecule. Based on the 20-kDa reduction in molecular size, we inferred that proteinase K removed the 2K fragment and the ~160 NH2-terminal aa residues of NS4B, whereas aa residues 160-190 of NS4B fused to eGFP were protected. This observation provides strong evidence for the existence of a second TMD in the region corresponding to pTMD 4 of DV NS4B and it implies the existence of a cytoplasmic loop preceding this TMD.


Figure 5
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  		FIGURE 5.
Computer topology predictions and deletion mapping analysis of DV NS4B. A, computer predictions of the topology of DV2-NGC 2K-NS4B by using multiple software packages. Black boxes represent the 2K fragment (starting with residue-23), which serves as a signal sequence for the NH2 terminus of NS4B. White boxes indicate pTMDs. The positions of the first and the last amino acid residue of the putative membrane spanning segments are given. B, schematic representation of the DV NS4B-eGFP fusion proteins used for deletion mapping analysis. Several plasmids expressing pTMDs fused alone or in combinations to eGFP were constructed. C, IF analysis of Huh-7/T7 cells fixed 8 h post-transfection with DV NS4B-eGFP fusion constructs. The names of the corresponding constructs are given below each panel. D, equilibrium density gradient analysis of lysates of Huh-7/T7 cells transfected with constructs expressing pTMDs fused to eGFP. Aliquots of each fraction were immunoblotted by using an antibody directed against eGFP. The percentage of signals detected in the LD fractions (1-4) and HD fractions (5-8) is given below each panel.

 
Western blot analysis of proteinase K-treated lysates of full-length 2K-4B-eGFP (residues -23 to 248) expressing cells revealed that most of the protein was protease sensitive indicating the cytoplasmic localization of eGFP. This result is in keeping with the notion that the region between residues 190 and 248 of NS4B indeed carries a TMD. However, a weak band of ~40 kDa was still visible after proteinase K treatment but not after Triton X-100 treatment. The size of this protected fragment indicates that approximately the last 100 amino acids of NS4B and the COOH-terminal eGFP were protected. This region includes pTMDs 4 and 5. Thus, it is possible that a small portion of the COOH terminus of NS4B resides in the ER lumen, whereas the majority is located on the cytoplasmic site. As described above, the smaller fragment that is not degraded by proteinase K even after Triton X-100 treatment most likely corresponds to a protease-resistant eGFP fragment.


Figure 6
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  		FIGURE 6.
Probing DV NS4B topology by protease protection assays. A, schematic representation of DV 2K-NS4B-eGFP fusion proteins. Several plasmids in which the coding region of eGFP was fused to the 3'-end of given pTMDs were constructed. Black boxes represent the 2K fragment, white boxes the pTMDs. B, Huh-7/T7 cells were transfected with the corresponding constructs. Postnuclear lysates were centrifuged at 20,000 x g for 10 min and resuspended pellets were left untreated, or were digested with proteinase K in the absence or presence of Triton X-100. Thereafter samples were subjected to SDS-PAGE followed by immunoblotting with GFP-specific antiserum. Molecular weight markers are depicted on the left.

 
Taken together, these results suggest that the 2K fragment and pTMD 4 span the membrane from the cytoplasmic to the luminal site, and pTMDs 3 and 5 from the luminal to the cytoplasmic site. pTMDs 1 and 2 most likely do not span the membrane, but seem to reside in the ER lumen. The COOH terminus of NS4B is predominantly on the cytoplasmic site, but may flip through the membrane with low frequency.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we determined the subcellular localization, membrane association, and membrane topology of DV NS4B.

Localization of NS4B in Infected Cells—By performing confocal immunofluorescence microscopy of DV2-infected cells we found that NS4B is localized in reticular structures and large cytoplasmic foci accumulating during viral replication. Dual-labeling experiments revealed that these structures seem to originate from the ER, and most likely not from the Golgi, the trans-Golgi, or from the intermediate compartment. Also viral proteins E and NS3, and viral dsRNA, an intermediate of viral replication, were found in these ER-derived structures, arguing that NS4B is part of the membrane-bound RC. NS5, the RNA-dependent RNA polymerase of the virus, was found predominantly in the nucleus of infected cells in agreement with previous reports (37, 38).

The results of our colocalization experiments are in part consistent with the observations of Munõz-Jordán and co-workers (32). They found that DV2 NS4B colocalizes with the ER marker calnexin in Vero cells transfected with a plasmid encoding for DV 2K-NS4B-HA. In contrast to our results, they did not observe cytoplasmic foci but rather a reticular staining pattern, arguing that cells transfected with this expression construct do not properly reflect the situation in infected cells. So far, it is unclear what the observed cytoplasmic foci are and how they develop, but comparable foci have also been identified in cells infected with other positive stranded RNA viruses, for example, the KUNV (42). This virus induces membrane structures called convoluted membranes and paracrystalline structures, representing the putative sites of viral polyprotein processing. In addition, proliferating ER and vesicles of about 100 nm in diameter described as small, spherical smooth membrane structures that seem to harbor the viral RCs were observed (9, 29, 43-46). It is possible that the cytoplasmic foci observed in our DV studies represent one or several of these membrane structures. The visible change in the ER architecture from the fine reticular network to foci-like structures in DV-infected cells over time and the extensive colocalization of dsRNA with NS3, NS4B, and E in these foci supports this possibility. Although there are hints that NS4B of some Flaviviruses is important for RNA replication (30, 31, 47, 48), KUNV NS4B was not detectable in partly purified replicase complexes, in contrast to NS1, NS2A, NS3, and NS4A (29). Moreover, KUNV NS4B was also not incorporated into convoluted membrane/paracrystalline structures, the presumable sites of polyprotein processing, in contrast to NS2B, NS3, and NS4A. However, the colocalization of DV NS4B with dsRNA as observed in our study suggests a role of this protein in DV replication. Further studies are in progress to unravel the role of the DV NS4B protein in the viral life cycle.


Figure 7
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  		FIGURE 7.
Model for the membrane topology of DV 2K-NS4B. 2K serves as a signal sequence for the translocation of the NH2 terminus of NS4B into the lumen of the ER. Our data indicate that predicted TMDs 1 and 2 do not span the membrane but rather reside in the ER lumen. TMDs 3, 4, and 5 span the membrane, and serve as internal signal sequences for membrane association. Glycosylation may occur at an internal N-glycosylation site in the predicted TMD 2.

 
It was described that NS4B of HCV is able to induce distinct membrane alterations in transfected cells (49). We and others could not detect comparable structures after transfection of Huh-7 and Vero cells with plasmids encoding for DV NS4B or KUNV NS4B (28, 32). These results suggest that the NS4B proteins of different genera of the Flaviviridae family carry out different functions.

Membrane Association and Topology of DV NS4B—Previous studies indicate that NS4B proteins of flaviviruses are targeted to the ER membrane by the 2K signal sequence located in the preceding NS4A protein (27). We also found that DV NS4B was stably associated with intracellular membranes. Moreover, by using several biochemical approaches we showed that this protein is integrated into membranes independent from the signal sequence residing in 2K, indicating that NS4B also comprises internal signal sequences. These observations are concordant with the results of in vitro translation experiments of yellow fever virus NS4B. It was suggested that portions of this protein might be capable of insertion into or association with microsomal membranes even in the absence of an NH2-terminal signal sequence (27).

To investigate the membrane association and the topology of DV NS4B in more detail, we first established a theoretical model derived from computer-based predictions. Three pTMDs clearly separated from each other by loop regions were predicted in the COOH-terminal half of NS4B by nearly all programs used, whereas the NH2-terminal half of the protein was more difficult to interpret. The three COOH-terminal pTMDs could be confirmed by several experimental approaches. They are located between amino acids 93-146, 146-190, and 190-248 of NS4B, and they are sufficient to target eGFP, a cytosolic protein, to intracellular membrane compartments. This shows that they can act as internal signal sequences of DV NS4B. Potential TMDs predicted by three of six computer programs in the NH2-terminal half of the protein (aa 1-56 and aa 56-93) were not sufficient to mediate membrane targeting of eGFP. Deletion mapping studies and proteinase K protection experiments lead to the model depicted in Fig. 7. The 2K fragment and pTMD 4 span the membrane from the cytoplasmic to the luminal site, and pTMDs 3 and 5 from the luminal to the cytoplasmic site. The pTMDs 1 and 2 most probably do not span the membrane. The NH2 terminus of DV NS4B is localized in the ER lumen where it is processed by the signalase of the host cell. The COOH terminus resides in the cytoplasm where cleavage by the viral protease occurs. We cannot exclude that the topology of NS4B expressed on its own differs from the topology of NS4B expressed in the context of the viral polyprotein. Attempts to determine NS4B topology by using DV-infected cells were not successful because the insertion of small epitope tags into different sites of NS4B led to a complete loss of viral RNA replication.4 Moreover, our DV NS4B-specific antibody is directed against the potential cytoplasmic loop region, and antibodies directed against other NS4B regions are not available. It is therefore not possible at this stage to determine NS4B topology in DV-infected cells.

Whereas it is difficult to envisage how the overall topology of NS4B would be affected when it is expressed out of the context of the polyprotein, it is possible that the luminal localization of the COOH terminus of a minor population of NS4B-eGFP fusion proteins is an artifact because of the non-natural sequence at the COOH terminus. On the other hand, if this topology holds true for authentic NS4B, our findings would indicate that the COOH-terminal region in a minor fraction of NS4B molecules slips post-translationally through the membrane after cleavage by the viral protease. Comparable post-translational reorientations of membrane proteins have been described for other viral proteins, for example, for the M protein of the gastroenteritis corona virus (50, 51), the hepatitis B virus large envelope protein (52-55), and the NH2 terminus of the HCV NS4B (26).

The best investigated function of flavivirus NS4B is its ability to block the IFN-{alpha}/beta-induced signal transduction cascade by interfering with phosphorylation of STAT1 (22, 32). Previous studies have shown that aa residues 54-102 of NS4B are required for IFN antagonism (32). Munõz-Jordán and co-workers (32) assumed that this part of the protein is localized in the cytoplasm between the first and the second TMD of NS4B. Their assumption was based on the topology model established for HCV NS4B and on computer predictions for the topology of NS4B of other members of the Flaviviridae family (26, 56). However, our results show that the DV NS4B region responsible for IFN-{alpha}/beta inhibition seems to be located in the ER lumen (Fig. 7). For this reason a direct interaction between DV NS4B and cellular cytoplasmic components involved in IFN signaling is not likely. We therefore hypothesize that NS4B-mediated inhibition of IFN signaling is achieved through an indirect effect, for example, by activation of cellular inhibitors of the Janus kinase/STAT pathway.

Apart from this inhibitory function some studies indicate that flavivirus NS4B plays an additional more direct role for RNA replication. Mutational analysis showed that DV4 NS4B could be involved in maintaining the balance between efficient replication in the mosquito vector and the human host (48). Furthermore, deletion studies and trans-complementation experiments with bovine viral diarrhea virus and KUNV indicate an important role of NS4B in viral replication (30, 31). Interestingly, in the case of HCV NS4B, which has negligible sequence identity of DV NS4B but a similar hydrophobicity profile, an ER or ER-derived membrane localization was found (25, 26, 57, 58). Membrane association of HCV NS4B occurs cotranslationally, and the protein behaves like an integral membrane protein (25). A comparison between our topology model and the topology model suggested for the HCV NS4B (26) revealed that the COOH-terminal region of HCV NS4B contains 4 TMDs, whereas the NH2-terminal part may contain an amphipathic helix (24). Several functions have been reported for NS4B of HCV, including translation inhibition (59, 60), modulation of NS5B enzymatic activity (61), and transformation of cells (62). In addition, it was described that HCV NS4B induces intracellular membrane rearrangements involved in the formation of the membranous web that is the site of HCV replication (49). In contrast, the exact function of DV NS4B has to be determined.

In conclusion, our experiments provide a first characterization of DV2 NS4B. Our topology model may be an excellent starting point for a detailed investigation of the functions of this protein in the viral life cycle.


   FOOTNOTES
 
* This work was supported by an unrestricted grant of the Bristol-Myers-Squibb Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 To whom correspondence may be addressed. E-mail: sandra_sparacio{at}med.uni-heidelberg.de.

2 To whom correspondence may be addressed: Im Neuenheimer Feld 345, 69120 Heidelberg, Germany. Tel.: 49-6221-56-7761; Fax: 49-6221-56-4570; E-mail: ralf_bartenschlager{at}med.uni-heidelberg.de.

3 The abbreviations used are: DV, Dengue virus; aa, amino acids; CLIMP, cytoskeleton linking membrane protein; dsRNA, double-stranded RNA; eGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GalT, galactosyltransferase; GST, glutathione S-transferase; HCV, hepatitis C virus; HD, high density; IFN-{alpha}/beta, interferon-{alpha}/beta; KUNV, Kunjin virus; LD, low density; NGC, New Guinea C-strain; NS, non-structural; PBS, phosphate-buffered saline; pTMD, predicted trans-membrane domain; RC, replication complex; ER, endoplasmic reticulum; STAT, transducers and activators of transcription; TMD, trans-membrane domain; HA, hemagglutinin. Back

4 S. Miller, S. Sparacio, and R. Bartenschlager, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Ulrike Herian for excellent technical assistance and our colleagues for helpful discussions during the course of this work. We also thank Volker Brass and Darius Moradpour for critical comments on the manuscript. We are grateful to Andrew Davidson for providing the DV-NGC encoding plasmid, and Eric Berger for providing the GalT antibody. We thank the Department of Parasitology at the University of Heidelberg for providing access to their confocal immunofluorescence microscope.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gubler, D. J. (2002) Trends Microbiol. 10, 100-103[CrossRef][Medline] [Order article via Infotrieve]
  2. Amberg, S. M., Nestorowicz, A., McCourt, D. W., and Rice, C. M. (1994) J. Virol. 68, 3794-3802[Abstract/Free Full Text]
  3. Cahour, A., Falgout, B., and Lai, C. J. (1992) J. Virol. 66, 1535-1542[Abstract/Free Full Text]
  4. Falgout, B., Pethel, M., Zhang, Y. M., and Lai, C. J. (1991) J. Virol. 65, 2467-2475[Abstract/Free Full Text]
  5. Falgout, B., and Markoff, L. (1995) J. Virol. 69, 7232-7243[Abstract]
  6. Murray, J. M., Aaskov, J. G., and Wright, P. J. (1993) J. Gen. Virol. 74, 175-182[Abstract/Free Full Text]
  7. Stadler, K., Allison, S. L., Schalich, J., and Heinz, F. X. (1997) J. Virol. 71, 8475-8481[Abstract]
  8. Mackenzie, J. M., Khromykh, A. A., Jones, M. K., and Westaway, E. G. (1998) Virology 245, 203-215[CrossRef][Medline] [Order article via Infotrieve]
  9. Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K., and Khromykh, A. A. (1997) J. Virol. 71, 6650-6661[Abstract]
  10. Benarroch, D., Egloff, M. P., Mulard, L., Guerreiro, C., Romette, J. L., and Canard, B. (2004) J. Biol. Chem. 279, 35638-35643[Abstract/Free Full Text]
  11. Chambers, T. J., Hahn, C. S., Galler, R., and Rice, C. M. (1990) Annu. Rev. Microbiol. 44, 649-688[CrossRef][Medline] [Order article via Infotrieve]
  12. Egloff, M. P., Benarroch, D., Selisko, B., Romette, J. L., and Canard, B. (2002) EMBO J. 21, 2757-2768[CrossRef][Medline] [Order article via Infotrieve]
  13. Tan, B. H., Fu, J., Sugrue, R. J., Yap, E. H., Chan, Y. C., and Tan, Y. H. (1996) Virology 216, 317-325[CrossRef][Medline] [Order article via Infotrieve]
  14. Chambers, T. J., Weir, R. C., Grakoui, A., McCourt, D. W., Bazan, J. F., Fletterick, R. J., and Rice, C. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8898-8902[Abstract/Free Full Text]
  15. Gorbalenya, A. E., Donchenko, A. P., Koonin, E. V., and Blinov, V. M. (1989) Nucleic Acids Res. 17, 3889-3897[Abstract/Free Full Text]
  16. Wengler, G. (1993) Virology 197, 265-273[CrossRef][Medline] [Order article via Infotrieve]
  17. Lindenbach, B. D., and Rice, C. M. (1997) J. Virol. 71, 9608-9617[Abstract]
  18. Lindenbach, B. D., and Rice, C. M. (1999) J. Virol. 73, 4611-4621[Abstract/Free Full Text]
  19. Chambers, T. J., McCourt, D. W., and Rice, C. M. (1989) Virology 169, 100-109[CrossRef][Medline] [Order article via Infotrieve]
  20. Guo, J. T., Hayashi, J., and Seeger, C. (2005) J. Virol. 79, 1343-1350[Abstract/Free Full Text]
  21. Liu, W. J., Wang, X. J., Mokhonov, V. V., Shi, P. Y., Randall, R., and Khromykh, A. A. (2005) J. Virol. 79, 1934-1942[Abstract/Free Full Text]
  22. Munoz-Jordan, J. L., Sanchez-Burgos, G. G., Laurent-Rolle, M., and Garcia-Sastre, A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14333-14338[Abstract/Free Full Text]
  23. Einav, S., Elazar, M., Danieli, T., and Glenn, J. S. (2004) J. Virol. 78, 11288-11295[Abstract/Free Full Text]
  24. Elazar, M., Liu, P., Rice, C. M., and Glenn, J. S. (2004) J. Virol. 78, 11393-11400[Abstract/Free Full Text]
  25. Hugle, T., Fehrmann, F., Bieck, E., Kohara, M., Krausslich, H. G., Rice, C. M., Blum, H. E., and Moradpour, D. (2001) Virology 284, 70-81[CrossRef][Medline] [Order article via Infotrieve]
  26. Lundin, M., Monne, M., Widell, A., Von Heijne, G., and Persson, M. A. (2003) J. Virol. 77, 5428-5438[Abstract/Free Full Text]
  27. Lin, C., Amberg, S. M., Chambers, T. J., and Rice, C. M. (1993) J. Virol. 67, 2327-2335[Abstract/Free Full Text]
  28. Westaway, E. G., Khromykh, A. A., Kenney, M. T., Mackenzie, J. M., and Jones, M. K. (1997) Virology 234, 31-41[CrossRef][Medline] [Order article via Infotrieve]
  29. Chu, P. W., and Westaway, E. G. (1992) Arch. Virol. 125, 177-191[CrossRef][Medline] [Order article via Infotrieve]
  30. Grassmann, C. W., Isken, O., Tautz, N., and Behrens, S. E. (2001) J. Virol. 75, 7791-7802[Abstract/Free Full Text]
  31. Khromykh, A. A., Sedlak, P. L., and Westaway, E. G. (2000) J. Virol. 74, 3253-3263[Abstract/Free Full Text]
  32. Munoz-Jordan, J. L., Laurent-Rolle, M., Ashour, J., Martinez-Sobrido, L., Ashok, M., Lipkin, W. I., and Garcia-Sastre, A. (2005) J. Virol. 79, 8004-8013[Abstract/Free Full Text]
  33. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., and Sato, J. (1982) Cancer Res. 42, 3858-3863[Abstract/Free Full Text]
  34. Appel, N., Pietschmann, T., and Bartenschlager, R. (2005) J. Virol. 79, 3187-3194[Abstract/Free Full Text]
  35. Sambrook, J., and Russel, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  36. Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and Fuerst, T. R. (1990) Nature 348, 91-92[CrossRef][Medline] [Order article via Infotrieve]
  37. Buckley, A., Gaidamovich, S., Turchinskaya, A., and Gould, E. A. (1992) J. Gen. Virol. 73, 1125-1130[Abstract/Free Full Text]
  38. Kapoor, M., Zhang, L., Ramachandra, M., Kusukawa, J., Ebner, K. E., and Padmanabhan, R. (1995) J. Biol. Chem. 270, 19100-19106[Abstract/Free Full Text]
  39. Kretzschmar, E., Bui, M., and Rose, J. K. (1996) Virology 220, 37-45[CrossRef][Medline] [Order article via Infotrieve]
  40. Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102[Abstract/Free Full Text]
  41. Preugschat, F., and Strauss, J. H. (1991) Virology 185, 689-697[CrossRef][Medline] [Order article via Infotrieve]
  42. Ng, M. L., Pedersen, J. S., Toh, B. H., and Westaway, E. G. (1983) Arch. Virol. 78, 177-190[CrossRef][Medline] [Order article via Infotrieve]
  43. Chu, P. W., Westaway, E. G., and Coia, G. (1992) J. Virol. Methods 37, 219-234[CrossRef][Medline] [Order article via Infotrieve]
  44. Leary, K., and Blair, C. D. (1980) J. Ultrastruct. Res. 72, 123-129[CrossRef][Medline] [Order article via Infotrieve]
  45. Mackenzie, J. M., Jones, M. K., and Young, P. R. (1996) Virology 220, 232-240[CrossRef][Medline] [Order article via Infotrieve]
  46. Ng, M. L., and Hong, S. S. (1989) Arch. Virol. 106, 103-120[CrossRef][Medline] [Order article via Infotrieve]
  47. Blaney, J. E., Jr., Manipon, G. G., Firestone, C. Y., Johnson, D. H., Hanson, C. T., Murphy, B. R., and Whitehead, S. S. (2003) Vaccine 21, 4317-4327[CrossRef][Medline] [Order article via Infotrieve]
  48. Hanley, K. A., Manlucu, L. R., Gilmore, L. E., Blaney, J. E., Jr., Hanson, C. T., Murphy, B. R., and Whitehead, S. S. (2003) Virology 312, 222-232[CrossRef][Medline] [Order article via Infotrieve]
  49. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D., and Bienz, K. (2002) J. Virol. 76, 5974-5984[Abstract/Free Full Text]
  50. Escors, D., Camafeita, E., Ortego, J., Laude, H., and Enjuanes, L. (2001) J. Virol. 75, 12228-12240[Abstract/Free Full Text]
  51. Risco, C., Anton, I. M., Sune, C., Pedregosa, A. M., Martin-Alonso, J. M., Parra, F., Carrascosa, J. L., and Enjuanes, L. (1995) J. Virol. 69, 5269-5277[Abstract]
  52. Bruss, V., Lu, X., Thomssen, R., and Gerlich, W. H. (1994) EMBO J. 13, 2273-2279[Medline] [Order article via Infotrieve]
  53. Lambert, C., and Prange, R. (2001) J. Biol. Chem. 276, 22265-22272[Abstract/Free Full Text]
  54. Ostapchuk, P., Hearing, P., and Ganem, D. (1994) EMBO J. 13, 1048-1057[Medline] [Order article via Infotrieve]
  55. Prange, R., and Streeck, R. E. (1995) EMBO J. 14, 247-256[Medline] [Order article via Infotrieve]
  56. Qu, L., McMullan, L. K., and Rice, C. M. (2001) J. Virol. 75, 10651-10662[Abstract/Free Full Text]
  57. Kim, M., Mackenzie, J. M., and Westaway, E. G. (2004) J. Virol. Methods 120, 179-187[CrossRef][Medline] [Order article via Infotrieve]
  58. Selby, M. J., Choo, Q. L., Berger, K., Kuo, G., Glazer, E., Eckart, M., Lee, C., Chien, D., Kuo, C., and Houghton, M. (1993) J. Gen. Virol. 74, 1103-1113[Abstract/Free Full Text]
  59. Florese, R. H., Nagano-Fujii, M., Iwanaga, Y., Hidajat, R., and Hotta, H. (2002) Virus Res. 90, 119-131[CrossRef][Medline] [Order article via Infotrieve]
  60. Kato, J., Kato, N., Yoshida, H., Ono-Nita, S. K., Shiratori, Y., and Omata, M. (2002) J. Med. Virol. 66, 187-199[CrossRef][Medline] [Order article via Infotrieve]
  61. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K. D., and McCarthy, J. E. (2002) J. Biol. Chem. 277, 45670-45679[Abstract/Free Full Text]
  62. Park, J. S., Yang, J. M., and Min, M. K. (2000) Biochem. Biophys. Res. Commun. 267, 581-587[CrossRef][Medline] [Order article via Infotrieve]

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